Upregulation of METTL3 in PCa correlates with poor survival

Recently, we have comprehensively dissected, genomically and transcriptomically, the m6A pathway (24 genes) as a whole in PCa using TCGA cohort [22]. We found that many of the m6A pathway genes were upregulated, while a few genes (including eraser FTO) downregulated, in primary PCa (pri-PCa) versus (vs.) normal tissues [22], indicating an overactivation of m6A pathway. To further investigate the potential roles of m6A modification in PCa evolution in detail, we focused on METTL3 as it’s the only enzymatic subunit in the Writer complex. Analysis of METTL3 mRNA levels in Oncomine database revealed that METTL3 was overexpressed in pri-PCa vs. normal/benign prostate tissues (Fig. 1A). This result was further confirmed by analyzing both the curated [23] and noncurated (i.e., provisional) TCGA cohorts (Fig. 1B). Interestingly, examination of large clinical RNA-seq datasets indicated a further increase of METTL3 levels in CRPC va. pri-PCa (Fig. 1C). In an attempt to understand the molecular basis underpinning METTL3’s dysregulation in PCa, we surveyed its mutational landscape. Although we did see an increase in METTL3 alteration in CRPC vs. pri-PCa cohorts, it was in total mutated at a very low frequency (Fig. S1A), suggesting that its mis-expression was not due to genomic alterations. Clinically, a strong positive correlation between METTL3 expression and tumor Gleason Score (GS) was observed (Fig. 1D), consistent with a gradual upregulation of METTL3 in GS high (> 7) vs. low (≤ 7) tumors (Fig. 1B). Also, the METTL3 mRNA levels adversely associated with PCa patients’ overall survival in multiple datasets (Fig. 1E). Experimentally, we determined METTL3 expression at both protein and mRNA levels in a panel of prostate normal and cancerous cell lines, finding a general overexpression in PCa cells vs. immortalized normal prostatic epithelial cells RWPE1 (Fig. S1B). Altogether, our data established a potential oncogenic role of METTL3 in PCa initiation and castration-resistant progression.

Fig. 1

Increased METTL3 expression in PCa correlates with multiple oncogenic pathways and worse patient survival. (A) Oncomine analysis showing increased METTL3 expression in PCa vs. normal or benign tissues. The bottom illustrates the details in three representative datasets. (B) Overexpression of METTL3 at mRNA level in both curated (2015 Cell) and noncurated pan-cancer PCa TCGA cohorts. (C) Comparison of METTL3 expression reveals an upregulation in CRPC vs. pri-PCa samples. D and E. High METTL3 mRNA levels correlate with increase GS (D) and worse patient overall survival (E) in indicated datasets. Survival p-value was determined using the Log-Rank test. F. GSEA of genes co-expressed with METTL3 in curated pri-PCa (TCGA) and CRPC (SU2C-PCF) cohorts. G-I. Fractionation of TCGA pri-PCa cohort (2015 Cell) into METTL3 high and low groups, and comparison of METTL3 expression (G, up), GS (G, bottom), patient’s survival outcome (H), and splicing landscape (I) showing METTL3 high group being more aggressive

A protumorigenic role of METTL3 is associated with multiple cancer-related pathways

Many roles of METTL3-mediated m6A signaling have been proposed in development and cancer [3, 4]. To comprehensively dissect the molecular functions of METTL3 in PCa, we performed a gene-coexpression analysis, coupled with gene set enrichment analysis (GSEA), to identify biological gene signatures and pathways that may be regulated by METTL3. Both curated TCGA pri-PCa [23] and CRPC [24] cohorts were examined and similar repertoires of biological pathways were identified (Fig. 1F and Table S1), indicating conserved roles of METTL3 in both treatment-naïve and treatment-resistant PCa. Globally, METTL3 positively regulated proliferation (evidenced by enrichment of pathways tied to cell cycle progression and DNA repair), MYC and PCa-related signatures, RNA metabolism (such as transcription and splicing), and stemness to eventually promote cancer development and progression (Fig. 1F and Fig. S1C). Supporting our analyses, the involvement of m6A pathway in all these processes have been documented in diverse biological contexts [4, 25, 26]. For example, it has been reported that METTL3 plays an oncogenic role in multiple cancer types [25] by regulating tumor stemness [27] and RNA stability and splicing [28, 29], among other mechanisms. Conversely, genes negatively correlated with METTL3 expression were enriched in pathways associated with extracellular matrix (ECM) and cell adhesion, differentiation, stress response and/or anti-tumor immunity (Fig. 1F and Table S1), indicating a cancer/metastasis-inhibiting function. To further corroborate these results, we fractionated TCGA pri-PCa cohort into two extremes (Fig. 1G) and asked whether high or low METTL3 levels in PCa was indeed associated with tumor aggressiveness. Expectedly, tumors expressing highly (vs. lowly) the METTL3 exhibited elevated GS (Fig. 1G, bottom), worse survival outcome (Fig. 1H), and distinct splicing landscapes (Fig. 1I). The total dysregulated splicing events (5.77-fold) and intron retention (IR; 31.62-fold) were specifically upregulated in the METTL3 high group (Fig. 1I). We have recently shown that the severity of RNA splicing disruption correlates with increased aggressiveness [30]. Collectively, higher METTL3 expression predicted a molecularly aggressive phenotype in PCa, consistent with recent reports [18, 21].

