Review Article Open Access

Cancer stem cells and autophagy: Facts and Perspectives

Ahmed Hama€ı1,2, Patrice Codogno1,2, and Maryam Mehrpour1,2,�

1INSERM U1151-CNRS UMR 8253 Institut Necker Enfants-Malades (INEM), 2Universit�e Paris Descartes,Sorbonne Paris Cit�e, Paris France

CONTENTSAbstract

2. INTRODUCTION

3. COMPLEXITYOFCANCERSTEMCELLS:TUMOR

HETEROGENEITY AND CSC PLASTICIY

3.1. Tumor heterogeneity: not all cells in a tumor are

equal

3.2. Clonal diversity of CSCs

3.3. Plasticity of CSCs

3.4. Tumor microenvironment

3.5. MicroRNAs and epigenetic regulation

3.6. Metastatic capacity of CSCs

4. PATHWAYSREGULATINGCSCMAINTENANCE

AND PLASTICITY

4.1. Embryonic and novel stem cell signaling path-

ways are active in CSCs

4.2. Role of IL-6 and RTK signaling in CSC

5. AUTOPHAGY IN CANCER CELLS

5.1. Tumor-suppressing and tumor-promoting func-

tions of autophagy

5.2. Autophagy and tumor cell metabolism

5.3. Autophagy and tumor cell dissemination and

metastasis

6. AUTOPHAGY IN STEM CELLS

7. AUTOPHAGY IN CANCER STEM CELLS

7.1. Cancer stem cell-associated microenvironment

and autophagy

7.1.1. Extracellular microenvironment

7.1.2. Cellular microenvironment

7.2. Autophagy, a hallmark of cancer stem cells

7.3. Hedgehog pathways

7.4. Notch pathways

7.5. Wnt pathways

7.6. Receptor tyrosine kinase pathways

7.7. IL-6/JAK/STAT3 pathways

8. THERAPEUTICMODULATIONOFAUTOPHAGY

IN CSC AND CONCLUDING REMARKS

ACKNOWLEDGEMENTS

References

Abstract: Themodulation of autophagy is now recognized as one of the hallmarks of cancer cells. Depending on the context andthe type of cancer, autophagy may suppress tumor growth or may allow cancer cells to overcome metabolic stress and thecytotoxicity of chemotherapy. Recent studies have shed light on the role of autophagy in normal stem cells and in cancer stemcells (CSC). TheCSCconcept has been a compelling but controversial idea formany years. Recently several independent groupsreported the in vivo existence of CSCs in brain, intestinal, and skin tumors. Specific targeting of CSCsmay improve the efficacy ofcancer therapy. In this review, we will first summarize briefly our current understanding of the CSC concept, regulation of tumorheterogeneity, and CSC plasticity. We will then provide an update on the role of autophagy and signaling pathways in CSCmaintenance and plasticity and, finally, will discuss the potential of targeting pathways involved in autophagy in CSCs fortreatment of cancer.

Keywords: macroautophagy, cancer stem cell models, signaling pathways, plasticity, therapeutic potential.

2. INTRODUCTIONThe concept that cancers result from aberrant versions

of normal developmental processes is very old (von

Hansemann, 1897). During the last 10 years, stem

cell biology concepts have been reapplied with a new

force to the field of cancer biology. Bonnet et al.

provide the first evidence for the existence, within

�Corresponding author: Maryam Mehrpour INSERM U1151, INEM,

14RueMariaHelenaVieiraDaSilva -CS 61431-75993Paris Cedex 14,

France Phone:C331 72 60 64 80;Mobile:C336 63 43 15 62; Fax:C331

72 60 63 99, E-mail: AhmedHama€ı: ahmed.hamai@inserm.fr,Maryam

Mehrpour: maryam.mehrpour@inserm.fr, Patrice Codogno:

patrice.codogno@inserm.fr

Received: June 3, 2014; Revised: July 7, 2014;

Accepted: July 7, 2014

Journal of Cancer Stem Cell Research (2014), 2:e1005�2013 Creative Commons. All rights reserved ISSN 2329-5872DOI: 10.14343/JCSCR.2014.2e1005http://cancerstemcellsresearch.com

human acute myeloid leukemia (AML), of a hierarchi-

cal organization reminiscent of a stem cell system [1].

Subsequently, the cancer stem cell (CSC, also known as

tumor-initiating or tumor-propagating cell) model

has been progressively applied to the study of many

other tumor types, especially solid epithelial carcino-

mas [2–16].

Tumors can be viewed as "caricatures" of normal

tissues where different cell types, generated as a result

of the processes of multi- lineage differentiation, and

are organized into hierarchical structures [17]. The

theory that tumor tissues are sustained in their long-

term growth by a subset of cancer cells endowed with

stem cell properties has significant experimental sup-

port. This subset of cancer cells displays two of the

hallmark properties of stem cells: capacity for self-

renewal (i.e., the ability to regenerate with intact

long-term proliferation and expansion potential) and

the ability to differentiate (i.e., the ability to develop

to more mature progeny thus generating cellular diver-

sity) [18, 19].

Using genetic lineage-tracing strategies three differ-

ent groups independently demonstrated the fate of CSCs

in vivo during tumor progression [20–22]. These studies

did not end the CSC debate, as lineage tracing has its

own limitations [23]. Driessens et al., in their elegant

study, found that benign papillomas are hierarchically

organized in vivo in support of the CSC hypothesis [21].

By using a multicolor conditional reporter assay,

Schepers et al. discovered that, in accordance with CSC

theory, most of the Lgr5C intestinal cells with CSC

properties differentiated into Lgr5� non-CSCs in vivo,

albeit Lgr5� cells were not being fate mapped in their

experiment [22]. Employing fate mapping in a mouse

model of glioma, Chen et al. identified a subset of

endogenous NestinC tumor cells that are the source of

new tumor cells after the drug temozolomide is admin-

istered to transiently arrest tumor growth [20]. The

NestinC cells are main driving force for tumor growth,

therapy resistance, and tumor relapse. More recently, an

international research team studied a group of patients

with myelodysplastic syndromes (MDS, a malignant

blood condition), which frequently develops into acute

myeloid leukemia, and found that rare Lin� CD34C

CD38� CD90C CD45RA� MDS cells function as MDS-

propagating cells in patients with low to intermediate

risk MDS [24].

Taken together, these studies define a common meth-

odological frame work for the experimental validation of

the CSC model. As a rule, a tumor is usually described as

following a CSC model based on the fulfillment of three

criteria:

1. Within tumor tissues, only a subset of cancer cells is

endowed with tumorigenic capacity when transplanted

into immunodeficient mice. These cells are characterized

by a distinctive repertoire of surfacemarkers (Table 1) and

can be reproducibly purified from other cells that do not

have this capacity.2. Tumors grown from tumorigenic cells containmixed

populations of both tumorigenic and non-tumorigenic

cancer cells, thus recreating the phenotypic heterogeneity

of the parent tumor.3. Tumors grown from tumorigenic cells can be serially

passaged indicative of self-renewal properties.

[25, 26].

Researchers prone that not all cancers appear to

follow the CSC model and that in such cancers,

although genetic and/or phenotypic heterogeneities are

present, all cells are equally tumorigenic and there is no

cellular hierarchy [25, 26]. However, two factors make

it difficult to know whether there is a cellular hierarchy

in this kind of cancers: CSC plasticity and transience of

markers. Further experimental investigation is needed

to know whether some cancers, including melanoma, do

not follow the CSC model.

Macroautophagy (hereafter referred to as autophagy,

see Box 1) is a lysosomal degradative process that

acts on cytoplasmic cellular components (such as

macromolecules, long-lived proteins, and damaged

organelles). Autophagy plays a major role under basal

conditions in cytoplasm quality control and cellular

homeostasis. In the response to stress, autophagy is

induced and acts to promote cell survival by providing

nutrients (e.g., amino acids, fatty acids, ATP). Indeed,

during period of starvation (such as glucose withdrawal,

growth factor or serum deprivation, or hypoxia),

the stimulation of the autophagic pathway fuels cells

with nutrients via lysosomal recycling to maintain

metabolic activity and to respond to the demand for

energy.

Autophagy is a multistep cellular process (see Box 2).

The master negative regulation of autophagy initiation is

mediated by the mammalian target of rapamycin complex

1 (mTORC1), which integrates multiple signaling path-

ways that are sensitive to the availability of amino acids,

ATP, growth factors, and the level of reactive oxygen

species (ROS). The modulation of autophagy is now

recognized as one of the hallmarks of cancer cells.

Depending on the type of cancer and the context, autop-

hagy can serve as a tumor suppressor or it can overcome

metabolic stress and the cytotoxicity of chemotherapy to

facilitate cancer cell survival. Recent studies have shed

light on the role of autophagy in tumor immune response

cross-talk with the microenvironment. In this review, we

will briefly describe recent advances in the characteriza-

tion of CSCs, focusing on the role of autophagy in cancer

stem cells, and then will discuss the implications of

modulating autophagy in therapies designed to target

CSCs.

