Targeting cancer stem cells: Modulating apoptosis and stemness - Chapter 2: Cancer stem cells; important players in tumor therapy resistance

Targeting cancer stem cells: Modulating apoptosis and stemness

Çolak, S.

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2016

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Final published version

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Çolak, S. (2016). Targeting cancer stem cells: Modulating apoptosis and stemness.

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S. Colak & J.P. Medema Published in FEBS Journal, 2014

Cancer stem cells; important players

in tumor therapy resistance

Abstract

Resistance to tumor therapy is an unsolved problem in cancer treatment. A plethora

of studies have attempted to explain this phenomenon and many mechanisms of

resistance have been suggested over the last decades. The concept of cancer stem cells

(CSCs), which describes tumors as hierarchically organized, has added a new level of

complexity to therapy failure. CSCs are the root of cancers and resist chemo- and

irra-diation therapy explaining cancer recurrence even many years after the therapy ended.

This review discusses briefly CSCs in cancers, gives an overview of the role of CSCs

in therapy resistance, and discusses the potential means of targeting these therapy

resistant tumor cells.

2

Introduction

Well over 40 years ago president Nixon signed the national cancer act and officially

started the war on cancer.

_{Despite this act and resulting significant improvements}

in therapy, the war has not ended and cancer is still one of the leading causes of death

worldwide. To illustrate, in 2012 14.1 million adults in the world were diagnosed with

cancer and in the same year an estimated 8.2 million people died from the disease

_The

main problem we face in cancer treatment is the presence or development of resistance

to therapy for which a multitude of distinct reasons have been defined. For instance,

acquisition of mutations in key signalling molecules, enhanced anti-apoptotic protein

expression, presence of quiescent and/or resistant tumor cells or high expression of drug

efflux pumps are all potential means that impair therapy efficacy.

_{In the last decade}

a lot of attention has gone to the role of a specific subset of cancer cells called cancer

stem cells (CSCs). In analogy with their normal counterparts, the stem cells, these cells

display a high level of therapy resistance and can effectively repopulate the tumor.

CSCs are the tumorigenic core of tumors

CSCs are defined based on their tumor forming capacity in xenograft studies.

_These

cells normally represent a minority of the tumor cells and can be identified by a long

list of markers, although most of these are not restricted solely to the CSC

popula-tion.

_{CSCs can be selected in vitro using spheroid growth in suspension and defined}

media compositions. Upon injection in mice CSCs, but not their more differentiated

counterparts, can very efficiently form tumors that resemble the original tumor from

which they were derived, including all differentiated cells. Moreover, re-isolation of

the CSCs from xenografts allows for serial transplantation to secondary and tertiary

mice, which is the gold-standard assay to prove that tumor cells are indeed CSCs.

CSCs were first defined in acute myeloid leukemia (AML) in 1994.

_CD34

_/CD38

expression has long been used to mark progenitor and pluripotent stem cells in the bone

marrow. Intriguingly, a similar subpopulation was detected in AML and

xenotrans-plantation of specifically this CD34

_/CD38

_{leukemia cells resulted in leukemia in mice}

that reproduced many features of human AML.

_{A decade later CSCs were detected in}

solid tumors. Breast, glioblastoma, prostate and colorectal tumors are only some of the

tumor types where CSCs were identified.

_{The variety of tumors in which CSCs}

were identified suggests that it is a common feature in most cancers, although some

observations indicate that it may not occur in all tumor types or alternatively at all

stages of disease.

As mentioned above, CSCs are highly tumorigenic and therefore are also referred to

as tumor-initiating cells. The name “CSC” does not refer to the fact that CSCs can be

derived from normal stem cells, but rather points to the idea that CSCs display

prop-erties normally attributed to stem cells. Firstly, stem cells have the capacity to

self-renew, i.e. to form a new stem cell upon division, and secondly stem cells can

differ-entiate into the more specialized cell types that make up a tissue.

_{Self-renewal and}

differentiation of stem cells is regulated by morphogenic pathways and interestingly

these signaling pathways are also highly active in many CSCs, suggesting that equal

regulatory principles exist in CSCs. One of the morphogenic pathways that is active in

stem cells is the Wnt signaling.

_{This pathway determines self-renewal and cell fate}

of hematopoietic stem cell (HSCs).