Attenuation of METTL3 inhibits PCa progression in vitro and in vivo

A critical role of METTL3 in mobility of AR− CRPC cells (i.e., DU145 and PC3) has been reported recently [21]. To comprehensively dissect the roles of METTL3-mediated m6A pathway in both AR+ and AR− cells, we knocked down METTL3 in LNCaP and PC3 cells. Two independent shRNA-mediated depletion of METTL3 (Fig. 2A and Fig. S2A) significantly reduced the total m6A levels in PCa cells (Fig. 2B), and concurrently led to a remarkable impairment of cell growth in these lines as evidenced by the colony formation assay (Fig. 2C). Moreover, trans-well (Fig. 2D) and wound-healing (Fig. 2E) assays revealed that attenuation of METTL3 significantly decreased cell mobility in PC3 and DU145 cells. Quantitation of apoptosis via Annexin V staining indicated that loss of METTL3 caused a significant increase in apoptosis (Fig. 2F and Fig. S2B) in both AR+ LNCaP and AR− lines, validating the reduced proliferation phenotype (Fig. 2C). Moreover, knockdown (KD) of METTL3 significantly inhibited PCa sphere formation (Fig. 2G), as well as the sphere size (Fig. S2C and Fig. S2D), indicating a requirement of METTL3 for optimal PCa stemness. Alternatively, we obtained similar results by depleting METTL3 with two independent siRNAs in PCa cells. Briefly, siRNA treatment significantly reduced METTL3 expression both at mRNA (Fig. S2E) and protein (Fig. S2F) levels, leading to a global decrease in RNA m6A methylation (Fig. S2G), clonal development capacity (Fig. S2H), viability (Fig. S2I), and migration (Fig. S2J) in both DU145 and PC3 cells. All these abovementioned in vitro assays strongly established a protumorigenic role of METTL3 in PCa. To next demonstrate the role of METTL3 in vivo, we generated subcutaneous tumors in BALB/c athymic nu−/nu− (nude) male mice implanted with PCa cells stably expressing control or METTL3-targeting shRNAs. As shown in Fig. 2H, METTL3 KD inhibited the growth of both PC3 and DU145 xenograft models. Quantitative real-time RT-PCR (qPCR; Fig. S2K) and immunohistochemistry analysis (Fig. S2L) confirmed the depletion of METTL3 in these tumors. Collectively, these data convincedly demonstrated that METTL3 is oncogenic in PCa development and progression.

Fig. 2

Knock-down of METTL3 significantly inhibits PCa progression in vitro and in vivo.A. Western blot analysis showing METTL3 abundance in three PCa cell lines transduced with scrambled (shNC) or METTL3-specific hairpin shRNAs (sh718 and sh812) and probed with indicated antibodies. GAPDH served as a loading control. B. Dot blot assay showing the global m6A levels in indicated cells treated with different shRNAs. Methylene blue (MB) staining served as a loading control. C. Knocking down METTL3 inhibits clonal development in indicated PCa cells. Representative images and relative quantification are shown for each cell line. Experimental details are as follows: LNCaP (30 K/well for 14 days), DU145 (8 K/well for 9 days), PC3 (5 K/well for 9 days). D and E. Cell mobility evaluated by Trans-well (40 K PC3 per well for 24 h and 70 K DU145 per well for 30 h; D) and wound-healing (24 h for PC3 cells and 36 h for DU145 cells; E) assays showing that METTL3 knock-down inhibits cancer cell migration. F. Flow cytometry analysis showing an increased cellular apoptosis in indicated PCa cell lines upon METTL3 depletion. G. Knocking down METTL3 inhibits sphere formation in both AR + and AR- PCa cell lines. H. Depletion of METTL3 inhibits the growth of PC3 and DU145 xenograft tumors in vivo (n = 5 for each group). Shown are the tumor growth curves (left), endpoint tumor images (middle), and tumor weight (right) of indicated PCa models

