A common regulatory variant in SLC35B4 influences the recurrence and survival of prostate cancer

Abstract

Single nucleotide polymorphisms (SNPs) within the regulatory elements of a gene can alter gene expression, making these SNPs of prime importance for candidate gene association studies. We aimed to determine whether such regulatory variants are associated with clinical outcomes in three cohorts of patients with prostate cancer. We used RegulomeDB to identify potential regulatory variants based on in silico predictions and reviewed genome-wide experimental findings. Overall, 131 putative regulatory SNPs with the highest confidence score on predicted functionality were investigated in two independent localized prostate cancer cohorts totalling 458 patients who underwent radical prostatectomy. The statistically significant SNPs identified in these two cohorts were then tested in an additional cohort of 504 patients with advanced prostate cancer. We identified one regulatory SNPs, rs1646724, that are consistently associated with increased risk of recurrence in localized disease (P = .003) and mortality in patients with advanced prostate cancer (P = .032) after adjusting for known clinicopathological factors. Further investigation revealed that rs1646724 may affect expression of SLC35B4, which encodes a glycosyltransferase, and that down-regulation of SLC35B4 by transfecting short hairpin RNA in DU145 human prostate cancer cell suppressed proliferation, migration and invasion. Furthermore, we found increased SLC35B4 expression correlated with more aggressive forms of prostate cancer and poor patient prognosis. Our study provides robust evidence that regulatory genetic variants can affect clinical outcomes.

Keywords: SLC35B4; multi-stage association study; prognosis; prostate cancer; regulatory variant.

Figure 1

Study design and key findings. The discovery stage 1, replication stage 2 and stage 3, and subsequent functional analyses are illustrated. One candidate susceptibility genes, SLC35B4, for prostate cancer progression were identified after replication testing and functional analyses

Figure 2

Impact of rs1646724 and rs4239504 on prostate cancer prognosis. Kaplan‐Meier estimates of BCR‐free survival by (A) rs1646724 and (B) rs4239504 genotype for patients with localized prostate cancer receiving RP in the discovery cohort, replication cohort and combined analysis. (C) Kaplan‐Meier estimates of overall survival by rs1646724 genotype for patients with advanced prostate cancer receiving ADT. Numbers in parentheses indicate the number of patients

Figure 3

Silencing SLC35B4 expression inhibits DU145 cell growth and migration. (A) Correlation of rs1646724 genotype with SLC35B4 expression. There is a trend towards increased SLC35B4 mRNA expression in carriers of the rs1646724 risk allele G. The numbers in parentheses indicate the number of cases. (B) Western blots showing that expression of SLC35B4 in DU145 cells is increased compared to that found in PC‐3 cells, and that expression was effectively down‐regulated by transfecting shRNA into DU145 cells. Down‐regulation of SLC35B4 inhibits DU145 cell (C) proliferation, (D) migration and (E) invasion. Data are expressed as the mean ± standard deviation (SD) of three independent experiments

Figure 4

Increased SLC35B4 expression is correlated with prostate cancer aggressiveness and poor patient prognosis. (A) Representative images of immunohistochemical staining for SLC35B4 expression in human benign prostate tissues as well as low‐, intermediate‐, and high‐grade adenocarcinomas. (B) Correlation of SLC35B4 expression with prostate cancer progression. More advanced prostate cancers and a higher Gleason score display a tendency towards increased SLC35B4 expression. (C) Increased SLC35B4 mRNA expression is correlated with reduced BCR‐free survival in patients with prostate cancer. Numbers in parentheses indicate the number of patients