Fig 2: METTL1 is upregulated in prostate cancer. A A schematic overview of the research workflow used to identify altered expression of RMPs associated with PCa. B Heatmap of average Z-scores of mRNA expression values in human primary (P) and metastatic (M) PCa samples compared to healthy tissue for significant differentially expressed RNA-modifying enzymes reveals METTL1 as the most overexpressed RMPs in PCa. Data from are from Grasso et al. [7] (n = N: 12, P: 49, M: 27); Taylor et al. [6] (n = N: 29, P: 131, M: 19); Varambally et al. [42] (n = N: 6, P: 7, M: 6); Lapointe et al. [44] (n = N: 9, P: 13, M: 4); Tomlins et al. [43] (n = N: 23, P: 32, M: 20) (left panel) datasets. Z-score averages for all datasets are also shown as “Aver”. Z-score values are indicated using numeric values. Grey Z-score values indicate no significant p-value. The right heatmap shows the average log2 fold change in mRNA expression values in Pten-cKO mice with prostate intraepithelial neoplasia (PIN) and invasive prostate carcinoma (Inv) compared to normal prostate tissue (right panel) (n = 4). C METTL1 and WDR4 expression are increased in primary (PT) and metastatic tumours (M) compared to normal tissues (N). Data are from Grasso et al. [7], Taylor et al. [6], and Varambally et al. [42] datasets. Log2-normalised gene expression values are shown. D High expression of METTL1 but not WDR4 is associated with poor patient prognosis. Kaplan–Meier curves representing biochemical recurrence-free survival (DFS) of patient groups selected according to gene expression, data from the Cambridge, Stockholm and Taylor cohorts [6, 46]. Data were retrieved from the camcAPP [48] and cBioportal. E Western blot from benign prostatic hyperplasia (BPH) (n = 7) and PCa patient samples (n = 14) (upper panel) and correlation analysis between METTL1 and WDR4 expression, AR and phospho-S6K (right panels). Statistical tests: ANOVA test (B, C), and log-rank Cox test (D). Data are represented as mean ± standard deviation (SD). Student’s t-test and Spearman’s correlation test (E)

Fig 3: WDR4 promotes the metastasis of bladder cancer cells in vitro and in vivo.A–F Cells were transfected with WDR4-specific siRNAs or the control siRNA. Interference with WDR4 expression significantly reduced the migration of UM-UC-3 (A, C) and 5637 (B, D) cells in the wound healing assay and the migration and invasion of UM-UC-3 (E) and 5637 (F) cells in the Transwell assays. WDR4 overexpression promoted the migration of UM-UC-3 cells in the wound healing assay (G, H) and migration and invasion in the Transwell assays (I). J Representative image of the popliteal LN metastasis mouse model. K Representative bioluminescence images of mice with LN metastases generated by WDR4 knockdown (WDR4 KD) and control knockdown (Control KD) UM-UC-3 cells. Representative image of excised popliteal LNs (L) and histogram of LN volumes in control KD and WDR4 KD mice (n = 6 per group) (M). N Hematoxylin-eosin (HE) staining of LNs from control KD and WDR4 KD mice. O Representative bioluminescence images of mice with LN metastases generated by WDR4 overexpression (WDR4) and control (Vector) UM-UC-3 cells. Representative image of excised popliteal LNs (P) and histogram of LN volumes in the vector and WDR4 groups (n = 6 per group) (Q). The data are presented as the means ± SDs, ***p < 0.001. R HE staining of LNs from the control vector and WDR4 overexpression groups. Scale bar of upper panel = 500 μm, Scale bar of lower panel = 50 μm.

Fig 4: WDR4 regulates bladder cancer progression by transcriptional repression of ARRB2.A Egr1-binding sites in the promoter of the ARRB2 gene; the three corresponding mutation sites are marked with red vertical lines. B After Egr1 knockdown or Egr1 overexpression in UM-UC-3 cells, the mRNA levels of ARRB2 were measured by RT‒qPCR. C The protein levels of ARRB2 were measured by WB. D The Egr1 overexpressing plasmid and the reporter plasmids containing the wild-type (WT) or mutated (MUT) of Egr1 binding sequence in the ARRB2 promoter were cotransfected into 293 T cells. A luciferase assay was performed to detect changes in relative luciferase activity. E Co-IP showed the interaction between endogenous WDR4 and Egr1 in UM-UC-3 and 5637 cells. F Co-IP showed the interaction between endogenous DDX20 and Egr1 in UM-UC-3 and 5637 cells. G IF staining and confocal observation of Egr1, DDX20 and WDR4 in tissue sections from bladder cancer patients. Scale bar 20 μm. H Co-IP showed the interaction between endogenous DDX20, WDR4 and Egr1 in WDR4 KD and Control KD UM-UC-3 cells. The data are presented as the means ± SDs, ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: Clinical significance of METTL1 and WDR4 upregulation in BC cells. (A) RT-qPCR data showing the relative expression levels of METTL1 and WDR4 in MCF7 and MCF10A cells. (B, C) IHC staining for METTL1 and WDR4 in BC TMAs. Scale bars: left, 200 μm; right, 40 μm. (D, E) Comparison of METTL1- and WDR4-staining scores at different BC stages. (F, G) Kaplan–Meier curves displaying the differences in DFS rates between patients classified based on their IHC scores for METTL1 and WDR4 expression. ns, not significant *p< 0.05; **p< 0.01; ***p< 0.001.