STATE-OF-THE-ART REVIEW

Functions of autophagy in the tumor microenvironment

and cancer metastasis
Erin E. Mowers1,2,3, Marina N. Sharifi1,2,4 and Kay F. Macleod1,2,5
1 The Ben May Department for Cancer Research, University of Chicago, IL, USA
2 The Committee on Cancer Biology, Chicago, IL, USA
3 Inter-disciplinary Scientist Training Program, Chicago, IL, USA
4 Medical Scientist Training Program, Chicago, IL, USA
5 The University of Chicago, IL, USA

Keywords Macro-autophagy is an ancient and highly conserved self-degradative pro-

autophagy; invasion; metastasis; mitophagy;
tumor cell migration; tumor
microenvironment; tumor stem cells
Correspondence
K. F. Macleod, Ben May Department for
Cancer Research, University of Chicago,
929 East 57th Street, Chicago, IL 60637,
USA
Fax: +1 773 702 4476
Tel: +1 773 834 8309
E-mail: kmacleod@uchicago.edu
(Received 3 December 2017, revised 7
January 2018, accepted 16 January 2018)
cess that plays a homeostatic role in normal cells by eliminating organelles,
pathogens, and protein aggregates. Autophagy, as it is routinely referred
to, also allows cells to maintain metabolic sufficiency and survive under
conditions of nutrient stress by recycling the by-products of autophagic
degradation, such as fatty acids, amino acids, and nucleotides. Tumor cells
are more reliant than normal cells on autophagy for survival in part due to
their rapid growth rate, altered metabolism, and nutrient-deprived growth
environment. How this dependence of tumor cells on autophagy affects
their progression to malignancy and metastatic disease is an area of
increasing research focus. Here, we review recent work identifying critical
functions for autophagy in tumor cell migration and invasion, tumor stem
cell maintenance and therapy resistance, and cross-talk between tumor cells
and their microenvironment.

doi:10.1111/febs.14388

Autophagy and cancer

The process of macro-autophagy involves the forma-
tion of double-membrane vesicles called autophago-
somes, around the cellular content that is to be
degraded, known as autophagic cargo [1,2]. Such cargo
typically includes organelles of all types, intracellular
pathogens, and protein aggregates. Induction of
autophagy is controlled at the transcriptional and
post-translational level (Fig. 1). Autophagy (Atg)
genes are transcriptionally regulated by the ATF4 and
MIT/TFE transcription factors [3,4], and as such are
induced by cellular stresses including amino acid depri-
vation and ER stress (ATF4) known to promote

Abbreviations
AMPK, adenosine monophosphate activated kinase; ATF4, activating transcription factor 4; ATG/Atg, human/mouse autophagy gene; ATP,
adenosine triphosphate; CBL, Casitas B-lineage lymphoma gene; CD44, cell differentiation antigen 44; CSC, cancer stem cell; CTL, cytotoxic
T lymphocyte; DAMP, damage-associated molecular pattern; DCIS, ductal carcinoma in situ; ECM, extracellular matrix; EMT, epithelial–
mesenchymal transition; ER, endoplasmic reticulum; FAK, focal adhesion kinase; FIP200, FAK-interacting protein 200 kD; GPT1, glutamate-
pyruvate transaminase-1; HSC, hematopoietic stem cell; IL-6, interleukin 6; JAK, janus kinase; JNK, c-Jun N-terminal kinase; LC3,
microtubule-associated light chain 3; MIT/TFE, microphthalmia-associated transcription factor/transcription factor E3; mTORC1, mammalian
target of rapamycin complex 1; mTOR, mammalian target of rapamycin; PDAC, pancreatic ductal carcinoma; PSC, pancreatic stellate cell;
PXN, paxillin; ROS, reactive oxygen species; SERM, selective estrogen receptor modulator; SRC, sarcoma oncoprotein encoded by c-SRC;
STAT, signal transducers and activators of transcription; TCA, tricarboxylic acid; TGF-b, transforming growth factor beta; TNF, tumor necrosis
factor; ULK1, Unc51-like kinase-1; VPS34, Vacuolar protein sorting-associated protein 34.

The FEBS Journal 285 (2018) 1751–1766 ª 2018 Federation of European Biochemical Societies 1751
Autophagy & Metastasis E. E. Mowers et al.

