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Turning Cancer Stem Cells Inside Out: An Exploration of Glioma Stem Cell Signaling Pathways

2009; Elsevier BV; Volume: 284; Issue: 25 Linguagem: Inglês

10.1074/jbc.r900013200

1083-351X

Zhizhong Li, Hui Wang, Christine E. Eyler, Anita B. Hjelmeland, Jeremy N. Rich,

Glioma Diagnosis and Treatment

Tumors are complex collections of heterogeneous cells with recruited vasculature, inflammatory cells, and stromal elements. Neoplastic cells frequently display a hierarchy in differentiation status. Recent studies suggest that brain tumors have a limited population of neoplastic cells called cancer stem cells with the capacity for sustained self-renewal and tumor propagation. Brain tumor stem cells contribute to therapeutic resistance and tumor angiogenesis. In this minireview, we summarize recent data regarding critical signaling pathways involved in brain tumor stem cell biology and discuss how targeting these molecules may contribute to the development of novel anti-glioma therapies. Tumors are complex collections of heterogeneous cells with recruited vasculature, inflammatory cells, and stromal elements. Neoplastic cells frequently display a hierarchy in differentiation status. Recent studies suggest that brain tumors have a limited population of neoplastic cells called cancer stem cells with the capacity for sustained self-renewal and tumor propagation. Brain tumor stem cells contribute to therapeutic resistance and tumor angiogenesis. In this minireview, we summarize recent data regarding critical signaling pathways involved in brain tumor stem cell biology and discuss how targeting these molecules may contribute to the development of novel anti-glioma therapies. Cancers can be considered organ systems with aberrant activation of developmental and wound response pathways. Recent evidence suggests that within some tumors there is a cell subpopulation with the special capacity for sustained self-renewal and tumor propagation in vivo. Cells fulfilling these criteria were originally reported in acute myeloid leukemia (1Bhatia M. Wang J.C. Kapp U. Bonnet D. Dick J.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5320-5325Crossref PubMed Scopus (727) Google Scholar), but similar populations were soon successively identified within various solid tumors (2Cho R.W. Clarke M.F. Curr. Opin. Genet. Dev. 2008; 18: 48-53Crossref PubMed Scopus (205) Google Scholar). The proper terminology regarding these cells remains unsettled, with most groups using terms such as CSCs, 2The abbreviations used are: CSCcancer stem cellNSCneural stem cellRTKreceptor tyrosine kinaseEGFRepidermal growth factor receptorBMPbone morphogenetic proteinBMPRBMP receptormiRNAmicroRNAVEGFvascular endothelial growth factor. tumor-initiating/propagating cells, and stem-like cancer cells. Although CSCs are a source of controversy, the concept recognizes the well described heterogeneity of tumor cells. Many critics contest the hypothesis on the grounds of a potential stem cell origin, challenge of current markers, or CSC frequency, none of which are implicit requirements of the CSC hypothesis (3Jordan C.T. Cell Stem Cell. 2009; 4: 203-205Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). cancer stem cell neural stem cell receptor tyrosine kinase epidermal growth factor receptor bone morphogenetic protein BMP receptor microRNA vascular endothelial growth factor. Malignant gliomas are essentially universally lethal despite conventional therapy, with surgical resection and chemoradiation limited to palliation. Glioma CSCs were among the first solid tumor CSCs described (4Singh S.K. Hawkins C. Clarke I.D. Squire J.A. Bayani J. Hide T. Henkelman R.M. Cusimano M.D. Dirks P.B. Nature. 