TRANSCRIPTIONAL AND POST-TRANSLATIONAL MECHANISMS CONTRIBUTE TO MAINTENANCE OF REST IN NEURAL TUMORS

The RE-1 silencing transcription factor (REST) is an important regulator of normal nervous system development. It negatively regulates neuronal lineage specification in neural progenitors by binding to its consensus RE-1 element(s) located in the regulatory region of its target neuronal differentiation genes. The developmentally coordinated down-regulation of REST mRNA and protein in neural progenitors triggers terminal neurogenesis. REST is overexpressed in pediatric neural tumors such as medulloblastoma and neuroblastoma and is associated with poor neuronal differentiation. High REST protein correlate with poor prognosis for patients with medulloblastoma, however similar studies have not been done with neuroblastoma patients. Mechanism(s) underlying elevated REST levels medulloblastoma and neuroblastoma are unclear, and is the focus of this thesis project. We discovered that transcriptional and post-translational mechanisms govern REST mis-regulation in medulloblastoma and neuroblastoma. In medulloblastoma, REST transcript is aberrantly elevated in a subset of patient samples. Using loss of function and gain of function experiments, we provide evidence that the Hairy Enhancer of Split (HES1) protein represses REST transcription in medulloblastoma cell lines, modulates the expression of neuronal differentiation genes, and alters the survival potential of these cells in vitro. We also show that REST directly represses its own expression in an auto-regulatory feedback loop. Interestingly, our studies identified a novel interaction between REST and HES1. We also observed their co-occupancy at the RE-1 sites, thereby suggesting potential for co-regulation of REST expression. Our pharmacological studies in neuroblastoma using retinoic acid revealed that REST levels are controlled by transcriptional and post-transcriptional mechanisms. Post-transcriptional mechanisms are mediated by modulation of E3 ligase or REST, SCFβ-TRCP, and contribute to resistance of some cells to retinoic acid treatment.

ANTI-TUMOR EFFECTS OF THE NOTCH PATHWAY IN GASTROINTESTINAL STROMAL TUMORS

Gastrointestinal Stromal Tumors (GIST) are sarcomas driven by gain-of-function mutations of KIT or PDGFRA. Although, the introduction of tyrosine kinase inhibitors has dramatically changed the history of this disease, evidences emerge that inhibition of KIT or PDGFRA are not sufficient to cure patients. The developmental pathway Notch has a critical role in the cell fate, regulating cell proliferation and differentiation. Dysregulation of Notch pathway has been implicated in a wide variety of cancers functioning as a tumor promoter or a tumor suppressor in a cell context dependent manner. Given that Notch activation deregulates the morphogenesis of mesenchymal cells in the GI track, that Notch acts as a tumor suppressor in neuroendocrine tumors, and finally that the cell of origin of GIST are the Interstitial Cell of Cajal that arise from a mesenchymal origin with some neuroendocrine features, we hypothesized that Notch pathway signaling may play a role in growth, survival and differentiation of GIST cells. To test this hypothesis, we genetically and pharmacologically manipulated the Notch pathway in human GIST cells. In this study, we demonstrated that constitutively active intracellular domain of Notch1 (ICN-1) expression potently induced growth arrest and downregulated KIT expression. We have performed a retrospective analysis of 15 primary GIST patients and found that high mRNA level of Hes1, a major target gene of Notch pathway, correlated with a significantly longer relapse-free survival. Therefore, we have established that treatment with the FDA approved histone deacetylase inhibitor SAHA (Vorinostat) caused dose-dependent upregulation of Notch1 expression and a parallel decrease in viability in these cells. Retroviral silencing of downstream targets of Notch with dominant negative Hes-1 as well as pharmacological inhibition of Notch pathway with a γ-secretase inhibitor partially rescued GIST cells from SAHA treatment. Taken together these results identify anti-tumor effect of Notch1 and a negative cross-talk between Notch1 and KIT pathways in GIST. Consequently, we propose that activation of this pathway with HDAC inhibitors may be a potential therapeutic strategy for GIST patients.

A Genomic Approach to Identify The Notch Pathway as a Putative Tumor Suppressor in Endometrial Cancer

Endometrial cancer is the most common gynecological malignancy and the fourth most frequently diagnosed cancer among women. The molecular changes that distinguish normal endometrium from endometrial carcinoma are not thoroughly understood. Identification of these changes could potentially aid in identifying at-risk women who are especially prone to develop endometrial cancer, such as obese women and women with Lynch Syndrome. A microarray analysis was performed using normal endometrium from thin and obese women and cancerous endometrium from obese women. We validated the differential expression of ten genes whose expression was significantly up-regulated or down-regulated using qRT-PCR. All of the genes had distinct expression levels depending on the endometrial carcinoma histotype. As a result, they could serve as molecular markers to distinguish between normal endometrium and endometrial cancer, as well as between low grade endometrial carcinomas and high grade endometrial carcinomas. Two of the ten genes validated, HEYL and HES1, are down-stream targets of the Notch signaling pathway. HEYL and HES1 were identified by microarray and qRT-PCR to have a significant decrease in expression in endometrial carcinomas compared to normal endometrium. We further analyzed the differential expression of other components of the Notch signaling pathway, Notch4 and Jagged1. They were also identified by qRT-PCR to be significantly down-regulated in endometrial carcinomas compared to normal endometrium. Therefore, we believe the Notch signaling pathway to act as a tumor suppressor in endometrial carcinomas.