_{High activity of this pathway is also observed in}

stem cells of other tissues like breast and colon.

_{Next to Wnt signaling, Notch}

signaling is shown to be essential for stem cell maintenance. Notch signaling is highly

active in HSC when compared to more differentiated cells and inhibition of Notch

signaling promotes differentiation of HSC.

_{Likewise, Hedgehog (HH) signaling}

regu-lates proliferation and self-renewal of stem cells and activation of HH signaling is able

to expand HSC and brain stem cells in vitro and in vivo.

_{In contrast to Wnt, Notch,}

and HH signaling BMP signaling is inhibiting stem cell expansion. Activation of BMP

signalling results in suppression of Wnt signaling and this controls stem cells numbers.

25-27

_{Combined these morphogenic pathways regulate stem cell fate and}

differentia-tion cues. Intriguingly, this regulatory network appears to extend to CSCs. In a high

throughput screening in breast CSCs salinomycin was identified as a compound that

eliminates CSCs.

_{This antibiotic was shown to inhibit Wnt signaling and as a result}

is capable to differentiate breast CSCs.

_{High Wnt pathway activity is also shown}

to be important for cell fate of CSCs from many tumors like CLL, breast, CRC,

squa-mous cell carcinoma, and lung cancer.

_{In these tumors inhibition of Wnt pathway}

activity, with e.g. salinomycin is detrimental for CSCs.

_{Beside Wnt signaling,}

Notch signaling can also regulate CSC self-renewal. Inhibition of Notch signals can

be achieved by neutralizing antibodies against DLL4, or treatment with a g-secretase

inhibitor (DBZ). GBM, CRC and breast cancer stem cells need high Notch activity

and inhibition of Notch results in loss of CSCs.

_{Furthermore, Hedgehog signalling}

is highly active in CSCs, which is shown to be required for self-renewal of CSCs in

various cancers like breast, lung, and CRC.

_{Morphogenic pathways and inhibitors}

are depicted in figure 1.

In addition to the analogous usage of morphogenic pathways, stem cells and CSCs

also appear to share high activity of DNA repair pathways. For example, HSC can

2

Figure 1: Targeting morphogenic pathways in CSCs

Morphogenic pathways Notch (blue), Wnt (green) and Hedgehog (red) signaling pathways are highly active and important for self-renewal of stem cell and CSCs. Blue: Notch signaling is activated via direct cell-cell contact. A cell expressing Notch ligand (e.g. DLL4) contacts with another cell that expresses Notch receptor. When bound by a Notch ligand, the intracellular domain of the Notch receptor (IC-Notch) is cleaved by γ-secretase (γsec) and is targeted to the nucleus to activate transcription of downstream target gene that enhance self-renewal of CSCs. This pathway can be inhibited with a DLL4 antibody that neutralizes Notch ligand DLL4. Also γ-secretase inhibitors like DBZ are efficient in blocking Notch signaling. Green: Wnt ligands binds to the Frizzled-LRP receptor and inhibit a cytoplasmic destruction complex (APC-GSK3β-Axin) of β-catenin, which then enters the nucleus to activate transcription of Wnt target genes that are known to be important for CSCs maintenance. Wnt signaling can be inhibited with salinomycin. Red: The Hedgehog pathway is activated by Hedgehog ligands binding to the Patched receptor, which releases its inhibition of the Smoothened (Smo) transmembrane receptor. Smoothened can then in turn activates Gli transcription factors, the final effectors of the Hedgehog pathway. The natural occuring compound cyclopamine is used to inhibit Smo receptor and thereby block Hedgehog signaling.

repair UV induced single-strand breaks faster when compared with more

differenti-ated cells

_{and this was reported in CSCs as well.}

_{It is important to mention that}

more and more evidence indicates that CSCs are not a fixed cell population, but rather

represents a state of tumor cells that appears inducible. Giving the right signals from

the micro-environment or introduction of new mutations can result in

de-differentia-tion of more differentiated tumor cells into CSCs.

