Abstract 1444: Validation of prostate cancer risk variants by CRISPR/Cas9 mediated genome editing at the MSMB locus
Authors: Wang Xing , Hayes James , Xu Xing , Mehtad Dipti , Gao Xiaoni , Lilja Hans , Klein Robert 
Published: July 2017

Abstract
Genome wide association studies (GWAS) have identified nuermous single nucleotide polymorphisms (SNPs) associated with prostate cancer risk. While numerous in silico analyses suggest that the majority of these SNPs may function through regulation of gene expression, wet lab experiments to distinguish the causative variants from their linkage disequilibrium (LD) partners is still needed. Here, we demonstrate how genome editing can investigate the role of individual polymorphisms on gene expression.
Here, we focus on the prostate cancer-associated SNP rs10993994, which is in the promoter of the MSMB gene that encodes for the prostate-secreted protein beta-MSP. This SNP is notable because it associates with seminal and serum levels of beta-MSP, PSA, and hK2; mRNA levels of MSMB and the nearby androgen receptor co-activator NCOA4 in the prostate; and mRNA levels of MSMB in the stomach. Intriguingly, rs10993994 is not a gastric cancer risk SNP suggesting that there may be other variants with prostate-specific function responsible for the observed prostate cancer association.
Using data from the FANTOM5 project, we show that rs7098889, in LD with rs10993994, is located in one of the most prostate-specific enhancers. We had previously shown that this SNP is associated with beta-MSP independent of rs10993994.
To distinguish the effects of these two SNPs on gene expression, we generated a series of double gRNA mediated CRISPR/Cas9 deletions of the relevant regulatory elements. Removing the element containing rs7098889 flanking region results in ~10 to 20 fold increase of MSMB expression in the prostate cancer LNCaP cell line but not in the gastric cancer AGS cell line. There is no change in NCOA4 in either cell line. Removing rs10993994 flanking region results in over 10 fold increase of MSMB expression and ~50% decrease of NCOA4 in LNCaP.
As both LNCaP and AGS are heterozygous for both SNPs, we then generated single clones in which only one allele was removed. For rs7098889, an over 300 fold increase of MSMB expression was observed in two out of the three clones with the T but not the C allele. Transcripts from high expressers all came from one allele. Similarly, single clone analysis showed 40 to 60 fold up-regulation of MSMB expression in two out of three clones with the C but not the T allele of rs10993994. This is consistent with the observation for rs7098889 since rs7098889T and rs10993994C are on the same haplotype.
Our work demonstrates dramatic and locus specific effect of individual polymorphisms on gene expression, which strongly support the notion that an important function of disease risk variants is through regulation of gene expression.
Citation Format: Xing Wang, James Hayes, Xing Xu, Dipti Mehtad, Xiaoni Gao, Hans Lilja, Robert Klein. Validation of prostate cancer risk variants by CRISPR/Cas9 mediated genome editing at the MSMB locus [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 1444. doi:10.1158/1538-7445.AM2017-1444