Reduced symmetric dimethylation stabilizes vimentin and promotes metastasis in MTAP‐ deficient lung cancer
2022; Springer Nature; Volume: 23; Issue: 8 Linguagem: Inglês
10.15252/embr.202154265
ISSN1469-3178
AutoresWen‐Hsin Chang, Yi‐Ju Chen, Yi‐Jing Hsiao, Ching‐Cheng Chiang, C Y Wang, Ya‐Ling Chang, Qi‐Sheng Hong, Chien–Yu Lin, Shr‐Uen Lin, Gee‐Chen Chang, Hsuan‐Yu Chen, Yu‐Ju Chen, Ching‐Hsien Chen, Pan‐Chyr Yang, Sung‐Liang Yu,
Tópico(s)Ubiquitin and proteasome pathways
ResumoArticle29 June 2022Open Access Source DataTransparent process Reduced symmetric dimethylation stabilizes vimentin and promotes metastasis in MTAP-deficient lung cancer Wen-Hsin Chang Wen-Hsin Chang orcid.org/0000-0002-8027-302X Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Yi-Ju Chen Yi-Ju Chen orcid.org/0000-0002-7203-9188 Institute of Chemistry, Academia Sinica, Taipei, Taiwan Contribution: Data curation, Formal analysis, Investigation, Visualization, Methodology Search for more papers by this author Yi-Jing Hsiao Yi-Jing Hsiao orcid.org/0000-0001-8780-8800 Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan Contribution: Formal analysis, Validation, Investigation, Methodology Search for more papers by this author Ching-Cheng Chiang Ching-Cheng Chiang Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan Contribution: Formal analysis, Validation, Investigation Search for more papers by this author Chia-Yu Wang Chia-Yu Wang orcid.org/0000-0002-0324-3302 Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan Contribution: Formal analysis, Validation, Investigation Search for more papers by this author Ya-Ling Chang Ya-Ling Chang orcid.org/0000-0003-0240-2647 Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan Contribution: Investigation Search for more papers by this author Qi-Sheng Hong Qi-Sheng Hong Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan Contribution: Investigation Search for more papers by this author Chien-Yu Lin Chien-Yu Lin Institute of Statistical Science, Academia Sinica, Taipei, Taiwan Contribution: Formal analysis Search for more papers by this author Shr-Uen Lin Shr-Uen Lin orcid.org/0000-0001-9266-387X Graduate Institute of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan Contribution: Investigation Search for more papers by this author Gee-Chen Chang Gee-Chen Chang orcid.org/0000-0002-1802-417X Division of Chest Medicine, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan School of Medicine, Chung Shan Medical University, Taichung, Taiwan Contribution: Resources Search for more papers by this author Hsuan-Yu Chen Hsuan-Yu Chen Institute of Statistical Science, Academia Sinica, Taipei, Taiwan Contribution: Formal analysis Search for more papers by this author Yu-Ju Chen Yu-Ju Chen orcid.org/0000-0002-3178-6697 Institute of Chemistry, Academia Sinica, Taipei, Taiwan Contribution: Resources, Formal analysis Search for more papers by this author Ching-Hsien Chen Corresponding Author Ching-Hsien Chen [email protected] orcid.org/0000-0002-4211-9988 Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, University of California Davis, Davis, CA, USA Division of Nephrology, Department of Internal Medicine, University of California Davis, Davis, CA, USA Comprehensive Cancer Center, University of California Davis, Davis, CA, USA Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Pan-Chyr Yang Corresponding Author Pan-Chyr Yang [email protected] orcid.org/0000-0001-6330-6048 Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan Contribution: Conceptualization, Supervision, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Sung-Liang Yu Corresponding Author Sung-Liang Yu [email protected] orcid.org/0000-0003-4535-9036 Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan Institute of Medical Device and Imaging, College of Medicine, National Taiwan University, Taipei, Taiwan Graduate Institute of Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Wen-Hsin Chang Wen-Hsin Chang orcid.org/0000-0002-8027-302X Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Yi-Ju Chen Yi-Ju Chen orcid.org/0000-0002-7203-9188 Institute of Chemistry, Academia Sinica, Taipei, Taiwan Contribution: Data curation, Formal analysis, Investigation, Visualization, Methodology Search for more papers by this author Yi-Jing Hsiao Yi-Jing Hsiao orcid.org/0000-0001-8780-8800 Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan Contribution: Formal analysis, Validation, Investigation, Methodology Search for more papers by this author Ching-Cheng Chiang Ching-Cheng Chiang Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan Contribution: Formal analysis, Validation, Investigation Search for more papers by this author Chia-Yu Wang Chia-Yu Wang orcid.org/0000-0002-0324-3302 Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan Contribution: Formal analysis, Validation, Investigation Search for more papers by this author Ya-Ling Chang Ya-Ling Chang orcid.org/0000-0003-0240-2647 Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan Contribution: Investigation Search for more papers by this author Qi-Sheng Hong Qi-Sheng Hong Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan Contribution: Investigation Search for more papers by this author Chien-Yu Lin Chien-Yu Lin Institute of Statistical Science, Academia Sinica, Taipei, Taiwan Contribution: Formal analysis Search for more papers by this author Shr-Uen Lin Shr-Uen Lin orcid.org/0000-0001-9266-387X Graduate Institute of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan Contribution: Investigation Search for more papers by this author Gee-Chen Chang Gee-Chen Chang orcid.org/0000-0002-1802-417X Division of Chest Medicine, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan School of Medicine, Chung Shan Medical University, Taichung, Taiwan Contribution: Resources Search for more papers by this author Hsuan-Yu Chen Hsuan-Yu Chen Institute of Statistical Science, Academia Sinica, Taipei, Taiwan Contribution: Formal analysis Search for more papers by this author Yu-Ju Chen Yu-Ju Chen orcid.org/0000-0002-3178-6697 Institute of Chemistry, Academia Sinica, Taipei, Taiwan Contribution: Resources, Formal analysis Search for more papers by this author Ching-Hsien Chen Corresponding Author Ching-Hsien Chen [email protected] orcid.org/0000-0002-4211-9988 Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, University of California Davis, Davis, CA, USA Division of Nephrology, Department of Internal Medicine, University of California Davis, Davis, CA, USA Comprehensive Cancer Center, University of California Davis, Davis, CA, USA Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Pan-Chyr Yang Corresponding Author Pan-Chyr Yang [email protected] orcid.org/0000-0001-6330-6048 Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan Contribution: Conceptualization, Supervision, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Sung-Liang Yu Corresponding Author Sung-Liang Yu [email protected] orcid.org/0000-0003-4535-9036 Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan Institute of Medical Device and Imaging, College of Medicine, National Taiwan University, Taipei, Taiwan Graduate Institute of Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Wen-Hsin Chang1, Yi-Ju Chen2, Yi-Jing Hsiao3, Ching-Cheng Chiang3, Chia-Yu Wang3, Ya-Ling Chang3, Qi-Sheng Hong3, Chien-Yu Lin4, Shr-Uen Lin5, Gee-Chen Chang6,7, Hsuan-Yu Chen4, Yu-Ju Chen2, Ching-Hsien Chen *,8,9,10, Pan-Chyr Yang *,1,11,12,† and Sung-Liang Yu *,3,13,14,15,16,† 1Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan 2Institute of Chemistry, Academia Sinica, Taipei, Taiwan 3Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan 4Institute of Statistical Science, Academia Sinica, Taipei, Taiwan 5Graduate Institute of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan 6Division of Chest Medicine, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan 7School of Medicine, Chung Shan Medical University, Taichung, Taiwan 8Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, University of California Davis, Davis, CA, USA 9Division of Nephrology, Department of Internal Medicine, University of California Davis, Davis, CA, USA 10Comprehensive Cancer Center, University of California Davis, Davis, CA, USA 11Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan 12Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 13Institute of Medical Device and Imaging, College of Medicine, National Taiwan University, Taipei, Taiwan 14Graduate Institute of Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan 15Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan 16Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan † These authors contributed equally to this work *Corresponding author. Tel: +1 530 752 6837; E-mail: [email