Not only signaling pathways, but also cell surface molecules are similarly expressed on

stem cells and CSCs. The pentaspan membrane glycoprotein CD133, also known as

Prominin-1, is expressed in normal stem cells, e.g. hematopoietic, neural, and intestinal

stem cells,

_{but CD133 was also used to identify CSCs from different tumor types.}

_{Moreover, in the last decade the G-protein coupled receptor Lgr5 received a lot}

of attention because high Lgr5 expression was reported to mark stem cells in various

organs. This target gene of the Wnt signaling pathway is exclusively expressed by stem

cells of various organs

_{and we and others have shown that Lgr5 also marks CSCs}

in various tumors.

_{Most of the currently used markers have no known role in CSC}

biology. In contrast, Aldehyde dehydrogenase isoform 1 (ALDH1) oxidizes aldehydes

to carboxylic acids and thus for instance catalyzes the conversion of retinol (vitamin

A) to retinoid acid. Inhibition of ALDH1 reduces retinoic acid levels and thereby

promotes HSC and breast CSCs self-renewal.

_{ALDH1 is highly expressed in many}

stem cells and CSCs, and ALDH1 expression and its activity were used to isolate stem

cells and CSCs.

_{Taken together, it appears that stem cells and CSCs are wired in}

the same way and share expression of several cell surface markers. Unfortunately, this

similarity extends to a more detrimental property, namely therapy resistance.

The extreme survivors; CSCs and their therapy resistance

The concept that CSCs selectively resist therapy stems from a multitude of

observa-tions in cell culture, animal models and cancer patients. In cell culture direct analysis

of apoptosis revealed that differentiated colon cancer cells are induced to die upon

chemotherapy treatment, while colon CSCs from the same cultures survive the toxic

insults.

_{This differential sensitivity was not due to proliferation differences between}

CSCs and more differentiated cells as it was also observed when using treatments that

are not dependent on cycling cells.

_{Moreover, these surviving CSCs can}

re-estab-lish the culture, confirming the idea that they are responsible for therapy failure.

Chemotherapy resistant CD133

_{CSCs were described in liver and lung cancer as}

well,

_{Similar observations were derived in pancreatic cancer where CSCs were}

2

this tumor type in vitro cell death was more pronounced in the differentiated CD133

-cells as compared to the CD133

_cells.

_{Finally, GBM CSCs and breast CSCs isolated}

from patient specimens, displayed selective resistance to various chemotherapies.

Next to the in vitro evidence, escape from therapy was also evident from xenograft

studies. Chemotherapy treatment of xenotransplanted CRCs resulted in an increase

in CD133

_{in the tumor.}

_{This indicates that CD133}

_{CSCs are more resistant to}

oxaliplatin in vivo when compared to differentiated CD133

_{cells. In vivo resistance}

of CRC CSCs is not restricted to oxaliplatin as mice bearing human CRC tumors

treated with irinotecan show an increase in cells that express ESA

_CD44

_CD166

_,

distinct markers for CSCs.

_{Moreover, in vivo gemcitabine treatment of}

xenotrans-planted pancreatic cancer induced the CD133

_{fraction, pointing to a gemcitabine}

resistant CSC population.

_{Finally, also in xenotransplants of AML enhanced}

CD34

_/CD38

_{CSCs were observed in vivo upon cytosine arabinoside treatment.}

Intriguingly, therapy resistance appears to be a general feature of these cells

and is not restricted to chemotherapy, but observed with radiotherapy as well.

Irradiated glioblastoma, either implanted subcutaneously or intracranially

showed an increase of CD133

_{cells when compared to non-irradiated tumors.}

This distinction was suggested to be clinically relevant as the authors extended

these findings to ex vivo irradiation of surgically removed glioblastomas.

Also for irradiation examples encompass other tumor types. MMTV-Wnt1 mice bearing

breast tumors showed an increase in the CSC (

Thy1

_CD24

_Lin

₎

_{fraction after}

irradi-ation and in the same study CSCs from primary human head and neck cancers proved

radioresistant.

_{Combined these data indicate that cell line or primary tumor-derived}

cells with CSC markers display decreased sensitivity to chemo and radiotherapy. It

is important to realize though that one potential caveat with this conclusion is the

fact that CSC markers are heavily debated, suggesting that the increases observed in

marker expression may not represent enhanced stemness(for a review see Medema

2013).

