TRRAP and the Maintenance of Stemness in Gliomas

Resistance of brain tumors to therapy and their eventual recurrence is attributed to stem-like cells. In this issue of Cell Stem Cell, a high-throughput kinome-wide screen (Wurdak et al., 2010Wurdak H. Zhu S. Romero A. Lorger M. Watson J. Chiang C.-y. Zhang J. Natu V.S. Lairson L.L. Walker J.R. et al.Cell Stem Cell. 2010; 6 (this issue): 37-47Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) has identified TRRAP, a kinase-related protein, as being required for stem cell character in gliomas. Resistance of brain tumors to therapy and their eventual recurrence is attributed to stem-like cells. In this issue of Cell Stem Cell, a high-throughput kinome-wide screen (Wurdak et al., 2010Wurdak H. Zhu S. Romero A. Lorger M. Watson J. Chiang C.-y. Zhang J. Natu V.S. Lairson L.L. Walker J.R. et al.Cell Stem Cell. 2010; 6 (this issue): 37-47Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) has identified TRRAP, a kinase-related protein, as being required for stem cell character in gliomas. The prognosis for treatment of glioblastomas remains dismal (a little more than 1 year), and research toward a better understanding of the biology of therapeutic resistance for this disease is actively ongoing. One proposed reason for the poor response to treatment is the existence of a small subpopulation of resistant cells within these tumors that survive treatment and repopulate the tumor. These resistant cells have properties similar to stem cells, and isolation of this subpopulation, referred to as brain tumor-initiating cells (BTICs) or brain tumor stem-like cells, was first achieved by utilizing the hematopoietic stem cell marker CD133 (Singh et al., 2004Singh 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 (6079) Google Scholar). In their study, the authors showed that this cell population from human glioblastoma samples could regenerate tumors with phenotypes comparable to those from the patient more efficiently than their CD133-negative counterparts. These stem-like cells contribute to the glioma's resistance to therapy (Bao et al., 2006Bao 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 (4873) Google Scholar). The data suggest that the stem cell-like properties of BTICs are important factors mediating tumor resistance and recurrence and that novel strategies that specifically target this population may be essential for improved therapeutic outcomes. Several signaling pathways including Notch, Shh, and PI3K are known to regulate the activity of normal neural stem cells (Stiles and Rowitch, 2008Stiles C.D. Rowitch D.H. Neuron. 2008; 58: 832-846Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar), implying that kinases regulate this phenotype. These signaling pathways similarly regulate the activity of BTICs and may represent one approach to directly target BTICs. The bone morphogenic proteins (BMPs) induce differentiation of neural stem cells into mature astrocytes and BMP4 induces differentiation of BTICs in gliomas (Piccirillo et al., 2006Piccirillo 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 (980) Google Scholar). By enforcing the differentiation of BTICs in this manner, one might predict that tumor recurrence would be less likely, because the stem cell properties of the tumor cells should be lost in the more mature progeny. This approach was successfully employed in the treatment of promyelocytic leukemia (Wang et al., 1998Wang Z.G. Delva L. Gaboli M. Rivi R. Giorgio M. Cordon-Cardo C. Grosveld F. Pandolfi P.P. Science. 1998; 279: 1547-1551Crossref PubMed Scopus (455) Google Scholar). In this issue of Cell Stem Cell, a manuscript from the Schultz laboratory (Wurdak et al., 2010Wurdak H. Zhu S. Romero A. Lorger M. Watson J. Chiang C.-y. Zhang J. Natu V.S. Lairson L.L. Walker J.R. et al.Cell Stem Cell. 2010; 6 (this issue): 37-47Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) provides further insight into this phenomenon and important experimental evidence to justify targeting of BTICs with differentiating factors. The authors used a phenotypic high-throughput shRNA library screen targeting the kinome to identify genes involved in the maintenance of “stemness” in gliomas, with BMP4 treatment as a positive control. The authors identified many expected kinases such as IGFR, as well as MAP and SRC family kinases. In addition, they identified TRRAP, an adaptor protein with homology to the PIKK kinases but lacking intrinsic kinase activity, as playing a role in the differentiation phenotype in glioblastoma stem-like cells. TRRAP is thought to regulate many biologic functions including chromatin remodeling, embryonic development, oncogenic transformation through c-Myc and E2F, as well as cell cycle progression. The authors report that knockdown of TRRAP in BTICs (grown as a monolayer on laminin) resulted in many phenotypic changes consistent with loss of stem cell characteristics (Figure 1) including decreased neurosphere formation in culture, decreased tumor formation upon transplantation into recipient mice, increased