DNA methylation is an epigenetic modification that plays a pivotal role in regulating gene expression and, consequently, influences a wide variety of biological processes and diseases. The advances in next-generation sequencing technologies allow for genome-wide profiling of methyl marks both at a single-nucleotide and at a single-cell resolution. These profiling approaches vary in many aspects, such as DNA input, resolution, coverage, and bioinformatics analysis. Thus, the selection of the most feasible method according with the project’s purpose requires in-depth knowledge of those techniques. Currently, high-throughput sequencing techniques are intensively used in epigenomics profiling, which ultimately aims to find novel biomarkers for detection, diagnosis prognosis, and prediction of response to therapy, as well as to discover new targets for personalized treatments. Here, we present, in brief, a portrayal of next-generation sequencing methodologies’ evolution for profiling DNA methylation, highlighting its potential for translational medicine and presenting significant findings in several diseases.

Background: The lack of sensitive and specific biomarkers for the early detection of prostate cancer (PCa) is a major hurdle to improve patient management. Methods: A metabolomics approach based on GC-MS was used to investigate the performance of volatile organic compounds (VOCs) in general and, more specifically, volatile carbonyl compounds (VCCs) present in urine as potential markers for PCa detection. Results: Results showed that PCa patients (n = 40) can be differentiated from cancer-free subjects (n = 42) based on their urinary volatile profile in both VOCs and VCCs models, unveiling significant differences in the levels of several metabolites. The models constructed were further validated using an external validation set (n = 18 PCa and n = 18 controls) to evaluate sensitivity, specificity and accuracy of the urinary volatile profile to discriminate PCa from controls. The VOCs model disclosed 78% sensitivity, 94% specificity and 86% accuracy, whereas the VCCs model achieved the same sensitivity, a specificity of 100% and an accuracy of 89%. Our findings unveil a panel of 6 volatile compounds significantly altered in PCa patients’ urine samples that was able to identify PCa, with a sensitivity of 89%, specificity of 83%, and accuracy of 86%. Conclusions: It is disclosed a biomarker panel with potential to be used as a non-invasive diagnostic tool for PCa.

Epitranscriptomics has gained ground in recent years, especially after the advent of techniques for accurately studying these mechanisms. Among all modifications occurring in RNA molecules, N6-methyladenosine (m⁶A) is the most frequent, especially among mRNAs. m⁶A has been demonstrated to play important roles in many physiological processes and several disease states, including various cancer models (from solid to liquid tumors). Tumor cells’ epitranscriptome is indeed disrupted in a way to promote cancer-prone features, by means of up/downregulating m⁶A-related players: the so-called writers, readers and erasers. These proteins modulate m⁶A establishment, removal and determine mRNAs fate, acting in a context-dependent manner, so that a single player may act as an oncogenic signal in one tumor model (methyltransferase like 3 (METTL3) in lung cancer) and as a tumor suppressor in another context (METTL3 in glioblastoma). Despite recent advances, however, little attention has been directed towards urological cancer. By means of a thorough analysis of the publicly available TCGA (The Cancer Genome Atlas) database, we disclosed the most relevant players in four major urogenital neoplasms—kidney, bladder, prostate and testicular cancer—for prognostic, subtype discrimination and survival purposes. In all tumor models assessed, the most promising player was shown to be Vir like m⁶A methyltransferase associated (VIRMA), which could constitute a potential target for personalized therapies.