_{Moreover, xenotransplantation models may not adequately represent the}

normal situation in patients

_{and select for distinct traits and/or markers.}

Neverthe-less, a first direct hint that this CSC resistance concept could explain minimal residual

disease and therapy failure in patients came from a study of the group of Luis Parada

who used a genetically modified mouse model to study endogenously growing tumors

in which CSCs could be traced using a Nestin reporter construct. Nestin

_{tumor cells,}

which represent a quiescent CSC population, could fully repopulate the tumor after

temozolomide chemotherapy, while selective deletion of these cells prevented tumor

outgrowth.

_{These data indicate that CSCs resist therapy and might be a}

poten-tial cause of tumor relapse. In line with this observation, an increasing list of

obser-vations in patients support the crucial role of CSCs in tumor relapse after therapy.

In patients with GBM, CRC, or breast cancer, increased CSC fractions using marker

expression were measured after chemotherapy treatment.

_{A more direct evidence}

for increases in true functional CSCs came from a study in breast cancer. In contrast to

other reports, here authors studied patient samples and performed functional assays.

Increase in mammosphere formation capacity was seen after chemotherapy

treat-ment,

_{proving that stemness rather showed a relative increase than decrease upon}

therapeutic intervention. The growing body of evidence that points to a role for CSCs

in resistance warrants a more detailed survey to increase our understanding of the

mechanisms that determine resistance in order to target these survivors of therapy.

Mechanisms behind therapy resistance

Normal stem cells contain multiple mechanisms to control cell death, which aids to

protect these crucial cells from cytotoxic insults. Elevated apoptosis resistance,

drug-efflux pumps, enhanced DNA repair efficiency, detoxification enzyme expression and

quiescence are all identified as pro-survival mechanisms. Intriguingly, all these

mecha-nisms appear to be hijacked by CSCs. For instance, mitochondrial apoptosis is

associ-ated with loss of mitochondrial membrane integrity, which is maintained by a strict

balance of anti-apoptotic BCL2 proteins (e.g. BCL2, BCLXL, and MCL1), pro-apoptotic

BCL2 family members (BAX and BAK) and BH3 proteins (e.g. BIM, BAD, and NOXA).

A cytotoxic insult-induced imbalance in the ratio of these molecules results in

permea-bilization of the mitochondrial outer membrane and subsequent activation of a caspase

cascade.

_{In stem cells, but also in CSCs, an elevated anti-apoptotic protein expression}

increases the threshold for apoptosis induction and thereby directly protects the cells

against apoptosis. For instance, in breast and AML CSCs, BCL2 and BCLXL are highly

expressed.

_{Similarly, in primary GBM cultures CD133}

_{CSCs had elevated}

expres-sion of BCL2 and BCLXL compared to their more differentiated CD133

_progeny.

_In

agreement with a role for apoptosis regulation in CSCs, direct proteomic analysis of

CRC CSCs and differentiated cells revealed “apoptosis” as one of the main molecular

pathways affected, involving differential expression of key anti-apoptotic proteins,

including BIRC6.

_{Combined this suggests that CSCs have an elevated anti-apoptotic}

threshold. Recent data confirm this idea using so-called BH3 profiling, an assay to

directly measure the apoptosis priming state of cells.

_{This revealed that CRC CSCs}

were less-primed as compared to differentiated cells, which at least in part explains

their resistance to conventional chemotherapy.

_{In agreement, sublethal doses of BH3}

2

mimetics can change this threshold and strongly sensitize CSCs to chemotherapy.

Besides an elevated apoptotic threshold, CSCs display high expression of drug efflux

pumps, like ATP-binding cassette (ABC) transporter family proteins.

_These

proteins are important for efflux of chemotherapy across the plasma membrane.

_Various

ABC transporter proteins are highly expressed in HSC and in AML CSCs (CD34

_/

CD38

_{) compared to the non stem (CD34}

_/CD38

_{) cells [81]. Also in GBM and}

mela-noma high expression of drug efflux pumps in CSCs are reported.

_{In the latter,}

expression of ABC transporter ABCB5 in fact serves as a marker for CSCs.

Surpris-ingly, in CRC a different scenario is reported. Here not CSCs, but rather the

differenti-ated cells express high levels of the drug efflux pump ABCB1. The authors suggested

that differentiated cells protect CSCs from chemotherapy treatment by forming a

protective rim around the CSCs.

