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Methods Mol Biol. Author manuscript; available in PMC 2017 August 03.
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Published in final edited form as:

Methods Mol Biol. 2017 ; 1594: 293–308. doi:10.1007/978-1-4939-6934-0_19.

Lysosomal Biology in Cancer

Colin Fennelly and Ravi K. Amaravadi

Abstract
Cells depend on the lysosome for sequestration and degradation of macromolecules in order to
maintain metabolic homeostasis. These membrane-enclosed organelles can receive intracellular
and extracellular cargo through endocytosis, phagocytosis, and autophagy. Lysosomes establish
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acidic environments to activate enzymes that are able to break down biomolecules engulfed
through these various pathways. Recent advances in methods to study the lysosome have allowed
the discovery of extended roles for the lysosome in various diseases, including cancer, making it
an attractive and targetable node for therapeutic intervention. This review focuses on key aspects
of lysosomal biology in the context of cancer and how these properties can be exploited for the
development of new therapeutic strategies. This will provide a contextual framework for how
advances in methodology could be applied in future translational research.

Keywords
Lysosome; Cancer; Drug therapy; Autophagy
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1 Introduction to the Lysosome

The lysosome is a membrane-enclosed organelle that functions as an essential part of the
digestive system of the cell. Christian de Duve first discovered lysosomes in 1955 and the
name was derived from the Greek term for digestive body. This organelle contains over 60
different types of hydrolases that can break down biological polymers such as proteins,
carbohydrates, lipids, and nucleic acids [1]. These enzymes require an acidic pH for optimal
function, which is achieved by using ATP hydrolysis to pump protons against their
electrochemical gradient into the lysosome by the vacuolar H+ ATPase (V-ATPase) [2, 3].
Due to their pH dependence, these enzymes are also called acid hydrolases. They are
produced in the endoplasmic reticulum (ER), trafficked to the golgi apparatus and tagged
with mannose-6-phosphate to be targeted to the lysosome [4]. Vesicular exchange between
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the trans-golgi network (TGN) and endosomes is involved in the transport of newly
synthesized proteins from the TGN to the endolysosomal compartment, and the reverse,
where some proteins are trafficked from the endolysosomal compartment back to the TGN.
Trafficking vesicles are categorized by their internal pH, where early endosomes are around
6–6.6, late endosomes are at 5 and lysosomes are the most acidic at pH 4.5. The mannose-6-
phosphate receptor marker is used to identify endosomes and lysosomes. Intracellular cargo
is delivered to the lysosomes through autophagy, whereas exogenous material can be
engulfed from outside the cell and delivered via endocytosis or phagocytosis. Endocytosis
occurs in either a clathrin-dependent or -independent manner. The lysosome serves as the
end organelle for these degradative endocytic pathways that begin at the plasma membrane.
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Once lysosomal substrates are broken down, their components can be recycled and reused
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by the cell as building blocks for macromolecules through various carrier-mediated transport
channels back into the cytosol [5, 6]. Other than the ubiquitin-proteosome system,
autophagy is the main degradation pathway for intracellular proteins. Unlike proteasomal
degradation, autophagy can accommodate organelles and cytoskeletal components in
addition to proteins. During chaperone-mediated autophagy, cytosolic proteins that contain
specific motifs are localized to the lysosome through the action of chaperones and the
lysosomal receptor LAMP-2A [7–9]. Another form of autophagy is microautophagy, which
involves the direct engulfment of cytoplasmic cargo at the lysosomal membrane [10]. The
most widely studied form of autophagy is macroautophagy, and therefore, hereafter our
discussion of autophagy will focus on this form. Autophagic degradation is accomplished
through the sequestration of soluble cargo into a double membrane structure—referred to as
a phagophore—to form an autophagosome that eventually fuses with the lysosome to
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complete the degradation process [11]. In addition to the main autophagy pathway, there are
the more recently recognized organelle-specific autophagy processes of lysophagy [12, 13],
mitophagy [14], ER-phagy [15], nucleophagy [16], and pexophagy [17]. This review will
outline how lysosomal biogenesis is regulated, our current understanding of the many roles
lysosomes play in cancer progression and cell death, examples of tool compounds that can
be used to modulate lysosomal function (Fig. 1), and a brief overview of efforts to translate
some of these findings into clinical trials.

2 Control of Lysosomal Biogenesis at the Transcriptional Level

Lysosome formation is typically thought of in terms of simply the vesicular trafficking of
key lysosomal proteins from the ER, golgi, endosomes, and eventually into lysosomes.
However, recent evidence suggests that lysosomal biogenesis is coordinated at the
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transcriptional level in a sophisticated manner, and can even play a critical role in cancer cell
metabolism [18, 19]. Transcription factor EB (TFEB) is a transcription factor that acts as a
master regulator for lysosomal biogenesis and drives the expression of over 500 genes
related to autophagy and autophagosome-lysosome fusion [20]. Other family members of
the TFE/MiTF family control this expression profile in different cellular contexts. Activation
of this expression profile called the CLEAR (coordinated lysosomal expression and
regulation) network occurs when TFEB translocates from the lysosomal membrane into the
nucleus. This system controls the expression of lysosomal enzymes required for the
breakdown of biomolecules and genes linked to the main trafficking pathways including
autophagy, endo/exocytosis, and phagocytosis [21]. Recent work using unbiased global
metabolite profiling revealed the MiT/TFE family critically supports the metabolism of
pancreatic ductal adenocarcinoma (PDA) [18]. The discovery of this expression profile for
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lysosomal biogenesis opens the door to new biomarkers and therapeutic targets.

3 Lysosomes and Cancer Progression

Besides its role in catabolism and recycling—i.e. feeding the cancer cell from the inside—
recent evidence indicates the lysosome is also a central node for metabolic growth signaling.
Cancer cells deviate from normal metabolism in order to acquire their idiosyncratic feature
of uncontrolled growth. This transformation results in rapid depletion of cellular nutrients,

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accumulation of aggregated proteins, and damaged organelles making certain cancer cells
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dependent on lysosomal recycling programs for survival and continued growth. Autophagic-
lysosomal degradation of macromolecules and organelles serves as a coping mechanism for
cancer cells to deal with these stresses while also providing a consistent supply of nutrients
to promote further growth. Additionally, lysosomes are not just degradative vesicles, but
signaling scaffolds for mTOR and AMPK signaling, as described later. They are arguably
the main nutrient sensing organelle in the cell. Targeting lysosomes can have pleiotropic
effects involving metabolism [22], reactive oxygen species (ROS) [23], DNA damage [24],
cell death [25, 26], and protein secretion [27].

Cancer cells depend on lysosome function and demonstrate changes in lysosomal volume
and subcellular localization during oncogenic transformation [28, 29]. Cathepsin proteases
are lysosomal hydrolases that can play dual roles in promoting and suppressing tumor
growth. They are observed as being upregulated and mislocalized in cancer [29, 30].
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Intracellular cathepsins are able to activate the intrinsic apoptotic pathway, but in contrast,
extracellular cathepsins promote tumor invasion through their ability to break down
basement membranes and activate other oncogenic proteins. In addition, cathepsins B, E,
and S have all been recognized as contributing to malignancy in different cancers [31–33].
Lysosomal membrane proteins like lysosome-associated membrane protein 1 (LAMP-1]
have been observed on the cell surface of highly metastatic colon cells, indicating a role for
these proteins in the extracellular matrix [34]. Other lysosomal membrane proteins such as
the V-ATPase have been shown to exert an influence on the tumor microenvironment by
pumping protons to the extracellular space [35]. The Na+/H+ exchanger has also been
associated with extracellular acidification and cancer cell invasion [36]. Another intriguing
aspect of lysosomes is their ability to secrete contents out of the cell by fusing with the
plasma membrane [4, 37]. For example, cells can expel ATP to the extracellular space with
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this secretory pathway to mediate cellular signaling through ATP receptors [38, 39]. The
observation that lysosomal exocytosis can play a role in cell signaling, proteolytic
extracellular matrix (ECM) remodeling and tumor invasion suggests that targeting lysosomal
exocytosis rather than individual cathepsins would be a more promising strategy [40].