Transcriptional control of autophagy

ER stress
mTORC1 AA deficiency

ATF4
MiT/TFE

Atg genes
Lysosomal genes

Fig. 1. Autophagy overview. Autophagy is exquisitely sensitive to amino acid deprivation in addition to other nutrient stresses. Amino acid
deprivation induces AMPK and suppresses mTORC1 to promote Ulk1 kinase activity as part of the preinitiation complex (Ulk1/FIP200/Atg13)
that in turn activates the lipid kinase activity of the initiation complex (Beclin1/VPS34 and associated proteins). This is turn recruits the
Atg12/Atg5/Atg16L conjugation complex that promotes processing and insertion of lipid conjugated LC3 into nascent phagophore
membranes. LC3 and related proteins interact with cargo to be turned over via autophagy and also promotes closure of the autophagosome
around the cargo. Fusion of these double-membrane autophagosomes with the lysosome results in proteolytic degradation of the cargo and
release from the autophagolysosome of amino acids, nucleotides and lipids derived from degraded cargo. In addition to induction of
autophagy by these post-translational mechanisms, autophagy is also stimulated by transcriptional control of Atg genes, notably by ATF4
and MiT/TFE transcription factors that are activated by amino acid deprivation among other stresses.

autophagy, as well as by other signals that inhibit
mTOR, a negative regulator of MIT/TFE factors
(Fig. 1) [4]. However, autophagy is primarily regulated
in a post-translational manner to permit a rapid
response to nutrient stress. The formation of
autophagosomes is regulated by a preinitiation com-
plex involving the Ulk1/Ulk2 serine/threonine kinase
that is sensitive to amino acid supply and cellular
energy status, as a result of being regulated negatively
by mTOR and positively by AMPK (Fig. 1) [5,6]. As
part of the preinitiation complex with ATG13 and
FIP200, Ulk1/2 phosphorylates Beclin1 to activate the
lipid kinase activity of Vps34 (a class III PI3K), the
catalytic component of the initiation complex, increas-
ing phospho-inositol-3-phosphate (PIP3) production
and recruiting further components of the autophagy
machinery to drive the process forward (Fig. 1) [1].
A key consequence of increased initiation complex
activity is the recruitment and activation of the conju-
gation complexes, including ATG5-ATG12/ATG16L
that in turn recruits processed LC3 and related mole-
cules to the expanding phagophore (Fig. 1) [1,6]. Pro-
cessed LC3 at nascent phagophores is key to selecting
cargo for degradation and proteins, or their cargo
adaptors, at cellular structures destined for degrada-
tion contain specific motifs known as LC3-interacting
region (LIR) motifs that promote their interaction
with LC3 (Fig. 1) [7,8]. Closure of the phagophore to
form a mature autophagosome requires LC3-related
proteins leading to fusion with the lysosome resulting
in degradation of autophagosomal cargo and recycling
of constituent amino acids, nucleotides, and fatty acids
(Fig. 1) [1,2,9]. Novel noncanonical functions for
autophagy proteins are emerging, including roles in

