21 research outputs found
The PP2A regulator IER5L supports prostate cancer progression
Prostate cancer exhibits high prevalence and accounts for a high number of cancer-related deaths. The discovery and characterization of molecular determinants of aggressive prostate cancer represents an active area of research. The Immediate Early Response (IER) family of genes, which regulate Protein Phosphatase 2A (PP2A) activity, has emerged among the factors that influence cancer biology. Here, we show that the less studied member of this family, Immediate Early Response 5 like (IER5L), is upregulated in aggressive prostate cancer. Interestingly, the upregulation of IER5L expression exhibits a robust association with metastatic disease in prostate and is recapitulated in other cancer types. In line with this observation, IER5L silencing reduces foci formation, migration and invasion ability in a variety of human and murine prostate cancer cell lines. In vivo, using zebrafish and immunocompromised mouse models, we demonstrate that IER5L-silencing reduces prostate cancer tumor growth, dissemination, and metastasis. Mechanistically, we characterize the transcriptomic and proteomic landscapes of IER5L-silenced cells. This approach allowed us to identify DNA replication and monomeric G protein regulators as downstream programs of IER5L through a pathway that is consistent with the regulation of PP2A. In sum, we report the alteration of IER5L in prostate cancer and beyond and provide biological and molecular evidence of its contribution to tumor aggressiveness.The work of A. Carracedo is supported by Fundación Cris Contra el Cáncer (PR_EX_2021-22), La Caixa Foundation under the agreement LCF/PR/HR17, the Basque Department of Industry, Tourism and Trade (Elkartek), the BBVA foundation (Becas Leonardo), the MICINN (PID2019-108787RB-I00; PID2022-141553OB-I0 (FEDER/EU); Severo Ochoa Excellence Accreditation CEX2021-001136-S), European Training Networks Project (H2020-MSCA-ITN-308 2016 721532), the AECC (GCTRA18006CARR), Vencer el Cáncer Foundation, the iDIFFER network of Excellence (RED2022-134792-T) and the European Research Council (Consolidator Grant 819242). CIBERONC was co-funded with FEDER funds and funded by ISCIII. AE was supported by a Juan de la Cierva Incorporación fellowship from the MCIN/AEI /10.13039/501100011033 and European Union NextGenerationEU/PRTR. IM is supported by Fundación Cris Contra el Cáncer (PR_TPD_2020-19). RRG and MG were supported by AECC Grant proyecto GCTRA18006CARR; and MICINN and FEDER funds (CIBERONC and PID2019-104948RB-I00; PID2022-143093OB-100). JW is supported by Finnish Cancer Foundations
METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer
Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N-7-methylguanosine (m(7)G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m(7)G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m(7)G tRNA methylation in cancer cell translation control and tumour biology
METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer
Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N7-methylguanosine (m7G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m7G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m7G tRNA methylation in cancer cell translation control and tumour biology
Additional file 18 of METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer [Dataset]
Additional file 18. Graphical abstract.Consejo Superior de Investigaciones CientĂficas (España)Peer reviewe
Additional file 12 of METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer [Dataset]
Additional file 12: Supplementary Table S3. METTL1 bound RNAs in HEK293T cells.Consejo Superior de Investigaciones CientĂficas (España)Peer reviewe
Additional file 9 of METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer [Dataset]
