Manipulating stem cells (both normal and cancer) has great potential for treating many human diseases. To understand the molecular mechanisms that regulate self-renewal, proliferation and differentiation of stem cells, we are taking two complementary approaches. One, we are using mouse models of brain cancer to identify and analyze molecular pathways that control cancer stem cells and to further refine the cancer stem cell hypothesis. Two, we are investigating the functions of known oncogenes and tumor suppressor genes in their regulation of normal stem cells during brain development. By straddling the fields of cancer biology and developmental biology with a focus on understanding the regulation of stem cells, we seek to identify cell intrinsic and extrinsic factors that control stem cell proliferation and differentiation, in both normal and cancer stem cells.
Targeting brain cancer stem cells
The discovery of cancer stem cells in human tumors opens the possibility for novel, targeted therapeutic agents that can significantly improve clinical outcomes for patients with aggressive cancers. Cancer stem cells are a subpopulation of cancer cells that are endowed with the defining characteristics of stem cells (self-renewal and multipotentiality) and the unique ability to initiate a tumor when transplanted. In theory, a single cancer stem cell can re-initiate tumor formation or form metastatic tumors. Recent studies that showed that cancer stem cells are more resistant to chemo- and radiation-therapies compared to non-stem cancer cells from the same tumor, supporting the hypothesis that targeting cancer stem cells will improve long-term outcomes for cancer patients.
In a recent study, we reported the isolation and characterization of cancer stem cells from a transgenic mouse model of malignant glioma (Tg(S100b-v-erbB; p53-/-)4496Waw), providing the initial proof of principle that mouse brain tumors also contain cancer stem cells (Harris et al., 2008). Combining the advantages of a genetically defined mouse model with the clinical relevance of primary human glioblastoma stem cell cultures, we are investigating molecules and pathways that can be used to specifically target cancer stem cells. To identify genes that are uniquely expressed in cancer stem cells compared to non-stem cancer cells and normal neural stem cells, we performed comparative transcriptome analyses using freshly FACS sorted normal and cancer stem cells from a mouse model of glioblastoma. From this analysis, we identified a gene signature, consisting of 45 genes, that is unique to cancer stem cells. We validated that some of the genes on this list are also expressed in human glioma patient samples in a pattern consistent with them being cancer stem cells. We are currently testing more candidate genes to both validate their expression in human glioma stem cells and to elucidate their molecular functions.
Testing the cancer stem cell hypothesis using mouse models of brain tumor
Using mouse models of brain cancer, we are testing the hypothesis that ablation of cancer stem cells is both necessary and sufficient to block existing tumor growth and inhibit recurrence. We have crossed our glioma and medulloblastoma models to other genetically engineered mice so that we selectively ablate specific populations of cancer cells expressing unique stem cell marker genes. By inducibly killing these cells in existing tumors, we can address whether ablation of these cells is both necessary and sufficient to block tumor growth. This important concept has not yet been proven, and the mouse models enable us to address this issue through genetic manipulations.
In parallel, we are also analyzing the molecular and cellular phenotypes of cancer stem cells during tumor progression. Using a mouse model of medulloblastoma, we observed drastic differences in cellular behaviors of tumor initiating cells in individual tumors, even though the initiating oncogenic event was identical in all tumors. This model provides us with an opportunity to examine molecular changes that accompany tumor progression and to scrutinize the heterogeneity of cancer stem cells in individual tumors of the same type and grade.
Oncogenes/tumor suppressor gene function in normal stem cells
We hypothesize that oncogenes and tumor suppressor genes that are commonly misregulated in human cancer have important regulatory functions in normal stem cells during development and in maintaining adult stem cell homeostasis. Currently, we are focusing on two oncogene families: Notch and ID (Inhibitor of Differentiation).
The Notch pathway has been studied for decades for its critical function during normal development. In the last several years, its function in tumorigenesis, particularly in hematological cancers, has indicated that activation of the Notch signaling pathway is a common and important oncogenic event in human cancers. Activation of Notch signaling has been reported in both human medulloblastomas and gliomas, and we have observed elevated Notch pathway signaling in S100β-verbB;p53-/- murine gliomas. To determine whether Notch signaling plays a role in regulating self-renewal of stem cells in the nervous system, we examined the effect of loss- and gain-of-function of the Notch pathway in neural stem cells during cortical and cerebellar development. Our study shows that activated Notch signaling expands the early neuroepithelium (expanding the stem cell number) and blocks stem-to-progenitor transition in maturing epithelium. Mice ectopically expressing activated form of Notch1 (N1-ICD) and Notch2 (N2-ICD) survive to postnatal stages but die around weaning, succumbing to ataxia and seizure. Depending on the cellular context, an activated Notch pathway can induce proliferation of stem cells (both self-renewal of early stem cells and block in transition to progenitors), block terminal differentiation, or induce apoptosis in the developing brain. Interestingly, when p53-dependent apoptosis in N1-ICD expressing mice is blocked, mutant mice develop medulloblastomas. Analysis of this model may provide important insight into the etiology and molecular characteristics of human medulloblastomas with elevated Notch signaling.
