Molecular Pathways

Cancer Stem Cells and Chemosensitivity

Marcello Maugeri-Sacc�a1, Paolo Vigneri2, and Ruggero De Maria1,3

AbstractCancer lethality is mainly due to the onset of distant metastases and refractoriness to chemotherapy.

Thus, the development of molecular targeted agents that can restore or increase chemosensitivity will

provide valuable therapeutic options for cancer patients. Growing evidence indicates that a cellular

subpopulation with stem cell–like features, commonly referred to as cancer stem cells (CSCs), is critical

for tumor generation and maintenance. Recent advances in stem cell biology are revealing that this

cellular fraction shares many properties with normal adult stem cells and is able to propagate the

parental tumor in animal models. CSCs seem to be protected against widely used chemotherapeutic

agents by means of different mechanisms, such as a marked proficiency in DNA damage repair, high

expression of ATP-binding cassette drug transporters, and activation of PI3K/AKT and Wnt pathways.

Moreover, microenvironmental stimuli such as those involved in the epithelial-mesenchymal transi-

tion and hypoxia indirectly contribute to chemoresistance by inducing in cancer cells a stem-like

phenotype. Understanding how CSCs overcome chemotherapy-induced death stimuli, and integrating

such knowledge into clinical research methodology, has become a priority in the process of identifying

innovative therapeutic strategies aimed at improving the outcome of cancer patients. Clin Cancer Res;

17(15); 4942–7. �2011 AACR.

Background

Adult stem cells are a rare and long-living cellular frac-tion that ensures tissue homeostasis by replacing senescentor damaged cells (1). The stem cell fate is regulated withinspecialized microarchitectonic structures, or niches, thatrespond to both local and systemic conditions (2). Whenrequired, stem cells divide asymmetrically, generating aslow-cycling daughter cell that retains the biological prop-erties of the mother, and a more active daughter cell thatproduces a progeny of more specialized cells undergoingterminal differentiation.

Recent advances in cancer biology support the criticalrole of an uncommon cellular population with stem-likefeatures in tumor generation and propagation (3–6). Thispopulation, commonly referred to as cancer stem cells(CSCs), displays three characteristics: (1) expression of arepertoire of markers common to stem and progenitorcells, (2) unlimited growth in vitro using media optimized

for normal stem cell cultures, and (3) the ability to repro-duce the parental tumor upon injection into immunocom-promised mice.

The concept that a transformed stem cell is the progeni-tor of an entire tumor population implies that cancers areorganized in a stringent hierarchy with a CSC at the apex ofthe pyramid (the hierarchical model), in a distortion of thefunctional architecture of a normal tissue. Consistent withthis hypothesis, increasing evidence suggests that CSCsaberrantly exploit molecules and pathways governing theself-renewal program. This is confirmed by the markedasymmetry in the distribution of self-renewal componentsbetween CSCs and their differentiated progeny (7, 8), andby the antitumor activity displayed by inhibitors of the self-renewal pathway in many preclinical models (9, 10).

It also appears that CSCs successfully secure appropriatemicroenvironmental stimuli by displacing normal stemcells from their niches. The interaction between CSCsand the microenvironment is bidirectional, as indicatedby the trans-differentiation process employed by CSCs togenerate vascular precursors (11, 12). Like their normalcounterpart, CSCs exhibit multifaceted defensive machin-ery that protects them from the effects of antiblastic com-pounds (13). In addition, the CSC fraction is probablyenriched after chemotherapy, as demonstrated by theincreased expression of stemness markers in patients withearly breast cancer who are receiving primary systemictherapy (14).

The mechanisms underlying chemoresistance can beschematically subdivided into two groups: CSC-intrinsicand CSC-extrinsic (or indirect). The first group includes

Authors' Affiliations: 1Department of Hematology, Oncology and Mole-cular Medicine, Istituto Superiore di Sanit�a, Rome, Italy; 2Department ofClinical and Molecular Bio-Medicine, University of Catania, Catania, Italy;3Mediterranean Institute of Oncology, Viagrande, Italy

Note: M. Maugeri-Sacc�a and P. Vigneri contributed equally to this work.

