Molecular strategies to reduce unnecessary repeat prostate biopsies of men with elevated serum PSA

Prostate cancer (PCa) is the most common cancer in men. It is a heterogeneous disease and currently there are no reliable biomarkers available to stratify men for prostate biopsy (PBx) and treatment. Hence, there is a risk of over-diagnosing insignificant disease, or under-diagnosing significant disease. We aimed to evaluate FDA approved Prostate Cancer gene 3 (PCA3, FDA approved) and N6-methyladenosine (m6A) for diagnostic properties. PCA3 is a long non-coding RNA (ncRNA) that is unstable, has an unclear biological role and is expensive to chemically treat to prevent degradation prior to analysis. Furthermore, the biological role of this RNA is unclear. Long ncRNAs are degraded into shorter forms, we explored whether this was the fatecase for PCA3. We identified a short segment of RNA within intron 1 of PCA3 bioinformatically which we termed PCA3 short RNA2 (PCA3-shRNA2). The and showed that PCA3-shRNA2 expression of this short RNA correlated to that of PCA3 in PCa cell lines, urinary samples and PBx tissue. PCA3-shRNA2 was overexpressed in urinary samples obtained from men with PCa compared to BPH, was regulated by testosterone and had a diagnostic accuracy similar to that of PCA3. We identified oncogenic mRNA targets of PCA3-shRNA2 and that are involved in oncogenesis and found that COPS2 was underexpressed in cancerous urinary samples. There are over a hundred RNA modifications described and methylation of N6-adenosine base is the most common methylated site. m6A is reversible and may be, involved in oncogenesis. and has recently been mapped throughout the transcriptome. We profiled m6A in PCa cell lines by immunoprecipitation and RNA sequencing, and found interesting oncogenic RNAs (e.g. PARG,) that were differentially expressed in LNCaP-LN3 cells. We identified a novel RNA within PCA3 that is stable, easy to measure, overexpressed in PCa samples and appeared to target oncogenic mRNAs. We profiled m6A in PCa cell lines and have identified important N6-adenosine methylated RNAs associated with PCa development. In conclusion PCA3-shRNA2 and m6A have evolving roles in cancer and may function well as biomarkers.