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The Side Story of Stem-like Glioma Cells
Cell Stem Cell
Previews The Side Story of Stem-like Glioma Cells Anders I. Persson1,* and William A. Weiss1,2,3,4,* 1Department
of Neurology of Pediatrics 3Department of Neurosurgery and Brain Tumor Research Center 4Helen Diller Family Comprehensive Cancer Center University of California, San Francisco, San Francisco, CA 94143, USA *Correspondence: [email protected] (A.I.P.), [email protected] (W.A.W.) DOI 10.1016/j.stem.2009.02.004 2Department
In this issue of Cell Stem Stell, Bleau et al. report that activity of the drug transporter ABCG2 defines a tumorigenic stem-like side population (SP) isolated from glioma. Surprisingly, temozolomide, the first-line chemotherapeutic used for treatment of glioma, increased this side population, and even more so when PTEN was deleted.
Glioblastoma multiforme (GBM), the most common form of primary brain tumor, is characterized by an intrinsic heterogeneity among cell types comprising the tumor mass, hence the name ‘‘multiforme.’’ The heterogeneity of cancer cell populations and how to target them therapeutically is a subject of great interest, and many recent studies converge to ask what role, if any, cancer stem cells (CSCs) play in cancer pathogenesis. CSCs appear to be resistant to chemotherapy, suggesting that these cells persist after treatment and drive recurrence (Reya et al., 2001). In this issue of Cell Stem Cell, Craig Jordan discusses the possibility that CSCs, defined as cancer cells that undergo self-renewal and are capable of recapitulating the entire tumor population, may not adhere to the lineage diagrams and stable phenotypes associated with ‘‘normal’’ stem cells, and might therefore be more effectively studied and targeted on the basis of their known functional properties (Jordan, 2009). Indeed, Holland and coworkers used just such a functional approach in their study. It is known that efflux of specific drugs by the membrane pump ABCG2 underlies chemotherapeutic resistance, and also that stem cells can be flow sorted as a side population (SP) because they use the same mechanism to efflux the DNA binding dye Hoechst 33342. It has also been demonstrated that the SP from glioma cell lines is enriched in tumorigenic cells with stem cell properties (Patrawala et al., 2005). In their study, Bleau et al. isolated SPs from PDGF-
induced murine gliomas (Shih et al., 2004) and human GBM samples and characterized the tumorigenic properties and drug sensitivity of these populations. In normal mouse brain, the SP was visualized as a discrete fraction and was found to consist mainly of endothelial cells that very actively excluded Hoechst 33342. In contrast, the SP examined in tumor samples expressed both endothelial and stem cell markers and contained cells that excluded dye over a significantly broader activity range. Notably, the endothelial cells in the tumor SPs exhibit reduced ABCG2 activity, a finding raising unanswered questions as to how reduced ABCG2 transporter function might contribute to the blood-brain barrier disruption observed as a typical feature of high-grade gliomas. The tumor-associated SP cells formed primary spheres in neurosphere medium. SP numbers, phenotype, and sphere formation ability were found to be increased in secondary spheres, suggesting that ABCG2 activity contributes to the selection and enrichment of SP cells. Bleau et al. next cultured the SP cells in neurosphere medium and examined how the SP phenotype was altered by phosphoinositide-30 kinase (PI3K)/Akt signaling and PTEN deletion. The PI3K/ Akt pathway is activated, and PTEN, a negative regulator of PI3K signaling, is mutated or deleted in the majority of GBM patients (Mclendon, 2008; Parsons et al., 2008). Loss of PTEN in conjunction with loss of p53 maintains both NSCs and glioma CSCs in an undifferentiated state, promoting self-renewal and inhibiting