Interestingly, we noticed that, in multiple assays (e.g., Fig. 2G and Fig. S2I), DU145 (vs. PC3) cells appeared to be more susceptible to METTL3 loss, especially in the tumor regeneration assay (Fig. 2H). Although both cell lines are AR−, PC3 is PTEN-null whereas DU145 expresses wide-type (WT) PTEN. We next investigated whether tumor suppressor PTEN plays a role in dictating biological response of these two lines to METTL3 depletion. Recently, there were two studies providing indirect link between METTL3/m6A signaling and PTEN regulation. Li et al. reported that METTL3-mediated m6A modification of HOXC10 in liver cancer promoted its expression, which in turn suppressed the transcription of PTEN [31]. In Lu et al. study, both METTL3 and m6A methylation were found markedly upregulated in lung resident mesenchymal stem cells (LR-MSCs), leading to an aberrant differentiation of LR-MSCs into myofibroblasts [32]. Molecularly, METTL3 binds the primary transcripts of miR21 (pri-miR21) to promote its m6A modification and maturation in LR-MSCs, causing elevation of mature miR-21. Resultantly, miR-21 targets PTEN mRNA for expression inhibition [32]. Notably, there was another study providing direct evidence that METTL3 regulates PTEN transcripts in prostate tissue. Li et al. indicated that METTL3 was aberrant upregulated in benign prostatic hyperplasia (BPH) samples and methylated PTEN mRNA for degradation through reader YTHDF2 [33].

Examination of gene expression changes in PTEN− PC3 cells upon METTL3 KD showed that PTEN and the two reported effectors (HOXC10 and miR-21) were not affected transcriptionally (Fig. S3A). Interestingly, in PTEN+ DU145 cells, METTL3 KD further increased the expression of PTEN but without affecting HOXC10 and miR-21 (Fig. S3B). Western blot assay confirmed the upregulation of PTEN protein only in DU145 cells upon METTL3 depletion (Fig. S3C). These data indicated that METTL3 loss-mediated inhibition of DU145 fitness might be, at least partially, through upregulation of PTEN. Next, we performed functional rescue experiments to see whether restoration of PTEN would sensitize PC3 cells to METTL3 loss. Overexpression (OE) of PTEN in METTL3-depleted cells significantly increased the expression of PTEN but not METTL3 (Fig. S3D). Functional colony formation (measuring cell growth; Fig. S3E) and sphere assay (measuring cancer stemness; Fig. S3F) showed that while METTL3 KD cells infected with or without Control-OE lentivirus displayed similar inhibitory effect on cell aggressiveness (comparing lane 2 and 3), PTEN-OE in PC3 cells further exacerbated the cell growth inhibition upon METTL3 KD (comparing lane 3 and 4). Importantly, the inhibition rates in colony formation and sphere assays increased from average 35% to 53% and 28% to 49%, respectively, in METTL3 KD PC3 cells without or with PTEN-OE (Fig. S3E and S3F). These data, together, strongly indicated that restoration of PTEN in PC3 made it more vulnerable to METTL3 loss. To further validate this idea that PTEN is functionally involved in the METTL3-mediated oncogenic effects in PCa, we also performed the same set of experiments in PTEN+ DU145 cells. As expected, similar results were observed, as PTEN-OE only increased the expression of PTEN but not the METTL3 mRNAs (Fig. S3G) and further sensitized DU145 to METTL3 KD in both colony formation (Fig. S3H) and sphere assay (Fig. S3I). Of clinical relevance, we analyzed the largest pri-PCa [23] and CRPC [34] cohorts to reveal that METTL3 is negatively correlated with PTEN at mRNA level in pri-PCa (spearman ρ = -0.22) samples (Fig. S3J). No correlation in CRPC samples was found (Fig. S3J), which was explainable by the fact that PTEN is frequently lost or mutated in CRPC patients (> 33% mutation rate; Fig. S3K). Altogether, our data established PTEN as a determinant in PCa cells with differential responses to METTL3 loss, in line with the previous report that PTEN is a downstream target of METTL3/m6A axis in prostate tissue [33].