The above points to the fact that CSCs employ means to avoid the impact of therapy,

which we can potentially circumvent using combination therapy. However a

poten-tially more challenging problem is the recent observation that CSCs may exist that

display quiescent properties. Selectivity of chemotherapy for cancer cells relies on

the fact that chemotherapy mainly kills cells that are highly proliferative. As rapid

uncontrolled proliferation is a standard feature of many tumor cells, chemotherapy is

thought to target tumor cells selectively over non-proliferating normal cells, consistent

with the observed toxicity in organs with rapidly dividing cells, such as bone marrow,

digestive tract, and hair follicles. In contrast, slow proliferating or quiescent normal

cells are largely protected from chemotherapy treatment. Importantly, this

resist-ance also extends to quiescent tumor cells.

In ovarian cancer CD24

_{CSCs are less}

proliferative and more resistant to chemotherapy when compared to CD24

_cells.

Recent data point to the existence of CSCs that are quiescent. These can be

identi-fied using the dye PKH26, which dilutes out when cells proliferate and therefore only

low or non-proliferative cells will retain the label. In primary melanoma cultures label

retaining cells were detected with a very low doubling time of around 4 weeks in vitro.

Although these cells are slow dividing they have increased sphere forming capacity in

vitro

suggesting that these label retaining cells are enriched in CSCs.

_{Such quiescent}

cells are also identified in pancreatic adenocarcinoma and shown to be enriched for

CSC markers like CD133, CD24

_/CD44

_{and ALDH. In agreement with this notion,}

these label retaining cells are more tumorigenic, indicating that cancer is not a disease

of homogeneously rapidly proliferating cells, but also contains quiescent cells that

can escape classical chemotherapy and subsequently induce regrowth of the tumor.

contain enhanced potential and/or time to repair the damage that is inflicted to them.

As many chemotherapeutic agents as well as radiotherapy work by inducing DNA

damage, cells that effectively repair DNA damage can potentially survive

chemo-therapy. Various reports have shown that CSCs, for instance from GBM, possess high

DNA repair activity, which makes them resistant to radiation and chemotherapy.

Similarly, in breast CSCs there is increased expression of DNA repair genes, indicating

that high DNA repair pathway activity may aid in making CSCs resistant to tumor

therapy. In conclusion, there are many ways for CSCs to resist tumor therapy. Figure

2 illustrates the reasons for therapy resistance in CSCs.

Killing CSCs, magic bullets or combination cocktails?

Although considered bad news, the efficient DNA repair of CSCs may also point to a

dependency for these mechanisms and as such offer a means to target these cells. For

example, CSCs in GBM have elevated activity of Chk1 and ATM and survive irradiation,

but inhibition of the cell cycle checkpoint kinases Chk1 and Chk2 is sufficient to sensitize

CSCs towards irradiation.

_{Recently, it has been reported that a combined Chk1 and}

PDK1 inhibition is required to kill CSCs in GBM.

_{Similarly, non-small cell lung cancer}

CSCs can be sensitized to chemotherapy by combining treatment with Chk1 and Chk2

inhibitors SB218078 or AZD7762.

_{Mechanistically, inhibition of Chk1 results in active}

Cdc2-cyclin B complex that is followed by mitotic catastrophe.

_{Although effective, these}

compounds are also relatively toxic and combination of the Chk inhibitor AZD7762 with

gemcitabine showed cardiac toxicity.

_{To overcome this toxicity an inhibitor of a}

down-stream target of Chk1, Wee1, was developed. In the presence of DNA damage Wee1 arrests

cells in G2 phase and allows cells to repair DNA before entering into mitosis. Interestingly,

Wee1 is reported to be overexpressed in GBM CSCs. In the same report, the authors show

that inhibition of Wee1 with PD0166285 sensitizes GBM CSCs towards irradiation.

Besides targeting the core of the repair machinery, a lot of effort is put into targeting the

execution machinery in cancer cells. Previously, we have used an inducible caspase-9 to

target colon CSCs. Upon activation of caspase-9, colon CSCs were killed efficiently in

vitro

and in vivo suggesting that activation of caspases are sufficient to efficiently kill

CSCs.