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E. E. Mowers et al.
antiviral immunity, secretory processes, and endocytic
trafficking that are distinct from autophagy but rely
on a select cadre of Atg gene products [10,11].
Together with the increasing variety of cargo that
autophagy seems capable of turning over, this has
complicated our ability to dissect at a mechanistic level
how autophagy influences disease processes, such as
cancer. In particular, the pleiotropic nature of autop-
hagy has made it challenging to determine whether
autophagy is tumor-promoting, and therefore a valid
therapeutic target, or indeed tumor-suppressive which
might limit its value as a target in cancer treatment
[12].
Much of the focus on the role of autophagy in can-
cer has been aimed at its role in tumor metabolism [9]
where its ability to modulate mitochondrial turnover
has been proposed as the main explanation for the
altered metabolic phenotype in tumors forming in
autophagy-deficient GEM models. Such models,
including Ras-driven lung and pancreatic cancers [13–
16], showed reduced fatty acid oxidation, a switch to
Warburg metabolism and increased ROS, consistent
with failure to degrade mitochondria in an appropriate
manner [12,17]. More recently, the role of autophagy
in modulating how the tumor microenvironment sus-
tains growing tumors has also come to the fore
[18,19]. Beyond its role in modulating tumor metabo-
lism, autophagy performs other functions that likely
contribute to its role in cancer and response to cancer
therapy [12].
Metastasis is a multistep process in which tumor
cells at the primary tumor site undergo epithelial-to-
mesenchymal transition (EMT) [20], acquire invasive
and other properties that allow them to escape
through the basement membrane into the vasculature,
in a part of the metastatic cascade known as intravasa-
tion [21]. This is followed by selective pressure on
escaped tumor cells to survive in the circulation where
they sometimes become lodged, or if they are able to
survive at all, use their invasive properties to extrava-
sate at a distant site [21]. Frequently, such tumor cells
alight in an environment that does not favor their
growth and where they are again subject to evolution-
ary pressures to survive [22]. However, even if tumor
cells survive such seemingly unfavorable conditions,
they may not be able to expand and can remain dor-
mant at such sites for years [22,23]. Recently, it has
emerged that primary tumors can release signals that
mobilize other cell types to establish a premetastatic
niche ahead of disseminating tumor cells landing there
to promote the survival and outgrowth of metastatic
lesions [24–26]. Throughout all these steps in the meta-
static cascade, tumor cells also need to evolve the
The FEBS Journal 285 (2018) 1751–1766 ª 2018 Federation of European Biochemical Societies
Autophagy & Metastasis
means to evade immune surveillance and T-cell-
mediated killing [27]. Intriguingly, many features of
the metastatic tumor cell, whether that be mesenchy-
mal properties or ability to escape immune surveil-
lance, are associated with a stem-like phenotype and
linked to drug resistance [20,28]. Autophagy is upregu-
lated in primary human breast, glioblastoma, mela-
noma, esophageal cancer, and hepatocellular
carcinoma upon progression to advanced metastatic
disease and expression of autophagy markers in these
cancers is associated with poor prognosis [29–32],
highlighting novel and important roles for autophagy
at different points in the metastatic cascade. Here, we
review recent work defining new functions of autop-
hagy in cancer metastasis including how autophagy
promotes acquisition of promigratory and invasive
properties, maintains tumor cell stemness and drug-
resistant phenotypes, and molds the coevolution of
tumors with their microenvironment.
Autophagic control of tumor cell
migration and invasion
Increased motility is required for tumor cells to escape
the primary tumor site and to successfully colonize sec-
ondary sites during metastasis [23,33,34]. This involves
protrusion of the plasma membrane in the forward
direction, adhesion of cellular integrins to the extracel-
lular matrix (ECM) and induced signaling through
focal adhesions [35,36], polymerization of the actin
cytoskeleton and contraction of the cell body [37], and
finally detachment of the cell from the ECM behind
the direction of movement [36]. Cells vary in how they
migrate (e.g., amoeboid versus mesenchymal) depend-
ing on cell type and microenvironment [36,38], but all
cell migration involves exquisite coordination of the
processes described above, in some fashion. Over the
past several years, increasing evidence has emerged to
demonstrate a direct role for autophagy in tumor cell
motility and invasion during metastasis [39–42], includ-
ing through turnover of components of the cell migra-
tion machinery [43,44] and ECM proteins [45], as well
as other roles such as modulation of the tumor cell
secretome [46].
Various studies have demonstrated a key role for
autophagy in focal adhesion dynamics during cell
migration and invasion [42–44, 47–49]. Inhibition of
focal adhesion kinase (FAK), such as occurs during
cell detachment from the substratum, resulted in
autophagic degradation of active SRC kinase. This
required c-CBL, a SRC-binding protein, and E3 ubiq-
uitin ligase that contains a LIR motif and interacts
directly with processed LC3B to promote autophagic
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turnover of SRC [42]. SRC turnover by autophagy
also required Ambra1, a multifunctional protein
known as an inhibitor of the Beclin1-VPS34 initiation
complex but that alternatively can interact with FAK
to limit active SRC at focal adhesions and promote
SRC degradation by autophagy [49]. Inhibition of
autophagy restored SRC expression at focal adhesions
in adhesion-stressed cells but was associated with cell
death [42] indicating that autophagic turnover of SRC
was required in response to cell detachment or FAK
inactivation (Fig. 2). Interestingly, FAK inhibition
increased autophagic flux suggesting that FAK activity
inhibits autophagy, possibly by sequestering or other-
wise interfering with FAK-interacting protein of 200
kD (FIP200) that although first identified as a protein
that interacts with and inhibits FAK kinase activity
[50], has subsequently been identified as the mam-
malian homolog of yeast autophagy gene Atg17
(Autophagy-related gene 17) and a key component of
the preinitiation complex in mammals that interacts
with ULK1 and ATG13 (Fig. 1) [51]. FAK may also
inhibit autophagy through its inhibitory interaction
FIP200
FAK
Amino acid
deprivation;
Low ATP Rho
SRC
Beclin1
LC3 ROCK1
NBR1
PXN
?
Focal
adhesion AUTOPHAGY
turnover
Autophagy cargo
Autophagy regulator
Fig. 2. Coordinate control of autophagy and cell migration.
Autophagy and cellular motility are coordinated at the molecular
level through reciprocal control of both processes by common
modulators including Focal adhesion kinase (FAK), FAK-Interacting
Protein 200 kD (FIP200), and Paxillin (PXN). Autophagy promotes
cell motility through turnover of focal adhesion complexes and
specifically via degradation of focal adhesion component, Paxillin
(PXN). Autophagy also promotes turnover of SRC in response to
FAK inhibition to prevent cell death in response to cell detachment.
Both FAK (through TSC2) and PXN (through ill-defined
mechanisms) feed back to modulate autophagic flux. Similarly,
there is a reciprocal interaction between autophagy and members
of the Rho family of small G proteins involved in cell migration.
Autophagy promotes Rho turnover via p62/Sqstm1 while ROCK1
that acts downstream of Rho stimulates autophagy through
phosphorylation of Beclin1.
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E. E. Mowers et al.
with Tuberous Schlerosis Complex 2 (TSC2) resulting
in activation of mTOR (Fig. 2) [52,53]. It will be
important in future studies to determine whether FAK
requires FIP200, TSC2, or other signaling mechanisms
to modulate autophagy and to assess the extent to
which FIP200 mediates cross-talk between autophagy
and cell migration.
Recent work from our laboratory demonstrated a
critical role for autophagy in the motility and invasion
of metastatic tumor cells in vitro and for tumor metas-
tasis in vivo [44]. Inhibition of autophagy reduced
tumor cell motility due to decreased focal adhesion
disassembly. This was attributed to accumulation of
Paxillin (PXN), a core component of focal adhesions
[44,48] and PXN was identified as a LC3-interacting
protein that contains a conserved LIR motif (Fig. 2)