Corresponding Author: Ruggero De Maria, Department of Hematology,Oncology and Molecular Medicine, Istituto Superiore di Sanit�a, VialeRegina Elena 299, 00161 Rome, Italy. Phone: 39-06-4990-3395; Fax:390-64-938-7087; E-mail: ruggero.demaria@iss.it

doi: 10.1158/1078-0432.CCR-10-2538

�2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 17(15) August 1, 20114942

proficient DNA repair machinery, high expression of ATP-binding cassette (ABC) drug transporters, and altered cellcycle kinetics. The latter group includes microenvironmen-tal influences that indirectly contribute to chemoresistance(Fig. 1).

CSC-intrinsic mechanisms of chemoresistancePreservation of the genetic code from exogenous or

endogenous injuries is critical for maintaining normalcellular function. After cells sense DNA damage, theybegin repair activities that restore the original sequenceof the genome. Alternatively, severe lesions lead to theelimination of irreversibly damaged cells by triggeringprogrammed cell death. Several partly overlapping repairsignals are involved in the maintenance of genomeintegrity (15). Each pathway corrects a specific form ofgenetic lesion that in turn reflects the type of damageinduced by the causal agents. Considerable evidenceindicates that embryonic (16) and adult (17) stem cellshave a greater ability to repair their genetic code thantheir offspring. However, aged stem cells tend to accu-mulate genetic/epigenetic mutations as a consequence ofa reduced ability to correct genetic lesions (18), whichmay account for the increased cancer incidence withaging. Although DNA damage repair is crucial for pre-

venting malignant transformation, transformed cancercells take advantage of improperly activated repairpathways, exploiting them to overcome chemotherapy-induced cell death.

Glioblastoma stem-like cells (GBM-SCs) are resistant tochemotherapy, independently of their ability to extrudecytotoxic drugs (19). After exposure to ionizing radiation,GBM-SCs activate ATM and ChK1 undergoing cell cyclearrest, and repair their DNAmore readily than do non-CSCs(20). Likewise, lung CSCs (LCSCs) exposed to genotoxicstress activate Chk1 and Chk2, whereas differentiated lungcancer cells are vulnerable to chemotherapy (Bartucci M.et al., unpublished data). When chemotherapy is combinedwith Chk1 inhibitors, LCSCs undergo cell death throughmitotic catastrophe. Hyperactivation of the ATR/Chk1 path-way also protects colon CSCs (CCSCs) from platinumderivatives, whereas this chemoresistant phenotype isreverted by the inhibition of ATR/Chk1 signaling (21).

The phosphatidylinositol-3 kinase PI3K/AKT pathwayis often deregulated in high-grade primary brain tumorsand mediates different protumorigenic activities (22).Moreover, an intricate connection links PI3K/AKT andDNA repair machinery (23). In glioblastoma cells, inhibi-tion of PI3K or AKT prolongs ionizing radiation-inducedDNA damage, as demonstrated by the delayed clearance of

Figure 1. CSC-intrinsicmechanisms of chemoresistance.A, high expression of MDR pumpsthat actively extrudechemotherapeutic agents ofnatural origin. B, proficientDNA repair machinery removesDNA adducts formed by alkylatingagents. C, chemotherapy killsrapidly proliferating cells whileslow-cycling/quiescent CSCsare spared from chemotherapy-induced cell death. D,microenvironmental stimuliassociated with EMT andhypoxia indirectly contributeto chemoresistance throughthe generation of cancerstem-like cells.

© 2011 American Association for Cancer Research

CSC

Stromal cell

Differentiated cancer cell

Chemotherapy

A B

D

C

MDR pumps

Extracellular space

ATP ADPIntracellular space

DNA repair

Quiescence

Microenvironmentalstimuli EMT

EMT

Hypoxia

O2

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g-H2AX foci (24). Accordingly, Akt inhibitors efficientlytarget GBM-SCs, determining a reduction of viable cells andabrogating neurosphere formation (25).

DNA sensor and repair pathways act in concert withapoptotic signaling to decide cell fate. Thus, the imbalanceof the apoptotic machinery toward an antiapoptotic statefavors cancer cell survival (26). Interleukin-4 (IL-4) isknown to amplify the expressionof antiapoptoticmediatorsin different epithelial cancers (27). The chemotherapy-resistant phenotype of CSCs seems to be sustained, at leastin part, by the release of IL-4 in a process that is abrogated byeither a neutralizing antibody or amutant formof IL-4 (28).Because IL-4 can be produced in a number of tumors (29,30), it is likely that other CSC types can exploit IL-4 tocounteract the cytotoxic activity of chemotherapeutic drugs.