RNA methylation at position N6 in adenosine (m⁶A) and its associated methyltransferase complex (MTC) are involved in tumorigenesis. We aimed to explore m⁶A biological function for long non-coding RNAs (lncRNAs) in prostate cancer (PCa) and its clinical significance. m⁶A and MTC levels in PCa cells were characterized by ELISA and western blot. Putative m⁶A-regulated lncRNAs were identified and validated by lncRNA profiler qPCR array and bioinformatics analysis, followed by m⁶A/RNA co-immunoprecipitation. Impact of m⁶A depletion on RNA stability was assessed by Actinomycin D assay. The association of m⁶A-levels with PCa prognosis was examined in clinical samples. Higher m⁶A-levels and VIRMA overexpression were detected in metastatic castration-resistant PCa (mCRPC) cells (p < 0.05). VIRMA knockdown in PC-3 cells significantly decreased m⁶A-levels (p = 0.0317), attenuated malignant phenotype and suppressed the expression of oncogenic lncRNAs CCAT1 and CCAT2 (p < 0.00001). VIRMA depletion and m⁶A reduction decreased the stability and abundance of CCAT1/2 transcripts. Higher expression of VIRMA, CCAT1, and CCAT2 as a group variable was an independent predictor of poor prognosis (HR = 9.083, CI95% 1.911–43.183, p = 0.006). VIRMA is a critical factor sustaining m⁶A-levels in PCa cells. VIRMA downregulation attenuates the aggressive phenotype of PCa by overall reduction of m⁶A-levels decreasing stability and abundance of oncogenic lncRNAs.

Upregulation of MYC and miRNAs deregulation are common in prostate cancer (PCa). Overactive MYC may cause miRNAs’ expression deregulation through transcriptional and post-transcriptional mechanisms and epigenetic alterations are also involved in miRNAs dysregulation. Herein, we aimed to elucidate the role of regulatory network between MYC and miRNAs in prostate carcinogenesis. MYC expression was found upregulated in PCa cases and matched precursor lesions. MicroRNA’s microarray analysis of PCa samples with opposed MYC levels identified miRNAs significantly overexpressed in high-MYC PCa. However, validation of miR-27a-5p in primary prostate tissues disclosed downregulation in PCa, instead, correlating with aberrant promoter methylation. In a series of castration-resistant PCa (CRPC) cases, miR-27a-5p was upregulated, along with promoter hypomethylation. MYC and miR-27a-5p expression levels in LNCaP and PC3 cells mirrored those observed in hormone-naíve PCa and CRPC, respectively. ChIP analysis showed that miR-27a-5p expression is only regulated by c-Myc in the absence of aberrant promoter methylation. MiR-27a-5p knockdown in PC3 cells promoted cell growth, whereas miRNA forced expression in LNCaP and stable MYC-knockdown PC3 cells attenuated the malignant phenotype, suggesting a tumor suppressive role for miR-27a-5p. Furthermore, miR-27a-5p upregulation decreased EGFR/Akt1/mTOR signaling. We concluded that miR-27a-5p is positively regulated by MYC, and its silencing due to aberrant promoter methylation occurs early in prostate carcinogenesis, concomitantly with loss of MYC regulatory activity. Our results further suggest that along PCa progression, miR-27a-5p promoter becomes hypomethylated, allowing for MYC to resume its regulatory activity. However, the altered cellular context averts miR-27a-5p from successfully accomplishing its tumor suppressive function at this stage of disease.

Testicular germ cell tumours (TGCTs) are a heterogeneous group of neoplasms, mostly affecting young men. Curability rates are high and adequate treatment relies on careful and accurate pathological and clinical assessment. Indeed, TGCTs' histopathological subtyping is critical for adequate therapeutic decision. Considering the limitation of currently available serum biomarkers, novel candidates have been proposed, most notably miR-371a-3p, which outperformed classical serum markers, but no detailed information concerning TGCT subtype was available. Thus, we carried out evaluation of miR-371a-3p expression levels among TGCT subtypes using a consecutive cohort of tissue samples. MiR-371a-3p discriminated TGCTs from control tissues with high sensitivity and specificity (AUC = 0.99). Furthermore, seminomas displayed higher miR-371a-3p expression levels compared to non-seminomatous TGCTs, which also showed significant differences among them. Nonetheless, prepubertal TGCTs depicted lower miR-371a-3p expression levels than postpubertal TGCTs. Globally, miR-371a-3p expression levels decreased in parallel with progressive cell differentiation. We concluded that miR-371a-3p is TGCTs-specific and it might be clinically useful for early detection and disease monitoring. This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology’.