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Table 1. Markers used for the identification of CSCs

Cancer Marker Expression in healthy tissue

AML CD38low Multiple stages of B and T cells

B cell malignancies CD19 Broadly on B lymphocytes

Breast ALDHC

CD24low Broadly on B cells; neuroblasts

CD44high Broadly in many tissues

CD49fCDLL1high DNERhigh

CD90

CD133

CD133CCXCR4C

Linlow

a6-integrin

Hedgehog-Gli activity

Colorectal ABCB5

ALDHC

b-catenin activity

CD24

CD26

CD29

CD44 Broadly in many tissues

CD133 Proliferative cells in multiple organs

CD166

EpCAM/ESA Pan-epithelial marker

LGR5

PrPc Broadly in many tissues

Glioma ALDHC

CD15, CD90

CD133high Proliferative cells in multiple organs

a6-integrin

MET

Nestin

SSEA-1C

Head and neck CD44high BMI-1 Broadly in many tissues

Hematopoietic malignancies CD34high Hematopoietic and endothelial progenitors

Liver CD13

CD24low

CD44high Broadly in many tissues

CD90 T cells, neurons

CD133 Proliferative cells in multiple organs

EpCAM

OV6

GEP

Lung ABCB2, ALDHC

CD44 Broadly in many tissues

CD90 T cells, neurons

CD117

CD133 Proliferative cells in multiple organs

CD24 Broadly on B cells; neuroblasts

Prostate ALDHC

CD44high

CD133

CD166

a2b1-integrin

a6-integrin

Trop 2

Pancreas ABCB2, ALDHC

c-Met

CD44 Broadly in many tissues

CD24low Broadly on B cells; neuroblasts

EpCAM/ESA Pan-epithelial marker

(Continued on the following page)

CSC and autophagy 3

J Cancer Stem Cell Res � http://cancerstemcellsresearch.com

3. COMPLEXITY OF CANCER STEM CELLS:TUMOR HETEROGENEITY AND CSCPLASTICIY

3.1 Tumor heterogeneity: not all cells in a tumor are

equal

Recent studies have revealed complexities of CSCs, such

clonal diversity and plasticity, and this has led to revision

of the original CSC model [27]. For a long time, tumor

heterogeneity was explained by a stochastic clonal model

(Figure 1), which is based on the notion that all tumor cells

are equipotent and can develop into tumors given genetic

and/or epigenetic changes. In the CSC model, not all cells

in tumors are equal [19]. Tumor tissues are characterized

by cellular hierarchy: Only a subset of tumor cells has the

ability for long-term self-renewal, and these cells have

tumorigenic potential much greater than that other cancer

cells. Tumor hierarchical organization consists of stem

cells, progenitors, and differentiated cells. Molecular het-

erogeneity is present not only among different tumors

same type (inter-tumor heterogeneity) but also among

different regions within the same tumor (intra-tumor

heterogeneity) [28].

3.2 Clonal diversity of CSCs

The models of CSC and clonal evolution are likely not

mutually exclusive. Instead, cancer cells that match the

characteristics predicted by one model or the other likely

coexist in a dynamic state. Both models predict that new

somatic mutations can generate clonal diversity, which

further increases tumor heterogeneity.

Table 1. Markers used for the identification of CSCs (continued )

Cancer Marker Expression in healthy tissue

CD133 Proliferative cells in multiple organs

CXCR4

Nestin

Nodal-Activin

Melanoma ABCB5 Keratinocyte progenitors

ALDHC

CD20 Broadly on B lymphocytes

CD133 Proliferative cells in multiple organs

CD271

Sarcoma CD44 Broadly in many tissues

CD105

STRO1

Table is adapted from refs: [19, 203, 204]

BOX 1 DIFFERENT FORMS OF AUTOPHAGY AND SELECTIVITYThe term of autophagy covers three mechanisms: macroautophagy, microautophagy, and chaperone-mediated

autophagy (CMA). Macro- and microautophagy are conserved in all eukaryotic cells, whereas CMA is restricted to

mammalian cells. Macroautophagy begins with the formation of a double-membrane-bound vacuole called the

autophagosome, which sequesters cytoplasmic material and delivers it to the lysosomal compartment. Micro-

autophagy is the direct engulfment of cytoplasmic material by the lysosomal membrane, and CMA is the lysosomal

importation of KFERQ-containing proteins, via their interaction with hsc70 and the lysosomal membrane protein

LAMP-2a. Macroautophagy and microautophagy are bulk processes, but, in many cases, they can be highly

selective of cellular structures, including peroxisomes (pexophagy), mitochondria (mitophagy), endoplasmic

reticulum (reticulophagy), ribosomes (ribophagy), midbody, micro-nuclei and part of the nuclei (nucleophagy),

lipid droplets (lipophagy), aggregate-prone proteins (aggrephagy), secretory granules (zymophagy), damaged

lysosomes (lysophagy), andmicroorganisms (xenophagy). The paradigm for selective autophagy is the recognition

of the cargo by autophagy receptors that connect the cargo to the autophagy machinery. Autophagy receptors (p62/

SQSTM1, NBR1, NDP52, optineurin) recognize ubiquitylated targets via ubiquitin-associated domains and the

protein LC3 via its LC3-interacting region (LIR) motif. In many autophagy receptors, phosphorylation consensus

sites are present in the vicinity of the LIR motif. Phosphorylation of these sites has been shown to increase the

affinity of the autophagy receptor for LC3. Defects in selective autophagy are accompanied by accumulation of

cargo and reduction in turnover of autophagy receptors. These defects disrupt tissue homeostasis and result in

diseases. As an example, accumulation of the autophagy receptor p62/SQSTM1 leads to activation of NF-kB and of

the transcription factor Nrf2, two events linked with tumorigenesis.

4 A. Hama€ı et al.

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3.3 Plasticity of CSCs

The concept of "CSC plasticity" posits that the majority of

tumor cells are stem cells with varying degrees of "stem-

ness" governed by microenvironmental cues and other

stochastic cell autonomous mechanisms [29–31].

Although, the underlying molecular mechanism for CSC

plasticity is unknown, the concept of plasticity links clonal

evolution and CSC models and demands that CSC hier-

archymust be seen as bidirectional balance between CSCs

and differentiated non-CSCs [32]. CSC plasticity could be

a stochastic process or might be orchestrated by micro-

environmental signals depending on the type and genetic

background of a tumor [23, 33].

The evidence that some cancer cells can undergo

reversible changes in their competence to form tumors

offers an alternative explanation for the increased tumor-

igenic potential of subsets of cancer cells that is indepen-

dent of the differentiation of CSCs. Some cancer cells can

reversibly transition between epithelial and mesenchymal

states, and there is evidence that breast cancer cells in the

mesenchymal state are more competent than those in the

epithelial state to form tumors [34]. However, Xie et al.

recently report that the epithelial–mesenchymal transition

(EMT) does not alter tumor-initiating capacity of breast

cancer cells [35].

Using the histone demethylase JARID1B as a biomark-

er, Roesch et al. characterized a distinct subpopulation of

slow-cycling melanoma cells that is required for contin-

uous tumor growth [36]. Cells that express JARID1B are

more competent to sustain tumor growth than JARID1B-

negative cells. Loss of JARID1B results in a reduction in

the number of melanoma cell metastases in the lung,

leading the authors to conclude that JARID1B might not

be necessary for tumor initiation but is probably required

for tumor progression and the initial formation of metas-

tases. Quintana et al. also reported that many markers

appear to be reversibly expressed by tumorigenic mela-

noma cells [37]. None of 22 heterogeneously expressed

markers, including CD271 and ABCB5, enriched tumor-

igenic cells. For example melanomas metastasized in

mice, irrespective of whether they arose from CD271� or

CD271C cells. Data suggest that there may not be a

hierarchy of tumorigenic and non-tumorigenic cells in

melanoma [37]. In skin squamous cell carcinoma,

CD34low cells can generate CD34hi cells on transplanta-

tion. However, CD34low cells that acquire CD34 expres-

sion do not generate secondary tumors with the same

efficiency as CD34hi cells isolated from the primary

tumors [38], suggesting that the reversibility of surface

marker expression does not imply that the fate of tumor

cells is also fully reversible.

Although lineage tracing studies provide strong evi-

dence in favor of the presence and differentiation of CSCs

in vivo in different solid tumors [20–22], a recent study

showed that Lgr5� cells can also act as the cell of origin for

intestinal adenomas and that these cells do not meet the

BOX 2 ROLE OF ATGS IN AUTOPHAGYThe discovery of autophagy-related (Atg) genes in yeast was a milestone in our understanding of how the

autophagosome is formed [202]. Fifteen ATG proteins constitute the core machinery of autophagosome formation.