The multidrug resistance (MDR) phenotype is a furthercritical hurdle for chemotherapy efficacy. ABC drug trans-porters are the main players in this phenomenon, becausethey actively extrude from cancer cells a variety of structu-rally and functionally unrelated drugs of natural origin(31). Both normal stem cells and their malignant counter-part express high levels of ABC pumps (32). In fact, theability of CSCs to actively exclude the HOECHST 33342dye has been exploited to facilitate their isolation andpurification. These CSCs, defined as the side population(SP) by the above-indicated assay, have been studied indifferent malignancies, including acute myeloid leukemia(AML) and neuroblastoma. The AML SP is characterized bya greater proficiency in extruding daunorubicin and mitox-antrone compared with the non-SP (33), and a similarpattern has been documented for neuroblastoma stem-likecells (34). Moreover, the doxorubicin-selected breast can-cer cell lineMCF-7/ADR acquires stem-like properties and amolecular portrait dominated by epithelial-to-mesenchy-mal transition (EMT)-related and self-renewal–relatedgenes (35). The gain of this stem-like state is coupled withthe overexpression of both MDR-linked genes and thecyclophosphamide-metabolizing enzyme aldehyde dehy-drogenase 1.

A further mechanism involved in CSCs’ resistance tochemotherapy is cell quiescence. In normal stem cells, aprolonged exit from the cell cycle ensures the longevity ofadult tissues by ensuring that stem cells do not exhaust theirproliferative potential (36). Quiescent stem cells efficientlyrepair DNA damage and reenter the cell cycle to reconsti-tute the damaged tissue after exposure to cytotoxic injury(37). In a malignant context, quiescent CSCs are mostlyspared by chemotherapy-induced cytotoxicity and aretherefore capable of reconstituting the original tumor.Initial evidence connecting quiescence to CSC chemoresis-tance comes from label-retaining approaches, indicatingthat pancreatic adenocarcinoma label-retaining cells(LRCs) encompass the operative criteria of CSCs and sur-vive 5-fluorouracil treatment, unlike their non-LRC coun-terpart (38). Similarly, putative ovarian CSCs display lowerproliferative activity, enhanced tumorigenicity in xenograftmodels, and increased resistance to cisplatin comparedwith the non-CSC fraction (39).

Indirect mechanisms of chemoresistanceThe interplay between CSCs and the microenvironment

is a dynamic process that leads to the continuous remodel-ing of both compartments. Experimental evidence con-firms the critical role of the EMT in the development ofcancer metastases and chemoresistance. Recent findingshave demonstrated that EMT is induced by the activationof a transcriptional complex influenced by different para-crine-acting signals, including the self-renewal–associatedpathways Hedgehog (40), Notch (41), and Wnt (42). Thiscomplex leads to radical cytoskeletal rearrangements cul-minating in a switch toward a mesenchymal-like pheno-type. Cells undergoing these morphofunctional changesare typically located at the tumor-stroma interface, wherethey gain prometastatic traits coupled with increased clo-nogenicity and enrichment in stem cell-associated markers(43).

In addition to the EMT, hypoxia is also emerging as acritical regulator of the CSC pool. Hypoxia derives fromdifferent cooperating factors, such as the chaotic and dys-functional vasculature that supplies malignant tumors, andpoor oxygen diffusion within rapidly expanding neo-plasms. Low oxygen tension activates the family ofhypoxia-inducible factors (HIFs), which trigger adaptivechanges at multiple levels, including the generation of newblood vessels in the attempt to ensure sufficient oxygen andnutrients (44). However, the abnormal architecture ofnewly formed vessels limits drugs perfusion, leading to asuboptimal concentration of chemotherapeutic agentswithin the tumor. Besides this mechanistic hypoxia-mediated drug resistance, direct evidence connects HIFfactors and CSCs. Cancer cells cultured under low oxygenconditions or low pH express higher levels of stemnessmarkers, acquire a stem-like phenotype, and overexpressstemness-related genes (45–47). Furthermore, based onfunctional similarities between adult stem cells and theirmalignant counterpart, it has been proposed that hypoxicareas within a tumor act as niches for CSCs (48).

