Abstract A43: DIRAS1 and DIRAS2 are tumor suppressor candidates that inhibit cell growth, motility, and proliferation while regulating autophagy in ovarian cancer.

Among the three members of the DIRAS family, DIRAS3 (also known as Aplasia Ras Homolog Member I; ARHI) has been best characterized. DIRAS3 is a maternally imprinted tumor suppressor gene that encodes a 26kDa GTPase which shares 60% homology to Ras and Rap. DIRAS3 is downregulated in many tumor types including ovarian cancer. DIRAS3 is downregulated by multiple mechanisms including loss of heterozygosity, transcriptional regulation by E2F1 and E2F4, hypermethylation of the second allele, and regulation by miRNAs 221 and 222. Re-expression of DIRAS3 at physiologic levels inhibits cancer cell growth, reduces motility, induces autophagy and promotes tumor dormancy. The mechanisms by which DIRAS3 induce autophagic cell death in vitro and tumor dormancy in vivo have been well characterized by previous work in our laboratory demonstrating that DIRAS3 is required for the induction of autophagy. Interestingly, mice do not have DIRAS3, as it was lost during evolutionary rearrangement of murine chromosomes 3 and 6, 60 million years ago, but murine cells can still undergo autophagy. Mice and humans express two closely related GTPases, DIRAS1 and DIRAS2. We are exploring the hypothesis that DIRAS1 and DIRAS2 replace the function of DIRAS3 in mice. These 22 kDa GTPases share 30-40% overall homology with H-Ras and 50-60% homology with DIRAS3, differing mostly by the truncation of their N-terminal extension. Although DIRAS1 and DIRAS2 have not previously been characterized in ovarian cancer, TCGA analysis suggests that higher mRNA expression of these genes results in a survival advantage for patients with high grade serous ovarian cancer. In this study we demonstrate that re-expression of DIRAS1 and DIRAS2 inhibit ovarian cancer cell growth in vitro and induce autophagy in both human and murine cells. This is demonstrated by the conversion of LC3-I to LC3-II by western blot analysis, the formation of LC3 punctae by fluorescence microscopy and visualization of double walled autophagosomes by electron microscopy. While the mechanism by which DIRAS1 and DIRAS2 induce autophagy is still being investigated, we have observed that overexpression results in inhibition of both the PI3K and Ras/MAPK signaling pathways. Additionally, DIRAS1 and DIRAS2 are required for murine autophagy induced by rapamyacin or starvation conditions in that autophagy is significantly decreased by siRNA knockdown of DIRAS1 and DIRAS2. This study explores the functional significance of DIRAS1 and DIRAS2 as tumor suppressors and investigates their role as surrogates to DIRAS3 (ARHI) in murine autophagy and tumor dormancy, providing a gateway to study this cellular process. Citation Format: Margie N. Sutton, Zhen Lu, Robert C. Bast, Jr. DIRAS1 and DIRAS2 are tumor suppressor candidates that inhibit cell growth, motility, and proliferation while regulating autophagy in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A43.