_{As described, anti-apoptotic proteins are highly expressed in various cancers and}

especially in CSCs. Targeting these anti-apoptotic proteins using small molecules that

have been developed therefore forms an attractive mechanism. For instance, ABT-737,

a small molecule inhibitor that targets BCL2, BCLXL, and BCLW tips the apoptotic

balance to a more pro-apoptotic state and reverts the resistance of colon CSCs.

2

Figure 2: Mechanisms of therapy resistance in CSCs

a) Four mechanisms that are used by CSCs to resist chemotherapy. Efficient DNA repair (orange), quies-cence (red), increase ABC transporter expression (green), and decreased mitochondrial priming (blue). b) Potential means of targeting therapy resistant CSCs.

the same specificity, seems to exert high toxicity for platelets, which depend on BCLXL

for survival.

_{Although more selective inhibitors, such as ABT-199 targeting only BCL2,}

have been developed to overcome this problem, our recent data indicates that also colon

CSCs are dependent on BCLXL for survival. In agreement inhibition of BCLXL with

ABT-737 or a BCLXL specific inhibitor (WEHI-539) is sufficient to kill colon CSCs.

Simi-larly, lung CSCs are shown to be dependent on BCLXL and also in these cells

inhibi-tion eliminates lung CSCs in vitro and in vivo.

_{Although this still raises the problem of}

toxicity, it is possible to use sublethal amounts of BCLXL inhibition, which is sufficient

to strongly sensitize CSCs towards chemotherapy.

_{It is currently not completely clear}

why colon CSCs acquire this dependency on BCLXL. One possible explanation is the

observation by Todaro and colleagues showing an autocrine loop of IL4/IL4R in colon

CSCs, which appears to maintain BCLXL levels and protect CSCs from chemotherapy.

One thing that allows for the identification of CSCs is the presence of several cell

surface markers. Various groups and companies therefore developed immunotoxins

that directly target such CSC markers. For instance, antibodies against for instance the

stem cell marker CD133 conjugated to paclitaxel or cytolethal distending toxin (Cdt)

target CD133 expressing cells and show in vitro and in vivo elimination of tumors.

Similarly, targeting of CD133

_{cells can be achieved by generation of CD133 specific}

Measlus viruses. These oncolytic viruses infect CD133 expressing cells and destroy

them by lysis.

_{Moreover, selective killing of CD133}

_{GBM cells was shown with}

CD133 antibodies coupled to singe walled carbon nanotubes (SWNTs). These

anti-CD133-SWNTs induce thermal destruction of cancer cells when it is combined with

nearIR laser light.

_{However, CD133 expression is not specific for CSCs but also}

expressed on normal stem cells, which should be protected from such therapies at

all times. To minimize toxicity and deliver drug to cancer selectivity photochemical

internalization (PCI) was developed. This technique makes it possible to release

drug in the tumor area specifically.

_{Next to CD133, there is an increasing effort}

to target other cell surface molecules including the stem cell marker Lgr5.

Neverthe-less, as is true for CD133, toxicity with such an approach can be expected.

Surpris-ingly, antibodies without toxins targeting other cell surface molecules are shown to

be efficient in killing CSCs as well. Antibodies against CD47 give promising effects

in various cancers. CD47 is a receptor that is involved in inhibition of so called

“eat-me” signals and is highly expressed on CSCs compared to more differentiated

cells. Blocking of this CD47 receptor with an antibody enables the phagocytosis of

AML CSCs and thereby blocks tumor growth.

_{In addition, CD47 inhibition also}

2

blocks tumor growth in solid cancers, like breast cancer, CRC, ovarian cancer and

GBM, which is also suggested to depend on facilitating phagocytosis of CSCs.

Next to phagocytosis induction, several antibodies have shown to delete essential

signals from CSCs. For instance, direct targeting of breast CSCs can be achieved by

using an antibody against CXCR1. The IL8 receptor CXCR1 is expressed almost

exclu-sively on CSCs and Repertaxin, an inhibitor of CXCR1/2, or anti-CXCR1 treatment

induces cell death in CXCR1

_{breast CSCs, which appears to be mediated by AKT}

signaling inhibition.