protected] author. Tel: +886 2 2356 2905; E-mail: [email protected] author. Tel: +886 2 2312 3456, ext 88697; E-mail: [email protected] EMBO Reports (2022)23:e54265https://doi.org/10.15252/embr.202154265 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The aggressive nature and poor prognosis of lung cancer led us to explore the mechanisms driving disease progression. Utilizing our invasive cell-based model, we identified methylthioadenosine phosphorylase (MTAP) and confirmed its suppressive effects on tumorigenesis and metastasis. Patients with low MTAP expression display worse overall and progression-free survival. Mechanistically, accumulation of methylthioadenosine substrate in MTAP-deficient cells reduce the level of protein arginine methyltransferase 5 (PRMT5)-mediated symmetric dimethylarginine (sDMA) modification on proteins. We identify vimentin as a dimethyl-protein whose dimethylation levels drop in response to MTAP deficiency. The sDMA modification on vimentin reduces its protein abundance but trivially affects its filamentous structure. In MTAP-deficient cells, lower sDMA modification prevents ubiquitination-mediated vimentin degradation, thereby stabilizing vimentin and contributing to cell invasion. MTAP and PRMT5 negatively correlate with vimentin in lung cancer samples. Taken together, we propose a mechanism for metastasis involving vimentin post-translational regulation. Synopsis Repression of MTAP-dependent symmetric dimethylation mediated by PRMT5 increases vimentin protein stability and leads to invasion and metastasis in MTAP-deficient lung cancer. MTAP loss promotes lung cancer metastasis. MTA accumulation in MTAP-deficient cancer cells inhibits PRMT5-mediated symmetric dimethylation on arginine residues of vimentin. Vimentin is destabilized by PRMT5-mediated symmetric dimethylation. Reduced dimethylation and stabilization of vimentin in MTAP-deficient cancer cells contributes to invasion and metastasis. Introduction Highly aggressive in nature, lung cancer poses many challenges in both detection and management (Herbst et al, 2018; Siegel et al, 2020). Although a variety of treatments, including surgery, radiation, and chemotherapy, are well administered, ongoing complications involving cancer drug resistance and metastasis abate their initial efficacy; therefore, there is an urgent need to identify intrinsic mechanisms in order to develop more effective and viable therapies. We previously established a series of lung adenocarcinoma cell lines with various degrees of invasiveness as a platform to study mechanisms of metastatic process (Chu et al, 1997; Chen et al, 2001). Although we have thus far discovered dysregulation in multiple genes responsible for metastasis by conducting expression microarray assays (Chen et al, 2012, 2014, 2016; Chang et al, 2016; Hsu et al, 2018), many alterations are yet to be elucidated completely. In the era of omics, genomic, transcriptomic and proteomic profiling have aided in delineating the molecular pathogenesis of cancer (Cancer Genome Atlas Research Network, 2014; Sanchez-Vega et al, 2018). In conjunction with the detection of structural variations and phosphoproteome, those multi-dimensional analyses further guide the development of genetic testing and therapeutic options (Sanchez-Vega et al, 2018; Chen et al, 2020). However, signaling pathways are driven by post-translational modifications (PTMs) on proteins under certain conditions. In addition to phosphorylation, other types of modification have recently gained attention given the crosstalk between different PTMs (Wu et al, 2019); therefore, a comprehensive screening for a specific PTM may shed new insight into novel therapeutic targets. Methylthioadenosine phosphorylase (MTAP) is an enzyme responsible for catalyzing the phosphorylation of methylthioadenosine (MTA), a by-product metabolite produced by the polyamine pathway, into methylthioribose-1-phosphate (MTR-1-P) and adenine for the salvage of methionine and adenine (Bertino et al, 2011). Clinically, approximately 15% of lung cancer patients carry MTAP gene deletion, and cancer cells with MTAP loss exhibit an increase of intracellular levels of MTA (Kryukov et al, 2016; Marjon et al, 2016; Mavrakis et al, 2016). Prior work reported that MTAP-deficient cells are preferentially vulnerable to the depletion of protein arginine methyltransferase 