Clinical-Translational Advances

It is possible to speculate that different mechanisms ofchemoresistance are preferentially, if not exclusively,responsible for distinct phases of cancer relapse and pro-gression. The temporal pattern of disease recurrence that ischaracteristic of many solid tumors suggests that the slowreplication kinetics of CSCs could account for the limitedefficacy of adjuvant chemotherapy in eradicating micro-scopic residual disease. Conversely, altered mechanisms ofDNA repair, overexpression of ABC transporters, andimproper activation of antiapoptotic signaling may bepredominant during metastatic progression, when differ-entiated tumor cells are killed by chemotherapy and resis-tant CSCs are forced to reenter the cell cycle to numericallyrestore the tumor population.

When chemotherapy-enhancing therapeutic approachesare considered, attention often turns to agents that interferewith DNA repair. Poly-ADP ribose polymerase (PARP)

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Clin Cancer Res; 17(15) August 1, 2011 Clinical Cancer Research4944

inhibitors are the prototype drugs of this class. The logicbehind the development of PARP inhibitors relies on theconcept of synthetic lethality, defined as the cooccurrenceof two genetic events that lead to cell death. To exploit thisconcept, cancer cells that are defective for a specific DNArepair pathway are exposed to compounds that inhibit adifferent signaling avenue that partially overlaps the defec-tive one. The combined (genetic and pharmacological)abrogation of two redundant DNA repair pathways resultsin an increased sensitivity of cancer cells to specific DNA-damaging agents. Different PARP inhibitors have demon-strated encouraging activity against tumors with inherentdefects in DNA repair, such as breast (49) and ovarian (50)carcinomas harboring BRCA1 or BRCA2 germline muta-tions. Chk1 inhibitors have also recently entered clinicaldevelopment in combination with gemcitabine, irinote-can, and cytarabine. In the case of Chk1 inhibitors, theprinciple of synthetic lethality involves the p53 tumorsuppressor: p53-defective cells are unable to undergo G1arrest, and as a result depend on Chk1 to activate cell cyclecheckpoints in response to DNA-damaging agents (51).Thus, in p53-defective CSCs, a synthetic lethality-drivenregimen should include a Chk1 inhibitor and a DNA-damaging agent, even though the ability of Chk1 inhibitorsto preferentially kill p53-deficient cells is still debated (52).Given the close relationship between DNA repair andapoptosis, compounds that target antiapoptotic proteins,such as Bcl-2 family member inhibitors, may be useful forp53 wild-type tumors. With this approach, one can selec-tively block sequential oncogenic activities by taking intoaccount the temporal and functional connections betweendifferent therapeutic targets.In addition to inhibiting various components of the

DNA repair pathway to potentiate the activity of alkylatingagents, investigators have developed inhibitors/modula-tors of ABC drug transporters as chemosensitizers toincrease the intracellular levels of ABC pump substrates,including taxanes, anthracyclines, and vinca alkaloids.After the failure of first- and second-generation ABC inhi-bitors, more-potent and specific third-generation antago-nists have been synthesized and are currently undergoingclinical development (32). Although direct proof of theantitumor activity of such compounds is still lacking, ABCinhibitors offer the possibility to block pumps distributedin different body sites, such as the blood-brain barrier, thusimproving drug biodistribution within sanctuary sites.A further strategy for eliminating CSCs entails the use of

differentiation-inducing agents that enhance chemosensi-tivity while depleting the CSC pool. The prodifferentiationactivity of the bone morphogenetic protein 4 (BMP4) onGBM-SCs may be exploited for the treatment of high-gradegliomas (53). Likewise, BMP4 was recently shown to pro-mote apoptosis, differentiation, and chemosensitization ofcolon CSCs through the inhibition of PI3K/AKT (54). Ofnote, the combined use of BMP4, 5-fluorouracil, andoxaliplatin can induce tumor eradication in a CSC-basedmodel of colon cancer. Because the sequential use ofdifferentiating agents and chemotherapy has shown con-

siderable efficacy in acute promyelocytic leukemia (55), itis likely that the increasing research on CSCs will promotethe use of similar strategies in solid cancers. In this context,it is conceivable that protective signals coming from themicroenvironment could counterbalance the activity ofdifferentiation-inducing agents. Thus, we envision thatcotargeting intrinsic and extrinsic mechanisms associatedwith CSC maintenance by combining differentiation-indu-cing agents with antiangiogenic compounds or inhibitorsof EMT/hypoxia-associated effectors may lead to a greaterdepletion of the CSC pool.

