978 3 319 42740 9 PDF

Current Cancer Research
Jin-Ming Yang Editor
Targeting
Autophagy
in Cancer
Therapy
Series Editor
Wafik El-Deiry
More information about this series at http://www.springer.com/series/7892

Jin-Ming Yang
Editor
Targeting Autophagy
in Cancer Therapy
Editor
Jin-Ming Yang
Department of Pharmacology
The Pennsylvania State University
College of Medicine
Hershey, PA, USA
ISSN 2199-2584 ISSN 2199-2592 (electronic)

ISBN 978-3-319-42738-6 ISBN 978-3-319-42740-9 (eBook)
DOI 10.1007/978-3-319-42740-9
Library of Congress Control Number: 2016949552
© Springer International Publishing Switzerland 2016

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Preface
Over the past decade or so, an ever-increasing body of scientific evidence points to
the functional role and unmistakable importance of autophagy in cancer. But can
autophagy be successfully exploited as a target in effective cancer therapy? It is now
widely believed that modulating the activity of autophagy through targeting regula-
tory components in the autophagy machinery may impact the development, pro-
gression, and therapeutic outcome of cancer. Therefore, autophagy has been
considered a novel and promising target for drug discovery/development and thera-
peutic intervention for cancer; in fact, targeting of autophagy as a therapeutic strat-
egy in cancer has already been explored in-depth and has shown great promise. The
purpose of this volume is to provide the latest updates on the current status and a
unique perspective on autophagy-based cancer therapy. This volume in the Springer
series, Current Cancer Research, will cover a wide range of topics, including an
overview of autophagy as a therapeutic target in cancer, autophagy modulators as
cancer therapeutic agents, implications of micro RNA-regulated autophagy in can-
cer therapy, modulation of autophagy through targeting PI3 kinase in cancer ther-
apy, targeting autophagy in cancer stem cells, and the roles of autophagy in cancer
immunotherapy. In addition, this volume presents a chapter on the application of
system biology and bioinformatics approaches to discovering cancer therapeutic
targets in the autophagy regulatory network. This comprehensive volume is intended
to be useful to a wide range of basic and clinical scientists, including cancer biolo-
gists, autophagy researchers, pharmacologists, and clinical oncologists who wish to
delve more deeply into this exciting new research area.
Although there are already several excellent books that cover the biology and
molecular biology of autophagy and their association with cancer development and
progression, this is the first book devoted solely to dealing with targeting autophagy
in cancer therapy. As the implications and importance of autophagy in cancer ther-
apy have been increasingly appreciated, this timely and unique volume assembled
by leading scientists in this field should prove its usefulness and value in under-
standing, exploring, developing, and promoting autophagy-based cancer therapy.
This volume has the following distinguishing features: (1) it is the first book solely
focusing on autophagy as a target in cancer therapy; (2) it is a comprehensive
v
vi Preface
discussion on the roles of autophagy in currently available cancer treatments; (3) it

is a timely complement to the book (volume 8): Autophagy and Cancer, 2013, in
this series. Finally, I want to sincerely thank all of the authors for their contribution.
It is my earnest hope that this volume will serve as a catalyst for further exploration
and investigation of autophagy-based cancer therapy.
Hershey, PA, USA Jin-Ming Yang

Contents
1 Autophagy as a Therapeutic Target in Cancer ...................................... 1

Jenny Mae Samson and Andrew Thorburn
2 Autophagy in Cancer Cells vs. Cancer Tissues:
Two Different Stories ................................................................................ 17
Chi Zhang, Tao Sheng, Sha Cao, Samira Issa-Boube,
Tongyu Tang, Xiwen Zhu, Ning Dong, Wei Du, and Ying Xu
3 Small-Molecule Regulators of Autophagy as Potential
Anti-cancer Therapy................................................................................. 39
Qing Li, Mi Zhou, and Renxiao Wang
4 Regulation of Autophagy by microRNAs:
Implications in Cancer Therapy .............................................................. 59
Hua Zhu and Jin-Ming Yang
5 Targeting PI3-Kinases in Modulating Autophagy
and Anti-cancer Therapy ......................................................................... 85
Zhixun Dou and Wei-Xing Zong
6 Adult and Cancer Stem Cells: Perspectives on Autophagic
Fate Determinations and Molecular Intervention ................................. 99
Kevin G. Chen and Richard Calderone
7 Role of Autophagy in Tumor Progression and Regression.................... 117
Bassam Janji and Salem Chouaib
Erratum to: Adult and Cancer Stem Cells: Perspectives

on Autophagic Fate Determinations and Molecular Intervention.............. E1
Index ................................................................................................................. 133
vii
Chapter 1
Autophagy as a Therapeutic Target in Cancer
Jenny Mae Samson and Andrew Thorburn
Abstract Autophagy is the process by which cellular material is delivered to the

lysosome for degradation and recycling. Macroautophagy involves delivery of mac-
romolecules and organelles to double membrane vesicles called autophagosomes
that fuse with lysosomes leading to degradation of the contents of the autophago-
somes. Chaperone-mediated autophagy involves direct recognition of specific pro-
teins by chaperone complexes that then directly deliver the protein target to the
lysosome. Microautophagy involves direct lysosomal capture of cytoplasmic mate-
rial. Of these three types, macroautophagy is by far the most studied and is known
to have multiple roles in cancer development, progression and response to therapy.
This has led to autophagy being widely viewed as a potential therapeutic target in
cancer. Important questions that must be answered include: Which tumors should or
should not be treated by direct autophagy inhibition? And, what is the best way to
target autophagy for cancer therapy? In this overview we summarize the back-
ground and some current ideas about the answers to such questions.
Keywords Autophagy • Apoptosis • Cancer therapy • ATG7 • BRAF • KRAS
Autophagy is the process through which proteins, organelles, and other cellular
contents are degraded in lysosomes. Macroautophagy involves the formation of
double membrane vesicles called autophagosomes that engulf and sequester cellu-
lar material. The autophagosomes then fuse with lysosomes, generating autopha-
golysosomes, in which the lysosomal hydrolases degrade the delivered material into
their macromolecular precursors for reuse. While the process of autophagy was first
described in the early 1960s, it is only in the past 10–15 years that its role in cellular
homeostasis (Kaur and Debnath 2015), as well as in many diseases (Kroemer 2015;
Rubinsztein et al. 2012) has been recognized. Two other types of autophagy that do
J.M. Samson • A. Thorburn (*)

Department of Pharmacology, University of Colorado School of Medicine,
Aurora, CO 80045, USA
e-mail: Andrew.Thorburn@ucdenver.edu
© Springer International Publishing Switzerland 2016 1

J.-M. Yang (ed.), Targeting Autophagy in Cancer Therapy,
Current Cancer Research, DOI 10.1007/978-3-319-42740-9_1
2 J.M. Samson and A. Thorburn
not involve autophagosomes have been characterized: chaperone-mediated autoph-

agy and microautophagy. Chaperone-mediated autophagy (CMA) involves the
direct recognition of proteins by heat shock protein hsc70 through an exposed
amino acid (KFERQ) motif and subsequent delivery of the bound pair to the lyso-
some through the lysosomal protein LAMP2A (Arias and Cuervo 2011; Kaushik
et al. 2011). Microautophagy is less well understood than either CMA or macroau-
tophagy and may involve components of the autophagic machinery and endocytic
pathways that allow direct engulfment of cytoplasmic material into the lysosome
(Sahu et al. 2011). Most of the work related to autophagy in the context of cancer
refers to macroautophagy, though recent work has demonstrated the importance of
CMA in tumor growth and progression. Hereafter we use the term “autophagy” to
mean macroautophagy.
As we will discuss, autophagy’s involvement in cancer is confusing and often-
times contradictory with both pro- and anti-tumor effects found in different contexts
(Hippert et al. 2006; White 2012; Galluzzi et al. 2015) and during cancer therapy
(Thorburn et al. 2014). In January 2016 a search of the ClinicalTrials.gov website
with the search term “autophagy” returned 60 clinical studies across the world. The
majority of these clinical studies deliberately attempt to inhibit autophagy during
cancer therapy usually together other anti-cancer treatments. The first cancer clini-
cal trials of autophagy inhibitors were reported in 2014 (Barnard et al. 2014;
Rangwala et al. 2014a, b; Rosenfeld et al. 2014; Vogl et al. 2014; Wolpin et al.
2014). These attempts to target autophagy in cancer therapy contrasts with only a
few examples where deliberate autophagy manipulation is being attempted to treat
other diseases (Kroemer 2015). Thus, despite the fact that arguments can be made
for and against inhibiting autophagy in cancer and for the utility of autophagy
manipulation in infectious disease, neurodegenerative disease, metabolic disease
and many others (Kroemer 2015), it is in cancer treatment where we are furthest
along in trying to apply these ideas in a clinical setting. It is also important to note
that many current anti-cancer treatments themselves induce autophagy (Shen et al.
2011; Levy and Thorburn 2011). Conversely, some microtubule-targeting drugs
such as paclitaxel inhibit autophagy (Veldhoen et al. 2013). This means that we are
routinely affecting autophagy in cancer patients through their course of treatment
whether we intend to or not. In this chapter, we focus on the deliberate targeting of
autophagy and provide an overview of arguments for and against the direct manipu-
lation of autophagy in cancer therapy.
Autophagy is regulated by a large set of evolutionarily conserved genes called
ATG genes (Mizushima et al. 2011). The ATG proteins represent a variety of types
of molecules including lipid and protein kinases and protein conjugating enzymes
and scaffolding proteins many of which may represent novel drug targets. Indeed
selective inhibitors of a lipid kinase, VPS34, (Bago et al. 2014; Dowdle et al. 2014;
Ronan et al. 2014) and the protein kinase ULK1 (Egan et al. 2015; Petherick et al.
2015) were recently shown to inhibit autophagy and to have anti-tumor effects. One
important source of confusion in the literature comes from the fact that all known
autophagy regulators (i.e. ATG proteins) have other cellular roles as well (Subramani
and Malhotra 2013). For example, loss of ATG7 inhibits autophagy, but ATG7 also
1 Autophagy as a Therapeutic Target in Cancer 3
regulates p53 via autophagy-independent mechanisms (Lee et al. 2012). So, if Atg7
deletion in a mouse model of cancer alters tumor growth (Guo et al. 2013a;
Karsli-Uzunbas et al. 2014; Strohecker et al. 2013; Xie et al. 2015; Rosenfeldt et al.
2013), is this due to autophagy being inhibited or could it be due to an effect on
p53? Similar examples arise with other essential autophagy regulators—e.g. ATG12
regulates apoptosis (Radoshevich et al. 2010; Rubinstein et al. 2011), ATG5 con-
trols MAP kinases (Martinez-Lopez et al. 2013) and mitotic catastrophe (Maskey
et al. 2013), while BECN1 controls cytokinesis (Thoresen et al. 2010). These effects
are all autophagy-independent and could also affect tumor cell growth/survival.
Without a known molecule that only regulates autophagy without affecting other
biological activities, current best practice for in vitro experiments is to target mul-
tiple autophagy regulators and ensure that they all have similar effects on the phe-
notype being studied before concluding that autophagy affects that phenotype
(Thorburn 2008, 2011). Such experimental rigor is more difficult in vivo but is, if
anything, even more important if we are to avoid misinterpretation of experimental
results. For example, it was believed that autophagy is critical for tuberculosis infec-
tion based on studies where mice lacking ATG5 were very susceptible to infection.
However, more extensive studies targeting multiple ATGs in mice demonstrated
that this susceptibility is not due to ATG5’s role in autophagy but rather a unique
function that is not seen when other ATGs are targeted (Kimmey et al. 2015).
Autophagy is often described as a mostly a non-selective process whereby any
cellular material in the vicinity of the forming autophagosomes can be sequestered
and eventually degraded. This idea is mistaken and oversimplified, as there are sev-
eral types of selective autophagy. In particular, there are specific autophagic mecha-
nisms for the degradation of mitochondria (mitophagy), intracellular bacteria
(xenophagy) (Randow and Youle 2014), the endoplasmic reticulum and contents of
the nucleus (Mochida et al. 2015), lipid droplets (lipophagy) (Singh et al. 2009), and
damaged lysosomes (Maejima et al. 2013). These specific forms of autophagy
potentially have important effects on tumors; for example, defective mitophagy has
been shown to promote breast cancer metastasis (Chourasia et al. 2015). Specific
proteins are also targeted for autophagic degradation, such as under conditions of
iron depletion, where specific targeting of ferritin to autophagosomes takes place to
allow release of iron (Mancias et al. 2014). Even in conditions where one might
think that non-selective autophagy would be favored, e.g. amino acid starvation
where autophagic degradation of any proteins would, at least in principle, provide
amino acids to the cell, autophagy is highly selective such that some proteins are
degraded while others are protected (Mathew et al. 2014). Thus, although we cur-
rently have a poor understanding of how cells determine which autophagy cargos
are degraded under different circumstances, it seems likely that autophagy is
largely—if not entirely—selective. This specificity in cargo delivery to autophago-
somes is critical in understanding the biological effects of autophagy. For instance,
it can explain how autophagy can promote apoptosis for one apoptosis inducer but
not another (Gump et al. 2014; Thorburn 2014). Although understanding selective
autophagy may be vital to effectively target autophagy therapeutically, at present we
have no way to selectively affect cargo-specific autophagy. All the current clinical
trials mentioned above use lysosome-targeted pharmacological agents to target

autophagy, namely chloroquine (CQ) or its derivative hydroxychloroquine (HCQ),
which both inhibit the lysosome. An important caveat to bear in mind is that CQ can
chemosensitize to other anti-cancer drugs through autophagy-independent mecha-
nisms as well as by inhibiting autophagy (Maycotte et al. 2012; Eng et al. 2016),
adding another layer to the complexity underlying the debate.
Autophagy is induced by diverse stresses such as nutrient deprivation, hypoxia,
metabolic stress and many others and in most cases the induction of autophagy
serves to protect cells from the insult. Thus, if cells are starved of amino acids they
rapidly induce autophagy and, if that autophagy induction is prevented using either
pharmacological inhibitors or genetic interference of the ATG genes that regulate
autophagy, many more cells die as a result of the amino acid starvation. Such exper-
iments clearly show that autophagy is protective in this context. Moreover, because
such effects are seen in response to a wide variety of pro-apoptotic stimuli, autoph-
agy is widely thought to protect against apoptosis. This protective effect is generally
the basis for the idea that autophagy inhibition will chemosensitize tumor cells to
other drugs that underlies the numerous clinical trials mentioned above (Thorburn
et al. 2014). Contrarily, early papers that considered autophagy’s roles in the cancer
chemotherapy response (e.g. to the anti-estrogen tamoxifen (Bursch et al. 1996), or
in apoptosis-deficient cells treated with DNA damaging drugs (Shimizu et al.
2004)), often concluded that the induction of autophagy by the therapeutic agent
caused tumor cell death. One of the first clear demonstrations that autophagy can
protect against chemotherapy came from studies in a Myc-driven lymphoma model
(Amaravadi et al. 2007). More recently, many studies with diverse anti-cancer drugs
including DNA damaging agents and other traditional cytotoxics as well as newer
“targeted” agents have tended to conclude that autophagy is primarily protective
against cancer therapy (Thorburn et al. 2014). In fact, it is clear that both in response
to physiological signals (e.g. during development) and exogenous pro-death stimuli,
autophagy can both promote and inhibit cell death/apoptosis (Fitzwalter and
Thorburn 2015).
As mentioned above, in the 60-odd ongoing clinical trials identified using the
search term “autophagy,” the majority are attempting to inhibit autophagy with
HCQ. The basis for these studies is twofold. First, an idea that inhibition of autoph-
agy will, by itself, inhibit tumor growth. Second the idea that autophagy inhibition
will make another anti-cancer treatment more effective. Let’s next consider the
rationales for both ideas.
1.1 Inhibiting Autophagy on Its Own for Anti-cancer

Treatment
Why think that autophagy inhibition could have an anti-tumor effect even in the
absence of other treatments? This concept is based on a large body of data showing
that direct interference with autophagy (e.g. by knocking down or knocking out
ATG genes) can, by itself, inhibit tumor growth and/or promote tumor cell death
(Guo et al. 2013b). The first such demonstration from Jay Debnath’s group showed
that autophagy was important for transformation by KRAS (Lock et al. 2011) and
many of the other studies identifying tumors that require autophagy have also tended
to focus on tumors with RAS pathway mutations. In fact, a series of studies in
genetically engineered mouse models from Eileen White and colleagues (e.g. Guo
et al. 2011, 2013a; Karsli-Uzunbas et al. 2014; Strohecker et al. 2013; Xie et al.
2015), Alec Kimmelman (Yang et al. 2011, 2014), Kevin Ryan (Rosenfeldt et al.
2013), and Josef Penninger (Rao et al. 2014) all focused on tumors driven by mutant
KRAS or BRAF and demonstrated anti-tumor effects upon genetic inhibition of
autophagy by knock out of an essential ATG. Recent studies in flies also showed
autophagy-dependence of RAS-driven tissue overgrowth, however, when tissue
growth was driven by the Notch pathway, autophagy had the opposite effect (Pérez
et al. 2015). This study is important because it establishes that autophagy’s roles in
controlling tissue growth can be different in different contexts. An important role
for BRAF mutation was demonstrated in pediatric brain tumors where brain tumor
cells with wild-type BRAF demonstrated no dependency on autophagy (Levy and
Thorburn 2012) (i.e. autophagy inhibition had little effect on tumor cell growth)
whereas similar tumor cells that harbored BRAF mutations displayed a high degree
of autophagy-dependence such that genetic or pharmacological inhibition of
autophagy was sufficient to kill them (Levy et al. 2014).
In some of the mouse studies, autophagy inhibition switched the tumor from an
adenoma or adenocarcinoma to a less aggressive tumor type called an oncocytoma
(Guo et al. 2013a; Strohecker et al. 2013). In humans, oncocytomas are known to
display defects in autophagy (Joshi et al. 2015). The majority of the tumor studies
listed above involved activation of an oncogene at the same time and in the same
cells that autophagy was inhibited by tissue-specific knockout of an essential ATG;
consequently in these cases tumor development and progression all took place with-
out the ability of the tumor cells to perform canonical autophagy. This observation
begs the question, what happens if a tumor is allowed to form first, then autophagy
is inhibited? Such studies are important because they mimic what a therapeutic
intervention might look like (if we had a perfectly effective inhibitor of autophagy
that worked as well as knockout of an essential ATG). In one study (Karsli-Uzunbas
et al. 2014) such an experiment was done. This work showed that although com-
plete, inducible knockout of ATG7 in adult mice is eventually toxic (the mice die of
infection or eventual neurodegeneration consistent with known functions of autoph-
agy that protect organisms), when autophagy was inhibited in the whole animal, this
blocked the growth and promoted regression, as well as switching to more benign
oncocytomas of pre-existing KRAS mutant lung tumors. An important concern
raised by this study is that because all the mice eventually died of neurodegenera-
tion and others were more susceptible to bacterial infection, we must be cautious
about autophagy inhibition as a therapeutic strategy. In humans we could presum-
ably never achieve as efficient and irreversible an inhibition of autophagy as we get
with the complete knockout of a gene in a mouse so such concerns may be allevi-
ated given two points: first, with pharmacological autophagy inhibitors that would
be used in people we could stop treatment to allow recovery from side effects, and
second, we would be unlikely to have as complete inhibition of the process.
These studies have led to the suggestion that KRAS mutant or BRAF mutant
tumors are the best candidates for autophagy inhibition therapy (Mancias and
Kimmelman 2011; Thorburn and Morgan 2015). However some studies have shown
that KRAS mutation does not always lead to tumor cells being more sensitive to
autophagy inhibition. In an aforementioned mouse study described above, it was
demonstrated that p53 status switched autophagy from being tumor promoting in
KRAS-driven pancreas cancer to being tumor inhibiting. Therefore, when KRAS-
driven pancreas tumors developed with germline loss of p53, autophagy inhibition
caused increase growth of the tumors while the same genetic manipulations demon-
strated an anti-tumor effect of autophagy inhibition in p53 wildtype mice (Rosenfeldt
et al. 2013). It is important to note that germline loss of p53 is not the way that p53
is inactivated during human pancreas cancer development, and that another study
where p53 loss occurred in a manner more analogous to what occurs during human
pancreas cancer found that p53 status did not alter the beneficial effect of autophagy
inhibition (Yang et al. 2014). The explanation for these differences is unknown but
imply an important role for p53 function during the development of a tumor in
determining whether autophagy promotes or inhibits tumor growth. Other evidence
suggests that RAS mutation by itself does not predict whether a tumor cell will be
inhibited or increased in its growth when autophagy is blocked. In genetically
defined human tumor cells where normal cells are immortalized then transformed
by sequential introduction of telomerase, inhibition of p53 and RB then transformed
with oncogenic HRAS, some cells showed that transformation was associated with
increased dependence on autophagy (i.e. autophagy inhibition reduced growth)
whereas other cells transformed in exactly the same stepwise fashion showed
increased growth when autophagy was inhibited (Morgan et al. 2014). More recent
analysis of a large number of RAS-mutant cell lines also concluded that growth of
RAS mutant cell lines was not necessarily inhibited when autophagy was blocked
(Eng et al. 2016). A recent study of pancreas tumors demonstrated a critical role for
autophagy that was linked not to RAS mutation per se (which nevertheless occurs
in the vast majority of pancreas tumors), but instead to increased activity of tran-
scription factors that drive autophagy and allow efficient tumor cell metabolism that
is necessary for sustaining cancer growth (Perera et al. 2015).
Although many studies have focused on RAS pathway driven tumors, an anti-
tumor effect of genetic inhibition of autophagy is also seen in murine tumors driven
by different oncogenic drivers (Huo et al. 2013; Wei et al. 2011, 2014). This raises
the question of whether some tumor cells are indeed highly dependent on autophagy
but that this dependency can occur with or without RAS mutation. A study in breast
cells (Maycotte et al. 2014) where over 100 different autophagy regulators were
targeted with pooled shRNAs attempted to circumvent the problem noted above
whereby non-autophagy functions of ATG genes confound conclusions of autoph-
agy being important for a biological effect. This is important because all the studies
described above where autophagy was targeted and shown to be critical for tumor
growth came to this conclusion after inhibiting only one or two ATGs.
The Maycotte et.al. study (Maycotte et al. 2014) found that some breast cancer
cell lines were highly dependent on autophagy for growth in the absence of added
stress such as amino acid starvation. These cells tended to lose shRNAs that target
positive regulators of autophagy. In other words, when autophagy was inhibited the
cells had a selective disadvantage for continued growth. Other breast cancer cell
lines could be grown for weeks with no apparent selection against shRNAs that
target autophagy suggesting that these cells don’t care about autophagy unless they
are stressed (e.g. by amino acid starvation). Importantly, only tumors grown from
autophagy-dependent tumor cells displayed any inhibition of growth in vivo when
autophagy was inhibited with CQ. These effects were associated with changes in
STAT3 signaling such that in autophagy-dependent breast cancer cells STAT3 sig-
naling and cell growth required autophagy, while in autophagy-independent breast
cancer cells STAT3 activity was not controlled by autophagy. In a follow-up paper
(Maycotte et al. 2015), it was shown that autophagy-dependent cells require autoph-
agy to promote secretion of the cytokine IL6, which is critical for promoting tumor
cell growth and cancer stem cell activity. In contrast, autophagy-independent cells
demonstrated no decrease in IL6 secretion when autophagy was inhibited, instead
secreting more IL6 when autophagy was inhibited. These effects were also associ-
ated with markedly different changes in gene expression patterns upon autophagy
inhibition between autophagy-dependent and autophagy-independent tumor cells.
Another study showed that autophagy-dependent secretion of IL6 and, most likely
of other signaling molecules, is critical for breast cancer cell invasion and metasta-
sis (Lock et al. 2014). Although we have a very poor understanding of the full nature
of the differences between autophagy-dependent and autophagy-independent can-
cer cells, these experiments suggest that the central differences of behavior in
response to targeting autophagy reveal themselves because autophagy controls
completely different and sometimes opposing pathways in different cancer cells.
These studies indicate that in some tumor cells (i.e. the ones that are highly depen-
dent on autophagy) continued tumor growth, survival and perhaps invasion all
depend on autophagy, making a strong argument for autophagy inhibition as a ther-
apeutic approach in cancer. However, it is imperative to understand that this only
occurs in some tumor cells. In others, not only might autophagy inhibition be inef-
fective, it may be counterproductive and actually increase tumor growth. It will be
critical to dissect the biology that underlies these differences if we are to know
which tumors to target and which not to target with autophagy inhibitors.
1.2 Inhibiting Autophagy to Make Other Treatments More

Effective
The majority of the clinical trials where autophagy is deliberately targeted involve
an autophagy inhibitor used in combination with another drug. A large amount of
literature describes chemosensitization effects of autophagy inhibition (Levy and
Thorburn 2011; Maycotte and Thorburn 2011; Thorburn et al. 2014; Rebecca and
Amaravadi 2015). Some of these effects may be due to the other anti-cancer drug
itself increasing autophagy. For example, mTOR inhibitors are potent inducers of
autophagy and it can be shown that co-ordinate inhibition of autophagy can sensi-
tize to mTOR inhibitors (Xie et al. 2013). The interpretation of such studies is that
the autophagy induced by the drug reduces its ability to kill the cancer cells, so that
the addition of an autophagy inhibitor (such as CQ) blocks this protection thus sen-
sitizing to the other drug. This finding has led to clinical studies of such combina-
tions (Rangwala et al. 2014a). However, as with the findings of opposing effects
when autophagy is targeted on its own in different contexts, recent work suggests
that the even the same combination of drugs in autophagy-dependent and -indepen-
dent tumors can show different effects. Thus, in the autophagy-dependent and
autophagy-independent breast cell lines described in the previous section (Maycotte
et al. 2014), the same drug combination (doxorubicin plus the autophagy inhibitor
chloroquine) was only synergistic in autophagy-dependent breast cancer cells and
was sometimes actually antagonistic in autophagy-independent breast cancer cells.
Similar results were found in autophagy-dependent BRAF mutant versus autophagy-
independent BRAF wild-type brain cancer cells (Levy et al. 2014). There are also
cases where specific anti-cancer drugs have been reported to require autophagy in
order to elicit their anti-tumor effect. Epidermal Growth Factor Receptor (EGFR)
signaling was reported to inhibit autophagy by phosphorylating and disrupting the
activity of the autophagy regulator Beclin 1 (BECN1) (Wei et al. 2013). Moreover,
EGFR inhibitors, which are commonly used to treat EGFR mutant tumors, were
found to restore this autophagy activity. The resultant anti-tumor effect was found
to require autophagy restoration, implying that in this case, adding on an autophagy
inhibitor would prevent the EGFR inhibitor from working. Such studies suggest that
choosing the correct drug to combine with autophagy inhibitors will be important
and, possibly even more critical, will be selecting such a combination for the appro-
priate tumor cells.
The aforementioned examples are attempting to increase the efficacy of a drug
that has at least some activity. One of the major problems in cancer therapy comes
when tumors acquire resistance to a drug, which may develop in myriad ways
(Holohan et al. 2013), including the increased expression of drug efflux pumps and
the reduced ability of the tumor cell to undergo apoptosis. For targeted therapies
such as kinase inhibitors that block specific signaling pathways, resistance com-
monly arises due to activation of the same pathway downstream of the inhibited
kinase or activation of a parallel signaling pathway. In some cases we are starting to
obtain evidence that autophagy inhibition can be used as a way to circumvent such
acquired resistance. The best examples to date come from studies in BRAF mutant
tumors. It has been shown that autophagy inhibitors can synergize with BRAF
inhibitors (Goodall et al. 2014). However, autophagy inhibitors may also be able to
do more: they can also overcome resistance to the BRAF inhibitor. In BRAF mutant
melanoma, the acquisition of resistance in the clinic to the RAF inhibitor vemu-
rafenib was shown to correlate with higher numbers of autophagosomes, suggesting
that increased autophagy occurs as the tumors evolve to become resistant against the
BRAF inhibitor and undergo more endoplasmic reticulum stress (Ma et al. 2014).
Moreover, in vitro experimental selection for vemurafenib resistance could be
reversed in this situation via autophagy inhibition. We also have at least one case
where such an effect—adding an autophagy inhibitor reverses resistance to the

BRAF inhibitor—may be true in a patient. In this case (Levy et al. 2014), a patient
with a BRAF mutant brain tumor was treated for almost a year with vemurafenib
but then had a recurrence indicating that her tumor had acquired resistance to the
drug. The patient was then treated with a combination of vemurafenib and CQ,
which caused tumor regression. Importantly, this particular patient was taken off the
BRAF inhibitor for periods of time while continuing treatment with CQ and this
caused the tumor to start growing again. Thus, in this patient, neither BRAF inhibi-
tor alone nor the autophagy inhibitor alone was effective at inhibiting tumor growth
and causing regression; only the combination works. These data are consistent with
the idea that it is the reversal of resistance that is the key benefit of autophagy inhibi-
tion. This patient also demonstrates that autophagy inhibition therapy can be done
for extensive periods of time (in this case more than 2 years as of the time of writ-
ing) without signs of toxicity due to the autophagy inhibitor. Therefore, the con-
cerns raised with the mice where inducible knockout of the Atg7 gene led to death
caused by neurodegeneration within a few months (Karsli-Uzunbas et al. 2014) may
be less significant in practice when we are incompletely inhibiting autophagy in the
clinic.
1.3 Potential Reasons Not to Inhibit Autophagy in Cancer

Therapy
The previous discussion argues that autophagy inhibition may be worthwhile as an

anti-cancer therapy alone or together with other drugs but only in some already
existing tumors. Other studies raise different issues that have been used to argue
against autophagy inhibition therapy. One possible reason is that autophagy may
serve to suppress the development of new cancers. The rationale for this argument
rests on the observation that several autophagy genes function as tumor suppressors
when they knocked out in mice. For instance, BECN1 homozygous deletion leads
to early embryonic lethality but heterozygous deletion causes increased incidence
of cancer (Qu et al. 2003; Yue et al. 2003), suggesting that BECN1 is a haploinsuf-
ficient tumor suppressor. In human tumors, this interpretation has been challenged
and it has been suggested that the apparent loss of BECN1 in human cancers is
primarily due to loss of an adjacent gene, BRCA1 (Laddha et al. 2014). However,
other studies suggest that such a bystander effect is not in play and that BECN1 is
functioning as a tumor suppressor in some human breast cancers (Tang et al. 2015).
In mice, mosaic deletion of ATG5 or liver-specific deletion of ATG7 leads to the
development of benign liver adenomas that do not progress to aggressive cancer
(Takamura et al. 2011). Deletion of other autophagy regulators in mice can also
cause spontaneous cancer development. Examples include BIF-1 (Takahashi et al.
2007), ATG4C (Marino et al. 2007) and UVRAG (Liang et al. 2006), although this
last example could be due to autophagy-independent functions in maintaining chro-
mosome stability (Zhao et al. 2012). Studies similar to these have led to the
suggestion that autophagy may suppress the development of cancer even when it
can promote cancer progression. In this case, one might expect that general inhibi-
tion of autophagy would cause pre-neoplastic lesions to progress faster.
The above examples consider the effect of autophagy in cancer to be primarily an
autonomous effect on the behavior of the tumor cell itself; that is, autophagy may
promote or inhibit growth of the cancer cell, cause it to be more or less likely to die,
or affect the cell’s ability to migrate and invade other tissues. Autophagy manipula-
tion in one cell may also alter the way that neighboring cells behave. This could
have repercussions for cancer development, progression, and response to therapy.
The best example here concerns how dying tumor cells do or do not affect and
engage the immune system. It was demonstrated that chemotherapy-induced immu-
nogenic cell death of cancer cells requires that autophagy be functional in the dying
tumor cells (Michaud et al. 2011). This effect was necessary for effective treatment
of the tumor in immune competent mice but not in immune deficient animals dem-
onstrating that the difference was due to how the immune system recognized the
dying cancer cells rather than an effect on the efficiency of tumor cell killing by the
chemotherapeutic itself. A mechanism was traced to a requirement for autophagy in
the release of ATP from the dying cells. In other circumstances autophagy may be
important in controlling the release of other immune stimulators such as the Damage
Associated Molecular Pattern (DAMP) molecule HMGB1 (Thorburn et al. 2009).
Autophagy may also be important in tumor antigen presentation (Li et al. 2012).
Together, these findings would tend to suggest that autophagy inhibition during
cancer therapy should reduce immunogenic tumor cell killing, i.e. arguing against
trying to target autophagy. However, even here the situation is complicated. It has
been shown that autophagy inhibition with CQ significantly enhances T cell-
mediated tumor killing after Interleukin 2 immunotherapy (Liang et al. 2012).
Hypoxia-induced autophagy impairs natural killer (NK) cell-mediated killing of
tumor cells and autophagy inhibition was shown to enhance tumor elimination by
NK cells in vivo (Baginska et al. 2013). Thus, as with the other competing effects
discussed above, the benefits and caveats of targeting autophagy are also manifested
when it comes to immunogenic tumor cell killing. These studies further emphasize
how crucial it will be to understand the full spectrum of effects—both good and
bad—that occur when autophagy is targeted during cancer therapy.
1.4 Conclusions
What should be clear from the above discussion (which is by no means definitive,
many other studies arguing both for and against autophagy as a therapeutic target in
cancer could have been discussed) is that there is no straightforward conclusion as
to how, or even whether, we should try to target autophagy as a therapeutic approach
to cancer. Numerous important questions remain to be answered and there is evi-
dence both for and against the idea of targeting autophagy that we need to make
sense of. Moreover, it is unclear how we should go about targeting autophagy.
Current clinical approaches focus on targeting the lysosome with drugs like HCQ,
and other, more potent lysosomal inhibitors are also being developed (Goodall et al.
2014; Mcafee et al. 2012). The ability of lysosome inhibitors to chemosensitize
through autophagy-independent mechanisms may also be useful (Maycotte et al.
2012; Eng et al. 2016). Earlier steps in the autophagy pathway can also be therapeu-
tically targeted (Bago et al. 2014; Dowdle et al. 2014; Ronan et al. 2014; Egan et al.
2015; Petherick et al. 2015). Will these be better than lysosome-targeted drugs for
cancer therapy? Perhaps the most critical issue is to determine which tumors should
be targeted and which should not. This is a pressing issue because accumulating
evidence suggests that not only might targeting autophagy be ineffective in some
tumors, in those tumors that are not highly dependent on autophagy, it may be coun-
terproductive to do so. If we try to inhibit autophagy in the wrong tumor, this may
not only fail to slow tumor growth, it might enhance growth. Targeting autophagy in
the wrong tumor may not only fail to make another drug more efficacious, it might
make that drug less effective. Added complexity comes when one considers how
altering autophagy in cancer cells may affect how other cells (e.g. immune cells)
recognize the tumor cells. It will require a much more sophisticated understanding
of how these effects work and how their balance determines the final outcome if we
are to effectively pursue autophagy as a therapeutic target in cancer.
Given this complexity, one might propose not even to try targeting autophagy in
cancer therapy. However, this is not an option; not only do we have ongoing clinical
trials whose interpretation will require that we better understand this process and
what it means for cancer cell behavior, we already know that even if we wanted to
avoid targeting autophagy we couldn’t do so. Most of our current anti-cancer treat-
ments themselves affect autophagy, so we are routinely affecting autophagy during
cancer therapy whether we like it or not. The way forward is to understand how the
various competing effects of autophagy on cancer treatment and cancer/tumor
behavior occur. Fortunately, the field is now poised to do so.
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Chapter 2
Autophagy in Cancer Cells vs. Cancer Tissues:
Two Different Stories
Chi Zhang, Tao Sheng, Sha Cao, Samira Issa-Boube, Tongyu Tang,