The lysosome is an important signaling hub that responds to both external and internal
stimuli to perceive the availability of nutrients, growth factor signals, and energy to maintain
metabolic homeostasis. One of the main regulators of cell growth and proliferation is
mammalian target of rapamycin complex 1 (mTORC1), which exerts its function directly
from the lysosomal membrane surface. mTORC1 is a multicomponent protein kinase
complex that includes mTOR, Regulatory Associated Protein of mTOR (RAPTOR), and
mLST8/GβL [41]. mTORC1 and its regulatory complexes detailed below, together integrate
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various nutritional and environmental cues including the presence of amino acids, growth
factors, glucose, hormones, and oxygen to drive anabolic processes such as protein, mRNA,
and lipid biosynthesis [22, 42, 43]. Active mTORC1 also phosphorylates ULK1 and ATG13
to inhibit their activity and block autophagy [44]. Additionally, TFEB has been recognized
as a target for mTORC1 suggesting this interaction directly influences expression of the
CLEAR network genes [45]. This ability to control biosynthetic and catabolic states makes
mTORC1 an important factor in metabolic signaling and mutations that lead to defective
mTORC1 regulation are commonly observed in human cancers [41, 46]. Oncogenic

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transformation is significantly enabled by mutations that lead to inactivation of key tumor

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suppressor genes including phosphatase and tensin homolog (PTEN), tuberous sclerosis
complex 1/2 (TSC1/TSC2), neurofibromin 1/2 (NF1/NF2), and liver kinase B1 (LKB1). In
all of these cases the downstream consequence of this inactivation is promotion of mTORC1
signaling [47]. mTORC1 kinase activity is stimulated by direct interaction with the GTPase
Ras homolog enriched in brain (RHEB) on the lysosome surface [48]. This interaction is
negatively regulated by the heterodimer TSC1/TSC2 and promoted by amino acids that
recruit mTORC1 to the lysosomal surface through Rag GTPases that are stabilized by the
Ragulator complex [49]. Recent investigations have also revealed that the V-ATPase can
mediate mTORC1 activation and autophagy [50]. This provides further evidence that
mTORC1 localization to the lysosomal surface is essential for its activation. Molecular
sensors for amino acids (Rags), growth factor inputs (Rheb), energy status (LKB1/AMPK1),
and lysosomal health (V-ATPase) all have to be aligned for full activation of mTORC1.
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Loss of the tumor suppressor PTEN has been shown to activate mTORC1 via protein kinase
B (AKT), which inhibits TSC1/TSC2 through phosphorylation [41]. TSC1/TSC2 can also be
suppressed when LKB1 is inactivated, preventing TSC1/TSC2 phosphorylation and
activation [41]. LKB1 regulates AMPK directly, and recent evidence indicates that AMPK is
also closely associated with the lysosomal surface. Another example of mTORC1-driven
oncogenesis is activation of eukaryotic translation initiation factor 4B (eIF4E) through
mTORC1-mediated inhibition of 4EBP1 [51]. This results in mRNA translation of cell cycle
regulatory genes and pro-tumorigenic genes such as the anti-apoptotic protein Mcl-1 that can
promote cancer cell survival in in vivo mouse models of lymphoma [52–54].

The lysosome’s role in catabolic recycling and metabolic growth decisions suggests there
may be therapeutic potential in targeting the lysosome. Great progress has been made in
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understanding the cell fates associated with lysosomal targeting, i.e. the role of the lysosome
in eliciting cell death.

4 Lysosomes and Cell Death

Lysosomes can play a role in each of the three major types of cancer cell death that include
apoptosis, autophagy, or necrosis [55]. A more recent form of cell death ferroptosis is also
dependent on the lysosome [56]. For apoptosis there are intrinsic and extrinsic pathways that
can be activated by different mechanisms. The intrinsic pathway involves mitochondrial
outer membrane permeabilization (MOMP) and cytochrome c release into the cytoplasm,
whereas the extrinsic is initiated by cell death receptors [57]. Both result in caspase
signaling cascades that are governed by the Bcl-2 family of proteins that ultimately regulate
MOMP. These proteins are classified in two categories: antiapoptotic (e.g. Bcl-2 and Bcl-xL)
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and proapoptotic (e.g. Bax and Bid) [58]. Damaged lysosomes allow proteolytic enzymes to
be released into the cytosol and initiate apoptosis [59]. Cathepsins B and D are known to
cleave Bid when ectopically in the cytosol, which results in MOMP followed by cytochrome
c release [60].

Cancer metabolism can create harsh byproducts such as ammonia, ROS, and hypoxia [61–
64]. To sustain oncogenic growth and cell survival autophagy can play a cytoprotective role

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that counteracts apoptosis by intercepting damaged mitochondria that could trigger

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apoptosis [65, 66]. Autophagy can have dual roles in the context of cancer and is also
recognized as a cell death pathway [67]. High drug doses can initiate apoptosis-independent
and autophagy-dependent cell death in vitro, although the relevance of autophagic cell death
in vivo has been called into question [68]. Necroptosis is a form of programmed necrosis
that serves as a backup role when apoptosis signaling is blocked by endogenous or
exogenous factors including viruses or mutations. The receptor-interacting serine/threonine
protein kinase 1 (RIPK1) and RIPK3 have been identified as regulators of apoptosis and
necrosis. RIPK1/3 can induce necroptosis via sphingomyelinase-mediated lysosomal
membrane permeabilization (LMP) [69]. Ferroptosis is an additional and unique form of cell
death that is dependent on iron and ROS [56]. It is distinct in that it has its own
morphological, genetic, and biochemical signatures. Misregulation of iron metabolism and
lipid peroxidation has been implicated in various pathologies including cancer [70, 71].
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One common theme that can impact multiple cell death pathways is LMP. This process has
been the topic of intense study for decades, and steadily methods to measure LMP in cancer
cells have become more reproducible and versatile. LMP permits the release of lysosomal
hydrolases into the cytosol and can contribute to the forms of cell death discussed above [28,
60]. Depending on the degree of permeabilization, LMP can either induce lysosomal cell
death through apoptosis or necrosis if the subsequent enzyme release is extensive enough
[72, 73]. For instance, LMP can initiate caspase cascades via the intrinsic apoptosis pathway
through cleavage of Bid and induction of Bax-mediated release of cytochrome c, but
cathepsins are able to mediate cell death in a caspase-independent manner as well [74].
Identifying stimuli that can cause the release of these lysosomal enzymes such as cathepsins
into the cytosolic lumen has potential applications for targeting the lysosome in cancer. LMP
can be evaluated in cells by detecting functional enzyme activity of lysosomal hydrolases
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present in the cytosol or visually by either tracking lysosomes with fluorescent dextran or
staining with antibody probes against galectins [75]. A small molecule screen done to
identify compounds able to induce p53-independent cell death found that the ones that were
effective worked through an LMP mechanism [76]. These findings and other studies have
lead to the proposal that transformed cells are more sensitive to lysosomal cell death and
further support the notion that targeting the lysosome can be an effective therapeutic strategy
[77].

5 Drugs That Target the Lysosome

There are at least five categories of drugs that target the lysosome. These include lysosomal
hydrolase inhibitors [78–82], heat shock protein 70 (HSP70) inhibitors, cationic amphiphilic
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compounds, V-ATPase inhibitors, and chloroquine derivatives that do not yet have a clear
mechanism of action.