differentiation, an effect attributed in part to activation of myc (Zheng et al., 2008). The authors found that PTEN deletion doubled the number of SP cells present in tumor-derived spheres, while PI3K inhibition led to loss of ABCG2 pump expression at the plasma membrane and a subsequent reduction of the SP fractions in both PTEN and wild-type cells. Collectively, these results suggest that PTEN-induced ABCG2 activity is responsible for the SP phenotype in glioma CSCs, extending and confirming previous results in other cell types (Takada et al., 2005; Zhou et al., 2007). Most notably, from a clinical perspective, Bleau et al. found that treating the SP spheres with the GBM chemotherapeutic temozolomide increased the SP fraction, and even more so in PTEN null spheres. These results led the authors to hypothesize that PTEN loss leads to increased temozolomide resistance. Temozolomide treatment also shortened tumor latency; mice grafted with tumorderived SP spheres cultured with temozolomide started to develop tumors after 25 days, while untreated transplants grew tumors after 40 days. Interestingly, temozolomide itself was not a substrate for ABCG2. The apparent increase in SP was instead traced to elevated activity of O6-methyl-guanine-DNA methyltransferase, a DNA repair enzyme known to mediate temozolomide resistance. To demonstrate relevance to human GBM, Bleau et al. next showed that blockade of either PI3K or Akt also downregulated the SP phenotype in primary patient-derived GBM SP spheres. Orthotopic xenografting
Cell Stem Cell 4, March 6, 2009 ª2009 Elsevier Inc. 191
Cell Stem Cell
Previews REFERENCES of SP cells from human GBM led to A B shortened latency for tumor formaBleau, A.-M., Hambardzumyan, D., Ozawa, tion compared to the main populaT., Fomchenko, E.I., Huse, J.T., Brennan, tion, consistent with SP cells being C.W., and Holland, E.C. (2009). Cell Stem Cell 4, this issue, 226–235. enriched for CSCs also in human tumors. Jordan, C. (2009). Cell Stem Cell 4, this issue, 203–205. Taken together, these findings PTEN Temozolomide offer important scientific insight, loss Mclendon, R. (2008). Nature 455, 1061– and yet seem paradoxical with 1068. respect to their therapeutic impliParsons, D.W., Jones, S., Zhang, X., Lin, cations. Bleau et al. (2009) showed J.C., Leary, R.J., Angenendt, P., Mankoo, P., Carter, H., Siu, I.M., Gallia, G.L., et al. that the abundance of the stem(2008). Science 321, 1807–1812. like glioma cells was readily modified through either acquisition of Patrawala, L., Calhoun, T., SchneiderBroussard, R., Zhou, J., Claypool, K., mutation (loss of PTEN) or in and Tang, D.G. (2005). Cancer Res. 65, response to treatment with temo6207–6219. Figure 1. Models to Describe How Loss of PTEN and zolomide (Figure 1). Importantly, Treatment with Temozolomide May Lead to Reya, T., Morrison, S.J., Clarke, M.F., and Chemoresistance in Glioma while temozolomide, the most Weissman, I.L. (2001). Nature 414, 105– (A) Loss of PTEN leads to increased ABCG2 activity (represented effective chemotherapy used for 111. by black squares) in a subset of cells, resulting in increased selfGBM, essentially sets the stage renewal and net chemoresistance. Shih, A.H., Dai, C., Hu, X., Rosenblum, (B) The alkylating agent temozolomide most effectively targets for recurrent drug-resistant diM.K., Koutcher, J.A., and Holland, E.C. non-SP cells, leading to relative expansion of a chemoresistant (2004). Cancer Res. 64, 4783–4789. sease, Bleau et al. also demon(ABCG2-positive) SP fraction. strate that inhibitors of PI3K, now Takada, T., Suzuki, H., Gotoh, Y., and Sugiyama, Y. (2005). Drug Metab. Dispos. in clinical trials in cancer, may be 33, 905–909. useful in blocking temozolomidedriven expansion of the SP. These find- turning ‘‘dial’’ rather than an on/off Zheng, H., Ying, H., Yan, H., Kimmelman, A.C., ings also show that the relative abun- ‘‘switch.’’ Clearly, future studies are Hiller, D.J., Chen, A.J., Perry, S.R., Tonon, G., Chu, G.C., Ding, Z., et al. (2008). Nature 455, dance and drug resistance of CSCs needed to understand why both loss of 1129–1133. can change during malignant pro- PTEN and temozolomide expand the SP, gression and in response to therapy. and how to translate these findings into Zhou, J., Wulfkuhle, J., Zhang, H., Gu, P., Yang, Y., Deng, J., Margolick, J.B., Liotta, L.A., Petricoin, E., Thus acquisition of stemness in cancer improved therapeutic approaches for 3rd, and Zhang, Y. (2007). Proc. Natl. Acad. Sci. USA 104, 16158–16163. is perhaps more likely a continually this deadly tumor.