[44]. The interaction between PXN and LC3B was pro-
moted by oncogenic SRC and required the Y40 resi-
due at position +1 of the LIR motif in PXN [44], a
site previously identified as a target of SRC phospho-
rylation [54]. Consistently, the ability of oncogenic
SRC to promote cell motility and invasion was depen-
dent on phosphorylation of Y40, interaction of PXN
with LC3, and functional autophagy (Fig. 2) [44]. The
targeting of PXN for autophagic degradation in the
highly metastatic tumor cells studied did not require
either of the cargo adaptors p62/Sqstm1 or Near
BRCA1 (NBR1) [44] but a different mechanism may
be at play in other cell types since in Ras-transformed
MCF10A breast epithelial cells, focal adhesion turn-
over by autophagy was specifically dependent on
NBR1 (Fig. 2) [43]. In addition, c-CBL has also been
reported to be required for targeting PXN to
autophagosomes for degradation [48], in addition to
its role in promoting SRC turnover [42]. Similar to
FAK that is both a regulator of autophagy and regu-
lated by autophagy, PXN is required for efficient
autophagosome formation in MEFs [55], is phospho-
rylated by Ulk1 and along with vinculin relocates from
focal adhesions to autophagosomes in response to
nutrient deprivation [55]. These studies highlight a crit-
ical role for autophagy in focal adhesion dynamics in
tumor cells and a reciprocal role for focal adhesion
components in modulating autophagy.
An intriguing reciprocal relationship also exists
between control of the Rho family of small GTPases
and autophagy during cell migration. RhoA, Rac1,
and CDC42 GTPases modulate cell motility by pro-
moting formation of membrane protrusions, lamel-
lopodia, and filopodia, respectively [36,56,57]. The
ability of rho1 to induce hemocyte migration during
wound healing in Drosophila was dependent on atg1
(autophagy-related gene-1) and ref(2)P the fly
E. E. Mowers et al.
homolog of cargo adaptor p62/sqstm1 [40]. Chemical
inhibition of autophagy prevented blood cell migration
to larval wound sites in flies while knockdown of be-
clin1 or ulk1 prevented mouse macrophages spreading
in response to inflammatory signals [40]. p62/Sqstm1
has since been shown to target mammalian RhoA to
the autophagosome for degradation [58] with the fail-
ure to turn over RhoA in cells knocked down for
ATG5 resulting in RhoA buildup at the midbody dur-
ing mitosis, cytokinesis defects, and aneuploidy [58].
Conversely, Rho signaling has been implicated in the
regulation of autophagy [59,60] with Rho-associated
kinase 1 (ROCK1) identified as a regulator of starva-
tion-induced but not basal autophagy [59]. Inhibition
of ROCK1 resulted in the formation of enlarged,
immature autophagosomes leading the authors to sug-
gest that ROCK1 promotes autophagy by limiting
time spent in early phagophore elongation phases of
autophagy [60]. ROCK1 is also activated by amino
acid deprivation leading to direct phosphorylation of
Beclin1 by ROCK1 on Thr119 causing disruption
of the Beclin1/Bcl-2 complex resulting in derepression
of autophagy (Fig. 2) [61,62]. Meanwhile, Rac1 plays
a role in modulating Rab7, a different small GTPase
that promotes maturation of late-stage autophago-
somes and lysosomal fusion [63]. The efficient cycling
of Rab7 GTPase activity as required for
autophagolysosome maturation in response to amino
acid starvation is controlled by armus, a RabGAP that
localizes to autophagosomes through direct interaction
with LC3 [64,65]. Rac1 also interacts with armus and
promotes autophagic recycling of E-cadherin during
EGF-stimulated cell scattering [64]. Failure to inacti-
vate Rac1 during amino acid starvation blocked
autophagy due to Rac1 binding to LC3 interfering
with the interaction of armus with LC3, thereby pre-
venting Rab7 localization to maturing autophagolyso-
somes [65]. Finally, RhoA, Rac1 and CDC42 were
also identified within an autophagy-centered human
gene interaction network built on known autophagic
responses to microenvironmental cues (Fig. 2) [66].
These and other studies highlight yet again how cell
migration is molecularly coordinated with control of
autophagy and vice versa.
Autophagy in tumor stem cells and
metastatic dormancy
Autophagy is required for normal tissue stem cell
maintenance and differentiation [67–69] with
hematopoietic stem cells dependent on autophagy for
survival [70,71] and muscle stem cells dependent on
autophagy to prevent senescence [72]. More
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Autophagy & Metastasis
specifically, mitophagy has been directly implicated in
preventing stem cell aging and promoting stem cell
self-renewal [73–75] such that defective mitophagy in
mammary stem cells inhibited preferential segregation
of younger mitochondria to daughter stem cells and
older mitochondria to daughter nonstem cells [73]. The
requirement for mitophagy in the self-renewal of
hematopoietic stem cells has been linked to elimination
of metabolically active mitochondria thereby maintain-
ing HSCs in a glycolytic state with low levels of oxida-
tive metabolism [76–78]. The balance between
glycolysis and oxidative metabolism is key to deter-
mining whether stem cells remain quiescent or undergo
differentiation [74,79–81]. By limiting mitochondrial
mass through mitophagy and stimulating glycolysis
while limiting oxidative metabolism, stem cells can
maintain their slow cycling, self-renewing state (Fig. 3)
[80]. By contrast, inhibition of glycolysis and enhance-
ment of mitochondrial respiration promotes differenti-
ation that involves mitochondrial remodeling, reduced
mitophagy, dispersed cytoplasmic localization, and
increased expression of enzymes involved in the TCA
cycle and the electron transport chain [79,81–84].
Cancer stem cells (CSCs) are defined functionally as
those relatively rare cells in a tumor that have the
capacity to self-renew and to differentiate to regenerate
all aspects of tumor heterogeneity [23,85,86]. Various
reports have correlated the expression of CSC mark-
ers, such as CD44, with increased invasiveness and
metastasis [87–91]. Indeed, stresses such as hypoxia
and TGF-b that promote epithelial-to-mesenchymal
transition (EMT) and a more motile tumor cell pheno-
type, also induce features of CSCs, including increased
self-renewal and upregulation of CD44 (Fig. 3)
[34,85,86,92–94]. Significantly, stresses such as hypoxia
and TGF-b also induce autophagy, alongside EMT
and stemness [32,95] and numerous studies have now
linked increased autophagy to maintenance of CSCs
(Fig. 3) [29–32,96]. In human breast ductal carcinoma
in situ (DCIS), increased autophagy was detected in
those rare tumor cells that showed tumor-initiating
capacity and migratory ability and CD44+ CSCs were
dependent on autophagic flux for their ability to form
mammospheres in vitro and tumors in vivo [29,96–98].
In esophageal squamous cell carcinoma (ESCC),
autophagy was required for the upregulation of CD44
that accompanied induction of EMT (Fig. 3) [30].
Here again, it was mitophagy that was specifically
upregulated in ESCC cells undergoing EMT [30], pos-
sibly in response to increased ROS production and
membrane depolarization that is known to activate
Parkin-dependent mitophagy [99]. Inhibition of Par-
kin-mediated mitophagy attenuated upregulation of
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Autophagy & Metastasis
Cancer stem cell
Self-renewal properties;
Mesenchymal gene expression;
Reduced mitochondrial mass.
Fig. 3. Requirement for autophagy in stem cell maintenance. Autophagy is required for maintenance of a stem-like phenotype both in
normal tissue stem cells but also in certain types of cancer stem cells. Stresses (hypoxia, TGF-b) that induce stem-like properties, including
self-renewal capability, EMT, upregulation of CD44, also induce autophagy that is required for many of these stem-like features, including
upregulation of CD44. Recent work has highlighted a specific role for mitophagy in promoting a stem-like state in cancers, possibly by
limiting ROS production and promoting glycolytic metabolism that may limit differentiation.
CD44 and led to cell death [30]. Similar to what has
been described for pluripotent stem cells, reduced
mitochondrial mass has also been reported to distin-
guish CSCs from non-CSCs (Fig. 3) [100,101]. How-
ever, quiescent PDAC cells with CSC properties were
recently described as relying on oxidative respiration
to maintain their stem cell state and were less depen-
dent on glycolysis than proliferating cells in the bulk
of the tumor [102]. Clearly, the role of mitophagy in
CSCs needs to be addressed more directly to determine