sensitivity to temozolomide and radiation therapy, and alteration of markers associated with differentiation such as loss of Nestin and Sox2 with gain of GFAP and TuJ1. The data imply that TRRAP loss depletes the stem-like pool of BTICs in vitro and mirrors the effects first reported for BMP4 (Piccirillo et al., 2006Piccirillo 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 (980) Google Scholar). Given the presented data on loss of function, one might expect overexpression of TRRAP to promote stem cell character and tumorigenic potential in these BTICs; in fact, TRRAP gene expression was elevated in the gliomas analyzed in this study. The effects on proliferation and differentiation induced by TRRAP knockdown appeared to be mediated in part by suppression of transcriptional activity for the mitotic cyclin A2 with associated epigenetic modifications at the cyclin A2 promoter and increased BMP/Smad signaling. The authors suggest that cyclin A2 may play a pivotal role in promoting the BTIC stem-like phenotype. There are data to support this possibility, because cyclin A2 overexpression was previously shown to confer anchorage-independent growth in cells (Barrett et al., 1995Barrett J.F. Lewis B.C. Hoang A.T. Alvarez Jr., R.J. Dang C.V. J. Biol. Chem. 1995; 270: 15923-15925Crossref PubMed Scopus (49) Google Scholar). Furthermore, connections may exist between cyclin A2 and differentiation of BTICs, as shown by the fact that the loss of cyclin A2 activity has been linked to stem cell differentiation (Chen et al., 2009Chen J. Wang F. Gao X. Zha D. Xue T. Cheng X. Zhong C. Han Y. Qiu J. Neurosci. Lett. 2009; 453: 166-169Crossref PubMed Scopus (5) Google Scholar). In addition, loss of cyclin A2 activity was recently shown to preferentially decrease the proliferation of hematopoietic and embryonic stem cells and to suppress their capacity to generate colony-forming units in comparison to their more differentiated counterparts (Kalaszczynska et al., 2009Kalaszczynska I. Geng Y. Iino T. Mizuno S. Choi Y. Kondratiuk I. Silver D.P. Wolgemuth D.J. Akashi K. Sicinski P. Cell. 2009; 138: 352-365Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). In the context of these previous reports combined with the Wurdak et al., 2010Wurdak H. Zhu S. Romero A. Lorger M. Watson J. Chiang C.-y. Zhang J. Natu V.S. Lairson L.L. Walker J.R. et al.Cell Stem Cell. 2010; 6 (this issue): 37-47Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar study, overall the data suggest that the decreased proliferation of BTICs observed after the loss of cyclin A2 may be connected to their differentiation status, potentially suggesting a role for cyclin A2 in differentiation of BTICs. Control of symmetrical and asymmetrical cell division is certainly part of stem cell character; however, this finding brings up the larger issue of how to deconvolute proliferation from stemness in experimental paradigms such as transplantation models or growth of neurospheres in cell culture. Another important question raised by this work is whether the function of TRRAP or cyclin A2 is a central feature of stem-like cells in all glioma subtypes. Gliomas are classified into several subtypes based on their gene expression and signal transduction profiles (Brennan et al., 2009Brennan C. Momota H. Hambardzumyan D. Ozawa T. Tandon A. Pedraza A. Holland E. PLoS ONE. 2009; 4: e7752Crossref PubMed Scopus (397) Google Scholar). In this context, if genes regulating BTIC character were analyzed in gliomas distinguished by subtype, would there be specific genes that would show up in one subtype or another? For example, would BTICs isolated from gliomas with EGFR amplification respond to TRRAP or cyclin A2 knockdown in tumors in a similar way as do the PDGFR-amplified subtypes? Could it be that within any given tumor there are several different stem-like cell types that are driven by different genes? The work by Wurdak et al., 2010Wurdak H. Zhu S. Romero A. Lorger M. Watson J. Chiang C.-y. Zhang J. Natu V.S. Lairson L.L. Walker J.R. et al.Cell Stem Cell. 2010; 6 (this issue): 37-47Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar certainly strengthens the notion that BTICs could be targeted specifically through manipulation of pathways that affect their differentiation and provides exciting new insight into the regulation of brain tumor stem cell biology. An RNAi Screen Identifies TRRAP as a Regulator of Brain Tumor-Initiating Cell DifferentiationWurdak et al.Cell Stem CellJanuary 08, 2010In BriefGlioblastoma multiforme (GBM) is a highly aggressive form of brain cancer associated with a very poor prognosis. Recently, the initiation and growth of GBM has been linked to brain tumor-initiating cells (BTICs), which are poorly differentiated and share features with neural stem cells (NSCs). Here we describe a kinome-wide RNA interference screen to identify factors that control the tumorigenicity of BTICs. We identified several genes whose silencing induces differentiation of BTICs derived from multiple GBM patients. Full-Text PDF Open Archive