Xiwen Zhu, Ning Dong, Wei Du, and Ying Xu
Abstract Autophagy has been considered strongly associated with cancer develop-
ment and possibly playing important roles in cancer progression. Here we present a
computational study of transcriptomic data of cancer tissues, totaling 6317 tissue
samples of 11 cancer types along with tissues of inflammatory diseases and cell line
based experiments for comparative purposes. Our study clearly revealed that some
widely held beliefs and speculations regarding autophagy in cancer may not be well
founded, knowing that many of the previous observations were made on cancer cells
cultured in man-made environments rather than actual cancer tissues. Our major find-
ings include: (i) the widely used assumption that cancer tissue cells are nutrient
depleted is not supported by our tissue-based gene-expression data analysis; (ii) the
11 cancer types studied fall into 2 distinct groups: those with low macro-autophagy
(LM) activities and those with high lysosome (HL) activities but induced by
C. Zhang • T. Sheng • S. Cao • S. Issa-Boube

Computational Systems Biology Lab, Department of Biochemistry and Molecular Biology,
and Institute of Bioinformatics, The University of Georgia, Athens, GA, USA
T. Tang
Department of Gastroenterology, First hospital of Jilin University, Changchun, China
X. Zhu
Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Chongqing Medical
University, Chongqing, China
N. Dong
Emergency Department, First hospital of Jilin University, Changchun, China
W. Du
College of Computer Science and Technology, Jilin University, Changchun, Jilin, China
Y. Xu (*)
College of Computer Science and Technology, Jilin University, Changchun, Jilin, China
School of Public Health, First hospital of Jilin University, Changchun, China
e-mail: xyn@uga.edu

18 C. Zhang et al.
icro-autophagy and chaperon-mediated autophagy; (ii) co-reduction in autophagy

m
and apoptosis are widely observed in cancer tissues; (iii) down-regulated autophagy
strongly correlates with up-regulated cell-cycle progression genes across all cancer
types, with one possible functional link detected that repressed autophagosome for-
mation may reduce the degradation of cellular organelles that are essential to cytoki-
nesis, hence contributing to cell cycle progression; (iv) significant correlation is
observed between autophagy and immune activities; (v) the down-regulated macro-
autophagy genes negatively correlate with the total mutation rates in cancer genomes
in LM cancers; and (vi) conditional correlation analyses point to a very unexpected
direction: cellular Fenton reactions may be the cause of the decreased macro-
autophagy and its co-expression with apoptosis, increased cell proliferation, genomic
mutation rate and even possibly immune response. The information derived here may
shed new light on elucidation of fundamental relationships between cancer and
autophagy as well as on how to take advantage of the derived relationship for
improved treatment of cancer.
2.1 E
xamining Autophagy in Cancer via Cancer Cell Line
Versus Cancer Tissues
Autophagy is a cellular survival process under nutrient deprivation and metabolic

stress. It degrades cellular proteins, other macromolecules, organelles and cyto-
plasm; and recycles the nutritious elements to support cell survival. Basal autophagy
is a constitutive process that plays a homeostatic function, acting in parallel with the
ubiquitin-directed proteasome degradation pathway to maintain the integrity of cel-
lular proteins and organelles. In terms of its role in cancer, the current understanding
is: autophagy has a role in supporting cancer cell survival under metabolic stress and
in hypoxic regions (Degenhardt et al. 2006). Interestingly, a few essential autophagy
genes are found to have high mutation rates across a few types of cancers. For
instance, allelic loss of beclin1 gene (BECN1, also known as ATG6) is reported to
be among commonly mutated genes in breast, ovarian and prostate cancers (Liang
et al. 1999), suggesting that these cancers try to avoid autophagy.
A widely accepted model is that autophagy plays a major survival role through-
out cancer initiation and early-stage development by helping cells overcome nutrient
deprivation and metabolic stress (Mathew et al. 2007), which are believed to take
place in cancer. Its role in more advanced cancers seems to be in stimulation of
necrotic cell death, leading to persistent inflammation and repeated wound-healing,
an environment that cancer development generally requires (Degenhardt et al. 2006).
On the other hand, autophagy is believed to have an important role in maintaining
genome integrity by limiting genome damages (Mathew et al. 2007), suggesting that
cancers may tend to repress autophagy, knowing that mutations are essential to can-
cer cell survival. To sort out these complex and conflicting relationships between
cancer and autophagy derived through cell line based studies as well as genome
2 Autophagy in Cancer Cells vs. Cancer Tissues: Two Different Stories 19
analyses of cancer tissue cells, we need to take a more systematic approach to study
a large number of actual cancer tissue samples (versus cancer cell lines).
It is noteworthy that there may be fundamental differences between cancer cell
line biology and the actual cancer biology. For example, while it has been specu-
lated that cancer cells tend to be low in ATP production based on cell line studies
(Lim et al. 2011) (may not be accurate), tissue-based analyses suggest that cancer
tissue cells may not be short of ATPs (Gottesman et al. 2002) and our recent study
provides an explanation of why this is the case (Hui Yan et al. 2016), suggesting that
nutrient deprivation might not necessarily be a valid assumption for autophagy
study in cancer tissues.
Here, we present a computational analysis of transcriptomic and genomic data of
cancer tissues in the TCGA database (Weinstein et al. 2013), covering 6317 samples
of 11 cancer types, aiming to gain a coherent understanding about the relationship
between autophagy and cancer. To put our study in a comparative setting, we have
also included transcriptomic data of 14 types of inflammatory diseases with 8 being
cancer prone and 6 being cancer independent along with control samples plus 12
cell-lined based datasets with experimentally induced autophagy along with con-
trols, to gain a deeper understanding about the actual roles played by autophagy in
disease tissues. The detailed information of the omic data used in our study is given
in the Sect. 2.9.
The rest of the chapter is organized as follows. Section 2.2 characterizes autoph-
agy in disease tissues and cell lines by comparing transcriptomic profiles of autoph-
agy related genes in both systems. Section 2.3 assesses the level of nutrient
deprivation and associated autophagy in cancer and inflammatory disease tissues.
Section 2.4 analyzes the interplay between autophagy and apoptosis. Section 2.5
discusses two novel associations between autophagy and cell cycle progression as
well as autophagy and immune system. Section 2.6 demonstrates the impact of
mutations of autophagy related genes. Section 2.7 discusses about general biologi-
cal processes that correlate with autophagy. Section 2.8 concludes our prediction of
functional roles of autophagy in cancer and inflammatory disease tissues. Section 2.9
covers the methods and data used in this chapter.
2.2 Gene Expression of Autophagy in Disease Tissues
We have conducted differential gene-expression analyses over a total of 6317 dis-

ease versus control tissue samples of 11 cancer types, 8 cancer-prone inflammatory
diseases and 6 cancer-independent inflammatory diseases plus 12 cell-based datas-
ets collected under serum depletion or other metabolic stress conditions for induc-
tion of autophagy. An inflammatory disease is considered as cancer-prone or
cancer-independent if the cancer occurrence rate is elevated with statistical signifi-
cance or not in the disease sites based on published statistics. The details of these
diseases and datasets are given in Sect. 2.9. A pathway is considered as up- or down-
regulated if it is enriched with up- or down-regulated genes assessed using a hyper-
geometric test with p-value = 0.05 as the statistical significance cut-off.
20 C. Zhang et al.
Three types of autophagy have been defined, namely macro-autophagy, micro-

autophagy and chaperon-mediated autophagy, which differ in both their induction
and execution processes (Glick et al. 2010). To the best of our knowledge, more
than 95 % published studies of autophagy in cancer are focused on macro-autophagy
(Mizushima 2007).
Eleven autophagy-related pathways are considered in our pathway enrichment
analyses, namely: (1) the core gene set of macro-autophagy induction, which con-
sists of the essential genes involved in autophagy initiation, nucleation and expan-
sion; (2) cargo recognition and selectivity genes, covering genes involved in
selective autophagy with a recognition function; (3) cytoplasm-to-vacuole targeting
pathway (CVT), which is similar to the bulk autophagy (Scott et al. 1996) except
that it is activated constitutively under normal growth conditions; (4) autophagy-
induction genes, consisting of early response genes that can lead to the induction of
autophagy; (5) nucleation assembly genes for the formation of an autophagosome
complex; (6) vesicle formation and autophagosome breakdown genes; (7) micro-
autophagy invagination pathway (Li et al. 2012); (8) chaperone mediated autophagy
genes; (9) lysosome genes; (10) lysosome degradation pathways; and (11) protea-
some genes for protein degradation in an autophagy independent manner. The path-
way enrichment results in all the aforementioned disease types are listed in Table 2.1.
Our finding is quite surprising as the analysis clearly shows that the expression pat-
terns of the 11 autophagy pathways are substantially different, specifically the macro-
autophagy pathways (i.e., the first 6 pathways above) in cancer cell lines versus cancer
tissues. Specifically, the macro-autophagy and lysosome related pathways are consis-
tently up-regulated in cell-line under nutrient deprivation conditions. However, the
macro-autophagy pathways, especially the autophagosome formation genes, including
MAP1LC3A, MAP1LC3B, MAP1LC3C, WIPI1, BNIP1, GABARAP, GABARAPL1,
GABARAPL2, PARK2, SRPX, LRRK2, ULK2, FYCO1, and TP53INP2, are consis-
tently down-regulated in 7 out of the 11 cancer types, namely THCA, BRCA, HNSC,
BLCA, COAD, LUAD and LUSC; and the lysosome pathway is also down-regulated
in COAD, LUAD and LUSC. The macro-autophagy recognition, induction and fusion
pathways are up-regulated in KICH and STAD; and nucleation assembly pathways are
up-regulated in KIRC. Interestingly, KICH, STAD, LICH and BRCA have their micro-
autophagy related genes up-regulated; and STAD has its chaperone-mediated autoph-
agy genes up-regulated while none of them are up-regulated in the aforementioned
cell-line datasets. Cancer types with up-regulation of at least one autophagy type,
namely KICH, STAD, KIRC, LICH and BRCA, all have up-regulated lysosome activ-
ity, hence providing a cross-validation between the two predictions and making the
them more trustworthy. Interestingly, proteasome genes are generally more up-
regulated in cancer types with down-regulated macro-autophagy, suggesting that they
may serve similar purposes and hence are mutually exclusive between macro-autophagy
and proteasome based protein degradation. Based on these observations, we classify
the 11 cancer types into 2 groups: those with low macro-autophagy (LM): BRCA,
HNSC, BLCA, COAD, LUAD and LUSC and those with high lysosome (HL): KICH,
STAD, KIRC, LIHC and THCA. It is noteworthy that the ATG genes, key autophagy
genes, are generally un-differentially expressed across all cancer types.
Table 2.1 Differential expressions of autophagy related pathways across different cancer cells, cancer tissues of different types, and disease tissues of different chronic
inflammatory diseases, where cell line represent the overall statistics of the 12 cell-line data, CII and CPI are for cancer-independent and cancer-prone chronic inflammatory
diseases, respectively
Cell line Cancer Inflammatory diseases
CII:
PSO, CII: CPI: CPI:
Metabolic AH, AST,CS, COPD,HCV, UC,CD,
stress KICH STAD KIRC LIHC THCA BRCA HNSC BLCA COAD LUAD LUSC IBS NAS CIR, IFP BE,AD
Cargo recognition Up Down Down Down Down Up Down
and selectivity
Core Up Down Down Down Down Down Down Up Down
CVT pathway Up Up Up Down Down Down Down Down Down Down
Induction and Up Up Up Down Up Down
fusion
Nucleation Up Up Down Down Down Down Down Down Down Up Down
assembly
Autophagosomal Up Down Down Down Down Down Down Down
structure
Micro-autophagy Up Up Up Up Up
invagination
Chaperone Up
mediated
autophagy
Lysosome Up Up Up Up Up Up Up Down Down Down Up Down Up Down
Lysosome Up Up Up Up Up Down Down Down Up Down Up Down
degradation
Proteasome Up Up Up Up Up Up
Detailed information of the data is given in Sect. 2.9
22 C. Zhang et al.
Down-regulated macro-autophagy and lysosome are also observed in four types

of cancer-prone inflammatory diseases, namely UC, CD, BE and AD while
up-regulated lysosome is observed in the other four types of cancer-prone inflammatory
diseases: COPD, IFP, HCV and CIR. In comparison, up-regulated macro-autophagy
and lysosome pathways are observed in three cancer-independent diseases: PSO, AH
and IBS; and down-regulated lysosome is found in the other three cancer-indepen-
dent diseases: AST, CS and NAS. These strongly suggest that there are no intrinsic
relations between cancer and autophagy.
In the remaining portion of the chapter, we focus on elucidation of the possible
reasons and functional roles of down-regulation of macro-autophagy and up-
regulation of lysosome in LM and HL diseases, respectively.
2.3 Nutrient Deprivation Is Unlikely in Cancer Tissue
It has been repeatedly observed that macro-autophagy can be induced in cancer

cells by nutrient deprivation (Mizushima et al. 2004). Hence it has been naturally
assumed that cancer tissue cells are also nutrient depleted based on such cell-line
studies coupled with observations that cancer tissue cells tend to have substantially
increased uptake of glucose (Adekola et al. 2012). However no experimental studies
have reliably established that cancer tissue cells are indeed nutrient depleted, to the
best of our knowledge. Actually, recent metabolomic studies of cancer tissues sug-
gest that the opposite may be true, i.e., cancer tissue cells are rich in nutrients and in
ATPs (Coller 2014). Our recent research provides a possible explanation of where
the plentiful ATPs may come from in cancer tissue cells (Hui Yan et al. 2016).
We have recently conducted a modeling analysis based on gene-expression data
of 6600+ tissue samples of 14 types of cancer versus controls, aiming to assess if
there may be Fenton reactions in mitochondria of cancer tissue cells, as strongly
suggested by various hints (Hui Yan et al. 2016). Fenton reaction: Fe2+ + H2O2
→ Fe3+ + OH− + •OH, a non-enzymatic reaction, can take place when the concentra-
tions of Fe2+ and H2O2 are sufficiently high, which is generally true for chronic
inflammatory sites (Winterbourn 1995). The products of the reaction are Fe3+, OH−
and •OH. When there are also plentiful reducing elements at or near the reaction
sites such as Vitamin C, sulfur or NADH, Fe3+ can be reduced to Fe2+, which will
enable the reaction to continue. We have developed a computational method to
demonstrate if a specific subcellular component may have elevated Fenton reactions
or not (Hui Yan et al. 2016), with its basic idea summarized below.
We can rewrite (continuous) Fenton reactions as: RA + H2O2 → OH− + •OH + X
with Fe2+ as the catalyst since Fe2+ is not consumed by the (continuous) reaction,
with RA and X representing the reducing element and its oxidized form, respec-
tively. We have identified marker genes for each of the five relevant quantities:
[RA], [H2O2], [OH−], [•OH] and [X] in three cellular compartments: cytosol, mito-
chondria, and extracellular region, whose expression levels reflect these quantities.
We have shown that in tissues without Fenton reactions, these five quantities (for
each cellular compartment) are largely independent of each other; and in tissues
having Fenton reactions, they are strongly correlated with each other as measured
via the Michaelis-Menton equation (Berg et al. 2002). Using this analysis tool, we
have demonstrated that all cancer tissues we studied have Fenton reactions in the
three cellular compartments with high statistical significance. Furthermore, we have
demonstrated that the OH− molecules continuously produced by the reaction in
mitochondria will lead to reduced concentration of mitochondrial protons, hence
leading to a proton gradient on the two sides of the inner membrane of mitochon-
dria, as well as proton influx via the ATP synthase and ATP production just like in
a respiration process. The difference is that there is no need for NADHs to push
their electrons through the electron transport chain to produce a proton gradient. In
sum, when mitochondrial Fenton reactions continue, they will produce ATPs just
like in respiration but it does not consume NADHs instead it consumes some reduc-
ing elements such as Vitamin C or sulfur.
Using this modeling approach, we also discovered that LM cancers generally
have higher levels of cytosolic and extracellular Fenton reactions in comparison
with the HL cancers, which tend to have higher mitochondrial but less increased
cytosolic Fenton reactions. Knowing that damaged mitochondria can be engulfed
by autophagosome and then degraded by lysosome, we posit that up-regulated
lysosome-degradation pathway may be involved in the removal of the oxidatively
damaged mitochondrial components (Zhou et al. 2011). As a comparison, the down-
regulated autophagosome-formation genes in the LM cancers (and inflammatory
diseases) are mostly over-expressed in cell line data. All these revealed that the dif-
ferentially expressed autophagy genes in both of the LM and HL cancers are highly
different to metabolic stress induced autophagy in cell lines.
We have also examined in cancer tissues and cell line experiments the expression
levels of nine sets of metabolic deprivation responsive genes, which are identified
through experiments independent of the expression data used here, under the condi-
tions of deprivation of methionine, leucine, glutamine, amino acids, glucose, and
serum (see Sect. 2.9), where the level of differential expression for each gene set
reflects the deprivation of a specific metabolite. On average, ~85% (p-value < 1e−30)
of the relevant marker genes are differentially expressed in the cell lines treated with
each such depletion while <15 % (p-value = 0.6) are differentially expressed in can-
cer tissues, suggesting a big difference in the level of metabolic deprivation between
the cell line based experiments and cancer tissues.
All these observations strongly suggest that nutrient depletion-induced macro-
autophagy is highly unlikely in cancer tissues.
2.4 Autophagy and Apoptosis
Cross-talks between autophagy and apoptosis have long been known and exten-
sively studied (Maiuri et al. 2007); and both are considered as having tumor sup-
pression functions (Su et al. 2013). We present a computational analysis of
expressions of genes involved in both autophagy and apoptosis to assess their
24 C. Zhang et al.
co-expression relations with other autophagy and apoptotic genes to elucidate

possible relationships between the two processes in cancer.
Our analyses have detected that a number of core macro-autophagy regulatory
genes are down-regulated in cancer tissues but up-regulated in cancer cell line data.
Similar patterns of down-regulated core apoptotic regulatory genes such as BCL2L1,
BAD, BAG1 and BCL2L11 are observed in tissues of most cancer types, but up-
regulated in cancer cell lines. Our co-expression analyses observed positive co-
expression among the down-regulated autophagy and apoptosis signaling genes and
negative co-expression between the autophagy and proteasome genes in LM cancers.
Interestingly, most of the autophagy co-expressed apoptosis genes are up-stream sig-
naling genes, proteasome and ubiquitination genes. The extrinsic and intrinsic apop-
totic pathways are largely independent to autophagy in cancer tissues.
We have also observed significant under-expression of several regulatory
genes involved in both autophagy and apoptosis such as (1) BH3 binding genes
(and complex) BECN1, BNIP3, UVRAG, VPS34, and BCL2L1; (2) DAPK
genes: DAPK1, DAPK2 and DAPK3; and (3) ATG5, in both LM and HL cancers.
Our co-expression analysis revealed that these genes in LM cancers are strongly
co-expressed with a number of proteasome genes, whose up-regulation tends to
be strongly associated with cytosolic Fenton reactions, hence supporting our
hypothesis that Fenton reaction may be a common reason for the differentially
expressed apoptosis regulation and down-regulated autophagy as detailed in
Discussion.
2.5 Novel Biological Processes Related to Autophagy
Gene co-expression networks are constructed for each disease type under study,
including both cancer and inflammatory diseases, to identify novel biological pro-
cesses that may associate with the observed down-regulation of macro-autophagy in
the LM diseases and up-regulation of lysosome in the HL diseases. We have previ-
ously developed a Mutual Rank (MR) based method to detect highly co-expressed
gene clusters, also referred to as co-expression modules in a global gene co-
expression network (Zhang et al. 2015) (see Sect. 2.9).
The method first applies a rank based statistic to detect the significant hub
genes in a given co-expression network and then identifies the co-expression
module surrounding each hub gene, where hub gene is intuitively defined as a
gene with substantially more interaction partners than its neighboring genes in the
given co-expression network. The method tends to identify strongly co-expressed
gene modules, allowing a gene to be part of multiple modules, hence sensitive to
identify novel biological processes correlated with specific targets, say autophagy
in the current study. Here, we have identified numerous co-expression modules
that are significantly enriched by autophagy genes. Functions of non-autophagy
genes in each module are functionally analyzed to reveal novel biological pro-
cesses strongly associated with autophagy in each disease class. In addition, we
Table 2.2 Information of the data analyzed in this chapter

Cancer Data
Cancer type label #Tumor #Control source Analyzed mutations
Bladder urothelial BLCA 408 19 TCGA TP53, PIK3CA,
carcinoma ARID1A, MUC17,
MUC16, TTN
Breast invasive BRCA 1095 113 TCGA TP53, PIK3CA, PTEN,
carcinoma MUC17, MUC16, TTN,
MUC4
Colon adenocarcinoma COAD 285 41 TCGA TP53, TTN
Head and neck HNSC 520 44 TCGA TP53, PIK3CA, NAV3,
squamous cell MUC17, MUC16, TTN,
carcinoma MUC4
Kidney chromophobe KICH 66 25 TCGA TP53
Kidney renal clear cell KIRC 533 72 TCGA ARID1A, PTEN, NAV3,
carcinoma MUC17, MUC16, TTN
Liver hepatocellular LIHC 371 50 TCGA TP53, MUC16, TTN
carcinoma
Lung adenocarcinoma LUAD 515 59 TCGA TP53, PIK3CA, NAV3,
KRAS, MUC17,
MUC16, TTN, MUC4
Lung squamous cell LUSC 501 51 TCGA TP53, PIK3CA, NAV3,
carcinoma MUC17, MUC16, TTN,
MUC4
Stomach STAD 238 33 TCGA TP53, PIK3CA,
adenocarcinoma ARID1A, PTEN, NAV3,
KRAS, MUC17,
MUC16, TTN, MUC4
Thyroid carcinoma THCA 505 59 TCGA
Inflammatory disease Disease Relevance to
types label #Disease #Control Data source cancer
Crohn’s disease CD 37 12 GSE16879 Risking
Ulcerative colitis UC 40 12 GSE16879 Risking
Liver cirrhosis CIR 13 10 GSE6764 Risking
Barrett’s esophagus BE 20 19 GSE26886 Risking
HCV infection HCV 16 2 GSE11190 Risking
Chronic obstructive COPD 35 63 GSE11784 Risking
pulmonary disease
Idiopathic pulmonary IFP 23 6 GSE21369 Risking
fibrosis
Atopic dermatitis AD 13 8 GSE32924 Risking
Psoriasis PSO 33 21 GSE14905 Independent
Asthmatics AST 42 28 GSE4302 Independent
Alcohol hepatitis AH 15 7 GSE28619 Independent
Non-alcoholic NAS 9 7 GSE63067 Independent
steatohepatitis
(continued)
26 C. Zhang et al.
Table 2.2 (continued)
Inflammatory disease Disease Relevance to
types label #Disease #Control Data source cancer
Irritable bowel syndrome IBS 28 77 GSE36701 Independent
Cutaneous sarcoidosis CS 15 5 GSE32887 Independent
Cell line experiments Cell type #Treated #Control Data source
Serum deprivation T98G cell 3 3 GSE1692
Glucose deprivation MCF7 cell 5 6 GSE19123
Starvation-induced autophagy Lymphoblastoid cell 3 3 GSE2435
line
Serum starvation Lymphoblastoid cell 6 6 GSE31040
line
Glucose deprivation HCT116 cell 9 9 GSE38061
Tunicamycin treatment PC3 cell 1 1 GSE38643
Induction of autophagy by PC3 cell 2 2 GSE46376
atorvastatin
GANT61 treatment ES2 and H4 2 2 GSE54936
Glucose deprivation A549 cell 2 2 GSE56843
4-hydroxytamoxifen IMR90 cell 6 6 GSE59522
treatment
Serum deprivation LoVo cell 3 3 GSE70976
Serum deprivation T cell 3 10 GSE7497
have also conducted a similar analysis but on co-expression modules enriched by

lysosome genes. These two classes of modules are referred to as autophagy- and
lysosome-centric modules in the following. Table 2.2 lists all such modules, along
with their annotated functions for each disease type under study.
2.5.1 Autophagy and Cell Cycle Control
We noted that cell cycle genes, specifically the G2-M transition genes, enrich at
least 30 % of the autophagy-centric co-expression modules in each LM cancer type
while G1-S transition genes and other cell cycle genes enrich ~20 % of the lysosome-
centric modules in HL cancers. Further analysis revealed that cell-cycle genes are
negatively correlated with the down-regulated autophagy genes in LM cancers and
positively correlated with the up-regulated lysosome genes in HL cancers. Figure 2.1
shows the co-expression networks among autophagy and cell cycle genes identified
in each cancer type.
Macro-autophagy may suppress cell-cycle progression through blocking the
transition from G2 to M (Kuo et al. 2011; Matsui et al. 2013). A recent study sug-
gests that autophagy may also have important roles in suppression of cytokinesis
(Kuo et al. 2011). Our analysis detected a consistent negative correlation between
Fig. 2.1 Co-expression network among down-regulated autophagy genes and up-regulated cell
cycle genes in COAD. The autophagy genes are represented by blue nodes and the cell cycle genes
are in orange. The red and green edges represent negative and positive co-expression, respectively,
and the width of the edge denotes the level of co-expression
the over-expressed cyclins CCNB1 and CCNB2, cyclin dependent kinases CDK1
and CDK2, a number of centrosomal protein genes, other G2-M transition genes
versus all the under-expressed macro-autophagy genes: ATG5, ATG7, ATG10,
ATG12, GABARAP, GABARAPL2, MAP1LC3B, and PARK2, all related to for-
mation and maturation of autophagosome in the following LM cancers: COAD,
LUAD and LUSC. With the knowledge that autophagic degradation may be
involved in the cleaning of midbody derivatives after cytokinesis (Pohl and
Jentsch 2009; Kuo et al. 2011), we speculate that suppression of the autophago-
some formation may preserve the organelles that are necessary for cell prolifera-
tion in cancer tissues.
28 C. Zhang et al.
Further analysis revealed that most of the up-regulated G1-S transition genes in
HL cancers are highly co-expressed with the over-expressed lysosome genes are
proteasome genes. The cyclins, cyclin dependent kinases, DNA polymerases and
other cell-cycle regulatory genes are largely independent of the lysosome genes. We
speculate that the co-expression between lysosome and proteasome just reflect a
normal relationship between the two protein degradation systems, which are both
up-regulated in cancer probably due to the increased damage to proteins possibly by
ROS (Waris and Ahsan 2006).
2.5.2 Autophagy and Immune Response
Our analysis has identified that the down-regulated genes involved in autophago-
some formation and maturation, such as MAP1LC3C, SNCA, ATG7, ATG4C,
ATG12, ATG5, OPTN, GABARAP, PARK2, and SH3GLB1 are significantly
Fig. 2.2 Co-expression network among autophagy and immune response genes in COAD. The
autophagy genes are represented by blue nodes and the immune response genes by orange nodes.
The green edges represent positive co-expressions and the width of an edge denotes the level of
co-expression
co-expressed with 81 under-expressed immune response genes in LM cancers.

Similarly, significant co-expression is observed between the down-regulated autoph-
agy genes and immune response genes in LM (and cancer prone) inflammatory dis-
eases. In comparison, 57 up-regulated lysosome genes are strongly co-expressed
with 178 up-regulated immune response genes in HL cancers. Interestingly, immune
response genes that are co-expressed with autophagy genes have substantial overlap
between LM and HL cancers but with opposite differential expression patterns, i.e.,
down vs. up-regulation. These immune response genes include CD markers, chemo-
kine ligands, chemokine receptors, interleukins, interleukin receptors and other
immune genes. The co-expression modules consisting of both autophagy and
immune response genes are shown in Fig. 2.2 for selected diseases.
From Fig. 2.2, we can see that these co-expression modules contain a large
number of genes related to multiple immune cell types such as CD4+ and CD8+
T cells, B cells, dendritic cells, natural killer cells and macrophages. This is con-
sistent with the observation that the co-expression modules enriched with
immune response and autophagy genes are also substantially enriched by lipid
binding, lipid metabolism and glycosaminoglycan metabolic genes as it is known
that increased lipid metabolism tends to trigger increased immune response
(Fritsche 2006) and the same with increased synthesis of cell-surface glycan
(Zhang 2006).
Previous studies have identified various invading microbes such as HBV and
H. Pylori have developed ways to evade autophagy by suppressing autophagosome
formation and fusion to lysosome (Tang et al. 2012), which is consistent with what
we observed in the LM cancers and cancer-prone inflammation. The autophagy eva-
sion mechanism has been observed in host cells causing a failed degradation of the
infected cells hence less antigen presenting (Paludan et al. 2005). We speculate that
normal autophagy in cancer tissue can degrade damaged macromolecules and
organelles to promote immune response through induction of antigen presenting.
Such a mechanism can be hindered in LM cancers by the suppressed autophago-
some formation that is possibly caused by extracellular and cytosolic Fenton reac-
tion, as discussed in Discussion.
2.6 Autophagy and Genomic Mutation
We have conducted a comparative analysis between the level of autophagy and

genomic mutation rate using cancer genomic sequences and matching transcrip-
tomic data in TCGA, to assess correlations between genomic mutations and the
expression levels of autophagy genes.
We have computed correlation between the somatic mutation rate and gene
expression level of autophagy-related genes for each cancer type to evaluate if
there is any correlation between the level of differentiation of autophagy genes
and the mutation rate at the whole genome level for each cancer type under
30 C. Zhang et al.
study. Interestingly, while correlations are detected, they are different for differ-
ent cancer types. We noted: the expression levels of lysosome genes are gener-
ally positively correlated with the mutation rate in KIRC, KICH and STAD
cancers while some sets of macro-autophagy genes are either positively or nega-
tively correlated with the mutation rate in COAD, LUAD, BLCA, and BRCA
with strong statistical significance. This correlation is insignificant in LICH,
THCA, LUSC, and HNSC.
Genes involved in autophagosome formation such as ATG2B, TP53INP2,
SQSTM1, FYCO1, GABARAPL1, GABARAPL2, GABARAPL3, MAP1LC3A,
MAP1LC3B and MAP1LC3C are negatively correlated with the mutation rates.
They are under-expressed in 8 out of the 11 cancer types, covering all LM cancers.
We noted that a number of pro-autophagy and pro-apoptosis signaling genes such as
ATG16L1, SKP2, BAX, BID, RPS6KB1, and PIK3R2 are positively correlated
with the mutation rate; and they are up-regulated in eight cancer types. Seven
autophagy core signaling genes: ATG3, ATG4A, ATG4C, ATG4D, ATG5, ATG7,
and ATG12 are positively correlated with the mutation rate but they are not signifi-
cantly differentially expressed. In addition, the proteasome genes are generally up-
regulated and positively correlated with the mutation rate in LM cancers. One
possible explanation is that increased proteasome activities tend to be associated
with increased Fenton reactions, which can lead to increased mutations, as further
discussed in Sect. 2.7.
We have also examined correlations between expressions of autophagy genes
and non-synonymous mutation of six specific cancer gene mutations: TP53,
KRAS, NAV3, PTEN, ARID1A, and PIK3CA and four frequently mutated
genes namely MUC4, TTN, MUC16, and MUC17. TP53 mutation rate is highly
positively correlated with the expression levels of a large number of autophagy
signaling genes such as ATG genes and BCL-2 genes in BRCA and
COAD. WIPI2, RB1CC1 and ATG9A, involved in regulation of the autophago-
some formation, are down-regulated in BRCA, COAD and STAD tissues har-
boring PI3KCA mutations, which are known to affect autophagy responses
through the PI3K pathway (Shanware et al. 2013). No significant association
has been observed between the expressions of autophagy genes and the other
examined gene mutations.
2.7 Discussion
The role of autophagy in cancer has long been debated as it is proposed to pro-
mote cancer cells’ survival under certain stresses and also possibly to have
tumor-suppression roles (Mathew et al. 2007). These studies have been pre-
dominantly conducted on cell lines with induced autophagy, hence naturally
raising a question: are such observations applicable to cancer tissues, knowing
that the environments in cancer tissues could be substantially different from cell
line studies?
We have recently conducted a comparative analysis of gene-expression of
cancer tissues and cancer cell lines of the matching cancer types, namely BLCA,
BRCA, COAD, KIRC, LIHC, LUAD, LUSC, PRAD, STAD and THCA, under
multiple conditions to assess the expression patterns of 896 well-characterized
biological pathways, covering 48.83 % (10,010/20,501) of human genes in the
two systems (Wei Du and Xu 2016). The following interesting and informative
observations are made: (1) 83.15 % of the pathways are found to have similar
expression patterns under multiple conditions across different cell lines for the
same cancer type; (2) 96.98 % of the pathways share similar expression patterns
across different gene-expression datasets for the same cancer type; and (3) only
20.42 % of the pathways share similar expression patterns between cancer tissue
samples and cell line datasets. This clearly raised a legitimate concern regarding
the applicability of cell-line based observations, particularly about autophagy
since our study has clearly shown that there are fundamental differences between
autophagy in the two systems.
From the comparative analyses presented throughout this chapter, we noted
that the following main differences between cancer tissues and cancer cell based
studies:
–– The 11 cancer types and 8 cancer prone inflammatory diseases studied here all
have reduced or unchanged macro-autophagy activities compared to their basal
level activities in the control samples, which is opposite to what has been
observed in cancer cell lines under induced metabolic stresses;
–– The 11 cancer types full into 2 classes, 1 with reduced macro-autophagy activi-
ties and the other with elevated lysosome activities associated with increased
micro-autophagy or chaperon-mediated autophagy; reduced macro-autophagy is
also observed in 4 cancer prone chronic inflammatory diseases;
–– The differentially expressed autophagy genes are largely independent of the
enzymes involved in energy metabolism across all the examined disease tissues.
Furthermore, there does not seem to be any nutrient-deprivation induced macro-
autophagy in cancer tissues;
–– Co-expression between altered autophagy activity levels and each of the follow-
ing: apoptosis, genomic mutation rate and to some level immune response seems
to have a strong influence from Fenton reactions in cancer tissues, which cancer
cells generally do not have.
Through co-expression analyses, we have identified numerous non-autophagy
genes that are strongly co-expressed with various autophagy genes. Out of these
genes, some could be the direct causes or results while others may co-occur with the
altered autophagy activities, both as results of some common causes. We have con-
ducted further computational analyses to assess if some of these co-expressed genes
with autophagy genes may be the result of one specific common cause, Fenton
reactions.
32 C. Zhang et al.
As discussed earlier, we consider Fenton reactions as one of the root causes

of malignant transformation from normal cells to neoplastic cells. Specifically,
we have demonstrated that all cancer tissues have Fenton reactions in cytosol,
mitochondria and extracellular space (Hui Yan et al. 2016) and cancer-prone
inflammatory tissue cells have the reaction in some but not all three subcellular
compartments (Chi Zhang and Xu 2016). By using the Michaelis-Menton equa-
tion, we have developed a computational procedure to assess if a specific sub-
cellular location may have Fenton reaction via estimating the four relevant
quantities modeled by Michaelis-Mention equation (see below) using gene-
expression data. Specifically, the following equation represents the model for
estimating the quantity of OH produced by Fenton reaction in a specific subcel-
lular location:
æ ö
ç åa X + a0 ÷
RA Fe
K cat
è ø
i i
[•OH ] = å XH 2 RA RA
i
RA
+ e,
XH 2 K1 K2 K3 K4
+ + +
åb X + b0 åc X + c0 æ öæ ö
ROS RA
1
ç åb X + b0 ÷ çåc X k + c0 ÷
j j k k ROS RA
øè ø
j j k
è
j k
j k

where XiFe, XjROS, and XkRA denote the gene expression of marker genes to estimate
the level of Fe2+, H2O2 and RA by the following linear models:
éë Fe 2 + ùû = åa i X iFe + a 0 + e Fe
i
[ H 2 O2 ] = åb j X ROS
j + b 0 + e ROS
j