2004; 432: 396-401Crossref PubMed Scopus (6142) Google Scholar) and remain one of the most widely used CSC models. Glioma CSCs share significant similarities with normal NSCs, including the expression of stem cell markers (CD133, Nestin, Musashi, and Sox2) and the capacity to differentiate into multiple lineages (5Bao S. Wu Q. McLendon R.E. Hao Y. Shi Q. Hjelmeland A.B. Dewhirst M.W. Bigner D.D. Rich J.N. Nature. 2006; 444: 756-760Crossref PubMed Scopus (4948) Google Scholar), but the overlap is incomplete. Notably, glioma CSCs are also highly resistant to chemoradiotherapies (5Bao S. Wu Q. McLendon R.E. Hao Y. Shi Q. Hjelmeland A.B. Dewhirst M.W. Bigner D.D. Rich J.N. Nature. 2006; 444: 756-760Crossref PubMed Scopus (4948) Google Scholar, 6Ma S. Lee T.K. Zheng B.J. Chan K.W. Guan X.Y. Oncogene. 2008; 27: 1749-1758Crossref PubMed Scopus (661) Google Scholar), underscoring the importance of developing more efficient therapies against CSCs and prompting researchers to elucidate the molecular mechanisms regulating CSCs. Here, we summarize recent findings regarding the signaling pathways that are critical to glioma CSC biology. CSC behaviors are constantly affected by external signals from their niche, including neighboring stromal, immune, and non-stem tumor cells. Extracellular and paracrine effects are mediated commonly from cell-surface ligand-receptor systems. Accumulating evidence has demonstrated that multiple glioma CSC functions hinge on major receptor-mediated pathways (Fig. 1). The RTK family mediates the effects of multiple oncogenic growth factor pathways, among which the EGFR is one of the best characterized in gliomas. Malignant glioma cells frequently have increased EGFR signaling as a result of either amplified EGFR copy number or expression of the constitutively active variant EGFRvIII. Transduction of murine Nestin+ NSCs with EGFRvIII induces glioma-like lesions (7Bachoo R.M. Maher E.A. Ligon K.L. Sharpless N.E. Chan S.S. You M.J. Tang Y. DeFrances J. Stover E. Weissleder R. Rowitch D.H. Louis D.N. DePinho R.A. Cancer Cell. 2002; 1: 269-277Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar). Similarly, a recent publication showed that the combination of AKT/phosphoinositide 3-hydroxykinase activation and constitutive EGFR activity is sufficient to transform murine NSCs (8Li L. Dutra A. Pak E. Labrie III, J.E. Gerstein R.M. Pandolfi P.P. Recht L.D. Ross A.H. Neuro-oncology. 2009; 11: 9-21Crossref PubMed Scopus (71) Google Scholar). Although the origin of glioma CSCs is under study (9Alcantara Llaguno S. Chen J. Kwon C.H. Jackson E.L. Li Y. Burns D.K. Alvarez-Buylla A. Parada L.F. Cancer Cell. 2009; 15: 45-56Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar), these data suggest a possible role for EGFR pathways in glioma and possibly CSC biology. In cell culture studies, the proliferation and neurosphere generation (an in vitro assay to assess the self-renewal of CSCs) of human glioma CSCs were dependent on the EGFR (10Soeda A. Inagaki A. Oka N. Ikegame Y. Aoki H. Yoshimura S.-i. Nakashima S. Kunisada T. Iwama T. J. Biol. Chem. 2008; 283: 10958-10966Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), like NSCs. The signal initiated by RTKs is transduced and amplified through downstream molecule cascades, such as the pro-survival AKT/phosphoinositide 3-hydroxykinase pathway. Upon activation by RTK pathways, AKT promotes survival, proliferation, invasion, and secretion of pro-angiogenic factors. We recently demonstrated that glioma CSCs are more dependent on AKT signals than matched non-stem glioma cells (54Eyler C.E. Foo W.C. LaFiura K.M. McLendon R.E. Hjelmeland A.B. Rich J.N. Stem Cells. 