A plasma biomarker panel of four MicroRNAs for the diagnosis of prostate cancer

Prostate cancer is diagnosed in over 1 million men every year globally, yet current diagnostic modalities are inadequate for identification of significant cancer and more reliable early diagnostic biomarkers are necessary for improved clinical management of prostate cancer patients. MicroRNAs (miRNAs) modulate important cellular processes/pathways contributing to cancer and are stably present in body fluids. In this study we profiled 372 cancer-associated miRNAs in plasma collected before (~60% patients) and after/during commencement of treatment (~40% patients), from age-matched prostate cancer patients and healthy controls, and observed elevated levels of 4 miRNAs - miR-4289, miR-326, miR-152-3p and miR-98-5p, which were validated in an independent cohort. The miRNA panel was able to differentiate between prostate cancer patients and controls (AUC = 0.88). Analysis of published miRNA transcriptomic data from clinical samples demonstrated low expression of miR-152-3p in tumour compared to adjacent non-malignant tissues. Overexpression of miR-152-3p increased proliferation and migration of prostate cancer cells, suggesting a role for this miRNA in prostate cancer pathogenesis, a concept that was supported by pathway analysis of predicted miR-152-3p target genes. In summary, a four miRNA panel, including miR-152-3p which likely targets genes with key roles in prostate cancer pathogenesis, has the potential to improve early prostate cancer diagnosis.Farhana Matin, Varinder Jeet, Leire Moya, Luke A. Selth, Suzanne Chambers, Australian Prostate Cancer BioResource, Judith A. Clements and Jyotsna Batr

A microsatellite repeat in PCA3 long non-coding RNA is associated with prostate cancer risk and aggressiveness.

Short tandem repeats (STRs) are repetitive sequences of a polymorphic stretch of two to six nucleotides. We hypothesized that STRs are associated with prostate cancer development and/or progression. We undertook RNA sequencing analysis of prostate tumors and adjacent non-malignant cells to identify polymorphic STRs that are readily expressed in these cells. Most of the expressed STRs in the clinical samples mapped to intronic and intergenic DNA. Our analysis indicated that three of these STRs (TAAA-ACTG2, TTTTG-TRIB1, and TG-PCA3) are polymorphic and differentially expressed in prostate tumors compared to adjacent non-malignant cells. TG-PCA3 STR expression was repressed by the anti-androgen drug enzalutamide in prostate cancer cells. Genetic analysis of prostate cancer patients and healthy controls (N > 2,000) showed a significant association of the most common 11 repeat allele of TG-PCA3 STR with prostate cancer risk (OR = 1.49; 95% CI 1.11-1.99; P = 0.008). A significant association was also observed with aggressive disease (OR = 2.00; 95% CI 1.06-3.76; P = 0.031) and high mortality rates (HR = 3.0; 95% CI 1.03-8.77; P = 0.045). We propose that TG-PCA3 STR has both diagnostic and prognostic potential for prostate cancer. We provided a proof of concept to be applied to other RNA sequencing datasets to identify disease-associated STRs for future clinical exploratory studies

Association Analysis of a Microsatellite Repeat in the TRIB1 Gene With Prostate Cancer Risk, Aggressiveness and Survival

With an estimated 1.1 million men worldwide diagnosed with prostate cancer yearly, effective and more specific biomarkers for early diagnosis could lead to better patient outcome. As such, novel genetic markers are sought for this purpose. The tribbles homologue 1 gene (TRIB1) has recently shown to have a role in prostate tumorigenesis and data-mining of prostate cancer expression data confirmed clinical significance of TRIB1 in prostate cancer. For the first time, a polymorphic microsatellite in this gene was studied for its potential association with prostate cancer risk and aggressiveness. Genomic DNA was extracted from a cohort of 1,152 prostate cancer patients and 1,196 cancer-free controls and the TTTTG-TRIB1 microsatellite was genotyped. The socio-demographic and clinical characteristics were analyzed using the non-parametric t-test and two-way ANOVA. Association of the TTTTG-TRIB1 microsatellite and prostate cancer risk and aggressiveness were analyzed by binary logistic regression and confirmed by bootstrapping. Total and prostate cancer mortality was analyzed using the Kaplan Meier test. Genotype and allele correlation with TRIB1 mRNA levels was analyzed using the non-parametric Kolmogorov–Smirnov test. To predict the effect that the TTTTG-TRIB1 polymorphisms had on the mRNA structure, the in silico RNA folding predictor tool, mfold, was used. By analyzing the publicly available data, we confirmed a significant over-expression of TRIB1 in prostate cancer compared to other cancer types, and an over-expression in prostate cancerous tissue compared to adjacent benign. Three alleles (three–five repeats) were observed for TTTTG-TRIB1. The three-repeat allele was associated with prostate cancer risk at the allele (OR = 1.16; P = 0.044) and genotypic levels (OR = 1.70; P = 0.006) and this association was age-independent. The four-repeat allele was inversely associated with prosatet cancer risk (OR = 0.57; P < 0.0001). TRIB1 expression was upregulated in tumors when compared to adjacent cancer-free tissue but was not allele specific. In silico analysis suggested that the TTTTG-TRIB1 alleles may alter TRIB1 mRNA structure. In summary, the three-repeat allele was significantly associated with prostate cancer risk, suggesting a biomarker potential for this microsatellite to predict prostate cancer. Further studies are needed to elucidate the functional role of this microsatellite in regulating TRIB1 expression, perhaps by affecting the TRIB1 mRNA structure and stability

KLK3 SNP-SNP interactions for prediction of prostate cancer aggressiveness.