Acid sphingomyelinase (ASM) is located in the lysosome and breaks down sphingomyelin
into ceramide, which is the substrate for the generation of other sphingolipids including
sphingomyelin and sphingosine 1-phosphate (S1P) [83]. It has been observed that cancer
cells display decreased levels of the proapoptotic lipid ceramide and increased levels of
proliferation promoting lipid S1P [84]. Cancer cells also exhibit lower ASM activity leading

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to higher sphingomyelin levels. Blocking ASM activity has shown to further elevate
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sphingomyelin levels and interrupt the function of the lysosomal membrane [85]. HSP70 is
expressed in many tumor types and can activate ASM, which is associated with increased
lysosomal integrity. Targeting HSP70 thereby inactivating ASM with small molecule
inhibitors like 2-phenylethynesulfonamide (PES) can increase LMP and cause cell death [86,
87]. Other drugs such as chloroquine (CQ), chlorpromazine, and amiodarone are cationic
amphiphilic agents that displace ASM from vesicular membranes in the lysosome and result
in lysosomal membrane permeabilization (LMP) and eventual tumor cell death [86].

HSP70 promotes tumor cell metastasis and survival by protecting lysosomal membrane
integrity. It can serve as a biomarker for poor prognosis due to its higher expression in many
cancers. The HSP70 modulator PES disrupts the protein interaction with p53 resulting in
massive accumulation of autophagosomes loaded with undigested cargo and cellular
apoptosis [88].
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Bafilomycin A1 is the prototypical inhibitor of the V-ATPase and prevents lysosomal

acidification and autophagic flux. It is similar to other compounds that are of microbial
origin including archazolid and cleistanthin A. However, other mechanisms of action have
been proposed for bafilomycin’s effects on the lysosome and autophagy. Bafilomycin A1
has also been shown to prevent autophagosome formation by activation of mTOR signaling,
suggesting that it may target both the early and late stages of autophagy [89]. This
impairment is mediated by dissociation of the Beclin1-Vps34 complex and encourages Bcl-2
interaction to drive autophagy inhibition and apoptotic cell death [37]. Interestingly,
Bafilomycin has also been shown to engage the mitochondria and induce translocation of
apoptosis inducing factor to the nucleus and provoke caspase-independent apoptosis [90].
Archazolid is another V-ATPase inhibitor and myxobacterial agent that has shown the ability
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to reduce the activity of the protease cathepsin B both in vitro and in vivo [91]. A member of
the manzamine alkaloids, manzamine A, was isolated from marine sponges of the genus
Haliclona, and demonstrated to have inhibiting effects on autophagy and the V-ATPase in
pancreatic cancer cells [92]. The diphyllin glycoside cleistanthin-A also has cytotoxic effects
on various tumor cell lines and targets the V-ATPase [93].

Salinomycin is a monocarboxylic polyether antibiotic that was isolated from a Streptomyces

albus strain and functions as an ionophore in the lysosome to facilitate the transport of
cations across cellular membranes (including lysosomal) [94–96]. Salinomycin has been
shown to impair autophagic flux in breast cancer cells [97] and even act in concert with
Gefitinib to induce apoptosis in colorectal cancer cells [98]. For the latter, this process was
dependent on ROS production and lead to loss of mitochondrial outer membrane potential
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and LMP. Other groups have also recognized oxidative stress as an important factor in
salinomycin-induced cell growth inhibition in prostate cancer cells [99]. Co-treatment of
salinomycin with doxorubicin or etoposide led to DNA damage and apoptosis in drug-
resistant cancer cells. This was also associated with enhanced expression of p53 and H2AX
as well as concurrent reduction in p21 [100]. In a different study, salinomycin suppressed
elevated p21 resulting from radiation treatment and promoted activation of H2AX and p53
resulting in DNA damage and G2 arrest [101]. Salinomycin is suggested to have selective
cytotoxic effects on cancer stem cells and also sensitize tumor cells to conventional

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chemotherapeutic drugs including methotrexate, adriamycin, and cisplatin in vitro and in

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vivo [102].

CQ accumulates in lysosomes and blocks autophagy by disrupting acidification and enzyme

function [103]. However, a definitive mechanism for CQ in mammalian cells remains
elusive. Other weak base compounds are known to also accumulate in lysosomes [104], but
none are known to inhibit autophagy. Interestingly, other drugs have been observed to
accumulate in lysosomes and it has been suggested that this mitigates the cytotoxic effect of
these compounds and aids in drug resistance [105–108]. A series of novel monomeric CQ
derivatives were tested in both lung and pancreatic cancer cells and proved to be eightfold
more potent than CQ [109]. Other efforts have identified the antimalarial agent quinacrine
(QN) as being much more effective at autophagy inhibition than CQ [110]. The synthesis of
novel monomeric QN analogs led to the generation of improved lysomotropic agents that
targeted the lysosome and elicited cell death in various cancer cell types in vitro [110].
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Another lysosomal agent, lucanthone, has been reported to inhibit lysosomal function and
induce apoptosis in a p53-independent manner [111]. Interestingly, these effects appear to be
dependent on cathepsin D. However, this agent has been suggested to block topoisomerase II
activity and inhibits AP endonuclease (APE1), an important enzyme in DNA base excision
repair suggesting it may not be specific for the lysosome [112].

6 Clinical Trials and Future Outlook

Over 40 clinical trials using hydroxychloroquine (HCQ) are being conducted worldwide in
humans and dogs [113]. Six phase I/II clinical trials have been performed in patients
diagnosed with refractory myeloma, glioblastoma, melanoma, and other cancers [114–119].
These trials also include combination therapies that were designed from preclinical studies
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[120–124]. Clinical trials to date have demonstrated that autophagy inhibition could be
achieved safely in patients. This was concluded from evidence of accumulated autophagic
vesicles in peripheral blood mononuclear cells and tumor cells. Even though high doses
were required to achieve this effect, treatment combinations were generally well tolerated
and there were not any signs of liver damage, metabolic dysfunction, or neurological
impairment [113]. However, there were some HCQ-cancer drug combinations that did result
in dose-limiting toxicities. In phase II clinical trials, patients previously treated for
metastatic pancreatic cancer were given HCQ alone and high doses were tolerated, but did
not demonstrate high therapeutic efficacy [119]. This suggests more potent compounds are
needed to generate the desired outcomes with the overall strategy of autophagy inhibition.

One of the limitations for clinical trials is biomarker availability for assessment of drug
efficacy. In the case of the autophagy inhibitor HCQ, the current methods include EM
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visualization of autophagic vesicle accumulation in peripheral blood mononuclear cells and

tumor cells along with LC3 western blotting and evaluation of total LC3 with
immunohistochemistry. Studies have been done to characterize secreted factors of tumor
cells exhibiting high autophagy and indicate that these could be potential candidates for
biomarkers [27]. There is growing evidence to encourage the concept that more potent
autophagy inhibitors could eventually be used synergistically with conventional
chemotherapy or radiotherapy [125]. Recent work has led to the dimerization of CQ to

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generate a CQ derivative (Lys05), which has proven to be far more potent as a single agent
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in vivo and in combination with B-Raf proto-oncogene serine/threonine protein kinase

(BRAF) inhibitors [126, 127]. Future studies will need to expand on the findings to date to
further elucidate the role of lysosomal function in tumor biology. Fortunately, there are many
opportunities to elicit an effect on lysosomal activity involving factors related to nutrient
sensing, kinase signaling, death signaling, and cell trafficking. Coupling functional studies
and molecular biology techniques will confirm the identification of new target candidates
and potential biomarkers. Capitalizing on other approaches involving high-throughout
readouts to analyze patient samples could also help detect what aspects of human cancer is
prone to lysosomal inhibition and lead to new clinical therapies.

