RNA m 6 A methylation regulates sorafenib resistance in liver cancer through FOXO 3‐mediated autophagy

Article5 May 2020Open Access RNA m6A methylation regulates sorafenib resistance in liver cancer through FOXO3-mediated autophagy Ziyou Lin Ziyou Lin Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Yi Niu Yi Niu National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Arabella Wan Arabella Wan Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Dongshi Chen Dongshi Chen Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Heng Liang Heng Liang National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Xijun Chen Xijun Chen Department of Abdominal Surgery, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China Search for more papers by this author Lei Sun Lei Sun National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Siyue Zhan Siyue Zhan National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Liutao Chen Liutao Chen School of Life Science, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Chao Cheng Chao Cheng Department of Thoracic Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Xiaolei Zhang Xiaolei Zhang National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Xianzhang Bu Xianzhang Bu National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Weiling He Corresponding Author Weiling He [email protected] Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Center for Precision Medicine, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Guohui Wan Corresponding Author Guohui Wan [email protected] orcid.org/0000-0001-5170-7282 Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Ziyou Lin Ziyou Lin Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Yi Niu Yi Niu National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Arabella Wan Arabella Wan Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Dongshi Chen Dongshi Chen Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA Search for more papers by this author Heng Liang Heng Liang National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Xijun Chen Xijun Chen Department of Abdominal Surgery, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China Search for more papers by this author Lei Sun Lei Sun National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Siyue Zhan Siyue Zhan National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Liutao Chen Liutao Chen School of Life Science, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Chao Cheng Chao Cheng Department of Thoracic Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Xiaolei Zhang Xiaolei Zhang National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Xianzhang Bu Xianzhang Bu National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Weiling He Corresponding Author Weiling He [email protected] Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Center for Precision Medicine, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Guohui Wan Corresponding Author Guohui Wan [email protected] orcid.org/0000-0001-5170-7282 Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Author Information Ziyou Lin1,2, Yi Niu2, Arabella Wan1, Dongshi Chen3, Heng Liang2, Xijun Chen4, Lei Sun2, Siyue Zhan2, Liutao Chen5, Chao Cheng6, Xiaolei Zhang2, Xianzhang Bu2, Weiling He *,1,2,7 and Guohui Wan *,1,2 1Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China 2National-Local Joint Engineering Laboratory of Druggability and New Drug Evaluation, National Engineering Research Center for New Drug and Druggability (cultivation), Guangdong Province Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China 3Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA 4Department of Abdominal Surgery, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China 5School of Life Science, Sun Yat-Sen University, Guangzhou, China 6Department of Thoracic Surgery, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China 7Center for Precision Medicine, Sun Yat-Sen University, Guangzhou, China *Corresponding author. Tel: +86 20 87755766; E-mail: [email protected] *Corresponding author. Tel: +86 20 39943495; E-mail: [email protected] The EMBO Journal (2020)39:e103181https://doi.org/10.15252/embj.2019103181 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 N6-methyladenosine (m6A) is an abundant nucleotide modification in mRNA, known to regulate mRNA stability, splicing, and translation, but it is unclear whether it is also has a physiological role in the intratumoral microenvironment and cancer drug resistance. Here, we find that METTL3, a primary m6A methyltransferase, is significantly down-regulated in human sorafenib-resistant hepatocellular carcinoma (HCC). Depletion of METTL3 under hypoxia promotes sorafenib resistance and expression of angiogenesis genes in cultured HCC cells and activates autophagy-associated pathways. Mechanistically, we have identified FOXO3 as a key downstream target of METTL3, with m6A modification of the FOXO3 mRNA 3′-untranslated region increasing its stability through a YTHDF1-dependent mechanism. Analysis of clinical samples furthermore showed that METTL3 and FOXO3 levels are tightly correlated in HCC patients. In mouse xenograft models, METTL3 