2008; 26: 3027-3036Crossref PubMed Scopus (203) Google Scholar). Pharmacologic inhibitors of AKT attenuate glioma CSC neurosphere formation, induce apoptosis, and substantially delay intracranial tumor formation. These data suggest that AKT inhibition may specifically target the CSC population to reduce tumor malignancy. BMPs are a family of growth factors named for their central roles in bone and cartilage formation (11Reddi A.H. Cytokine Growth Factor Rev. 1997; 8: 11-20Crossref PubMed Scopus (255) Google Scholar). Most BMPs elicit their actions through binding to cell-surface receptor kinases (the BMPRs). The canonical effectors of BMPRs are the Smad proteins. Activating phosphorylation of receptor Smad1/5/8 enables these proteins to bind to the co-activator Smad4, translocate into the nucleus, and regulate transcription. BMPs are crucial factors that regulate proliferation and apoptosis in NSCs and usually promote the differentiation of NSCs (12Panchision D.M. McKay R.D. Curr. Opin. Genet. Dev. 2002; 12: 478-487Crossref PubMed Scopus (172) Google Scholar). Interestingly, a prodifferentiation BMP response mechanism seems to be preserved in some glioma CSCs, as CSCs express BMPRs, and BMPs inhibit the proliferation of these cells (13Piccirillo S.G. Reynolds B.A. Zanetti N. Lamorte G. Binda E. Broggi G. Brem H. Olivi A. Dimeco F. Vescovi A.L. Nature. 2006; 444: 761-765Crossref PubMed Scopus (984) Google Scholar). BMP ligands deplete the CSC population by inducing the differentiation of CSCs into astroglial and neuron-like cells. Treating CSCs with BMPs in vivo markedly delays tumor growth and reduces tumor invasion. These data suggest that selective activation of BMP pathways may reduce the tumorigenic capacity of CSCs. Similar to the previous report, Lee et al. (14Lee J. Son M.J. Woolard K. Donin N.M. Li A. Cheng C.H. Kotliarova S. Kotliarov Y. Walling J. Ahn S. Kim M. Totonchy M. Cusack T. Ene C. Ma H. Su Q. Zenklusen J.C. Zhang W. Maric D. Fine H.A. Cancer Cell. 2008; 13: 69-80Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar) reported that glioma CSCs differentiate upon BMP treatment. However, CSCs from one patient displayed enhanced proliferation rather than differentiation upon BMP treatment. This CSC sample displayed a fetal BMPR expression pattern due to epigenetic silencing of Bmpr1b. Restoration of BMPR1B expression sensitized CSCs to BMP-mediated differentiation, suggesting that the expression pattern of BMPR may determine BMP-mediated CSC behavior. Thus, an individual tumor's genomic and epigenetic characteristics may determine the response to anti-CSC treatment. The binding of Hedgehog ligands to their receptors activates transducers termed GLIs (named for their discovery in gliomas), which then translocate into the nucleus to activate or repress downstream targets. The Hedgehog pathway is one of the key regulators of embryogenesis and is critical for several different types of normal stem cells, including NSCs (15Park Y. Rangel C. Reynolds M.M. Caldwell M.C. Johns M. Nayak M. Welsh C.J. McDermott S. Datta S. Dev. Biol. 2003; 253: 247-257Crossref PubMed Scopus (137) Google Scholar, 16Wechsler-Reya R.J. Scott M.P. Neuron. 1999; 22: 103-114Abstract Full Text Full Text PDF PubMed Scopus (1074) Google Scholar). Mutations in the Hedgehog pathway are associated with medulloblastomas, primary brain tumors common in children, and several genetic medulloblastoma models incorporate aberrant Hedgehog signaling. Hedgehog signaling is also active in gliomas and contributes to CSC function (17Dahmane N. Sanchez P. Gitton Y. Palma V. Sun T. Beyna M. Weiner H. Ruiz i Altaba A. Development (Camb.). 2001; 128: 5201-5212PubMed Google Scholar, 18Clement V. Sanchez P. de Tribolet N. Radovanovic I. Ruiz i Altaba A. Curr. Biol. 2007; 17: 165-172Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar, 19Bar E.E. Chaudhry A. Lin A. Fan X. Schreck K. Matsui W. Piccirillo S. Vescovi A.L. DiMeco F. Olivi A. Eberhart C.G. Stem Cells. 