References
1. Schröder BA, Wrocklage C, Hasilik A, Saftig P. The proteome of lysosomes. Proteomics. 2010;
Author Manuscript

10(22):4053–4076. [PubMed: 20957757]

2. Mindell JA. Lysosomal acidification mechanisms. Annu Rev Physiol. 2012; 74:69–86. [PubMed:
22335796]
3. Ohkuma S, Moriyama Y, Takano T. Identification and characterization of a proton pump on
‘lysosomes by fluorescein isothiocyanate-dextran fluorescence. Proc Natl Acad Sci. 1982; 79(9):
2758–2762. [PubMed: 6178109]
4. Repnik U, Cesen MH, Turk B. The endolysosomal system in cell death and survival. Cold Spring
Harb Perspect Biol. 2013; 5(1):a008755.doi: 10.1101/cshperspect.a008755 [PubMed: 23284043]
5. Rong Y, McPhee CK, Deng S, Huang L, Chen L, Liu M, Tracy K, Baehrecke EH, Yu L, Lenardo
MJ. Spinster is required for autophagic lysosome reformation and mTOR reactivation following
starvation. Proc Natl Acad Sci U S A. 2011; 108(19):7826–7831. [PubMed: 21518918]
6. Lloyd JB. Metabolite efflux and influx across the lysosome membrane. Subcell Biochem. 1996;
27:361–386. [PubMed: 8993166]
7. Dice JF. Peptide sequences that target cytosolic proteins for lysosomal proteolysis. Trends Biochem
Author Manuscript

Sci. 1990; 15(8):305–309. [PubMed: 2204156]

8. Cuervo AM, Dice JF. A receptor for the selective uptake and degradation of proteins by lysosomes.
Science. 1996; 273(5274):501–503. [PubMed: 8662539]
9. Kaushik S, Cuervo AM. Chaperone-mediated autophagy: a unique way to enter the lysosome world.
Trends Cell Biol. 2012; 8(22):407–417. DOI: 10.1016/j.tcb.2012.05.006
10. Li WW, Li J, Bao JK. Microautophagy: lesser-known self-eating. Cell Mol Life Sci. 2012; 69(7):
1125–1136. [PubMed: 22080117]
11. Burman C, Ktistakis NT. Autophagosome formation in mammalian cells. Semin Immunopathol.
2010; 6(27):421–429.
12. Maejima I, Takahashi A, Omori H, Kimura T, Takabatake Y, Saitoh T, Yamamoto A, Hamasaki M,
Noda T, Isaka Y, Yoshimori T. Autophagy sequesters damaged lysosomes to control lysosomal
biogenesis and kidney injury. EMBO J. 2013; 32(17):2336–2347. DOI: 10.1038/emboj.2013.171
[PubMed: 23921551]
13. Okamoto K. Organellophagy: eliminating cellular building blocks via selective autophagy. J Cell
Biol. 2014; 205(4):435–445. DOI: 10.1083/jcb.201402054 [PubMed: 24862571]
Author Manuscript

14. Baumann K. Mitophagy receptors unravelled. Nat Rev Mol Cell Biol. 2015; 16(10):580. [PubMed:
26350072]
15. Khaminets A, Heinrich T, Mari M, Grumati P, Huebner AK, Akutsu M, Liebmann L, Stolz A,
Nietzsche N, Koch S, Mauthe M, Katona I, Qualmann B, Weis J, Reggiori F, Kurth I, Hübner CA,
Dikic I. Regulation of endoplasmic reticulum turnover by selective autophagy. Nature. 2015;
522(7556):354–358. [PubMed: 26040720]
16. Mijaljica D, Devenish RJ. Nucleophagy at a glance. J Cell Sci. 2013; 129(pt 19):4325–4330.

Methods Mol Biol. Author manuscript; available in PMC 2017 August 03.
 Fennelly and Amaravadi Page 9

17. Sakaia Y, Okua M, van der Klei IJ, Kiel JA. Pexophagy: autophagic degradation of peroxisomes.
Mol Cell Res. 2006; 1763(12):1767–1775.
Author Manuscript

18. Perera RM, Stoykova S, Nicolay BN, Ross KN, Fitamant J, Boukhali M, Lengrand J, Deshpande
V, Selig MK, Ferrone CR, Settleman J, Stephanopoulosn G, Dyson NJ, Zoncu R, Ramaswamy S,
Haas W, Bardeesy N. Transcriptional control of autophagy–lysosome function drives pancreatic
cancer metabolism. Nature. 2015; 524(7565):361–365. [PubMed: 26168401]
19. Sardiello M, Palmieri M, di Ronza A, Medina DL, Valenza M, Gennarino VA, Di Malta C,
Donaudy F, Embrione V, Polishchuk RS, Banfi S, Parenti G, Cattaneo E, Ballabio A. A gene
network regulating lysosomal biogenesis and function. Science. 2009; 325(5939):473–477.
[PubMed: 19556463]
20. Settembre C, Di Malta C, Polito VA, Garcia Arencibia M, Vetrini F, Erdin S, Erdin SU, Huynh T,
Medina D, Colella P, Sardiello M, Rubinsztein DC, Ballabio A. TFEB links autophagy to
lysosomal biogenesis. Science. 2011; 332(6036):1429–1433. [PubMed: 21617040]
21. Palmieri M, Impey S, Kang H, di Ronza A, Pelz C, Sardiello M, Ballabio A. Characterization of
the CLEAR network reveals an integrated control of cellular clearance pathways. Hum Mol Genet.
2011; 19(20):3852–3866. DOI: 10.1093/hmg/ddr306
Author Manuscript

22. Settembre C, Fraldi A, Medina DL, Ballabio A. Signals from the lysosome: a control centre for
cellular clearance and energy metabolism. Nat Rev Mol Cell Biol. 2013; 14(5):283–296. DOI:
10.1038/nrm3565 [PubMed: 23609508]
23. Lee J, Giordano S, Zhang J. Autophagy, mitochondria and oxidative stress: cross-talk and redox
signalling. Biochem J. 2012; 441(2):523–540. DOI: 10.1042/BJ20111451 [PubMed: 22187934]
24. Kroemer G, Marino G, Levine B. Autophagy and the integrated stress response. Mol Cell. 2010;
40(2):280–293. DOI: 10.1016/j.molcel.2010.09.023 [PubMed: 20965422]
25. Amaravadi RK, Thompson CB. The roles of therapy-induced autophagy and necrosis in cancer
treatment. Clin Cancer Res. 2007; 13(24):7271–7279. DOI: 10.1158/1078-0432.CCR-07-1595
[PubMed: 18094407]
26. Yu F, Chen Z, Wang B, Jin Z, Hou Y, Ma S, Liu X. The role of lysosome in cell death regulation.
Tumour Biol. 2016; 37(2):1427–1436. [PubMed: 26631036]
27. Kraya AA, Piao S, Xu X, Zhang G, Herlyn M, Gimotty P, Levine B, Amaravadi RK, Speicher DW.
Identification of secreted proteins that reflect autophagy dynamics within tumor cells. Autophagy.
2015; 11(1):60–74. DOI: 10.4161/15548627.2014.984273 [PubMed: 25484078]
Author Manuscript

28. Kirkegaard T, Jäättelä M. Lysosomal involvement in cell death and cancer. Biochem Biophys Acta.
2009; 1793(4):746–754. [PubMed: 18948147]
29. Kallunki T, Olsen OD, Jäättelä M. Cancer-associated lysosomal changes: friends or foes?
Oncogene. 2013; 32(16):1995–2004. [PubMed: 22777359]
30. Palermo C, Joyce JA. Cysteine cathepsin proteases as pharmacological targets in cancer. Trends
Pharmacol Sci. 2008; 29(1):22–28. [PubMed: 18037508]
31. Withana NP, Blum G, Sameni M, Slaney C, Anbalagan A, Olive MB, Bidwell BN, Edgington L,
Wang L, Moin K, Sloane BF, Anderson RL, Bogyo MS, Parker BS. Cathepsin B inhibition limits
bone metastasis in breast cancer. Cancer Res. 2012; 72(5):1199–1209. [PubMed: 22266111]
32. Small DM, Burden RE, Jaworski J, Hegarty SM, Spence S, Burrows JF, McFarlane C,
Kissenpfennig A, McCarthy HO, Johnston JA, Walker B, Scott CJ. Cathepsin S from both tumor
and tumor-associated cells promote cancer growth and neovascularization. Int J Cancer. 2013;
133(9):2102–2112. [PubMed: 23629809]
33. Keliher EJ, Reiner T, Earley S, Klubnick J, Tassa C, Lee AJ, Ramaswamy S, Bardeesy N, Hanahan
Author Manuscript