depletion significantly enhances sorafenib resistance of HCC by abolishing the identified METTL3-mediated FOXO3 mRNA stabilization, and overexpression of FOXO3 restores m6A-dependent sorafenib sensitivity. Collectively, our work reveals a critical function for METTL3-mediated m6A modification in the hypoxic tumor microenvironment and identifies FOXO3 as an important target of m6A modification in the resistance of HCC to sorafenib therapy. Synopsis METTL3 depletion in the hypoxic tumor microenvironment promotes sorafenib resistance, tumor progression and induction of autophagy. Stabilization of FOXO3 mRNA through METTL3-mediated m6A modification is critical to prevent induction of autophagy and resistance. METTL3, a primary m6A methyltransferase, is downregulated in sorafenib-resistant hepatocellular carcinoma (HCC). Depletion of METTL3 enhances sorafenib resistance in HCC under intratumoral environment. FOXO3 is a critical target of METTL3 during sorafenib resistance. m6A modification of FOXO3 promotes mRNA stability in an YTHDF1-dependent manner. Introduction Hepatocellular carcinoma (HCC) is a primary liver malignancy in patients with chronic liver disease and cirrhosis (Villanueva, 2019), and compared with most solid tumors its incidence is increasing worldwide year by year due to hepatitis viruses (HBV and HCV) infection and alcohol use (Siegel et al, 2019). In the early stages, curative treatments such as tumor resection, ablation, and liver transplantation can be used to treat HCC (Yang et al, 2019). Patients with advanced HCC are administrated with topical treatment and systemic chemotherapy as they are no longer eligible for curative therapies. Sorafenib, the only FDA-approved first-line treatment for advanced HCC, is a multi-target kinase inhibitor for Raf kinases, vascular endothelial growth factor, and platelet-derived growth factor receptors, and improves survival with a median OS rate of 6.5 months compared to the placebo group (Cheng et al, 2009). However, patients with advanced HCC predominantly develop resistance to sorafenib treatment and their survival benefit is limited to 3–5 months with severe side effects (Llovet et al, 2008). A hypoxic microenvironment is a common feature of human advanced solid tumors linked to resistance. The median O2 partial pressure in HCC is 6 mm Hg compared with 30 mm Hg in normal liver (Vaupel et al, 2007), and the homeostatic response to the intratumoral hypoxic environment is mediated by hypoxia-inducible factor (HIF-1), consisting of HIF-1α and HIF-1β subunits (Koh & Powis, 2012). Examining the underlying mechanisms of acquired resistance toward sorafenib in the intratumoral hypoxic microenvironment may provide a new approach to develop individualized therapeutic strategies for coping with sorafenib resistance and to investigate potential combination therapies for the advanced HCC. Recently, the role of N6-methyladenosine (m6A) regulation in various biological processes has been an emerging focus of investigation. m6A is a predominantly internal modification of RNA in mammalian cells, with the features of being dynamic and reversible (Niu et al, 2018). The functional components of the RNA methyltransferase complex include METTL3 (methyltransferase-like 3), METTL14 (methyltransferase-like 14), and WTAP (Wilms tumor 1-associated protein), while the RNA demethylases include FTO (fat mass and obesity-associated protein) and ALKBH5 (a-ketoglutarate-dependent dioxygenase alkB homolog 5). m6A binding proteins with a YT521-B homology (YTH) domain, including YTHDC1, YTHDC2, YTHDF1, YTHDF2, and YTHDF3, recognize m6A in a methylation-dependent manner (Zaccara et al, 2019). Furthermore, the m6A methyltransferase function of METTL3 strictly requires METTL14 as a co-activator. METTL3 and METTL14 form a m6A holoenzyme complex, where METTL3 functions as the catalytic subunit, while METTL14 binds to RNA substrates, stabilizes the structure of the complex, and activates METTL3 via allostery (Sledz & Jinek, 2016; Wang et al, 2016a,b). Emerging evidence has shown that m6A modification plays important roles in various cellular processes including RNA stability (Schwartz et al, 2014; Wang et al, 2014), translation (Wang et al, 2015; Zhou et al, 2015), structure (Liu et al, 2015), localization (Fustin et al, 2013), alternative polyadenylation, and splicing (Haussmann et al, 2016; Lence et al, 2016). Dysregulation of m6A methylation has been frequently reported in various human cancers (Cui et al, 2017; Li et al, 2017; Liu et al, 2018; Niu et al, 2019). For example, we previously showed that FTO induced breast cancer progression through demethylation of BNIP3 in a YTHDF2-independent manner (Niu et al, 2019). Targeting key regulators of the m6A modification has been discussed as a potential therapeutic approach for cancer treatment (Niu et al, 2018). However, the role of the m6A modification in drug resistance of HCC has not been fully described. Thus, examining the m6A regulation in sorafenib-resistant HCC under intratumoral hypoxic microenvironment can provide more comprehensive insights into the molecular mechanisms of the occurrence of sorafenib resistance in HCC. Herein, we