5 (PRMT5), and MTA metabolite selectively inhibits the enzymatic activity of PRMT5 (Kryukov et al, 2016; Marjon et al, 2016; Mavrakis et al, 2016). PRMTs are classified into three types: all of the type I, II, and III PRMTs can catalyze the formation of monomethylarginine (mMA); the following generation of asymmetric dimethylarginine (aDMA) is catalyzed by type I PRMTs, and symmetric dimethylarginine (sDMA) is generated by type II PRMTs including PRMT5. The general biological functions of PRMTs include, but are not limited to, gene transcription, chromatin structure remodeling, RNA metabolism, and signal transduction, depending on the substrate proteins they methylate (Guccione & Richard, 2019). Although MTAP deletion sensitizes cancer cells to PRMT5 depletion, pharmacologic inhibition of PRMT5 by a specific small-molecule inhibitor shows only modest selective tumor cytotoxicity (Kryukov et al, 2016; Marjon et al, 2016; Mavrakis et al, 2016). Thus, identification of viable molecular targets is of utmost importance for developing effective therapeutic options for MTAP-loss cancer. Herein, we investigated the role of MTAP in lung cancer metastasis, and elucidated its underlying mechanisms by using a methylproteomic screen. We further identified vimentin as a downstream metastatic executor in MTAP-deficient cells. The aim of this study is to provide potential therapeutic targets for lung cancer. Results MTAP deficiency confers the tumorigenesis and metastatic ability of lung adenocarcinoma To unveil invasion/metastasis-related gene expression alterations, we utilized our previous transcriptomic profiles from an isogenic invasion cell model (Chen et al, 2001) and copy number variation data from the Cancer Genome Atlas (TCGA) to identify potential gene candidates whose altered expression levels are associated with cancer cell invasion ability and copy number variations. Among the intersected genes, MTAP was downregulated (log2 ratio = −6.09) in highly invasive CL1-5 compared to less invasive CL1-0 cells, and its deletion frequency is 12.1% (62/511) in a TCGA cohort (Fig 1A). We confirmed decreased expression levels of MTAP mRNA and protein in CL1-5 cells (Fig 1B), and found that compared to the PBMCs from healthy individuals, CL1-0 displayed half level of copy number, whereas CL1-5 had a severe copy number loss (Fig EV1A). In addition, MTAP mRNA expression was almost undetectable in 42.9% (6/14) of all tested lung cancer cell lines in our lab (Fig EV1B). Although a high percentage of MTAP deletion was found in several clinicopathologic cancer studies (Bertino et al, 2011; Su et al, 2014; Kryukov et al, 2016; Woollard et al, 2016), MTAP gene deletion associated with the invasive and/or metastatic processes is the first time to be mentioned. Figure 1. MTAP loss promotes invasion/metastasis of lung adenocarcinoma A. The Venn diagram showing 207 differentially expressed genes (DEGs) between CL1-0 and CL1-5 cells (fold change greater than 5-fold in Log2 scale) and 66 genes with copy number variation (CNV, Frequency ≧5%) in lung adenocarcinoma (LUAD) from TCGA database (PanCancer Atlas) and the 4 intersected genes. B. Comparisons of MTAP mRNA and protein expression between CL1-0 and CL1-5 cells detected by RT-qPCR (top, mean ± SD, Student t test, n = 3, biological replicates, *P < 0.05) and Western blot assays using anti-MTAP antibody (bottom, representative of three independent experiments). C. Cell invasion abilities of MTAP-overexpressing CL1-5 or MTAP-knockout CL1-0 and H1650 were determined by Boyden chamber invasion assays (mean ± SD, Student t test, n = 3, biological replicates, *P < 0.05). sgCTRL indicates cells transduced with a nontargeting control sgRNA, and sgMTAP indicates cells transduced with a sgRNA targeting MTAP. Bottom: the protein expression levels of V5-MTAP and endogenous MTAP were detected by Western blots. D. Migration assays using a single-cell tracking, time-lapse video microscopy system. Representative trajectories and quantification of averaged velocity of cells (mean ± SD, Student t test, n = 20, technical replicates, *P < 0.05). Data shown are representative of three independent experiments. E. Effect of MTAP overexpression on lung cancer metastasis in vivo was demonstrated by orthotopic implantation assays. Top: representative photographs of lungs and H&E staining of the lung sections. The primary tumors are indicated by arrowheads and the metastatic nodules are indicated by arrows. Bottom: quantification of averaged primary tumor sizes, metastatic incidence and nodule number. F. Kaplan–Meier analyses of overall survival (top) and progression-free survival (bottom) for 101 patients with lung adenocarcinoma grouped into high- or low-MTAP mRNA expression measured by RT-qPCR. P values were obtained by log-rank test. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Expression and effect of MTAP on cell proliferation, colony forming, tumorigenesis, invasion, morphology, and metastasis in lung cancer cells A. Comparison of MTAP DNA copy number between PBMCs from healthy donors, CL1-0, and CL1-5 cells detected by real-time genomic PCR (mean ± SD, Student t test, n = 3, biological replicates, *P < 0.05). Ribonuclease P (RNaseP) served as the internal control. B. Relative mRNA expression of MTAP was detected in 14 lung cancer cell lines assayed by RT-qPCR (mean ± SD, n = 3, biological replicates). TATA-binding protein (TBP) served as the internal control. C. Effect of MTAP on cell proliferation was determined by cell collection and counting at indicated time points (mean ± SD, Student t test, n = 3, technical replicates, *P < 0.05). Data shown are representative of three independent experiments. D. Effect of MTAP on anchorage-independent colony formation. Left: quantification of colonies stained by crystal violet and counted under phase microscopy (mean ± SD, Student t test, n = 4, biological replicates, *P < 0.05). Right: images of anchorage-independent colony formation assays of CL1-5 Mock control and MTAP-overexpressing cells. E. CL1-5 Mock and MTAP-overexpressing transfectants were subcutaneously injected into the NOD/SCID mice to determine the tumorigenesis ability. Left: the tumor volumes measured at indicated days (mean ± SD, Student t test, n = 8, biological replicates, *P < 0.05). Right: representative images of tumors and the tumor weights measured at 27 days postinjection (mean ± SD, Student t test, n = 8, biological replicates, *P < 0.05). Scale bar, 5 mm. F. Cell invasion abilities of MTAP-overexpressing A549 and H322M were determined by Matrigel invasion assays (mean ± SD, Student t test, n = 3, biological replicates, *P < 0.05). Bottom: the protein expression levels of V5-MTAP were detected by Western blots. G. Effect of MTAP expression on cell morphology. Cells were examined by a phase contrast microscope. Scale bar, 50 μm. Data shown are representative of three independent experiments. H. Micrometastatic analysis of MTAP-overexpressing cells. NOD/SCID mice were intravenously injected with CL1-5 Mock control and MTAP-overexpressing cells, and sacrificed at 10 weeks post injection. Left: the appearances and the representative hematoxylin and eosin (H&E) staining of the lung sections from mice injected with CL1-5 Mock and MTAP transfectants. Scale bar, 50 μm. Right: the gross pulmonary metastasis nodules were quantified under dissecting microscope (Mock, n = 11; MTAP, n = 9; biological replicates, mean ± SE, Student t test, *P < 0.05). Download figure Download PowerPoint Despite a previous report showing an inhibitory effect of MTAP on breast cancer cell growth (Christopher et al, 2002), the functional roles of MTAP in lung cancer remain unclear. We found that MTAP overexpression suppressed both cell proliferation and colonization capacity (Fig EV1C and D), and mice bearing subcutaneous MTAP-overexpressing tumors exhibited smaller size and weight than those in mock control group (Fig EV1E). To prove that MTAP functions as a metastasis suppressor, we established both MTAP-knockout and -overexpressing cells to examine their invasive and migratory abilities. As we expected, overexpression of MTAP inhibited both phenotypes, while MTAP knockout elevated invasion and migration abilities compared to control cells (Figs 1C and D, and EV1F). Moreover, the morphology of MTAP-deficient cells tended to be mesenchymal-like (Fig EV1G). We further carried out intravenous injection and orthotopic implantation assays to determine the role of MTAP in metastasis. An intravenous tail injection assay showed mice injected with MTAP-overexpressing cells had less pulmonary metastasis nodules than those with mock control cells did (Fig EV1H). In an orthotopic xenograft model, smaller primary tumors and less metastatic tumor