Risk classification for prostate cancer (PCa) aggressiveness and underlying mechanisms remain inadequate. Interactions between single nucleotide polymorphisms (SNPs) may provide a solution to fill these gaps. To identify SNP-SNP interactions in the four pathways (the angiogenesis-, mitochondria-, miRNA-, and androgen metabolism-related pathways) associated with PCa aggressiveness, we tested 8587 SNPs for 20,729 cases from the PCa consortium. We identified 3 KLK3 SNPs, and 1083 (P < 3.5 × 10-9) and 3145 (P < 1 × 10-5) SNP-SNP interaction pairs significantly associated with PCa aggressiveness. These SNP pairs associated with PCa aggressiveness were more significant than each of their constituent SNP individual effects. The majority (98.6%) of the 3145 pairs involved KLK3. The 3 most common gene-gene interactions were KLK3-COL4A1:COL4A2, KLK3-CDH13, and KLK3-TGFBR3. Predictions from the SNP interaction-based polygenic risk score based on 24 SNP pairs are promising. The prevalence of PCa aggressiveness was 49.8%, 21.9%, and 7.0% for the PCa cases from our cohort with the top 1%, middle 50%, and bottom 1% risk profiles. Potential biological functions of the identified KLK3 SNP-SNP interactions were supported by gene expression and protein-protein interaction results. Our findings suggest KLK3 SNP interactions may play an important role in PCa aggressiveness

Evaluating Approaches for Constructing Polygenic Risk Scores for Prostate Cancer in Men of African and European Ancestry

Genome-wide polygenic risk scores (GW-PRSs) have been reported to have better predictive ability than PRSs based on genome-wide significance thresholds across numerous traits. We compared the predictive ability of several GW-PRS approaches to a recently developed PRS of 269 established prostate cancer-risk variants from multi-ancestry GWASs and fine-mapping studies (PRS269). GW-PRS models were trained with a large and diverse prostate cancer GWAS of 107,247 cases and 127,006 controls that we previously used to develop the multi-ancestry PRS269. Resulting models were independently tested in 1,586 cases and 1,047 controls of African ancestry from the California Uganda Study and 8,046 cases and 191,825 controls of European ancestry from the UK Biobank and further validated in 13,643 cases and 210,214 controls of European ancestry and 6,353 cases and 53,362 controls of African ancestry from the Million Veteran Program. In the testing data, the best performing GW-PRS approach had AUCs of 0.656 (95% CI = 0.635-0.677) in African and 0.844 (95% CI = 0.840-0.848) in European ancestry men and corresponding prostate cancer ORs of 1.83 (95% CI = 1.67-2.00) and 2.19 (95% CI = 2.14-2.25), respectively, for each SD unit increase in the GW-PRS. Compared to the GW-PRS, in African and European ancestry men, the PRS269 had larger or similar AUCs (AUC = 0.679, 95% CI = 0.659-0.700 and AUC = 0.845, 95% CI = 0.841-0.849, respectively) and comparable prostate cancer ORs (OR = 2.05, 95% CI = 1.87-2.26 and OR = 2.21, 95% CI = 2.16-2.26, respectively). Findings were similar in the validation studies. This investigation suggests that current GW-PRS approaches may not improve the ability to predict prostate cancer risk compared to the PRS269 developed from multi-ancestry GWASs and fine-mapping

A genetic risk score to personalize prostate cancer screening, applied to population data.

Background: A polygenic hazard score (PHS)—the weighted sum of 54 SNP genotypes—was previously validated for association with clinically significant prostate cancer and for improved prostate cancer screening accuracy. Here, we assess the potential impact of PHS-informed screening. Methods: UK population incidence data (Cancer Research UK) and data from the Cluster Randomized Trial of PSA Testing for Prostate Cancer were combined to estimate age-specific clinically significant prostate cancer incidence (Gleason≥7, stage T3-T4, PSA ≥10, or nodal/distant metastases). Using hazard ratios estimated from the ProtecT prostate cancer trial, age-specific incidence rates were calculated for various PHS risk percentiles. Risk-equivalent age—when someone with a given PHS percentile has prostate cancer risk equivalent to an average 50-year-old man (50-years-standard risk)—was derived from PHS and incidence data. Positive predictive value (PPV) of PSA testing for clinically significant prostate cancer was calculated using PHS-adjusted age groups. Results: The expected age at diagnosis of clinically significant prostate cancer differs by 19 years between the 1st and 99th PHS percentiles: men with PHS in the 1st and 99th percentiles reach the 50-years-standard risk level at ages 60 and 41, respectively. PPV of PSA was higher for men with higher PHS-adjusted age. Conclusions: PHS provides individualized estimates of risk-equivalent age for clinically significant prostate cancer. Screening initiation could be adjusted by a man’s PHS. Impact: Personalized genetic risk assessments could inform prostate cancer screening decisions

Polygenic hazard score is associated with prostate cancer in multi-ethnic populations.