[ RA ] = åck X kRA + c0 + e RA ,
k
where ai, bi, and ci are regression parameters and ε, εFe, εROS, and εRA are errors.
We have checked if the observed significant co-expressions between autoph-
agy and other biological processes: cell cycle process, mutation rate, and
immune response, are possibly causally linked with each other or are common
results of Fenton reactions. Statistically, we have checked the co-expression
level between A (autophagy) and B (one of the other biological processes),
denoted as cor(A, B), and compared this with the same co-expression level but
under condition of C (Fenton reaction), denoted as cor(A, B | C). The contribu-
tion of C to the co-expression between A and B is evaluated by the Ratio of
Significant Conditional Dependence (RSCD) defined as the ratio of the number
of significantly correlated gene pairs from A and B versus the number of sig-
nificantly correlated gene pairs from A and B under condition of C (see
Fig. 2.3 Distribution of the RSCD (A,B|C) values with A = down-regulated autophagosome for-
mation genes, B = the top 400 biological processes co-expressed with the autophagosome forma-
tion genes, and C = cytosolic Fenton reaction in three LM cancer types
Sect. 2.9). Smaller RSCD values indicate higher impact of C on the correlation

between A and B.
We have computed the RSCD values for A = down-regulated autophago-
some formation genes, B = the top 400 biological processes co-expressed with
A, and C = cytosolic Fenton reaction in the LM cancer types. Histograms of
the RSCD values for COAD, LUAD and LUSC are plotted in Fig. 2.3. From
the figure, we can see that the RSCD values in the three cancer types are con-
sistently distributed as bimodal distributions with one large peak for low
RSCD values (<0.6) and a small peak for high RSCD (>0.6) values, strongly
suggesting most of the biological processes co-expressed with the down-reg-
ulation of autophagosome formation genes are dependent on cytosolic Fenton
reactions. Further analysis revealed that the biological processes with low
RSCD values are quite consistent among the three cancer types, namely apop-
tosis, cell cycle, DNA binding, ion binding, mitochondria, nucleotide synthe-
sis, Golgi apparatus, mRNA transcription, translation and organelles. It is
noteworthy that the centrosome genes have the highest RSCD values among
all the proliferation related pathways, substantiating our hypothesis that other
than being the common results of cytosolic Fenton reactions, decreased
autophagosome formation may directly influence cell cycle process.
Considering the biological properties of A, B, and C, we speculate that the
best explanation of the observations is that cytosolic Fenton reactions are a
common reason for the down-regulated autophagosome formation and other
co-expressed biological processes.
Interestingly, most of the immune related genes and pathways do not seem to
have influence from cytosolic Fenton reactions as revealed by the above analy-
sis (with high RSCD values). Hence we then conducted a similar analysis but
34 C. Zhang et al.
using extracellular Fenton reactions, and have the following results. The aver-
age RSCD value of the immune and inflammation related pathways conditional
to cytosolic Fenton reaction is around 0.8 in the three cancer types while the
average RSCD value of the pathways conditional to extracellular Fenton reac-
tion is 0.5, suggesting a significant contribution of extracellular Fenton reaction
on the correlation between immune response and autophagy but still directly
interactions between them exist there. Possible explanations of the observation
include (1) the macro-autophagy contribute to cytokine production as in infec-
tious diseases, hence suppressed autophagosome cause less cytokine releasing
and immune surveillance (Harris 2011); (2) the macro-autophagy is suppressed
by signaling pathways of certain interleukins include IL-4, IL-10 and IL-13 as
a result of deregulated immune response (Lapaquette et al. 2015); and (3)
extracellular Fenton reaction is a common reason for the immune response and
suppressed autophagosome in the LM cancers.
2.8 Conclusion
Our comparative analysis of transcriptomic data of cancer tissues versus cancer cell
lines revealed that (1) cancer tissues generally do not have metabolic stress and its
induced macro-autophagy; (2) while some cancer tissues have increased lysosome
activity, it is largely induced micro-autophagy or chaperon-mediated autophagy for
degradation of oxidatively damaged macromolecules and organelles due to Fenton
reactions; and (3) various cellular processes are found to be co-expressed with
autophagy genes; the majority of them may represent co-occurring events with
autophagy as common results of Fenton reactions, rather than causal relations with
autophagy. Overall, new information is revealed, which is clearly subject to further
experimental validation and may possibly lead to improved understanding about the
biology of autophagy in cancer tissues.
2.9 Material and Methods
2.9.1 Data Used
We have conducted a differential gene expression analysis measured using nor-

malized fold change in 11 TCGA cancer types: namely bladder urothelial carci-
noma (BLCA), breast invasive carcinoma (BRCA), colon adenocarcinoma
(COAD), head and neck squamous cell carcinoma (HNSC), kidney chromophobe
(KICH), kidney renal clear cell carcinoma (KIRC), liver hepatocellular carci-
noma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma
(LUSC), stomach adenocarcinoma (STAD), and thyroid carcinoma (THCA);
eight cancer-prone inflammatory diseases: cirrhosis (CIR), Barrett's esophagus

(BE), ulcerative colitis (UC), Crohn’s disease (CD), chronic HCV infection
(HCV), idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary dis-
ease (COPD), and atopic dermatitis (AD) that have significant increased risk for
cancer development; and six cancer-independent inflammatory diseases namely
alcohol hepatitis (AH), non-alcoholic steatohepatitis (NAS), cystitis (CS), asth-
matics (AST), and psoriasis (PSO) whose occurrence does not increase the risk
of cancer development as reported in literature; and 12 datasets of cell line-based
gene-expression data collected under serum depletion or other metabolic stress
conditions to induce autophagy.
RNA-seq and genomic data of these tissue and cell samples are collected from
TCGA and GEO databases. Detailed information of the datasets are listed in
Table 2.2. The RNA-seq data are normalized using the RSEM method while all the
microarray data are measured by UA133 plus 2.0 array and normalized by the
RMA method.
2.9.2 G
ene Differential Expression and Pathway
Enrichment Analysis
Differentially expressed genes in cancer and inflammatory disease are assessed by

using the Mann-Whitney test with p-value adjusted by the FDR method and FDR = 0.05
is used as the significance cut-off. Average Fold Change (FC) is used on cell line gene
expression under autophagy-inducing condition versus controls for determination of
differentially expressed genes due to their limited sample size. We use log (FC) = 0.5
and −0.5 as the cut-off for over and under expression, respectively.
Eleven autophagy related pathways are manually generated, as detailed in the main
text. 2775 pathways covering major biological processes including cell prolifera-
tion, apoptosis, and immune response among a few others are collected from the
MsigDB database. In addition, nine sets of metabolic deprivation marker genes
responsive to glucose, leucine, methionine, glutamate, other amino acids as well as
serum deprivation are also retrieved from the MsigDB chemical perturbation
responsive gene sets.
Pathway enrichment is assessed using a hypergeometric test and the p-values are
adjusted by the FDR method with FDR = 0.05 as the significance cut-off.
2.9.3 Gene Co-expression Analysis
We have previously developed a Rank-based gene co-expression module extraction

method (Zhang et al. 2015). The method first identifies hub genes in a given gene
network and then expand the hubs to co-expression modules. The method is applied
36 C. Zhang et al.
to the cancer and inflammatory disease data to identify co-expression modules that
are significantly enriched by autophagy genes. The co-expression modules enriched
by down-regulated macro-autophagy genes in LM diseases and up-regulated lysosome
genes in HL diseases are specifically analyzed to elucidate biological processes
related to the differentially expressed autophagy genes.
2.9.4 Correlate Gene Expression Data to Genomic Mutations
We define the total mutation rate of each sample as the total number of non-synonyms
point mutations. Pearson correlation coefficient between the expression level of each
gene and the mutation rate for each disease type is calculated. Significance of the
correlation is assessed by using the t-test. Six cancer genes and four frequently
mutated genes are selected and analyzed. Association between each mutation and
autophagy gene is tested by comparing the gene expression level in sample with the
mutation versus the mutation free samples by Mann Whitney test. FDR = 0.05 is used
as the significance cut-off.
2.9.5 Ratio of Significant Conditional Dependence
The Ratio of Significant Conditional Dependence (RSCD) is defined by the ratio of

the number of significantly correlated gene pairs from sets A and B versus the number
of significantly correlated gene pairs from A and B under condition C. The RSCD
is defined by following:
# {p.cor ( Ga ,Gb ) a Ga Î A, Gb Î B}
RSCD ( A, B | C ) = ,
# {p.cor ( Ga , Gb | C ) < a | Ga Î A, Gb Î B}

where Ga and Gb are a pair of genes from pathway A and B, p. cor is the p-value of
the co-expression and α is the significance level. We use Pearson correlation to evaluate
the co-expression level between gene pairs. The Pearson correlation product-moment
function is applied to access the p-value for each correlation (and conditional corre-
lation) and a = 0.05 is used as the statistical significance cut-off. There is a signifi-
cant contribution by C to the correlation between A and B if the conditional correlation
level Cor (A, B | C) is substantially lower than Cor (A, B), and there is no significant
contribution by C to the correlation between A and B if Cor (A, B | C) is comparable
with Cor (A, B). Hence smaller RSCD values imply higher impact of C on the
correlation between A and B.
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Chapter 3
Small-Molecule Regulators of Autophagy
as Potential Anti-cancer Therapy
Qing Li, Mi Zhou, and Renxiao Wang
Abstract Autophagy is an evolutionary conserved lysosomal pathway functioned

in the turnover of cellular macromolecules and organelles. It is known that autoph-
agy can have a cytoprotective effect in tumor cells under therapeutic treatment.
Autophagy inhibitors thus may be used as auxiliary drugs to augment the anti-tumor
activity of cancer therapies. On the other hand, autophagy is a cytotoxic event that
can kill tumor cells. Autophagy inducers that increase the level of autophagy thus
may be developed as a new class of anti-cancer therapy. This chapter will describe
the known pathway of autophagy and its relationship to cancer. The focus of this
chapter is to give a summary of the known small-molecule regulators of autophagy,
including inhibitors and inducers, discovered as potential therapies for cancer
treatment.
Keywords Autophagy • Cell death • Autophagy inhibitor • Autophagy inducer •

Anti-cancer treatment
3.1 Introduction
Autophagy plays an essential role in normal physiology. Under normal conditions,

autophagy occurs at basal levels to maintain cellular homeostasis by removing long-
lived or misfolded proteins and clearing damaged or dysfunctional organelles.
Under starved conditions, autophagy can be induced to digest dysfunctional proteins
and organelles more rapidly, which will help cell to survive (Green and Levine 2014).
It is also well known that autophagy may protect cell by overcoming adversities,
Q. Li • M. Zhou • R. Wang (*)

State Key Laboratory of Bioorganic and Natural Products Chemistry,
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences,
345 Lingling Road, Shanghai 200032, P.R. China
e-mail: wangrx@mail.sioc.ac.cn

40 Q. Li et al.
such as starvation, chemotherapies or radiotherapies. Autophagy can also exhibit

cytotoxicity in certain condition, e.g. when apoptosis is blocked (Gewirtz 2014).
In this chapter, we will briefly introduce the molecular mechanism of autophagy
and its dual role in anti-cancer therapy development. We will also review the public
reported small-molecule regulators of autophagy, including autophagy inhibitors
and autophagy inducers, discovered as potential anti-cancer therapies.
3.2 The Process of Autophagy
The known pathway of autophagy includes induction, formation and elongation of

isolation membrane, autophagosome completion, fusion of autophagosome and
lysosome, and degradation in autolysosome (Fig. 3.1). Autophagy is regulated by a
“pre-initiation” ULK complex, which includes ULK1, FIP200 and ATG13. The
ULK complex then activates Class III PI3K complex, which requires the disruption
of binding of anti-apoptotic Bcl-2 proteins to Beclin 1 and is also regulated by
AMPK. The Class III PI3K complex generates Phosphatidylinositol 3-phosphate
Fig. 3.1 The process of autophagy. The process of autophagy starts with the formation of an isola-
tion membrane, which is regulated by the initiation Class III PI3K complex. The Class III PI3K
complex is activated by the pre-initiation ULK complex which is negatively and positively regu-
lated by upstream kinases mTOR and AMPK, respectively. The activated Class III PI3K complex
generates PI3P at the site of nucleation of the isolation membrane. This event leads to the binding
of proteins involved in the “elongation reaction” to the isolation membrane, resulting in formation
of autophagosome. Then, autophagosome fuses with lysosome to form autolysosome, in which its
contents undergo degradation and recycling
3 Small-Molecule Regulators of Autophagy as Potential Anti-cancer Therapy 41
(PI3P) at the site of nucleation of the isolation membrane (also called the phago-
phore). The elongation reaction of the isolation membrane is a complicated process
to form LC3-II. LC3-II generated by the ATG4-dependent proteolytic cleavage of
LC3, and required of ATG7, ATG3, and the ATG12/ATG5/ATG16L complex, which
is associated with the mature autophagosome. The autophagosome fuses with a
lysosome to form an autolysosome, in which the surrounded contents are degraded
and released into the cytoplasm for recycling (Marino et al. 2014).
In the context of nutrient starvation, AMPK is activated and/or mTORC1 is
inhibited, which in turn activated the ULK complex to engage autophagy. During
starvation-induced autophagy, AMPK is required to release negative regulators of
the Beclin 1-VPS34 initiation complex, such as Bcl-2/Bcl-xL (Wirth et al. 2013).
3.3 Role of Autophagy in Cancer
Autophagy has been shown to act as a tumor suppressor, but its role in cancer treat-
ment is still controversial (Maycotte and Thorburn 2011). Almost all of traditional
anti-cancer therapies, such as anti-cancer drugs and ionizing radiation, affect
autophagy. Most of anti-cancer drugs increase autophagy, which protects treated
tumor cells to survive. However, it also has been reported that autophagy is a cell
death mechanism when apoptosis is blocked, known as autophagic cell death
(Maycotte and Thorburn 2011; Thorburn et al. 2014).
3.3.1 Tumor Promotion
In tumorigenesis, the rapid growth of tumor tissue puts cancer cells under harsh and
continuous metabolic stress that results in nutrient deprivation, growth factor limita-
tion, and hypoxia (Zhou and Wang 2013). Nutrient deprivation induces autophagy by
mTORC1 inhibition and AMPK activation. Autophagy can also be induced by
hypoxia, it has been found to localize to hypoxic tumor regions, supporting cell sur-
vival through elimination of autophagic substrate p62, damaged mitochondria and
reactive oxygen species (ROS) (Maycotte and Thorburn 2011; Zhou and Wang 2013).
Autophagy may promote tumor cell metastasis by preventing anoikis. When
cells are detached from the extracellular matrix (ECM), they may undergo anoikis.
However, metastatic tumor cells may escape from anoikis and invade other organs
(Frisch and Screaton 2001). Loss of clonogenic capacity is a foundational factor
during tumorigenesis, autophagy can be induced to reduce clonogenic capacity after
anoikis (Fung et al. 2008).
In tumor treatment, autophagy has been proposed as a protective mechanism to
resist chemo- or radio-therapy and to help residual tumor cells to enter dormancy. It
is well known that autophagy can function as a survival mechanism which is
activated after cancer treatment. In certain instances, tumor can relapse and metas-
42 Q. Li et al.
tasize after primary tumor treatment in many years later, suggesting residual tumor
cells may remain in a dormant state. A recent study showed overexpression of tumor
suppressor aplasia Ras homolog member I (ARHI) promotes the formation of dor-
mant tumors, which was reduced by autophagy inhibitor CQ (Sosa et al. 2013).
3.3.2 Tumor Suppression
Autophagy occurs at basal levels during nutrient rich conditions. The basal autoph-
agy has been shown to be a tumor suppressor mechanism. Cell-cycle check-points
are inactivated in tumor cells, but autophagy limits the accumulation of DNA dam-
age and suppresses the mutation rate. It confirms the role for autophagy in protect-
ing the genome in a cellular spontaneous mechanism of tumor suppression (Mathew
et al. 2007).
A direct link between autophagy and tumor suppression is the discovery that
Beclin 1 could function as a tumor suppressor (Liang et al. 1999). The autophagy
gene Beclin 1 is mono-allelic deleted in 40–75 % of cases of human sporadic
breast, ovarian, and prostate cancer. Disruption of Beclin 1 increases the frequency
of spontaneous malignancies and accelerates the development of hepatitis B virus-
induced premalignant lesions in a targeted mutant mouse model (Qu et al. 2003).
In addition, animals deficient in autophagy-related Atg4C show an increased sus-
ceptibility to develop fibrosarcomas induced by chemical carcinogens (Marino
et al. 2007).
In apoptosis-deficient cancer cells, autophagy has been induced to maintain
cell metabolism and viability during nutrient starvation and protect cells from
necrosis. Finally, if the nutrient deprivation persists, continuous autophagy may
lead to autophagic cell death, which is type II programmed cell death (PCD) (the
others are type I PCD apoptosis and type III PCD necrosis) (Clarke 1990).
Autophagic cell death can be suppressed by autophagy inhibitors (e.g., 3-methyl-
adenine and wortmannin) or genetic knockout/knockdown of essential autophagy
genes (Shimizu et al. 2014). A recent study indicated that JNK activation is cru-
cial for the autophagic cell death of Bax/Bak double knockout cells (Shimizu
et al. 2010).
3.4 Small-Molecule Regulators of Autophagy
A good number of small-molecule regulators of autophagy have been reported in

literature. They have been used either as chemical tools in basic research on autoph-
agy, or developed as drug candidates for cancer treatment (Zhou and Wang 2013;
Baek et al. 2012; Fleming et al. 2011; Wu and Yan 2011; Levy and Thorburn 2011;
Nagelkerke et al. 2015).
3.4.1 Autophagy Inhibitors
There is extensive and relatively definite evidence showed that the level of autoph-
agy increased in tumor cells. Considering the tumor promotion mechanism of
autophagy, many compounds have been developed to treat cancers based on their
autophagy inhibition function (Table 3.1).
Multiple clinical trials are currently on-going at every phase by combining
autophagy inhibitors with various conventional treatment methods in order to
enhance the response to treatment (Gewirtz 2014; Kumar et al. 2015).
3.4.1.1 Class III PI3K Inhibitors
The Class III PI3K, Vps34, shows the positive relationship with autophagy and
generates PI3P at the site of nucleation of the isolation membrane by forming a
complex with Beclin 1 and other cofactors (Green and Levine 2014). A number of
PI3K inhibitors have been developed as autophagy inhibitors, including wortman-
nin, LY294002, 3-methyladenine (3-MA), and SAR405.
Wortmannin, a steroid metabolite of the fungi Penicillium funiculosum, is a non-
specific covalent PI3K inhibitor (Powis et al. 1994). LY294002 is a morpholino
derivative of quercetin (Vlahos et al. 1994). Wortmannin derivative PX-866 and
LY294002 (Arg-Gly-Asp-Ser)-conjugated SF1126 were shown to be active against
various cancer xenografts (Maira et al. 2009). Treatment with wortmannin or
LY294002 resulted in a strong inhibition of proteolysis in amino acids-deprivation
rat hepatocytes (Blommaart et al. 1997). 3-MA inhibited endogenous protein degra-
dation by about 60 % at 5 mM, and suppressed the formation of autophagosomes
(Seglen and Gordon 1982). These three PI3K inhibitors act on PI3K nonselectively,
regarding as tools to study PI3K/mTOR pathway and autophagy.
SAR405, a derivative of pyrimidinones, is a first reported selective inhibitor of
class III PI3K Vps34. Inhibition of Vps34 by SAR405 affects late endosome-
lysosome compartments and prevents autophagy, co-treatment with SAR405 and
mTOR inhibitor everolimus results in synergistic anti-proliferative activity in renal
tumor cell lines (Ronan et al. 2014).
3.4.1.2 Compounds Disrupting Lysosomal Homeostasis
Lysosome is a membrane-bound cell organelle found in most animal cells. It con-

tains hydrolytic enzymes capable of breaking down virtually all kinds of biomole-
cules. In the late stage of autophagy, lysosomes fuse with autophagosomes to digest
the contents of autophagosomes.
Chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) are widely used
as the first choice drug for malaria treatment. CQ seems to exert its effects through
the weak-base feature by enriching in acidic lysosomes, and thereby destroyed lyso-
44 Q. Li et al.
Table 3.1 Small-molecule inhibitors of autophagy

Mechanism and Mechanism and
Compound references Compound references
Pan-PI3K inhibitor Pan-PI3K inhibitor
(Powis et al. 1994; (Maira et al. 2009;
Maira et al. 2009; LY294002 Blommaart et al.
Blommaart et al. 1997; Vlahos et al.
Wortmannin
1997) 1994)
Pan-PI3K inhibitor Class III PI3K
(Maira et al. 2009; inhibitor (Ronan
Seglen and Gordon et al. 2014)
3-methyladenine
1982) SAR405
(3-MA)
Disrupts lysosomal Disrupts lysosomal
homeostasis homeostasis
(Homewood et al. (Homewood et al.
Chloroquine (CQ) Hydroxychloroquine
1972; Fukuda et al. 1972)
2015; Balic et al. (HCQ)
2014; Kimura et al.
2013)
ATPase inhibitor, Disrupts lysosomal
disrupts lysosomal homeostasis (Cerny
homeostasis (Harada et al. 2004; Lu et al.
Bafilomycin A1 et al. 1996; Mauvezin Vacuolin-1 2014)
and Neufeld 2015)
Disrupts lysosomal Disrupts lysosomal
Thymoquinone
homeostasis (Chen homeostasis
Matrine et al. 2006; Liu et al. (Racoma et al.
2010; Wang et al. 2013)
2013)
Cathepsin inhibitor, Cathepsin inhibitor,
disrupts lysosomal disrupts lysosomal
homeostasis (Tamai homeostasis (Tanida
E-64d Pepstatin A
et al. 1986; Tanida et al. 2005;
et al. 2005) Umezawa et al.
1970)
Topoisomerase Casein kinase
inhibitor, disrupts activator, inhibits
lysosomal the transcription of
homeostasis (Bases Pyrvinium autophagy genes
Lucanthone and Mendez 1997; (Thorne et al. 2010;
Carew et al. 2011) Deng et al. 2013)
ATG4B inhibitor, Macrolide antibiotic
inhibits formation of (Nakamura et al.
NSC185058 LC3-II (Akin et al. 2010)
2014)
Clarithromycin
(continued)

Compound references Compound references
Macrolide antibiotic
(Renna et al. 2011)
Azithromycin
somal function (Homewood et al. 1972). It shows the antitumor activity in many
kind of tumor cells, such as endometrial cancer cells (Fukuda et al. 2015), pancre-
atic cancer stem cells (Balic et al. 2014). Inhibition of autophagy by CQ could
sensitize cisplatin-tolerant cancer cells, as well as injure kidney cells in chemo-
therapy, leading to acute kidney injury (Kimura et al. 2013).
Bafilomycin A1 is an inhibitor of V-ATPase, which is necessary for acidification
of the endocytic compartments (Harada et al. 1996). It can also disrupt autophagic
flux by inhibiting calcium ATPase-dependent autophagosome-lysosome fusion
(Mauvezin and Neufeld 2015).
Vacuolin-1 has been discovered in an image-based phenotypic screen for inhibi-
tors of the secretory pathway, by blocking the Ca2+-dependent exocytosis of lyso-
somes (Cerny et al. 2004). Treatment with vacuolin-1 alkalinized lysosomal pH and
decreased lysosomal Ca2+ content in HeLa cells (Lu et al. 2014).
Matrine, derived from traditional Chinese medicine Sophora flavescens, has
been reported to improve the immune function and life quality of cancer patients by
combining standard therapies (Chen et al. 2006). It can also inhibit proliferation and
induce apoptosis of pancreatic cancer cells (Liu et al. 2010). Recently matrine has
been reported to block autophagic degradation by impairing the activities of lyso-
somal proteases, and elevating pH values in endosomes/lysosomes (Wang et al.
2013).
Thymoquinone, derived from Nigella sativa seed, was reported to inhibit prolif-
eration in glioblastoma cells. It induced lysosomal membrane permeabilization,
resulting in a leakage of cathepsin B into the cytosol, which mediates caspase-
independent cell death (Racoma et al. 2013).
Cathepsins are proteases distributed in almost all mammalian cells, with func-
tions in tumor progression (Nomura and Katunuma 2005). Most of the members of
cathepsins become activated at the low pH level in lysosomes. Their activities are
closely linked with the lysosomal function. Cathepsins inhibitors E64d (Tamai et al.
1986) and pepstatin (Umezawa et al. 1970) are frequently used in autophagy-related
research as autophagy inhibitors (Tanida et al. 2005).
Lucanthone, an inhibitor of topoisomerase, has been used as an adjuvant in radi-
ation therapy (Bases and Mendez 1997). It induces lysosomal membrane permeabi-
lization to break lysosomal homeostasis, and possesses significantly more potent
activity in breast cancer models compared with CQ (Carew et al. 2011).
46 Q. Li et al.
3.4.1.3 Others Types of Autophagy Inhibitors
An FDA-approved antihelminthic drug pyrvinium shows wide-ranging anti-cancer

activity during glucose starvation. It binds casein kinase 1 and increases the kinase
activity of casein kinase 1α, which is a negative regulator of Wnt1 pathway (Thorne
et al. 2010). Pyrvinium was reported to inhibit autophagy by suppressing the tran-
scription of autophagy genes, such as Beclin1 and Vps34. The inhibition of
autophagy by pyrvinium increases the anti-cancer activity of 2-deoxy-D-glucose
(Deng et al. 2013).
ATG4B is an essential cysteine proteinase to activate LC3 produce LC3-II.
Knockdown of Atg4b in Osteosarcoma Saos-2 cells resulted in a failure of forming
tumors in mouse models. The antagonist of ATG4B NSC185058 shows a negative
impact on the development of Saos-2 osteosarcoma tumors in vivo (Akin et al.
2014).
Macrolide antibiotic clarithromycin and aithromycin were reported to block
autophagy, whose mechanisms still remain unclear. Clarithromycin increased the
anti-tumor activity of thalidomide against multiple myeloma cells (Nakamura et al.
2010). Azithromycin mediated autophagy increases the risk of infection with
drug-resistant pathogens (Renna et al. 2011).
3.4.2 Autophagy Inducers
Based on the role of autophagy in tumor suppression, many compounds are used as
anti-cancer reagents by inducing autophagy (Fulda and Kogel 2015) (Table 3.2). It
should be noted that not all autophagy inducers may be used as anti-cancer reagents.
It is because some of them induce protective autophagy, which leads to tumor resis-
tance. These compounds are combined with autophagy inhibitors to treat cancer
usually, so they are not discussed in this chapter.
3.4.2.1 mTOR Inhibitors
mTOR (mammalian Target Of Rapamycin) senses cellular nutrient and energy levels,
and negatively regulates autophagy. mTOR forms two distinct signaling complexes,
mTORC1 and mTORC2. The molecular mechanism of how mTORC2 is regulated
by its upstream effectors is largely unknown. mTORC1 (hereafter mTOR) is a mas-
ter regulator of cellular metabolism and autophagy, regulated by the growth factor/
PI3K/AKT signaling pathway. It integrates nutrient and growth factors which signal
to promote anabolic metabolism, such as protein synthesis and lipid synthesis, and
to inhibit catabolic pathways, such as lysosomal biogenesis and autophagy (Laplante
and Sabatini 2012).
Inhibition of mTOR leads to activation of ULK1, which then phosphorylates
other critical subunits of the ULK1 complex, ATG13 and FIP200 (Jung et al.
Table 3.2 small-molecule inducers of autophagy

Compound references Compound References
mTOR inhibitor mTOR inhibitor
(Kim and Guan (Kim and Guan
2015; Nam et al. 2015; Albert et al.
2013) 2006; Cao et al.
2006)
Rapamycin Everolimus
Antifungal drug, Topoisomerase
inhibits mTOR/ inhibitor, inhibits
AKT/PI3K signaling mTOR activity
pathway (Liu et al. (Ristic et al. 2014;
2014) Idarubicin Plumbridge and
Brown 1978)
Itraconazole
AMPK activator Tyrosine kinase
Metformin (Takahashi et al. inhibitor, activates
2014) AMPK (Yu et al.
Nilotinib 2013)
Protein kinase C Disrupts calcium
inhibitor, activates homeostasis
AMPK (Kumar et al. (Hoyer-Hansen
2014) et al. 2007;
Ionomycin Hoyer-Hansen and
Rottlerin
Jaattela 2007)
Disrupts calcium Disrupts calcium
homeostasis homeostasis (Wong
(Hoyer-Hansen et al. et al. 2013)
2007; Hoyer-Hansen
and Jaattela 2007) Saikosaponin-d
Thapsigargin
Disrupts calcium Disrupts calcium
homeostasis homeostasis
(Rubiolo et al. 2014; (Salabei et al. 2012)
Azad et al. 2008) Verapamil
Yessotoxin
HDACi, induces HDACi, induces
Valproic acid ROS-dependent autophagic cell
autophagy (Shao Vorinostat death (Shao et al.
et al. 2004; Fu et al. 2004; Zhang et al.
2010) 2005; Yamamoto
et al. 2008; Wei
et al. 2010)
Sodium HDACi, induces HDACi, induces
butyrate autophagic cell death autophagic cell
(Shao et al. 2004; death (Watanabe
Hamer et al. 2008) FK228 et al. 2009)
(continued)
48 Q. Li et al.

Compound references Compound References
Arsenic trioxide (As2O3) Induces autophagic Sodium arsenite Induces ROS-
cell death (Miller (NaAsO2) dependent
et al. 2002; autophagic cell death
Goussetis et al. (Miller et al. 2002;
2010) Zhu et al. 2014; You
et al. 2015)
EHMT2 inhibitor, Bcl-2 inhibitor,
induces autophagic induces autophagy
cell death (Kim et al. by releasing Beclin
2013a) 1 (Shimizu et al.
Gossypol
2004; Voss et al.
BIX-01294 2010)
Bcl-2 inhibitor, Bcl-2 inhibitor,
induces autophagy induces autophagic
by releasing Beclin 1 Obatoclax cell death (Heidari
Apogossypolone (Zhang et al. 2010; et al. 2010;
Arnold et al. 2008; Bonapace et al.
He et al. 2014; Niu 2010)
et al. 2014)
Tyrosine kinase Induces autophagic
inhibitor, induces cell death (Wang
autophagic cell death et al. 2014;
(Chen et al. 2015; Gemcitabine Donadelli et al.
Lapatinib Chen et al. 2014) 2011)
Induces ATG-5- Salinomycin Cation ionophore,
dependent autophagy activates autophagic
(Leng et al. 2013) flux
Ursolic
acid
2009). The growth factor/PI3K/AKT/mTOR pathway is the main pathway regu-

lated by mTOR, its activation is associated with malignant transformation and
apoptotic resistance, and hence represents a cell survival mechanism (Polivka and
Janku 2014).
In earlier studies, mTOR inhibitors, such as rapamycin and everolimus, were
reported to induce autophagy in various model systems. The induction of autoph-
agy by mTOR inhibitors are more inclined to protect cancer cells survival, not
death (Kim and Guan 2015). However, recent reports showed rapamycin-induced
autophagy may sensitize cancer cells to radiotherapy. Everolimus inhibited
radiation-induced AKT/mTOR signaling pathway and enhances the cytotoxic
effects of radiation in breast cancer cell models (Albert et al. 2006). It increased
the radio-sensitization of PTEN-null prostate cancer cells, and enhances radiation-
induced mortality in apoptosis deficient cells, which means everolimus may induce
autophagic cell death in certain condition (Cao et al. 2006). In addition, persistent
activation of autophagy by mTOR inhibitor rapamycin leads radio-resistant cancer
cells into senescence in head and neck cancer cells and a xenograft model (Nam
et al. 2013).
Itraconazole, a traditional broad-spectrum antifungal drug, inhibited cell prolif-

eration and induced autophagic progression in glioblastoma cells by repression of
AKT/mTOR signaling pathway. Its anti-proliferative activity was inhibited by the
blockage of autophagy, suggesting autophagy is responsible for itraconazole-
induced inhibition of proliferation (Liu et al. 2014).
Idarubicin is a DNA-binding antileukemic drug (Plumbridge and Brown 1978),
has been showed to induce apoptosis and cytotoxic autophagy through mTOR
repression. Autophagy inhibitor wortmannin or CQ partially reduced the cytotoxic-
ity of idarubicin in the acute lymphocytic leukemia REH cells (Ristic et al. 2014).
3.4.2.2 AMPK Activators
AMPK are known to induce autophagy. Under starvation condition, AMPK induc-
tion and/or mTOR inhibition will lead to autophagy by ULK1 phosphorylation.
AMPK can also act directly on the Beclin 1/Vps34 complex (Kim et al. 2013b).
Metformin, a prescribed drug for type 2 diabetes, activated AMPK and reduced
cell proliferation, leading to the induction of apoptosis and autophagy. Inhibition of
autophagy by knockdown of Beclin 1 or by 3-MA suppressed the anti-proliferative
effects of metformin on endometrial cancer cells, indicating that the anti-proliferative
effects of metformin are partially or completely dependent on autophagy (Takahashi
et al. 2014).
Nilotinib, a tyrosine kinase inhibitor, significantly reduced cell viability in hepa-
tocellular carcinoma cell lines through autophagy by AMPK activation instead of
apoptosis. Knock-down of Atg5 reduced the effect of nilotinib on autophagy and
cell death significantly, co-treatment of nilotinib with a known AMPK activator
metformin enhanced the effect of nilotinib on autophagy and cell death (Yu et al.
2013).
Rottlerin, a protein kinase C inhibitor, showed anti-cancer activity in prostate
cancer. It induced early stage autophagy via AMPK activation and apoptosis via
inhibiting PI3K/AKT/mTOR pathway in human prostate cancer stem cells.
Co-treatment with autophagy inhibitors bafilomycin or 3-MA inhibited rottlerin-
induced apoptosis. It illustrated that autophagy was required in rottlerin mediated
prostate cancer treatment (Kumar et al. 2014).
3.4.2.3 Compounds Disrupting Calcium Homeostasis
Calcium (Ca2+) is one of the most important cellular second messengers. The disor-
der of Ca2+ homeostasis can evoke different types of cell death in cancer cells.
Autophagic cell death can be induced by vitamin D3, ATP, ionomycin and thapsi-
gargin, which increased the cytosolic Ca2+ in MCF-7 breast cancer cells (Hoyer-
Hansen et al. 2007). One of the best investigated mechanisms of calcium-mediated
autophagy induction is mTOR-mediated endoplasmic reticulum (ER) stress and
unfolded protein response (UPR) activation (Hoyer-Hansen and Jaattela 2007).
50 Q. Li et al.
Saikosaponin-d is an inhibitor of Ca2+ ATPase which induces autophagy by acti-