D, DePinho RA, Castro CM, Weissleder R. Targeting cathepsin E in pancreatic cancer by a small
molecule allows in vivo detection. Neoplasia. 2013; 15(7):684–683. DOI: 10.1593/neo.13276
[PubMed: 23814481]
34. Furuta K, Ikeda M, Nakayama Y, Nakamura K, Tanaka M, Hamasaki N, Himeno M, Hamilton SR,
August JT. Expression of lysosome-associated membrane proteins in human colorectal neoplasms
and inflammatory diseases. Am J Pathol. 2001; 159(2):449–455. [PubMed: 11485903]
35. Dettmer J, Hong-Hermesdorf A, Stierhof YD, Schumacher K. Vacuolar H+-ATPase activity is
required for endocytic and secretory trafficking in Arabidopsis. Plant Cell. 2006; 18(3):715–730.
DOI: 10.1105/tpc.105.037978 [PubMed: 16461582]

Methods Mol Biol. Author manuscript; available in PMC 2017 August 03.
 Fennelly and Amaravadi Page 10

36. Cardone RA, Casavola V, Reshkin SJ. The role of disturbed pH dynamics and the Na+/H+
exchanger in metastasis. Nat Rev Cancer. 2005; 5(10):786–795. [PubMed: 16175178]
Author Manuscript

37. Piao S, Amaravadi RK. Targeting the lysosome in cancer. Ann N Y Acad Sci. 2016; 1371(1):45–
54. DOI: 10.1111/nyas.12953 [PubMed: 26599426]
38. Zhang Z, Chen G, Zhou W, Song A, Xu T, Luo Q, Wang W, Gu XS, Duan S. Regulated ATP
release from astrocytes through lysosome exocytosis. Nat Cell Biol. 2007; 9(8):945–953.
[PubMed: 17618272]
39. Ralevic V, Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev. 1998; 50(3):413–
492. [PubMed: 9755289]
40. Hämälistö S, Jäättelä M. Lysosomes in cancer-living on the edge (of the cell). Curr Opin Cell Biol.
2016; 39:69–79. [PubMed: 26921697]
41. Sabatini DM. mTOR and cancer: insights into a complex relationship. Nat Rev Cancer. 2006; 6(9):
729–734. [PubMed: 16915295]
42. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012; 149(2):274–
293. DOI: 10.1016/j.cell.2012.03.017 [PubMed: 22500797]
43. Efeyan A, Zoncu R, Chang S, Gumper I, Snitkin H, Wolfson RL, Kirak O, Sabatini DD, Sabatini
Author Manuscript

DM. Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and
survival. Nature. 2013; 493(7434):679–683. DOI: 10.1038/nature11745 [PubMed: 23263183]
44. Ganley IG, Lam du H, Wang J, Ding X, Chen S, Jiang X. ULK-Atg13-FIP200 complexes mediate
mTOR signaling to the autophagy machinery. J Biol Chem. 2009; 20(7):1992–2003. DOI:
10.1091/mbc.E08
45. Roczniak-Ferguson A, Petit CS, Froehlich F, Qian S, Ky J, Angarola B, Walther TC, Ferguson SM.
The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome
homeostasis. Sci Signal. 2012; 5(228):ra42.doi: 10.1126/scisignal.2002790 [PubMed: 22692423]
46. Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell. 2007; 12(1):9–22.
DOI: 10.1016/j.ccr.2007.05.008 [PubMed: 17613433]
47. Leone RD, Amaravadi RK. Autophagy: a targetable linchpin of cancer cell metabolism. Trends
Endocrinol Metab. 2013; 24(4):209–217. DOI: 10.1016/j.tem.2013.01.008 [PubMed: 23474062]
48. Long X, Lin Y, Ortiz-Vega S, Yonezawa K, Avruch J. Rheb binds and regulates the mTOR kinase.
Curr Biol. 2005; 15(8):702–713. DOI: 10.1016/j.cub.2005.02.053 [PubMed: 15854902]
Author Manuscript

49. Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, Tempst P, Sabatini
DM. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell
growth machinery. Cell. 2002; 110(2):163–175. [PubMed: 12150925]
50. Meo-Evoli N, Almacellas E, Massucci FA, Gentilella A, Ambrosio S, Kozma SC, Thomas G,
Tauler A. V-ATPase—a master effector of E2F1-mediated lysosomal trafficking, mTORC1
activation and autophagy. Oncotarget. 2015; 6(28):28057–28070. [PubMed: 26356814]
51. Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and
ageing. Nat Rev Mol Cell Biol. 2011; 12(1):21–35. DOI: 10.1038/nrm3025 [PubMed: 21157483]
52. Hsieh AC, Costa M, Zollo O, Davis C, Feldman ME, Testa JR, Meyuhas O, Shokat KM, Ruggero
D. Genetic dissection of the oncogenic mTOR pathway reveals drug-gable addiction to
translational control via 4EBP-eIF4E. Cancer Cell. 2010; 17(3):249–261. DOI: 10.1016/j.ccr.
2010.01.021 [PubMed: 20227039]
53. Wendel HG, De Stanchina E, Fridman JS, Malina A, Ray S, Kogan S, Cordon-Cardo C, Pelletier J,
Lowe SW. Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy. Nature. 2004;
428(6980):332–337. [PubMed: 15029198]
Author Manuscript

54. Wendel HG, Silva RL, Malina A, Mills JR, Zhu H, Ueda T, Watanabe-Fukunaga R, Fukunaga R,
Teruya-Feldstein J, Pelletier J, Lowe SW. Dissecting eIF4E action in tumorigenesis. Genes Dev.
2007; 21(24):3232–3237. DOI: 10.1101/gad.1604407 [PubMed: 18055695]
55. Duprez L, Wirawan E, Vanden Berghe T, Vandenabeele P. Major cell death pathways at a glance.
Microbes Infect. 2009; 11(13):1050–1062. [PubMed: 19733681]
56. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ,
Cantley AM, Yang WS, Morrison B 3rd, Stockwell BR. Ferroptosis: an iron-dependent form of
non-apoptotic cell death. Cell. 2012; 149(5):1060–1072. DOI: 10.1016/j.cell.2012.03.042
[PubMed: 22632970]

Methods Mol Biol. Author manuscript; available in PMC 2017 August 03.
 Fennelly and Amaravadi Page 11

57. Elmore S. Apoptosis—a review of programmed cell death. Toxicol Pathol. 2007; 35(4):495–516.
[PubMed: 17562483]
Author Manuscript

58. Dupreza L, Wirawana E, Vanden Berghea T, Vandenabeele P. Major cell death pathways at a
glance. Microbes Infect. 2009; 11(13):1050–1062. [PubMed: 19733681]
59. Guicciardi ME, Leist M, Gores GJ. Lysosomes in cell death. Oncogene. 2004; 23(16):2881–2890.
[PubMed: 15077151]
60. Boya P, Kroemer G. Lysosomal membrane permeabilization in cell death. Oncogene. 2008; 27(50):
6434–6451. DOI: 10.1038/onc.2008.310 [PubMed: 18955971]
61. Eng CH, Yu K, Lucas J, White E, Abraham RT. Ammonia derived from glutaminolysis is a
diffusible regulator of autophagy. Sci Signal. 2010; 3(119):ra31. [PubMed: 20424262]
62. Zhang H, Bosch-Marce M, Shimoda LA, Tan YS, Baek JH, Wesley JB, Gonzalez FJ, Semenza GL.
Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. J Biol
Chem. 2008; 283(16):10892–10903. [PubMed: 18281291]
63. Noman MZ, Janji B, Kaminska B, Van Moer K, Pierson S, Przanowski P, Buart S, Berchem G,
Romero P, Mami-Chouaib F, Chouaib S. Blocking hypoxia-induced autophagy in tumors restores
cytotoxic T-cell activity and promotes regression. Cancer Res. 2011; 18(71):5976–5986.
Author Manuscript