In mammalian cells, these ATGs are hierarchically recruited to form a phagophore, or isolation membrane, that

subsequently elongates to form the autophagosome. Recent studies have shown that ATG assembly takes place at

the point of contact between the endoplasmic reticulummembrane and the outer membrane of mitochondria. Other

membranes, such as those of the endosomes and the Golgi apparatus, and the plasma membrane also contribute to

the biogenesis of the autophagosome. Several functional modules involving ATG have been identified during the

phases of autophagosome formation from the initiation step to the elongation/closure step. Five functional modules

have been defined: the ULK1 complex (ULK1 is the mammalian homolog of yeast Atg1), class III phosphati-

dylinositol 3-kinase (PIK3C3/Vps34) complex I (which contains PIK3C3 and Beclin 1, the latter is the mammalian

homolog of the yeast Atg6). The activation of ULK1 complex is down-regulated by mTORC1, which integrates

multiple signaling pathways that are sensitive to the availability of amino acids, ATP, growth factors, and the level

of ROS. The two complexes ULK1 and PIK3C3 are involved in the initiation of autophagy. Phosphatidylinositol 3-

phosphate (PtdIns3P), which is produced by the enzymatic activity of PIK3C3, recruitsWIPI1/2 (homologs of yeast

Atg18) at the phagophore, and DFCP1 at the endoplasmic reticulum site of autophagosome formation known as the

omegasome. These two PtdIns3P-binding proteins characterize the third functional cassette. One of the functions of

WIPI2 is to control the transport of the multispanning membrane ATG9 from the phagophore to a peripheral

endosome/Golgi localization. The ATG9 protein makes up the fourth functional cassette. In yeast, the fusion of

Atg9-containing vesicles is important in the very early stages of autophagy. In mammalian cells, the trafficking of

ATG9 to the phagophore is also an early event that occurs soon after autophagy induction. The last functional

module consists of the two ubiquitin-like conjugation systems: ATG12–ATG5 and the LC3–phosphatidyletha-

nolamine (LC3 is the mammalian homolog of yeast Atg8), which are involved in the elongation and closure of the

autophagosomal membrane.

CSC and autophagy 5

J Cancer Stem Cell Res � http://cancerstemcellsresearch.com

criteria of the CSC model [39]. This counter challenging

evidence may possibly support the concept of CSC plas-

ticity as it can be taken to suggest that non-CSCs can be de-

differentiated into CSCs since Lgr5� cells can give rise to

Lgr5C cells in vivo.

It is also important to note that nearly all existing reports

about reversible transition between CSCs (tumorigenic)

and non-CSCs (non-tumorigenic) states come from stud-

ies performed using immortalized cell lines [23]. As

certain high passage cell lines have been found to show

endogenous phenotypic plasticity, it is critical to investi-

gating the extent of such plasticity in spontaneously

arising tumors in vivo [32]. In support of in vivo CSC

plasticity, Friedmann-Morvinski et al. reported that most

differentiated cells in the central nervous system undergo

de-differentiation upon defined genetic alterations to gen-

erate a neural stem cells or a progenitor state capable of

initiating and maintaining tumor progression. These cells

can also give rise to the heterogeneous populations

observed in malignant gliomas [40]. More in depth in

vivo lineage tracingwithmulticolor labelling of non-CSCs

and CSCs may elucidate differentiation hierarchies and

confirm CSC plasticity.

Various factors clearly have significant roles in main-

taining CSC plasticity as well as solid tumor heterogene-

ity: 1. genetic instability or DNA mutations, 2. the tumor

microenvironment and CSC niche, and 3. microRNAs and

other epigenetic factors. Excellent recent reviews have

highlighted these various factors [23, 32, 41]. It seems

plausible that all these factors are essential for tumor

heterogeneity and for CSC plasticity.

3.4 Tumor microenvironment

Tumormicroenvironments consist of several types of cells

in direct contact with tumor cells and secreted extracel-

lular factors that may be critical for self-renewal and

maintenance of CSCs and tumor progression. Several

studies indicate that a dynamic interaction between tumor

cells and stromal cells, including immune cells, cancer-

associated fibroblast (CAFs), endothelial cells, tumor-

associated macrophages (TAMs), regulates tumor pro-

gression by acting on stemness proprieties or CSC

Founder cellStem or progenitor cellStem or progenitor cell

TumorHeterogenity

Mutation

Stochastic model Original CSC model CSC plasticity model

genetic & epigentic factors

genetic & epigentic factors

: CSC : progenitor

Figure 1. Cartoon representation of differentmodels of tumorigenesis.The stochastic or clonalmodel assumes that every cell within a tumor

has equal tumorigenic potential. From a common ancestor, different subclones emerge due to selection and distinct mutations. The original CSC

hypothesis posited that transforming genetic or epigenetic factors in a single cell result in stem-like characteristics that allowing this cell to divide

symmetrically (to self-renew) or asymmetrically (to generate differentiated cells in order to propagate and form a tumor). A combination of the

CSC model and clonal evolution has been proposed to account for clonal diversity of CSCs. The CSC plasticity model is based on the original

CSCmodel but introduces the concept of dynamic stemness, whereby a non-CSC can acquire stemness and become a CSC. CSCs and non-CSCs

thus exhibit reversibility of stemness as indicated by special arrows. Thismodel emphasizes the need for a combinatorial therapeutic approach to

target both CSC and non-CSC populations.

6 A. Hama€ı et al.

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populations. One example of an interaction between

CAFs and tumoral stem cells was reported in ovarian

cancer. This interaction results in the activation of the

hedgehog (Hh) pathway in aldehyde dehydrogenases

positive (ALDHC) ovarian CSCs and the induction of

bone morphogenic proteins (BMPs) 2 and 4, stimulating

proliferation [42]. CAFs and TAMs are a source of

multiple growth factors and chemokines/cytokines

including TGF-b, IL-6, IL-8, and oncostatin M (OSM)

that induce the emergence of CSCs associated with EMT

in several types of cancers including that of the breast

[43–45].

A recent study revealed synergistic interactions

between endothelial cells and CSCs that promote the

malignant properties of glioblastoma CSCs in an IL-8-

dependent manner [46]. Endothelial cells not only secrete

high levels of IL-8 but also inducedCSCs to upregulate the

IL-8 cognate receptors CXCR1 and CXCR2, which col-

lectively enhances CSC migration, growth, and stemness

properties. Accordingly, in both in vitro and in vivo studies

of glioblastoma, Hedgehog and NOTCH ligands (Hh,

Jagged-1, respectively) are released from tumor endothe-

lial cells and activate the Hh and NOTCH pathways in

tumor cells promoting stem-like characteristics [47, 48].

In similarmanner, endothelial cells promote the colorectal

CSC phenotype through a soluble form of Jagged-1 [49].

More importantly, these studies revealed the activity of the

Hh network in the niche of CSCs in which glioma cells

induce, via VEGF pathway, the secretion of Sonic Hh by

endothelial cells resulting in the paracrine-mediated

activation of Hh pathway in glioma cells. Although the

origin of CSCs remains controversial, these data support

cell plasticity in which cancer cells may acquire CSC

phenotypes through re-programming or de-differentia-

tion process [50, 51]. In further support, it has been

reported that glioblastoma stem-like cells are able to

differentiate into vascular endothelial like cells or vas-

cular pericytes, raising the possibility that these differ-

entiated cells arise from CSCs and generate a CSC niche

[52–54].

3.5 MicroRNAs and epigenetic regulation

MicroRNAs (miRNAs) regulate functions of both normal

stem cells and CSCs [55–58], and dysregulation of

miRNA expression has been implicated in tumorigenesis

[59].Yu et al. provided the first evidence for the significant

downregulation of let-7 in CSCs [60]. A wide range

of cancer types are characterized by overexpression of

Lin 28, a factor that binds to miRNAs including let-7.

Blockage of let-7 miRNA biogenesis and subsequent de-

repression of let-7 miRNA target genes by Lin28 plays

important roles in cancer progression and metastasis.

Lin28 is activated in CSCs (for review see [61]). The

Struhl laboratory has provided evidence for the role of

miR-200 family members in regulating CSC plasticity in

breast cancer patients [62, 63]. Overexpression of miR-

200b and miR-200c strongly inhibits the proliferation and

tumorigenicity of breast cancer cells. This affect is medi-

ated by regulation of polycomb repressor complex (PRC)

proteins [57, 62]. This complex influences chromatin

structure and regulates transcriptional activity of a number

of important loci including the Ink4a locus, which encodes

the tumor suppressor proteins p16INK4a and p19ARF (also

called p14ARF when referring specifically to the human

version). Proteins in PRC1 cooperate with PRC2 to serve

as transcriptional repressors by binding to histones dis-

playing histone H3 lysine (H3K27) trimethylation [64].

The CSC population overexpresses enhancer of zeste

homolog 2 (EZH2), one of the key members of PRC2,

responsible for trimethylating H3K27 in various cancers

including breast, pancreas, prostate, and ovary [65, 66].

EZH2 is essential for maintenance of an intact CSC

population. Furthermore, BMI-1 (for B cell specificMolo-

ney murine leukemia virus integration site 1) and EZH2

are overexpressed in CSCs and necessary for initiation of

various solid tumors such as head and neck cancers and

glioblastoma [67]. BMI-1 complexeswith other PRC1 and

PRC2 proteins to repress the expression of genes bearing

H3K27 histone marks including those encoding INK4a/

ARF and HOX [68, 69]. BMI-1 expression is negatively

regulated by miR-200c.

The Weinberg laboratory reported that microenviron-

mental signals, such as TGFb, enhance CSC plasticity. In

response to signals, the promoter of ZEB1 (for zinc-finger

E box-binding homeobox 1) converts from bivalent to

active chromatin by reductions in the presence of

H3K27me3 repressive histone modifications. This in turn

induces non-CSC to CSC conversion in breast cancer [70,

71]. miR-200 and ZEB1 have antagonizing effects, and

inhibition of miR-200 increases ZEB1 mRNA levels and

the CD44low-to-CD44hi transition in HME-flopc cells.