2007; 25: 2524-2533Crossref PubMed Scopus (530) Google Scholar). Hedgehog ligands are required for CSC self-renewal as well as tumorigenesis. Treatment of glioma CSCs with the Hedgehog inhibitor cyclopamine or transduction with GLI RNA interference inhibits proliferation and self-renewal while increasing apoptosis. The importance of Hedgehog signaling is further underscored by the observation that Hedgehog inhibition augments the efficacy of the standard-of-care chemotherapy agent temozolomide to abolish CSC proliferation and induce cell death. In vivo, Hedgehog pathway inhibition blocks CSC tumor growth (18Clement V. Sanchez P. de Tribolet N. Radovanovic I. Ruiz i Altaba A. Curr. Biol. 2007; 17: 165-172Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar). Bar et al. (19Bar E.E. Chaudhry A. Lin A. Fan X. Schreck K. Matsui W. Piccirillo S. Vescovi A.L. DiMeco F. Olivi A. Eberhart C.G. Stem Cells. 2007; 25: 2524-2533Crossref PubMed Scopus (530) Google Scholar) also showed that cyclopamine treatment depletes CSCs, as viable cells after treatment failed to propagate tumors in vivo. In agreement with our study (5Bao S. Wu Q. McLendon R.E. Hao Y. Shi Q. Hjelmeland A.B. Dewhirst M.W. Bigner D.D. Rich J.N. Nature. 2006; 444: 756-760Crossref PubMed Scopus (4948) Google Scholar), they found that CSCs respond poorly to radiotherapy. However, cyclopamine treatment improves the effects of radiation on CSC survival (19Bar E.E. Chaudhry A. Lin A. Fan X. Schreck K. Matsui W. Piccirillo S. Vescovi A.L. DiMeco F. Olivi A. Eberhart C.G. Stem Cells. 2007; 25: 2524-2533Crossref PubMed Scopus (530) Google Scholar). Together, these data suggest that the Hedgehog pathway is important for glioma CSCs and that pharmacologic inhibitors may improve traditional therapy efficiency against gliomas. Notch proteins are single-pass transmembrane receptors that mediate cell-cell communication. Upon ligand binding, Notch is cleaved by the γ-secretase complex to release its intracellular domain from the cell membrane. Cleaved Notch translocates into the nucleus to function as a transcription factor. The importance of the Notch signaling pathway is evident by its high conservation throughout evolution. During development, Notch promotes the proliferation of normal NSCs while suppressing their differentiation (20Gaiano N. Fishell G. Annu. Rev. Neurosci. 2002; 25: 471-490Crossref PubMed Scopus (505) Google Scholar, 21Solecki D.J. Liu X.L. Tomoda T. Fang Y. Hatten M.E. Neuron. 2001; 31: 557-568Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Notch is required for neural progenitors both in vitro and in vivo (22Androutsellis-Theotokis A. Leker R.R. Soldner F. Hoeppner D.J. Ravin R. Poser S.W. Rueger M.A. Bae S.K. Kittappa R. McKay R.D. Nature. 2006; 442: 823-826Crossref PubMed Scopus (860) Google Scholar). Notch signaling has been implicated in brain tumor biology as well. Expression of Notch-1 and its ligands Delta-like-1 and Jagged-1 is critical to high grade gliomas and medulloblastomas (23Purow B.W. Haque R.M. Noel M.W. Su Q. Burdick M.J. Lee J. Sundaresan T. Pastorino S. Park J.K. Mikolaenko I. Maric D. Eberhart C.G. Fine H.A. Cancer Res. 2005; 65: 2353-2363Crossref PubMed Scopus (482) Google Scholar, 24Fan X. Mikolaenko I. Elhassan I. Ni X. Wang Y. Ball D. Brat D.J. Perry A. Eberhart C.G. Cancer Res. 2004; 64: 7787-7793Crossref PubMed Scopus (344) Google Scholar, 25Hallahan A.R. Pritchard J.I. Hansen S. Benson M. Stoeck J. Hatton B.A. Russell T.L. Ellenbogen R.G. Bernstein I.D. Beachy P.A. Olson J.M. Cancer Res. 2004; 64: 7794-7800Crossref PubMed Scopus (371) Google Scholar). The potential role of Notch signaling in brain tumor CSCs was first