Additional file 9: Supplementary Figure S9. Characterization of Mettl1 deletion in murine PCa. A) Schematic overview of Mettl1 systemic knockout and Mettl1flox/flox mouse model generation. B) mRNA levels of Mettl1 in prostatic tissue from five-month-old Pten-KO/Mettl1+/+, Pten-KO/Mettl1+/-, and Pten-KO/Mettl1fl/fl mice. Mean ± SD, n=3. C) 5’tRNA fragment accumulation in Pten-KO/Mettl1+/+, Pten-KO/Mettl1+/- and Pten-KO/Mettl1fl/fl tumours. The right bar chart shows means ± SD, n ≥5. D) Haematoxylin and eosin staining of Pten-KO/Mettl1+/+, Pten-KO/Mettl1+/-, and Pten-KO/Mettl1fl/fl tumour sections from the ventral, dorsal, and anterior lobes. E) Representative images of immunostained sections for METTL1 and markers of proliferation (Ki67), M1-like (iNOS) and M2-like (CD68, Arg1) macrophages, and CD8+ T cells (CD8) from Pten-KO/Mettl1+/+, Pten-KO/Mettl1+/-, and Pten-KO/Mettl1fl/fl prostate tumours. Arrows indicate positive cells. F) Prostate tumour volume from Pten-KO/Mettl1+/+ and Pten-KO/Mettl1+/- five-month-old mice reflects reduced tumour burden after Mettl1 deletion. Ventral (V), dorsal (D) and anterior (A) lobes. Mean ± SD, n≥3. G) Systemic deletion of Mettl1 resulted in reduced tumour proliferation (Ki67+ cells) and increased immune infiltration of iNOS+ (M1-like) macrophages and CD8+ T cells. Staining of tumours from Pten-KO/Mettl1+/+ and Pten-KO/Mettl1+/- five-month-old mice. Mean ± SD, n≥3, >10 images per biological replicate. H) Tumour growth of allografts derived from Pten-KO/Mettl1+/+ and Pten-KO/Mettl1flox/flox tumour cells injected into NSG male mice reflects not significant tumour formation reduction in the absence of Mettl1. Mean ± SEM, n=9. I) Prostate lobe volumes in Pten-KO/Mettl1flox/flox (fl/fl) mice and Pten-KO/Mettl1+/+ (+/+) mice treated with anti-CD8 antibodies or IgG control. Mean ± SD, (n≥6). J) Prostate lobe volumes in Pten-KO/Mettl1flox/flox (fl/fl) mice and Pten-KO/Mettl1+/+ (+/+) mice treated with anti-PD1+anti-CTLA4 antibodies or IgG control. Normal prostate lobe volumes (from wild-type mice) are also shown. The bar chart on the left shows the volume in separated lobes. The bar chart on the right shows the sum of the volume of all lobes. Mean ± SD, (fl/fl and +/+ n≥6; Normal n=5). Scale bars represent 10 mm (D), 50 ÎĽm (E). Statistical tests: One-tailed Student’s t-test (G), Mann-Whitney test (C, F, H, I, J), *p<0.05, **p<0.01, ***p<0.001.Consejo Superior de Investigaciones CientĂficas (España)Peer reviewe
Additional file 8 of METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer [Dataset]
Additional file 8: Supplementary Figure S8. Immune cell characterisation and correlation with METTL1 expression in human PCa. A) Complete cytokine array analysis of PC3 METTL1 KO compared to WT cells’ c.m. shows differential expression of inflammatory cytokines. Mean ± SD of log2 fold change, n = 4. B) tSNE analysis of the expression markers of M1-, M2-like and Mø macrophages exposed to PC3 METTL1 KO and WT cells’ c.m. of a second biological replicate (KO2 and WT2). C) Phagocytic capacity of macrophages exposed to PC3 METTL1 KO and WT cells’ c.m. Mean ± SEM, n = 3. D) Polarisation of primary peripheral blood monocytes (PMBCs) exposed to PC3 METTL1 KO and WT cells’ c.m. The upper panel shows a schematic of the workflow used. The lower panels show the quantification of the percentage of positive cells. n = 3 technical replicates of 2 biological replicates. E) Activation of T cells from a second blood donor and co-culture with macrophages exposed to PC3 WT and METTL1 KO cells’ c.m. T cells were stained with CFSE on day 0 and measured on day 3. Means ± SD, n = 3 for two biological replicates. F) Representative immunohistochemical staining for METTL1 and CD86 (M1-like macrophages), CD163 (M2-likemacrophages), and CD8 T cells in human PCa biopsies. The scale bar represents 50 ÎĽm. G, H) METTL1 mRNA expression inversely correlates with M1-like macrophage infiltration using the CIBERSORT deconvolution algorithm with TCGA and Taylor datasets (G), and TAM infiltration analysed using TIMER (H). Statistical tests: One-tailed t-test: *p < 0.05, **p < 0.01, ***p < 0.001 (D, E), Pearson’s correlation (r), p-value (pV), and linear regression with 95% confidence (bands) are shown (G, H).Consejo Superior de Investigaciones CientĂficas (España)Peer reviewe
Additional file 6 of METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer [Dataset]