This is abrogated when ZEB1 levels are reduced, which

indicates that ZEB1 is a key miR-200 target. miR-200c

represses the expression of BMI-1, and loss of miR-200b

increases levels of suppressor of zeste 12 homolog

(SUZ12) expression and H3K27 methylation [57]. Inter-

estingly, ectopic expression of SUZ12 in transformed cells

generates CSCs [63]. In addition, miR-200c levels are

regulated by a complicated loop involving BMI-1 and

ZEB1 [72].

These findings revealed that miR-200 family members

are essential in regulation of CSC formation and function

via PRC1 and PRC2 complexes. miR-200 is also involved

in modulating the expression of SRY (sex determining

region Y)-box 2 (SOX2), Kruppel-like factor 4 (KLF4),

BMI-1, and SUZ12 genes, to regulate stemness in cancer

cells [57, 73]. The Tang laboratory found that miR-34a is a

key negative regulator of CD44C prostate cancer cells. In

addition, miR-34a directly targets stem cell-related genes

like CD44 and NOTCH, implying that these miRNAs

maintain the CSC phenotype by halting their differentia-

tion [74].

CSC and autophagy 7

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3.6 Metastatic capacity of CSCs

There is much discussion regarding the metastasis of

cancer stem cells. Some studies have indicated that sub-

populations of tumorigenic pancreatic cancer [75] and

colon cancer [76] cells are enriched for the capacity to

metastasize. The Wicha laboratory found that breast can-

cer cell lines contain functional CSCs with metastatic

capacity and a distinct molecular signature [77]. Using

a molecular tracking approach, Dieter et al. identified a

population of extensively self-renewing, long-term tumor-

initiating cells (LT-TICs) [78]. The vast majority of LT-

TIC clones are able to form metastases, suggesting that

this subpopulation may be responsible not only for tumor

regeneration, but also for tumor dissemination. We have

recently shown that a subpopulation of CD44C CSCs that

express cellular prion protein promote EMT via ERK2,

thereby contributing to metastatic capacity of colorectal

cancer [15].

4. PATHWAYS REGULATING CSCMAINTENANCE AND PLASTICITY

4.1 Embryonic and novel stem cell signaling pathways

are active in CSCs

Normal stem cells and CSCs share signaling pathways

such as Wnt, Notch, and hedgehog [79]. Certain signal-

ing pathways are novel to CSCs and are involved in

maintenance and plasticity. Interleukin-6 (IL-6), Janus

Kinase 1 (JAK1), signal transducer and activator of

transcription 3 (STAT-3) signaling and their crosstalk

with the receptor tyrosine kinase (RTK) pathway and

stemness related transcription factors play central roles

in regulating CSC plasticity in solid cancer (Figure 2

and 3) [43, 62, 80].

4.2 Role of IL-6 and RTK signaling in CSC

Introduction of an activatingmutant of STAT3 is sufficient

to induce transformation of immortalized cells [81], sug-

gesting that activation of the JAK-STAT signaling path-

way is sufficient to mediate tumorigenesis. More recent

studies have suggested that STAT3 activation is important

for the tumorigenic ability of CSCs in glioblastoma [82,

83], lung [84], and colon cancers [85, 86]. Moreover, IL-6

plays a crucial role in maintaining CSC plasticity [80]. As

downstream targets, IL-6 regulates CSC-associated

OCT4, SOX2,NANOG, andCD44 gene expression (Figure

2), which converts non-CSCs into CSCs [43].

Recent proteomic data indicate an alliance of PI3K and

STAT3 in cancers. In glioblastoma stem cells, STAT3 is

phosphorylated on residue Y705 and activated in a PI3K-

dependent manner. A dominant negative mutant of

STAT3 interferes with PI3K-induced oncogenic transfor-

mation. Phosphorylation of STAT3 ismediated by the Tec

family kinaseBMX [87]. TheRTKpathways aremediated

by the proto-oncogenemembers of the EGFR/ErbB(Her2)

family, which are highly expressed and deregulated in

many cancers including glioblastomas and breast and head

and neck squamous cell carcinomas [88–90].Activation of

EGFR promotes the acquisition and maintenance of var-

ious CSC characteristics including the expression of self-

renewal markers (CD44, BMI-1, Oct-4, NANOG,

CXCR4, and SDF-1) and capacity to form tumorspheres.

Accordingly, EGFR/Her2 targeting with Gefitinib, erlo-

tinib, lapitinib, or anti-ErbB2 monoclonal antibody tras-

tuzumab results in the decrease in cancer initiating cells

and the decrease in their resistance to chemotherapy drugs

[91]. The EGF-RTK signaling pathway acts as a link

between Hedgehog and IL-6 signaling [92]. PI3K activat-

ed viaEGFR signaling can turn onGli activator (Gli A) via

the PI3K-AKT-mTORC1-S6K pathway to induce the

expression of CSC-specific genes or expression of ligands,

such as Jagged 2, that activate classical stem cell signaling

pathways.

Hypoxia (or low oxygen (O2) levels) is believed to be

involved in tumor progression and metastasis. Hypoxia

provides tumor cells with cues for maintenance of a stem-

like state and may help to drive the linkage between EMT

and CSCs. Hypoxia has been shown to dramatically

increase the CD133C stem cell fraction in a variety of

cancers and consequently drives the CSC phenotype.

Hypoxia-inducible factors (HIFs), particularly HIF1a and

HIF2a, are key mediators of the cancer hypoxia response

and high expression levels of HIFs correlate with a poor

prognosis in various tumor types [93]. HIF2a acts as a

transcriptional activator of stemness-specific genesOCT4,

SOX2, CD44, KLF-4, andMYC, among other [51]. Unlike

of HIF2a, HIF1a is activated via PI3K-AKT to induce

expression of Twist1, an EMT modulator, and polycomb

repressor BMI-1 to support the downregulation of p16 and

p19 that are required for the differentiation of CSCs. It is

noteworthy that these newly discovered signaling path-

ways act in synergistic manners with the overall result of

inducing stemness. Further elucidation of the complex

crosstalk among these pathways will increase our under-

standing of CSC plasticity.

5. AUTOPHAGY IN CANCER CELLS5.1 Tumor-suppressing and tumor-promoting

functions of autophagy

The modulation of autophagy is now recognized as one

of the hallmarks of cancer cells. There is accumulating

evidence that autophagy plays a role in each of the

various stages of tumorigenesis. Depending on the type

of cancer and the context, autophagy fulfils a dual role,

having both tumor-promoting and tumor-suppressing

properties. Recent studies have shed light on the role

of autophagy in the immune response to tumor growth.

Readers interested in a more detailed discussion of the

role of tumor suppressors and oncogenes in the regula-

tion of autophagy should see several recent reviews

[94, 95]. The first link between autophagy and tumor

suppression came from the discovery that monoallelic

8 A. Hama€ı et al.

J Cancer Stem Cell Res � http://cancerstemcellsresearch.com

disruption of BECN1 on chromosome 17q21 is observed

in 40% to 75% of human breast, ovarian, and prostate

tumors [96, 97]. In addition, the heterozygous deletion

of BECN1 in mice increases spontaneous malignancies

[98], whereas restoration of its expression in breast

cancer cells inhibits tumor growth in a mouse xenograft

model [96]. However, recently data suggests that, con-

trary to these previous reports, BECN1 is not signifi-

cantly mutated in human cancer and therefore is not a

classic tumor-suppressor gene [99].

Autophagy is stimulated by tumor suppressors (PTEN,

TSC1/TSC2, LBK1, DAPK, p19ARF) and inhibited by

WntJag/Delta

Notch Fz Receptor

β-catenin

Hh

Oct4, CD44, Jag1Survivin, Myc

Nucleus

Cytoplasm PITCH1Smo

GliAGliR

GliAJag2, SnailCyclinD, Myc

NICD

miR-200c

K27me3K27me3

K27me3

PRC1 PRC2

BMI-1

p19ARF, HOX

Bmi-1

CBXs

PHCs RING1A/B

PRC1

EZH2/1

Suz-12 EED

PRC2

Oct4, Sox2Nanog, CD44Klf-4, Myc, CCND1

IL-6

IL-6RJAK

Stat3

Stat3

S6KmTOR

RTK (EGFR)

GAB1

PI3K

AKTBMX

HIF2α

HIF1α HIF2α

Twist

self-renewalexpression of stemness markers

self-renewal expression of stemness markers

BMI-1-mediated regulationof CSC self-renewal and EMT

HES, ZEB1,Snail

Hypoxia

EGF

SuFu

Gli1/2/3

+Hh-Hh

Figure 2. Representation of the signaling pathways involved inCSCmaintenance, self-renewal, andplasticity. CSCs usemany of the same

signaling pathways that are found in normal stem cells such as Notch, Wnt, and hedgehog (Hh). In addition, pathways involving IL-6, EGF, and

hypoxia are also important in CSCs. See text for details of newly discovered signaling. Crosstalk between pathways and epigenetic regulation are

shown. In Notch signaling, four Notch receptors and five ligands, including jagged (jag) and delta like (delta), are important. Upon ligand

binding, the receptor is first cleaved in the extracellular domain by ADAM (a disintegrin metalloproteinase) and subsequently within

transmembrane domain by gamma-secretase complex to release the notch intracellular domain (NICD). The NICD translocates into the

nucleus replacing co-repressors with co-activators to induce expression of target genes.