nodules were observed in mice receiving MTAP-overexpressing cells (Fig 1E), indicating that MTAP inhibits the metastatic potential of lung cancer. In light of an inhibitory effect of MTAP on tumorigenesis and metastasis, we next evaluated the clinical relevance of MTAP gene in patient outcome. MTAP expression in tumor specimens from 101 Taiwanese diagnosed with lung adenocarcinoma was detected by using RT-qPCR assays. The clinical characteristics of these patients were summarized in Appendix Table S1. Patients with low MTAP expression showed worse overall survival and progression-free survival as compared to those patients with high MTAP expression (Fig 1F and Appendix Table S2). Multivariate Cox regression analysis further confirmed that MTAP expression is an independent prognostic factor of overall survival (Appendix Table S3). These results suggest that MTAP functions as a tumor and metastasis suppressor. MTAP deletion inhibits PRMT5-mediated sDMA of vimentin to promote cancer invasion We next performed targeted metabolomics analysis to verify the catalytic activity of ectopically expressed MTAP in lung cancer cells. The metabolic profiling of MTAP-overexpressing and mock control CL1-5 cells showed that MTA level was diminished by abundant MTAP, concomitant with an increase of downstream metabolites in methionine and adenine salvage cycles (Fig EV2A) and variations in metabolites involved in other pathways (Appendix Table S4), implying that ectopic expression of MTAP is functional. To investigate the molecular mechanism of MTAP-mediated suppression of lung cancer metastasis, 1,042 differentially expressed genes with 2-fold change from the mRNA profiling of mock and MTAP-overexpressing CL1-5 cells were subjected to pathway analysis. In addition to IGF-related pathways as we described previously (Xu et al, 2019), cell adhesion/cytoskeleton remodeling and invasion-related pathways were significantly affected by MTAP (Fig EV2B), but this transcriptomic data were not enriched in the epithelial–mesenchymal transition (EMT) signature (Fig EV2C). Click here to expand this figure. Figure EV2. MTAP-mediated metabolism, signaling pathways, and methylproteome A. MTAP-mediated metabolic alterations in polyamine, methionine, and adenine salvage pathways. The numbers are the average fold change intensities and associated errors for metabolites of CL1-5 MTAP/CL1-5 Mock (Student t test, n = 3, biological replicates, *P < 0.05). B. Top 20 ranking MTAP-altered pathways and cellular processes identified by the MetaCore analytical suite (version 20.4 build 70,300). Blue bars: IGF-related pathways; Orange bars: cell adhesion/cytoskeleton remodeling and invasion-related pathways. C. Gene Set Enrichment Analysis (GSEA) of expression microarray data from CL1-5 Mock and MTAP-overexpressing cells were performed using the gene set of HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION. D. Schematic diagram of the identification of differentially symmetrically dimethylated proteins. E. In vitro methylation of vimentin by PRMT5. Tritiated proteins were separated by SDS-PAGE, stained with Coomassie blue (left), dried and analyzed by fluorography (right). Histone H3 and H4 proteins were used as positive controls. *: tritiated vimentin. F. Immunoprecipitation analysis for vimentin dimethylation in CL1-5 Mock and MTAP-overexpressing cells. Data shown are representative of three independent experiments. G. Immunoprecipitation analysis of the association between endogenous vimentin and endogenous PRMT5 in CL1-5 Mock and MTAP-overexpressing cells. Data shown are representative of three independent experiments. H. CL1-5 MTAP-overexpressing and Mock cells were transfected with vimentin siRNAs for 72 h and analyzed by Boyden chamber invasion assays (mean ± SD, Student t test, n = 3, biological replicates, *P < 0.05). The silence efficiency of vimentin was examined by Western blot. Source data are available online for this figure. Download figure Download PowerPoint We noticed that the invasion-suppressive ability of enzyme-defective D220A mutant MTAP which fails to catalyze MTA (Appleby et al, 1999; Xu et al, 2019) was attenuated (Fig 2A), suggesting that the enzymatic activity of MTAP is critical for its cancer suppressive function. Given the altered enzymatic activity of PRMTs in response
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