The ability to easily expand in vitro CSCs from severalsolid tumors has radically modified the preclinical modelsof human cancer based on cancer cell delivery in immu-nocompromised mice. Many standard cancer cell linesgenerate tumors whose phenotype appears extremely dif-ferent from human tumors. It is likely that the orthotopictransplantation of CSCs in the appropriate murine back-ground will allowmore reliable testing of anticancer agentsby taking into account the specific molecular settings of thetumor-initiating cells of each human malignancy (56, 57).Such models are very flexible and may allow investigatorsto test potential approaches for both adjuvant and meta-static therapies.

In this regard, the discovery of CSCs has also broughtinto question the general approach that is presentlyemployed to validate novel pharmacological compounds.Currently, anticancer drugs are initially tested in metastaticpatients and, if found effective, are then moved to theadjuvant setting. However, all evidence concerning CSCssuggests that this approach may be conceptually wrong,and that employing parameters of rapid tumor response,evaluated in metastatic disease, may underestimate thebenefit of multiple drugs. On the one hand, it is likelythat inhibitors of paracrine-acting pathways could offerconsiderable opportunities as adjuvant therapies targetingminimal residual disease. On the other hand, compoundsproducing rapid tumor shrinkage may be more suitable forthe metastatic or neoadjuvant setting. The lack of benefitfrom bevacizumab (58) and cetuximab (59) as adjuvanttherapy in colorectal cancer patients, despite their pivotalrole in the management of metastatic disease, corroboratesthis hypothesis. Likewise, a phase II study comparingFOLFOX or FOLFIRI plus bevacizumab with or withoutthe Smoothened inhibitor GDC-0449 in patients withmetastatic colorectal cancer failed to reach its primaryendpoint (60), despite the encouraging activity of GDC-0449 in tumors with activating mutations of the Hedgehogpathway (61). Similar unsatisfactory results were observedin a randomized placebo-controlled phase II trial withGDC-0449 as maintenance therapy in advanced ovariancancer (62).

Finally, it is worth noting that efforts to develop che-motherapy-enhancing agents aimed at eliminating CSCsmust take safety issues into account. To this end, researchefforts should be oriented toward a deeper characterizationof adult stem cells in order to avoid, or at least minimize,the inhibition of crucial mechanisms for normal stem

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cell maintenance. This aspect is even more relevant whenanti-CSC drugs are considered for the treatment of pediatricpatients and young adults.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Giuseppe Loreto for technical assistance.

Grant Support

Italian Association for Cancer Research, Italian Ministry of Health, andItalian Ministry for University and Research (FIRB_RBAP10KJC5 to R. DeMaria).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 1, 2011; revised April 20, 2011; accepted April 20, 2011;published OnlineFirst May 27, 2011.

References1. Reya T, Morrison SJ, ClarkeMF,Weissman IL. Stem cells, cancer, and

cancer stem cells. Nature 2001;414:105–11.2. Voog J, Jones DL. Stem cells and the niche: a dynamic duo. Cell Stem

Cell 2010;6:103–15.3. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a

hierarchy that originates from a primitive hematopoietic cell. Nat Med1997;3:730–7.

4. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF.Prospective identification of tumorigenic breast cancer cells. ProcNatl Acad Sci USA 2003;100:3983–8.

5. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, PeschleC, et al. Identification and expansion of human colon-cancer-initiatingcells. Nature 2007;445:111–5.

6. Eramo A, Lotti F, Sette G, Pilozzi E, Biffoni M, Di Virgilio A, et al.Identification and expansion of the tumorigenic lung cancer stem cellpopulation. Cell Death Differ 2008;15:504–14.

7. Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, et al.Identification of pancreatic cancer stem cells. Cancer Res2007;67:1030–7.

8. Hoey T, Yen WC, Axelrod F, Basi J, Donigian L, Dylla S, et al. DLL4blockade inhibits tumor growth and reduces tumor-initiating cellfrequency. Cell Stem Cell 2009;5:168–77.

9. Bar EE, Chaudhry A, Lin A, Fan X, Schreck K, Matsui W, et al.Cyclopamine-mediated hedgehog pathway inhibition depletesstem-like cancer cells in glioblastoma. Stem Cells 2007;25:2524–33.