LOCKing in Cellular Potential Helle F. Jørgensen1,* and Amanda G. Fisher1,* 1MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK *Correspondence: [email protected] (H.F.J.), [email protected] (A.G.F.) DOI 10.1016/j.stem.2009.02.007
Recently in Nature Genetics, Wen et al. (2009) report that H3K9me2 modification is more abundant in differentiated cells than in embryonic stem cells and that it decorates chromatin in repressive blocks. A spectrum of differentiated cell types that constitute higher organisms arise from pluripotent cells that are found in the inner cell mass of blastocyst-stage embryos. Cellular potential is gradually lost as cells acquire specific fates during development (Figure 1A), although under certain experimental conditions, their potential can be reset (via reprogramming, Figure 1A).
The exchange of cellular potential for functional specialization takes place—in almost all cases—without loss of genetic material, a result that has led to speculations that progressive silencing of the genome could underwrite directional differentiation (Spemann, 1938). Epigenetic mechanisms capable of modulating the accessibility of the genome to DNA-
192 Cell Stem Cell 4, March 6, 2009 ª2009 Elsevier Inc.
binding transcription factors have been implicated in this process (Reik, 2007), particularly dynamic changes in DNA methylation and covalent modification to histone tails. The importance of these epigenetic mechanisms for normal development is underscored by the prevalence of lethality among embryos lacking DNA and histone methyltransferases.
Previews The Side Story of Stem-like Glioma Cells Anders I. Persson1,* and William A. Weiss1,2,3,4,* 1Department
of Neurology of Pediatrics 3Department of Neurosurgery and Brain Tumor Research Center 4Helen Diller Family Comprehensive Cancer Center University of California, San Francisco, San Francisco, CA 94143, USA *Correspondence: [email protected] (A.I.P.), [email protected] (W.A.W.) DOI 10.1016/j.stem.2009.02.004 2Department
In this issue of Cell Stem Stell, Bleau et al. report that activity of the drug transporter ABCG2 defines a tumorigenic stem-like side population (SP) isolated from glioma. Surprisingly, temozolomide, the first-line chemotherapeutic used for treatment of glioma, increased this side population, and even more so when PTEN was deleted.