whether reduced mitochondrial mass arising from
increased mitophagy in response to EMT, cancer ther-
apy or other challenges, is indeed required to establish
and/or maintain tumor stem cell properties.
The ‘reawakening’ of tumor cells at distant sites
leading to outgrowth of macrometastatic disease many
years after primary tumors were “successfully” treated
has led to the concept of metastatic dormancy
[22,23,103,104]. Various studies have shown a role for
autophagy in promoting tumor cell survival during
tumor dormancy [105]. For example, inhibition of
autophagy blocked re-emergence of ovarian tumors
after dormancy induced by the ARHI tumor suppres-
sor [105]. Preclinical studies also showed that autop-
hagy inhibition in the El-Myc mouse model of B-cell
lymphoma following treatment with alkylating agents
blocked tumor recurrence validating a role for autop-
hagy in promoting both tumor dormancy and drug
resistance [106]. Autophagy may promote the dor-
mancy of disseminated tumor cells simply by supplying
key amino acids and other nutrients or alternatively,
as discussed above, autophagy may play a more
instructive role by eliminating mitochondria, modulat-
ing redox balance, and actively promoting the stem
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E. E. Mowers et al.
EMT
Hypoxia Non-stem cell
TGF-
Increased
mitophagy
Decreased
mitophagy Differentiated phenotype;
Epithelial gene expression;
High mitochondrial mass.
cell state [22,107,108]. Indeed, CSC markers are upreg-
ulated on disseminated tumor cells in the bone marrow
of breast cancer patients [109] and such observations
have led to the suggestion that dormant tumor cells
are in fact CSCs that depend on autophagy to survive
at secondary sites over extended periods of time and
expand later as metastatic lesions made up of both
CSCs and nonstem tumor cells representing the full
heterogeneity of rapidly growing tumors [22,105]. If as
suggested, dormant tumor cells are preferentially reli-
ant on autophagy for survival, then this provides a
rationale for combining autophagy inhibition with
conventional therapeutic responses to eliminate dor-
mant tumor cells and prevent subsequent metastatic
outgrowth.
Autophagy contributes to the therapy resistance of
numerous cancers, including resistance to conventional
genotoxic therapies [110,111], prostate cancer to
androgen ablation therapy [112], breast cancer to
SERMs [113–115], GIST to Imatinib [116,117], lung
cancer to tyrosine kinase inhibitors [118–120], glioblas-
toma to temozolomide [121], myeloma to bortezomib
[122,123], melanoma and brain cancers to B-Raf inhi-
bitors [124–126], as well as resistance of various can-
cers to PI3K inhibitors [127]. Thus, there is a
compelling rationale to combine these therapeutic
approaches with agents that inhibit autophagy, such as
chloroquine, an antimalarial drug that inhibits autop-
hagy by increasing the pH of the lysosome and block-
ing lysosomal proteases [128,129]. Early clinical trials
combining chloroquine with more conventional thera-
pies, such as temozolomide for glioblastoma treatment
showed promise with autophagy inhibition more than
doubling survival times [128,130,131]. One particularly
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E. E. Mowers et al.
successful trial applying chloroquine to conventional
doxorubicin treatment of non-Hodgkins lymphoma in
dogs demonstrated both that effective chloroquine
doses were well-tolerated and also improved overall
drug response and progression-free survival [132].
More recent studies have reported efficacy of adjuvant
chloroquine treatment in clinical trials for an extended
range of human cancers, as reviewed in more detail
elsewhere [128,129].
However, the ability to target autophagy in cancer
has also been limited by the high doses of agents such
as chloroquine required to elicit an effect in humans
[128,129,133], the unpredictable effects of lysosomal
inhibitors used to block autophagy on the activity of
mTORC1 that is regulated at the lysosome also
[134,135], the unknown off-target effects of com-
pounds such as chloroquine [128] and the possible
adverse consequences of systemic autophagy inhibition
[136]. An additional hurdle to assessing the efficacy of
autophagy inhibition in cancer treatment is the lack of
reliable markers of autophagy in human tumors [129].
Nevertheless, the development of improved derivatives
of chloroquine that are efficacious at lower doses [133]
and simultaneously target both autophagy and
mTORC1 activity at the lysosome [137], in addition to
development of targeted inhibitors of ULK1 [138,139],
VPS34 [140], and other enzymes required for autop-
hagy [141] holds significant promise for our ability to
overcome the tumor-promoting effects of autophagy in
cancer therapy resistance and metastatic dormancy
[12].
Autophagy in the tumor
microenvironment
Cancer cells coevolve with their tumor microenviron-
ment and the role of autophagy in modulating how
the cancer cell interacts with other cell types in the sur-
rounding milieu is emerging as a key topic in deter-
mining whether autophagy inhibition is likely to be
effective in cancer treatment [129]. How autophagy
modulates the interaction of tumor cells with compo-
nents of both the innate and adaptive immune systems
is particularly complex, as has been reviewed exten-
sively elsewhere [142,143]. Autophagy promotes the
release of damage-associated molecular patterns
(DAMPs) and ATP from dying tumor cells thereby
recruiting CD8+ cytotoxic T lymphocytes (CTLs) that
synergize with conventional therapeutics to eliminate
cancers [144,145]. Autophagy also promotes tumor
immune surveillance through trafficking of tumor-
antigens through the lysosome for cross-presentation
on dendritic cells and by suppressing infiltration of
The FEBS Journal 285 (2018) 1751–1766 ª 2018 Federation of European Biochemical Societies
Autophagy & Metastasis
tumor-promoting FoxP3+ T-regulatory cells [145,146].
These antitumor activities of autophagy in eliciting an
immune response would argue that inhibiting autop-
hagy is not justified in combination with conventional
cancer therapy. However, the role of autophagy in
modulating tumor immunity may be context depen-
dent with other studies using less immunogenic tumor
models indicating that antitumor immunity is not
adversely affected by autophagy inhibition [147] and
that in fact autophagy promotes escape from immune
surveillance in some models [148,149] possibly through
indirect mechanisms relying on the role of autophagy in
promoting EMT and secretion of immune-modulatory
cytokines [150]. A recent study has identified a novel
role for autophagy in suppressing the antitumor activ-
ity of certain types macrophages in a mouse model of
pancreatic ductal carcinoma (PDAC) [151]. Inhibiting
autophagy through inducible expression of a dominant
negative Atg4B allele (Atg4BCA) decreased PDAC
tumor growth and induced tumor regression of already
formed tumors in an autochtonous model of PDAC
[151]. However, when autophagy was similarly inhib-
ited in an orthotopic transplant model using primary
tumor cells from the autochtonous model, tumor
growth was inhibited but tumor regression of existing
tumors was not observed [151]. Inhibiting autophagy
by expression of Atg4BCA caused macrophage accu-
mulation in autochtonous PDAC tumors while deple-
tion of macrophages prevented tumor regression
induced by autophagy inhibition without affecting
rates of tumor cell growth. These findings indicate
strongly that autophagy plays both a cell intrinsic role
promoting tumor cell growth and also has a noncell
autonomous effect suppressing the antitumor activities
of macrophages [151]. It was not determined how
autophagy inhibition resulted in macrophage recruit-
ment to the tumor although production of DAMPs by
dying tumor cells as a result of autophagy inhibition
could provide an explanation. As reviewed elsewhere
[142] [143], further investigation is required to clarify
when and how autophagy modulates the interaction of
tumor cells with immune cells in the tumor microenvi-
ronment.
Several recent reports have illuminated how autop-
hagy can be induced in certain other nontumor cells
that make up the tumor microenvironment to promote
tumor cell growth and invasion [18,152]. Examination
of the interaction between pancreatic ductal adenocar-
cinoma (PDAC) cells and pancreatic stellate cells
(PSCs), a specialized type of fibroblast that makes up
a large part of the desmoplastic microenvironment in
PDAC, revealed that autophagy was upregulated
specifically in PSCs when grown with pancreatic tumor
1757
Autophagy & Metastasis E. E. Mowers et al.

