Deciphering Epitranscriptomic Signature: the Role of 5-Methylcytosine in Prostate Cancer

Abstract

Background: Prostate Cancer (PCa) ranks as the 2nd most incident cancer and 5th most leading cause of cancer-related deaths worldwide. Its heterogeneous nature presents a significant hurdle in treatment and patient management. Thus, there remains a critical need to discover new molecular signatures to improve patient stratification and optimize therapeutic interventions. 5-Methylcytosine (m5C) is one of the most prevalent RNA modifications in mammals. Its regulatory proteins, capable of adding, reading or removing the methylation mark, have been implicated in cancer, including PCa. Furthermore, this modification was described as a potential predictive biomarker for treatment decision making in other malignancies. Nonetheless, understanding of the role of m5C modification in PCa is still scarce. Hence, the primary aim of this study is to comprehensively characterize m5C modification and its associated regulatory proteins in PCa. Methods: An in-silico analysis was performed using The Cancer Genome Atlas (TCGA), where m5C-related proteins were evaluated in PCa. The transcript levels of the most deregulated genes, YTHDF2, NOP2, NSUN2 and NSUN6 were assessed in a series of PCa patients treated with radical prostatectomy at IPO Porto. Immunohistochemical (IHC) assays for NSUN2 protein were carried out in the same series, and the results were analyzed according to standard clinicopathological variables, namely tumor grade and stage. Additionally, NSUN2 and NOP2 transcript and protein levels were evaluated in different PCa and non-malignant cell lines. The NSUN2 gene was successfully silenced by shRNA in C4-2B cell line. Moreover, characterization of transcript and protein levels associated with Epithelial-Mesenchymal Transition (EMT) was performed in C4-2B cells with NSUN2 shRNA. Results: Several m5C players, including NSUN2, are significantly increased in the TCGA CaP series compared to normal samples. Regarding IPO Porto PCa patients, higher NSUN2 transcript levels were significantly associated with cases of less differentiated tumors and more advanced stages. There was also a significant association between higher NSUN2 IHC scores and intensities and higher group gradings, as well as more advanced stages, in agreement with the transcript analysis. In addition, PCa cell lines showed contrasting NSUN2 transcript levels compared to non-malignant lines, being especially higher in the C4-2B cell line. Transfection of NSUN2 shRNA successfully promoted a decrease in NSUN2 expression in C4-2B cells. EMT-related proteins showed increased levels in the NSUN2 knockdown cell line, while some of the proteins associated with the epithelial phenotype were decreased. However, NSUN2 does not appear to have a relevant role in EMT in PCa. Conclusions: High levels of NSUN2 protein were associated with less differentiated PCa, as well as more advanced tumor stages. The results of this study indicate an involvement of this protein in PCa, suggesting a potentially oncogenic function in this neoplasm.