Glioblastoma multiforme (GBM), the most common form of primary brain tumor, is characterized by an intrinsic heterogeneity among cell types comprising the tumor mass, hence the name ‘‘multiforme.’’ The heterogeneity of cancer cell populations and how to target them therapeutically is a subject of great interest, and many recent studies converge to ask what role, if any, cancer stem cells (CSCs) play in cancer pathogenesis. CSCs appear to be resistant to chemotherapy, suggesting that these cells persist after treatment and drive recurrence (Reya et al., 2001). In this issue of Cell Stem Cell, Craig Jordan discusses the possibility that CSCs, defined as cancer cells that undergo self-renewal and are capable of recapitulating the entire tumor population, may not adhere to the lineage diagrams and stable phenotypes associated with ‘‘normal’’ stem cells, and might therefore be more effectively studied and targeted on the basis of their known functional properties (Jordan, 2009). Indeed, Holland and coworkers used just such a functional approach in their study. It is known that efflux of specific drugs by the membrane pump ABCG2 underlies chemotherapeutic resistance, and also that stem cells can be flow sorted as a side population (SP) because they use the same mechanism to efflux the DNA binding dye Hoechst 33342. It has also been demonstrated that the SP from glioma cell lines is enriched in tumorigenic cells with stem cell properties (Patrawala et al., 2005). In their study, Bleau et al. isolated SPs from PDGF-
induced murine gliomas (Shih et al., 2004) and human GBM samples and characterized the tumorigenic properties and drug sensitivity of these populations. In normal mouse brain, the SP was visualized as a discrete fraction and was found to consist mainly of endothelial cells that very actively excluded Hoechst 33342. In contrast, the SP examined in tumor samples expressed both endothelial and stem cell markers and contained cells that excluded dye over a significantly broader activity range. Notably, the endothelial cells in the tumor SPs exhibit reduced ABCG2 activity, a finding raising unanswered questions as to how reduced ABCG2 transporter function might contribute to the blood-brain barrier disruption observed as a typical feature of high-grade gliomas. The tumor-associated SP cells formed primary spheres in neurosphere medium. SP numbers, phenotype, and sphere formation ability were found to be increased in secondary spheres, suggesting that ABCG2 activity contributes to the selection and enrichment of SP cells. Bleau et al. next cultured the SP cells in neurosphere medium and examined how the SP phenotype was altered by phosphoinositide-30 kinase (PI3K)/Akt signaling and PTEN deletion. The PI3K/ Akt pathway is activated, and PTEN, a negative regulator of PI3K signaling, is mutated or deleted in the majority of GBM patients (Mclendon, 2008; Parsons et al., 2008). Loss of PTEN in conjunction with loss of p53 maintains both NSCs and glioma CSCs in an undifferentiated state, promoting self-renewal and inhibiting
differentiation, an effect attributed in part to activation of myc (Zheng et al., 2008). The authors found that PTEN deletion doubled the number of SP cells present in tumor-derived spheres, while PI3K inhibition led to loss of ABCG2 pump expression at the plasma membrane and a subsequent reduction of the SP fractions in both PTEN and wild-type cells. Collectively, these results suggest that PTEN-induced ABCG2 activity is responsible for the SP phenotype in glioma CSCs, extending and confirming previous results in other cell types (Takada et al., 2005; Zhou et al., 2007). Most notably, from a clinical perspective, Bleau et al. found that treating the SP spheres with the GBM chemotherapeutic temozolomide increased the SP fraction, and even more so in PTEN null spheres. These results led the authors to hypothesize that PTEN loss leads to increased temozolomide resistance. Temozolomide treatment also shortened tumor latency; mice grafted with tumorderived SP spheres cultured with temozolomide started to develop tumors after 25 days, while untreated transplants grew tumors after 40 days. Interestingly, temozolomide itself was not a substrate for ABCG2. The apparent increase in SP was instead traced to elevated activity of O6-methyl-guanine-DNA methyltransferase, a DNA repair enzyme known to mediate temozolomide resistance. To demonstrate relevance to human GBM, Bleau et al. next showed that blockade of either PI3K or Akt also downregulated the SP phenotype in primary patient-derived GBM SP spheres. Orthotopic xenografting