vating the Ca2+/calmodulin-dependent AMPK/mTOR signaling pathway, ER stress
and UPR. It leaded to autophagic cell death especially in apoptosis-resistant MEFs
cells, which either lack Caspases 3, 7 or 8 or have the Bax/Bak double knockout
(Wong et al. 2013).
Yessotoxin has been shown to modulate Ca2+ gating resulted in increasing cyto-
solic calcium in human lymphocytes. In glioma cells, it induced autophagic cell
death mediated by BNIP3 (Rubiolo et al. 2014). BNIP3 inhibits the mTOR pathway
through RHEB, which has been shown to induce cell death by autophagy inhibitor
3-MA but not by caspase inhibitor z-VAD-fmk (Azad et al. 2008).
Verapamil is an L-type Ca2+ channel antagonist. Treatment with verapamil did
not affect cell viability in vascular smooth muscle cells, but inhibited cell prolifera-
tion and induced morphological alterations, such as karyokinesis and accumulated
perinuclear vacuoles due to enhanced mitochondrial damage and upregulated
autophagy (Salabei et al. 2012).
3.4.2.4 Histone Deacetylase Inhibitors
Histone deacetylase (HDAC) is a class of enzymes that remove acetyl groups from
an lysine on histone, allowing the histones to wrap the DNA more tightly, leading to
chromatin remodeling and transcriptional suppression of key apoptosis and cell
cycle regulatory genes (Jazirehi 2010).
It has been reported that HDAC inhibitors preferentially kill transformed cells or
cancer cells. According to their chemical structures, HDAC inhibitors can be classi-
fied into several groups, including (i) short-chain fatty acids, such as sodium butyr-
ate and valproic acid ; (ii) hydroxamic acids, such as vorinostat; and (iii) cyclic
tetrapeptides, such as FK228 (Shao et al. 2004).
Valproic acid, a widely used anti-epilepsy drug, induces autophagy by ROS-
dependent pathway in glioma cells. Combination with other autophagy inducers
(such as rapamycin, LY294002) increased valproic acid-induced autophagic cell
death (Fu et al. 2010). Sodium butyrate exerts potent effects on the inhibition of
inflammation and carcinogenesis in colon tissue (Hamer et al. 2008). It induces
mitochondria-mediated apoptosis and autophagic cell death in HeLa cells (Shao
et al. 2004).
Vorinostat (also known as suberoylanilide Hydroxamic Acid) is approved by FDA to
treat cutaneous T cell lymphoma (Zhang et al. 2005). It was reported to induce apoptosis
and autophagic cell death in chondrosarcoma cell lines and HeLa cells (Shao et al. 2004;
Yamamoto et al. 2008). Combination with vorinostat and BH3-mimetic GX15-070 has
synergistic effects in acute myeloid leukemia (AML) cell lines and primary AML cells
by activating both apoptosis and autophagy (Wei et al. 2010).
Another HDAC inhibitor FK228 can also induce autophagy. Disrupting autophagy
with CQ enhanced FK228-induced cell death, which means FK228-induced autophagy
is cytoprotective (Watanabe et al. 2009).
3.4.2.5 BH3 Mimetics
Anti-apoptotic Bcl-2 proteins show its anti-apoptotic activity by binding pro-

apoptotic protein Bax/Bak, they inhibit autophagy by binding Beclin 1 through their
BH3 domain. Consequently, BH3 mimetics are able to activate apoptosis and
autophagy (Pattingre and Levine 2006). They have a role in control of autophagic
cell death depends on the autophagy genes Beclin 1 and Atg5 (Shimizu et al. 2004).
Bcl-2 inhibitor gossypol was reported to induce autophagic cell death by releas-
ing Beclin 1 in malignant glioma, it potentiated the anti-cancer activity of temozolo-
mide, which was used as a first-line treatment of glioblastoma multiforme (Voss
et al. 2010). Apogossypolone is a derivative of gossypol, exhibits a higher antitumor
activity and lower toxicity than gossypol (Zhang et al. 2010; Arnold et al. 2008).
It can also inhibit the binding of Bcl-2 to Beclin 1, it induces autophagy and radio-
sensitizing in nasopharyngeal carcinoma cells in vitro and in vivo (He et al. 2014).
Moreover, it reduced Bcl-2 expression, and enhanced the expression of Bax and
Beclin 1 in MCF-7 cells (Niu et al. 2014).
Obatoclax (also known as GX15-070) has been reported to overcome glucocorticoid
resistance in acute lymphoblastic leukemia (ALL) by inducing apoptosis and autophagy,
which can be inhibited by downregulation of ATG5 or Beclin 1 (Heidari et al. 2010). In
childhood ALL cells, obatoclax plays a role in autophagy-dependent necroptosis, which
is required for overcoming glucocorticoid resistance (Bonapace et al. 2010).
3.4.2.6 Other Types of Autophagy Inducers
Inorganic arsenic is a worldwide environmental pollutant. It is widely known as

carcinogens that induce cancers in many human tissues (Miller et al. 2002). Arsenic
trioxide (As2O3) has been used to treat acute promyelocytic leukemia, and also acts
as a potent inducer of autophagy in leukemia cells. Treatment with autophagy inhib-
itors or knockdown of Beclin 1 or Atg7 resulted in the decreased inhibition of arse-
nic trioxide on leukemic cell lines and primary leukemic progenitors from AML
patients (Goussetis et al. 2010). Another trivalent arsenicals sodium arsenite
(NaAsO2) has showed the positive relationship with the incidence of type 2 diabe-
tes. This phenomenon may be attributed to the ability of sodium arsenite in inducing
ROS-dependent autophagic cell death in pancreatic β-cells. Autophagy inhibitor
3-MA protected the cells against sodium arsenite cytotoxicity, and autophagy
inducer rapamycin further decreased the cell viability of sodium arsenite-treated
INS-1 rat insulinoma cells (Zhu et al. 2014). Sodium arsenite showed an anti-
proliferative effect on DU145 prostate cancer cells in xenograft mice, it induced
both apoptosis and autophagic cell death via ROS (You et al. 2015).
BIX-01294, a selective inhibitor of euchromatic histone-lysine N-methyltransferase
2 (EHMT2), induced autophagic cell death via EHMT2 dysfunction and intracellular
ROS production, and increased autophagy-dependent and caspase-independent cell
death in primary human breast and colon cancer cells (Kim et al. 2013a).
52 Q. Li et al.
Lapatinib, a tyrosine kinase inhibitor, has been widely accepted in the treatment
of breast cancer. In breast cancer cells, it induced apoptosis and protective autophagy
related with lapatinib resistance (Chen et al. 2015). But in human hepatoma cells,
researchers found that lapatinib induced autophagy, a higher percent of dead cells
and a lower percent of hypodiploid cells, that suggesting non-apoptotic cell death
but autophagic cell death in lapatinib-treated hepatoma cells (Chen et al. 2014).
Gemcitabine (GEM) is currently the first-line treatment for pancreatic cancer.
GEM elevated autophagic progress by the MEK/ERK signaling pathway. The
autophagic activity was reduced in GEM-resistant human pancreatic cell line
KLM1-R compared to GEM-sensitive KLM1 cells, suggesting autophagy was
required in GEM-mediated pancreatic cancer treatment (Wang et al. 2014).
Combination with GEM and cannabinoid triggers autophagic cell death in pancre-
atic cancer cells through a ROS-mediated mechanism (Donadelli et al. 2011).
Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, has been
showed potent anti-cancer activity. UA was reported to promote cervical cell lines
TC-1 cell death, not through apoptosis but ATG5-dependent autophagy. Treatment
with autophagy inhibitor and Atg5 knockdown increased the survival of TC-1 cells
treated with UA (Leng et al. 2013).
Salinomycin, a cation ionophore, has been showed to reduce the viability of breast
cancer stem-like/progenitor cells by inhibiting autophagy (Yue et al. 2013). However,
it has been reported to induce autophagy in human non-small cell lung cancer cells
(Li et al. 2013). In recent study by Li et al. (Jangamreddy et al. 2015), treatment with
lower concentration of salinomycin activates autophagic flux in prostate cancer cells
while murine embryonic fibroblasts (MEFs) show an inhibition of autophagic flux.
But it inhibits autophagic flux in both cell types at a higher concentration, which
means salinomycin seems like autophagy inducer instead of inhibitor.
3.5 Summary
Autophagy is a catabolic mechanism mediated by lysosomal degradation, which is

required for cellular homeostasis. Disruption of autophagy is associated with vari-
ous human diseases. Autophagy is regulated by a series of proteins, such as some
kinases, some of which in turn may be used as the molecular targets for the develop-
ment of autophagy inhibitors and inducers. The most notable characteristics of
autophagy related to cancer is its dual role, i.e. it can act as a tumor suppressing
mechanism as well as a tumor promoting mechanism. Using autophagy inhibitors
as an adjuvant therapy in combination with cytotoxic anti-cancer drugs is a promis-
ing way to overcome resistance in cancer therapies. On the other hand, a number of
compounds achieve their anti-cancer activities by inducing autophagic cell death,
presenting another possible strategy for developing novel anti-cancer drugs.
Considering the complex context of autophagy, it is important to specify the indica-
tion and potential side effects of each type of small-molecule autophagy regulator in
anti-cancer drug discovery.
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Chapter 4
Regulation of Autophagy by microRNAs:
Implications in Cancer Therapy
Hua Zhu and Jin-Ming Yang
Abstract As an emerging hallmark of cancer, aberrant energy metabolism has

drawn increasing attention in both basic research and clinical study. Autophagy is
one of the main mechanisms for cells to maintain metabolic homeostasis, and can-
cer cells often display altered autophagic activity. Thus, autophagy is now pursued
as a target for anti-cancer therapies. The current approaches to modulating autoph-
agy include manipulation of either expressions or functions of the proteins that are
key components of autophagic pathways. As a main post-transcriptional regulatory
factor, microRNAs play important roles in various physiological and pathophysio-
logical processes including cancers. Since miR-30a was first reported to regulate
autophagy through targeting 3′ untranslated region (3′ UTR) of Beclin-1, a key
autophagy regulatory gene, numerous miRNAs involved in autophagy regulation
have been reported. Here we overview the current knowledge regarding the roles of
miRNAs in regulation of autophagy and their implication in cancer therapy.
Keywords Autophagy • microRNA • Post-transcriptional regulation • Cancer

therapy • Cancer metabolism
4.1 Introduction
Autophagy is one of the key processes in maintaining cellular homeostasis. In

response to various stresses such as metabolic and therapeutic stress, autophagy
promotes the elimination of abnormal proteins or organelles and the refurbishment
of energy for cell survival. Most of the time, there is a basal level of autophagy
responsible for degradation of cytoplasmic aggregates. However, prolonged
H. Zhu
Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University
Wexner Medical Center, 460 W 12th Avenue, Columbus, OH 43210, USA
J.-M. Yang (*)
Department of Pharmacology, The Penn State Hershey Cancer Institute, The Pennsylvania
State University, College of Medicine and Milton S. Hershey Medical Center,
500 University Drive, Hershey, PA 17033, USA
e-mail: juy16@psu.edu

60 H. Zhu and J.-M. Yang
autophagy under stressful condition (e.g., nutrient deprivation) could result in cell
death, i.e., cannibalistic cell death. Dysregulation of autophagy is implicated in
many pathological conditions including neurodegenerative diseases and cancer.
Therefore, understanding the molecular mechanism of autophagy regulation would
be important for development of novel therapeutic strategies for those diseases.
MicroRNA (miRNA) a group of non-coding RNAs able to target to 3′ untranslated
region (3′ UTR) of mRNAs to suppress protein expression. Increasing evidences
have demonstrated that miRNAs have important roles in a variety of physiological
and pathophysiological processes. For instance, connections between miRNAs and
autophagy have been revealed in many studies. miRNAs can manipulate autophagy
by targeting the expression of key proteins involved in autophagy regulation. It has
become clear that there is an abundance of miRNAs, which are either tumor sup-
pressive or tumor promotive, are involved in autophagic control and impact cancer
development and progression. In the chapter, we will discuss the role of miRNA in
regulation of autophagy and the related therapies for cancer treatment.
4.2 Autophagy and Its Regulatory Proteins
4.2.1 Autophagy Is an Intrinsic Process to Maintain

Cellular Homeostasis
The cycle of protein synthesis and degradation is essential in sustaining cellular

homeostasis. Autophagy is maintained at a basal level under physiological condi-
tions (Mizushima et al. 2008; Denton et al. 2009). Increases in autophagic activity
often occur as a response to various stresses such as glucose deprivation and hypoxia
(Kroemer et al. 2010), serving as a survival or protective mechanism. Prolonged
autophagy, however, can eventually result in cell death (cannibalistic cell death)
(Pan et al. 2013). Autophagy, a process of “eating of self” (Glick et al. 2010; Levine
and Kroemer 2008, 2009), is a means of cell survival through eliminating protein
aggregates and removing damaged cell organelles (Gottlieb et al. 2010). It was first
clearly demonstrated in the rat livers exposed to the pancreatic hormone glucagon
(Deter and De Duve 1967) that the mitochondria and other cytoplasmic material
were subjected to lysosome-mediated degradation in double-membrane structures
referred to as autophagosomes (Zhang and Baehrecke 2015; Glick et al. 2010).
Various forms of autophagy have been identified, including macro- or micro-
autophagy, and chaperone-mediated autophagy (Mizushima et al. 2008; Glick et al.
2010; Yang and Klionsky 2010). Macroautophagy is the most extensively defined
type that is characterized by the formation of autophagosome. Autophagy prevents
dysfunctional intracellular proteins from accumulating, subsequently recycling the
proteins to produce source of energy. Therefore, autophagy is appreciated as an
intrinsic pathway to promote cell survival under stress conditions. However, as
mentioned above, prolonged autophagy can cause cell death. Indeed, autophagy
was originally considered as a type of programmed cell death that plays an
4 Regulation of Autophagy by microRNAs: Implications in Cancer Therapy 61
important role in controlling the balance of organism health (Wang and Klionsky
2003; Zhang and Baehrecke 2015), in addition to other interactive types of cell
death such as apoptosis and necrosis (Gordy and He 2012; Amaravadi et al. 2007;
Schweichel and Merker 1973). Differently, autophagy is an intracellular procedure
in which defective or excess cytoplasmic components are degraded (Wu et al. 2015).
Autophagy consists of five stages: induction, vesicle nucleation, elongation,
retrieval, and fusion for cargo degradation (Sui et al. 2011; Jing et al. 2015).
During autophagy, various organelles such as the endoplasmic reticulum, mito-
chondria, or plasma membrane are utilized to form autophagosomal vacuoles (Puri
et al. 2013; Longatti et al. 2012; Hailey et al. 2010; Ravikumar et al. 2010; Maiuri
et al. 2007; Levine and Klionsky 2004). The vacuoles fuse with the lysosomes to
initiate protein degradation via lysosomal hydrolases (Gottlieb et al. 2010; Maiuri
et al. 2007; Levine and Klionsky 2004). Microautophagy occurs when there is an
uptake of whole organelles or cytoplasm directly at vacuole surface. These two
processes are seemingly nonspecific for bulk degradation, although other more
selective degradation pathways are seen in chaperone-mediated autophagy (Wang
and Klionsky 2003).
4.2.2 Regulation of Autophagy by ATG
Genetic studies in yeast uncovered a set of autophagy-related, termed ATG genes,

which are required for autophagosome formation (Feng et al. 2014; Nakatogawa
et al. 2009). Further investigations revealed the preservation of the ATG genes
across yeast and humans (Mizushima and Komatsu 2011). The formation of
autophagosome involves ATG genes (such as ATG5, ATG7, and ATG10) and anti-
apoptotic proteins (e.g., Bcl-2/Bcl-xL) (Mizushima et al. 2008). Other important
regulators of autophagy include mammalian target of rapamycin (mTOR) and
AMP-responsive protein kinase (AMPK) (Sui et al. 2015). Autophagy is closely
associated with nutrition status of cells because it is inhibited by mTOR, a meta-
bolic sensor, and activated by AMPK (Zhang and Baehrecke 2015; Gottlieb et al.
2010). mTOR senses amino acid levels and insulin signaling to inhibit autophagy
through phosphorylating unc-51-like kinase ½ (ULK1/2) and ATG proteins
(Klionsky 2007). On the other hand, AMPK can exert a negative effect on mTOR
signaling through TSC1/2 phosphorylation in response to an increased AMP/ATP
ratio or mitogen-activated protein kinases (MAPKs) (Sui et al. 2011; Li et al. 2013;
Radogna et al. 2015; Russell et al. 2014). Nutrient deficiency inhibits mTOR and
promotes the binding of dephosphorylated ATG13 to ATG1, triggering the creation
of autophagosomal membranes (Jing et al. 2015).
Induction of autophagy is initiated by the activation of the ULK complex (ULK ½,
ATG13, FIP200 and ATG101) (Jing et al. 2015). Subsequently, vesicle nucleation is
induced by the formation of a complex of Beclin-1 and a myristylated kinase to acti-
vate Vps34 class III phosphatidylinositol 3-kinase (PI3K) (Jing et al. 2015). Elongation
is mediated by the ubiquitin-like conjugation systems with a number of ATG proteins
(ATG12-ATG5-ATG16L and ATG8-phosphatidylethanolamine system), along with

the retrieval and fusion process regulated by ATG2, ATG9, ATG18 and the UV
irradiation resistance-associated tumor suppressor gene (UVRAG). This process is
further facilitated by the microtubule-associated protein 1 light chain 3 (LC3),
which couples with phosphatidylethanolamine (PE) to form a membrane-bound
conjugation system (Su et al. 2015). Finally, the autophagosome fuses with lyso-
some to form autophagolysosome via ATG9 and vacuole membrane protein 1
(VMP1), where intracellular proteins are degraded by the lyososomal cathepsins
(Wang and Klionsky 2003; Gottlieb et al. 2010; Yang and Klionsky 2010; Jing et al.
2015; Levine and Klionsky 2004; Ichimura et al. 2000; Mizushima et al. 1999;
Kaminskyy and Zhivotovsky 2012). Recycled macromolecules and organelles are
then turned into their original building blocks of amino acid and fatty acid
(Mizushima and Komatsu 2011; Li and Vierstra 2012).
4.2.3 Other Autophagy-Regulatory Proteins
There are a variety of other proteins that participate in autophagy regulation

(Gibbings et al. 2013). For instance, apoptosis repressor with caspase recruit domain
(ARC) is an anti-autophagic protein (Bo et al. 2014). Immunity-related GTPase
family M gene (IRGM) manipulates autophagy to alter the innate immune response
(Singh et al. 2010). Histone acetyltransferases (HATs) and deacetylases (HDACs)
are critical in protein acetylation to promote or suppress gene transcription (Jing
et al. 2015; Sui et al. 2015). Selective autophagy can be facilitated by the SQSTM1/
p62-like receptors (SLRs), which identify and target substrates for autophagosomes
(Gibbings et al. 2013).
The Bcl-2 protein family is a group of key autophagy regulators. Bcl-2 can bind
to Beclin-1 to inhibit autophagy (Jing et al. 2015). The interaction between Bcl-2
and Beclin-1 determines the coordination of autophagy and apoptosis (Kang et al.
2011). Beclin-1 is a 60 kDa-coiled protein, and its aberrant expression has been
observed in certain diseases including cancer and neurodegenerative disease (Wang
and Klionsky 2003). Beclin-1 is frequently monoallelically mutated in various can-
cers such as breast or ovarian cancers, with decreased expression of the protein in
several types of tumors (Itakura et al. 2012; Yue et al. 2003). A study showed that
Beclin-1 is required in the embryonic phase and for autophagy but not for apoptosis
(Yue et al. 2003).
4.2.4 Implication of Autophagy in Human Diseases
Dysregulation of autophagy is related to pathogenesis of various human diseases

(Klionsky 2007; Zhou et al. 2012; Lavandero et al. 2013) such as aging, cancer
(Radogna et al. 2015), and neurodegenerative disease (Wu et al. 2015; Zhu et al.
2009). Beclin-1 has been reported as a candidate tumor suppressor (Wang and
Klionsky 2003; Yue et al. 2003), and mutation or inactivation of one allele of
Beclin-1 is frequently detected in many types of human cancers (Gottlieb et al.
2010). Induction of autophagy is believed to be beneficial in certain pathological
conditions. However, whether autophagy is a gateway to or a protection from cell
death remains to be fully elucidated. While some studies propose that hyper-
activation of autophagy may segue into apoptosis or necrosis (Maiuri et al. 2007;
Denton et al. 2015; Berry and Baehrecke 2008; Clarke and Puyal 2012), others
suggest that autophagy is an adaptive response to counter cell death (Singh et al.
2010; Brest et al. 2011). It is likely that autophagy plays roles in both of cell death
and survival, similar to inflammation that can act as an organism-protective
immune reaction but when overactive, it can result in cannibalistic cell death
(Mizushima 2007). Understanding the mechanisms of the crosstalk between
autophagy and other cellular processes (e.g., apoptosis, necrosis) would be impor-
tant for further dissecting the contribution of autophagy to pathogenesis of some
human diseases.
4.3 Molecular Basis of miRNAs
4.3.1 MicroRNAs in Human Genome
Eukaryotic cells utilize protein regulatory mechanisms that have been relatively
conserved through evolution (Zhao and Srivastava 2007). Researches in the past a
few decades have progressed toward the direction of studying small non-coding
RNAs in the regulatory of cellular activity. Four of these RNAs have been identi-
fied: microRNA (miRNA), short interfering RNAs (siRNA), repeat-associated
small interfering RNAs (rasiRNAs) and piwi-interacting RNAs (piRNAs) (Aravin
and Tuschl 2005; Kim 2006). Among these small non-coding RNAs, miRNAs are
the most phylogenetically conserved in various physiological processes—one of the
first miRNAs discovered, let-7, is conserved from worms to mammals (Pasquinelli
et al. 2000; Reinhart et al. 2000). MicroRNAs are small RNAs of about 22 nucleo-
tides in length that can regulate specific messenger RNAs (mRNAs) (Zhao and
Srivastava 2007; Clark et al. 2015). There are more than 1000 miRNAs encoded in
the human genome and are expressed in a tissue-specific manner (Zhao and
Srivastava 2007). Each strand has a specific function in silencing post-transcriptional
gene expression (Clark et al. 2015). They have been found to regulate approxi-
mately 50 % of all protein-coding genes and facilitate many cellular processes
including apoptosis, differentiation, and cell proliferation (Zhai et al. 2013a; Krol
et al. 2010; Huang et al. 2011a). Recent studies have implied a great potential of
targeting miRNA as a novel therapeutic strategy for diseases such as cancer, neuro-
degenerative diseases, heart disease, and many others (Zhao and Srivastava 2007;
Clark et al. 2015; Ding et al. 2015; Gupta et al. 2015).
Fig. 4.1 Biogenesis of microRNAs and their intracellular functions
4.3.2 Biogenesis and Function of miRNAs
As shown in Fig. 4.1, majority of miRNA is transcribed by RNA polymerase II in

the nucleus. Transcripts are then processed in the nucleus by a Class 2 ribonuclease
III enzyme, Drosha, to become one or more precursor miRNAs (pre-miRNAs) with
a hairpin structure of about 70 nucleotides (Lee et al. 2003). At the 3′ end, there is
a 2-nucleotide overhang that is recognized by exportin 5 for export to the cytoplasm
(Leung 2015). An enzyme of the RNAse III family, Dicer, processes the pre-
miRNAs into an approximately 22-nucleotide duplex, and then incorporated into
RISC (Gibbings et al. 2013; Zhao and Srivastava 2007; Leung 2015; Bernstein et al.
2001; Jing et al. 2005). EIF2C complex is required for protecting the miRNAs from
degradation by cytoplasmic RNases (Gibbings et al. 2013). The duplex is then
loaded by an Ago protein that retains a specific single-stranded mature miRNA
(Leung 2015). When miRNA-mRNA complementarity is identified by the ‘seed
sequence’, AGO binds the protein GW182 to activate RISC for translation suppres-
sion or deadenylation/degradation of mRNAs (Leung 2015).
The processing bodies (P-Bodies) located in the cytoplasm are rich in Ago pro-
teins. Fluorescent microscopy observations indicate that P-Bodies are full of Ago
and mRNA decay factors, insinuating that miRNAs and their complementary
mRNAs are processed in P-Bodies (Cougot et al. 2004; Liu et al. 2005; Sen and
Blau 2005). However, the quantity of the splicer Ago2 in P-Bodies accounts for
<1 % of cytoplasmic Ago2, indicating that mRNA decay occurs elsewhere in the
cytoplasm as well (Leung 2015). Ago2 is phosphorylated at Ser-387 by mitogen-
activated protein kinase (MAPK), which is essential for its localization to P-bodies
to repress translation. Precise complementarity of miRNA-mRNA allows Ago2 pro-
tein to cleave the targeted mRNA (Leung 2015). During stress, some Ago proteins
and polymerases can re-localize to the cytoplasmic structures known as stress gran-
ules (Leung et al. 2006, 2011). Because Ago proteins are crucial in orchestrating
miRNA activities, post-translational modifications of these proteins (e.g., under
hypoxia or immune stimulation) can largely alter miRNA actions (Leung 2015).
Therefore, a better understanding of protein modifications in the RISC complex and
their upstream signaling would help further elucidate the miRNA regulatory
pathways.
4.3.3 Regulation of Messenger RNA by microRNAs
miRNAs are generated based on nucleolytic targets for distinct genes (Weil et al.
2015) through binding to 3′-untranslated region (3′ UTR) of matching mRNAs. The
end caps of mRNA are essential for strand stability (Weil et al. 2015). The 5′ cap
regulates nuclear export (Visa et al. 1996; Lewis and Izaurralde 1997), prevent deg-
radation by exonucleases (Evdokimova et al. 2001; Gao et al. 2000), and promote
translation (Sonenberg and Gingras 1998). In the 3′ end, structures including the
poly(A) tail can increase or decrease the stability of mRNAs. It was believed that
absence of poly(A) tail results in mRNA destabilization (Weil et al. 2015).
3′-untranslated region (3′ UTR) contains putative miRNA target sites for down-
regulation of mRNAs (van Solinge et al. 2015). Of particular interest, the AU-rich
elements (AREs) are observed in the 3′ UTR of many mRNAs with short half-lives,
such as proto-oncogenes and cytokines (Jing et al. 2005; Weil et al. 2015). There are
various proteins able to bind to ARE and responsible for the stability of mRNAs
(Jing et al. 2005). It has been reported that miR-16 holds the complementary
sequence (UAAAUAUU) to the ARE of 3′ UTR (Jing et al. 2005). Down-regulation
or overexpression of miR-16 respectively increases or decreases mRNA stability,
suggesting that mRNA degradation is dependent on ARE pairing with miRNA (Jing
et al. 2005). Various ARE sequences are present in mRNAs, allowing for simultane-
ous miRNA manipulation (Jing et al. 2005).
Two modes of complementary mRNA silencing are mediated by miRNAs: by
mRNA decay or by translational repression (Ameres and Zamore 2013). miRNAs
form the RNA-induced silencing complex (RISC), a ribonucleoprotein complex
(Leung 2015; Wilson and Doudna 2013) that includes Argonaute (Ago) proteins to
facilitate gene-silencing pathways (Hock and Meister 2008). In mammals, Ago1
through Ago4 are all active in the miRNA pathway. When miRNAs are precisely
complementary to the target mRNA through partial base pairing (usually through
the ‘seed’ sequence, nucleotides 2–7), the RISC cleaves the mRNA at the position
facing nucleotides 10 and 11 of the small RNA (Leung 2015; Iwakawa and Tomari
2015; Lai 2002; Stark et al. 2003; Lewis et al. 2005). Current evidence suggests that
each miRNA may affect specific targets independently or cooperatively (Zhao and
Srivastava 2007). There are miRNA clusters that reside in corresponding introns of
paralogous genes, and miRNAs of the same family can regulate sequential events
(Zhao and Srivastava 2007).
However, the complementary base-pairing of miRNA-mRNA is not the only cri-
terion for targeting mRNA for silencing (Zhao and Srivastava 2007). Additional
effector proteins are recruited to induce other modes of mRNA silencing or degra-
dation distinct from endonucleolytic cleavage (Zhao and Srivastava 2007; Iwakawa
and Tomari 2015). miRNAs can also induce mRNA destabilization through the
GW182 protein and Ago, which attracts deadenylases onto target mRNAs (Iwakawa
and Tomari 2015). GW182 is known to play a role in both of mRNA decay and
translational repression (Iwakawa and Tomari 2015). miRNAs can also promote the
accessibility of mRNA poly(A) tail to deadenylases (Iyer et al. 2006). Decapping
factors, the decapping complex (DCP2) and activators (DCP1, RCK/p54/DDX6),
are then recruited and attached to the target mRNAs. Finally, RISC proceeds with
the decapping and decay of target mRNAs, which is promoted by the binding of
4E-T to eIF4E (Artavanis-Tsakonas et al. 1999; Johnston et al. 2005; Johnston and
Hobert 2003). Degradation occurs in the traditional 5′-to-3′ fashion in the decay
pathway (Lai 2002; Du and Zamore 2005). It has been proposed that mRNA decay
is the dominant mechanism of mRNA silencing over translational repression
(Iwakawa and Tomari 2015). Further studies are warranted to determine the physi-
ological importance of both of the miRNA functions in cells.
4.4 MicroRNA and Autophagy: Connections

in Cancer Biology
4.4.1 miRNA in Cancers
Alternations in gene expression via DNA methylation, histone acetylation, and

microRNAs have been implicated in carcinogenesis and progression (Sui et al.
2015). Involvement of miRNAs in tumor formation was first reported in 2002 (Dai
and Tan 2015), and since then it has been widely investigated. Studies have shown
opposing spectrums of miRNA functions: miRNAs can act either as tumor suppres-
sors or as oncogenes. Targeting miRNA has been emerging as a novel therapeutic
strategy in cancer treatment. Mechanisms of miRNA-based cancer therapeutics
include: inducing apoptosis, suppressing tumor angiogenesis, and down-regulating
adenosine triphosphate (ATP)-binding cassette (ABC) transporters (Dai and Tan
2015). Post-transcriptional modification of autophagy by miRNAs is a new area of
investigation (Zhai et al. 2013a). The role of miRNA in regulating autophagy and its
impact on anticancer treatment will be discussed in this chapter.
4.4.2 The Role of miRNAs in Various Steps of Autophagy
Dysregulation of programmed cell death is a hallmark of cancer development and

progression (Dai and Tan 2015). The first connection between miRNA and autoph-
agy was made by Zhu et al. who reported that miR-30a could negatively affect
autophagic activity by regulating beclin-1 expression in cancer cells (Zhu et al.
2009). Further researches showed that various miRNAs could affect different stages
of autophagy. For example, induction of autophagy via the ULK complex is affected
by a variety of miRNAs including miR-20a, miR-101 and miR-106a/b, which can
directly target ULK1/2 (Pan et al. 2013; Ciuffreda et al. 2010; Wu et al. 2012).
Several binding sites for miR-885-3p were found in ATG13, ATG9A, and ATG2B
(Huang et al. 2011b). Multiple components in the mTOR signaling including RHEB
and RICTOR are modulated by miR-155 (Wang et al. 2013a). AMPK, which can
inhibit mTOR, is targeted by miR-148b (Zhao et al. 2013). The Class III PI3K/
Beclin-1 complex in vesicle nucleation is regulated by miR-30a, miR-519a, miR-
216a, and miR-376b (Pan et al. 2013; Su et al. 2015; Menghini et al. 2014; Huang
et al. 2012; Korkmaz et al. 2012; Mikhaylova et al. 2012). Furthermore, the expres-
sion of PI3K catalytic unit is thought to be silenced by miR-338-5p (PIK3C3 or
Vps34) (Su et al. 2015). Phosphatase and tensin homolog (PTEN), a known inhibi-
tor of PI3K, is a target for a number of miRNAs, including miR-21, miR-214, miR-
216a, miR-217, miR-221, miR-222, miR-26a, and miR-18a (Su et al. 2015; Dai and
Tan 2015). In the elongation step, UVRAG is modulated by miR-374a and miR-630
(Xu et al. 2013). A pathway of the ubiquitin-like conjugation system involving the
Atg12 and Atg5 covalent conjugation that requires Atg7 and Atg10 is modulated by
a number of miRNAs. MiR-375 and miR-17 target 3′ UTR of Atg7 gene to regulate
its expression (Su et al. 2015; Comincini et al. 2013; Chang et al. 2012a). miR-204
can inhibit autophagy and suppress cancer progression via the LC3 conjugation
system (Su et al. 2015). Other possible miRNAs involved in modulators of autoph-
agy include miR-30a, miR-181a, miR-374a and miR-630 (Frankel and Lund 2012).
Finally, the autophagosome fusion to lysosomes can be regulated by miR-34a and
miR-130a (Christoffersen et al. 2010). MiR-21, miR-155, and miR-221/222 influ-
ence programmed cell death in similar types of cancer cells. Specifically, miR-21
can confer radio-sensitivity through inhibition of PI3K/AKT pathway (Chen et al.
2014) and regulate anti-apoptotic Bcl-2 in glioma cells (Liu et al. 2014). Furthermore,
single miRNA may play a role in multiple pathways. An example of this case is
miR-30a, which can target Beclin-1, ATG5, and p53 (Su et al. 2015). The regulation
of autophagy by various microRNAs is summarized in Fig. 4.2.
4.4.3 Oncogenic miRNA (Oncomir)
Oncogenic miRNAs are often implicated in modulation of autophagic pathways.

miR-30a can bind to 3′ UTR of Beclin-1 to suppress its expression and autophagy
(Zhu et al. 2009; Chen et al. 2014). Downregulation of miR-30a and miR-19a-5p
Fig. 4.2 Summary of identified microRNAs that target key proteins of autophagic pathway for
regulation of autophagy
may decrease tumor cell chemosensitivity by activating autophagy and suppressing

apoptosis (Xu et al. 2012; Zou et al. 2012). In nasopharyngeal cancer and cervical
cancer cells, knockout of the endogenous miR-155 targeting mTOR signaling com-
ponents inhibits hypoxia-induced autophagy (Wan et al. 2014). miR-31 and miR-
34c respond to oxidative stress by activating cytoprotective autophagy, sustaining
cancer progression and metastasis (Pavlides et al. 2010). A therapeutic strategy has
been proposed using anti-miR-21 to augment autophagy and increase apoptosis in
cancer treated with radiation (Liu et al. 2014).
4.4.4 Tumor Suppressive miRNA
Tumor suppressive miRNAs may have equally important roles in regulating autoph-
agic pathways. When miR-23b is overexpressed in pancreatic cancer cells, its target
ATG12 is found to be down-regulated, which leads to lower autophagic activity.
This phenomenon has also been observed in bladder cancer, where overexpression
of miR-23b correlates to a longer overall survival rate (Majid et al. 2013; Wang
et al. 2013b). Tumor suppressor miR-101 can inhibit etoposide- or rapamycin-
induced autophagy in MCF-7 breast cancer cells (Frankel et al. 2011), and its
expression is down-regulated in various types of cancer, including breast, liver, and
prostate cancer. In castration-resistant mesenchymal prostate cancer cells,
down-regulation of miR-205 led to increased cytotoxicity of cisplatin and interfered

with the adaptive ability of cancer cells to cisplatin treatment (Pennati et al. 2014).
Replacing miR-375 in liver cancers inhibited hypoxia-induced autophagy by target-
ing ATG7 and ATG4D (Chang et al. 2012b). Mimicking the tumor suppressive miR-
NAs or augmenting their effects in autophagy may have important implication in
therapeutic intervention against cancer.
4.4.5 Regulation of miRNAs by Autophagy
While miRNAs can control autophagy, this important cellular process may also
regulate miRNAs to maintain their homeostasis. Gibbings et al. first reported that
the key components of miRNA biogenesis complexes, Dicer and Ago2, are selec-
tively degraded by the NDP52-mediated autophagy (Gibbings et al. 2012). Cells
with low autophagic activity exhibits increased Ago2 and Dicer, with less Ago-
binding to miRNAs. As the inactive Dicer-Ago2 complexes can suppress the active
complexes, autophagy is an important means in eliminating the inactive complexes
to promote miRNA activity (Gibbings et al. 2013). Autophagy has also been shown
to degrade specific miRNAs or the RISC components. Down-regulation of autopha-
gic activity may be associated with up-regulation of oncogenic miRNA and carci-
nogenesis (Jing et al. 2015).
4.4.6 Role of Autophagy and miRNA in Cancer Therapy
During tumor development, autophagic activity may fluctuate in different stages