cells but not when cocultured with normal ductal
epithelia. This in turn promoted protein catabolism in
PSCs generating alanine to fuel the TCA cycle, oxygen
consumption, and lipid synthesis in the adjacent tumor
cells (Fig. 4) [18]. The effects of increased alanine
uptake by tumor cells and its conversion to pyruvate
to fuel TCA cycle and lipid synthesis allowed glucose-
derived carbon to be more efficiently used to promote
serine and glycine biosynthesis with knock-on effects
for nucleotide production. These effects of coculture of
tumor cells with PSCs or PSC-conditioned media was
blocked by inhibiting autophagy in PSCs or by knock-
ing down the GPT1 alanine transaminase (that con-
verts alanine to pyruvate) in tumor cells. The growth
stimulatory effects of PSCs on PDAC growth were
observed both in vitro and in vivo in both subcuta-
neous and orthotopic mouse models using human
PDAC cells and both human and mouse PSCs [18].
Consistent with a role in promoting tumor growth,
there was selection against expression of the autophagy
inhibitory activity of Atg4BCA in PSCs in an auto-
chtonous mouse model of PDAC [151].
Complementary work highlighted the role played by
autophagy in activating PSCs to produce promigratory
and invasive cytokines such as IL-6, in addition to
extracellular matrix proteins [153]. Conversely, inhibi-
tion of autophagy in PSCs resulted in adoption of a
quiescent state, reduced expression of IL-6 and ECM
proteins, attenuated migration/invasion properties and
reduced metastasis to the liver of coinjected PDAC
cells in an orthotopic mouse model (Fig. 4) [153].
Autophagy is known to promote secretion of IL-6 and
other factors [46,154,155] and thus inhibiting
autophagy as a means to limit tumor cell migration
and invasion, in addition to tumor cell growth is
well-justified.
In pancreatic cancer, the desmoplastic reaction that
is dependent on PSC activation is a barrier to therapy
both by inducing hypoxia and stem-like phenotypes in
the tumor cells, and by physically preventing access of
the vasculature and drugs to the tumor [156]. Efforts
to improve delivery of drugs to PDAC tumors, by
degrading the desmoplastic ‘fortress’ around these
tumors [157], have failed and this has been attributed
to selection for more aggressive tumor cells and
increased vasculature leading to easier egress of Tregs,
reduced tumor immune surveillance, and easier escape
of more aggressive mesenchymal tumor cells into the