studied in medulloblastomas, with CSCs expressing high levels of Notch and displaying sensitivity to Notch pathway inhibitors (26Fan X. Matsui W. Khaki L. Stearns D. Chun J. Li Y.M. Eberhart C.G. Cancer Res. 2006; 66: 7445-7452Crossref PubMed Scopus (544) Google Scholar). Notch functions were later linked to glioma CSCs, as Notch signaling increases expression of the stem cell marker Nestin in gliomas. Notch expression in a K-Ras-induced mouse glioblastoma model generates proliferative lesions in the NSC-rich subependymal zone, ultimately leading to glioma formation (27Shih A.H. Holland E.C. Neoplasia. 2006; 8: 1072-1082Crossref PubMed Scopus (163) Google Scholar). In addition, activation of Notch signaling in the glioma cell lines increases the formation of neurosphere-like colonies (28Zhang X.P. Zheng G. Zou L. Liu H.L. Hou L.H. Zhou P. Yin D.D. Zheng Q.J. Liang L. Zhang S.Z. Feng L. Yao L.B. Yang A.G. Han H. Chen J.Y. Mol. Cell. Biochem. 2008; 307: 101-108Crossref PubMed Scopus (122) Google Scholar). Transcription factors, epigenetic regulators, and miRNAs are extremely powerful regulators of normal cells and cancer cells. They are capable of simultaneous regulation of multiple downstream targets and are implicated in glioma CSC survival, proliferation, and maintenance (Fig. 1). The c-Myc oncoprotein has been extensively studied for its instrumental role in the proliferation of both normal stem cells and tumor cells. Recently, inducible pluripotent stem cells were generated from differentiated cells through the introduction of several transcription factors, including c-myc (29Takahashi K. Yamanaka S. Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (19304) Google Scholar), supporting a role in core stem cell machinery. c-Myc may thus serve as a critical link between "stemness" and malignancy. c-Myc expression correlates with the grade of malignancy in gliomas (30Herms J.W. von Loewenich F.D. Behnke J. Markakis E. Kretzschmar H.A. Surg. Neurol. 1999; 51: 536-542Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). We recently determined that glioma CSCs express elevated levels of c-Myc and that c-Myc is required both for maintenance of glioma CSCs in vitro and for their tumorigenic capacity in vivo (31Wang J. Wang H. Li Z. Wu Q. Lathia J.D. McLendon R.E. Hjelmeland A.B. Rich J.N. PLoS ONE. 2008; 3: e3769Crossref PubMed Scopus (326) Google Scholar). These data derived from human patient samples are supported by mouse glioma models. Conditional overexpression of c-Myc in mouse astroglia (a subtype of NSCs) results in growing tumors that resemble human gliomas (32Jensen N.A. Pedersen K.M. Lihme F. Rask L. Nielsen J.V. Rasmussen T.E. Mitchelmore C. J. Biol. Chem. 2003; 278: 8300-8308Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). c-Myc additionally prevents differentiation and promotes self-renewal of tumor neurospheres derived from a p53/Pten double knock-out mouse model (33Zheng H. Ying H. Yan H. Kimmelman A.C. Hiller D.J. Chen A.J. Perry S.R. Tonon G. Chu G.C. Ding Z. Stommel J.M. Dunn K.L. Wiedemeyer R. You M.J. Brennan C. Wang Y.A. Ligon K.L. Wong W.H. Chin L. DePinho R.A. Nature. 2008; 455: 1129-1133Crossref PubMed Scopus (578) Google Scholar). Oct4, along with Sox2 and Nanog, is believed to be a core component in controlling the balance between self-renewal and differentiation in embryonic stem cells. Oct4 is also one of the factors that generates inducible pluripotent stem cells (29Takahashi K. Yamanaka S. Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (19304) Google Scholar). Oct4 is highly expressed in many human glioma specimens and cell lines, and its expression correlates with glioma grade (34Du Z. Jia D. Liu S. Wang F. Li G. Zhang Y. Cao X. Ling E.