(Guo et al. 2013; Jiang and Mizushima 2014; Rao et al. 2014). Autophagy may act
as a tumor suppressor in the initial stages of tumoregenesis and therefore, but it is
often down-regulated (Jing et al. 2015). Beclin-1, a key autophagy regulator, is con-
sidered as a tumor suppressor in breast cancer (Liang et al. 1999). However, many
cancer cells exhibit increased autophagic activity in response to therapeutic or meta-
bolic stress, implying a cyto-protective role of autophagy. It was observed in pan-
creatic cancer cells that inhibiting autophagy can elevate the amount of intracellular
reactive oxygen species, which increase DNA damage (Yang et al. 2011).
Interestingly, apoptosis is usually inhibited in cancer of all stages and as a result,
tumor size is increased and drug resistance occurs. Using autophagy as an alterna-
tive method to facilitate cell death in non-apoptotic cancer cells is a new therapeutic
strategy being explored (Li et al. 2013). Modulation of autophagy by miRNAs has
been used to decrease drug resistance of cancer cells (Pan et al. 2013). miR-30a can
inhibit Beclin-1-dependent autophagy and increase the sensitivity to imatinib in
chronic myeloid leukemia (CML) cells (Yu et al. 2012a). miR-30a and a number
of other miRNAs can sensitize tumor cells to cisplatin both in vitro and in vivo
(Jing et al. 2015). Future research on miRNA-based therapeutic strategies should
focus on tumor type, tumor environment, and disease context (Jing et al. 2015).
4.5 Targeting miRNA-Mediated Autophagy

in Cancer Treatment
4.5.1 Current Status
Autophagy can act either as a tumor promoter or tumor suppressor, depending on

context. Inhibiting the tumor suppressor role of autophagy may pave a way for
growth of precancerous cells; on the other hand, malignant cells may require
autophagy to survive under various stressful conditions (Li et al. 2013; Shintani and
Klionsky 2004). To certain extent, the dual functions of autophagy in cancer may
complicate the application of modulating autophagy in therapeutic intervention.
Still, emerging evidence suggest that targeting miRNAs may hold promises as a
novel strategy in cancer therapy (Table 4.1).
4.5.2 Breast Cancer
miR-30a inhibits autophagy by down-regulating Beclin-1 expression and suppress-

ing tumor growth (Zhu et al. 2009). Similarly, miR-376b targets Beclin-1 and
ATG4 to suppress starvation-induced autophagy (Zhai et al. 2013a; Korkmaz et al.
2012). In the HER2/neu+ MCF-7 breast cancer cells, autophagic cell death can be
induced by miR-221/222, which inhibits p27kip1 to regulate PI3K/Akt pathway
(Miller et al. 2008). miR-148b can regulate the PI3K pathway that involves the
catalytic subunit p110α, reducing breast cancer aggressiveness (Cimino et al.
2013). Also, in MCF-7 cells, miR-101 acts as a potent inhibitor of basal, etoposide-
induced or rapamycin-induced autophagy (Frankel et al. 2011). Suppression of
miR-21 in the HER2+ breast cancer cells induced PTEN expression and increased
trastuzumab sensitivity (Braconi et al. 2010). In the Cav(−/−) breast tumor stromal
cells, there was an upregulation of both miR-31 and miR-34 that is associated with
autophagy induction that supports cancer cell survival (Zhai et al. 2013a). miR-
200c-mediated inhibition of autophagy can enhance radio-sensitivity in breast can-
cer cells (Sun et al. 2015).
4.5.3 Prostate Cancer
Increased levels of miR-205 in castration-resistant mesenchymal prostate cancer

cells can cause an impairment of autophagy and an increase of cisplatin toxicity
(Pennati et al. 2014). In the prostate cancer tissues and serum from patients, miR-
212 was found down-regulated. Further study demonstrated that miR-212 could
suppress the starvation-induced autophagy by targeting sirtuin 1 (SIRT1) and induce
cellular senescence and anti-angiogenic effect (Ramalinga et al. 2015).
Table 4.1 Roles of miRs in autophagy in cancer

Cancer types miR(s) Target of miR(s) Effects References
Breast cancer miR-30a Beclin-1 Slows cancer Zhu et al.
progression (2009)
miR-376b Beclin-1 and Down-regulate Zhai et al.
ATG4 starvation-induced (2013a),
autophagy Korkmaz et al.
2012
miR- p27kip1 Induce autophagic Miller et al.
221/222 cell death in HER2/ (2008)
neu+ MCF-7 breast
cancer cells
Inhibit p27kip1 to
regulate PI3K/Akt
down stream
miR-148b PI3K pathway Reducing breast Cimino et al.
cancer (2013)
aggressiveness
miR-101 MCF-7 cells Potent inhibitor of Frankel et al.
basal, etoposide- (2011)
induced and
rapamycin-induced
autophagy
miR-21 HER2+ breast Suppression of Braconi et al.
cancer cells miR-21 induce (2010)
PTEN expression
and increased
trastuzumab
sensitivity
miR-31 and Cav(−/−) breast Promote autophagy Zhai et al.
miR-34 tumor stromal (2013a)
cells
miR-200c Breast cancer Inhibition of Sun et al. (2015)
cells autophagy and
enhancement of
radiosensitivity
Prostate cancer miR-205 Castration- Induce autophagy Pennati et al.
resistant impairment that (2014)
mesenchymal potentiated cisplatin
prostate cancer toxicity
cells
miR-212 SIRT1 Suppress starvation- Ramalinga et al.
induced autophagy (2015)
Ovarian cancer miR-214 PTEN Positively regulate Yang et al.
autophagy (2008)
miR-29b Expression of Negatively regulate Dai et al. (2014)
high MAPK and autophagy
ATG9a protein
levels
(continued)

Lung cancer miR-99a mTOR Suppress the Oneyama et al.
tumorigenicity of (2011)
cancer cells
miR-503 Non-small-cell Suppress Yang et al.
lung cancer proliferation and (2014)
(NSCLC) metastasis
miR- Class I PI3K Inhibit metastasis Yu et al. (2014)
193a-5p pathway
miR-17-5p Beclin-1 Confer paclitaxel Chatterjee et al.
resistance (2014)
miR-143 Non-small-cell Halt cell proliferation Wei et al.
lung cancer (2015)
(NSCLC) and Modulate autophagy
H1299 cells
miR-7 Epidermal growth Induce autophagy Tazawa et al.
factor receptor (2012)
(EGFR)
Colorectal cancer miR-30b Human colorectal Regulate the PI3K Liao et al.
cancer cells pathway (2014)
miR-18a HCT116 cells Increase autophagy Qased et al.
in response to (2013)
radiation
Apoptosis of colon Seoudi et al.
cancer cells (2012), Fujiya
et al. (2014)
mTOR Suppression Qased et al.
(2013)
miR-22 Cancer cells Enhance sensitivity Zhang et al.
to 5-fluorouracil by (2015a)
inhibiting autophagy
and promoting
apoptosis
miR-502 RAB1B Hinder autophagy Adlakha and
Saini (2011),
Zhai et al.
(2013b)
miR- LC3BII and Bcl2 Suppress autophagy Sumbul et al.
204-5p (2014))
Renal cell miR-204 LC3 Inhibit autophagy Su et al. (2015),
carcinoma and suppress renal Mikhaylova
clear cell carcinoma et al. (2012),
development Hall et al.
(2014)
(continued)

Hepatocellular miR-101 Hepatocellular Activate apoptosis, Yu et al.
carcinoma carcinoma (HCC) synergize with (2012a), Xu
and oncogene doxorubicin or et al. (2014)
eZH2 fluorouracil
miR- mTOR Reduce cell invasion Xu et al. (2012),
199a-3p and sensitizes HCC Fornari et al.
to doxorubicin (2010)
miR-26b AMPK Enhances Zhao et al.
chemosensitivity of (2014)
the cancer cells
miR-375 ATG7 Inhibit autophagy Chang et al.
under hypoxic (2012a, b)
conditions
miR- Cells treated with Promote autophagy Stiuso et al.
423-5p sorafenib (2015)
miR-224 HCC Induce autophagy Lan et al.
(2014a, b)
Pancreatic cancer miR-23b / Pancreatic cancer Increase autophagic Wang et al.
miR-630 cells activity, promote (2013b, c),
ATG 12 expression Donadelli and
and increase Palmieri (2013)
radioresistance
miR-155 Pancreatic cancer Protect from Shahbazi et al.
cells programmed cell (2013)
death
miR-216a Beclin-1 Increase the Zhang et al.
radiosensitivity of (2015b)
pancreatic cancer
cells
miR-182 Pancreatic cancer Suppression of Bcl2 Peng et al.
cells (2013)
Glioma miR-17 ATG7 Increase the Comincini et al.
sensitivity of cancer (2013)
cells to chemotherapy
and radiation
miR-663 Cancer cells Regulate the PI3K Shi et al. (2014)
pathway
miR-21 Human glioma Suppress Gwak et al.
cells LN18 and radiosensitivity (2012)
LN428
4.5.4 Ovarian Cancer
Cisplatin resistance in ovarian cancer cells can be promoted by miR-214, which

targets PTEN to positively regulate autophagy (Yang et al. 2008). The negative cor-
relation of low miR-29b expression to high MAPK and ATG9a protein levels was
associated with poor prognosis in patients with ovarian cancer (Dai et al. 2014).
4.5.5 Lung Cancer
Lung tumorigenesis can be suppressed by miR-99a that targets mTOR (Oneyama

et al. 2011). The ectopic expression of miR-503 in non-small-cell lung cancer
(NSCLC) leads to suppression of proliferation and metastasis of tumor cell (Yang
et al. 2014). Metastasis can also be inhibited by miR-193a-5p-mediated inactivation
of the class I PI3K pathway (Yu et al. 2014). Down-regulation of miR-17-5p can
confer paclitaxel resistance through altering Beclin-1 expression (Chatterjee et al.
2014). Cell proliferation in NSCLC can be halted by miR-143, which modulates
autophagy in tumor cells (Wei et al. 2015). Ectopic expression of miR-7 induces
autophagy by limiting the expression of epidermal growth factor receptor (EGFR),
and this also occurs in esophageal cancer (Tazawa et al. 2012).
4.5.6 Colorectal Cancer and Renal Cell Carcinoma
miR-30b directly regulates the PI3K pathway in human colorectal cancer cells,
thereby modulating autophagy (Liao et al. 2014). Oncogenic miR-18a increases
autophagy in HCT116 cells by interacting with the ataxia telangiectasia mutated
(ATM) gene in response to radiation (Qased et al. 2013). However, prolonging this
action can lead to apoptosis in colon cancer cells (Seoudi et al. 2012; Fujiya et al.
2014). miR-18a can also suppress mTOR to impact autophagy (Qased et al. 2013).
miR-22 can enhance tumor cell sensitivity to 5-fluorouracil by inhibiting autophagy
and promoting apoptosis (Zhang et al. 2015a). miR-502 can block autophagy by
decreasing RAB1B, a GTPase (Adlakha and Saini 2011; Zhai et al. 2013b). Tumor
suppressor miR-204-5p can suppress the activity of LC3B-II in autophagy (Sumbul
et al. 2014). Via the LC3 conjugation system, miR-204 can inhibit autophagy and
suppress the development of renal clear cell carcinoma (Su et al. 2015; Mikhaylova
et al. 2012; Hall et al. 2014).
4.5.7 Hepatocellular Carcinoma
miR-101, which is considered as a tumor suppressor, can inhibit autophagy in hepa-

tocellular carcinoma (HCC) cells and target the oncogene EZH2 to activate apopto-
sis, thereby effectively synergizing with doxorubicin or fluorouracil (Yu et al.
2012a; Xu et al. 2014). miR-199a-3p can regulate mTOR and autophagy to reduce
cell invasion and sensitize HCC to doxorubicin (Xu et al. 2012; Fornari et al. 2010).
miR-26b can modulate autophagy through directly affecting AMPK, thereby
enhancing chemosensitivity of the cancer cells (Zhao et al. 2014). Down-regulation
of miR-375 can inhibit autophagy through targeting ATG7 in tumor cells subjected
to hypoxic (Chang et al. 2012a, b). On the other hand, miR-423-5p can promote
autophagy in cells treated with sorafenib (Stiuso et al. 2015). In a study by Lan
et al., using amiodarone as an autophagy inducer, autophagy-mediated degradation
of miR-224 suppressed HCC tumorigenesis (Lan et al. 2014a, b).
4.5.8 Pancreatic Cancer
Autophagic activity was found to be increased in pancreatic cancer cells when miR-
23b or miR-630 expression was decreased, with an up-regulation of ATG12 expres-
sion and increased resistance to radiation therapy (Wang et al. 2013b, c; Donadelli
and Palmieri 2013). Oncogenic miR-155 can protect pancreatic cancer cells from
programmed cell death (both apoptosis and autophagy) by targeting p53 pathway
(Shahbazi et al. 2013). miR-216a can inhibit the Beclin-1-mediated autophagy and
increase the sensitivity of pancreatic cancer cells to radiation therapy (Zhang et al.
2015b). Up-regulation of miR-182 is correlative with the suppression of Bcl2 in
pancreatic cancer cells (Peng et al. 2013).
4.5.9 Glioma
It has been reported that miR-17 has the ability to enhance the sensitivity of glioma
cells to chemotherapy and radiotherapy through interfering with the E1-like
enzyme, ATG7 (Comincini et al. 2013). miR-663 can directly regulate the PI3K-
mediated autophagy signaling (Shi et al. 2014). Overexpression of oncogenic miR-
21 can suppress radiosensitivity in human glioma cell lines LN18 and LN428
(Gwak et al. 2012).
4.5.10 Perspective
Although the roles of the miRNA-regulated autophagy in cancer development and

progression remains to be further elucidated, a growing body of evidence indicate
that targeting miRNAs to modulate autophagy may have important implication in
cancer treatment. Many recent studies have demonstrated the potential of using
miRNA mimics or anti-miRs as anticancer therapeutics. The miRNA-based therapy
may complement conventional treatments by reinforce their efficacy. Increased cell
death resulting from suppression of autophagy was observed in breast cancer cells
treated with miR-101 (Frankel et al. 2011). Cisplatin treatment induces autophagy
in various types of cancer, accompanied by down-regulations of several miRNAs
(Claerhout et al. 2010; Harhaji-Trajkovic et al. 2009; Ren et al. 2010). Apoptosis in
tumor cells induced by cisplatin can be enhanced by miR-30a through suppression
of Beclin-1 (Zou et al. 2012). Additionally, miR-30a can enhance the therapeutic
efficacy in imatinib-resistant chronic myelogenous leukemia (Yu et al. 2012b), and

is considered a putative biomarker in a variety of cancer. Contrastingly, there are
known miRNAs that can increase drug resistance and radio-resistance in cancer
cells, for instance, miR-221/222 in breast cancer cells (Miller et al. 2008) and miR-
21 in glioblastoma cells (Maiuri et al. 2007).
A common barrier of miRNA-based therapy is the drug delivery problem. This
includes inadequate cellular uptake, short half-life, and rapid renal clearance of
miRNAs (Aliabadi et al. 2012). A number of strategies are being employed to over-
come these issues. Chemical modification (Bader et al. 2011; Janssen et al. 2013),
biodegradable nanocarriers (Chen et al. 2014; Aliabadi et al. 2012; Bouchie 2013;
Daka and Peer 2012), and conjugations of miRNAs to conventional cytotoxins
(Schroeder et al. 2012), are being developed at present. Future studies on autophagy-
related miRNAs should take into account the following issues: (1) are the miRNAs
a single tumor-specific or present in multiple tumors? (2) are they tumor suppressive
or oncogenic? (3) are they promoting or suppressing autophagy, and in which spe-
cific step do they regulate autophagy? (4) what are the specific targets for these
miRNAs? (5) how can we take advantage of the knowledge of miRNAs for treat-
ment of different cancer?. The increasing understanding of the molecular mecha-
nisms behind miRNA regulation of autophagy will help overcome various barriers
in developing the miRNA-based cancer therapy against multi-drug resistance,
radio-resistance, cancer metastasis, and other malignant phenotypes.
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Chapter 5
Targeting PI3-Kinases in Modulating
Autophagy and Anti-cancer Therapy
Zhixun Dou and Wei-Xing Zong
Abstract Phosphoinositide 3-kinases (hereafter referred to as PI3-kinases) are

lipid kinases that phosphorylate the 3′-hydroxyl group of inositol lipids. The gener-
ated phospholipids are critical signaling molecules that recruit proteins to specific
intracellular membranes leading to localized activation of these proteins. PI3-
kinases regulate many cellular activities, and are closely linked to human diseases,
including cancer. One molecular event regulated by PI3-kinases is autophagy, an
evolutionarily conserved membrane trafficking process that degrades and recycles
cellular constituents to maintain cell and tissue homeostasis. Over the past two
decades, our understanding of PI3-kinases has progressed from pan-PI3-kinase
inhibitor studies to isoform-specific genetic knockout and systems biology interac-
tome analyses. Our view of autophagy has emerged from unicellular yeast vesicle
trafficking to mammalian physiology and human diseases. In this chapter we sum-
marize the major discoveries on autophagy regulation by PI3-kinases and discuss
the therapeutic potentials of targeting PI3-kinases in modulating autophagy and in
cancer therapy.
Keywords Autophagy • PI3K • Cancer therapy • Chloroquine • p110α • p110β •

Vps34
Z. Dou, Ph.D.
Epigenetics Program, Department of Cell and Developmental Biology, Perelman School
of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
e-mail: zdou@upenn.edu
W.-X. Zong, Ph.D. (*)
Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University,
Piscataway, NJ 08854, USA
Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, USA
e-mail: weixing.zong@pharmacy.rutgers.edu

86 Z. Dou and W.-X. Zong
5.1 Introduction to PI3-Kinases
In late 1980s, Lewis Cantley and coworkers discovered a new lipid kinase that phos-
phorylates the 3′-hydroxyl group of inositol lipids to produce a new lipid PtdIns(3)
P (Whitman et al. 1988). The lipid showed slight different biochemical signatures
from PtdIns(4)P and PtdIns(5)P, which were the known lipids at that time. The
importance of PI3-kinase was not clear until the discovery that it associates with
growth factor receptors, such as G protein-coupled receptor (GPCR), and that
growth factor, including insulin, stimulation results in production of a novel lipid
PtdIns(3,4,5)P3 from PtdIns(4,5)P2 (Auger et al. 1989; Ruderman et al. 1990;
Traynor-Kaplan et al. 1988). In early 1990s, the first PI3-kinase, p110α, was cloned,
and it was found to be in a complex with p85 (Hiles et al. 1992). Later years in the
1990s have witnessed an expansion of the PI3-kinase field, as more subunits and
isoforms were cloned (Vanhaesebroeck et al. 2012). It has since become clear that
PI3-kinases are central players of cellular metabolism, growth, proliferation, and
survival. The research on PI3-kinases was largely facilitated by the discovery of
wortmannin, a steroid metabolite of the fungus Talaromyces wortmannii, as a non-
specific covalent inhibitor of all PI3-kinase isoforms (termed as pan-PI3-kinase
inhibitor). In conjugation with other available molecular biology tools, the use of
wortmannin has helped identify numerous cellular activities regulated by PI3-
kinases, including autophagy (Engelman et al. 2006).
Based on substrate specificity and sequence homology, PI3-kinases are grouped
into three classes: Class I, Class II, and Class III (Engelman et al. 2006) (Table 5.1).
Class I PI3-kinases are among the first identified PI3-kinases, and are comprised of
a 110 kDa catalytic subunit and a regulatory subunit. When this group of enzymes
were first discovered, they showed in vitro activity to phosphorylate PtdIns,
PtdIns(4)P, and PtdIns(4,5)P2. In vivo, activation of Class I PI3-kinases by receptor
tyrosine kinases (RTKs) and GPCRs induces production of PtdIns(3,4,5)P3, but not
PtdIns(3)P or PtdIns(3,4)P2 (Hawkins et al. 1992; Stephens et al. 1991). Hence,
PtdIns(4,5)P2 is more likely to be the preferred in vivo substrate of Class I
Table 5.1 Components and catalytic preference of three classes of PI3-kinases

Class Catalytic subunit Regulatory subunit In vivo substrate Catalytic product
IA p110α p85α PtdIns(4,5)P2 PtdIns(3,4,5)P3
p85β
p110β p50α
p55α
p110δ p55γ
IB p110γ p101 PtdIns(4,5)P2 PtdIns(3,4,5)P3
p84/p87
II PIK3C2α None PtdIns PtdIns(3)P
PIK3C2β
PIK3C2γ
III Vps34 Vps15 PtdIns PtdIns(3)P
5 Targeting PI3-Kinases in Modulating Autophagy and Anti-cancer Therapy 87
PI3-kinases. Class I PI3Ks are further divided into two classes, Class IA and Class
IB. Class IA PI3Ks respond to both RTKs and GPCRs, and are composed of a
110 kDa catalytic subunit (p110α, β, δ) and an 85 kDa regulatory subunit (p85). In
some cases, a p50 or p55 subunit is present as the regulatory subunit. Class IB
PI3Ks respond only to GPCRs and consist of the p110γ catalytic subunit and the
p101 or p84/p87 regulatory subunit. Among the p110 catalytic subunits, p110α and
p110β isoforms are ubiquitously expressed, whereas the p110δ and p110γ expres-
sions are restricted in hematopoietic cells (Engelman et al. 2006).
Class II PI3-kinases are comprised of three members, PIK3C2α, β, and γ. The
preferred in vivo substrate is PtdIns. Class II PI3-kinases are monomeric and lack a
regulatory subunit. PIK3C2α and PIK3C2β are ubiquitously expressed, whereas
PIK3C2γ is highly expressed in the liver and prostate (Engelman et al. 2006).
Class III PI3K consists of only one member, Vps34. It was first identified in a
yeast mutant that is defective in vacuole protein sorting (Herman and Emr 1990;
Schu et al. 1993) and is the only PI3-kinase evolutionary conserved from yeast to
mammals. Vps34 phosphorylates PtdIns into PtdIns(3)P. Vps34 associates with its
regulatory subunit Vps15, and is ubiquitously expressed in mammalian tissues.
5.2 Class I PI3-Kinases as Essential Regulators of Growth,

Proliferation, and Cancer
The functional significance of Class I PI3-kinases is suggested by the early evidence

that they are associated with growth factor receptors, and thus may be involved in cell
stimulation, growth, and metabolism. In support of this notion, inhibition of PI3-
kinases by pharmacological inhibitors or genetic approaches leads to impaired cellu-
lar activities signaled through growth factor receptors (Auger et al. 1989; Cantley
2002; Ruderman et al. 1990). The identification of p85 regulatory subunits also helped
the understanding of the regulation of PI3-kinase activity. p85 and p110α exist as a
heterodimer, with a strong binding affinity. While p85 does not possess PI3-kinase
activity, p110α was shown to be a bona-fide PI3-kinase. The p85 regulatory subunit is
composed of an N-terminal SH3 domain, a BH domain, and the p110 binding domain
flanked by two SH2 domains (Cantley 2002; Engelman et al. 2006). Since SH2
domains bind to phosphorylated-tyrosine peptides, this provides a clue to the recruit-
ment of PI3-kinases to RTKs. The p110 catalytic subunit possesses an N-terminal p85
binding domain, a Ras binding domain, a C2 domain, a helical domain, and a
C-terminal catalytic domain. The interaction of p110s with p85s stabilizes p110s, and
inactivates their catalytic activity. In vivo, p110s are obligated heterodimers with p85s
(Geering et al. 2007). Upon RTK activation, the SH2 domains of p85 associate with
activated receptor and recruits p110s to the localized plasma membrane. The p110-p85
interaction with receptor relieves p110s from the inhibition posed by p85, which pro-
duce PtdIns(3,4,5)P3 at the localized plasma membrane. The mechanisms of recruit-
ment and activation of p110-p85 by GPCRs are distinct from those of RTKs, and are
involved in direct GPCR-p110 interaction (Engelman et al. 2006).
The Class I PI3-kinases mediate growth factor signaling mainly through the
localized production of PtdIns(3,4,5)P3 at the plasma membrane. Several other PH
domain-containing proteins regulated by PtdIns(3,4,5)P3 have been identified,
including Akt/PKB, PDK1, Rac, and SGK3 (Engelman et al. 2006). Akt, also known
as PKB, was the first identified effector of PI3-kinase (Burgering and Coffer 1995;
Franke et al. 1995; Stephens et al. 1998). Akt possesses a pleckstrin homology (PH)
domain, which binds to PtdIns(3,4)P2 and PtdIns(3,4,5)P3. The catalytic product of
PI3-kinases recruits Akt to the plasma membrane, where it is phosphorylated by
PDK1 at Thr308, a step required for its activation. Akt regulates several signaling
pathways, including the activation of glucose transporters that mediates glucose
uptake, FOXO family transcription factors that regulates cell cycle and metabolism,
and tuberous sclerosis complex (TSC) that regulates activation of mammalian target
of rapamycin (mTOR) (Cantley 2002; Engelman et al. 2006). mTOR complex acti-
vation leads to phosphorylation of S6 and 4-EBP which ultimately promote protein
synthesis and cell growth (Laplante and Sabatini 2012).
Class I PI3-kinases and their signaling pathways are widely mutated in human
cancer (Wong et al. 2010). Several pieces of evidence directly connect PI3-kinase
pathways to cancer. First, oncogenic Ras can directly interact with and activate PI3-
kinase via the Ras-binding domain on p110α. In vivo mouse models demonstrate
that the interaction of Ras and p110α is required for tumorigenesis (Gupta et al.
2007). Second, the tumor suppressor PTEN is a phosphatase for PtdIns(3,4,5)P3 that
functions to antagonize PI3-kinase signalings (Maehama and Dixon 1998; Myers
et al. 1998). Loss of PTEN was found in many cancers. Third, p110α activation
mutations have been identified in a large number of cancer patients. The p110α
hyper-activation mutations are the most frequent onco-protein activation event in
all human cancers. They bypass the need for growth factor stimulation, resulting in
uncontrolled activation of cell growth and proliferation (Engelman et al. 2006;
Wong et al. 2010). Given the strong connection of Class I PI3-kinases and cancer,
there is tremendous pharmaceutical interest in developing PI3-kinase inhibitors as
targeted anti-cancer therapies. Several drugs are in clinical trials and have shown
promising results in treating cancers bearing PI3-kinase mutations.
5.3 PI3-Kinases and Autophagy: Class I and Class III PI3-

Kinases Differentially Regulate Autophagy
The connection between PI3-kinases and autophagy was first reported by Blommaart
et al. in 1997 (Blommaart et al. 1997). Using rat hepatocytes as a model, the authors
investigated autophagy triggered by amino acid starvation, in the presence of two
structurally unrelated pan-PI3-kinase inhibitors, wortmaninn and LY294002. Both
inhibitors strongly suppressed autophagy, at the autophagy membrane sequestration
step, while did not affect lysosomal pH. In addition, the authors demonstrated that
3-methyladenine, a commonly used inhibitor of autophagy membrane sequestration,
is an inhibitor of PI3-kinases (Blommaart et al. 1997). Petiot et al. made a similar
In vitro lipid
delivery showed Atg14 and KO of Class I PI3-
distinct classes Rubicon found kinase p110α and
Pan-PI3-Kinase of PI3-kinases Beclin 1-Vps34 to be key p110β revealed Selective
inhibitors shown differentially interaction partners of the p110β as a Vps34
to inhibit regulate established in Beclin 1-Vps34 positive regulator inhibitor
autophagy. autophagy. mammals. complex. of autophagy. designed.
1997 1999 2000 2001 2006 2009 2010 2012 2014 2015
Role of Beclin 1 Link between UVRAG First Vps34 KO Vps34 KO Crystal

in mammalian Vps34 and identified as a mouse made. mouse structure of
autophagy autophagy component of demonstrated yeast Vps34
revealed. established in the Beclin 1- Vps34 crystal deficient complex II
yeast. Vps34 complex. structure resolved. autophagy. resolved.
Fig. 5.1 Key events related to PI3-kinases in modulating autophagy are highlighted
observation that pan-PI3-kinase inhibitors and 3-methyladenine suppress autophagy

(Petiot et al. 2000), and further evaluated the effects of distinct classes of PI3-kinases
in autophagy regulation. Through direct intracellular delivery of phospho-lipids, the
authors discovered that the product of Class I PI3-kinase, PtdIns(3,4)P2 and
PtdIns(3,4,5)P3, inhibited autophagy, whereas the product of Class III PI3-kinase,
PtdIns(3)P, stimulated autophagy. PtdIns(4)P and PtdIns(4,5)P2 had little or no effect
on autophagy. In addition, 3-methyladenine was demonstrated to be an inhibitor of
Class III PI3-kinase (Petiot et al. 2000). These early studies indicated that Class I PI3-
kinases inhibit autophagy, whereas Class III PI3-kinase promotes autophagy (Fig. 5.1).
In 2001, the role of Class III PI3-kinase, Vps34, in autophagy was established in
yeast (Kihara et al. 2001b). Vps34 was co-precipitated with Vps30/Atg6.
Furthermore, Vps34 was shown to be in two distinct complexes: one consists of
Vps34, Vps15, Vps30/Atg6, and Atg14, which regulates autophagy (termed as
complex I), and the other consists of Vps34, Vps15, Vps30/Atg6, and Vps38, which
regulates carboxypeptidase Y (CPY) sorting (termed as complex II). Genetic dele-
tion of Vps34 or Vps15 impairs both autophagy and CPY sorting (Kihara et al.
2001b). This work provided the first genetic evidence that Vps34 is involved in
autophagy (Fig. 5.1).
In 1999, Beclin 1, the first mammalian specific autophagy gene, was identified
(Liang et al. 1999). Beclin 1 is the mammalian ortholog of yeast Vps30/Atg6. Its
ectopic expression was able to rescue the autophagy defect in Vps30/Atg6 deficient
yeast. Monoallelic deletion of Beclin 1 gene was found to be associated with human
breast cancer, opening up a functional link between autophagy and cancer (Liang
et al. 1999) (Fig. 5.1). Given the interaction of yeast Atg6 and Vps34, Tamotsu
Yoshimori and coworkers first demonstrated a Beclin 1-Vps34 interaction in mam-
malian cells (Kihara et al. 2001a) (Fig. 5.1). The work also showed that while all
Beclin 1 associates with Vps34, only 50 % of Vps34 associates with Beclin 1

(Kihara et al. 2001a). This is consistent with the current understanding that Vps34
has functions other than autophagy.
Vps34 exists in a stable complex with Vps15. Vps15 itself does not possess PI3-
kinase activity but is an essential regulatory subunit for Vps34. Approximately 50 %
of the Vps34-Vps15 complex interacts with Beclin 1, which is likely to be the pool
involved in autophagy (Kihara et al. 2001a). Like in yeast, the Beclin 1-Vps34-
Vps15 core complex interacts with distinct partners which positively or negatively
regulate autophagy.
The Atg14-containing pool of the Beclin 1-Vps34-Vps15 complex is termed
complex I, which specifically regulates autophagy (Fig. 5.1). Atg14 stimulates
Vps34 catalytic activity and is required for autophagosome formation (Matsunaga
et al. 2009; Sun et al. 2008; Zhong et al. 2009). Atg14 is able to sense curved mem-
brane (Fan et al. 2011), and localizes to the precursor membrane of autophagosome
(Matsunaga et al. 2010), which directs autophagosome formation from ER and
mitochondrial contact regions, termed as mitochondria-associated ER membrane
(Hamasaki et al. 2013).
The UVRAG-containing Beclin 1-Vps34-Vps15 complex pool is termed com-
plex II, which regulates autophagosome maturation (Fig. 5.1). UVRAG is the ortho-
log of yeast Vps38, which mediates CPY sorting. Mammalian UVRAG stimulates
Vps34 catalytic activity and primarily functions at the endosome and lysosome
steps, which promotes endosomal and lysosomal activities and hence autophagy
flux (Liang et al. 2006, 2008). The crystal structure of Vps34 was resolved in 2010
(Miller et al. 2010) and that of the yeast Vps34 complex II in 2015 (Rostislavleva
et al. 2015) (Fig. 5.1). Another interacting partner in the complex, Rubicon, is a
negative regulator of autophagy (Fig. 5.1). Rubicon primarily localizes to the late
endosome, and inhibits Vps34 kinase activity, thus suppressing autophagosome
maturation (Matsunaga et al. 2009; Zhong et al. 2009).
In addition to the above major components, several other proteins have been
described to interact with the Beclin 1-Vps34-Vps15 core complex, although at a
weaker affinity or in a condition-dependent manner, such as Ambra1, Bif-1, NRBF2,
and Bcl-2 (Funderburk et al. 2010).
5.4 PI3-Kinases and Autophagy: Views from Genetically

Modified Mice
While early studies using PI3-kinase inhibitors paved the road for autophagy study,
it should be noted that most of the inhibitors used are not specific for the various
PI3-kinase isoforms. For example, wortmaninn and LY294002, the first two inhibi-
tors shown to inhibit autophagy, are pan-PI3-kinase inhibitors. 3-methyladenine
inhibits both Class I and Class III PI3-kinases (Wu et al. 2010). Several PI3-kinase
knockout mouse lines have been generated to elucidate the physiological roles of
PI3-kinases, including the two ubiquitously expressed Class I isoforms p110α and
p110β, and the Class III PI3-kinase Vps34 (Fig. 5.1). As whole body knockout of
these isoforms are embryonic lethal, most of the information was obtained from
tissue-specific knockout strategy.
The Class I PI3-kinase p110α and p110β subunits are downstream of growth fac-
tor receptors that stimulate cell growth and proliferation. This is mainly mediated
through the Akt-mTOR pathway that activates nutrient uptake and macromolecule
synthesis. Therefore the Class I PI3-kinases were expected to suppress autophagy.
While this is indeed the case for p110α, genetic ablation of p110β impairs autoph-
agy (Dou et al. 2010). Further mechanistic studies revealed that p110β does not act
on the Akt-mTOR pathway to regulate autophagy. Rather, p110β stimulates Vps34
catalytic activity through a scaffolding mechanism that does not require the cata-
lytic activity of p110β (Dou et al. 2010).
The kinase-independent function of p110β has been well noticed. While p110β
knockout mice are embryonic lethal, mice bearing both alleles of kinase-dead mutants
of p110β are viable (Ciraolo et al. 2008). Further, the kinase-dead p110β mutant is
able to rescue some of the defects of p110β knockout cells with respect to cell growth,
endocytosis, and mTOR activity (Jia et al. 2008). These observations support a scaf-
fold function of p110β. p110β is known to be a Rab5 effector (Christoforidis et al.
1999). Through competition of Rab5 GAP that stimulates hydrolysis of GTP, p110β
keeps Rab5 at its GTP-bound form, which is the active form (Dou et al. 2013). The
GTP-bond active Rab5 interacts with the Beclin 1-Vps34 complex, and stimulates
autophagy through activation of Vps34 (Ravikumar et al. 2008).
A direct evidence of Vps34 in autophagy in mammals was established by the
conditional genetic ablation of Vps34, which showed deficient autophagy in mouse
embryonic fibroblast, liver, and heart (Jaber et al. 2012) (Fig. 5.1). Consistent with
the results in yeast, deletion of Vps34 leads to defects in both autophagy and endo-
somal trafficking. Electron microscopy imaging demonstrated that Vps34 knockout
cells do not form double-membrane autophagosome, and show abnormal endo-
somes and multivesicular bodies. Functional analysis proved that Vps34 deletion
led to impaired autophagy flux, both in vitro and in vivo. In liver, loss of Vps34
largely phenocopies Atg7 deficiency, including accumulation of lipid droplets, pro-
tein aggregations, loss of glycogen, and impaired starvation-induced autophago-
some formation and autophagy flux (Jaber et al. 2012). Similarly, Vps34 was also
deleted in immune cells, where deficient autophagy is observed in the absence of
Vps34 (Willinger and Flavell 2012).
5.5 PI3-Kinases Integrate Nutrient and Growth Factor

Signals to Modulate Autophagy
Autophagy as an evolutionarily conserved stress response and cell renewal process

is tightly regulated at the molecular level. Both positive and negative signals balance
the fine-tuning machinery of autophagy, at the steps of autophagosome initiation,
Fed condition Nutrient starvation Growth factor limitation