64. Bellot G, Garcia-Medina R, Gounon P, Chiche J, Roux D, Pouyssegur J, Mazure NM. Hypoxia-
induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L
via their BH3 domains. Mol Cell Biol. 2009; 29(10):2570–2581. DOI: 10.1128/MCB.00166-09
[PubMed: 19273585]
65. Green DR, Galluzzi L, Kroemer G. Mitochondria and the autophagy-inflammation-cell death axis
in organismal aging. Science. 2011; 333(6046):1109–1112. DOI: 10.1126/science.1201940
[PubMed: 21868666]
66. Geisler S, Holmström KM, Skujat D, Fiesel FC, Rothfuss OC, Kahle PJ, Springer W. PINK1/
Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat Cell Biol. 2010;
12(2):119–131. [PubMed: 20098416]
67. White E, Mehnert JM, Chan CS. Autophagy, metabolism, and cancer. Clin Cancer Res. 2015;
21(22):5037–5046. DOI: 10.1158/1078-0432.CCR-15-0490 [PubMed: 26567363]
68. Amaravadi RK. Autophagy-induced tumor dormancy in ovarian cancer. J Clin Invest. 2008;
118(12):3837–3840. DOI: 10.1172/JCI37667 [PubMed: 19033653]
Author Manuscript

69. Vandenabeele P, Galluzzi L, Berghe TV, Kroemer G. Molecular mechanisms of necroptosis: an

ordered cellular explosion. Nat Rev Mol Cell Biol. 2010; 11(10):700–714. [PubMed: 20823910]
70. Yang WS, Stockwell BR. Ferroptosis: death by lipid peroxidation. Trends Cell Biol. 2016; 26(3):
165–176. [PubMed: 26653790]
71. Xie Y, Hou W, Song X, Yu Y, Huang J, Sun X, Kang R, Tang D. Ferroptosis: process and function.
Cell Death Differ. 2016; 23(3):369–379. DOI: 10.1038/cdd.2015.158 [PubMed: 26794443]
72. Foghsgaard L, Wissing D, Mauch D, Lademann U, Bastholm L, Boes M, Elling F, Leist M,
Jäätteläa M. Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by
tumor necrosis factor. J Cell Biol. 2001; 5(153):999–1010.
73. Vancompernolle K, Van Herreweghe F, Pynaert G, Van de Craen M, De Vos K, Totty N, Sterling A,
Fiers W, Vandenabeele P, Grooten J. Atractyloside-induced release of cathepsin B, a protease with
caspase-processing activity. FEBS Lett. 1998; 3(438):150–158.
74. Imamura K, Ogura T, Kishimoto A, Kaminishi M, Esumi H. Cell cycle regulation via p53
phosphorylation by a 5′-AMP activated protein kinase activator, 5-aminoimidazole-4-
carboxamide-1-beta-D-ribofuranoside, in a human hepatocellular carcinoma cell line. Biochem
Author Manuscript

Biophys Res Commun. 2001; 287(2):562–567. [PubMed: 11554766]

75. Aits S, Jäättelä M, Nylandsted J. Methods for the quantification of lysosomal membrane
permeabilization: a hallmark of lysosomal cell death. Methods Cell Biol. 2015; 126:261–285.
[PubMed: 25665450]
76. Erdal H, Berndtsson M, Castron J, Brunk U, Shoshan M, Linder S. Induction of lysosomal
membrane permeabilization by compounds that activate p53-independent apoptosis. Proc Natl
Acad Sci U S A. 2005; 102(1):192–197. [PubMed: 15618392]

Methods Mol Biol. Author manuscript; available in PMC 2017 August 03.
 Fennelly and Amaravadi Page 12

77. Fehrenbacher N, Gyrd-Hansen M, Poulsen B, Felbor U, Kallunki T, Boes M, Weber E, Leist M,

Jäättelä M. Sensitization to the lysosomal cell death pathway upon immortalization and
Author Manuscript

transformation. Cancer Res. 2004; 64(15):5301–5310. [PubMed: 15289336]

78. Maynadier M, Vezenkov LL, Amblard M, Martin V, Gandreuil C, Vaillant O, Gary-Bobo M, Basile
I, Hernandez JF, Garcia M, Martinez J. Dipeptide mimic oligomer transporter mediates
intracellular delivery of Cathepsin D inhibitors: a potential target for cancer therapy. J Control
Release. 2013; 171(2):251–257. [PubMed: 23899821]
79. Kos J, Mitrović A, Mirković B. The current stage of cathepsin B inhibitors as potential anticancer
agents. Future Med Chem. 2014; 6(11):1355–1371. [PubMed: 25163003]
80. Duong LT, Wesolowski GA, Leung P, Oballa R, Pickarski M. Efficacy of a cathepsin K inhibitor in
a preclinical model for prevention and treatment of breast cancer bone metastasis. Mol Cancer
Ther. 2014; 13(12):2898–2909. DOI: 10.1158/1535-7163.MCT-14-0253 [PubMed: 25249554]
81. Tsai JY, Lee MJ, Chang MD, Wang HC, Lin CC, Huang H. Effects of novel human cathepsin S
inhibitors on cell migration in human cancer cells. J Enzyme Inhib Med Chem. 2014; 29(4):538–
546. [PubMed: 24083411]
82. Lankelma JM, Voorend DM, Barwari T, Koetsveld J, Van der Spek AH, De Porto AP, Van Rooijen
Author Manuscript

G, Van Noorden CJ. Cathepsin L, target in cancer treatment? Life Sci. 2010; 86(7–8):225–233.
[PubMed: 19958782]
83. Petersen NH, Olsen OD, Groth-Pedersen L, Ellegaard AM, Bilgin M, Redmer S, Ostenfeld MS,
Ulanet D, Dovmark TH, Lonborg A, Vindelov SD, Hanahan D, Arenz C, Ejsing CS, Kirkegaard T,
Rohde M, Nylandsted J, Jaattela M. Transformation-associated changes in sphingolipid
metabolism sensitize cells to lysosomal cell death induced by inhibitors of acid sphingomyelinase.
Cancer Cell. 2013; 24(3):379–393. DOI: 10.1016/j.ccr.2013.08.003 [PubMed: 24029234]
84. Savić R, Schuchman EH. Use of acid sphingomyelinase for cancer therapy. Adv Cancer Res. 2013;
117:91–115. [PubMed: 23290778]
85. Smith EL, Schuchman EH. Acid sphingomyelinase overexpression enhances the antineoplastic
effects of irradiation in vitro and in vivo. Mol Ther. 2008; 16(9):1565–1571. DOI: 10.1038/mt.
2008.145 [PubMed: 18628757]
86. Saftig P, Sandhoff K. Cancer: killing from the inside. Nature. 2013; 7471(502):312–313.
87. Leu JI, Pimkina J, Pandey P, Murphy ME, George DL. HSP70 inhibition by the small-molecule 2-
phenylethynesulfonamide impairs protein clearance pathways in tumor cells. Mol Cancer Res.
Author Manuscript

2011; 9(7):936–947. DOI: 10.1158/1541-7786.MCR-11-0019 [PubMed: 21636681]

88. Granato M, Lacconi V, Peddis M, Lotti LV, Di Renzo L, Gonnella R, Santarelli R, Trivedi P, Frati
L, D’Orazi G, Faggioni A, Cirone M. HSP70 inhibition by 2-phenylethynesulfonamide induces
lysosomal cathepsin D release and immunogenic cell death in primary effusion lymphoma. Cell
Death Dis. 2013; 4:e730.doi: 10.1038/cddis.2013.263 [PubMed: 23868063]
89. Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y. Bafilomycin A1
prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and
lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct. 1998; 1(23):33–42.
90. Yuan N, Song L, Zhang S, Lin W, Cao Y, Xu F, Fang Y, Wang Z, Zhang H, Li X, Wang Z, Cai J,
Wang J, Zhang Y, Mao X, Zhao W, Hu S, Chen S, Wang J. Bafilomycin A1 targets both autophagy
and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia. Haematologica. 2015;
3(100):345–356. DOI: 10.3324/haematol.2014.113324
91. Kubisch R, Frohlich T, Arnold GJ, Schreiner L, von Schwarzenberg K, Roidl A, Vollmar AM,
Wagner E. V-ATPase inhibition by archazolid leads to lysosomal dysfunction resulting in impaired
Author Manuscript

cathepsin B activation in vivo. Int J Cancer. 2014; 134(10):2478–2488. DOI: 10.1002/ijc.28562