Wnt signaling begins when one of the Wnt proteins binds to a Frizzled (Fz) family receptor. Co-receptors, such as lipoprotein receptor-related

protein (LRP)-5/6, receptor tyrosine kinase (Ryk), and ROR2, may facilitate Wnt signaling. Upon activation of the receptor, Fz and LRP recruit

theDisheveled protein (Dvl) to the intracellular side of the cell. Dvl then recruits glycogen synthetase kinase 3 (GSK3) to phosphorylate LRP and

inhibit phosphorylation of beta-catenin. The inhibition of the phosphorylation of beta-catenin releases it from conjugation to ubiquitin. Thus, the

beta-catenin is released from the "degradation complex" and translocates into the nucleus increase the activity of target genes. Several kinases

including GSK3 connect the Wnt and Hh pathways.

In the absence of Hh, the transmembrane protein Patched (PITCH1) inhibits signaling from the Smoothened molecule (Smo). In this state,

full-length Gli is phosphorylated then cleaved to its repressor form (GliR) and enters the nucleus where it represses transcription of target

genes. Upon Hh binding to PITCH1, the complex is internalized, allowing Smo activation. Cleavage of Gli is inhibited and the full-length

activator form (GliA) enters the nucleus and activates transcription of target genes. A complex ofmolecules regulates the cytoplasmic/nuclear

translocation of Gli.

BMI-1 acts as a major regulator of CSC self-renewal and plasticity. BMI-1 expression is negatively regulated by miRNAs such as mir-200c.

BMI-1 forms an essential component of polycomb regulatory complex 1 (PRC1). PRC1 has a fundamental role in the organization of

chromatin structure, which, in turn, regulates the expression of a cassette of genes involved in stem-cell behavior. These genes include the

INK4A/ARF and HOX. Proteins in the PRC1 complex cooperate with PRC2 complex proteins to serve as transcriptional repressors by binding

to histones displaying histone H3 lysine (H3K27) trimethylation. BMI-1 may also cooperate with TWIST1 to regulate the EMT, a state

associated with CSCs and metastasis.

CSC and autophagy 9

J Cancer Stem Cell Res � http://cancerstemcellsresearch.com

oncogenes (AKT/PI3K, BCL-2, RAS, c-MYC, tyrosine

kinase receptors). The proto-oncogene c-Myc has been

shown to induce autophagy independently of its onco-

genic property. Factors encoded by these tumor sup-

pressors have inhibitory effects on mTORC1 activity

and those encoded by the oncogenes inhibit ULK1

initiation complex or Beclin-1 (for review see [95]). In

ensuring cellular homeostasis in healthy cells, autop-

hagy prevents excessive ROS production and the result-

ing DNA damage and genomic instability, which can

lead to tumoral transformation. Indeed, it has been

shown that tumors formed by autophagy-deficient cells

(by biallelic loss of Atg5 or monoallelic loss of Beclin1

[96]) display genomic instability and unrepaired DNA

damage, resulting from ROS generation and impairment

of autophagy-mediated removal of damaged organelles,

especially mitochondria [100–102]. Clinical observa-

tions show that, low or deficient Beclin-1 expression is

associated with shorter overall survival in some cancers,

whereas elevated Beclin-1 levels appear to be associated

with a more favorable prognosis [103]. Interestingly, it

has been recently demonstrated that STAT3 inhibits

Beclin-1 expression by directly binding to the BECN1

promoter and recruitment of histone deacetylase 3

(HDAC3) in non-small cell lung cancer cells [104]. An

inverse correlation between phosphorylated STAT3 and

Beclin-1 was reported in glioma samples [105]. In

addition, mutations of other ATG genes, such as ATG2B,

ATG5, ATG9B, and ATG12, are found in gastric and

colorectal cancers. Mice deficient in Atg4C, a cysteine

protease required for the processing of LC3, have

increased susceptibility to chemically induced fibrosar-

comas [106], and mice deficient for Atg5 or Atg7

develop benign liver tumors [107]. Overall, the data

suggest that a defective autophagic process is clearly

linked to cancer development. A systematic review and

meta-analysis of the prognostic value of BECN1 and

LC3B indicated that the BECN1 appears to be predictive

of favorable prognosis in gastric cancer, breast cancer,

and lymphoma and that LC3B may predict unfavorable

prognosis of breast cancer. Due to the limited number of

patients and retrospective design of the original studies,

more powerful prospective cohorts are required to verify

these conclusions [108].

The most commonly mutated tumor suppressor gene is

p53. p53 plays opposing roles in the regulation of

ULK1111111ULK1

mTOR

cell growth

Protein synthesis

AUTOPHAGY

RTK

WntNotch

Hedgehog

Cytoplasm Stat3

PI3K BMX

Hypoxia

PKR

eIF2A

AKT

CSC self-renewal and EMT expression of stemness markers

Nucleus

EGF

Stat3

NotchTwist

PKR

AUTOPHAGY

Conventional STAT3 activation

eIF2A

Figure 3. Autophagy and cancer stemness pathways interconnect. Autophagy is a multistep cellular process (see Box 2). The master

negative regulator of autophagy initiation is the mammalian target of rapamycin complex 1 (mTORC1), which integrates multiple signaling

pathways.Activation of theEGFR-class I PI3K-AKT-mTORsignaling axis inhibits autophagy. EGFR signaling acts as a link betweenHedgehog

and IL-6 signaling. STAT3 is a master regulator of IL-6 signaling. Recently, an alliance of PI3K and STAT3mediated by the TEC kinase BMX

was discovered. STAT3 is phosphorylated on residue Y705 and activated in a PI3K-dependent manner. New data identify STAT3 as an

autophagy regulator. In the cytoplasm, STAT3 interacts with the PKR kinase to inhibit eIF2A phosphorylation and reduce autophagy. STAT3

activation disrupts the interaction with PKR kinase resulting in eIF2A activation and subsequently induction of autophagy. This non-canonical

function of STAT3 has an important role in normal cells, and we suggest that it might also affect CSCs.

10 A. Hama€ı et al.

J Cancer Stem Cell Res � http://cancerstemcellsresearch.com

autophagy that depend on its subcellular location. It acts as

a pro-autophagic nuclear factor and an anti-autophagic

cytoplasmic factor, reflecting the spatial control of p53

signaling (for review [109]). Autophagy may also prevent

cancer by regulating the cellular level of p62 (also known

as SQSTM1) [110]. Accumulation of p62 in autophagy-

deficient cells promotes tumorigenesis. p62 serves as a

cytoplasmic scaffold protein and activates pro-survival

and pro-inflammatory transcription factors NF-kB and

NRF2; these factors upregulate expression of antioxidant

genes to protect cells during oxidative stress [111, 112].

Interestingly, p62 mediates activation of mTORC1 by

interacting with Raptor.

Once a tumor is established, autophagy can contribute

to tumor progression, by allowing tumor cells to survive

stressful conditions (e.g., hypoxia, acidosis, nutrient

deprivation) and by sustaining the deep metabolic reor-

ganization that cancer cells undergo after oncogenic trans-

formation. The first genetic clue that autophagy is

necessary for the progression and survival of tumors came

from the observation that the deletion of BECN1 is only

observed on one allele in human cancers; this suggests that

tumor cells require functional autophagy for malignant

transformation [113]. In addition, deletion of the essential

autophagygeneFIP200 impairs oncogene-induced tumor-

igenesis in a mouse model of breast cancer [114]. In solid

tumors, autophagy localizes to the hypoxic regions or the

poorly vascularized regions, and its inhibition induces the

cell death specifically in these regions [102]. This induc-

tion of autophagy in hypoxic regions is regulated inde-

pendently by HIF-1a and the AMPK signaling pathway.

Stabilized HIF-1a induces the BH3-only protein BNIP3,

which disrupts the interaction between Beclin-1 and Bcl-2

orBcl-xL, allowingBeclin-1 to induce autophagy [115]. In

contrast, AMPK mediates mTOR kinase inhibition, thus

turning on autophagy [116]. A mouse model of RAS-

mediated non-small cell lung cancer, in which the tumor-

suppressive and tumor-promoting function of autophagy

can be visualized, it has been demonstrated that autophagy

also promotes tumor growth by suppressing the p53

response, maintaining mitochondrial function, and sus-

taining metabolic homeostasis and survival in stress. In a

mouse model of RAS-mediated lung tumor autophagy

prevents diversion of tumor progression to benign onco-

cytomas [117]. In mammary cancers, partial autophagy

impairment due to allelic loss ofBecn1 increases apoptosis

and significantly delays mammary tumor development

following PALB2 or BRCA1/BRCA2 loss but only when

p53 is present [118]. Thus, autophagy may promote mam-

mary tumor growth by suppressing p53 activation induced

by DNA damage.

5.2 Autophagy and tumor cell metabolism

The "Warburg effect" is a hallmark of tumors: Cancer cells

upregulate aerobic glycolysis and reduce the activity of

mitochondrial electron chain transport thus increasing the

rate of energy production (despite reducing its efficiency).