discovered that the m6A modification was decreased in sorafenib-resistant HCC and in sorafenib-resistant liver tumor cells. Depletion of METTL3, the primary m6A methyltransferase, enhanced HCC resistance to sorafenib through activating angiogenesis and autophagy pathways. Using MeRIP-Seq analysis and functional studies, we identified FOXO3 as a critical downstream target of METTL3-mediated m6A modification. Our results showed that METTL3 promoted FOXO3 mRNA methylation in the 3′UTR region and enhanced its mRNA stability in an YTHDF1-dependent manner. Overexpression of FOXO3 rescued the sorafenib-resistant phenotype induced by METTL3 depletion. We further validated the effects of the METTL3/FOXO3 axis on sorafenib resistance through both in vitro and in vivo experiments. Taken together, our study reveals a connection of m6A modification, sorafenib resistance, and autophagy under hypoxia, and provides insights into the multiple molecular mechanisms of sorafenib resistance in HCC, as well as expanding the understanding of therapy resistance. Results Down-regulation of METTL3 in sorafenib-resistant HCCs To investigate the molecular mechanism of sorafenib resistance in HCC, we obtained liver tumors with acquired sorafenib resistance (n = 3) from patients with long-term sorafenib treatment and performed transcriptome sequencing to examine the differentially expressed genes by comparison to sorafenib-sensitive liver tumors (n = 3). We identified 819 genes with significant up-regulation and 775 genes with significant down-regulation in the sorafenib-resistant liver tumors compared to the sorafenib-sensitive liver tumors (Appendix Fig S1A and Table EV2). Gene enrichment analysis with KOBAS highlighted dysregulation of several signaling pathways involved in drug resistance, including cellular response to chemical stimulus, response to organic substance, and response to nitrogen compound (Fig 1A). A novel group of transferase was identified to play roles in sorafenib resistance in liver cancer. 13 genes overlapped in these four signaling pathways (Fig 1B). We identified that METTL3, the primary component of m6A methyltransferase, was significantly down-regulated in sorafenib-resistant HCC (Fig 1C). We further validated down-regulation of METTL3 in sorafenib-resistant liver tumors by RT–PCR (Fig 1D). In a clinical outcome analysis, we found that down-regulation of METTL3 was associated with lower survival rates in HCC patients (Appendix Fig S1B). By analyzing the pathological stage plot in liver cancer in the TCGA database, we found that METTL3 expression level was significantly down-regulated in advanced stages of HCCs (Fig 1E). These results indicate that down-regulation of METTL3 may be implicated in sorafenib resistance in HCC. Figure 1. METTL3 was down-regulated in the sorafenib-resistant HCCs A. Gene enrichment analysis on differentially expressed genes between sorafenib-sensitive and sorafenib-resistant human liver tumors. B. Venn diagrams show overlapped genes in signaling pathways response to sorafenib. C. Heatmap of overlapped expressed genes in response to sorafenib resistance. D. The mRNA expression level of METTL3 in sorafenib-sensitive (n = 3) and sorafenib-resistant (n = 3) human liver tumors. E. Expression of METTL3 was significantly down-regulated in advanced stage of liver cancer from TCGA. F. METTL3 RNA levels between naïve HepG-2 cells (n = 3) and sorafenib-resistant HepG-2 cells (n = 10). G. The IC50 of sorafenib-resistant HepG-2 cells treated with sorafenib under hypoxic condition (1% O2). H. The protein level of METTL3 in sorafenib-resistant HepG-2 cells treated with sorafenib under hypoxic condition (1% O2). I. The global RNA m6A level in naïve HepG-2 and sorafenib-resistant HepG-2 determined by dot blotting assay under hypoxic condition (1% O2). Data information: In all relevant panels, ***P < 0.001; ****P < 0.0001; two-tailed t-test. Data are presented as mean ± SD and are representative of three independent experiments. Source data are available online for this figure. Source Data for Figure 1 [embj2019103181-sup-0005-SDataFig1.pdf] Download figure Download PowerPoint To confirm the role of METTL3 in sorafenib resistance, we systematically analyzed the GSE62813 database for the change of METTL3 expression in HepG-2 cells and sorafenib-resistant HepG-2 cells, and found that METTL3 was significantly down-regulated in sorafenib-resistant HCC cells (Fig 1F). To validate the role of METTL3 in acquired resistance, we generated a sorafenib-resistant HepG2 cell line in vitro by exposing the cells with sorafenib at 5% of IC concentration for 3 days and gradually increased the concentration by 5% of IC until reaching the IC50 concentration. We confirmed the acquired resistance of these HepG2 cells toward sorafenib by comparing to the naïve HepG-2 cells (Fig 1G). Importantly, we found that METTL3 was markedly reduced in sorafenib-resistant HepG-2 cells with consistently decreased global RNA m6A level (Fig 1H and I), indicating a potential role of METTL3 in mediating resistance toward sorafenib in HCC cells. Moreover, we noticed that hypoxic condition extensively existed in HCC, as we observed