Growth Growth
factor factor
PIP3
p110-p85 Akt
Akt
AMPK
mTOR p110β
mTOR TIP60 mTOR Rab5
Vps34 Vps34 Vps34
complex complex ULK1 complex
Nutrient ULK1 ULK1
Autophagy Autophagy Autophagy
Active Inactive
Fig. 5.2 Schematics of autophagy regulation by Class I and Class III PI3-kinases. In fed condi-
tion, Class I PI3-kinases are activated while Class III PI3-kinase is inactivated, resulting in sup-
pressed autophagy. Upon nutrient or growth factor starvation, PI3-kinases are differentially
regulated to promote autophagy
maturation, degradation and lysosome recycling. In this perspective, PI3-kinases

are not only the key machinery of autophagy, but are also the critical signal trans-
ducers of environmental clues that instruct differential cellular responses.
Amino acids are potent activators of mTOR (Laplante and Sabatini 2012),
which efficiently suppresses autophagy (Fig. 5.2). Amino acid deprivation inhib-
its mTOR and stimulates autophagy. mTOR regulates autophagy through multi-
ple mechanisms (Fig. 5.2). It has been shown to directly phosphorylate and
inhibit ULK1/Atg1 complex (Kim et al. 2011). mTOR can also directly phos-
phorylates Atg14 at five Ser/Thr sites, and inhibits the Atg14-containing Vps34
complex I. In support of mTOR-mediated inhibitory Atg14 phosphorylation,
mutation of the phosphorylation sites on Atg14 results in an enhanced Atg14-
pool of Vps34 kinase activity and autophagy even under fed condition (Fig. 5.2)
(Yuan et al. 2013).
Glucose is another essential mediator of autophagy. Glucose scarcity results in
reduced cellular ATP and increased ADP and AMP, which activates AMP-
activated protein kinase (AMPK) (Mihaylova and Shaw 2011). AMPK directly
phosphorylates ULK1/Atg1 and stimulates the kinase activity of the complex,
thereby promoting autophagy (Kim et al. 2011; Mihaylova and Shaw 2011)
(Fig. 5.2). In addition, AMPK directly phosphorylates multiple players of the
Vps34 complex, including Vps34, Beclin 1, Atg14, and UVRAG (Kim et al.
2013) (Fig. 5.2). Upon glucose withdrawal, while total Vps34 kinase activity is
reduced, Atg14- and UVRAG-associated Vps34 activity is induced. Beclin 1
phosphorylation by AMPK is necessary to promote the activity of Atg14-
containing Vps34, which is required for glucose starvation-induced autophagy.
Further, AMPK also phosphorylates Vps34 that does not participate in autoph-
agy, leading to a reduction of Vps34 kinase activity of this pool (Kim et al. 2013).
These observations support a central role of Vps34 in autophagy triggered by
glucose starvation.
While autophagy in unicellular organisms is mainly regulated by nutrient avail-

ability, metazoan cells depend on growth factors to regulate nutrient uptake, growth,
proliferation, and metabolism. An essential player transducing growth factor signals
is PI3-kinase. Growth factors and their signalings are able to regulate autophagy
through several mechanisms. One is through the activation of the Class I PI3-kinase-
Akt-mTOR signaling pathway. Upon growth factor limitation, mTOR is suppressed,
resulting in activation of autophagy. The other is through Akt-mediated phosphory-
lation of autophagy molecules (Fig. 5.2). Akt directly phosphorylates Beclin 1 to
suppress autophagy (Wang et al. 2012). Mutation of residues on Beclin 1 phos-
phorylated by Akt induces autophagy (Wang et al. 2012). Akt can also signal
through GSK3-TIP60 to regulate ULK1 acetylation (Lin et al. 2012). Growth factor
limitation leads to decreased Akt activity and enhanced ULK1 acetylation and
autophagy (Lin et al. 2012). It is interesting to note that there exist mechanisms in
multi-cellular organisms to regulate autophagy in a manner that is more proximal to
growth factor receptors (Fig. 5.2). Active EGFR has been shown to directly bind to
Beclin 1 and lead to tyrosine phosphorylation of Beclin 1 at multiple residues,
which inhibits Beclin 1 and Vps34 activity and autophagy (Wei et al. 2013). Another
mechanism connecting growth factor receptors and the Vps34 complex is through
the Class I PI3-kinase p110β subunit. In response to growth factor deprivation,
p110β dissociates from the growth factor receptor complex, and translocates to the
Rab5-positive subcellular compartments to stimulate Rab5-GTP and Vps34 activ-
ity, leading to enhanced autophagy (Dou et al. 2013). These mechanisms provide a
more direct and rapid way in multi-cellular organisms to activate autophagy when a
cell senses growth factor scarcity.
5.6 PI3-Kinase Inhibitors in Targeting Cancer

and Autophagy
The Class I PI3-kinase-Akt-mTOR pathway is frequently mutated in human cancers

(Engelman et al. 2006; Wong et al. 2010). It is also often interconnected with other
oncogenic signaling events. Much effort has been put on developing pharmacologi-
cal inhibitors for anti-cancer therapy. Several classes of agents targeting the Class I
PI3-kinase-Akt-mTOR pathway have been approved by the US Food and Drug
Administration (FDA) (for a complete database of clinical trials, visit ClinicalTrials.
gov) or are in clinical trials. These include isoform specific PI3-kinase inhibitors,
pan-Class I PI3-kinase inhibitors, mTOR inhibitors, Akt inhibitors, and inhibitors
that target both Class I PI3-kinases and mTOR.
The design of isoform-specific PI3-kinase inhibitors is based on the discovery
that many tumors harbor specific PI3-kinase mutations. The most common example
is the prevalent p110α hotspot mutations, such as the H1047R, E542K and E545K
mutations. Thus, inhibitors specifically blocking p110α catalytic activity should be
effective against these types of tumors. In addition to p110α inhibitors, p110δ selec-
tive inhibitors have shown very promising outcome for the treatment of hematopoi-
etic malignancies (Fruman and Rommel 2014).
Several potential caveats for isoform specific Class I PI3-kinase inhibitors are
noted. One issue is the presence of feedback loops. The growth factor receptor-
Class I PI3-kinase-Akt-mTOR signaling negatively regulate the growth factor
receptor complex, leading to reduced growth factor receptor activity, to prevent
over-activation. Inhibition of specific isoform of PI3-kinase, such as p110α, results
in reduced Akt-mTOR signaling, thus relieves the inhibition of the growth factor
receptor, resulting in signalings through other mechanisms, such as p110β and Ras
pathways. To overcome this, pan-Class I PI3-kinase inhibitors that targets multiple
isoforms were designed and are in clinical trials. Such inhibitors also show high
efficacy against PTEN deficient cancers, which signal through both p110α and
p110β isoforms. Inhibitors that simultaneously target PI3-kinases and mTOR are
also in clinical trials. While these combinatorial approaches should be more effec-
tive than single selective inhibitors, potential side effects should be taken into con-
sideration, because Class I PI3-kinases are critical for a variety of normal cell and
tissue activities, including heart contractility and glucose metabolism (Fruman and
Rommel 2014).
Autophagy is often induced as a stress response during oncogenesis and upon
many therapeutic treatments to confer survival advantage cancer cells (Galluzzi
et al. 2015). Inhibition of autophagy has been considered a reasonable way to treat
cancer especially in combination with other therapeutics to overcome resistance
(Amaravadi et al. 2011). A well-pursued autophagy inhibitory drug in cancer treat-
ment is chloroquine, as well as its derivative hydroxy-chloroquine (HCQ).
Chloroquine is a well-tolerated FDA-approved drug for treating malaria, rheuma-
toid arthritis, and lupus. Pre-clinical studies support the notion that combination
therapy with HCQ is an efficient anti-cancer strategy. In recent years, clinical trials
with HCQ are actively ongoing for treatment of several cancers, in combination
with other therapies, including radiation, mTOR inhibitors, BRAF inhibitors, and
others (Rangwala et al. 2014; Rosenfeld et al. 2014). Besides chloroquine and HCQ,
other autophagy inhibitors are being pursued. Lys05, a chloroquine-based lyso-
somal inhibitor, is tenfold more potent than HCQ in inhibiting autophagy (McAfee
et al. 2012). Unlike HCQ which does not show anti-tumor activity by itself, Lys05
has anti-tumor activity as a single agent (McAfee et al. 2012). Another set of drug
candidates are inhibitors specific to Class III PI3-kinase (Vps34). Several new lines
of Vps34 inhibitors have been synthesized, which show specific inhibitory effect on
Vps34 but not other PI3-kinases. Such inhibitors potently inhibit autophagy and
endosomal trafficking (Dowdle et al. 2014; Ronan et al. 2014). Cautions are noted
for the potential side effects of Vps34 inhibitors. Deletion of Vps34 in mouse heart
results in contractility dysfunction and heart failure, and ablation of Vps34 in liver
results in fatty liver accompanied by metabolic dysfunctions (Jaber et al. 2012).
Other inhibitors are also being pursued, including small molecules inhibiting ULK1
and Atg7. The safety and antitumor activities of these compounds awaits further
evaluation. Nonetheless, as PI3-kinase signaling and autophagy are largely involved
in human cancers and are heavily interconnected with each other, more effective
and selective approaches to target these molecular events in combination with other
emerging therapeutics will offer new opportunities to eradicate the most deadly
malignant disease.
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Chapter 6
Adult and Cancer Stem Cells: Perspectives
on Autophagic Fate Determinations
and Molecular Intervention
Abstract Autophagy is a highly conserved mechanism for the maintenance of cel-

lular homeostasis and functionality in pluripotent stem cells, adult stem cells, and
normal somatic cells. Cytoprotective roles of autophagy are essential for eliminating
damaged subcellular organelles like mitochondria and protein aggregates, thereby
reducing reactive oxygen species (ROS) levels and promoting normal or cancer cell
survival. We clarify multiple autophagic inducers, default pathway sensors, and reg-
ulators in various stages of stem cells. Of note, with autophagy deficiency, there are
two major autophagy-associated outcomes, including pro-autophagic cell survival
and death. Clearly, the fates of autophagic determination are tightly regulated by
their microenvironments, cell types, and the interplay among multiple cell death
machineries related to autophagy, apoptosis, and necrosis. Based on the above fun-
damental autophagic differences among various cell types, we propose a new con-
cept, balanced autophagy, which sheds light on an equilibrium state between
pro-autophagic cell survival and death. We further suggest new strategies targeting
therapeutic-resistant cancer stem cells that emphasize the modulatory effects of
pro-autophagic cell death in intractable cancer cells.
Keywords Autophagy • Mitophagy • Adult stem cells • Cancer stem cells •

Mitochondria
The original version of this chapter was revised. An erratum to this chapter can be found at
DOI 10.1007/978-3-319-42740-9_8
K.G. Chen (*)
Department of Microbiology and Immunology, Georgetown University Medical Center,
Washington, DC 20057, USA
NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National
Institutes of Health, Bethesda, MD 20892, USA
e-mail: kgc26@georgetown.edu
R. Calderone

100 K.G. Chen and R. Calderone
6.1 Introduction
Autophagy is how eukaryotic cells remove severely injured subcellular organelles,

free-radical impaired proteins, aggregated macromolecules, and other cytotoxic
substances (Kroemer et al. 2010; Rubinsztein et al. 2005, 2011; Ravikumar et al.
2010; Cheng et al. 2013). Autophagic processes are usually implemented in several
sequential steps (i.e., induction, vesicle nucleation, and expansion), involving mul-
tiple protein complexes (Rubinsztein et al. 2011). Briefly, cytoplasmic substrates
such as damaged organelles or protein aggregates are isolated in a double-
membraned vesicle (known as an autophagosome), which are subsequently fused to
lysosomes to form autolysosomes where autophagic cargoes are degraded.
Moreover, the degraded small molecules or peptides can be recycled into the cyto-
plasm as new energy resources.
There are various autophagic mechanisms based on the types of cytoplasmic
substrates (of autophagosomes), which include subcellular organelles (such as
mitochondria, peroxisomes, and endoplasmic reticulum), various sizes of vesicles,
and part of the cell nucleus (Kroemer et al. 2010). Ultrastructural analysis reveals
that the diagnostic feature of autophagy is the formation of either autophagosomes
or autophagolysosomes. Other associated changes include mitochondrial damage,
dilation of the rough endoplasmic reticulum (ER) and Golgi complex, and cyto-
plasmic lipid droplets (Kroemer et al. 2010; Rubinsztein et al. 2005, 2011;
Ravikumar et al. 2010). Specific autophagosomes containing melanosomes
(termed melano-autophagosomes) were also reported in melanoma cells induced
by cytotoxic drugs (Chen et al. 2009). Here, we focus on mitophagosome-related
autophagy, a specific autophagy that involves damaged mitochondria digested by
macroautophagy.
Mitochondria-based mitophagy is of particular interest. Because mitochondria
are the critical sites for oxidative phosphorylation, a predominant resource of
energy (ATP) production in eukaryotic cells. Under oxidative stress and pathologi-
cal conditions, “bad” mitochondria discharge highly reactive oxygen species
(ROS). Cytotoxic mitochondrial metabolites such as ROS are able to induce mito-
chondrial DNA mutations, mitochondrial dysfunction, cytoplasmic and nuclear
damages, subsequently leading to mitochondria-related diseases. Mitochondrial
damage also triggers programmed cell death or apoptosis. Current data support that
mitophagy is an efficient way to halt many pathological-condition-driven processes
and to increase the turnover of functional mitochondria. Moreover, mitophagy may
be the most efficient type of autophagy against physiological or pathological aging
in neurons, cardiomyocytes, and many types of mammalian cells including cancer
cells (Cheng et al. 2013; Kubli and Gustafsson 2012; Zhang 2013; Ding and Yin
2012; Chen and Chan 2009).
Nonetheless, the fates of mitophagy in vivo are not well elucidated due to a lack
of powerful live imaging assays in humans. A growing body of evidence suggests a
pivotal role for mitophagy in the maintenance of the stemness of adult stem cells.
6 Autophagic Fates in Adult and Cancer Stem Cells 101
However. it is not well understood how mitophagy is regulated in cancer-initiating

cells, which are also known as “cancer stem cells.” The cancer stem cell hypothesis
is an elusive but potentially important theory in cancer therapies (Clarke et al. 2006;
Tan et al. 2006). It is important to verify how cancer stem cells evolve to utilize
mitochondria to balance ATP production under various stress conditions. A deep
understanding of these complicated natures of mitophagy would enable new strate-
gies to combat aggressive cancer.
Here, we briefly review the evolution of diverse autophagic mechanisms in vari-
ous organisms, dissect essential autophagic machineries with a focus on mitophagy,
and evaluate the functionality of mitophagy based on its defense mechanisms. We
further elucidate major autophagic inducers, energetic sensors, transducers, and
molecular regulators of oxidative stress-based mitophagy, particularly in adult and
cancer stem cells. With respect to cancer therapeutics based on targeting mitophagy,
we concisely analyze current treatments. Finally, we propose new therapeutic con-
cepts and strategies concerning how to treat intractable cancer via precision inter-
vention of autophagy dynamics.
6.2 Evolutionarily Conserved Mitophagic Machineries
In yeasts, autophagy is an ancient method to obtain recycled energy-rich molecules

during nutrient deprivation. Among many model organisms, stimulation of autoph-
agy activity via caloric restriction, Sirtuin 1 activation, and pharmacological inter-
ventions (e.g. treatments with rapamycin, resveratrol, or spermidine) increases life
span in these organisms (Rubinsztein et al. 2011). Genetically, short-lived mutants in
Saccharomyces cerevisiae were associated with autophagy defects (Matecic et al.
2010). Upon dietary restriction, activation of the translational repressor 4E-BP pro-
longs life span by refining mitochondrial function, likely through inactivation of S6K
and eIF4F in the cytoplasm in yeasts and Drosophila (Wang et al. 2008a; Zid et al.
2009). Transgenic expression of the autophagy-related gene Atg8a in the brain of
Drosophila directly augmented autophagic activity in neurons, which decreased
insoluble ubiquitinated proteins and increased cellular resistance to hydrogen perox-
ide (H2O2), thus increasing life span up to 56 % in female Drosophila (Simonsen
et al. 2008).
In mammalian cells, caloric restriction, decreased protein synthesis, reduced
ROS, and augmented autophagic activity play an important role in prolonging life
span. Genetic inhibition of autophagy-responsive genes diminishes autophagic
potentials and induces degenerative changes in various tissues, thereby accelerating
physiological and pathological aging (Ravikumar et al. 2010). In recent years,
mutations of autophagy-related genes were found to underlie miscellaneous human
diseases, including static encephalopathy of childhood with neural degeneration in
adulthood (SENDA), Vici syndrome, hereditary spastic paraparesis, Parkinson’s
disease, lysosomal storage disorders, Crohn’s disease, and different types of cancer
(Jiang and Mizushima 2014). Given that autophagy is associated with diverse
human diseases, there is a great need to precisely define the functionality of autoph-
agy, especially mitophagy, in mammalian cells.
6.3 Functions of Mitophagy
It is generally believed that autophagy, a critical regulator of mitochondrial homeo-

stasis, confers cytoprotective effects in various tissues or organs by degrading detri-
mental cargoes, bacteria, and viruses (Kroemer et al. 2010; Jiang et al. 2010;
Ravikumar et al. 2005; Zhang and Cuervo 2008). Damaged mitochondria with a
missing membrane potential discharge ROS and noxious apoptotic intermediates,
which are subjected to removal by mitophagy, thus maintaining physiologically rel-
evant numbers of mitochondria. Exemplified in apoptotic T-cells, the maintenance
of mitochondrial turnover is essential for promoting mature T-cell survival (Kovacs
et al. 2012). The molecular basis of this mitophagy was often facilitated by serine/
threonine protein kinases-interventional phosphorylation of mitochondria and sub-
sequently by the E3 ubiquitin ligases-mediated ubiquitination of mitochondrial
membrane proteins. For instance, in neurons, the bad mitochondria are usually
tagged by the PTEN induced putative kinase 1 (PINK1), ubiquinated by the E3
ubiquitin ligase Parkin, and then degraded by (macro)autophagy. Thus, deletion of
PINK1 results in deficiency of Complex I and II of mitochondria in the stratum of
knockout mice (Gautier et al. 2008), suggesting a potential role of PINK1 in brain
function as well as neurogenesis.
Genetic manipulation of autophagy-related (ATG or Atg) genes has been fre-
quently used to identify potential mitophagy functions. Tissues with deletion of
ATG genes regularly exhibit some aging-related phenotypes, including intracellular
accumulation of inclusion bodies, lysosomes containing the aging lipofuscin pig-
ment, and morphologically damaged mitochondria. For example, Atg7-knockout in
mesoendodermal lineages (such as liver, skeletal muscle, and islet β cells), mito-
chondrial dysfunction was a common feature, which includes accumulation of
deformed and swelling mitochondria (Ebato et al. 2008; Jung et al. 2008; Masiero
et al. 2009; Komatsu et al. 2006, 2007; Wu et al. 2009). Other noticeable changes
were reflected in subcellular organelles (e.g. accumulation of peroxisomes, ER dis-
tension, and vacuolar changes), mesodermally muscle-related changes (e.g. muscle
atrophy and sarcopenia), and altered endocrine processes (e.g. degeneration of islets
and impaired glucose tolerance). In addition, pronounced molecular signatures
include ubiquitin-positive inclusions in hepatocytes, accrual of an autophagy recep-
tor (p62/STQM or p62) concomitant with NRF2 activation, and an increase in ubiq-
uitinated protein aggregates colocalized with p62 (Ebato et al. 2008; Jung et al.
2008; Masiero et al. 2009; Wu et al. 2009; Komatsu et al. 2010). These structural
and biochemical changes that resulted from Atg gene deletion in mice are consistent
with the autophagic phenotypes displayed from yeasts to human beings. Thus,
mitophagy is a default program that may be accurately regulated by different induc-
ers, energetic sensors, and signal transducers.
6.4 Mitophagy: Inducers and Energetic Sensors
Collectively, inducers, metabolic or energetic sensors, transducers, and regulators

function cooperatively in multiple pathways. Nutrient starvation, caloric restriction,
hypoxia, and oxidative stress are potent autophagy inducers. Specific energetic sen-
sors (on membrane and in the nuclei) receive signals from both exogenous and
endogenous inducers (e.g. starvation, low glucose, oxidative stress, and low ATP).
These sensors are able to transactivate core transducers such as 5′ adenosine
monophosphate-activated protein kinase (AMPK). Consecutively, signal transduc-
tion pathways activate regulatory networks, which intertwine transcriptional pro-
grams with proteomics to positively or negatively regulate the input signals (Fig. 6.1).
Nutrient Starvation and Caloric Restriction as Generic Inducers: Caloric restric-
tion or reduced nutrient consumption has been proven to be an effective anti-aging
intervention in many model organisms including primates (Colman et al. 2009).
Caloric restriction induces autophagy through multiple pathways that include activa-
tion of AMPK, SIRT1 (an NAD+-dependent deacetylase), PHA4, and the inhibition of
the IGF1-mTOR pathway (Egan et al. 2011; Canto et al. 2010; Kenyon 2010; Hansen
et al. 2008). These manifold pathway-driven anti-aging mechanisms are perhaps car-
ried out in a spatial-temporal manner, which significantly increase the autophagic
diversity and complexity. Interestingly, caloric restriction or starvation induces meta-
bolic stress and activates autophagy in a cancer microenvironment, which is partially
guided by the ROS-mediated AMPK activation (Li et al. 2013). These data provide a
strong link between nutrient starvation and oxidative stress in autophagy.
Oxidative Stress Inducers: The tumor suppressor role of autophagy has been con-
firmed in genetically modified mouse models with targeted deletion of Beclin1 (Qu
et al. 2003; White et al. 2010; Yue et al. 2003). The underlying mechanism includes
autophagy that removes damaged mitochondria and reduces high levels of ROS,
thus minimizing DNA damage, enhancing chromosomal stability, and mitigating
protein quality control (Mathew et al. 2007, 2009). Interestingly, ROS (e.g. hydro-
gen peroxide) induces becline-1-independent autophagy, known as non-canonical
autophagy. Moreover, hydrogen peroxide was also shown to direct oxidation of
ATG4, a protease, upon starvation (Scherz-Shouval and Elazar 2011). Thus, autoph-
agy inhibits oxidative stress through both canonical and non-canonical autophagy
pathways, therefore offering efficient quality control of mitochondrial activity.
Mitochondrial fusion and asymmetric fission offer alternative quality control to
remove damaged mitochondria (as indicated by low membrane potential, ΔΨm) and
to enable the functional ones to enter the next fusion-fission cycle (Twig et al. 2008).
Given that asymmetrical fission is compromised or discontinued in cells, it would
certainly increase the accumulation of endogenous ROS rendering cells bioenergetic
deficient (e.g. defective oxidative phosphorylation). However, it is not clear whether
mitophagy deficiency-induced defective oxidative phosphorylation would encourage
cancer cells to adapt to the lower energy-producing glycolysis. Nonetheless, the
above mitophagy mechanisms might provide new insights into the Warburg effect,
which describes how cancer cells utilize low-efficiency glycolysis as the predomi-
nant energy source, regardless of the availability of aerobic and anaerobic (or
hypoxic) conditions (Vander Heiden et al. 2010; Gottlieb and Vousden 2010).
Hypoxia, one of the cancer hallmarks, represents a blood supply deficiency related to
oxygen deprivation in a tumor mass. Hypoxia has been an active area of cancer
research because it confers cancer therapeutic resistance and thus is an adverse prog-
nostic factor in cancer therapy (Shannon et al. 2003).
Hypoxia as a Mitophagic Inducer: Under hypoxic conditions, induction of autoph-
agy enables cancer cells to survive in certain cellular stress conditions. In general,
this process seems to be regulated by HIF1α (Thiery et al. 2009). Specifically,
HIF1α induces autophagy-associated expression of BNIP3 and BNIP3L (Mazure
and Pouyssegur 2009), which are responsible for stimulating mitophagy to cope
with ROS. Under clinically relevant hypoxia, AMPK-independent autophagy is able
to support human tumor cells resistant to radiotherapy (Chaachouay et al. 2015).
Recently, HIF1α has also been shown to regulate the viability of prostate cancer
stem cells via mTOR signaling (Marhold et al. 2015). Furthermore, an AMP/ATP-
AMPK-TSC-mTOR pathway was implicated in the control of HIF1α-independent
hypoxia-induced autophagy (Papandreou et al. 2008; Yu et al. 2011). These studies
highlight the importance of intracellular energy sensors such as AMP and ATP in
the regulation of autophagic response (Fig. 6.1).
AMP/ATP as intracellular Sensors: Energy utilization by cancer cells has been an
enigma since the discovery of the Warburg effect. The role of ATP signaling in
autophagy is not well understood. Remarkably, the energy levels in cells can be
accurately measured by the AMP/ATP ratios. An increase in AMP/ATP ratios and
nutrient depletion were found to induce AMPK activation, which subsequently acti-
vates autophagy via Unc-51-like autophagy activating kinase 1 (ULK1) and the
inhibition of mTOR (Alers et al. 2012). Moreover, exhaustion of cellular ATP in
Fig. 6.1 (continued) LKB1 might function as a cellular context-dependent switch between a regu-
lator of AMP/ATP ratios and a modulator of AMPK-dependent and AMPK-independent pathways
(g). Under clinical relevant hypoxia, HIF1α stimulates the AMPK-independent autophagy through
inhibition of mTOR, which renders adult and cancer stem cells resistant to the detrimental effects
of DNA-damaging agents and ROS (h). HIF1α also induces autophagy-related BNIP3 and BNIP3L
expression to enhance its protective effects (c). Finally, frequent oncogenic, genotoxic, and oxida-
tive stress stabilize and activate p53, which might also stimulate or inhibit autophagy by activating
or inhibiting AMPK respectively and by coordinately regulating downstream transcriptional pro-
grams (i, c). Abbreviations: ACD autophagy-induced cell death or autophagic cell death, AMP
adenosine monophosphate, AMPK 5′ adenosine monophosphate-activated protein kinase, ATG
autophagy-related genes, ATP adenosine triphosphate, HIF1α hypoxia-inducible factor 1-alpha,
LKB1 serine/Threonine Kinase 11 encoded by the STK11 gene, MAPK mitogen-activated protein
kinases, mTOR the mammalian target of rapamycin, p53 tumor protein p53, PM plasma mem-
brane, ULK1 Unc-51 like autophagy activating kinase 1. Pathway diagram descriptions: “?” unde-
fined signal transduction pathways, “↑” increased ratios; lines or curves with arrowheads indicating
enhanced effects or activation, whereas lines or curves with round ends denoting decreased effects
or inhibition; the thickness of the lines (or curves) corresponding to the magnitude of the effects
(i.e., thicker lines indicate stronger effects); and the dotted lines (or curves) specifying reduced or
weak contributions to downstream outcomes
Fig. 6.1 An integrative model of energy states with autophagic fate outcomes in adult and cancer
cells: cellular and molecular mechanisms by which adult and cancer stem cells sense both intracel-
lular (a) and extracellular energetic changes (b) integrate mitochondrial autophagy pathways with
core transcriptional programs (such as hypoxic, oxidative stress, and cell cycle control) (c). The
proposed model emphasizes the prevalence of glycolysis in stem cells (including pluripotent stem
cells, adult and cancer stem cells). Altered ATP energetic states ultimately alter proteomics and
transcriptional networks to determine autophagic cell fates in different types of stem cells. These
interconnections provide the possibility to precisely formulate autophagy-related cancer therapies
using anti-autophagic cell survival or pro-autophagic cell death strategies. In this model, we desig-
nate that cellular lineage differentiation is an energy-consuming process that requires sufficient
ATP production through the oxidative phosphorylation of mitochondria via the tricarboxylic acid
(TCA). The reactive oxygen species (ROS) byproducts generated during this process might coop-
eratively stimulate lineage differentiation (d). ROS might also suppress cancer cell metastasis by
inhibiting the epithelial-mesenchymal transition (EMT). Moreover, a low energy-consuming state
is required for stem cell quiescence and self-renewal (e). Under the conditions of autophagy-
induced cell death (ACD) conditions, mitophagic events produce extremely low or no ATP, thus
incapable of maintaining the mitochondrial membrane potential (f). With respect to the regulation,
diverse functionality of LKB1 might directly or indirectly control ATP levels, cell cycle check-
points, AMPK, and mTOR, thus placing this factor as one of the central regulators. Intriguingly,
defective mitochondria was found in Lkb1-deficient bone marrow cells (Gurumurthy

et al. 2010). Lkb1 is a new cell-cycle restrictive checkpoint that is independent of its
regulation of AMPK and mTOR signaling. Further inactivation of Lkb1 led to rapid
depletion of hematopoietic stem cells (Gurumurthy et al. 2010). These data validate
AMP/ATP dynamics as major intracellular autophagic sensors. Moreover, AMP/
ATP signals might control a tentative autophagic switch between an LKB1-ATP-
AMPK-dependent and -independent pathways (Fig. 6.1).
Interestingly, the immune system exemplifies autophagy regulation of ATP. In
activated CD4+ T cells, suppressed autophagy led to reduced energy metabolism
characterized by reduced ATP production, glycolysis, and fatty acid utilization
(Hubbard et al. 2010). Autophagy appears to provide metabolite resources needed
to produce ATP (Lum et al. 2005), which facilitates cell survival under sustained
withdrawal of growth factors in bone marrow hematopoietic cells. Thus, autophagy
might play an essential role in the maintenance of intracellular ATP levels during
the course of apoptosis concomitant with caspase activation. A stable intracellular
level of ATP may be indispensable for secretion of “find-me” signals (e.g.
lysophosphatidylcholine) as well as “eat-me” signals (e.g. phosphatidylserine). In
addition, cancer chemotherapy-induced autophagy also promotes ATP release from
cancer cells (Fig. 6.1). This process can be blocked by the inhibition of autophagy.
Does ATP modulate antitumor immune response in T-cells? Indeed, extracellular
ATP released from tumor cells recruits dendritic cells and triggers a T-cell response
to tumor cells (Michaud et al. 2011). On the other hand, ATP-mediated immunore-
sponse, together with T-cell mediated autophagy, may enhance cancer cell survival
by evading conventional therapeutic regimens (Buchser et al. 2012).
6.5 Stem Cells and Mitophagy
Numerous types of stem cells exist in mammalian life, ranging from embryonic
development to adulthood under both physiological and pathological conditions.
Briefly, stem cells can be classified into two major groups (i.e., pluripotent and
multipotent stem cells). Pluripotent stem cells, including embryonic stem cells
(ESCs) and induced pluripotent stem cells (iPSCs), which can potentially produce
any cell or tissue type in mammals (Chen et al. 2014a). With respect to multipotent
stem cells, they share some basic features of pluripotent stem cells. However, mul-
tipotent adult stem cells only have limited diversification potential, usually differen-
tiating to two or more cell types (Wagers and Weissman 2004).
Embryonic Stem Cells: In mammalian development, ESCs generate the embryo
and ultimately the fetus. The inner cell mass (ICM), an ESC cluster inside the blas-
tocyst from preimplantation-stage embryos, could be isolated and cultured in cell
culture dishes in vitro (Evans and Kaufman 1981; Thomson et al. 1998; Chen et al.
2014b). These ESCs possess the capacities of self-renewal (i.e. replicating them-
selves) and differentiation to all cell types of the three germ layers (i.e. ectoderm,
mesoderm, and endoderm). The ectoderm, the outermost germ layer of cells derived
from the ICM, develops into the nervous system, sensory organs, and skin. The
endoderm generates respiratory and digestive organs (e.g. lung, liver, and pancreas),
whereas the mesoderm gives rise to bone, muscle, connective tissue, kidney, and
hematopoietic cells.
Induced Pluripotent Stem Cells: These iPSCs are derived by directly reprogram-
ming somatic cells using multiple transcriptional factors (e.g. Oct4, Sox2, Klf4, and
c-Myc). The introduction of four specific genes encoding the above transcription
factors into somatic cells (e.g. fibroblasts) could convert these cells into a pluripo-
tent state, similar to ESCs (Takahashi and Yamanaka 2006). Essentially, iPSCs can
be propagated indefinitely in vitro and differentiated into all cell types of the three
germ layers. Hence, iPSCs hold great promise in the fields of regenerative medicine,
disease modeling, drug discovery, and cancer research (Robinton and Daley 2012).
Multipotent Adult Stem Cells: Multipotent adult stem cells, also known as somatic
stem cells, are undifferentiated cells that are found within differentiated tissues or
organs. Adult stem cells exist in the bone marrow, brain, liver, heart, and many other
organs. The primary roles of adult stem cells in a living organism may be associated
with cellular maintenance, tissue repair, and cell replacement. Interestingly, adult
stem cells also possess the capacities of self-renewal and differentiation into some
specialized cell types. The origins of adult stem cells are different in terms of their
tissue or organ types. The adult brain contains stem cells that are able to differenti-
ate into astrocytes, oligodendrocytes, and neurons. Some tissues or organs may have
one or more types of adult stem cells. For example, the bone marrow comprises at
least two stem cells niches that nurture hematopoietic stem cells (that form all types
of blood cells in the body) and skeletal stem cells (that generate cartilage, bone,
hematopoiesis-supportive stroma, and marrow adipocytes) (Morrison and Scadden
2014; Bianco and Robey 2015). Encouragingly, hematopoietic stem cells from the
bone marrow have been used in allogenic bone marrow transplants for more than 40
years. It is conceivable that many other types of adult stem cells could be also useful
for stem cell-based therapies in the future.
Nonetheless, adult stem cells only exist as a rare population of cells within a tis-
sue or organ. Due to a lack of definitive surface markers, isolation of these stem
cells represents a big challenge. Additionally, large-scale amplification of these
adult stem cells may be also an obstacle that impedes future stem-cell based thera-
pies. Moreover, there is little known about how adult stem cells are regulated both
in vivo and in vitro. Some adult stem cells as well as cancer stem cells typically exist
in a quiescent state with a longer lifespan in specified stem cell niches. Autophagy
is thought to be critical for maintaining stem cell homeostasis in cells that have
undertaken tissue regeneration and cellular reprogramming (Pan et al. 2013;
Phadwal et al. 2013). The exact role of autophagy in the regulation of pluripotent,
adult, and cancer stem cells remains to be determined.
The Role of Mitophagy in Pluripotent Stem Cells: Mitophagy has been implicated in
facilitating reprogramming of somatic cells to iPSCs. Vazquez-Martin et al. reported
that reprogramming of somatic cells (e.g. mouse fibroblasts) by the Yamanaka fac-
tors (i.e., Oct4, Klf4, and Sox2) to the pluripotent state was drastically reduced by
95 % by pharmacologically induced mitochondrial fusion using the mitochondrial
division inhibitor mdivi-1 (Vazquez-Martin et al. 2012). Mechanistically, mitochon-