Thus, the enhanced conversion of glucose to lactate is

observed in tumor cells, even in the presence of normal

levels of oxygen. Aerobic glycolysis supports various

biosynthetic pathways and, consequently, the metabolic

requirements for proliferation. The PI3K pathway is con-

sidered to be a master regulator of the glycolytic pheno-

type through AKT1 and mTOR signaling and subsequent

downstreamHIF-1 activation. TheAMP-activated protein

kinase (AMPK) pathway is also important. AMPK is

considered a metabolic checkpoint as it can control cell

proliferation when activated under energetic stress, and

activation of AMKP results in the inhibition of mTOR

activity and subsequent autophagy induction.

Oncogenic transformation (e.g., due to RAS, C-MYC,

AKT) is characterized by a deep metabolic reprogram-

ming resulting in upregulation of aerobic glycolysis in

favor of the activity of oxidative phosphorylation [119].

Thus, using preferentially glucose, the metabolic demand

of cancer cells is higher than that of normal cells, and

autophagy may help overcome cellular energy deficits

[120]. Indeed, human cancer cell lines bearing activating

mutations in RAS commonly have high levels of basal

autophagy. In a subset of these cancer cells, downregula-

tion of essential autophagy proteins (for example, by

knockout of ATG5 or by treatment of cells with an shRNA

targeting ATG7) abolishes their tumorigenicity and

reduces glucose metabolism indicating the high depen-

dence of autophagy for their survival [102, 121–123].

Autophagy may contribute to metabolism rearrangement

by targeting the degradation of damaged mitochondria

resulting in the decrease of oxidative phosphorylation.

Both RAS and c-MYC impair acetyl-CoA production (by

blocking its generation from the decarboxylation of pyru-

vate and the b-oxidation of fatty acids [122]) cancer cellshave to maintain oxidative metabolism for their survival

[123]. Thus, autophagy might maintain functional mito-

chondria by providing substrates to replenish the mito-

chondrial TCA cycle by amino acids (such as glutamine

resulting in high-energy metabolites), through protein or

lipid degradation, or through the degradation ofmembrane

organelles or lipid droplets and sugars [122].

5.3 Autophagy and tumor cell dissemination and

metastasis

Autophagy may also promote tumor cell dissemination

and metastasis by protecting these cells from anoikis,

which is a form of programmed cell death induced by the

detachment of anchorage-dependent cells from the extra-

cellular matrix. Indeed, it has been demonstrated that this

detachment from the extracellular matrix is a critical step

in dissemination. In addition, metastasis induces pro-

survival autophagy in epithelial cells to protect them

from anoikis, and depletion of ATG protein enhances

cell death and reduces clonogenic recovery after anoikis

[124, 125].

CSC and autophagy 11

J Cancer Stem Cell Res � http://cancerstemcellsresearch.com

6. AUTOPHAGY IN STEM CELLSBoth embryonic and adult stem cells are unique cell

populations characterized by their ability to self-renew

and differentiate into various cells in the body. Embry-

onic stem cells ensure appropriate development, where-

as adult stem cells enable tissue renewal and cellular

homeostasis. Therefore, it was expected that autophagy

would play a crucial role in the quality control mechan-

isms and maintenance of cellular homeostasis in various

stem cells given their relatively long life in organisms.

Accumulating evidence has provided insights into the

crucial role of autophagy in embryonic stem cells

(ESCs), and tissue stem cells that persist throughout

the adult life including hematopoietic stem cells (HSCs),

cardiac or neural stem cells, and mesenchymal stem

cells (MSCs). Autophagy regulates self-renewal, plur-

ipotency, differentiation, and quiescence (for review see

[126–128]). Recent advances in metabolomic analysis

and studies in mouse models indicate that metabolic

reprogramming (i.e., stem cell-specific programs that

may drive the dependency of stem cells on specific

nutrients, such as glucose, fatty acids and glutamine,

as well as impose changes to the wiring of metabolic

pathways to maintain stemness) is also required for the

maintenance of stem cell self-renewal [129]. Various

types of stem cells heavily rely on anaerobic glycolysis.

Maintenance of the stem cell pool and of self-renewing

stem cells are regulated by bioenergetic signaling that

involves the AKT–mTOR pathway, fatty acid metabo-

lism, and glutamine metabolism.

7. AUTOPHAGY IN CANCER STEM CELLS7.1 Cancer stem cell-associated microenvironment

and autophagy

Sharing the same properties as ‘natural’ stem cells, accu-

mulating evidence also support the essential role of autop-

hagy in the maintenance and the self-renewal of cancer

stem cells (CSCs) in several histological types of cancer

including breast, glioblastoma, and pancreatic cancers

[130–133]. Firstly, autophagy has been predicted to ensure

a cytoprotective adaptive mechanism to overcome tumor

microenvironmental stress in solid epithelial tumors. The

molecular tumor microenvironment is characterized by

hypoxia or oxygen-limiting areas, oxidative stress, deple-

tion of nutrients or growth factors, and high levels of

metabolic derivatives including L-lactate, resulting in

extracellular acidosis [42, 134].

7.1.1 Extracellular microenvironment

CSCs are frequently located at the hypoxic core of solid

tumors including breast, pancreatic, and colorectal cancers

as well as glioblastomamultiforme (a vascularized tumor)

[135, 136]. In addition, hypoxia appears to play a critical

role in the formation of the CSC niches [137]. For exam-

ple, anti-angiogenic treatments of patients with breast

cancer or glioblastoma results in hypoxia-mediated autop-

hagy that promotes tumor cell survival and results in an

increase in the population of CSCs [138, 139]. In the case

of the pre-malignant lesions, breast ductal carcinoma in

situ (DCIS), the capacity of CSC-like precursor cells to

survive in the hypoxic and nutrient-deprived environment

of the intraductal niche is dependent on the activation of

autophagy (which is associated with up-regulation of

BECLIN-1, ATG5, MAP1LC3A/B) [140, 141]. As

described above, the effects of hypoxia are mainly medi-

ated by the transcriptional regulators HIF-1a and HIF-2a.Multiple recent experimental studies have revealed that

these two factors are required for maintaining the pheno-

type and function of CSCs in glioblastoma, neuroblasto-

ma, renal cancer, and non-small cell lung cancer (for

review see [142]). They stimulate autophagy by inducing

expression of autophagy-regulating genes including

BNIP/BNIP3L (belonging to BH3-only family). By dis-

rupting the interaction between Beclin-1 and Bcl-2, these

factors allowBeclin-1 to induce autophagy and to promote

survival [115]). In addition, hypoxia-induced BNIP tar-

gets mitophagy resulting in the clearance of damaged

mitochondria and reducing ROS production [143]. High

levels of intracellular ROS promote both the loss of self-

renewal and differentiation and the radiosensitization of

tumor-initiating cells for several types of cancer, including

breast, glioma, and prostate [144–146], indicating that

redox status plays an important role in stem cell mainte-

nance (for review see [147, 148]). The autophagic degra-

dation of mitochondria leads to low levels and activities of

mitochondria and promotes glycolysis, which charac-

terizes both stem cells and CSCs (the Warburg effect or

Warburg-like glycolytic metabolism) in aid of mitochon-

dria-driven oxidative phosphorylation [149].

Although not focused specifically on CSCs, studies

have demonstrated that deletion of FIP200 or other

autophagy genes in oncogene-driven mammary tumor

mouse models of human breast cancer significantly

suppresses mammary tumorigenesis and progression

[114, 150]. Proliferation of tumor cells was reduced in

vitro and in vivo, and there was a significant decrease in

anchorage-independent growth in soft agar. This was

correlated with a compromised glycolysis resulting in an

impaired glucose uptake and intracellular lactate pro-

duction. Collectively, these studies highlight the active

contribution of autophagy to malignant transformation

and in the acquisition or the maintenance of stemness by

CSC-like cell populations through support for the met-

abolic shift.

7.1.2 Cellular microenvironment

Although not formally determined, it is likely that autop-

hagy play a crucial role in the microenvironment of CSCs.

We know this indirectly for certain parameters such as the

autophagy-mediated unconventional secretion and

expression of pro-inflammatory cytokines [151, 152], as

these cytokines are able to modulate the autophagic

response by the activation of the IKK/NF-kB signaling

[153] and the JAK/STAT3 pathway [154]. In addition,

12 A. Hama€ı et al.

J Cancer Stem Cell Res � http://cancerstemcellsresearch.com

several groups of researchers have demonstrated that

autophagic CAFs exposed to oxidative stress (H2O2, ROS)

produce energy-rich glutamine, L-lactate, and ketones to

"fuel" the mitochondrial activity of cancer cells by their

conversion into acetyl-CoA, which is associated with the

CSC phenotype [152]. Indeed, the use of L-lactate and

ketones by cancer cells promotes a "stemness-like" tran-

scriptional profile that is specifically associated with

tumor recurrence, metastasis, and poor clinical outcome.

This seems to bemediated by acetyl-CoA,which promotes

mitochondrial-dependent ATP production and acetylation

of proteins (including histones), both implicated in

the epigenetic control of gene expression. Interestingly,

evidence is accumulating that acetylation and post-

transcriptional modifications, including phosphorylation,

ubiquitination, and lipidation, are involved in the complex

regulatory network that controls autophagy, and these

modifications likely constitute the connection between

autophagy and cellular metabolism (see for review

[155]). However, it remains to be determined how autop-

hagy contributes to the metabolic shift of cancer cells and

the acquisition of ‘stemness’ byCSC-like cell populations,

although all are linked to acetyl-CoA status.

