Abstract 2562: Bladder cancer GWAS signal at 4p16.3 affects response of TMEM129 to chemically-induced endoplasmic reticulum stress
Authors: Banday A. Rouf , Kiss Eniko , Yan Wusheng , Middlebrooks Candace , Prokunina-Olsson Ludmila 
Published: July 2016

Background: SNP rs798766 within the 4p16.3 region was associated with bladder cancer risk in several GWAS. This SNP is located within a linkage disequilibrium block that includes TMEM129, TACC3, FGFR3 and SLBP. We aimed to explore molecular mechanisms of this GWAS signal.

Methods: Expression analysis of all isoforms of TMEM129, TACC3, FGFR3 and SLBP was performed in the Cancer Genome Atlas (TCGA) bladder cancer dataset (N = 412). Expression was analyzed by linear regression in relation to rs798766 genotypes adjusting for age, sex, race, DNA CpG methylation status and copy number variation (CNV). Expression in additional bladder tumors (N = 42) was tested with TaqMan expression assays and with splicing form-specific in situ hybridization (ISH) assays in a tissue microarray that included 7 normal-tumor bladder tissue pairs. Allele-specific TMEM129 exontrap assays in 5 bladder cancer cell lines were used to evaluate alternative splicing. TMEM129 isoforms were cloned and overexpressed in cell lines for functional characterization by confocal microscopy, reporter assays, RNA-seq and qRT-PCR arrays to explore their effects on cell signaling and cell viability. Endoplasmic reticulum (ER) stress was induced by treating bladder cancer cell lines with chemicals dithiothreitol (DTT), tunicamycin and MG132.

Results: Of all the isoforms of four genes tested the strongest association with rs798766 genotypes was observed for TMEM129-b transcript (padj = 1.65E-06); expression was decreased with bladder cancer risk allele. Exontrap analysis revealed that allele G of a SNP rs2236786 located within exon 3 of TMEM129 (r2 = 1.0 with GWAS SNP rs798766) is responsible for alternative splicing of TMEM129. Allele-specific alternative splicing also results in lower expression of TMEM129-b and creation of a new TMEM129-a2 transcript. TMEM129-a2 is predicted to undergo nonsense mediated decay (NMD) except when under ER stress. TMEM129 is an E3 ligase and its protein isoforms are predicted to be functionally different. Confocal imaging showed that all isoforms are located in ER and thus can compete with each other. Overexpression of TMEM129 isoforms resulted in differential activation of genes from the main ER stress response pathways - IRE1, PERK and ATF6. Chemically-induced ER stress increased expression of all TMEM129 isoforms, although the induction was lower in cell lines with risk G allele of rs2236786.

Conclusions: The risk of bladder cancer is known to be increased by environmental exposures. Our results suggest that bladder cancer risk could be modulated by a genetic variant that affects the function of TMEM129, an E3 ligase. This mechanism could be important for response to environmentally-induced ER stress and subsequent development of bladder cancer.

Citation Format: A. Rouf Banday, Eniko Kiss, Wusheng Yan, Candace Middlebrooks, Ludmila Prokunina-Olsson. Bladder cancer GWAS signal at 4p16.3 affects response of TMEM129 to chemically-induced endoplasmic reticulum stress. [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 2562.