Author Summary Family history has long been recognized as an important risk factor for prostate cancer. Beginning in 2006, researchers have identified several genetic variants that are associated with prostate cancer risk. Intriguingly, the majority of prostate cancer risk variants do not reside in genes. Determining the genes involved in the develop- ment of disease, therefore, has proved challenging. In this study we interrogate a known prostate cancer risk poly- morphism on chromosome 10—rs10993994. We report that this variant is significantly associated with the RNA expression levels of two genes—MSMB and NCOA4. When these expression changes are modeled in a cell line, prostate cells that were previously non-tumorigenic acquire a property known as anchorage independence, a characteristic of cancer cells. Notably, the prostate risk variant is not associated with expression or functional changes in breast or colon cells. In addition, the effects are most pronounced in normal rather than tumor prostate tissue. Overall, these findings help define the genes driving prostate cancer risk at chromosome 10. More generally, the discoveries demonstrate the importance of consider- ing several target genes, as well as the importance of cellular context (tissue type and histological state), in future analyses of other genetic risk regions. receptor co-activator [10,11] and is within 16 kb telomeric of rs10993994. Given its proximity to the risk variant and its activity in the prostate gland [12], NCOA4 has also been considered a candidate gene involved in the mechanism of disease risk [8]. Gene expression is a heritable trait [13–16] and represents a powerful avenue for connecting risk variants with their target genes. Studies have demonstrated that variation at intergenic or intronic disease-associated loci can act through gene regulatory mechanisms [17–20]. Because regulatory elements can interact with many genes [21], and since both MSMB and NCOA4 are strong candidates for prostate cancer risk, we evaluated the relationship between risk allele status and transcript abundance of these genes across both normal and tumor prostate tissues. We also tested the functional consequences of altering the expression levels of these candidate genes in immortalized prostate epithelial cells. Results A total of 180 individuals of European ancestry were genotyped for the rs10993994 polymorphism, and MSMB and NCOA4 mRNA levels were quantified in tissue isolated from radical prostatectomy surgical specimens. Samples were derived from two cohorts- the Gelb Center at Dana-Farber Cancer Institute (DFCI) (N = 121 – histologically normal and tumor prostate tissue) and the Physicians’ Health Study (PHS) [22] (N = 59 – prostate tumor tissue only). In the DFCI cohort, transcript levels were measured in both normal and tumor prostate tissue using a quantitative competitive PCR strategy (Methods). Two MSMB and five NCOA4 isoforms annotated in the Ensembl database (build 52) were evaluated (Figure 1A). Each isoform of MSMB and NCOA4 was expressed in both normal and tumor prostatic tissue. Expression levels of MSMB and NCOA4 transcripts were significantly higher in normal compared with tumor tissue (p,0.0001), consistent with previously published reports [23–25]. In the PHS cohort, only tumor tissue was isolated from radical prostatectomy specimens. For this cohort the probes used to measure expression captured both MSMB isoforms and all but one of the NCOA4 isoforms. MSMB and NCOA4 Implicated in Prostate Cancer Risk The T (risk) allele is significantly associated with transcript levels of both MSMB and NCOA4 in histologically normal prostate tissue (N = 84). While the T allele is associated with decreased expression of both MSMB isoforms (p-value range, 0.0033–0.0042), it is associated with increased expression of all five isoforms of NCOA4 (p-value range, 6.761027–0.0055) (Figure 1B). In tumor tissue, MSMB retains its association with risk allele status (p-value range, 0.016–0.053, Figure 1C, Figure S1). NCOA4 expression levels, however, are no longer associated with genotype in tumor tissue (p.0.30, Figure 1C, Figure S1). The associations are specific to the genes in this region. Expression levels of TIMM23, the next closest gene to the risk locus and 1.4 kb telomeric to NCOA4, are not correlated with genotype status in either normal or tumor tissue. (p.0.30, Figure S2). Because rs10993994 is not a risk allele in colon or breast cancers, we reasoned that the association between genotype and disease- relevant genes may be specific to prostate tissue and not observed in other tissue types. If an association between genotype and expression of MSMB or NCOA4 were observed in tissue other than prostate, then that gene may be less likely to be involved in prostate cancer risk. MSMB and NCOA4 expression levels were measured in histologically normal colon (N = 72) and breast (N = 56) tissue samples. While breast tissue expresses both genes, colon tissue only expresses NCOA4. Unlike prostate tissue, neither breast nor colon tissue demonstrates convincing or consistent associations with genotype across isoforms (Figure S3). To evaluate the functional implications of the genetic findings, we tested the effect of increasing NCOA4 and suppressing MSMB expression levels in immortalized prostate epithelial cells (LHSAR) [26]. Specifically, we assessed the ability of NCOA4 and MSMB to promote anchorage-independent growth, a phenotype strongly associated with cell transformation [27–30]. Suppression of MSMB expression in LHSAR cells led to a significant increase in anchorage-independent colony growth (p-values 0.0023–0.0001; Figure 2A, Figure S4). Overexpression of NCOA4 in LHSAR cells also resulted in robust anchorage-independent colony growth (p-value 0.0074; Figure 2B, Figure S4). To assess whether these alterations were specific for prostate epithelial cells, similar functional studies were performed in immortalized human mammary epithelial cells [31]. In contrast to what was observed in prostate epithelial cells, manipulating expression levels of MSMB or NCOA4 did not result in any consistently significant change in the anchorage-independent growth in mammary epithelial cells (Figure S5). Together, these observations implicate a role for both NCOA4 and MSMB in the transformation of prostate epithelial cells. Discussion Genetic data presented here demonstrate that the chromosome 10q11 prostate cancer risk locus is associated with decreased levels of MSMB and increased levels of NCOA4 RNA expression. Strikingly, the functional data fully corroborate the genetic data. When MSMB is knocked down or NCOA4 is overexpressed in immortalized prostate epithelial cells, the cells become anchorage independent. Our data suggest that both MSMB and NCOA4 mediate prostate tumorigenesis, and this study is the first, to our knowledge, to implicate these genes in actual human prostate tissue. As expected in a cohort comprised of subjects who underwent radical prostatectomy, a large majority of individuals included in the analysis were diagnosed with low- or intermediate- risk prostate cancer. Despite a relatively homogenous cohort, the results presented here are likely generalizable to most prostate cancer cases since rs10993994 appears to confer risk for all levels of prostate cancer aggressiveness [1,3,32,33,34]. PLoS Genetics | www.plosgenetics.org 2 November 2010 | Volume 6 | Issue 11 | e1001204