METTL3 promotes prostate tumorigenesis in an m6A-dependent manner

METTL3 forms an obligate heterodimer with METTL14 to exert its methyltransferase activity [3]. To determine whether METTL3’s function of accelerating PCa aggressiveness was dependent on its m6A catalytic activity, we successfully established stable cell lines expressing WT METTL3 or its catalytic-dead mutant (aa395-398, DPPW→APPA). A lack of methyltransferase activity of this mutant has been previously described [35, 36]. Overexpression of the WT, but not the mutant, METTL3 (Fig. 3A) resulted in a significant increase in global m6A levels in both DU145 and PC3 cells by dot-blot assay (Fig. 3B), which was further confirmed by quantification of RNA methylation status via EpiQuik m6A Quantification Kit (Fig. 3C). A series of cell-based assays in two different PCa cell lines showed that only transduction of WT (compared to the mock or mutant-expressing) METTL3 lentivirus further promoted cell clonal development (Fig. 3D), proliferation (cell counting; Fig. 3E), and migration ability (trans-well assay; Fig. 3F). Importantly, xenograft tumor assay indicated that the catalytic-dead METTL3 (vs. WT) failed to promote DU145 tumor development in vivo, as evidenced by indistinguishable difference in tumor growth dynamics (Fig. 3G) and weight (Fig. 3H) between mutant and control groups. Collectively, these results strongly indicated that the m6A catalytic activity of METTL3 is required for its role in advancing prostate tumorigenesis.

Fig. 3

METTL3 overexpression promotes PCa progression in a m6A-dependent way. (A) Western blot analysis showing overexpression of WT or catalytically dead (i.e., mutant) METTL3 in PCa cells. GAPDH served as a loading control. (B) Dot blot assay showing the global m6A levels in indicated cells treated with different overexpressing constructs. Methylene blue (MB) staining served as a loading control. (C) Relative m6A levels in indicated conditions detected via EpiQuik m6A quantification kit. D-F. Overexpression of WT, but not the mutant, METTL3 promotes CRPC clonal development (D), proliferation (E), and Trans-well migration (F) in both DU145 and PC3 cells in vitro. G and H. The m6A enzymatic activity is required for METTL3 in enhancing PCa development in vivo. Shown are the tumor growth curves (G), endpoint tumor images and tumor weight (H) of DU145 model. In D, 5 K PC3 and 8 K DU145 were initially seeded and visualized at day 10. In F, 40 K PC3 per well for 24 h and 70 K DU145 per well for 30 h were used

Identification of global METTL3 targets in PCa cells

To directly unravel the mechanisms of action of METTL3 in PCa cells, we utilized a multi-omics approach. We first performed RNA-seq analysis in PC3 cells with or without METTL3-KD, finding a set of 177 differentially expressed genes (DEGs; 66 up- and 111 down-regulated) (Fig. 4A and Table S2). Gene ontology (GO) analysis indicated that genes upregulated in METTL3-KD cells were enriched in pathways tied to inflammation/immunity and cell death (Fig. 4B), whereas genes downregulated were enriched in pathways associated with migration/adhesion, differentiation and proliferation, in line with an oncogenic role of METTL3. The m6A writer complex binds RNA to decorate m6A modification [26]. Next, we performed RIP-seq experiments in both AR+ LNCaP and AR− PC3 cells with an METTL3-specific antibody. As shown in Figs. 4C, 382 and 738 potential METTL3 target genes were identified in PC3 and LNCaP, respectively, with 849 genes being shared between these two cell lines (Fig. 4D and Table S3). Consistently, GO analysis of genes bound by METTL3 revealed, overall, a similar pattern of biological pathways enriched in LNCaP and PC3 cells (Fig. 4E), highlighting that METTL3 may play similar functional roles in both AR+ and AR− cells. To further identify genes that were directly regulated by METTL3 in PCa cells, we integrated the RNA-seq and RIP-seq data by overlapping DEGs and genes bound by METTL3 protein (Fig. 4D), and identified 11 key genes (Fig. 4F). Among them, SYNE2 [37] and HEG1 [38] have been reported to be regulated by m6A modification in different context, indicating validity of our data. We chose RRBP1 (reticulum ribosome-binding protein 1) for further investigation as it showed the most significant reduction in mRNA expression upon METTL3-KD.