Pancreatic ductal
adenocarcinoma (PDAC)

Metabolites
Autophagy (eg. alanine);
Inducing signals,
Pro-migratory
IL-6? Other?
cytokines
(eg. IL-6)

Pancreatic stellate
cell (PSC)

Fig. 4. Autophagy in the tumor microenvironment supports tumor cell growth through supply of nutrients and other factors. The growth of
pancreatic ductal adenocarcinoma (PDAC) is supported by a highly complex and dense desmoplastic stroma that includes pancreatic stellate
cells (PSCs). Autophagy plays a critical role in the tumor-promoting activity of PSCs that supply associated PDAC tumor cells with the
breakdown products of autophagy, including amino acids such as alanine. Inhibition of alanine metabolism in tumor cells neutralized the
protumor activity of PSCs. Autophagy is induced in PSCs by their association with PDAC cells although how tumor cells signal to PSCs to
induce autophagy is not yet clear. Autophagy in PSCs was also required for production of proinflammatory cytokines such as IL-6, that may
be feeding back to both promote tumor cell motility and mobilization of metabolites systemically in the animal, but also may also contribute
to the signal that induces autophagy in PSCs in the first place. Additional metabolites and factors may also contribute to additional aspects
of how autophagy in the tumor microenvironment promotes tumor growth and metastasis.

