Genetic and functional analyses implicate the NUDT11, HNF1B, and SLC22A3 genes in prostate cancer pathogenesis

Abstract

One of the central goals of human genetics is to discover the genes and pathways driving human traits. To date, most of the common risk alleles discovered through genome-wide association studies (GWAS) map to nonprotein-coding regions. Because of our relatively poorer understanding of this part of the genome, the functional consequences of trait-associated variants pose a considerable challenge. To identify the genes through which risk loci act, we hypothesized that the risk variants are regulatory elements. For each of 12 known risk polymorphisms, we evaluated the correlation between risk allele status and transcript abundance for all annotated protein-coding transcripts within a 1-Mb interval. A total of 103 transcripts were evaluated in 662 prostate tissue samples [normal (n = 407) and tumor (n = 255)] from 483 individuals [European Americans (n = 233), Japanese (n = 127), and African Americans (n = 123)]. In a pooled analysis, 4 of the 12 risk variants were strongly associated with five transcripts (NUDT11, MSMB, NCOA4, SLC22A3, and HNF1B) in histologically normal tissue (P ≤ 0.001). Although associations were also observed in tumor tissue, they tended to be more attenuated. Previously, we showed that MSMB and NCOA4 participate in prostate cancer pathogenesis. Suppressing the expression of NUDT11, SLC22A3, and HNF1B influences cellular phenotypes associated with tumor-related properties in prostate cancer cells. Taken together, the data suggest that these transcripts contribute to prostate cancer pathogenesis.

Fig. 1.

RNA expression of significantly associated genes in normal prostate tissue of EA, Japanese, and AA individuals. Each distribution is summarized as a boxplot. The horizontal line within the box represents the median of the distribution, and the hinges of the box represent the 25th and 75th percentiles. The P value for each graph denotes the significance of association between expression and genotype. (A) Expression in histologically normal EA tissue (n = 200). (B) Expression in histologically normal Japanese tissue (n = 84). (C) Expression in histologically normal AA tissue (n = 123).

Fig. 2.

Suppression of risk loci genes has differential effects on cell viability of prostate cell lines. The indicated genes were suppressed by lentivirally mediated delivery of shRNA and viability measured 6 d after selection using Cell Titer Glo (Promega) assay. Specifically, SLC22A3, NUDT11, and HNF1B were each suppressed with two different shRNAs targeting different sites of the genes (Materials and Methods). shRNA targeting luciferase (sh_Luc) was used as a negative control. Data represent the average and SD of at least two independent experiments. Cell viability of LNCaP (A), RWPE (B), and LHSAR (D) cell lines was significantly inhibited by suppression of all targeted candidate genes. Cell viability of PC3 cells (C) was not significantly affected by suppression of targeted candidate genes, compared with Luciferase control.

Fig. 3.

Suppression of risk loci genes is associated with decreased anchorage-independent growth of prostate cancer cell lines. Soft agar colony formation assays were performed using LNCaP (A) (P values = 0.03, sh_SLC22A3_1; 0.49, sh_SLC22A3_2; 0.01, sh_NUDT11_1; 0.02, sh_NUDT11_2; 0.01, sh_HNF1B_1; 0.03 sh_HNF1B_2; 0.007, sh_MSMB) and PC3 (B) (P values = 0.03, sh_NUDT11_1; 0.01, sh_NUDT11_2; 0.08, sh_HNF1B_1; 0.25 sh_HNF1B_2; 0.02, sh_MSMB) cells that were infected with lentivirus expressing the indicated shRNA. Specifically, SLC22A3, NUDT11, and HNF1B were suppressed with two different shRNAs targeting different sites of the genes (Materials and Methods). shRNA targeting luciferase (sh_Luc) was used as a negative control. shRNA targeting MSMB (sh_MSMB) was used as a positive control, as previously shown to increase anchorage-independent colony formation. P values, paired two-tailed t test. Data represent the average and SD of at least two independent experiments.