drial division by mdivi-1 discriminatorily impedes the self-assembly of a dynamin
family member (termed DRP1) of the large GTPases, thereby preventing the renewal
of functional mitochondria via mitochondrial fission. Furthermore, Sox2-dependent
temporal inhibition of mTOR activates autophagy, which is also a key step to initiat-
ing cellular reprogramming (Wang et al. 2013). Additionally, human iPSCs have
been generated from patients carrying m.3243A > G, the most common mitochon-
drial DNA mutation found in many human diseases. The iPSC-derived neurons and
various tissues showed specific complex I defects in the respiratory chain of mito-
chondria. Complex I was subjected to degrading via mitophagy as indicated by
explicit expression of both PINK1 and Parkin in perinuclearly sequestered autopha-
gosomes (Hamalainen et al. 2013). Taken together, the above studies provide new
insights into the role of autophagy in the regulation of reprogramming efficiency.
Clearly, mitochondrial fusion, a surveillance mechanism to ensure sufficient num-
bers of functional mitochondria and Complex I activity, is essential for mitochon-
drial homeostasis and normal cell differentiation.
The Role of Mitophagy in Adult Stem Cells: Adult stem cells or progenitors are
found in the tissues derived from the three-lineage differentiation. The involvement
of autophagy in the maintenance of self-renewal and differentiation of these stem
cells might be essential for these cells to meet the minimal metabolic requirements
(Ito and Suda 2014). With respect to adult stem cells derived from neuroectoderm,
p53 negatively regulates the self-renewal and proliferation of adult neural stem cells
(NSCs) through the cell cycle regulator p21 (CDKN1A) (Meletis et al. 2006). A
recent report indicates that deletion of RB1-Inducible Coiled-Coil 1 (RB1CC1), a
gene essential for autophagy induction in mammalian cells, leads to the loss of
postnatal NSCs and impaired neuronal differentiation in the postnatal brain (Wang
et al. 2008b). Interestingly, p53-dependent apoptosis and cell cycle arrest were asso-
ciated with postnatal NSC death, but not with the impaired neuronal differentiation.
Thus, down-regulation of an oxidative state by N-acetylcysteine was able to salvage
the weakened neuronal differentiation (Wang et al. 2008b). Moreover, after insulin
withdrawal, adult hippocampal NSC had a caspase-independent autophagic cell
death related to the essential autophagy gene Atg7 (Yu et al. 2008). Systemic Atg7
ablation in mice instigated neurodegeneration, increased susceptibility to infection,
and reduced survival to 3 months (Karsli-Uzunbas et al. 2014).
Regarding adult stem cells from mesoendoderm, ex vivo cytokine withdrawal
and in vivo caloric restriction induce a protective autophagy against metabolic
stress, which is mediated by FOXO3A in mouse hematopoietic stem cells (Warr
et al. 2013). Hematopoietic stem cell compartment sustains FOXO3A-driven gene
expression profiles that poise hematopoietic stem cells for rapid induction of
autophagy upon the emergence of an energy crisis. Endoderm-derived bipotent liver
progenitors are believed to activate liver regeneration in the adult liver upon hepa-
tectomy or massive liver damage. However, the mechanism that maintains the pro-
genitors’ stemness is not well studied. A recent report revealed that autophagy was
also required for the maintenance of liver progenitor cells under both physiological
and pathological conditions (Cheng et al. 2015). Collectively, these data support the
essential roles of autophagy in ensuring adult stem cell survival and death under
specific conditions. These results also offer consistent examples that shed light on
the molecular interplay among oxidative states, autophagy, autophagic cell death,
and apoptosis.
The Role of Mitophagy in Cancer Stem Cells: There are likely two-compartment
autophagy mechanisms: autophagy metabolism in cancer cells and in tumor micro-
environments (including normal tissues and adult stem cells). In cancer cells,
canonical autophagy is mediated by Beclin 1 and essential for the tumorigenicity of
mammary cancer stem-cell like progenitors (Gong et al. 2013). Autophagy is essen-
tial for glucose homeostasis and the maintenance of lung tumors (Karsli-Uzunbas
et al. 2014). Noticeably, autophagy is also able to suppress tumor progression by
maintaining tumor cells in a quiescent state. Moreover, the tumor suppressor protein
p53 induces autophagy under genomic stress conditions. The p53-dependent activa-
tion of autophagy is coordinately regulated by mTOR and AMPK (Feng et al. 2005,
2007; Crighton et al. 2006). Essentially, it is the nuclear p53 that transactivates
autophagy-related genes (Tasdemir et al. 2008a). Under certain conditions, p53 is
also a negative regulator of autophagy. Genetic or pharmacological inhibition of the
tumor suppressor protein p53 activates autophagy (Fleming et al. 2011; Tasdemir
et al. 2008b, c). Many autophagic inducers induce autophagy through the E3 ubiq-
uitin ligase MDM2-mediated degradation of p53. Under nutrient depletion and
hypoxic conditions, increased autophagy activity maintains higher ATP levels, thus
enhancing the survival of p53-deficient cancer cells. However, cytoplasmic p53,
instead of nuclear p53, was able to repress enhanced autophagy in p53 (−/−) cells
(Tasdemir et al. 2008a). Thus, p53 plays a dual role in the regulation of autophagy,
in which p53 protein modifications, intracellular localizations, and functional states
are critical for this distinct regulation. Verification of the detailed cellular and
molecular context related to p53 is an important step to understand the complexity
of autophagy regulation in malignant tumors.
With regard to autophagy in tumor microenvironments, an increasing body of
evidence indicates that there are complicated signaling interactions among normal
tissues, adult stem cells, and cancer stem cells. The interactions between endocrine
and paracrine signals are implicated in the regulation of pluripotent stem cell niches
(Chen et al. 2014a). Presumably, parallel endocrine-paracrine signals would also
regulate cancer stem cell survival and growth (Fig. 6.1). Another possible mecha-
nism is that endocrine-paracrine interactive signals regulate normal adult stem cells,
stromal cells, immunological response cells, and fibroblast-like cells at the periphery
of cancer stem cell niches. It is conceivable that increased autophagy may interfere
with the function of endocrine cells (such as insulin-producing β cells) and hormonal
response of microenvironmental stem cell niches in a non-cell-autonomous way.
Nevertheless, caution must be taken when we study a rare population of multipo-
tent and cancer stem-like cells with the capacity of self-renewal and tumor-initiating
in vitro and in animal models. For example, breast cancer stem-like cells can be
propagated as suspended colonies termed “mammospheres.” Enhanced autophagic
expression of Beclin 1 was found in aldehyde dehydrogenase 1-positive (ALDH1+)
cells within mammospheres (Gong et al. 2013). These results may reflect a preven-
tive response of cancer stem cell-like cells under altered growth conditions in vitro,
not necessarily the properties of mammary cancer stem cells. Cancer stem cell-like
cells usually have different degrees of quiescence, sometime referred to as slow-
cycling cells (Roesch et al. 2010). These cellular behaviors would make it difficult
to distinguish them from normal adult stem cells or normal tissues. Furthermore,
such cellular quiescence may also benefit cancer stem cell to survive in a low energy
state, potentially facilitating the development of intractable cancer properties.
Unraveling these autophagy-related behaviors is particularly important when we
consider a strategy for treating cancer patients by targeting cancer stem cells-based
autophagy.
6.6 Cancer Therapeutics Targeting Mitophagy
It is clear now that the inhibition of autophagy may lead to at least three different
outcomes (i.e., cell differentiation or survival, cellular quiescence, and autophagic
cell death) (Fig. 6.1d–f). Autophagy-mediated differentiation and cell survival ren-
der cancer cells resistant to oxidative stress, apoptosis, and necrosis. Therefore, inhi-
bition of autophagy sensitizes cancer cells to DNA-damaging agents such as cisplatin
and 5-fluorouracil in esophageal and colorectal cancer cells (Li et al. 2010; Liu et al.
2011). It has been noted that the cyclosporine A analogue SDZ PSC-833, a potent
multidrug resistance (MDR) inhibitor, has a pro-autophagic cell death effect in pig-
mented melanoma cells (Chen et al. 2009). However, there is not a well-defined
interphase between pro-survival and pro-death autophagy, which we designate here
as “balanced autophagy.” We reason that treating cancer patients by targeting differ-
ent phases of autophagy may achieve correspondingly different outcomes.
Anti-autophagy Based Cancer Therapy: Inhibition of autophagy in cancer seems an
emerging cancer therapy (Cheng et al. 2013). There is a broad spectrum of small
molecules that belong to autophagy stage-specific inhibitors or modulators. For
example, class III PI3K (Vps34) inhibitors (e.g. 3-methyladenine, wortmannin, and
LY294002) interfere with early-stage autophagic recruitment to the membranes and
with lysosomal structures. Chloroquine, hydroxychloroquine, and bafilomycin A1
belong to late-stage inhibitors of autophagy. Both chloroquine and hydroxychloro-
quine are lysosomotropic drugs that inhibit lysosomal acidification (Ruiz-Irastorza
et al. 2010). Of note, bafilomycin A1, a specific inhibitor of vacuolar-H+-ATPases,
inhibits the acidification of endocytic structures (Shacka et al. 2006). Lysosomal
inhibition of vacuolar-H+-ATPase activity by proton pump inhibitors increases both
extracellular and lysosomal organelle pH. As a result, these proton pump inhibitors
significantly escalate cytoplasmic retention and nuclear import of some chemother-
apeutic agents, thus vividly sensitizing solid tumor cells to the effects of cisplatin,
5-fluorouracil, and vinblastine (Luciani et al. 2004). Therefore, lysosomotropic
drugs and proton pump inhibitors might share an antitumor mechanism through the
inhibition of lysosomal acidification-based autophagy.
Microtubule-stabilizing (e.g. taxanes) and disrupting agents (e.g. colchicine and

Vinca alkaloids) interfere with subcellular organelle transport along microtubules,
thereby indirectly inhibiting the fusion between autophagosomes and lysosomes.
We need to point out that the majority of these inhibitors including chloroquine and
microtubule-stabilizing and -destabilizing agents are widely used anticancer drugs.
These cytotoxic drugs frequently cause multidrug resistant phenotypes in cancer
cells, which are mediated by a cluster of ATP-binding cassette (ABC) transporters,
particularly, ABCB1, ABCG2, and ABCC1 (Chen and Sikic 2012; Gottesman et al.
2002). Nonetheless, some anti-malaria-based drugs such as chloroquine and quina-
crine have been assessed in humans and showed greater cytotoxicity, which should
be modified for future clinical trials, perhaps in combination with other pathway
inhibitors to enhance autophagic effects. Finally, ionizing radiation preferentially
induces expression of LC3, Atg5, and Atg12 in CD133+ glioma initiating stem cells,
but not in CD133− cells (Lomonaco et al. 2009), suggesting the utility of the induc-
tion of a specific anti-autophagy in eradicating cancer stem cells, particularly in
those quiescent and slow-cycling stem cells.
Balanced Autophagy Initiates Homeostasis and Sustains Cell Dormancy: Balanced
autophagy represents an equilibrium state, often with cell dormancy, between pro-
autophagic cell survival and cell death. Fundamentally, cell dormancy with revers-
ible cell cycle arrest or slow-cycling cells, low metabolic rates, reduced protein
synthesis, and autophagic activation provide essential resources for cancer stem cell
survival, repair, and self-renewal. As discussed in previous sections, a well-charac-
terized signaling pathway of dormancy is the inhibition of the mTOR pathway and
subsequently decreased biosynthesis. These properties are essential for cancer stem
cells to develop therapeutic resistance (Fig. 6.1e). It is imperative to accurately
define the cellular and molecular states of cancer, which enable us to determine
potential therapeutic resistance, tolerance, and predictive response.
Pro-autophagic Cell Death Based Cancer Therapy: Teleologically, we may con-
sider pro-autophagic cell death to enhance cancer therapies under certain circum-
stances (Fig. 6.1f). In pigment-producing melanoma cells, the use of the MDR
inhibitor SDZ PSC-833 promotes melanoautophagic cell death (Chen et al. 2009).
This study provides the rationale for the combined use of potent MDR inhibitors
with other pro-autophagic death inducers to treat patients with drug-resistant cancer
stem cells.
Managements of Oxidative Stress and Metastasis of Cancer: Metastatic cancer cells
with upregulated autophagy exhibit an apoptosis-resistant phenotype (e.g. resistant
to TRAIL-induced apoptosis) when compared to non-metastasizing cells (Glinsky
and Glinsky 1996; Han et al. 2008). Administration of inhibitors of Beclin1 and
ATG7 restore TRAIL-induced apoptotic cell death in cancer (Han et al. 2008; He
et al. 2012). The influence of oxidative stress levels on therapeutic response in meta-
static cancer cells is not well elucidated. A recent study found that metastatic mela-
noma cells had high levels of oxidative stress that is associated with low viability of
this type of metastasizing cells. Therefore, antioxidant treatments of metastatic
melanoma in the mouse model promote melanoma cell survival (Piskounova et al.
2015). This study raises the possibility of using pro-oxidants to modulate oxidative
stress in cancer, which might prevent cancer metastasis in future clinical trials.
6.7 Conclusions
The cytoprotective roles of autophagy may function as a double-edge sword, which

promotes both normal and cancer cell survival. We show that autophagic inducers,
sensors, transducers, and regulators function cooperatively in multiple default path-
ways. Noticeably, the inhibition of autophagy may cause three possible autophagic
outcomes, including pro-autophagic cell survival, quiescence, and death. Moreover,
the cellular determination of a given autophagic outcome is dependent on cellular
context and complicated spatial-temporal relationships among autophagy, apoptosis,
and necrosis. Importantly, these different outcomes provide a fundamental basis for
clinical intervention of cancer and should be considered when designing a therapeutic
regimen. Finally, we propose new concepts such as balanced autophagy related to the
quiescent state of cancer stem cells and the rationale of pro-autophagic cell death, a
previously unappreciated strategy, for future cancer therapy.Acknowledgments We
would like to thank our colleague Professor R. Padmanabhan for discussion and Ms.
Verma Walker, NIH Library Editing Service, for reviewing the manuscript.
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Chapter 7
Role of Autophagy in Tumor Progression
and Regression
Bassam Janji and Salem Chouaib
Abstract Depending on tumor type, stage, and genetic context, autophagy can play
an opposite role in cancer by promoting tumor progression or regression. It is now
well established that autophagy limits tumor initiation, however, it promotes the pro-
gression of well-established tumors. In the context of tumor progression and immune
response, experimental evidence indicate that autophagy plays a key role in maintain-
ing survival of tumor cells under stress condition such as hypoxia. Indeed, by activat-
ing autophagy, tumor cells are able to escape immunosurveillance by activating
several overlapping mechanisms in cancer cells. Such findings have inspired signifi-
cant interest to develop autophagy inhibitor molecules as an entirely new approach to
cancer treatment. While much remains to be learned mechanistically, it is now widely
established that modulation of this process will be an attractive avenue for future anti-
cancer therapeutic approaches. In this chapter, we will briefly describe the role of
autophagy in tumor regression in the context of inflammation, necrosis, oxidative
stress and genomic instability. We will also focus on recent reports highlighting the
role of autophagy in the impairment of the anti-tumor immune response. In keeping
with this, we believe that targeting autophagy may represent a conceptual realm for
new anti-tumor strategies aiming to block immune escape.
Keywords Autophagy • Tumor immunity • Inflammation • Hypoxia • Tumor pro-

gression • Tumor regression • Tumor therapy
B. Janji, Ph.D. (*)

Laboratory of Experimental Cancer Research, Department of Oncology,
Luxembourg Institute of Health, Luxembourg City, Luxembourg
e-mail: bassam.janji@lih.lu
S. Chouaib, Ph.D.
INSERM U1186, Gustave Roussy Cancer Center, Villejuif, France

118 B. Janji and S. Chouaib
7.1 Autophagy Regulation in Physiological and Pathological

Conditions
Autophagy acts as a catabolic process crucial for cellular homeostasis and mainte-
nance of cell integrity under stressful conditions (Mizushima 2007; Yang and
Klionsky 2010). Autophagy is a degradation mechanism of cell components which
allows the recycling of essential amino acids, nucleotides, and fatty acids necessary
for energy and macromolecule biosynthesis (Corcelle et al. 2009; Glick et al. 2010).
During cancer progression, autophagy can be induced by different stresses, particu-
larly hypoxia, nutrient deprivation, or extracellular matrix detachment (Rosenfeldt
and Ryan 2009; Yang and Klionsky 2009). The autophagic process is characterized
by the formation of phagophore or isolation membrane mainly dependent on
Beclin-1 (BECN1) complexes. Following this so-called nucleation stage, the phag-
ophore is elongated by several Autophagy-related proteins (ATG) and the
Microtubule-associated protein 1A/1B-light chain 3 (LC3)-I is lipidated into
LC3-II. Maturation of the phagophore, through the action of LC3-II and BECN1
proteins, enables the sequestration of cell constituents into well-characterized
vesicles named autophagosomes. Fusion of autophagosomes with lysosomes leads
to the formation of autolysosomes and the degradation of their contents by lyso-
somal hydrolases (Kang et al. 2011).
Under physiological conditions, autophagy is constitutively executed at basal
level in all cells to promote cell homeostasis. However, in tumor cells autophagy is
activated in response to various cellular stresses and environmental factors includ-
ing hypoxia (Mathew and White 2011). Therefore, the major consensus that emerge
is that autophagy can act as tumor suppressor and tumor promoter. Such opposite
role of autophagy in cancer seems to be related to the stage of the tumor. In fact,
autophagy clearly suppresses the initiation and the development of tumors, how-
ever, it is considered as a key survival pathway in response to stress, and many
established tumors require autophagy to survive.
7.2 Autophagy as a Tumor Regression Mechanism
The role of autophagy in tumor suppression relies on its effect on several oncogenic
pathways such as the activation of the PI3K/Akt pathway via activating PI3K muta-
tions, AKT amplifications, or PTEN loss of function. (Guertin and Sabatini 2007).
Moreover, the amplification of the apoptosis inhibitor Bcl-2 has been reported in
some circumstances to inhibit autophagy through its binding to beclin1 (Sinha and
Levine 2008; Maiuri et al. 2007). The involvement of p53 in the regulation of autoph-
agy seems to be complex. Indeed, the activation of p53 by nutrient deprivation or
genotoxic stress leads to the activation of autophagy through the inhibition of mTOR
or by the activation of DRAM (damage-regulated autophagy modulator) (Balaburski
et al. 2010; Crighton et al. 2006; Feng et al. 2005). However, consistent with the role
7 Role of Autophagy in Tumor Progression and Regression 119
of autophagy as tumor suppressor, the functional loss of p53 was expected to decrease
autophagy or suppress basal autophagy. The later effect seems to depend on the cyto-
plasmic, not the nuclear, pool of p53 (Tasdemir et al. 2008).
In addition to the indirect evidence described above, several direct evidences
support the tumor suppressing properties of autophagy. Indeed the autophagy exe-
cution protein Beclin1 is a haplo-insufficient tumor suppressor protein. Mono-
allelic deletion of BECLIN1 are reported in sporadic human breast and ovarian
carcinoma (Aita et al. 1999), and heterozygous deletion of BECLIN1 predisposed
mice to a variety of tumors including mammary neoplastic lesions, lung adenocar-
cinomas, hepatocellular carcinomas and B cell lymphomas (Qu et al. 2003). These
results indicate that functional autophagy may be constraining tumor initiation
(Liang et al. 1999). Similarly, homozygote deletion of ATG5 was shown to predis-
posed mice specifically to liver tumors with high penetrance (Takamura et al. 2011).
The tumor suppressive properties of autophagy have been extensively investigated.
Below we will provide some mechanistic insights into the tumor-suppressive
functions of autophagy.
7.2.1 Autophagy Inhibition Regulates Tumor Necrosis

and Inflammation
It has been reported that autophagy can modulate the inflammatory microenvironment
that play a major role in tumor development and considered as a common future of
early cancer development. Thus, experimental evidence suggest that autophagy-defi-
cient tumors displayed an increased level of necrosis and inflammation. The activation
of autophagy in tumor cells inhibits necrotic cell death which subsequently stimulates
a robust inflammatory response in vivo (Kono and Rock 2008). In addition, it has been
proposed that the impairment of both apoptosis and autophagy promotes necrotic cell
death, in vitro and in vivo, associated with an inflammatory response and an acceler-
ated tumor growth (Degenhardt et al. 2006). These results highlight that autophagy
regulates necrosis-induced cell death and inflammation. Furthermore, autophagy also
prevents necroptosis which is a form of caspase-independent cell death mediated by
cell death ligands (i.e. TNF-α and FasL) (Degterev and Yuan 2008; Shen and Codogno
2012). Indeed, autophagy is essential to overcome zVAD-induced necroptosis in L929
cells. Activation of PI3K-Akt-mTOR pathway, a well-known autophagy inhibitor
pathway, can sensitize L929 cells to zVAD-induced necroptosis, while amino-acid
and serum starvation protect these cells (Wu et al. 2009). Similarly, autophagy
prevents poly-(ADP-ribose) polymerase (PARP)-mediated cell death. Such cyto-
protective role of autophagy in PARP-mediated necrosis was illustrated by showing
that DNA damages induced by doxorubicin in fibroblasts lead to PARP-1 activation
and autophagy induction which protects cells against necrosis. Targeting autophagic
genes ATG5 or BECLIN1, sensitizes cells to doxorubicin-induced necrotic cell
death (Munoz-Gamez et al. 2009).
Autophagy is also a key process for the maintenance of intracellular ATP level
required for the secretion of lysophosphatidylcholine (LPC). Secretion of LPC is
associated with the acute phase of the inflammatory response and is involved in the
development of chronic inflammation. It has been shown that autophagy-deficient
cells fail to generate phosphatidylserine on the outer membrane surface—an impor-
tant anti-inflammatory pro-apoptotic marker. Such observation could explain why
defect in autophagy stimulates inflammatory response subsequently to insufficient
clearance of dead cells (Pierdominici et al. 2012).
Following autophagy inhibition, the accumulation of the autophagy cargo pro-
tein p62/SQSTM1 activates the pro-inflammatory transcription factor NF-kB and
the stress-responsive transcription factor NRF2, thus favoring inflammation and tis-
sue injury (Levine et al. 2011). The transcription factors NF-kB family members
regulate the expression of a broad range of genes involved in development, prolif-
eration, and survival of tumor cells. The activation of these transcription factors
leads to the regulation of inflammation and innate and adaptive immune responses
(Smale 2011). As the activation of NF-kB is mediated by the IkB kinase (IKK)
complexes, it has been reported that IKK complexes are targets for degradation by
autophagy when the heat shock protein 90 (Hsp90) function is inhibited (Xu et al.
2011). Another mechanism of regulation of NF-kB by autophagy is mediated by the
Kelch-like ECH-associated protein 1 (Keap1). Keap1 interacts with the kinase
domain of IKKβ through its C-terminal domain. This domain is also required for the
binding of Keap1 to the transcription factor NRF2, which controls the expression of
some antioxidant genes. In response to tumor necrosis factor (TNF), Keap1 nega-
tively regulates the activation of NF-kB through inhibition of the IKKβ phosphory-
lation and induction of IKKβ degradation by autophagy pathway (Fan et al. 2010).
The E3 ubiquitin ligase Ro52 is another signaling molecule that targets IKKβ for
degradation through the autophagy pathway. In response to distinct stimuli, specific
interactions of Hsp90, Keap1 and Ro52 with IKKs regulate NF-kB activity through
their ability to activate or repress the degradation of IKKs by autophagy (Trocoli
and Djavaheri-Mergny 2011). It has been suggested that the crosstalk between
NF-kB and autophagy regulates inflammasome activity leading to the modulation
of the activation of caspase-1 and subsequently the secretion of potent pro-inflam-
matory cytokines (Strowig et al. 2012). Based on the studies discussed above, it
appears that autophagy is an important modulator of cancer pathogenesis through
its ability to regulate inflammation.
7.2.2 Autophagy Prevents Oxidative Stress and Genomic

Instability
The role of autophagy in cancer suppression has been reported by several in vivo
studies (White et al. 2010). Thus, Beclin1-defective mice showed an increased sus-
ceptibility to develop cancer (Qu et al. 2003; Yue et al. 2003). This could be related
to the involvement of autophagy in the management of oxidative stress and in the
maintenance of the genomic integrity. In this regard, it has been described that
autophagy can limit DNA damage, chromosomal instability and aneuploidy
(Mathew et al. 2007). Several studies suggested that the ubiquitin- and LC3-binding
protein p62 may play a determinant role (Komatsu et al. 2007; Mathew et al. 2009).
Indeed, the inability of autophagy-deficient cells to degrade p62 lead to the aberrant
accumulation of this protein, which is sufficient to promote tumorigenesis (Mathew
et al. 2009). Thus, p62 activates the transcription factor NRF2 through the direct
inhibition of Keap1 (Komatsu et al. 2010; Lau et al. 2010). However, the role of
NRF2 in DNA damage promotion is not clearly understood so far. In addition, p62
may act as an important NF-kB modulator in tumorigenesis (Duran et al. 2008).
This study highlights that the increase in DNA damage in autophagy-deficient cells
is associated with high levels of damaged mitochondria and reactive oxygen species
(ROS), accumulation of ER chaperones and protein disulfide isomerases. DNA
alterations were suppressed by ROS scavengers, confirming the essential role of
autophagy in oxidative stress management and, subsequently, in protein quality
control (Mathew et al. 2009).
Excessive exposure to ROS alters the function of multiple cellular macromole-
cules by oxidation (e.g. nucleic acids, lipids, proteins). However, oxidative stress is
closely linked to mitochondria dysfunction. Since autophagy is the only process
allowing the mitochondrial turnover by a mechanism so-called mitophagy, prevent-
ing the accumulation of damaged mitochondria highly reduces the risk of oxidative
stress. Moreover, mitochondria produce the bulk of ATP required for vital cellular
functions (e.g. DNA replication, mitosis, transcription). In this regard, the ability of
autophagy to control proteins/organelles quality and to maintain cellular energy
homeostasis highlights its antitumorigenic activity (Jin 2006). Such a role has been
demonstrated in autophagy-defective cells, where the presence of damaged proteins
is crucial in DNA replication, mitosis or centrosome function. Moreover, autophagy
defective cells displaying defect in mitochondrial clearance and subsequently an
alteration in ATP production may also alter DNA replication or repair by affecting
the arrest of the replication forks and the generation of breakage/fusion/bridge
cycles responsible for gene amplification (Jin and White 2008). Finally, the implica-
tion of autophagy in the physiological protein turnover may also influence the
occurrence of DNA damage. Indeed, cell cycle progression is driven by the periodic
activity of proteins including Cyclin-dependent kinases (CDKs), Cyclins, CDKs
inhibitors. Therefore, it stands to reason that a deregulation in the physiological
protein turnover in autophagy-deficient cells may alter the correct sequence of the
cell cycle progression (Jin and White 2008). Taken together, it has become clear that
autophagy helps normal cells to overcome several types of stresses (e.g. metabolic,
oncogenic), that directly limits their oncogenic transformation. In contrast, such
management of cellular stresses is also observed in cancer cells, and leads in this
case to cancer promotion (Rosenfeldt and Ryan 2011).
Senescence is an irreversible cell cycle arrest associated with an active metabolism,
which can limit the proliferation of abnormal cells. In this context, autophagy is also
able to mitigate the accumulation of genomic alteration by inducing the mitotic
senescence transition. Young et al. reported an accumulation of autophagosomes in
Ras-induced IMR90 senescent fibroblasts, suggesting that autophagy is required for

tumor senescence. In addition, targeting ATG5/7 delayed the senescent phenotype,
while induction of autophagy clearly enhanced the protein turnover that contributed
to synthesis of pro-senescence cytokines (e.g. IL-6, IL-8) (Young et al. 2009). This
study suggests that autophagy not only facilitates the entry into senescence but also
reinforces the senescent phenotype of cells.
7.2.3 Autophagy Contributes to Tumor Cell Death
The role of autophagy in promoting tumor cell death has been proposed based on
the observation that apoptosis occurs concomitantly with features of autophagy
(Kroemer and Levine 2008) and that prolonged stress and progressive autophagy
can lead to cell death (Mathew and White 2007). Together with apoptosis (type I
cell death) and necrosis (type III cell death) (Schweichel and Merker 1973), autoph-
agy was first described as type II cell death. The relevance of autophagic cell death
in development has been established in lower eukaryotes and invertebrates such as
Dictyostelium discoideum and Drosophila melanogaster (Denton et al. 2009; Kosta
et al. 2004). Evidence has been reported that mammalian development does not
require autophagy, as newborn mice lacking essential autophagy genes show any
anatomical or histological defects and no impairment of the cell death (Mizushima
et al. 2008). This evidence is supported by the fact that the depletion of autophagy
genes in human or mice mammalian cells induces apoptosis rather than protects cell
against death induced by different stresses (Boya et al. 2005; Gonzalez-Polo et al.
2005). The role of autophagy in cell death induction is not clear, and needs further
investigation. However, the more convincing evidence highlighting the role of
autophagy in cell death has been reported in mammal's neuronal cells. Indeed, fol-
lowing insulin starvation, hippocampal neural stem cells undergo autophagic cell
death, while targeting autophagy by silencing ATG7 blocks this process. It is worthy
to note that autophagic cell death occurs only in cells with functional apoptosis and
is caspase-independent (Yu et al. 2008). Currently, the majority of experimental
evidence showing autophagic cell death in mammalian cells were mainly conducted
in vitro and in cells defective in apoptosis machinery. It has been shown that DAPK
(death associated protein kinase) plays an important role in the regulation of both
autophagy and apoptosis. Indeed, DAPK induces autophagy by phosphorylation of
Beclin1, and is associated with the induction of apoptosis. However this type of
DAPK-dependent autophagic death is caspase dependent, and it remains to be elu-
cidated whether DAPK-mediated cell death is a real autophagic cell death, or
whether autophagy only assists in the apoptosis execution phase (Gozuacik et al.
2008). It has been proposed that cells rather die with autophagy, and not by autoph-
agy as they showed that none of 1400 compounds, evaluated for their ability to
induce autophagic puncta and increase autophagic flux, killed tumor cells through
the induction of autophagy (Shen and Codogno 2012). Moreover a careful determi-
nation of the autophagic flux is needed to differentiate autophagic cell death from
other forms of non-apoptotic programmed-cell death, such as necroptosis. These

examples illustrate that autophagy may be involved in lethal signaling although the
role of autophagy itself in cell killing remains unclear. Thus, further studies are
required in order to define the exact role of autophagic cell death mechanism.
7.3 Autophagy Modulates the Anti-tumor Immune Response
Recently, autophagy has emerged as a new critical mechanism activated in tumor

cells in hypoxic microenvironment that mediates tumor resistance to innate and
adaptive anti-tumor immune responses. Several reports demonstrate that autophagy
activation not only enables tumor cells to survive stress conditions during cancer
development but also provides them an intrinsic resistance mechanism to escape
anti-tumor immune response.
7.3.1 Role of Autophagy in Tumor Cell Resistance to CTL-

Mediated Killing
The first evidence for such a role of autophagy was provided by Noman et al. who
demonstrated that hypoxic lung carcinoma cells can evade cytolytic T lymphocyte
(CTL)-mediated lysis through autophagy induction (Noman et al. 2011, 2012).
Indeed, inhibition of autophagy using small interfering RNA (siRNA) directed
against ATG5 or BECN1 restored tumor cells sensibility to CTL-mediated lysis
which correlated with a decrease in hypoxia-dependent induction of the phosphory-
lation of Signal Transducer and Activator of Transcription (STAT)-3. This result
allowed the prediction that blocking autophagy would inhibit pSTAT3-dependent
survival mechanism making tumor cells more susceptible to CTL attack under
hypoxia. However, considering the degradation role of autophagy, it is difficult to
perceive that autophagy is involved in the stabilization of pSTAT3 under hypoxia.
Focusing on the crosstalk between the adaptor protein p62/SQSTM1, the ubiquitin-
proteasome system (UPS) and autophagy, this study revealed that the induction of
hypoxia inducible factor (HIF)-1α has two effects in tumor cells: (i) HIF-1α triggers
the phosphorylation of Src which subsequently phosphorylates the tyrosine residue
Y705 of STAT3 (ii) HIF-1α activates autophagy by a mechanism implicating the
increased expression of BCL2/adenovirus E1B 19 kDa protein-interacting protein
(BNIP)3/BNIP3L and the dissociation of the BECN1-BCL2 (B cell lymphoma 2)
complex. Autophagy activation results in degradation of the p62 protein. Knowing
that p62 is the receptor/adaptor protein responsible for targeting pSTAT3 to the
UPS, the autophagy-dependent degradation of p62 leads to the accumulation of
pSTAT3. When autophagy is inhibited in tumor cells, the degradation of p62 is
blocked and therefore accumulates in tumor cells. This accumulation accelerates the
UPS-dependent degradation of pSTAT3 (Noman et al. 2009) (Fig. 7.1a).
Epithelial to mesenchymal transition (EMT) is a trans-differentiation process

necessary for the morphogenesis of tissue during embryonic development (Nieto
2013). While its role in cancer cell invasion, metastasis and drug resistance is well
established, recent report described that autophagy can be activated in tumor cells
undergoing EMT and that such EMT-induced autophagy represents another mecha-
nism of cancer cell resistance to CTL-mediated lysis (Akalay et al. 2013a, b). In this
study, the authors showed that the induction of EMT program by overexpression of
SNAI1 in breast cancer cells coincides with a drastic change in cell morphology and
the activation of autophagy flux most likely through the overexpression of BECN1 in
mesenchymal cells. Although the exact molecular mechanism by which the EMT
affects the expression of BECN1 remained to be addressed, several lines of evi-
dence indicate that this may be related to SNAI1- or EMT-dependent repression of
microRNA(s) involved in modulation of BECN1 expression (Siemens et al. 2011;
Yu et al. 2012). This result extended the role of SNAI1 as a regulator of autophagy
and paves the way to investigate the functional role of EMT-induced autophagy in
tumor cells. In this context, results described in this study showed that targeting
BECN1 in mesenchymal cells was sufficient to restore CTL-mediated tumor cell
lysis, without affecting the mesenchymal morphology and the expression of EMT
markers. This finding implies that autophagy is a downstream target of the EMT
program in breast cancer cells. Overall, this study suggests that EMT-induced
autophagy is a novel mechanism by which tumor cells regulate CTL reactivity and
impede their cytotoxic activity, and further points to the complex relationship
between the tumor and the immune system (Fig. 7.1b).
7.3.2 Role of Autophagy in Tumor Cell Resistance

to NK-Mediated Killing
It is now well established that several resistance mechanisms are regulated in tumor
cells to escape immune surveillance in hypoxic tumor microenvironment. Recent
evidence described how tumor cells can escape natural killer (NK)-mediated
immune surveillance by activating autophagy under hypoxia (Baginska et al. 2013;
Viry et al. 2014). Indeed, NK cells recognize and kill their targets by several mecha-
nisms including the release of cytotoxic granules containing perforin (PRF1) and
serine protease granzyme B (GZMB). It has been recently proposed that PRF1 and
GZMB enter target cells by endocytosis and traffic to large endosomes named
“gigantosomes” (Thiery et al. 2010, 2011). Subsequently, PRF1 is involved in the
formation of pores in the membrane of the “gigantosome”, leading to the gradual
release of GZMB and the initiation of apoptotic cell death. The formation of amphi-
somes following the fusion between autophagic vacuoles and early endosomes
appears to be necessary in some cases for the generation of autolysosomes. In this
report (Baginska et al. 2013), the authors described that the pro-apoptotic protein
GZMB is selectively degraded upon autophagy activation in hypoxic cells, thereby
blocking NK-mediated target cell apoptosis (Fig. 7.1c). In line with this, they
showed that GZMB is detected in autophagosomes and provided evidence that
a b c
CTL CTL NK cell
Extracellular GZMB
space
Cytosol Tumor cell
UPS
degradation of Autophagy
P p62 pSTAT3 by p62 induction GZMB- GZMB
STAT3 p62
mediated degradation
lysis through
autophagy
BECN1
STAT3 p62 BECN1
BECN1
degradation BECN1
Autophagy
through
induction
autophagy
?
Autophagy Epithelial EMT Mesenchymal HIF-1α

induction
HIF-1α BECN1
Hypoxia or EMT-induced autophagy
Fig. 7.1 Autophagy activation in tumor cell acts as an intrinsic resistance mechanism against anti-
tumor immune response. The tumor microenvironment and/or EMT program activate autophagy in
target cells. The induction of autophagy operates as a cell resistance mechanism leading to tumor
escape from CTL- or NK-mediated lysis. (a) Hypoxic stress leads to the accumulation of HIF-1α.
HIF-1α activates autophagy and simultaneously increases the phosphorylation level of STAT3 at the
Tyr705 residue. As an autophagic substrate, p62/SQSTM1 is degraded in the autophagosomes fol-
lowing their fusion with lysosomes. As p62 is involved in targeting pSTAT3 to the UPS, its degrada-
tion leads to the accumulation of pSTAT3 in cells and such accumulation constitutes a cell survival
mechanism. In autophagy-defective cells, p62 is no longer degraded, and its accumulation acceler-
ates the UPS-dependent degradation of pSTAT3 and thereby restores CTL-mediated tumor cell lysis.
(b) The acquisition of an EMT phenotype confers resistance to CTL-mediated lysis through autoph-
agy induction. The increase in mesenchymal markers following the activation of EMT program leads
to the up-regulation of BECN1 by a yet undefined mechanism. Such upregulation induces autophagy
and impairs CTL-mediated tumor cell lysis. In mesenchymal cells, targeting BECN1 is sufficient to
restore CTL-mediated lysis. (c) Following the recognition of their targets, NK cells secrete cytotoxic
granules containing PRF1, GZMB, and other hydrolytic enzymes that enter target cells, traffic to
enlarged endosomes, and initiate tumor cell death. Under hypoxia, excessive autophagy in target cells
leads to the fusion of autophagosomes with vesicles containing GZMB leading to its specific degradation
by autophagy, thereby inhibiting NK-mediated lysis. Targeting autophagy prevents the degradation
of GZMB and thereby restores NK-mediated tumor cell killing
GZMB level is significantly decreased in hypoxic compared to normoxic target

