Genetic variants in the Hippo pathway predict biochemical recurrence after radical prostatectomy for localized prostate cancer

Abstract

While localized prostate cancer is potentially curative, many patients still show biochemical recurrence (BCR) after curative treatments such as radical prostatectomy (RP). The Hippo pathway has recently been shown to be an evolutionarily conserved regulator of tissue growth, and its perturbation can trigger tumorigenesis. We hypothesize that genetic variants of the Hippo pathway may influence clinical outcomes in localized prostate cancer patients. We genotyped 53 tagging single-nucleotide polymorphisms (SNPs) from seven core Hippo pathway genes in 246 localized prostate cancer patients treated with RP. Kaplan-Meier analysis and Cox proportional hazard models were utilized to identify significant SNPs that correlated with BCR. For replication, five associated SNPs were genotyped in an independent cohort of 212 patients. After adjusting for known clinicopathologic factors, the association between STK3 rs7827435 and BCR (P = 0.018) was replicated in the second stage (P = 0.026; Pcombined = 0.001). Additional integrated in silico analysis provided evidence that rs7827435 affects STK3 expression, which in turn is significantly correlated with tumor aggressiveness and patient prognosis. In conclusion, genetic variants of the Hippo pathway contribute to the variable outcomes of prostate cancer, and the discovery of these biomarkers provides a molecular approach for prognostic risk assessment.

Figure 1. Kaplan-Meier survival curves of BCR-free survival by STK3 rs7827435 genotypes for localized prostate cancer patients receiving RP in the discovery cohort (left), replication cohort (middle), and combined analysis (right).

Numbers in parentheses indicate the number of patients.

Figure 2. Summary of the functional analyses for the STK3 rs7827435 locus.

(A) STK3 shows significant eQTL association with rs7827435 genotype in prostate tissues (GTEx data set). Numbers in parentheses indicate the number of cases. (B) Regulatory annotation of variants within the LD block containing STK3 rs7827435. In the LD block with the lead SNP rs7827435, ENCODE data showed evidence of enhancer elements coinciding with rs7827435 and linked variants in GM12878 B-lymphoblastoid cells, HD.CD184EC hESC-derived CD184+ endoderm cultured cells, and several additional cell types. In addition, multiple regulatory motifs are predicted to be affected. (C) Expanded view of the ENCODE and GTEx data for the LD block containing the STK3 rs7827435. The H3K4Me1, H3K4Me3, and H3K27Ac tracks show the genome-wide levels of enrichment of the mono-methylation of lysine 4, tri-methylation of lysine 4, and acetylation of lysine 27 of the H3 histone protein, as determined by the ChIP-seq assays. These levels are thought to be associated with enhancer and promoter regions. Chromatin State Segmentation track displays chromatin state segmentations by integrating ChIP-seq data using a Hidden Markov Model for GM12878 B-lymphoblastoid cells, K562 leukemia cells, HepG2 hepatocellular carcinoma cells, HMEC normal mammary epithelial cells, NHEK epidermal keratinocytes, and NHLF normal lung fibroblast cells. The chromatin state regions predicted for promoters and enhancers are highlighted. DNase clusters track shows DNase hypersensitivity areas. Tnx Factor track shows regions of transcription factor binding of DNA, as assayed by ChIP-seq experiments. The results of STK3 gene-centric eQTL analysis for prostate tissues using GTEx data are visualized as a regional plot. Closed circles highlight where rs7827435 and its linked SNPs are placed.

Figure 3. Correlation of STK3 mRNA expression with prostate cancer progression.

The associations between STK3 expression and prostate cancer aggressiveness were analyzed using TCGA data. More advanced prostate cancers, high pathologic stage (A) and pathologic Gleason score (B), display significantly lower STK3 DNA methylation and higher STK3 mRNA expression. Numbers in parentheses indicate the number of patients. rho: Spearman's rank correlation coefficient. P: P-value. (C) STK3 shows higher levels of gene expression in tumors with increased DNA copy number at 8q22. −1: hemizygous deletion; 0: diploid; 1: gain; 2: amplification. (D) Kaplan-Meier curves of recurrence-free survival according to the alterations in STK3. Patients were dichotomized with or without STK3 amplification and mRNA upregulation.