10. Farnie G, Clarke RB, Spence K, Pinnock N, Brennan K, Anderson NG,et al. Novel cell culture technique for primary ductal carcinoma in situ:role of Notch and epidermal growth factor receptor signaling path-ways. J Natl Cancer Inst 2007;99:616–27.

11. Ricci-Vitiani L, Pallini R, Biffoni M, TodaroM, Invernici G, Cenci T, et al.Tumour vascularization via endothelial differentiation of glioblastomastem-like cells. Nature 2010;468:824–8.

12. Wang R, Chadalavada K, Wilshire J, Kowalik U, Hovinga KE, Geber A,et al. Glioblastoma stem-like cells give rise to tumour endothelium.Nature 2010;468:829–33.

13. Eyler CE, Rich JN. Survival of the fittest: cancer stem cells in ther-apeutic resistance and angiogenesis. J Clin Oncol 2008;26:2839–45.

14. Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu MF, et al.Intrinsic resistance of tumorigenic breast cancer cells to chemother-apy. J Natl Cancer Inst 2008;100:672–9.

15. Hoeijmakers JH. Genome maintenance mechanisms for preventingcancer. Nature 2001;411:366–74.

16. Maynard S, Swistowska AM, Lee JW, Liu Y, Liu ST, Da Cruz AB, et al.Human embryonic stem cells have enhanced repair of multiple formsof DNA damage. Stem Cells 2008;26:2266–74.

17. Bracker TU, Giebel B, Spanholtz J, Sorg UR, Klein-Hitpass L, Moritz T,et al. Stringent regulation of DNA repair during human hematopoieticdifferentiation: a gene expression and functional analysis. Stem Cells2006;24:722–30.

18. Rossi DJ, Bryder D, Seita J, Nussenzweig A, Hoeijmakers J, Weiss-man IL. Deficiencies in DNA damage repair limit the function ofhaematopoietic stem cells with age. Nature 2007;447:725–9.

19. Eramo A, Ricci-Vitiani L, Zeuner A, Pallini R, Lotti F, Sette G, et al.Chemotherapy resistance of glioblastoma stem cells. Cell Death Differ2006;13:1238–41.

20. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, et al.Glioma stem cells promote radioresistance by preferential activationof the DNA damage response. Nature 2006;444:756–60.

21. Gallmeier E, Hermann PC, Mueller MT, Machado JG, Ziesch A, DeToni EN, et al. Inhibition of ataxia telangiectasia- and Rad3-relatedfunction abrogates the in vitro and in vivo tumorigenicity of humancolon cancer cells through depletion of the CD133(þ) tumor-initiatingcell fraction. Stem Cells 2011;29:418–29.

22. Hambardzumyan D, Squatrito M, Carbajal E, Holland EC. Gliomaformation, cancer stem cells, and akt signaling. Stem Cell Rev2008;4:203–10.

23. Puc J, Keniry M, Li HS, Pandita TK, Choudhury AD, Memeo L, et al.Lack of PTEN sequesters CHK1 and initiates genetic instability.Cancer Cell 2005;7:193–204.

24. Kao GD, Jiang Z, Fernandes AM, Gupta AK, Maity A. Inhibition ofphosphatidylinositol-3-OH kinase/Akt signaling impairs DNA repair inglioblastoma cells following ionizing radiation. J Biol Chem2007;282:21206–12.

25. Eyler CE, Foo WC, LaFiura KM, McLendon RE, Hjelmeland AB, RichJN. Brain cancer stem cells display preferential sensitivity to Aktinhibition. Stem Cells 2008;26:3027–36.

26. Signore M, Ricci-Vitiani L, De Maria R. Targeting apoptosis pathwaysin cancer stem cells. Cancer Lett Epub 2011.

27. Todaro M, Lombardo Y, Francipane MG, Alea MP, Cammareri P,Iovino F, et al. Apoptosis resistance in epithelial tumors is mediated bytumor-cell-derived interleukin-4. Cell Death Differ 2008;15:762–72.

28. Todaro M, Alea MP, Di Stefano AB, Cammareri P, Vermeulen L,Iovino F, et al. Colon cancer stem cells dictate tumor growth andresist cell death by production of interleukin-4. Cell Stem Cell 2007;1:389–402.

29. Conticello C, Pedini F, Zeuner A, Patti M, Zerilli M, Stassi G, et al. IL-4protects tumor cells from anti-CD95 and chemotherapeutic agents viaup-regulation of antiapoptotic proteins. J Immunol 2004;172:5467–77.