cells. Furthermore, targeting autophagy genetically or inhibiting lysosomal hydro-
lases by pharmacological approaches restored GZMB level which ultimately leads
to the recovery of hypoxic cells lysis by NK cells in vitro and in vivo. Based on
these results, the authors stated that tumor regression can be achieved by inhibiting
autophagy in hypoxic cancer cells, thus enabling their NK-mediated lysis (Baginska
et al. 2013; Viry et al. 2014).
Overall, studies described above underline the activation of autophagy as a key
mechanism in tumor escape from immune cell attack within the tumor microenvi-
ronment. However, an important issue that arises from these studies is whether
hypoxia is the only microenvironmental factor involved in the induction of autoph-
agy in tumor cells. An interesting recent report provided strong evidence that lym-
phoid effectors not only provide lytic signals but also promote autophagy in the
remaining target cells, a process called cell-mediated autophagy (C-MA) (Buchser
et al. 2012). Thus, C-MA has been reported in different human epithelial tumors
after interaction with immune cells at high ratio of effectors to targets. Importantly,
it has been showed that C-MA not only acts as a mechanism of resistance to immune
cell-mediated lysis but also limits the cytotoxic activity of stress factors such as
γ-radiation (Buchser et al. 2012).
These studies highlight that the activation of autophagy plays a critical role in
tumor cell escape from both adaptive and innate immunity. Therefore, targeting
autophagy has been proposed to improve CTL- and NK-based immunotherapy in
experimental mouse model (Baginska et al. 2013; Noman et al. 2011, 2012). Intense
research efforts are currently focusing on the development of autophagy inhibitors
that could improve tumor immunotherapy.
7.4 Targeting Autophagy in Cancer Therapy
Currently, there are several clinical trials registered in the National Cancer Institute
(www.cancer.gov/clinicaltrials) exploring anti-autophagy strategies in a variety of
human cancers. Most of these trials are ongoing, with minimal published results avail-
able, and nearly all use Hydroxychloroquine (HCQ) or Chloroquine (CQ). It is worthy
to note that CQ or HCQ are lysosomotropic agents that act at the level of the lysosome
by inhibiting acidification, thereby impairing autophagosome degradation. These
clinical trials were initiated based on the fact that autophagy is induced as a survival
mechanism in a variety of tumor cells and preclinical models by several types of che-
motherapeutic agents. Because only a subpopulation of tumor cells undergo autoph-
agy, it is unlikely that autophagy inhibitors are used in cancer therapy as single agent.
Indeed, most of these clinical trials used HCQ in combination with other anti-cancer
therapies. While these preclinical data are generally supportive of incorporating anti-
autophagy therapies in cancer treatment trials, it has been observed, in some circum-
stances, that inhibition of autophagy decreases therapeutic efficacy. Understanding
the circumstances in which autophagy inhibition impairs the therapeutic effect will be
of great importance. Importantly, while CQ and HCQ are effective inhibitors of

autophagy in vitro, whether they will do so at doses used in current clinical trials is
still uncertain. An important issue related to the use of these autophagy inhibitors
concerns the micromolar concentration that is required to inhibit autophagy and show
anti-tumor efficacy in preclinical models. While this is theoretically achievable at tol-
erated doses after prolonged dosing, it should be better optimized in clinic (Tett et al.
1993; Munster et al. 2002). Trials combining HCQ as neoadjuvant treatment will pro-
vide tumor tissues available for analysis both before and after HCQ treatment.
However, the effectiveness of HCQ in the inhibition of autophagy still prove difficult,
as HCQ is often combined with other therapies (chemotherapy and radiotherapy) that
are also known to modulate autophagy. Alternative biomarkers to predict for autoph-
agy activation as well as autophagy dependence are currently an area of intense inves-
tigation (Kimmelman 2011). A recently reported phase I trial of HCQ in combination
with adjuvant temozolomide and radiation in patients with glioblastoma found that
the maximum tolerated dose of HCQ was 600 mg per day, and this dose achieved
concentrations of HCQ required for autophagy inhibition in preclinical studies. In this
trial, investigators observed a dose-dependent inhibition of autophagy, as indicated by
increases in autophagic vesicles (revealed by electron microscopy), and detected ele-
vations in LC3-II in peripheral blood mononuclear cells. In addition, in a phase I trial
of 2-deoxyglucose, an agent that blocks glucose metabolism, autophagy occurred in
association with a reduction in p62/SQSTM1 in peripheral blood mononuclear cells
(Stein et al. 2010). These data suggest the potential interest of such biomarkers in the
evaluation of autophagy modulation during therapy and in the correlation with treat-
ment outcome.
CQ inhibits the last step of autophagy at the level of the lysosome, thereby
impacting lysosomal function. Therefore, its effects are not entirely specific to
autophagy. Currently, there is a great deal of interest in developing new inhibitors of
autophagy. In this regards, and given the complexity of the autophagic process,
multiple proteins involved in this process could be good candidates for developing
others autophagy inhibitors. It is likely that kinases would be prime candidates for
inhibition such as Vps34, a class III PI3K, which has a critical early role in autopha-
gosome development. This is particularly attractive, as there has been significant
success in designing effective class I PI3K inhibitors (Wong et al. 2010). However,
one potential issue which needs to be considered is that Vps34 has roles in other
aspects of endosome trafficking, and this may lead to unwanted effects and toxicity
(Backer 2008). The mammalian orthologs of yeast ATG1, ULK1/2, which acts
downstream from AMPK and the TOR complex, have been recently shown as criti-
cal proteins for autophagy activation (Hara et al. 2008; Egan et al. 2011). Other
potential targets for autophagy inhibitors would be LC3 proteases, such as ATG4b,
which are necessary for LC3 processing. However, whichever approach is taken, the
delicate balance between potency and toxicity must be determined to achieve a
clinical success. While there are still uncertainties of how autophagy inhibition will
fare as an anti-cancer therapy, the preclinical data generally support this approach.
The current clinical trials will hopefully provide insight into whether this will be a
viable therapeutic paradigm (Kimmelman 2011).
Acknowledgments A part of research results presented in this chapter was generated at

Luxembourg Institute of Health and Gustave Roussy Cancer Center. Research projects related to
these results were funded by grants from Luxembourg Ministry of Culture, Higher Education and
Research (Grant LHCE 2013 11 05); Fondation Cancer Luxembourg; FNRS Televie (7.4517.14;
7.4571.15 and 7.4664.15) and “Equipe labélisée la ligue contre le cancer”.
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ERRATUM TO
Adult and Cancer Stem Cells: Perspectives

on Autophagic Fate Determinations
and Molecular Intervention
© Springer International Publishing Switzerland 2016

DOI 10.1007/978-3-319-42740-9_8
Due to a typesetting error, the author name Richard Calderone was wrong in the
initial version published online and in print. The correct name, as appears now
in both the print and online version of the book, is:
The updated online version of the original chapter can be found at

http://dx.doi.org/10.1007/978-3-319-42740-9_6
K.G. Chen (*)

NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke,
National Institutes of Health, Bethesda, MD 20892, USA
e-mail: kgc26@georgetown.edu
R. Calderone
© Springer International Publishing Switzerland 2016 E1

Index
A canonical autophagy, 5
Acute myeloid leukemia (AML) cell lines, 50 chemosensitization effects, 7
5’-Adenosine monophosphate-activated CQ, 9
protein kinase (AMPK), 103 cytokine IL6, 7
Aithromycin, 46 direct interference, 4
AKT, 88 EGFR signaling, 8
Akt-mediated phosphorylation, 93 genetic inhibition, 6
Akt-mTOR pathway, 91 human tumor cells, 6
Aldehyde dehydrogenase 1-positive IL6, 7
(ALDH1+) cells, 109–110 autophagy-dependent cells, 7
AMP/ATP, 104 autophagy-dependent secretion, 7
AMP-activated protein kinase (AMPK), 41, autophagy-independent cells, 7
92, 127 inhibition, 5
AMPK activators, 49 kinase inhibitors, 8
AMP-responsive protein kinase (AMPK), 61 KRAS, 5
Anoikis, 41 KRAS mutant, 6
Anti-aging intervention, 103 KRAS mutation, 6
Anti-apoptotic Bcl-2 proteins, 51 KRAS-driven pancreas tumors, 6
Anti-autophagy Based Cancer Therapy, 110 mouse studies, 5
Anti-cancer therapy, 86 mTOR inhibitors, 7
PI3K (see Phospoinositide 3-kinases (PI3K)) myriad ways, 8
Anti-cancer treatment, 7, 39 neurodegeneration, 5
acquired resistance, 8 oncocytoma, 5
amino acid starvation, 7 p53, 6
autophagy inhibitor, 8 pancreas tumors, 6
autophagy-dependent BRAF mutant versus pharmacological autophagy inhibitors, 5
autophagy-independent BRAF RAS mutation, 6
wild-type brain cancer cells, 8 RAS pathway driven tumors, 6
BRAF inhibitor, 8 RAS-mutant cell lines, 6
BRAF mutant, 6 shRNAs, 7
BRAF mutant tumors, 8 small-molecule regulators (see Small-
BRAF mutation, 5 molecule regulators)
breast cancer cell lines, 6 STAT3 signaling, 7
breast cells, 6 telomerase, 6

Current Cancer Research, DOI 10.1007/978-3-319-42740-9
134 Index
Anti-cancer treatment (cont.) dysfunctional proteins and organelles, 39

therapeutic intervention, 5 exogenous pro-death stimuli, 4
vemurafenib, 8, 9 genomic mutation, 29–30
Anti-estrogen tamoxifen, 4 HCQ, 4
Antihelminthic drug, 46 and immune response, 28–29
Anti-proliferative activity, 49 inhibition, 4
Anti-tumor immune response, 123–126 anti-tumor effect (see Anti-cancer
Aplasia Ras homolog member I (ARHI), 42 treatment, autophagy)
Apogossypolone, 51 in cancer therapy, 9–10
Apoptosis, 75 inhibitors, 2
apoptosis-deficient cells, 4 lysosome, 11
ATG12 regulates, 3 macroautophagy, 1
cancer cells vs. tissues, 23–24 mechanisms, 3
inhibit cell death, 4 microautophagy, 2
Arsenic trioxide (As2O3), 51 and microRNA connections in cancer
Ataxia telangiectasia mutated (ATM) gene, 74 biology, 66–69
ATG genes, 2–3, 102 microtubule-targeting drugs, 2
ATG proteins, 61 Myc-driven lymphoma model, 4
Atg14, 90 non-selective autophagy, 3
ATG4b, 46, 127 non-selective process, 3
ATG5, 24 physiological signals, 4
ATG6, 18 PI3K (see Phospoinositide 3-kinases (PI3K))
ATG7, 122 pro- and anti-tumor effects, 2
in adult mice, 5 pro-apoptotic stimuli, 4
inhibits autophagy, 2 process, 40–41
liver-specific deletion, 9 protective effect, 4
ATP-binding cassette (ABC) transporters, 111 proteins, 3
Autophagic cell fates, 104–105 radiotherapies, 40
Autophagosome formation, 30 regulation by ATG genes, 61–62
Autophagosomes, 1–3, 8, 41, 100 regulators, 2, 3
Autophagy, 9–10, 18–19, 39, 41–42, 59, 86, 118 role in cancer therapy, 69
anti-cancer treatments, 2, 11 small-molecule regulators
apoptosis, 3, 23–24 (see Small-molecule regulators)
ATG genes, 2–3 starvation, 40
autophagosomes, 1, 3 in tumor cell resistance to CTL-mediated
in cancer killing, 123–124
tumor promotion, 41–42 in tumor cell resistance to NK-mediated
tumor suppression, 42 killing, 124–126
in cancer cells vs. tissues (see Cancer tumor suppressor and promoter, 118
cells vs. tissues) types, 1
cancer chemotherapy response, 4 Autophagy inducers
catabolic mechanism, 52 AMPK activators, 49
and cell cycle control, 26–28 As2O3, 51
cellular homeostasis, 39 BH3 mimetics, 51
characteristics, 52 BIX-01294, 51
chemotherapies, 40 calcium homeostasis, 49–50
Class III PI3K complex, 40 carcinogens, 51
clinical studies, 2 GEM, 52
clinical trials, 4, 11 HDAC, 50
CMA, 2 inorganic arsenic, 51
CQ, 4 Lapatinib, 52
degradation, 3 mTOR, 46–49
description, 1 NaAsO2, 51
disruption, 52 salinomycin, 52
by diverse stresses, 4 small-molecule inducers, 46
Index 135
tumor suppression, 46 biological processes, 32

UA, 52 cancer cell line biology and actual cancer
Autophagy inhibitor biology, 19
antihelminthic drug, 46 characterized biological pathways, 31
ATG4B, 46 co-expression analyses, 31
azithromycin, 46 comparative analysis, gene-expression, 31
Class III PI3K, 43 energy metabolism, 31
clinical trials, 43 environments, 31
lysosomal homeostasis, 43–45 Fenton reaction, 32
macrolide antibiotic clarithromycin and gene co-expression analysis, 35–36
aithromycin, 46 gene differential expression, 35
pyrvinium, 46 gene expression of autophagy, 19–22
small-molecule inhibitors, 44–45 genome analyses, 18–19
tumor promotion mechanism, 43 genome integrity, 18
Autophagy-induced cell death (ACD), 104–105 genomic mutation, 29–30
Autophagy-induction genes, 20 hypoxic regions, 18
Autophagy-related proteins (ATG), 118 information of datasets, 34–35
Azithromycin, 46 metabolic stress, 18
Michaelis-Menton equation, 32
necrotic cell death, 18
B novel biological processes, 24–29
Bafilomycin A1, 45 nutrient deprivation, 18, 22–23
Basal autophagy, 18 pathway enrichment analysis, 35
Bcl-2 inhibitor gossypol, 51 RMA method, 35
Bcl-2 protein family, 62 RNA-seq and genomic data, 35
Beclin 1 (BECN1), 3, 8, 9, 18, 89, 118, RSCD, 32–34, 36
124, 125 RSEM method, 35
Beclin 1-Vps34-Vps15, 90 TCGA and GEO databases, 35
BECLIN1, 119 TCGA cancer types, 34
BECN1-BCL2 (B cell lymphoma 2) TCGA database, 19
complex, 123 total mutation rate, 36
BH3 binding genes, 24 transcriptomic and genomic data, 19
BH3 mimetics, 51 tumor-suppression roles, 30
BH3-mimetic GX15-070, 50 Cancer progression, 118
BIX-01294, 51 Cancer stem cells, 101, 107
BRAF Cancer therapy, 9, 10
autophagy inhibitors, 8 autophagy inhibition
autophagy-dependence, 5 autonomous effect, 10
autophagy-dependent vs. autophagy- BECN1, 9
independent, 8 BRCA1, 9
brain tumors, 5 chemotherapy-induced immunogenic
and KRAS, 5, 6 cell death, 10
mutant brain tumor, 9 chromosome stability, 9
BRAF inhibitor, 8 DAMP molecule HMGB1, 10
Breast cancer, 70 immunogenic tumor cell killing, 10
Breast cancer cell lines, 6 Interleukin 2 immunotherapy, 10
liver-specific deletion of ATG7, 9
manipulation, 10
C mechanism, 10
Calcium (Ca2+) homeostasis, 49–50 mosaic deletion of ATG5, 9
Cancer cells vs. tissues NK cell, 10
apoptosis, 23–24 NK cells, 10
ATP production, 19 targeting autophagy in, 126–127
basal autophagy, 18 Cannibalistic cell death, 60, 63
beclin1 gene, 18 Canonical autophagy, 5
136 Index
Carboxypeptidase Y (CPY), 89 LY294002, 88

Carcinogens, 51 NRBF2, 90
Cathepsins, 45 phospho-lipids, 89
Cell cycle control, 26–28 rat hepatocytes, 88
and autophagy Rubicon, 90
cell-cycle regulatory genes, 28 UVRAG-containing Beclin 1-Vps34-
co-expression networks, 26, 27 Vps15, 90
cyclin dependent kinases, 27, 28 Vps15, 90
cyclins, 27, 28 Vps30/Atg6, 89
DNA polymerases, 28 Vps34, 89, 90
G1-S transition genes, 26 wortmaninn, 88
G2-M transition genes, 26 classification, 87
LM cancers, 27 growth factor receptors, 87
lysosome and proteasome, 28 in vivo, 86
macro-autophagy, 26 isoforms, 91
negative correlation, 26 mediate growth factor, 88
suppression of cytokinesis, 26 mTOR complex, 88
up-regulated G1-S transition genes in oncogenic Ras, 88
HL cancers, 28 p110 catalytic subunit possesses, 87
Cell death p110-p85 interaction, 87
caspase-independent, 45 p110α activation mutations, 88
mechanism, 41 p110α exist, 87
tumor promotion, 41–42 p85 regulatory subunits, 87
tumor suppression, 42 PH domain-containing proteins, 88
types, 49 PtdIns(4,5)P2, 86
Cell survival mechanism, 48 RTK activation, 87
Cell-mediated autophagy (C-MA), 126 RTKs and GPCRs, 86
Cell-surface glycan, 29 SH2 domains, 87
Cellular homeostasis, 39, 60–61, 118 and signaling pathways, 88
Chaperone-mediated autophagy (CMA), 2, 20 targeted anti-cancer therapies, 88
Chemosensitization effects tumor suppressor PTEN, 88
autophagy inhibition, 7 Class II PI3-kinases
Chemosensitize tumor cells, 4 members, 87
Chemotherapies, 40 Class III PI3K complex, 40
Chemotherapy-induced autophagy, 106 Class III PI3K inhibitors, 43
Chloroquine (CQ), 4, 7–10, 43 Class III PI3-kinases
FDA-approved drug, 94 member, 87
HCQ, 94 Vps34, 87
Clarithromycin, 46 Colorectal Cancer, 74
Class I PI3-kinase-Akt-mTOR pathway, 93 Crohn's disease, 101
Class I PI3-kinases, 88–90 CTL-mediated tumor cell, 125
AKT, 88 Cyclic tetrapeptides, 50
and cancer, 88 Cyclin-dependent kinases (CDKs), 121
catalytic subunit and regulatory subunit, 86 Cytokine IL6, 7
and class III Cytolytic T lymphocyte (CTL), 123
3-methyladenine, 88, 89 Cytoplasm-to-vacuole targeting pathway
Ambra1, 90 (CVT), 20
Atg14, 90
autophagy regulation, 89
Bcl-2, 90 D
Beclin 1, 89 Damage Associated Molecular Pattern
Beclin 1-Vps34-Vps15, 90 (DAMP) molecule HMGB1, 10
Bif-1, 90 DAPK (death associated protein kinase), 122
inhibit and promotes autophagy, 89 DAPK genes, 24
Index 137
Decapping complex and activators, 66 up-regulated lysosome, 22

Dictyostelium discoideum, 122 up-regulated macro-autophagy, 22
Diverse stresses, 4 Genomic mutation
Down-regulated lysosome, 22 and autophagy, 29–30
Down-regulated macro-autophagy, 22 Gigantosomes, 124
DRAM (damage-regulated autophagy Glioma, 75
modulator), 118 Glucose, 92
Drosophila, 101 Granzyme B (GZMB), 124, 125
Drosophila melanogaster, 122 Growth factor receptors, 86
Dysregulation of autophagy, 60, 62
H
E HBV, 29
Ectoderm, 106 HCQ, 127
EIF2C complex, 64 Hepatocellular carcinoma,
Embryonic stem cells (ESCs), 106 74–75
Epidermal growth factor receptor (EGFR), 8, 74 Hereditary spastic paraparesis, 101
Epithelial to mesenchymal transition High lysosome (HL), 20
(EMT), 124 H. Pylori, 29
Everolimus, 48 Histone deacetylase (HDAC) inhibitors, 50
Extracellular matrix (ECM), 41 Hydroxamic acids, 50
Hydroxy-chloroquine (HCQ), 4, 43, 126
cancer treatment, 94
F clinical trials, 94
FDR method, 35 Lys05, 94
Fenton reactions, 22–24, 29, 32 pre-clinical studies, 94
in mitochondria, 22 Hypoxia, 104
FK228, 50 Hypoxia inducible factor (HIF)-1α, 123
FOXO family transcription factors, 88 Hypoxic lung carcinoma cells, 123
FOXO3A, 108 Hypoxic stress, 125
G I
G protein-coupled receptor (GPCR), 86 Idarubicin, 49
G1-S transition genes, 26 IkB kinase (IKK) complexes, 120
G2-M transition genes, 26 IL6
Gemcitabine (GEM), 52 autophagy-dependent secretion, 7
Gene co-expression analysis, cytokine, 7
35–36 Immune response, 28, 29
Gene expression in disease tissues and autophagy
cancer types, 20 autophagosome formation and
cancer-prone/cancer-independent, 19 maturation, 28
chaperone-mediated autophagy, 20 cancer-prone inflammation, 29
disease types, 20, 21 CD markers, 29
down-regulated lysosome, 22 cell types, 29
down-regulated macro-autophagy, 22 cell-surface glycan, 29
HL, 20 chemokine ligands, 29
LM, 20 chemokine receptors, 29
lysosome pathway, 20 co-expression modules, 28, 29
macro-autophagy, 20 down vs. up-regulation, 29
micro-autophagy, 20 down-regulated autophagy genes, 29
pathway enrichment analyses, 20, 21 down-regulated genes, 28
proteasome genes, 20 H. Pylori, 29
up-/down-regulated genes, 19 HBV, 29
138 Index
Immune response (cont.) M

interleukin receptors, 29 Macro-autophagy, 1, 20, 60
interleukins, 29 Mammalian target of rapamycin (mTOR),
LM and HL cancers, 29 46–49, 88
LM cancers, 29 activation of ULK1, 46
up-regulated lysosome genes, 29 anabolic metabolism, 46
Immunity-related GTPase family M gene anti-proliferative activity, 49
(IRGM), 62 cell survival mechanism, 48
Implication in human diseases, 62–63 description, 46
Induced pluripotent stem cells (iPSCs), and Everolimus, 48
106, 107 Idarubicin, 49
Inner cell mass (ICM), 106 Itraconazole, 49
Interleukin 2 immunotherapy, 10 master regulator, cellular metabolism, 46
Isoform-specific PI3-kinase inhibitors, 93 nutrient and growth factors, 46
Itraconazole, 49 radio-resistant cancer cells, 48
rapamycin-induced autophagy, 48
Mammospheres, 109
K Mann-Whitney test, 35
Kelch-like ECH-associated protein 1 Matrine, 45
(Keap1), 120 Melano-autophagosomes, 100
KRAS Metastatic cancer cells, 111
and BRAF, 5, 6 Metformin, 49
mutant lung tumors, 5 3-Methyladenine (3-MA), 43, 44, 49–51
pancreas cancer, 6 Michaelis-Menton equation, 23, 32
Micro-autophagy, 2, 20, 61
Microtubule-targeting drugs, 2
L miRNA
Lapatinib, 52 based cancer therapeutics, 66
LC3-II, 41 biogenesis and function, 64–65
Lipid PtdIns(3)P, 86 in cancers, 66
Lipophagy, 3 in human genome, 63–66
LM cancers, 23 regulation by autophagy, 69
Low macro-autophagy (LM), 20 regulation of messenger, 65–66
Lucanthone, 45 role in autophagy, 67
Lung Cancer, 74 role in cancer therapy, 69
LY294002, 43, 44, 50 tumor suppressive, 68–69
Lysophosphatidylcholine (LPC), 120 Mitochondria-associated ER membrane, 90
Lysosomal storage disorders, 101 Mitogen-activated protein kinases (MAPKs),
Lysosome 61, 65
antitumor activity, 45 Mitophagy, 3, 100, 121
autophagosomes, 43 cancer therapeutics targeting, 110–112
Bafilomycin A1, 45 functions, 102
Cathepsins, 45 inducers and energetic sensors, 103–106
CQ, 43 role in adult stem cells, 108
HCQ, 43 role in cancer stem cells, 109
Lucanthone, 45 role in pluripotent stem cells, 107, 108
Matrine, 45 stem cells and, 106–110
membrane-bound cell organelle, 43 MsigDB database, 35
Thymoquinone, 45 mTOR
Vacuolin-1, 45 activators, 92
Lysosome degradation pathways, 20 amino acid deprivation inhibits, 92
Lysosome genes, 20 inhibitors, 7, 43
Lysosome pathway, 20 mTORC1 inhibition, 41
Index 139
mTOR-mediated endoplasmic reticulum (ER) P

stress, 49 P110α, 86–88, 91, 93, 94
Multidrug resistance (MDR) inhibitor, 110 P110β, 87, 91, 93, 94
Multipotent adult stem cells, 107 Paclitaxel, 2
Murine embryonic fibroblasts (MEFs), 52 Pan-Class I PI3-kinase inhibitors, 94
Mutual Rank (MR) based method, 24 Pancreas tumors, 6
Myc-driven lymphoma model, 4 Pancreatic cancer, 75
pan-PI3-kinase inhibitor, 86
Parkinson's disease, 101
N Pearson correlation, 36
Natural killer (NK) cell, 10 Pharmacological autophagy inhibitors, 5
Necrotic cell death, 18 Phosphatase and tensin homolog (PTEN), 67
Neurodegeneration, 5 Phospoinositide 3-kinases (PI3K)
Nigella sativa, 45 class I, 87–88
Nilotinib, 49 class I and III, 88–90
Non-canonical autophagy, 103 classes, 86–87
Non-small-cell lung cancer (NSCLC), 74 genetically modified mice, 90–91
Novel biological processes GPCR, 86
autophagy- and lysosome-centric growth factor receptors, 86
modules, 26 inhibitors, 43
and cell cycle control, 26–28 inhibitors in targeting cancer and
co-expressed gene modules, 24 autophagy, 93–94
co-expression modules, 24 isoforms, 86
gene co-expression networks, 24 lipid PtdIns(3)P, 86
immune response, 28–29 nutrient and growth factor signals, 91–93
MR based method, 24 p110α, 86
non-autophagy genes, 24 research, 86
rank based statistic, 24 PKB, 88
Nutrient deprivation Poly-(ADP-ribose) polymerase (PARP)-
ATPs, 22 mediated cell death, 119
cancer tissues and cell line experiments, 23 Pro-autophagic Cell Death Based Cancer
cell-line studies, 22 Therapy, 111
down-regulated autophagosome-formation Progenitors, 109
genes, 23 Programmed cell death (PCD), 42
Fenton reactions, 22, 23 Prostate cancer, 70
gene-expression data, 22 Proteasome genes, 20
HL cancer, 23 PtdIns(4)P, 86
LM cancer, 23 PtdIns(5)P, 86
metabolomic studies of cancer tissues, 22 PTEN induced putative kinase 1 (PINK1), 102
Michaelis-Menton equation, 23 Pyrvinium, 46
nutrient depletion-induced macro-
autophagy, 23
up-regulated lysosome-degradation R
pathway, 23 Rab5 GAP, 91
Radiotherapies, 40
Rank-based gene co-expression module
O extraction method, 35
Obatoclax (GX15-070), 51 Rat hepatocytes, 88
Oncocytoma, 5 Ratio of Significant Conditional Dependence
Oncogenic miRNA (Oncomir), 67–68 (RSCD), 33
Ovarian cancer, 73 average, 34
Oxidative stress, 121 biological processes, 33
Oxidative stress inducers, 103 definition, 32, 36
140 Index
Ratio of Significant Conditional Dependence TOR complex, 127

(RSCD) (cont.) Total mutation rate, 36
distribution, 33 Tricarboxylic acid (TCA), 104–105
Fenton reaction, 34 Tuberous sclerosis complex (TSC), 88
high values, 33 Tumor development, 69
histograms, 33 Tumor necrosis factor (TNF), 120
Pearson correlation, 36 Tumor promoter or suppressor, 70
values, 33 Tumor promotion, 41–42
RB1-Inducible Coiled-Coil 1 (RB1CC1), 108 Tumor regression mechanism, autophagy as,
Reactive oxygen species (ROS), 100 118–123
Receptor tyrosine kinases (RTKs), 86 contributes to tumor cell death, 122–123
Regulatory proteins, 60–63 prevents oxidative stress and genomic
Renal cell carcinoma, 74 instability, 120–122
RMA method, 35 tumor necrosis and inflammation,
RNA-induced silencing complex (RISC), 65 119–120
Rottlerin, 49 Tumor suppression, 42
RSEM method, 35 Tumor suppressor and promoter, 118
Rubicon, 90 Tumorigenesis, 41
Type 2 diabetes, 51
S
Saccharomyces cerevisiae, 101 U
Saikosaponin-d, 50 Ubiquitin-proteasome system (UPS), 123
Salinomycin, 52 ULK complex, 40
SAR405, 43, 44 ULK1 acetylation, 93
Short-chain fatty acids, 50 Unc-51-like autophagy activating kinase 1
shRNAs, 7 (ULK1), 104
Signal Transducer and Activator of Unfolded protein response (UPR) activation, 49
Transcription (STAT)-3, 123 Ursolic acid (UA), 52
Skeletal stem cells, 107 US Food and Drug Administration (FDA), 93
Slow-cycling cells, 110 UVRAG, 90
Small interfering RNA (siRNA), 123 UVRAG-containing Beclin 1-Vps34-Vps15, 90
Small-molecule regulators
autophagy inducers, 46–52
autophagy inhibitors, 43–46 V
Sodium arsenite (NaAsO2), 51 Vacuolin-1, 45
Sodium butyrate, 50 Valproic acid, 50
Somatic stem cells, 107 V-ATPase, 45
Sophora flavescens, 45 Vemurafenib, 8, 9
SQSTM1/p62-like receptors (SLRs), 62 Verapamil, 50
ßVps30/Atg6, 89 Vici syndrome, 101
Starvation, 40 Vorinostat, 50
STAT3 signaling, 7 Vps15, 90
Static encephalopathy of childhood with Vps34, 43, 89, 90
neural degeneration in adulthood AMPK, 92
(SENDA), 101 Atg6, 89
Stem cells-based autophagy, 110 Atg14, 90
Stress granules, 65 Atg14-containing Vps34 complex I, 92
Beclin 1, 90, 93
Class III PI3-kinase, 91
T complex II, 90
TCGA cancer types, 34 complex with Vps15, 90
TCGA database, 19 CPY, 89
Telomerase, 6 direct evidence, 91
Thymoquinone, 45 drug candidates, 94
Index 141
genetic ablation, 91 W
genetic evidence, 89 Warburg effect, 103
GTP-bond active Rab5 interacts, 91 Wortmannin, 43, 44
immune cells, 91
knockout cells, 91
p110β, 91 X
phosphorylates PtdIns, 87 Xenophagy, 3
and Rab5-GTP, 93
regulates autophagy, 89
side effects, 94 Y
UVRAG, 90, 92 Yessotoxin, 50

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Current Cancer Research

More information about this series at http://www.springer.com/series/7892

ISSN 2199-2584 ISSN 2199-2592 (electronic)

Library of Congress Control Number: 2016949552

© Springer International Publishing Switzerland 2016

Printed on acid-free paper

This Springer imprint is published by Springer Nature

discussion on the roles of autophagy in currently available cancer treatments; (3) it

Hershey, PA, USA Jin-Ming Yang

1 Autophagy as a Therapeutic Target in Cancer ...................................... 1

Erratum to: Adult and Cancer Stem Cells: Perspectives

Index ................................................................................................................. 133

Jenny Mae Samson and Andrew Thorburn

Abstract Autophagy is the process by which cellular material is delivered to the

Keywords Autophagy • Apoptosis • Cancer therapy • ATG7 • BRAF • KRAS

J.M. Samson • A. Thorburn (*)

© Springer International Publishing Switzerland 2016 1

not involve autophagosomes have been characterized: chaperone-mediated autoph-

trials mentioned above use lysosome-targeted pharmacological agents to target

1.1 Inhibiting Autophagy on Its Own for Anti-cancer

1.2 Inhibiting Autophagy to Make Other Treatments More

where such an effect—adding an autophagy inhibitor reverses resistance to the

1.3 Potential Reasons Not to Inhibit Autophagy in Cancer

The previous discussion argues that autophagy inhibition may be worthwhile as an

Chi Zhang, Tao Sheng, Sha Cao, Samira Issa-Boube, Tongyu Tang,

C. Zhang • T. Sheng • S. Cao • S. Issa-Boube

© Springer International Publishing Switzerland 2016 17

­ icro-­autophagy and chaperon-mediated autophagy; (ii) co-reduction in autophagy

Autophagy is a cellular survival process under nutrient deprivation and metabolic

2.2 Gene Expression of Autophagy in Disease Tissues

We have conducted differential gene-expression analyses over a total of 6317 dis-

Three types of autophagy have been defined, namely macro-autophagy, micro-­

Down-regulated macro-autophagy and lysosome are also observed in four types

2.3 Nutrient Deprivation Is Unlikely in Cancer Tissue

It has been repeatedly observed that macro-autophagy can be induced in cancer

2.4 Autophagy and Apoptosis

co-­expression relations with other autophagy and apoptotic genes to elucidate

2.5 Novel Biological Processes Related to Autophagy

Table 2.2 Information of the data analyzed in this chapter

have also conducted a similar analysis but on co-expression modules enriched by

2.5.1 Autophagy and Cell Cycle Control

2.5.2 Autophagy and Immune Response

co-­expressed with 81 under-expressed immune response genes in LM cancers.

2.6 Autophagy and Genomic Mutation

We have conducted a comparative analysis between the level of autophagy and

As discussed earlier, we consider Fenton reactions as one of the root causes

Sect. 2.9). Smaller RSCD values indicate higher impact of C on the correlation

2.9 Material and Methods

2.9.1 Data Used

We have conducted a differential gene expression analysis measured using nor-

eight cancer-prone inflammatory diseases: cirrhosis (CIR), Barrett's esophagus

Differentially expressed genes in cancer and inflammatory disease are assessed by

2.9.3 Gene Co-expression Analysis

We have previously developed a Rank-based gene co-expression module extraction

2.9.4 Correlate Gene Expression Data to Genomic Mutations

2.9.5 Ratio of Significant Conditional Dependence

The Ratio of Significant Conditional Dependence (RSCD) is defined by the ratio of

Qing Li, Mi Zhou, and Renxiao Wang

Abstract Autophagy is an evolutionary conserved lysosomal pathway functioned

icro-autophagy and chaperon-mediated autophagy; (ii) co-reduction in autophagy

2.2 Gene Expression of Autophagy in Disease Tissues

Three types of autophagy have been defined, namely macro-autophagy, micro-

2.3 Nutrient Deprivation Is Unlikely in Cancer Tissue

2.4 Autophagy and Apoptosis

co-expression relations with other autophagy and apoptotic genes to elucidate

2.5 Novel Biological Processes Related to Autophagy

2.5.1 Autophagy and Cell Cycle Control

2.5.2 Autophagy and Immune Response

co-expressed with 81 under-expressed immune response genes in LM cancers.

2.6 Autophagy and Genomic Mutation

2.9 Material and Methods

2.9.3 Gene Co-expression Analysis

2.9.4 Correlate Gene Expression Data to Genomic Mutations

2.9.5 Ratio of Significant Conditional Dependence