-A. Hao A. Glia. 2008; 57: 724-733Crossref Scopus (131) Google Scholar). The direct role of Oct4 in glioma CSCs is not well understood, but overexpression of Oct4 in rat C6 glioma cells increases the expression level of the stemness marker Nestin. These data suggest that Oct4 may inhibit the differentiation of glioma CSCs (34Du Z. Jia D. Liu S. Wang F. Li G. Zhang Y. Cao X. Ling E.-A. Hao A. Glia. 2008; 57: 724-733Crossref Scopus (131) Google Scholar) and contribute to CSC maintenance. Olig2 is a transcription factor that is almost exclusively expressed in the central nervous system. During brain development, Olig2 is expressed in neural progenitor cells that give rise to oligodendrocytes and certain neuronal subtypes (35Lu Q.R. Sun T. Zhu Z. Ma N. Garcia M. Stiles C.D. Rowitch D.H. Cell. 2002; 109: 75-86Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar, 36Novitch B.G. Chen A.I. Jessell T.M. Neuron. 2001; 31: 773-789Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 37Takebayashi H. Yoshida S. Sugimori M. Kosako H. Kominami R. Nakafuku M. Nabeshima Y. Mech. Dev. 2000; 99: 143-148Crossref PubMed Scopus (318) Google Scholar, 38Zhou Q. Anderson D.J. Cell. 2002; 109: 61-73Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar). Pathological analysis revealed that Olig2 is expressed in almost all adult astrocytomas and is required for tumor initiation, suggesting a link to gliomas and CSCs (39Ligon K.L. Alberta J.A. Kho A.T. Weiss J. Kwaan M.R. Nutt C.L. Louis D.N. Stiles C.D. Rowitch D.H. J. Neuropathol. Exp. Neurol. 2004; 63: 499-509Crossref PubMed Scopus (341) Google Scholar, 40Ligon K.L. Huillard E. Mehta S. Kesari S. Liu H. Alberta J.A. Bachoo R.M. Kane M. Louis D.N. DePinho R.A. Anderson D.J. Stiles C.D. Rowitch D.H. Neuron. 2007; 53: 503-517Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Functionally, Olig2 is required in both normal NSCs and glioma CSCs. Olig2 sustains the replication-competent state of neural progenitors and is necessary for the multilineage differentiation potential of neural progenitors (38Zhou Q. Anderson D.J. Cell. 2002; 109: 61-73Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar, 41Lee S.K. Lee B. Ruiz E.C. Pfaff S.L. Genes Dev. 2005; 19: 282-294Crossref PubMed Scopus (172) Google Scholar). Olig2 function is mediated through the proliferative regulation of glioma CSCs in part through suppression of p21WAF1/CIP1 (40Ligon K.L. Huillard E. Mehta S. Kesari S. Liu H. Alberta J.A. Bachoo R.M. Kane M. Louis D.N. DePinho R.A. Anderson D.J. Stiles C.D. Rowitch D.H. Neuron. 2007; 53: 503-517Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). bmi1 belongs to the Polycomb group genes, which usually function as epigenetic silencers. Bmi1 has been implicated in determining stem cell fate in multiple tissues and is a positive regulator of NSC self-renewal (42Dirks P. Cancer Cell. 2007; 12: 295-297Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). bmi1 is also a known oncogene that is frequently overexpressed in many cancer types, including gliomas. Using cells from wild-type and bmi1 knock-out mice, researchers demonstrated that Bmi1 is required for transforming both differentiated astrocytes and NSCs (43Bruggeman S.W. Hulsman D. Tanger E. Buckle T. Blom M. Zevenhoven J. van Tellingen O. van Lohuizen M. Cancer Cell. 2007; 12: 328-341Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Whereas transformed wild-type NSCs give rise to high grade gliomas in vivo, bmi1-deficient NSCs are able to initiate only less malignant tumors. bmi1-deficient tumors have fewer cells expressing the NSC marker Nestin, implying that they may have fewer CSCs. Both transformed and non-transformed bmi1-deficient NSCs demonstrate impaired capability of neurogenesis, highlighting a role of Bmi1 in controlling differentiation of normal and