Additional file 6: Supplementary Figure S6. METTL1 mediated methylation promotes cell proliferation, and tumour growth in vivo. A) Proliferation of DU145 METTL1 KO and WT cell clones. Mean ± SD, n = 6. The thick dotted line represents the average growth of the WT and KO cells. B) Proliferation of METTL1-silenced 22Rv1 cells. Mean ± SD, n = 3. C) Cell cycle analysis of PC3 METTL1 KO, WT, and parental cells showing decreased cell cycle progression in METTL1 KO cells. Mean ± SD, n = 3. The dotted lines indicate the mean of three biological replicates. D) Representative images of BrdU staining of three clones of PC3 METTL1 KO and WT cells. Quantification is shown in Fig. 5. E) Schematic representation of generation of single-cell-derived spheroids, and representative images of single-cell-derived spheroids (lower panel). Quantification is shown in Fig. 5. F) METTL1 depletion affects tumour growth in vivo. Immunohistochemical staining for METTL1, Ki67, and cleaved caspase 3 (Cl-Casp3) in PC3 METTL1 KO and WT cell-derived xenografts. Right charts show the quantification of Ki67 + and Cl-Casp3 + cells per microscopic field. Mean ± SD, n = 5, and ten images per biological replicate. G-I) Protein expression (G) and m7G methylation levels (H, I) of a second PC3 METTL1 KO cell clone (KO2) ectopically expressing an HA-tagged wild-type (WT) or catalytic dead mutant (AFPA) version of METTL1. METTL1 KO2 cells were infected with the empty vector (eV) as a control. Methylene blue staining was used as the loading control (H, bottom panel). Mean ± SD, n = 3 (H). J, K) Proliferation (J) and spheroid formation capacity (K) of PC3 METTL1 KO2 cells re-expressing METTL1 (WT) or catalytic dead mutant (AFPA) compared with METTL1 KO2 (eV). Mean ± SD, n = 6. L) WDR4 mRNA expression and cell growth of PC3 cells expressing doxycycline-inducible SCR shRNA or two shRNA against WDR4 with or without doxycycline induction. Mean ± SD are represented (n = 6). M) WDR4 mRNA expression and tumour growth of xenografts of cells expressing doxycycline-inducible SCR shRNA or a shRNA against WDR4 with or without doxycycline induction. Mean ± SEM are represented (n = 10 for shWD2, n = 5 for SCR, for each Dox condition). Scale bar represents 100 ÎĽm (D), 50 ÎĽm (F). Statistical tests: Two-way ANOVA (A, I, J), one-way ANOVA (C), and one-tailed Student’s t-test (B, F, K, L, M). **p < 0.01, ***p < 0.001, ****p < 0.0001.Consejo Superior de Investigaciones CientĂficas (España)Peer reviewe
Additional file 17 of METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer [Dataset]
Additional file 17: Supplementary Table S8. Correlation of differentially expressed mRNAs and newly synthesized proteins in WT and METTL1 KO PC3 cells.Consejo Superior de Investigaciones CientĂficas (España)Peer reviewe
Additional file 3 of METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer [Dataset]
Additional file 3: Supplementary Figure S3. tRNAs are methylated at guanine-7 by METTL1. A) Western blot showing the expression of doxycycline-inducible HA-METTL1 in PC3 cells. B, C) Density distribution of reads per million (RPM) identified after PAR-CLIP analysis of METTL1-bound tRNAs (B) and other RNA species (C) in PC3 (control) and HA-METTL1 expressing PC3 cells. The red line indicates the third lower quartile of total RPMs. D) tRNAs are the most common RNA species that bind Flag-METTL1 in HEK293T cells. Density distribution of reads per million (RPM) identified after PAR-CLIP analysis of METTL1-bound RNAs: The red line indicates the third lower quartile of total RPMs. Data were retrieved from Bao et al. study. E) tRNAs comprise half of the reads of all RNAs bound to METTL1 after PAR-CLIP analysis in HEK293 cells. F) Boxplot representing the median with interquartile range of reads per million (RPM) per transcript bound to METTL1 in HEK293T cells. G) Schematic representation of genomic editing introduced by CRISPR-Cas9 technology in the PC3 METTL1 KO clones used in this study. H) METTL1 mRNA expression levels in METTL1 KO, and WT control clones used in this study. Mean ± SD, n = 3. I) Graphics representing the experimental workflow followed to generate AlkAniline-seq libraries. J) Normalised cleavage signals for METTL1-methylated tRNAs Cys and Ile, and METTL1 non-methylated tRNAs His and Glu in PC3 WT and METTL1 KO cells.Consejo Superior de Investigaciones Cientificas (CSIC)Peer reviewe