7.2 Autophagy, a hallmark of cancer stem cells

We recently showed that autophagy is also crucial for the

maintenance and tumorigenicity of CSCs in breast cancer

[130, 156]. From breast tumor cells isolated from patients

and from human breast cancer cell lines, mammospheres

that are enriched in breast CSCs display a significantly

higher level of autophagy flux than the adherent counter-

parts, both at basal levels and under starvation conditions.

Furthermore, cells positive for aldehyde dehydrogenase 1

(ALDH1, a cancer stem cell marker, see Table 1) derived

from mammospheres constitutively present higher levels

of autophagy flux than do the bulk tumor cell populations

both at basal levels and under starvation conditions. This

robust autophagic flux is associated with up-regulation of

Beclin-1 at the protein and mRNA levels in breast CSCs/

progenitor cells. The specific depletion of BECN1 or

ATG7 or treatmentwith salinomycin (a chemical inhibitor

of autophagic flux [157]) dramatically reduces both the

size and formation efficiency of mammospheres, reflect-

ing the alteration of the proliferation and the self-renewal

ability, respectively, of CSCs/progenitor cells. More

importantly, the suppression of autophagy in breast

CSCs/progenitor cells by Beclin-1 depletion inhibits their

tumorigenicity in nude mice. Our data are consistent with

those reported by Wei et al., indicating that the suppres-

sion of autophagy by the deletion of FIP200, an important

regulator of autophagy in mammalian cells, inhibits mam-

mary tumorigenesis [114]. Moreover, different autop-

hagy-associated factors such as DRAM1 and p62 (also

known as sequestosome1 (SQSTM1) are highly expressed

in glioblastomas that exhibit a mesenchymal gene signa-

ture and aggressive phenotypes associated with a poor

overall survival [132]. The enhanced expression of

DRAM1 and p62 also correlate with up-regulated expres-

sion of the mesenchymal marker MET, activation of

MAPKs, and enhanced motility and invasion of glioma

stem cells [132]. Since impairment of the autophagic

response dramatically decreases the CD44CCD24�/Low

breast CSC-like cell population [131], crosstalk likely

exists between autophagy and stemness-associated path-

ways that support survival and CSC-like properties. Crit-

ical pathways include classical stem cell signaling

pathways involvingWnt/b-catenin, Notch, and Hedgehog(Hh) and the novel RTK and IL-6/JAK/STAT3 pathways.

7.3 Hedgehog pathways

To date, a relationship between Hh and autophagy path-

ways has been reported in the neuroblastoma cell line

SHSY5Y [158], pancreatic ductal adenocarcinoma cells

[159], the cervical HeLa cell line [160], a chondrosarcoma

cell line [161], and a hepatocarcinoma cell line [162].

Under starvation conditions, the inhibition of the Hh

pathway by theHh antagonist cyclopamine reduces autop-

hagic flux in tumor cells and is associated with a decrease

in Atg5 expression, suggesting transcriptionally mediated

regulation of key autophagic genes by Hh signaling. This

same study also reported the presence of the multiple non-

consensus GLI binding sites in the human ATG5 promoter

[158]. More recently, however, it was shown that Hh

signaling negatively regulates autophagy in pancreatic

cancer cells [159]. In support of this, treatment of cultured

human hepatocarcinoma cells with an Hh signaling ligand

(recombinant Sonic Hh) or either of the agonists SAG or

purmorphamine prevents the induction of autophagy; in

contrast, GANT61 (a small molecule inhibitor of Gli1 and

Gli2) induces autophagy [162]. Hh inhibition-induced

autophagy is associated with up-regulation of Bnip3, lead-

ing to Bnip3-mediated displacement of Bcl-2 fromBeclin-

1 and induction of apoptosis. More importantly, in a tumor

xenograft mouse model, administration of GANT61 inhi-

bits tumor formation in vivo and decreases tumor volume;

this effect is partially blocked by the autophagy inhibitor 3-

MA [162]. It is crucial that the connection between the Hh

pathway and autophagy, in particular in cancer stem cells,

must tobeclarified.Ofnote,Hhhas recentlybeenproposed

to be a general positivemetabolic regulator in cancer [163]

and might act in concert with autophagy to influence the

survival and the maintenance of CSCs.

7.4 Notch pathways

The Notch signaling pathway (Figure 2) is critical in

CSCs from different cancers including breast, pancreas,

melanoma, lung, and glioblastoma [164–168]. Aberrant

activation of Notch signaling, characterized by high

levels of Notch-1, Notch-2, and Jagged-1 ligand, is

associated with CSC features [169, 170]. Accordingly,

the inhibition of Notch signaling depletes the CSC pop-

ulation and reduces their sphere-forming ability, which

CSC and autophagy 13

J Cancer Stem Cell Res � http://cancerstemcellsresearch.com

reflects stem cell self-renewal ability, and their tumori-

genicity in immunodeficient mice [166]. Moreover, it

was reported that the activation of Notch signaling path-

ways is mechanistically linked with the acquisition of the

EMTphenotype and the generation of CSCs in pancreatic

cancer [171]. In addition, known specific chemical inhi-

bitors of the Notch pathway, sulforaphane, quercetin,

curcumin, and isoflavone are also able to inhibit activa-

tion of the Notch pathway cancers of the pancreas and

breast models [172]. However, the molecular mechanism

through which Notch signaling regulates CSC features

remains undefined and its action on autophagy in CSCs

has not yet been explored. Notch target genes Akt,mTOR,

ERK, NF-kB, p53, and VEGF, are known to modulate or

regulate autophagy [173, 174], may also have effects on

the CSC phenotype.

7.5 Wnt pathways

The Wnt/b-catenin signaling pathway also plays a crucialrole in the acquisition of the CSC phenotype [175, 176].

The inhibition of the Wnt/b-catenin signaling pathway

results in a decrease in sphere-forming ability of tumor

cells derived from breast cancer and melanoma, reflecting

the self-renewal of CSCs, reduces tumorigenic capacity

(or tumor-initiating potential) of these cells in mice, and

enhances their sensitivity to drug-mediated apoptosis

[177, 178]. Wnt/b-catenin signaling regulates OCT4 at

transcriptional level the expression.OCT4, in concert with

other transcription factors such as Nanog and Sox2, con-

trols the self-renewal and the maintenance of CSCs [179].

The Wnt/b-catenin signaling pathway is also involved in

the induction of EMT, which is closely related to the

acquisition of a stemness phenotype [180–182]. b-cateninis also known to facilitate adaptation to microenviron-

mental conditions including hypoxia [183] and oxidative

stress [184], and it has been clearly demonstrated thatWnt/

b-catenin signaling negatively regulates both basal and

stress-induced autophagy and p62 expression via T cell-

specific transcription factor 4 (TCF4) both in vitro and in

vivo [185]. Furthermore, during nutrient deprivation,

b-catenin is selectively degraded via the formation of a

b-catenin–LC3 complex, attenuating b-catenin/TCF-driven transcription and proliferation to favor adaptation

during metabolic stress. Thus, these findings reveal a

regulatory feedback mechanism that places b-catenin at

a key cellular integration point coordinating proliferation

with autophagy.

Autophagy has also been shown to negatively regulate

Wnt signaling by promotingDisheveled (Dvl) degradation

in starvation condition [186]. The degradation of Dvl is

mediated by its ubiquitylation, which promotes p62 asso-

ciation and subsequently its p62-mediated binding of LC3.

Dvl also interacts directly with LC3 through its LC3-

interacting region (LIR) [187]. There is an inverse corre-

lation between Dvl expression and autophagy markers in

late stages of human colon cancer, indicating that the

impairment of autophagy may contribute to aberrant Wnt

activation during cancer development [186]. Thus, these

findings, which might be extended to CSCs, support a

regulatory feedback model between Wnt signaling and

autophagy that functions to integrate information from

different signaling pathways.

7.6 Receptor tyrosine kinase pathways

During autophagy, EGFR activates the ras and raf-1

mitogen-activated protein kinase pathways and the

PI3K/AKT pathway. These pathways activate mTOR

signaling resulting in the inhibition of autophagy [95]. It

was recently demonstrated that elevation of autophagy in

gefitinib-treated breast cancer cells is correlated with

downregulation of AKT and ERK1/2 signaling early in

the course of treatment [188]. More importantly, active

EGFR is able to interact with Beclin-1 leading to its multi-

site tyrosine phosphorylation, enhanced binding to inhi-

bitors, decreased Beclin-1-associatedVPS34 kinase activ-

ity, and subsequently suppression of autophagy (Wei, Y.

et al., 2013 cell). Interestingly, this same study showed that

in non-small-cell lung carcinoma tumor xenografts, the

expression of a tyrosine phosphomimetic Beclin-1 mutant

leads to reduced autophagy, enhanced tumor growth,

tumor dedifferentiation, and resistance to EGFR tyrosine

kinase inhibitor therapy. Thus, oncogenic receptor tyro-

sine kinases are able to directly regulate the core autop-

hagy machinery, which may contribute to tumor

progression and chemoresistance. As illustrated in

Figure 2, the EGFR signaling pathway acts as a link

between Hh and IL-6 signaling via mTOR signaling and

nonreceptor tyrosine kinase BMX/STAT3 signaling,

respectively [23].