[PubMed: 24166050]
92. Kallifatidis G, Hoepfner D, Jaeg T, Guzman EA, Wright AE. The marine natural product
manzamine A targets vacuolar ATPases and inhibits autophagy in pancreatic cancer cells. Mar
Drugs. 2013; 11(9):3500–3516. DOI: 10.3390/md11093500 [PubMed: 24048269]
93. Zhao Y, Lu Y, Ma J, Zhu L. Synthesis and evaluation of cleistanthin A derivatives as potent
vacuolar H+-ATPase inhibitors. Chem Biol Drug Des. 2015; 86(4):691–696. [PubMed: 25677205]
94. Zhou S, Wang F, Wong ET, Fonkem E, Hsieh T, Wu JM, Wu E. Salinomycin—a novel anti-cancer
agent with known anticoccidial activities. Curr Med Chem. 2014; 20(33):4095–4101.

Methods Mol Biol. Author manuscript; available in PMC 2017 August 03.
 Fennelly and Amaravadi Page 13

95. Fuchs D, Heinold A, Opelz G, Daniel V, Naujokat C. Salinomycin induces apoptosis and
overcomes apoptosis resistance in human cancer cells. Biochem Biophys Res Commun. 2009;
Author Manuscript

390(3):743–749. [PubMed: 19835841]

96. Kim KY, Yu SN, Lee SY, Chun SS, Choi YL, Park YM, Song CS, Chatterjee B, Ahn SC.
Salinomycin-induced apoptosis of human prostate cancer cells due to accumulated reactive oxygen
species and mitochondrial membrane depolarization. Biochem Biophys Res Commun. 2011;
413(1):80–86. [PubMed: 21871443]
97. Yue W, Hamai A, Tonelli G, Bauvy C, Nicolas V, Tharinger H, Codogno P, Mehrpour M.
Inhibition of the autophagic flux by salinomycin in breast cancer stem-like/progenitor cells
interferes with their maintenance. Autophagy. 2013; 9(5):714–729. DOI: 10.4161/auto.23997
[PubMed: 23519090]
98. Zou ZZ, Nie PP, Li YW, Hou BX, Rui-Li, Shi XP, Ma ZK, Han BW, Luo XY. Synergistic induction
of apoptosis by salinomycin and gefitinib through lysosomal and mitochondrial dependent
pathway overcomes gefitinib resistance in colorectal cancer. Oncotarget. 2015; :1–19. DOI:
10.18632/oncotarget.5628 [PubMed: 25595910]
99. Ketola K, Hilvo M, Hyotylainen T, Vuoristo A, Ruskeepaa AL, Oresic M, Kallioniemi O, Iljin K.
Author Manuscript

Salinomycin inhibits prostate cancer growth and migration via induction of oxidative stress. Br J
Cancer. 2012; 106(1):99–106. DOI: 10.1038/bjc.2011.530 [PubMed: 22215106]
100. Kim JH, Chae M, Kim WK, Kim YJ, Kang HS, Kim HS, Yoon S. Salinomycin sensitizes cancer
cells to the effects of doxorubicin and etoposide treatment by increasing DNA damage and
reducing p21 protein. Br J Pharmacol. 2011; 162(3):773–784. DOI: 10.1111/j.
1476-5381.2010.01089.x [PubMed: 20973777]
101. Kim WK, Kim JH, Yoon K, Kim S, Ro J, Kang HS, Yoon S. Salinomycin, a p-glycoprotein
inhibitor, sensitizes radiation-treated cancer cells by increasing DNA damage and inducing G2
arrest. Invest New Drugs. 2012; 30(4):1311–1318. [PubMed: 21573958]
102. Tang QL, Zhao ZQ, Li JC, Liang Y, Yin JQ, Zou CY, Xie XB, Zeng YX, Shen JN, Kang T, Wang
J. Salinomycin inhibits osteosarcoma by targeting its tumor stem cells. Cancer Lett. 2011;
311(1):113–121. [PubMed: 21835542]
103. Steinman RM, Mellman IS, Muller WA, Cohn ZA. Endocytosis and the recycling of plasma
membrane. J Cell Biol. 1983; 96(1):1–27. [PubMed: 6298247]
104. Fu D, Zhou J, Zhu WS, Manley PW, Wang YK, Hood T, Wylie A, Xie XS. Imaging the
Author Manuscript

intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative

hyperspectral stimulated Raman scattering. Nat Chem. 2014; 6(7):614–622. [PubMed:
24950332]
105. Zhitomirsky B, Assaraf YG. Lysosomal sequestration of hydrophobic weak base
chemotherapeutics triggers lysosomal biogenesis and lysosome-dependent cancer multidrug
resistance. Oncotarget. 2015; 6(2):1143–1156. [PubMed: 25544758]
106. Duvvuri M, Gong Y, Chatterji D, Krise JP. Weak base permeability characteristics influence the
intracellular sequestration site in the multidrug-resistant human leukemic cell line HL-60. J Biol
Chem. 2004; 279(31):32367–32372. DOI: 10.1074/jbc.M400735200 [PubMed: 15181006]
107. Gorden BH, Saha J, Khammanivong A, Schwartz GK, Dickerson EB. Lysosomal drug
sequestration as a mechanism of drug resistance in vascular sarcoma cells marked by high
CSF-1R expression. Vasc Cell. 2014; 6(20):1–14. [PubMed: 24472220]
108. Wang E, Lee MD, Dunn KW. Lysosomal accumulation of drugs in drug-sensitive MES-SA but
not multidrug-resistant MES-SA/Dx5 uterine sarcoma cells. J Cell Physiol. 2000; 184(2):263–
Author Manuscript

274. [PubMed: 10867652]

109. Nordstrom LU, Sironi J, Aranda E, Maisonet J, Perez-Soler R, Wu P, Schwartz EL. Discovery of
autophagy inhibitors with antiproliferative activity in lung and pancreatic cancer cells. ACS Med
Chem Lett. 2015; 6(2):134–139. DOI: 10.1021/ml500348p [PubMed: 25699157]
110. Wang T, Goodall ML, Gonzales P, Sepulveda M, Martin KR, Gately S, MacKeigan JP. Synthesis
of improved lysomotropic autophagy inhibitors. J Med Chem. 2015; 58(7):3025–3035. [PubMed:
25793774]

Methods Mol Biol. Author manuscript; available in PMC 2017 August 03.
 Fennelly and Amaravadi Page 14

111. Carew JS, Espitia CM, Esquivel JA, Mahalingam D, Kelly KR, Reddy G, Giles FJ, Nawrocki ST.
Lucanthone is a novel inhibitor of autophagy that induces cathepsin D-mediated apoptosis. J Biol
Author Manuscript

Chem. 2011; 286(8):6602–6613. [PubMed: 21148553]