malignant stem cells. Although it remains unclear whether Bmi1 is also critical for CSCs derived from human gliomas, Bmi1 helps to maintain CSCs from hepatocellular carcinoma (44Chiba T. Miyagi S. Saraya A. Aoki R. Seki A. Morita Y. Yonemitsu Y. Yokosuka O. Taniguchi H. Nakauchi H. Iwama A. Cancer Res. 2008; 68: 7742-7749Crossref PubMed Scopus (178) Google Scholar). miRNAs are small noncoding RNAs that can silence target genes through post-transcriptional mechanisms on target mRNAs. miRNAs are powerful intracellular regulators because a single miRNA can regulate many distinct mRNAs. In cancer biology, miRNAs can function as "oncogenes," e.g. the miR-17–92 polycistron, or "tumor suppressors," e.g. the miR-15/miR-16 cluster. miRNA regulation appears to be critically important in glioma cell behaviors. For instance, miRNA-21 was shown to be significantly overexpressed in glioblastomas, and inhibition of its function induces apoptosis (45Chan J.A. Krichevsky A.M. Kosik K.S. Cancer Res. 2005; 65: 6029-6033Crossref PubMed Scopus (2240) Google Scholar). Two recent reports directly investigated the roles of miRNAs in glioma CSCs. The levels of miR-124 and miR-137 are reduced in grade III and IV malignant gliomas in comparison with normal brain (46Silber J. Lim D.A. Petritsch C. Persson A.I. Maunakea A.K. Yu M. Vandenberg S.R. Ginzinger D.G. James C.D. Costello J.F. Bergers G. Weiss W.A. Alvarez-Buylla A. Hodgson J.G. BMC Med. 2008; 6: 14Crossref PubMed Scopus (787) Google Scholar). Overexpression of these two miRNAs inhibits proliferation while inducing differentiation of glioma CSCs, indicating a tumor suppressor role for these two miRNAs in CSCs. Similarly, another miRNA, miR-451, is expressed at lower levels in CD133+ glioma CSCs in comparison with CD133− non-stem glioma cells (47Gal H. Pandi G. Kanner A.A. Ram Z. Lithwick-Yanai G. Amariglio N. Rechavi G. Givol D. Biochem. Biophys. Res. Commun. 2008; 376: 86-90Crossref PubMed Scopus (212) Google Scholar). External expression of miR-451 inhibits the growth of glioma CSCs and disrupts neurosphere formation. The relationship between CSCs and their microenvironment is reciprocal. CSCs not only receive signals from extracellular sources through receptors but also actively transmit signals to manipulate the environment. In this way, CSCs are able to modulate the same microenvironment that produces signals regulating CSCs. Angiogenesis is the best studied example of such cross-talk (Fig. 2). Active angiogenesis is a hallmark of solid tumors and plays a key role in providing oxygen and nutrition as well as facilitating metastasis. Malignant gliomas are characterized by florid angiogenesis, with neovascularization significantly correlated with enhanced tumor aggressiveness, degree of tumor malignancy, and poor clinical prognosis. Given the importance of CSCs in glioma maintenance, it is not surprising that CSCs and angiogenesis are tightly connected. We showed that, in comparison with matched non-stem cancer cells, glioma CSCs have a stronger capacity for promoting angiogenesis, partially through amplified secretion of VEGF, one of the most important pro-angiogenic factors (48Bao S. Wu Q. Sathornsumetee S. Hao Y. Li Z. Hjelmeland A.B. Shi Q. McLendon R.E. Bigner D.D. Rich J.N. Cancer Res. 2006; 66: 7843-7848Crossref PubMed Scopus (1095) Google Scholar). Treating glioma CSCs with the VEGF-neutralizing antibody bevacizumab attenuates their ability to promote angiogenesis both in vitro and in vivo, which in turn markedly inhibits the tumorigenesis of CSCs. The mechanism underlying the specific up-regulation of VEGF in CSCs is still unclear, but it has been suggested that environmental factors such as hypoxia and acidosis play important roles in this process. Alternatively, VEGF