Fig. 2

PI3K-AKT-mTORC pathway mediate upregulation of METTL1 expression in PCa. A Correlation analysis between METTL1 and PTEN expression in human primary prostate tumours expression datasets. Plotted values correspond to the log₂-normalised gene expression values for each patient in the indicated dataset. The black line represents linear regression, grey area indicates the limits of the confidence intervals. Pearson’s correlation coefficient (R) and p-values are indicated. Grasso n = 88; Taylor n = 183; TCGA n = 497. B, C METTL1 expression is regulated downstream of AKT signalling. Western blot (B) and RT-qPCR (C) analyses of METTL1 expression upon PI3K pathway inhibition in DU145 cells. DMSO as vehicle (Veh), BKM-120 (BKM) as pan-PI3K inhibitor, MK2206 (MK) as AKT inhibitor, rapamycin (RAPA) as mTORC1 inhibitor, and Torin (TOR) as mTORC1/2 inhibitor. For western blotting, cells were treated for 48 h, and for RT-qPCR, for 8 h. Means ± SD are shown (n = 2) (B) and (n = 6) (C). D Stratification of patients with a worse prognosis according to high METTL1 levels and low PTEN expression. Kaplan–Meier curves representing the disease-free survival (DFS) of patient groups selected according to Q1 (Pten^L) and Q4 (Pten^H) quartile expression of PTEN, and METTL1 high (Met^H, log₂-normalised expression > 8.72) and METTL1 low (Met^L, log₂-normalised expression < 8.72) in recurrent and disease-free (DF) tumour samples from the TCGA dataset. E–G Mettl1 is highly expressed in mouse prostate tumours. Mettl1 expression analysis in Pten-KO mice at 3 and 6 months of age compared to wild-type mice (WT) at the same ages by western blot (E, F) and RT-qPCR (G). * in (E) indicates an unspecific band. Means ± SD are shown for three (F) and five replicates (G). H, J Increased 7-guanine tRNA methylation in RNA from prostate tumours compared with that in normal prostate. North-dot blot of m⁷G levels in long RNAs (> 200 nucleotides long RNAs) and tRNAs extracted from prostatic tissue of Pten-KO mice with intraepithelial prostatic neoplasia (at 3 months of age) or invasive tumour (at 6 months), and wild-type (WT) mice of the same age. Methylene blue staining was used as the loading control (H, bottom panel). Means ± SD are represented (n = 4) (J). I, K Increased 7-guanine tRNA methylation in the urine of mice bearing PCa tumours. North-dot blot of m.⁷G levels from the urines of Pten-KO mice with invasive tumour (at 6 months), and wild-type (WT) mice of the same age. Means ± SD are shown (n > 3). L Representative images of immunostained sections for Mettl1 and markers for luminal (AR), and basal (K14) cells in Pten-KO prostates (anterior lobes) at initiation (3 m) and in invasive carcinoma (6 m) and in age-matched wild-type (WT) prostates. Scale bars represent 50 μm. Statistical tests: one-tailed Student’s t-test (C), log-rank test (D), Mann–Whitney test (F, G, J, K). *p < 0.05, **p < 0.01, ***p < 0.001