Id genes encode helix-loop-helix (HLH) transcription factors that are thought to inhibit differentiation and promote proliferation. Mutational analyses of Id genes clearly demonstrate their essential functions in many tissues during mammalian development. Recently, we have found that two members of this gene family, Id2 and Id4, can either stimulate or inhibit proliferation of neural stem/progenitor cells, depending on the cell type in which they are expressed. Id4 is required for cell cycle progression and expansion of the early telencephalic neuroepithelium. However, in postmitotic neurons of the cortex, Id4 is required to block aberrant cell cycle re-entry (Yun et al, 2004). Similarly, although there are many reports that indicate that Id2 promotes proliferation, we have recently observed that over-expression of Id2 in the neuroepithelium has the surprising effect of reducing neuroepithelial expansion, leading to a smaller brain size. Consistently, neurospheres (neural stem cells) derived from Id4-/- or Id2-overexpressing mice do not proliferate or form secondary spheres (self-renewal) as robustly as those derived from littermate controls. We hypothesize that Id2 and Id4 regulate self-renewal and/or transition of stem-to-progenitor cells in the developing cortical neuroepithelium.
We are currently testing this hypothesis using genetically engineered mice and also identifying downstream target genes of Id2 and Id4. Interestingly, we observed that Id4 but not Id2 is more highly expressed in cancer stem cells compared to non-stem cancer cells of mouse malignant glioma. Hence, we hypothesize that Id4 may be a regulator of both normal and glioma stem cells and a potential therapeutic target for cancer stem cells.
Principal Investigator: Kyuson Yun, Ph.D.
Postdoctoral Fellow: Sivaraman Natarajan, Ph.D., Kin-Hoe Chow, Ph.D.
Research Assistant I: Pamelia Fraungruber, B.S.
Research Administrative Assistant: Patricia Cherry
Dowell KG, Simons AK, Zack Wang ZZ, Yun K, Hibbs MA. 2013. Cell-type-specific predictive network yields novel insights into mouse embryonic stem cell self-renewal and cell fate. PLoS ONE 8(2):e56810.
Park HJ, Hong M, Bronson RT, Israel MA, Frankel WN, Yun K. 2013. Elevated Id2 expression results in precocious neural stem cell depletion and abnormal brain development. Stem Cells :EPub ahead of print.
Li Y, Hibbs MA, Gard AL, Shylo NA, Yun K. 2012. Genome-wide analysis of N1ICD/RBPJ targets in vivo reveals direct transcriptional regulation of Wnt, SHH, and Hippo pathway effectors by Notch1. Stem Cells 30(4):741-752.
Zhang L, Lapierre A, Roy B, Lim M, Zhu J, Wang W, Sampson SB, Yun K, Lyons B, Li Y, Lin DT. 2012. Imaging glioma initiation in vivo through a polished and reinforced thin-skull cranial window. J Vis Exp 69(pii):4201.
Harris MA, Yang H, Low BE, Mukherje J, Guha A, Bronson RT, Shultz LD, Israel MA, Yun K. 2008. Cancer stem cells are enriched in the side-population cells in a mouse model of glioma. Cancer Research 68: 10051-10059.
Yun K, Tennent B. 2008. Cancer stem cells. Drug Discov Today: Disease Models 4(2):47-52.
Nam JS, Park E, Turcotte TJ, Palencia S, Zhan X, Lee J, Yun K, Funk WD, Yoon JK. 2007. Mouse R-spondin2 is requred for apical ectodermal ridge maintenance in the hindlimb. Dev Biol 311(1):124-135. PMCID: PMC2692258
Yun K, Mantani A, Garel S, Rubenstein J, Israel M. 2004. Id4 regulates neural progenitor proliferation and differentiation in vivo. Development 131(21):5441-5448.
Andrews G, Yun K, Rubenstein J, Mastick G. 2003. Dlx transcription factors regulate differentiation of dopaminergic neurons of the ventral thalamus. Mol Cell Neurosci 23(1):107-120.
Yun K, Garel S, Fischman S, Rubenstein JLR. 2003. Patterning of the lateral ganglionic eminence by the Gsh1 and Gsh2 homeobox genes regulates striatal and olfactory bulb histogenesis and the growth of axons through the basal ganglia. J Comp Neruol 461(2):151-165.
Garel S, Yun K, Grosschedl R, Rubenstein JLR. 2002. The early topography of thalamocortical projection is shifted in Ebf1 and Dlx 1/2 mutant mice. Development 129(24):5621-5634.
Yun K, Fischman S, Johnson J, Hrabe de Angelis M, Weinmaster G, Rubenstein JLR. 2002. Modulation of the Notch signaling pathway by Mash1 and Dlx1/2 regulates sequential specification and differentiation of progenitor cell types in the subcortical telencephalon. Development 129(21):5029-5040.
Yun K, Potter S, Rubenstein JLR. 2001. Gsh2 and Pax6 play complementary roles in dorsoventral patterning of the mammalian telencephalon. Development 128(6):193-205.
Anderson S, Mione M, Yun K, Rubenstein JL. 1999. Differential origins of neocortical projection and local circuit neurons: role of Dlx genes in neocortical interneuronogenesis. Cereb Cortex 9(6):646-654.
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