Genetic models for cancer have been evaluated using almost exclusively European data, which could exacerbate health disparities. A polygenic hazard score (PHS1) is associated with age at prostate cancer diagnosis and improves screening accuracy in Europeans. Here, we evaluate performance of PHS2 (PHS1, adapted for OncoArray) in a multi-ethnic dataset of 80,491 men (49,916 cases, 30,575 controls). PHS2 is associated with age at diagnosis of any and aggressive (Gleason score ≥ 7, stage T3-T4, PSA ≥ 10 ng/mL, or nodal/distant metastasis) cancer and prostate-cancer-specific death. Associations with cancer are significant within European (n = 71,856), Asian (n = 2,382), and African (n = 6,253) genetic ancestries (p < 10-180). Comparing the 80th/20th PHS2 percentiles, hazard ratios for prostate cancer, aggressive cancer, and prostate-cancer-specific death are 5.32, 5.88, and 5.68, respectively. Within European, Asian, and African ancestries, hazard ratios for prostate cancer are: 5.54, 4.49, and 2.54, respectively. PHS2 risk-stratifies men for any, aggressive, and fatal prostate cancer in a multi-ethnic dataset

The kallikrein 14 gene is down-regulated by androgen receptor signalling and harbours genetic variation that is associated with prostate tumour aggressiveness

Kallikrein 14 (KLK14) has been proposed as a useful prognostic marker in prostate cancer, with expression reported to be associated with tumour characteristics such as higher stage and Gleason score. KLK14 tumour expression has also shown the potential to predict prostate cancer patients at risk of disease recurrence after radical prostatectomy. The KLKs are a remarkably hormone-responsive family of genes, although detailed studies of androgen regulation of KLK14 in prostate cancer have not been undertaken to date. Using in vitro studies, we have demonstrated that unlike many other prostatic KLK genes that are strictly androgen responsive, KLK14 is more broadly expressed and inversely androgen regulated in prostate cancer cells. Given these results and evidence that KLK14 may play a role in prostate cancer prognosis, we also investigated whether common genetic variants in the KLK14 locus are associated with risk and/or aggressiveness of prostate cancer in approximately 1200 prostate cancer cases and 1300 male controls. Of 41 single nucleotide polymorphisms assessed, three were associated with higher Gleason score (≥7): rs17728459 and rs4802765, both located upstream of KLK14, and rs35287116, which encodes a p.Gln33Arg substitution in the KLK14 signal peptide region. Our findings provide further support for KLK14 as a marker of prognosis in prostate cancer

MicroRNA-3162-5p-Mediated Crosstalk between Kallikrein Family Members Including Prostate-Specific Antigen in Prostate Cancer

BACKGROUND: MicroRNAs mediate biological processes through preferential binding to the 3′ untranslated region (3′ UTR) of target genes. Studies have shown their association with prostate cancer (PCa) risk through single-nucleotide polymorphisms (SNPs), known as miRSNPs. In a European cohort, 22 PCa risk-associated miRSNPs have been identified. The most significant miRSNP in the 3′ UTR of Kallikrein-related peptidase 3 (KLK3) created a binding site for miR-3162-5p. Here we investigated the miR-3162-5p–KLK interaction and the clinical implication of miR-3162-5p in PCa. METHODS: We tested the role of miR-3162-5p in PCa etiology using IncuCyte live-cell imaging and anchorage-independent growth assays. The effect of miR-3162-5p on KLK and androgen receptor (AR) expression was measured by RT-quantitative (q)PCR and target pulldown assays. KLK3 proteolytic activity was determined by DELFIA® immunoassay. Mass spectrometry identified pathways affected by miR-3162-5p. miR-3162-5p expression was measured in clinical samples using RT-qPCR. RESULTS: miR-3162-5p affected proliferation, migration, and colony formation of LNCaP cells by regulating the expression of KLK2–4 and AR by direct targeting. KLK3 protein expression was regulated by miR-3162-5p consistent with lower KLK3 proteolytic activity observed in LNCaP-conditioned media. KLK/AR pulldown and mass spectrometry analysis showed a potential role of miR-3162-5p in metabolic pathways via KLK/AR and additional targets. Increased miR-3162-5p expression was observed in prostate tumor tissues with higher Gleason grade. CONCLUSIONS: Our study provides an insight into possible involvement of miR-3162-5p in PCa etiology by targeting KLKs and AR. It highlights clinical utility of miR-3162-5p and its interactive axis as a new class of biomarkers and therapeutic targets for PCa.No Full Tex