30. TodaroM, Zerilli M, Ricci-Vitiani L, Bini M, Perez AleaM, Maria FlorenaA, et al. Autocrine production of interleukin-4 and interleukin-10 isrequired for survival and growth of thyroid cancer cells. Cancer Res2006;66:1491–9.

31. Wu CP, Calcagno AM, Ambudkar SV. Reversal of ABC drug trans-porter-mediated multidrug resistance in cancer cells: evaluation ofcurrent strategies. Curr Mol Pharmacol 2008;1:93–105.

32. Moitra K, Lou H, Dean M. Multidrug efflux pumps and cancer stemcells: insights into multidrug resistance and therapeutic development.Clin Pharmacol Ther 2011;89:491–502.

33. Wulf GG, Wang RY, Kuehnle I, Weidner D, Marini F, Brenner MK, et al.A leukemic stem cell with intrinsic drug efflux capacity in acutemyeloid leukemia. Blood 2001;98:1166–73.

34. Hirschmann-Jax C, Foster AE, Wulf GG, Nuchtern JG, Jax TW, GobelU, et al. A distinct "side population" of cells with high drug effluxcapacity in human tumor cells. Proc Natl Acad Sci USA2004;101:14228–33.

35. Calcagno AM, Salcido CD, Gillet JP, Wu CP, Fostel JM, Mumau MD,et al. Prolonged drug selection of breast cancer cells and enrichmentof cancer stem cell characteristics. J Natl Cancer Inst 2010;102:1637–52.

Maugeri-Sacc�a et al.

Clin Cancer Res; 17(15) August 1, 2011 Clinical Cancer Research4946

36. Essers MA, Offner S, Blanco-Bose WE, Waibler Z, Kalinke U, Duch-osal MA, et al. IFNalpha activates dormant haematopoietic stem cellsin vivo. Nature 2009;458:904–8.

37. Dekaney CM, Gulati AS, Garrison AP, Helmrath MA, Henning SJ.Regeneration of intestinal stem/progenitor cells following doxorubicintreatment of mice. Am J Physiol Gastrointest Liver Physiol 2009;297:G461–70.

38. Dembinski JL, Krauss S. Characterization and functional analysis of aslow cycling stem cell-like subpopulation in pancreas adenocarci-noma. Clin Exp Metastasis 2009;26:611–23.

39. Gao MQ, Choi YP, Kang S, Youn JH, Cho NH. CD24þ cells fromhierarchically organized ovarian cancer are enriched in cancer stemcells. Oncogene 2010;29:2672–80.

40. Ohta H, Aoyagi K, Fukaya M, Danjoh I, Ohta A, Isohata N, et al. Crosstalk between hedgehog and epithelial-mesenchymal transition path-ways in gastric pit cells and in diffuse-type gastric cancers. Br JCancer 2009;100:389–98.

41. Wang Z, Li Y, Kong D, Banerjee S, Ahmad A, Azmi AS, et al.Acquisition of epithelial-mesenchymal transition phenotype of gem-citabine-resistant pancreatic cancer cells is linked with activation ofthe notch signaling pathway. Cancer Res 2009;69:2400–7.

42. Gupta S, Iljin K, Sara H, Mpindi JP, Mirtti T, Vainio P, et al. FZD4 as amediator of ERG oncogene-induced WNT signaling and epithelial-to-mesenchymal transition in human prostate cancer cells. Cancer Res2010;70:6735–45.

43. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. Theepithelial-mesenchymal transition generates cells with properties ofstem cells. Cell 2008;133:704–15.

44. Zhou J, Schmid T, Schnitzer S, Br€une B. Tumor hypoxia and cancerprogression. Cancer Lett 2006;237:10–21.

45. Li Z, Bao S,WuQ,Wang H, Eyler C, Sathornsumetee S, et al. Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells.Cancer Cell 2009;15:501–13.

46. Heddleston JM, Li Z, McLendon RE, Hjelmeland AB, Rich JN. Thehypoxic microenvironment maintains glioblastoma stem cells andpromotes reprogramming towards a cancer stem cell phenotype. CellCycle 2009;8:3274–84.

47. Hjelmeland AB, Wu Q, Heddleston JM, Choudhary GS, MacSwords J,Lathia JD, et al. Acidic stress promotes a glioma stem cell phenotype.Cell Death Differ 2011;18:829–40.