Fig. 4

Identification of the genome-wide METTL3 targets in PCa cells. (A) Heatmap of differentially expressed genes (DEGs) identified in PC3 cells by RNA-seq (fold change (FC) ≥ 1.5 and FDR < 0.1). (B) GO analysis of DEGs as shown in A. (C) Volcano plot of METTL3-bound genes identified in AR + LNCaP and AR− PC3 cells by RIP-seq (FC ≥ 2 and FDR < 0.1). (D) Overlap between DEGs and METTL3-bound genes in indicated contexts. (E) GO analysis of METTL3-bound genes showing that a significant proportion of enriched pathways are commonly enriched in both LNCaP and PC3 cells. (F) List of the 11 overlapped genes from D

METTL3 accelerates PCa progression through upregulating oncogenic RRBP1

In line with an upregulation of METTL3 in pri-PCa and CRPC, similar pattern was found for RRBP1 mRNA levels (Fig. 5A), implying a positive relationship between them. qPCR analysis confirmed a reduction in RRBP1 expression at both mRNA (Fig. 5B) and protein (Fig. 5C) levels upon METTL3-KD. Notably, the enzymatic activity of METTL3 was required for this phenotype, as only the exogenous expression of WT, but not the mutant, METTL3 strongly upregulated RRBP1 protein expression (Fig. 5D). To further demonstrate RRBP1 as a direct substrate bound by METTL3, we performed RIP-qPCR to reveal that METTL3 strongly bound RRBP1 transcripts in both DU145 and PC3 cells (Fig. 5E) and the m6A modification on RRBP1 mRNA by m6A-qPCR was significantly reduced upon METTL3-KD (Fig. 5F). Analysis of RRBP1 sequences and METTL3 RIP-seq peaks unraveled multiple consensus m6A motifs (RRACH) in its 3’-UTR region (Fig. 5G). Mutagenesis assay was performed with luciferase (Luc) reporters containing either a WT or a mutated (MUT) 3’-UTR placed after the coding region of a firefly luciferase. Results indicated that the luciferase activity of WT, but not the MUT, reporter was significantly reduced in DU145 cells when METTL3 was knocked down (vs. shNC control) (Fig. 5H), suggesting a positive regulation of m6A on RRBP1 mRNA. To further dissect the fate of RRBP1 mRNA upon m6A methylation, we determined its mRNA stability after transcriptional inhibition with Actinomycin D. Attenuation of METTL3 markedly accelerated RRBP1 mRNA decay in both DU145 and PC3 cells (Fig. 5I), suggesting that METTL3 upregulated RRBP1 through mRNA stability in a m6A-dependent manner. Altogether, these results established RRBP1 as a direct target of METTL3 in PCa cells.

Fig. 5

Oncogenic RRBP1 is a direct and functional target of METTL3-mediated m6A signaling in promoting PCa aggressiveness. A. Overexpression of RRBP1 at mRNA level during PCa progression, as analyzed in TCGA (pri-PCa vs. N) and CRPC (vs. pri-PCa) cohorts. B. qPCR analysis showing a reduced RRBP1 expression in PC3 and DU145 cells upon METTL3 depletion. C and D. Western blot analysis of RRBP1 protein levels in indicated PCa cells with METTL3-KD (C) or METTL3 overexpression (D). Tubulin served as a loading control. E. RIP-qPCR analysis showing an enrichment of METTL3 binding at RRBP1 transcripts in PCa cells. F. m6A-qPCR analysis showing much reduced m6A modifications in RRBP1 transcripts in PCa cells upon METTL3-KD. G. Schematic of the potential m6A sites in the RRBP1 3’-UTR. Shown below is the experimental design of constructing luciferase reporters containing WT or mutant 3’-UTR sequences of RRBP1.H. Luciferase reporter assay using the WT or mutated 3’-UTR constructs in DU145 cells with or without METTL3-KD. The firefly luciferase activity was normalized to Renilla luciferase activity. I. mRNA stability assay showing the kinetics of RRBP1 expression in PCa cells with or without METTL3-KD after treatment with actinomycin D (10 µg/mL) for indicated time points. J. Efficient siRNA-mediated KD of RRBP1 both at the mRNA levels (left) and protein levels (right) in DU145 cells. Tubulin served as a loading control. K and L. Knocking down RRBP1 inhibits clonal development (8 K/well for 10 days, K) and migration (50 K/well for 30 h, L) in DU145 cells. M-O. RRBP1 is a key downstream effector of METTL3/m6A pathway in PCa. Reducing the RRBP1 protein into baseline level (M) significantly counteracts the pro-proliferative (N) and pro-migratory (O) effects of METTL3 overexpression in PCa cells. Experiments were performed in METTL3 overexpressing DU145 cells treated with or without siRNA targeting RRBP1. Empty vector-overexpressing cells (OE-NC) transfected with siNC was used as a baseline control