_{Intriguingly, PI3K/AKT signaling addiction in colon CSCs was}

also reported in colon CSCs, where a CD44v6-positive subset was identified that is

exclusively metastatic. These cells express high levels of PI3K, which, if inhibited, alter

the viability of the cells and impede the capacity to migrate,

_{suggesting that PI3K}

signaling is crucial for CSCs.

As described, CSCs require signaling through morphogenic pathways for their

main-tenance, suggesting that these may be attractive targets for therapy as well. In

agree-ment, a screen for CSC sensitizing compounds identified salinomycin, which inhibits

Wnt signaling and eliminates breast and CLL CSCs.

_{As CSCs in CML, AML and}

skin tumors are dependent on the Wnt pathway, inhibition can be clinically relevant.

_{Furthermore, inhibition of Notch signalling pathway using a neutralizing}

anti-body against DLL4 results in less tumor engraftment in secondary tumors suggesting

in vivo

differentiation of CSCs. Importantly, DLL4 antibody was also able to

sensi-tize the tumor to irinotecan in vivo.

_{Inhibition of Notch signaling was also}

suffi-cient to deplete GBM CSCs and sensitize ovarian CSCs to chemotherapy.

_Lastly,

HH signaling can be inhibited by using cyclopamine and this Smoothened

antago-nist sensitizes AML CSCs to Ara-c treatment.

_{Similarly, in GBM and in}

pancre-atic cancer decreases in CSCs are observed after treatment with Smoothened

inhibi-tors cyclopamine or CUR199691.

_{These data point to a crucial role for HH}

signalling in cancer stemness and this is confirmed by knockdown of Smoothened,

which results in loss of CML CSCs.

_{Antibodies can also be used to target CSC}

niche. Blood vessels maintain GBM CSCs in a stem like state. Targeting

microenvi-ronment with Bevacizumab, an antibody against VEGF is able to differentiate GBM

CSCs.

_{In 2006 Jin et al showed that using an antibody against CD44 decreased}

homing of AML cells and thereby promoting the AML CSCs to differentiate to a

more mature cancer cell progeny. This antibody inhibited AML growth in mice.

reported to happen with bone morphogenetic protein 4 (BMP4) as well. In CRC BMP4

expression is exclusively expressed by differentiated cancer cells and shown to induce

differentiation of CSCs and sensitization to oxaliplatin in vivo.

_{In addition, BMP4}

also forces GBM CSCs to differentiate and thereby inhibits their tumorigenicity.

Not only inhibition of morphogenic pathways, but activation of signaling pathways

can change CSCs cell fate as well. Activation of the unfolded protein response (UPR)

induces differentiation of stem cells in the mouse intestine.

_{In line with this,}

salu-brinal induces UPR in colon CSCs and forces them to differentiate. In addition to

inducing differentiation, UPR sensitizes cells to chemotherapy in vitro and in vivo

(M.C.B. Wielenga and S. Colak unpublished observations). Although targeting CSCs

by forcing them to differentiate or by the induction of apoptosis seem to be a

attrac-tive therapeutic options, the suggested flexibility of the system is a clear caveat. Even

when CSCs are eliminated within a tumor, differentiated cells can de-differentiated

and take the place of the CSCs that were deleted.

_{Targeting the cues that induce}

de-differentiation or simply attacking both CSCs and more-differentiated cells needs

to be achieved to eradicate a tumor.

_{Altogether, direct CSCs targeting or CSC}

differ-entiation therapy are promising means to improve tumor therapy. Further studies are

needed to investigate the most promising combination treatments that does not give

severe toxicities.

Summary

Identification of CSCs in many tumors allowed for a better understanding as to why

even many years after therapy tumors can relapse. There is increasing evidence that

targeting these CSCs is important to improve therapies. Here we reviewed mechanisms

that make CSCs resistant to therapy A better understanding of these mechanisms

and the way CSCs retain their tumorigenic stem cell capacities is crucial. The exiting

new insight will undoubtedly provide new therapeutic tools in the years to come.

Acknowledgements

We thank the members of the laboratory for useful discussion. JPM is

sponsored by grants from the Netherlands Organization for Scientific

Research (NWO; Gravitation-Cancer Genomics Center The Netherlands

Zwaartekracht), from the Dutch Cancer Society (UVA2009-4416 and

UVA2012-5735), from MLDS (FP13-07) and Alpe dHuzes/KWF (CONNECTION).

2

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