1758 The FEBS Journal 285 (2018) 1751–1766 ª 2018 Federation of European Biochemical Societies
E. E. Mowers et al.
periphery [158,159]. Thus, efforts to limit autophagy in
PSCs may provide a more promising alternative in
PDAC treatment since it would both limit the ability
of PSCs to supply tumor cells with nutrients such as
alanine [18], as well as proinvasive factors, such as IL-
6 [153] but importantly autophagy inhibition would
also be predicted to limit acquisition of stem-like,
drug-resistant features and other malignant properties
by the tumor itself, as described above (Fig. 3).
Genetic analysis of the role of autophagy in tumor
growth in Drosophila has further emphasized the sig-
nificance of autophagy in the tumor microenvironment
[152]. Here, it was shown that autophagy is upregu-
lated systemically in flies bearing malignant RasV12;
scrib-/- tumors in their eye-antennal imaginal discs but
not with benign RasV12 tumors [152]. Ablation of
autophagy in the tumor had only a modest effect on
tumor growth but inhibition of autophagy in either
the nontumor epithelia surrounding the tumor or in
the entire animal had a marked effect on both tumor
growth and invasiveness [152]. Indeed, transplant of
quiescent RasV12;scrib-/-;Atg13-/- autophagy-deficient
tumors into autophagy competent host flies could res-
cue tumor growth while transplant into autophagy-
deficient host flies could not [152]. In this model,
induction of autophagy in the tumor microenviron-
ment, which was referred to as non-cell-autonomous
autophagy (NAA) was independent of tumor growth
since dominant negative PI3K blocked tumor cell
growth but the tumor was still able to induce autop-
hagy in neighboring nontumor cells. Intriguingly, the
ability of the tumor to induce NAA was dependent on
tumor-specific activation of TNF, JAK/STAT, JNK,
and Hippo pathway signaling via Yorkie [152] with
Yorkie transcriptional targets upd1 and upd3 (both IL-
6 like cytokines in flies) able to induce NAA [152].
The Yorkie target ImpL2, an insulin/IGF antagonist
secreted by overproliferating tissues was previously
shown to cause systemic tissue wasting in flies
[160,161] but was not required to induce NAA in flies
with RasV12;scrib-/- tumors [152]. In line with the work
from Kimmelman and colleagues [18], silencing of the
amino acid transporter slimfast in tumors, reduced
tumor growth although uptake of a specific amino acid
or other possible mediators of the non-cell-autono-
mous/autophagy-dependent signal feeding tumor cells
was not identified [152].
These various studies collectively demonstrate that
malignant tumors induce autophagy in nontumor cells
making up their microenvironment in a manner that
further promotes tumor growth and progression
(Fig. 4). Intriguingly, the fly studies suggest that tumor
growth can induce autophagy systemically in tissues
The FEBS Journal 285 (2018) 1751–1766 ª 2018 Federation of European Biochemical Societies
Autophagy & Metastasis
throughout the animal and it remains to be determined
which factors drive this phenomenon but likely signals
that circulate, again perhaps IL-6. Critically, these
reports demonstrate a key role for autophagy in non-
tumor cells in producing metabolites that fuel tumor
cell growth. In the case of mammalian pancreas cancer
studies, this appears to be driven by autophagy-depen-
dent production of alanine from associated nontumor
cells that is taken up by the tumor and fed into the
TCA cycle and lipid biosynthetic pathways among
other [18] with a similar mechanism identified in flies
where slimfast was required for the positive effects of
NAA on tumor growth [152]. Production of alanine
by cancer-associated fibroblasts in PDAC predomi-
nantly affected tumor growth rate [18] but autophagy
in nontumor cells also drove tumor cell invasion and
metastasis [152,153], perhaps through production of
promigratory cytokines, such as IL-6.
These studies open up new perspectives on the role
of autophagy in cancer progression and metastasis.
Particularly intriguing is the possibility that diet and
metabolic stresses inducing autophagy in distant
organs could influence tumor growth and metastasis at
another site or indeed that tumor growth may influ-
ence systemic metabolism by promoting autophagy in
key organs, such as the liver. Also, there is the possi-
bility that other metabolites in addition to alanine,
produced by protein catabolism during autophagy in
nontumor tissue may play a signaling or metabolic
role in promoting tumor progression and metastasis.
These are all avenues of future investigation.
Acknowledgments
This work was supported by NIH/NCI RO1
CA162405 (KFM), NIH/NCI T32 CA009594 (MNS),
NIH/GM T32 GM007281 (EEM).
Author contributions
All authors reviewed the literature, discussed the key
topics to include, and contributed to all sections of the
manuscript.
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