7.7 IL-6/JAK/STAT3 pathways

It has been clearly demonstrated in breast cancer that the

IL-6/JAK/STAT3 pathway is important for CSC plasticity

as it promotes the selection and expansion of CD44high/

CD24low cells [189, 190]. Like IL-6, OSM (which belongs

to the same family of pro-inflammatory cytokines) is a

robust stimulator of STAT3 and promotes emergence of

CSCs, and EMT programs [44, 191]. Recent data have

identified STAT3 as a cytoplasmic autophagy regulator.

STAT3 interacts with the PKR kinase in the cytoplasm

resulting in the inhibition of eIF2A phosphorylation and in

the reduction of the activity of the autophagic pathway

(Figure 3). Upon activation, STAT3 dissociates from PKR

resulting in activation of eIF2A and subsequent induction

of autophagy [154, 192]. Additionally, genetic or phar-

macological inactivation of STAT3 reduces LC3-II levels

in pancreatic cancer cells, again indicative of a role for

STAT3 in transcriptional regulation of autophagy [193].

Since STAT3 is activated by both inflammatory and

oxidative stress signaling, STAT3 might play a vital role

in the regulation of autophagy through its contributions to

the positive feedback loop between ROS and autophagy,

14 A. Hama€ı et al.

J Cancer Stem Cell Res � http://cancerstemcellsresearch.com

Table 2. Compounds targeting CSC through pathways involved in autophagy (ATG) regulation

Class Drug Tumor type Target

Modulation

of ATG Ref

Autophagy inducers

mTOR inhibitors Rapamycin Malignant glioma,

Pancreatic

cancer, Breast

cancer, Glioma

cancer cells,

Colon cancer

cells

MTORC1 ND [195, 206, 207]

[205]

[205, 208]

Torin, Resveratrol,

Metformin

Breast cancer,

Lymphoma

AMPK ND [199, 209]

Akt inhibitors SZL-P1-4 Several cancer cell

lines, Prostate

cancer

SKP2–SCF E3

ligase activity and

Akt

ND [210]

Proteasome inhibitors Combined MG-

132 and acid

retinoic

Neuroblastma cell

lines

Proteasome ND [211]

Tyrosine kinase inhibitors Erlotinib

associated or not

with

cyclopamine

Glioblastoma EGFR and

Hedgehog

ND [213]

Gefitinib,

PD153035,

Imatinib

mesylate

Nasopharyngeal

carcinoma,CML

EGFR/PI3K ND

[212]

[214]

[215]

HDAC inhibitors Vorinostat,

Sodium butyrate

Neuroblastoma

cells,

Endometrial

tumor cells

HDAC ND [216, 217]

IL6 Curcumin, Ursolic

acid, Resveratrol

Colon cancer cells,

Glioblastoma

STAT3 ND [85, 218]

[219]

Monoclonal antibodies Cetuximab Colon cancer MET signaling ND [215]

EGFR

Hypoxia YC-1, AEW541 Gefitinib-resistant

lung cancer

HIF1a ND [220]

Hypoxia/

starvation

Pancreatic cancer [221]

Farnesyl transferase

inhibitors

BMS-214662 CML Farnesyl transferase ND [222]

Notch Inhibitor Arsenic trioxide Glioma cancer

cells

ND [223]

Hedgehog inhibitors Robotnikinin Glioma cancer

cells, CML

Sonic Hh ND [224, 225] for

review [79]

Cyclopamine,

Synthetic small

molecules HPI

1–4

smo

Gli1/2(HPI-1)

Gli2 (HPI-2 and

HPI-3) Cilia (HPI-

4)

Chloroquine CXCL12/CXCR4

ERK and STAT3

[197]

Wnt signaling Curcumin Breast cancer, Rat

glioma cancer

cells,

Esophageal

Unknown [226, 227]

(Continued on the following page)

CSC and autophagy 15

J Cancer Stem Cell Res � http://cancerstemcellsresearch.com

which is crucial in CSCs. In addition, the phosphorylation

and activation of STAT3mediated bymTORC1have been

reported [194]. Activation of the mTORC1–STAT3 path-

way, which is connected with autophagy signaling, is

required for the viability and the maintenance of breast

cancer stem-like cells [195]. It remains to be determined,

however, how autophagy mediated by this pathway con-

tributes to the maintenance and the plasticity of CSCs.

8. THERAPEUTIC MODULATION OFAUTOPHAGY IN CSC AND CONCLUDINGREMARKS

In addition to its evolutionarily conserved role in promot-

ing cell survival during metabolic stress, autophagy also

plays an essential role in determining how cancer cells

respond to therapy and changing environmental stimuli

[95, 196]. Drug resistance that results in tumor relapse

remains amajor obstacle to improvement of cancer patient

survival, and drug resistance may be due at least in part to

the existence of CSCs. Table 2 summarizes the numerous

studies that have investigated compounds that likely target

CSCs through pathways involved in autophagy regulation;

these compounds include inhibitors of mTOR, Akt, the

proteasome, tyrosine kinases, HDAC, IL6 signaling, hyp-

oxia, farnesyl transferase, Notch, Hh signaling, and Wnt

signaling. Only a few studies, including ours [157], have

reported positive effects of therapeutic agents on modu-

lation of autophagy in CSCs (Table 2, modulation of ATG

column). Depending on the type of cancer and the com-

pound, inhibition or induction of autophagy has been

observed. For example, in one study, it was shown that

the autophagy inhibitor chloroquine targets pancreatic

CSCs via inhibition of CXCR4 and hedgehog signaling

independently of autophagy [197].

For complete eradication of cancer, it may be necessary

to target both CSCs and non-CSC populations. The cross-

talk between tumor microenvironment and CSCs is

believed to be the key source for cancer cell differentiation

and bidirectional conversion [70]. The factors responsible

for bidirectional conversion of cancer cells have yet to be

identified, and the relationship between this conversion

and autophagy is not understood. Taking into account of

the concept CSC plasticity, cancer therapy should both

target CSCs and also prevent CSC generation from non-

CSCs (Figure 1). It also remains to be determined whether

CSCs display ‘oncogene addiction’. This phenomenon is

characterized by an absolute dependence of tumor cells on

continued oncogene expression for their survival, and it

has been proposed that other tumorigenic cell populations

show this dependence.

A few drugs, such as salinomycin [198] and metformin

[199], have been identified that target CSCs in vitro and in

Table 2. Compounds targeting CSC through pathways involved in autophagy (ATG) regulation (continued )

Class Drug Tumor type Target

Modulation

of ATG Ref

cancer, Breast

cancerPhosphosulindac

(OXT-328)

b-catenin

degradation

[228, 229]

[230]

EB1089 Gastric cancer

cells, Prostate

cancer cells

Analog of vitamin D ND [223, 231]

Resveratrol Breast and

pancreatic

cancer cells

Antioxidant ND [232]

Breast cancer Fatty acid synthetase ND [233, 234]

Autophagy inhibitors

Others Chloroquine Pancreatic cancer

cells

Inhibition of

lysosomal

degradation

[221]

Salinomycin Mammary

carcinoma cells,

Several breast

cancer cell lines

Stat3, Jak2 andDNA

methyltransferase1

[235]

Inhibition of

lysosomal

[157]

3MA Pancreatic cancer

cells

Inhibition of

autophagosome

formation

[221]

Wnt Signaling Salinomycin,

Nigericin

Chronic

lymphocytic

leukemia cells

LRP6 ND [236]

OMP-111118R5 Fzd ND [237]

16 A. Hama€ı et al.

J Cancer Stem Cell Res � http://cancerstemcellsresearch.com

vivo. These compounds successfully eliminate CSCs and

have been used alone or in combination with conventional

chemotherapeutic drugs (for review see [200]). Recently,

a pharmacological inhibitor of BMI-1, PTC-209, was

shown to effectively inhibit CSC self-renewal in vitro

and to effectively block tumor growth inmouse xenografts

[201]. Tumors failed to grow back following withdrawal

of treatment, indicating that PTC-209 has irreversible

effects. Furthermore unlike of salinomycin, PTC-209

inhibits tumor growth in the absence of apparent systemic

toxicity. Further investigations of the molecular mecha-

nism of CSC plasticity, themode of action of CSC specific

drugs, and the role of autophagy are required before highly

effective therapies can be identified for patients in all

stages of cancer. Previous preclinical studies have sug-

gested that CSC-targeting agents will have the greatest

efficacywhen they are administered early or in an adjuvant

setting alongside a primary therapy [69]. It will therefore

be interesting to explore the efficacy of PTC-209 in such

settings.

ACKNOWLEDGEMENTSThis work was supported by institutional funding from

the Institut National de la Sante et de la Recherche

Medicale (INSERM), and grants from the Agence Natio-

nale de la Recherche (ANR) and the Agence Nationale de

la Recherche (ANR). We sincerely apologize to authors

whose original articles or important review papers in

autophagy and cancer stem cell research areas could not

be cited owing to space restrictions.

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