Cell Stem Cell 4, March 6, 2009 ª2009 Elsevier Inc. 191
Cell Stem Cell
Previews REFERENCES of SP cells from human GBM led to A B shortened latency for tumor formaBleau, A.-M., Hambardzumyan, D., Ozawa, tion compared to the main populaT., Fomchenko, E.I., Huse, J.T., Brennan, tion, consistent with SP cells being C.W., and Holland, E.C. (2009). Cell Stem Cell 4, this issue, 226–235. enriched for CSCs also in human tumors. Jordan, C. (2009). Cell Stem Cell 4, this issue, 203–205. Taken together, these findings PTEN Temozolomide offer important scientific insight, loss Mclendon, R. (2008). Nature 455, 1061– and yet seem paradoxical with 1068. respect to their therapeutic impliParsons, D.W., Jones, S., Zhang, X., Lin, cations. Bleau et al. (2009) showed J.C., Leary, R.J., Angenendt, P., Mankoo, P., Carter, H., Siu, I.M., Gallia, G.L., et al. that the abundance of the stem(2008). Science 321, 1807–1812. like glioma cells was readily modified through either acquisition of Patrawala, L., Calhoun, T., SchneiderBroussard, R., Zhou, J., Claypool, K., mutation (loss of PTEN) or in and Tang, D.G. (2005). Cancer Res. 65, response to treatment with temo6207–6219. Figure 1. Models to Describe How Loss of PTEN and zolomide (Figure 1). Importantly, Treatment with Temozolomide May Lead to Reya, T., Morrison, S.J., Clarke, M.F., and Chemoresistance in Glioma while temozolomide, the most Weissman, I.L. (2001). Nature 414, 105– (A) Loss of PTEN leads to increased ABCG2 activity (represented effective chemotherapy used for 111. by black squares) in a subset of cells, resulting in increased selfGBM, essentially sets the stage renewal and net chemoresistance. Shih, A.H., Dai, C., Hu, X., Rosenblum, (B) The alkylating agent temozolomide most effectively targets for recurrent drug-resistant diM.K., Koutcher, J.A., and Holland, E.C. non-SP cells, leading to relative expansion of a chemoresistant (2004). Cancer Res. 64, 4783–4789. sease, Bleau et al. also demon(ABCG2-positive) SP fraction. strate that inhibitors of PI3K, now Takada, T., Suzuki, H., Gotoh, Y., and Sugiyama, Y. (2005). Drug Metab. Dispos. in clinical trials in cancer, may be 33, 905–909. useful in blocking temozolomidedriven expansion of the SP. These find- turning ‘‘dial’’ rather than an on/off Zheng, H., Ying, H., Yan, H., Kimmelman, A.C., ings also show that the relative abun- ‘‘switch.’’ Clearly, future studies are Hiller, D.J., Chen, A.J., Perry, S.R., Tonon, G., Chu, G.C., Ding, Z., et al. (2008). Nature 455, dance and drug resistance of CSCs needed to understand why both loss of 1129–1133. can change during malignant pro- PTEN and temozolomide expand the SP, gression and in response to therapy. and how to translate these findings into Zhou, J., Wulfkuhle, J., Zhang, H., Gu, P., Yang, Y., Deng, J., Margolick, J.B., Liotta, L.A., Petricoin, E., Thus acquisition of stemness in cancer improved therapeutic approaches for 3rd, and Zhang, Y. (2007). Proc. Natl. Acad. Sci. USA 104, 16158–16163. is perhaps more likely a continually this deadly tumor.
LOCKing in Cellular Potential Helle F. Jørgensen1,* and Amanda G. Fisher1,* 1MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK *Correspondence: [email protected] (H.F.J.), [email protected] (A.G.F.) DOI 10.1016/j.stem.2009.02.007
Recently in Nature Genetics, Wen et al. (2009) report that H3K9me2 modification is more abundant in differentiated cells than in embryonic stem cells and that it decorates chromatin in repressive blocks. A spectrum of differentiated cell types that constitute higher organisms arise from pluripotent cells that are found in the inner cell mass of blastocyst-stage embryos. Cellular potential is gradually lost as cells acquire specific fates during development (Figure 1A), although under certain experimental conditions, their potential can be reset (via reprogramming, Figure 1A).
The exchange of cellular potential for functional specialization takes place—in almost all cases—without loss of genetic material, a result that has led to speculations that progressive silencing of the genome could underwrite directional differentiation (Spemann, 1938). Epigenetic mechanisms capable of modulating the accessibility of the genome to DNA-
192 Cell Stem Cell 4, March 6, 2009 ª2009 Elsevier Inc.
binding transcription factors have been implicated in this process (Reik, 2007), particularly dynamic changes in DNA methylation and covalent modification to histone tails. The importance of these epigenetic mechanisms for normal development is underscored by the prevalence of lethality among embryos lacking DNA and histone methyltransferases.