48. Gilbertson RJ, Rich JN. Making a tumour's bed: glioblastoma stemcells and the vascular niche. Nat Rev Cancer 2007;7:733–6.

49. Tutt A, Robson M, Garber JE, Domchek SM, Audeh MW, Weitzel JN,et al. Oral poly(ADP-ribose) polymerase inhibitor olaparib in patientswith BRCA1 or BRCA2 mutations and advanced breast cancer: aproof-of-concept trial. Lancet 2010;376:235–44.

50. Audeh MW, Carmichael J, Penson RT, Friedlander M, Powell B, Bell-McGuinn KM, et al. Oral poly(ADP-ribose) polymerase inhibitor ola-

parib in patients with BRCA1 or BRCA2 mutations and recurrentovarian cancer: a proof-of-concept trial. Lancet 2010;376:245–51.

51. Zhou BB, Elledge SJ. The DNA damage response: putting check-points in perspective. Nature 2000;408:433–9.

52. Zenvirt S, Kravchenko-Balasha N, Levitzki A. Status of p53 in humancancer cells does not predict efficacy of CHK1 kinase inhibitorscombined with chemotherapeutic agents. Oncogene 2010;29:6149–59.

53. Piccirillo SG, Reynolds BA, Zanetti N, Lamorte G, Binda E, Broggi G,et al. Bone morphogenetic proteins inhibit the tumorigenic potential ofhuman brain tumour-initiating cells. Nature 2006;444:761–5.

54. Lombardo Y, Scopelliti A, Cammareri P, Todaro M, Iovino F, Ricci-Vitiani L, et al. Bonemorphogenetic protein 4 induces differentiation ofcolorectal cancer stem cells and increases their responseto chemotherapy in mice. Gastroenterology 2011;140:297–309.

55. Sanz MA, Martín G, Gonz�alez M, Le�on A, Ray�on C, Rivas C, et al.Risk-adapted treatment of acute promyelocytic leukemia withall-trans-retinoic acid and anthracycline monochemotherapy: amulticenter study by the PETHEMA group. Blood 2004;103:1237–43.

56. Baiocchi M, Biffoni M, Ricci-Vitiani L, Pilozzi E, De Maria R. Newmodels for cancer research: human cancer stem cell xenografts. CurrOpin Pharmacol 2010;10:380–4.

57. Todaro M, Iovino F, Eterno V, Cammareri P, Gambara G, Espina V,et al. Tumorigenic and metastatic activity of human thyroid cancerstem cells. Cancer Res 2010;70:8874–85.

58. Allegra CJ, Yothers G, O'Connell MJ, Sharif S, Petrelli NJ, ColangeloLH, et al. Phase III trial assessing bevacizumab in stages II and IIIcarcinoma of the colon: results of NSABP protocol C-08. J Clin Oncol2011;29:11–6.

59. Alberts SR, Sargent DJ, Smyrk TC, Shields AF, Chan E, Goldberg RM,et al. Adjuvant mFOLFOX6 with or without cetuxiumab (Cmab) inKRASwild-type (WT) patients (pts) with resected stage III colon cancer(CC): results from NCCTG intergroup phase III trial N0147 [abstractCRA3507]. J Clin Oncol 2010;28 Suppl 15:262–s.

60. Berlin J, Bendell J, Hart LL, Firdaus I, Gore I, Hermann RC, et al. Aphase 2, randomized, double-blind, placebo-controlled study ofhedgehog pathway inhibitor (HPI) GDC-0449 in patients with pre-viously untreated metastatic colorectal cancer (MCRC) [abstractLBA21]. Ann Oncol 2010;21 Suppl 8:viii10.

61. Von Hoff DD, LoRusso PM, Rudin CM, Reddy JC, Yauch RL, Tibes R,et al. Inhibition of the hedgehog pathway in advanced basal-cellcarcinoma. N Engl J Med 2009;361:1164–72.

62. Kaye SB, Fehrenbacher L, Holloway R, Horowitz N, Karlan B, Amit A,et al. A phase 2, randomized, placebo-controlled study of Hedgehog(HH) pathway inhibitor GDC-0449 as maintenance therapy in patientswith ovarian cancer in 2nd or 3rd complete remission (CR) [abstractLBA25]. Ann Oncol 2010;21 Suppl 8:viii11.

Cancer Stem Cells and Chemosensitivity

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