Abstract 5126: Allele-specific effect of rs2294008 on mRNA and protein expression of the prostate stem cell antigen (PSCA) in human normal and tumor bladder tissue

Abstract

Abstract Background: A recent GWAS identified a SNP rs2294008 within the prostate stem cell antigen (PSCA) at 8q24.3 as a risk factor for bladder cancer, [OR=1.13 (1.09-1.17), p=4E-11, Rothman, 2010]. PSCA encodes a GPI-anchored cell membrane glycoprotein expressed in the prostate, bladder, stomach and pancreas. A transcript with the non-risk C allele of rs2294008 generates a protein of 114 amino acids, while the risk T allele creates a novel upstream translation start site (acg->aTg) that extends the N-terminal leader peptide by 9 amino acids, to generate a protein of 123 amino acids. A humanized monoclonal anti-PSCA antibody is already used in clinical trials for prostate and pancreatic cancer, but the functional roles of PSCA and its genetic variants remain unknown. Methods: We performed RNA-sequencing in 3 matched normal-tumor bladder tissue samples and mRNA expression analysis in 42 tumor and 41 normal bladder tissue samples (24 matched normal-tumor pairs). Genotyping and AEI (Allelic Expression Imbalance) was performed using an allelic discrimination genotyping assay. PSCA isoforms with T and C alleles were cloned into a pFC14A CMV Flexi expression vector. Protein PSCA expression in normal/tumor bladder tissues and transfected cells was analyzed with Western blot and immunohistochemistry (IHC). Results and Conclusion: RNA-sequencing detected PSCA expression in all bladder tissue samples. Analysis of three samples heterozygous for rs2294008 showed evidence of strong AEI - 90% of all PSCA transcripts carried the risk T allele, while only 10% showed the non-risk C allele. Similar pattern of expression was observed for 11 other transcribed SNPs within the PSCA, but not for SNPs located in neighboring genes, Ly6K and JRK, implying that the AEI is specific for PSCA. By using an allele-specific expression assay that measured allelic ratio of rs2294008 in PSCA transcripts, we also confirmed AEI detected by RNA-seq. PSCA mRNA expression was strongly increased in individuals with risk T allele in both normal (ptrend =0.0155 for rs2294008) and tumor samples rs2294008 (ptrend=0.0054), which is further indicative of effect of AEI. IHC on a panel of paired normal-tumor bladder tissue microarrays (TMA) confirmed mRNA expression results and showed PSCA protein expression only in carriers of risk genotype (TT) but not in non-risk genotype (CC), both in tumor and normal bladder tissue sections. Analysis of recombinant PSCA expression in transfected cells showed stronger surface expression of the risk T allele, compared to C allele. In conclusion, we show preferential mRNA and protein expression of PSCA in individuals with risk allele T of rs2294008. Thus, genotyping information for PSCA rs2294008 may emerge as an important decisive factor for response to PSCA antibody mediated treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5126. doi:1538-7445.AM2012-5126