112. Luo M, Kelley MR. Inhibition of the human apurinic/apyrimidinic endonuclease (Ape1) repair
activity and sensitization of breast cancer cells to DNA alkylating agents with lucanthone.
Anticancer Res. 2004; 24(4):2127–2134. [PubMed: 15330152]
113. Rebecca VW, Amaravadi RK. Emerging strategies to effectively target autophagy in cancer.
Oncogene. 2016; 35(1):1–11. DOI: 10.1038/onc.2015.99 [PubMed: 25893285]
114. Mahalingam D, Mita M, Sarantopoulos J, Wood L, Amaravadi RK, Davis LE, Mita AC, Curiel
TJ, Espitia CM, Nawrocki ST, Giles FJ, Carew JS. Combined autophagy and HDAC inhibition: a
phase I safety, tolerability, pharmacokinetic, and pharmacodynamic analysis of
hydroxychloroquine in combination with the HDAC inhibitor vorinostat in patients with
advanced solid tumors. Autophagy. 2014; 10(8):1403–1414. DOI: 10.4161/auto.29231 [PubMed:
24991835]
115. Rangwala R, Chang YC, Hu J, Algazy KM, Evans TL, Fecher LA, Schuchter LM, Torigian DA,
Panosian JT, Troxel AB, Tan KS, Heitjan DF, DeMichele AM, Vaughn DJ, Redlinger M, Alavi
Author Manuscript

A, Kaiser J, Pontiggia L, Davis LE, O’Dwyer PJ, Amaravadi RK. Combined MTOR and
autophagy inhibition: phase I trial of hydroxychloroquine and temsirolimus in patients with
advanced solid tumors and melanoma. Autophagy. 2014; 10(8):1391–1402. DOI: 10.4161/auto.
29119 [PubMed: 24991838]
116. Barnard RA, Wittenburg LA, Amaravadi RK, Gustafson DL, Thorburn A, Thamm DH. Phase I
clinical trial and pharmacodynamic evaluation of combination hydroxy-chloroquine and
doxorubicin treatment in pet dogs treated for spontaneously occurring lymphoma. Autophagy.
2014; 10(8):1415–1425. DOI: 10.4161/auto.29165 [PubMed: 24991836]
117. Rangwala R, Leone R, Chang YC, Fecher LA, Schuchter LM, Kramer A, Tan KS, Heitjan DF,
Rodgers G, Gallagher M, Piao S, Troxel AB, Evans TL, DeMichele AM, Nathanson KL,
O’Dwyer PJ, Kaiser J, Pontiggia L, Davis LE, Amaravadi RK. Phase I trial of
hydroxychloroquine with dose-intense temozolomide in patients with advanced solid tumors and
melanoma. Autophagy. 2014; 10(8):1369–1379. DOI: 10.4161/auto.29118 [PubMed: 24991839]
118. Rosenfeld MR, Ye X, Supko JG, Desideri S, Grossman SA, Brem S, Mikkelson T, Wang D,
Chang YC, Hu J, McAfee Q, Fisher J, Troxel AB, Piao S, Heitjan DF, Tan KS, Pontiggia L,
Author Manuscript

O’Dwyer PJ, Davis LE, Amaravadi RK. A phase I/II trial of hydroxychloroquine in conjunction
with radiation therapy and concurrent and adjuvant temozolomide in patients with newly
diagnosed glioblastoma multiforme. Autophagy. 2014; 10(8):1359–1368. DOI: 10.4161/auto.
28984 [PubMed: 24991840]
119. Wolpin BM, Rubinson DA, Wang X, Chan JA, Cleary JM, Enzinger PC, Fuchs CS, McCleary NJ,
Meyerhardt JA, Ng K, Schrag D, Sikora AL, Spicer BA, Killion L, Mamon H, Kimmelman AC.
Phase II and pharmacodynamic study of autophagy inhibition using hydroxychloroquine in
patients with metastatic pancreatic adenocarcinoma. Oncologist. 2014; 19(6):637–638. DOI:
10.1634/theoncologist.2014-0086 [PubMed: 24821822]
120. Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI, Thomas-Tikhonenko A,
Thompson CB. Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced
model of lymphoma. J Clin Invest. 2007; 117(2):326–336. DOI: 10.1172/JCI28833 [PubMed:
17235397]
121. Bray K, Mathew R, Lau A, Kamphorst JJ, Fan J, Chen J, Chen HY, Ghavami A, Stein M, DiPaola
RS, Zhang D, Rabinowitz JD, White E. Autophagy suppresses RIP kinase-dependent necrosis
Author Manuscript

enabling survival to mTOR inhibition. PLoS One. 2012; 7(7):e41831.doi: 10.1371/journal.pone.

0041831 [PubMed: 22848625]
122. Xie X, White EP, Mehnert JM. Coordinate autophagy and mTOR pathway inhibition enhances
cell death in melanoma. PLoS One. 2013; 8(1):e55096.doi: 10.1371/journal.pone.0055096
[PubMed: 23383069]
123. Carew JS, Nawrocki ST, Kahue CN, Zhang H, Yang C, Chung L, Houghton JA, Huang P, Giles
FJ, Cleveland JL. Targeting autophagy augments the anticancer activity of the histone deacetylase
inhibitor SAHA to overcome Bcr-Abl–mediated drug resistance. Blood. 2007; 110(1):313–322.
[PubMed: 17363733]

Methods Mol Biol. Author manuscript; available in PMC 2017 August 03.
 Fennelly and Amaravadi Page 15

124. Qiu L, Yao M, Gao M, Zhao Q. Doxorubicin and chloroquine coencapsulated liposomes:
preparation and improved cytotoxicity on human breast cancer cells. J Liposome Res. 2012;
Author Manuscript

22(3):245–253. [PubMed: 22607110]

125. Amaravadi RK, Lippincott-Schwartz J, Yin XM, Weiss WA, Takebe N, Timmer W, DiPaola RS,
Lotze MT, White E. Principles and current strategies for targeting autophagy for cancer
treatment. Clin Cancer Res. 2011; 17(4):654–666. DOI: 10.1158/1078-0432.CCR-10-2634
[PubMed: 21325294]
126. McAfee Q, Zhang Z, Samanta A, Levi SM, Ma XH, Piao S, Lynch JP, Uehara T, Sepulveda AR,
Davis LE, Winkler JD, Amaravadi RK. Autophagy inhibitor Lys05 has single-agent antitumor
activity and reproduces the phenotype of a genetic autophagy deficiency. Proc Natl Acad Sci U S
A. 2012; 109(21):8253–8258. [PubMed: 22566612]
127. Amaravadi RKWJ. Lys05: a new lysosomal autophagy inhibitor. Autophagy. 2012; 8(9):1383–
1384. [PubMed: 22878685]
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Fig. 1.
The key elements involved in lysosomal biology in cancer. Factors that can play roles in
cancer progression are in red. Vesicular trafficking funnels cellular contents into the
lysosome from various pathways including endocytosis and autophagy. TFEB regulates
expression of autophagy and lysosomal genes. Elevated autophagy has proven to aid in
oncogenesis by providing macromolecules for sustained growth and eliminating damaged
proteins, organelles, and hazardous waste including ammonia and ROS. Cellular contents
can also be secreted to the extracellular space through exocytosis. Lysosomal cathepsins
have been observed in the extracellular space where they are free to engage the ECM for
remodeling to promote tumor invasion. Lysosomal membrane proteins such as the V-ATPase
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and LAMP2 have also been observed promoting metastasis by exerting an influence on the
tumor microenvironment from the plasma membrane. MTORC1 is known to positively
regulate growth and feed into other oncogenic signaling pathways. HSP70 supports
lysosomal membrane integrity by activating ASM and can even be used as a biomarker for
prognosis. Various drugs (green) that target the lysosome or cellular components involved
with the lysosome can destabilize the organelle leading to LMP and activation of cell death
pathways (yellow). aa amino acid, ASM acid sphingomyelinase, Baf Bafilomycin A 1, CQ
chloroquine, ECM extracellular matrix, HSP70 70-kDa heat shock proteins, LAMP
lysosome associated membrane glycoproteins, LMP lysosomal membrane permeabilization,
mTORC1 mammalian target of rapamycin complex 1/mechanistic target of rapamycin
complex 1, PES phenylethynesulfonamide, QN quinacrine, ROS reactive oxygen species,
TFEB transcription factor EB, V-ATPase Vacuolar-type H+ATPase
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Methods Mol Biol. Author manuscript; available in PMC 2017 August 03.