Abstract 2457: Investigating the functional significance of novel, recurrent noncoding mutations of TBC1D12 in bladder cancer

Abstract

Abstract Somatic noncoding mutations remain widely unexplored despite examples of critical importance such as driver mutations in the promoter of the TERT gene. We recently identified novel somatic mutations in the regulatory Kozak sequence (just upstream of the translation start site) of TBC1D12 in ~15% of bladder cancer patients, making it one of the most frequently mutated noncoding regions in any cancer (Araya et. al 2016). TBC1D12 was not previously implicated in cancer, and little is currently known about its function. It is a member of the TBC1 superfamily, suggesting a role in intracellular trafficking as a RAB-specific GTPase-activating protein (GAP). To investigate the functional implications of TBC1D12 mutations in bladder cancer, we undertook a combined computational and experimental approach. We analyzed clinical correlates, identified downstream effector candidates, and assessed the impact of these mutations on gene expression. To determine the clinical relevance of the TBC1D12 mutation, we did correlation and survival analyses of clinical data of bladder cancer patients from TCGA. We found that TBC1D12 mutations confer slightly worse survival but were not linked to other clinical correlates. To identify potential downstream effectors, we used reverse phase protein array (RPPA) data from 127 patients to search for proteins that were differentially expressed or phosphorylated between patients with or without TBC1D12 mutations. We found differential expression of GSK3 and differential phosphorylation of p90RSK and c-Jun, suggesting three potential downstream effector candidates. Hierarchical clustering of RPPA data for these three proteins revealed 4 subgroups with distinct molecular phenotypes. Furthermore, most of the TBC1D12 mutants were clustered within one molecular subtype. To experimentally test these downstream effector candidates and identify new ones, we modulated the levels of TBC1D12 in bladder cancer cell line Ku-19-19 using overexpression plasmids or siRNAs. After validating that these transfections were effective in changing TBC1D12 expression, we performed RNA-Seq experiments. Results from this study are expected to be presented at the meeting. In summary, these studies suggest that the novel and recurrent noncoding mutations in TBC1D12 may affect patient survival and alter the expression or post-translational modification of downstream targets. Potential molecular mechanisms associated with the presence of TBC1D12 mutations will be discussed, and a series of follow-up studies are underway. These studies also demonstrate that applying both computational and experimental approaches can help develop and test evidence-based hypotheses, contributing to an understanding of the clinical and functional impact of TBC1D12. Citation Format: Angela S. Li, Jason A. Reuter, Can Cenik, Michael P. Synder. Investigating the functional significance of novel, recurrent noncoding mutations of TBC1D12 in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2457. doi:10.1158/1538-7445.AM2017-2457