Exploration of the TranslationalTherapeutic Potential of Stem Cell B

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The Neuro-Oncology Branch Laboratory of translational science is divided into three major areas: Project 2: Exploration of the Translational/Therapeutic Potential of Stem Cell Biology in Gliomas Phenotypic characteristics and the multitude of genetic aberrations found within repeatedly in vitro passaged cancer cell lines often bear little resemblance to those found within the corresponding primary human tumors. Not only does this explain why both in vitro and in vivo cancer cell line-based preclinical therapeutic screening models have been poorly predictive for identifying clinically useful therapeutic agents, but may also have led to some important misinterpretations regarding the relevance of aberrant signaling pathways within cell lines compared to primary tumors. This realization led us on a search for a more biologically relevant model system for exploring glioma biology and for the screening of new therapeutic agents. Through multiple approaches including extensive cell biology tools, in vivo tumor model studies, global gene expression assays, and genome-wide SNP analysis, we demonstrated that TSCs closely mimic the genotype, gene-expression profile and biology of their parental primary tumors. These TSCs established in our laboratories has been recognized as accepted standard brain tumor cells in vitro and distributed into numerous laboratories. By collaboration with prominent brain tumor clinic centers, we now have established tens of TSC cultures and xenograft tumors from GBM patients. Xenograft tumors derived from these TSCs explicitly and precisely maintain the characteristics of parental tumors in patients. These TSCs and corresponding xenograft tumors in mice are long-sought in vitro and in vivo preclinical therapeutic models and may be used as an important landmark on the road to "personalized" therapy for each patient. Molecular understanding of deregulated differentiation pathways in TSCs: The delicate balance between stem cell self-renewal and differentiation is controlled by various cell intrinsic and extrinsic factors that are critical for normal tissue homeostasis. Despite extensive phenotypic and functional similarities between TSCs and normal stem cells, the differentiation potentials of TSCs are not entirely normal. Elucidation of the differentiation pathways operative in both normal stem cells and TSCs will be critical for fully understanding tumorigenesis and will likely lead to novel therapeutic targets. We have identified a set of deregulated differentiation pathways in TSCs derived from human primary glioblastoma. We demonstrated that both bone morphogenetic protein (BMP)-mediated and ciliary neurotrophic factor (CNTF)-mediated Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathways elicit distinct biological consequences in adult brain derived TSCs compared to normal neural stem cells (NSCs). Like very early embryonic NSCs, 0308 TSCs proliferate in response to BMP and elicit marginal STAT3 activation after CNTF challenge. However, unlike normal NSCs in later developmental stages that acquire responsiveness to CNTF-triggered STAT3 activation in association with increased expression of BMP receptor 1B (BMPR1B), 0308 cells do not express BMPR1B secondary to Enhancer of Zeste homolog 2 (EZH2)-dependent BMPR1B promoter hypermethylation. Forced expression of BMPR1B in 0308 TICs either by transgene expression or demethylation of the promoter fully restores their differentiation capabilities and induces loss of their tumorigenicity not only via a BMP-mediated pathway but also by CNTF-mediated Jak/STAT activation. A survey of 54 primary human glioblastomas reveals that approximately 20% have suppressed expression of BMPR1B associated with promoter hypermethylation. Taken together, these data implicate that deregulation of the BMP developmental differentiation pathway in a subset of glioblastoma TSCs contributes to their tumorigenic phenotype by not only desensitizing TIC to normal differentiation cues, but also by converting otherwise cytostatic signals to pro-proliferative signals. Extensive in vitro and in vivo characterization of GBM TSCs by using differentiation-inducing agents such as retinoic acid demonstrated that these TSCs differentiate efficiently and stop proliferation. We have demonstrated that retinoic acid treatment achieve cyctostatic effect by decreasing the proportion of CD133 positive cells, a putative marker for brain TSCs, from tumors and by inducing differentiation into astroglial lineage. Interestingly, a subset of GBM TICs pretreated with radiation and chemotherapeutic agents in vivo, do not reveal significant retinoic acid-mediated differentiation. Elucidation of underlying molecular mechanism will provide important clue for predicting sensitivity of differentiation therapeutic approach.Characterization of TSCs in aspect of differentiation-inducing agents further revealed the limitation of traditional glioma cell lines grown in serum. For example, retinoic acid treatment and CNTF exposure potently induce differentiation in most GBM TICs but not of traditional cell lines. This prompted us to question whether many of potential tumor suppressors and/or cytostatic genes previously studied in cell lines, were not recognized. Given the ever increasing number of potential TSGs and oncognes in glioblastoma TSCs identified from bioinformatics approach and technical expertise of stem cell culture accumulated in the laboratories, we have set up screening systems to study the function of these genes in stem cell cultures