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"IDENTIFICATION OF GLOBAL ALTERATION OF TRANSLATIONAL REGULATION IN GLIOMA IN VIVO"

by
Karim Helmy
M.D./Ph.D. Program
B.S., Stanford University, Stanford, CA


Thesis Advisor: Eric Holland, M.D., Ph.D.
Department of Cancer Biology and Genetics
Memorial Sloan-Kettering Cancer Center

Wednesday, May 25, 2011
MSB E-609B, 1:00 P.M.


Abstract

Malignant gliomas are a collection of primary brain tumors that are characterized by invasive growth and resistance to therapy, contributing for poor clinical prognosis. Understanding mechanisms of gene regulation in glioma under homeostatic conditions and after therapeutic stress may instruct novel approaches to therapy. Post-transcriptional regulation of gene expression contributes importantly to the protein output of a cell, however, methods for measuring translational regulation in complex in vivo systems are lacking. Here we describe a sensitive method for measuring translational regulation in defined cell populations from heterogeneous tissue in vivo. We adapted translating ribosome affinity purification (TRAP) methodology to measure translational efficiencies in the Olig2-positive tumor cell population in a genetically engineered mouse model (GEMM) of glioma. Global measurement of paired ribosome-bound and total cellular mRNA populations from tumor cells in vivo identified a broad distribution of translational efficiencies amongst mRNA species that was highly reproducible across biological samples. Comparison of the translation state of glioma cells to non-transformed oligodendrocyte progenitor cells (OPCs) in normal brain identified global alteration of translation in tumor, and specifically of genes involved in cell division and lipid metabolism. Furthermore, investigation of alteration in steady state translational efficiencies upon genomic loss of PTEN, one of the most frequently mutated and deleted tumor suppressors in glioma, identified differential translation of proteins involved in cellular respiration and oxidative phosphorylation, biological functions canonically associated with PI3K/Akt signaling. To investigate the dynamic nature of translational regulation in glioma in vivo, we treated glioma-bearing mice with ionizing radiation (IR), first-line therapy for patients with glioma. Prior in vitro studies have suggested that translational regulation may be more efficient than transcriptional regulation for modulating gene expression following acute stress, when rapid changes in protein levels are critical. Surprisingly, IR-induced changes in ribosome-associated RNA closely mirrored changes in total RNA, demonstrating translational regulation as much less dynamic than transcriptional regulation in the glioma radiation response. Application of this method in additional cell types and disease models and in different conditions of stress will extend our knowledge of the scope and dynamic nature of this important mode of gene regulation.


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