Next, we investigated the role of RRBP1 in PCa. siRNA-mediated KD of RRBP1 significantly reduced its expression at both mRNA and protein levels (Fig. 5J), and concurrently suppressed cell proliferation (Fig. 5K) and migration (Fig. 5L). These results suggested an oncogenic role for RRBP1 in PCa progression. To further test whether RRBP1 was a key downstream effector of METTL3-mediated m6A pathway, we performed RRBP1-KD experiments in METTL3-overexpresssing PCa cells. Our data clearly showed that reducing the RRBP1 protein expression to a baseline level (comparable to cells without METTL3 overexpression; Fig. 5M) significantly counteracted the pro-proliferative (Fig. 5N) and pro-migratory (Fig. 5O) effects of METTL3 overexpression in DU145 cells. Altogether, our findings highlighted that METTL3 accelerates PCa progression via targeting RRBP1 mRNA for stabilization in a m6A-dependent manner.

An m6A landscape in human PCa tissues reveals clinical significance of the METTL3/RRBP1 axis

A global m6A landscape in clinical PCa samples is lacking, although there are few studies that have identified multiple METTL3 targets at individual gene basis, with the aid of general m6A methylation patterns in particular PCa cell lines. Here, we for the first time performed m6A-seq on two pairs of matched cancer/paracancer tissues. By HOMER algorithm, we showed that transcripts pulled-down by an m6A-specific antibody markedly enriched for the known m6A consensus RRACH motif (Fig. 6A), validating our experimental pipeline. Moreover, m6A peaks were predominately located in the vicinity of 5’UTR to 1st Exon and CDS to 3’UTR regions (Fig. 6B), similar to previously reported m6A maps generated in other tissues [39]. No obvious difference was found in the global m6A distribution pattern among cancer vs. paracancer tissues (Fig. 6C). Pair-wise comparison of m6A peaks in tumor vs. nontumor tissues identified 5180 methylated peaks in 2,910 genes and 3710 peaks in 2025 genes for patient 1 and 4, respectively (Fig. 6D and Table S4). To determine the cellular pathways that m6A might influence in clinical PCa, we conducted GO analysis on genes containing differential m6A peaks. Globally, m6A regulated mRNAs encoded a variety of pathways linked to a spectrum of important biological functions including development, cell adhesion and ECM (Fig. 6D), consistent with reported diverse roles of m6A pathway [3]. Specifically, besides that several GO terms were commonly enriched in two patients, a large part of enriched GO terms was still quite distinct, suggesting heterogeneity of m6A landscape among PCa patients. This is in line with the intrinsic property of heterogeneity of PCa. Our data therefore provides a useful resource for further mining m6A pattern in clinical PCa.

Fig. 6

The clinical significance of the METTL3/RRBP1 axis in human PCa. A. Two paris of matched PCa and paracancer tissues were used for m6A-seq analysis. Shown are the concesus m6A RRACH motifs identified in two clinical prostate tumor samples. B and C. Statistics of m6A-seq peaks. Shown are the profiles of m6A peak density along mRNA transcript (B) and the genomic peak locations (C). D. GO analysis of genes bearing differential m6A peaks in tumor vs. nontumor tissues. Shown are the top 20 enriched pathways in two patient tumors, with a small proportion of GO pathways being commonly shared. E. Survival analysis of TCGA pan-cancer cohort based on genes that were co-upregulated or co-downregulated in both mRNA expression and m6A levels in tumor tissues. F. Distribution and differential enrichment of m6A peaks across RRBP1 transcripts in tumor (T) and normal (N) samples. G. Positive correlations between RRBP1 and METTL3 mRNA levels in TCGA pri-PCa (left) and CRPC (right) cohorts

To further demonstrate the clinical relevance of RNA m6A modification in PCa, we established signatures denoting to genes co-upregulated or co-downregulated in both mRNA expression and m