Epigenetic Dysregulation of Tumor Suppressor Genes in CP/CPPS: Assessing the Diagnostic and Prognostic Utilities of Liquid Biopsies

Abstract

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a prevalent disorder affecting about 90% of patients with prostatitis syndrome. CP/CPPS can potentially progress to prostate cancer (PCa) in older age. However, the underlying molecular mechanisms of this disease remain unclear. Tumor suppressor genes (TSGs) are essential in preventing the malignant transformation of cells, and their epigenetic dysregulation is commonly observed in cancer. Therefore, we hypothesize that epigenetic aberrations in TSGs may also occur in CP/CPPS and contribute to the development of PCa. This study aims to analyze the epigenetic status of TSGs in CP/CPPS by using liquid biopsies such as ejaculate (EJ) and post-prostatic massage urine (ExU), specifically the promoter methylation and messenger ribonucleic acid (mRNA) expression status of selected PCa-associated TSGs, and to determine which type of liquid biopsy is promising for the establishment of biomarkers. We utilized pyrosequencing and real-time polymerase chain reaction (RT-PCR) to investigate the promoter methylation and mRNA expression levels of a group of candidate prostatic TSGs. Specially, we analyzed CDKN2A, EDNRB, PTGS2, BMP4, BMP7, PITX2, and GSTP1 in somatic cells isolated from ejaculate (EJ) and post-prostatic massage urine (ExU) from 30 healthy men and 50 patients diagnosed with CP/CPPS. In addition, to examine the source of hypermethylation and down-regulated mRNA expression of CDKN2A in liquid biopsies, we further sorted epithelial cells and leukocytes individually using the magnetic-activated cell sorting system (MACS) from fresh EJ and ExU samples collected from 34 CP/CPPS patients and 26 healthy donors. The efficiency of cell sorting was assessed by immunofluorescent staining (IF) for Prostate-specific antigen (PSA), Epithelial cell adhesion molecule (EpCAM), and Protein tyrosine phosphatase, receptor type, C (CD45). Furthermore, we analyzed the correlations between our experimental data and the clinical parameters. Lastly, we evaluated the influence of exosomes isolated from ExU supernatant on the polarization of the monocyte cell line THP-1 and the expression levels of CDKN2A. Our results showed that the promoter methylation levels of EDNRB, CDKN2A, PTGS2, BMP7, and BMP4 were significantly higher in the somatic cells of the EJ sample from CP/CPPS patients. Additionally, the mRNA expression levels of CDKN2A and PTGS2 were significantly downregulated in CP/CPPS patients' somatic cells. In the ExU samples, we observed that EDNRB and CDKN2A were more highly methylated, and CDKN2A and PTGS2 were expressed at lower levels in CP/CPPS patients compared to healthy controls. Given the sensitivity and specificity of CDKN2A in demonstrating the differences in methylation and expression levels between CP/CPPS patients and healthy controls, we selected it for further analysis in a MACS-cell sorting experiment. Our results showed that CP/CPPS patients exhibited significantly higher levels of CDKN2A methylation in both sorted epithelial cells and leukocytes and lower mRNA expression levels in epithelial cells and leukocytes of EJ. Importantly, we also demonstrated the feasibility of the MACS system in liquid biopsies from CP/CPPS patients and healthy controls containing a low amount of cells. Furthermore, we found significant correlations between our experimental results and clinical parameters such as prostate volume, PSA, NIH Chronic Prostatitis Symptom Index (NIH-CPSI), International Prostate Symptom Score (IPSS), and sperm motility. Finally, we investigated the impact of exosomes derived from ExU supernatant on the polarization of M0 macrophages. Our findings suggest that ExU exosomes may promote the polarization of M0 macrophages towards the M1 phenotype. This study significantly contributes to identifying potential biomarkers for the prognosis of CP/CPPS from liquid biopsies, suggesting a potential link between CP/CPPS and prostate cancer. Specifically, the study highlights the close correlation between the methylation and downregulation of TSGs in CP/CPPS and various clinical parameters, indicating the potential role of TSGs in the assessment of CP/CPPS severity and prognosis, as well as the fertility of affected individuals. Additionally, the study introduces a practical approach for isolating epithelial cells and leukocytes from liquid biopsies, facilitating more in-depth research into CP/CPPS. Finally, these findings lay the groundwork for future investigations to elucidate the molecular mechanisms underlying CP/CPPS and its potential progression to prostate cancer.