Abstract 733: Analysis of the hypoxic transcriptome in cells and solid tumors reveals a novel spliced isoform of the key regulator of mRNA translation, eIF2B5
Authors: Brady Lauren K. , Shekher Rohil , Popov Vladimir , Ivan Mircea , Radovich Milan , Koumenis Constantinos 
Published: July 2016

Abstract
Solid tumors exhibit a range of oxygen concentrations, in part due to abnormal vasculature and limited diffusion of oxygen which lead to hypoxia. Tumor hypoxia is associated with resistance to treatment, metastasis and poor patient outcome for many types of cancer. Assessing the impact of hypoxia in the tumor microenvironment and understanding how it contributes to tumorigenesis and survival is a challenging research goal for several reasons; a chief obstacle has been developing methods to analyze the transcriptome within precise regions of the tumor microenvironment in vivo. To address this task, we used Laser-Capture-Microdissection (LCM) of tumors labeled with the hypoxia probe, EF5, to specifically isolate normoxic and hypoxic regions of tumors from a murine model of head and neck cancer. We then carried out deep RNA-sequencing of the in vivo samples and corresponding in vitro samples grown in 0.5% oxygen conditions.
Here we present the first described study of the hypoxic transcriptome in tumors at base-pair resolution. Our goal is to use these data to evaluate the potential of previously unstudied hypoxia-mediated pathways, such as the regulation of mRNA splicing, to be targeted therapeutically. We identified global changes in several classes of splicing in hypoxic compared to normoxic head and neck cancer cells, including changes in expression of last exons, retained introns and 3’ untranslated regions. Many of the alternatively spliced isoforms are in pathways central to hypoxic adaptation, such as glycolysis, cell cycle and translation. Among these genes, we uncovered a previously undescribed splicing event in the master regulator of translation, EIF2B5. The full-length protein encoded by EIF2B5 (eIF2Bå), is a necessary catalytic component of the eIF2B complex, which binds eIF2á and exchanges GDP for GTP to initiate translation. Strikingly, hypoxia induces retention of intron 12 in EIF2B5, which leads to insertion of an early stop codon in frame with the coding sequence; the resulting 65kDa protein isoform lacks a functional guanine nucleotide exchange (GEF) domain compared to the full-length isoform, but retains the region known to bind eIF2B complex subunits. Thus, we predict that the 65kDa isoform of eIF2Bå, may act in opposition to full-length eIF2Bå and inhibit translation initiation in conditions of oxygen deprivation. We are investigating EIF2B5, and other select hypoxia-regulated isoforms, to evaluate how alternative splicing impacts cell growth and survival in hypoxic tumors.
Citation Format: Lauren K. Brady, Rohil Shekher, Vladimir Popov, Mircea Ivan, Milan Radovich, Constantinos Koumenis. Analysis of the hypoxic transcriptome in cells and solid tumors reveals a novel spliced isoform of the key regulator of mRNA translation, eIF2B5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 733.