could also be induced by oncogene activation, e.g. the loss of PTEN and/or activation of EGFR could result in high VEGF expression in gliomas. The extraordinary pro-angiogenic influence of CSCs is likely critical for their maintenance not only because blood vessels provide oxygen and nutrition but also because, like normal NSCs, glioma CSCs are situated in "vascular niches." Both normal NSCs and CSCs are believed to be physically located in specialized microenvironmental niches, which regulate self-renewal and differentiation. NSCs are located in niches defined by the vasculature (49Tavazoie M. Van der Veken L. Silva-Vargas V. Louissaint M. Colonna L. Zaidi B. Garcia-Verdugo J.M. Doetsch F. Cell Stem Cell. 2008; 3: 279-288Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 50Shen Q. Wang Y. Kokovay E. Lin G. Chuang S.M. Goderie S.K. Roysam B. Temple S. Cell Stem Cell. 2008; 3: 289-300Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar), and this property is shared by medulloblastoma CSCs (51Calabrese C. Poppleton H. Kocak M. Hogg T.L. Fuller C. Hamner B. Oh E.Y. Gaber M.W. Finklestein D. Allen M. Frank A. Bayazitov I.T. Zakharenko S.S. Gajjar A. Davidoff A. Gilbertson R.J. Cancer Cell. 2007; 11: 69-82Abstract Full Text Full Text PDF PubMed Scopus (1780) Google Scholar). Co-injection of CSCs and endothelial cells accelerates tumor initiation and growth. This reciprocal relationship between angiogenesis and glioma CSCs may help explain the preliminary success of anti-angiogenesis therapy on malignant gliomas. Clinical trials of anti-angiogenic drugs like bevacizumab and cediranib have demonstrated promising preliminary results in glioblastoma patients (52Vredenburgh J.J. Desjardins A. Herndon 2nd, J.E. Dowell J.M. Reardon D.A. Quinn J.A. Rich J.N. Sathornsumetee S. Gururangan S. Wagner M. Bigner D.D. Friedman A.H. Friedman H.S. Clin. Cancer Res. 2007; 13: 1253-1259Crossref PubMed Scopus (931) Google Scholar, 53Batchelor T.T. Sorensen A.G. di Tomaso E. Zhang W.T. Duda D.G. Cohen K.S. Kozak K.R. Cahill D.P. Chen P.J. Zhu M. Ancukiewicz M. Mrugala M.M. Plotkin S. Drappatz J. Louis D.N. Ivy P. Scadden D.T. Benner T. Loeffler J.S. Wen P.Y. Jain R.K. Cancer Cell. 2007; 11: 83-95Abstract Full Text Full Text PDF PubMed Scopus (1520) Google Scholar). It is possible that these drugs might directly disrupt the maintenance of CSCs, thus effectively eliminating the roots of tumor progression. The existence of glioma CSCs prompts a refocusing of our views of cancer biology. Rather than a bulk of equally potent neoplastic cells, gliomas appear to have a cellular hierarchy. Within this hierarchy, glioma CSCs represent cells with an extraordinary capacity for tumorigenesis, making them attractive targets for anti-glioma therapies. Unfortunately, glioma CSCs are also highly resistant to traditional therapies, making the development of novel treatments against CSCs an urgent task. Remarkable insights have been gained through recent studies of signaling pathways that are differentially regulated in CSCs and non-stem cancer cells. In addition to the signaling discussed above, other oncogenic signals (e.g. Ras/mitogen-activated protein kinase (MAPK) pathway) or tumor suppressors (e.g. p53) may play important roles in glioma CSCs. CSCs may be more dependent on oncogenic signaling motifs, so selectively and effectively targeting these oncogenes may be a promising anti-CSC therapeutic strategy. Blood vessels provide not only nutrition/oxygen to cancer cells but also a favorable microenvironment for the maintenance of CSCs. Thus, anti-angiogenic agents appear promising because they may inhibit tumor growth in two ways: 1) direct interruption of nutrient-supplying vasculature and 2) indirect CSC inhibition by disruption of vascular niches.