an elevated expression level of HIF-1α in liver tumors when comparing to non-tumor liver tissues (Appendix Fig S1C and D). We established a HCC subcutaneous tumor model in nude mice with SMMC-7721 cells and found that 80% of tumors exhibited a higher level of hypoxia (Appendix Fig S1E and F). We also performed an orthotopic liver tumor xenograft with Bel-7402 cells to validate the hypoxic condition in HCC. Higher levels of HIF-1α were detected in tumor tissue compared to non-tumor tissue (Appendix Fig S1G and G1), indicating oxygen deprivation was a common feature of HCC. To examine the role of METTL3 in HCC, our following functional experiments in vitro were conducted at low oxygen levels (1% O2) to mimic the intratumoral microenvironment in HCC. Knockdown of METTL3 enhanced sorafenib resistance in HCC To evaluate whether the global m6A level is related to tumorigenesis in liver cancer, we generated a series of various expression levels of METTL3 in the normal liver cell line WRL68 (Appendix Fig S2A and B). The normal liver cells formed an enlarged colony after silencing METTL3, but had no meaningful change after overexpressing METTL3 (Fig 2A and B). Neither METTL3 knockdown nor METTL3 overexpression in WRL68 cells induced sensitivity toward sorafenib treatment, indicating that sorafenib was not toxic to normal liver cells regardless of various levels of METTL3 expression (Appendix Fig S2C). However, knockdown of METTL3 in WRL68 cells moderately promoted cell growth, while overexpression of METTL3 in WRL68 cells had no effect in a cell proliferation assay and cell number counting (Appendix Fig S2D and E). To further explore the role of the m6A modification in HCC toward sorafenib resistance, we constructed six stable METTL3-depeleted HCC cell lines (SMMC-7721 #sh1 #sh2, Bel-7402 #sh1 #sh2, and HepG-2 #sh1 #sh2) and confirmed the knockdown effects in both protein and RNA expression levels (Appendix Fig S2F and G). We showed the decrease of global RNA m6A level in these six stable METTL3-depleted cell lines by dot blot assay (Appendix Fig S2H), and the increased sensitivity (IC50) of METTL3-knockdown SMMC-7721, Bel-7402, and HepG-2 cells to treatment with sorafenib (Fig 2C and D, Appendix Fig S2I). We further confirmed the resistant phenotypes of METTL3-knockdown HCC cells toward sorafenib by cell survival assays (Appendix Fig S2J–L) under hypoxia. We noticed a moderately increased IC50 for sorafenib treatment of METTL3-knockdown HCCs under normoxic condition (21% O2) (Appendix Fig S2M–O), excluding that the potential molecular mechanism of METTL3-mediated sorafenib resistance in HCC was mainly caused by hypoxia. Figure 2. Knockdown of METTL3 enhanced sorafenib resistance in HCC A, B. Clonogenic survival of METTL3 knockdown (A) and METTL3 overexpression (B) in normal liver cell line WRL68 cells for 7 days in normoxic condition (21% O2) and quantification of clusters in A-1 and B-1, respectively. C, D. The IC50 of METTL3-knockdown SMMC-7721 cells (C) and Bel-7402 cells (D) after treated with sorafenib for 24 h under hypoxic condition (1% O2). E. Overexpression of wild-type METTL3 or catalytic mutant METTL3 in sorafenib-resistant HepG-2 cells. F. The global RNA m6A level in sorafenib-resistant HepG-2 cells with wild-type METTL3 overexpression or catalytic mutant METTL3 overexpression by dot blotting assay. G–I. Rescue of shRNA-resistant wild-type METTL3 but not catalytic mutant METTL3 sensitized METTL3-knockdown SMMC-7721 cells (G), Bel-7402 cells (H), and sorafenib-resistant HepG-2 cells (I) to sorafenib treatment. The IC50 of the cells was measured after treated with sorafenib for 24 h under hypoxic condition (1% O2). J. Cell survival assay of sorafenib-resistant HepG-2 cells with wild-type METTL3 overexpression or catalytic mutant METTL3 overexpression after treated with sorafenib for 24 h under hypoxic condition (1% O2). Scale bar, 1 mm. K. Capillary-like structures in HUVECs treated with the tumor-conditioned medium (TCM) from control HCCs and METTL3-knockdown HCCs cultured under hypoxic conditions for 48 h. Quantification of the number of tubes with ImageJ in K-1. L, M. The relative mRNA expression levels of angiogenesis genes were detected in SMMC-7721 cells (L) and Bel-7402 cells (M) with METTL3 knockdown by RT–PCR assay. N. The protein levels of VEGF-A in METTL3-knockdown HCCs. O. The protein levels of PDGF-B in METTL3-knockdown HCCs. Data information: In all relevant panels, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; two-tailed t-test. Data are presented as mean ± SD and are representative of three independent experiments. Source data are available online for this figure. Source Data for Figure 2 [embj2019103181-sup-0006-SDataFig2.pdf] Download figure Download PowerPoint Moreover, to determine the role of m6A in sorafenib resistance in HCC, we performed rescue experiments in HCC cells with stable METTL3 depletion and sorafenib-resistant HepG-2 cells using wild-type METTL3 and a catalytic mutant METTL3 (resistant to METTL3 shRNA) (Appendix Fig S2P and Q) (Fig 2E and F). Our results showed that rescue of shRNA-resistant wild-type METTL3, but not catalytic mutant METTL3, sensitizes METTL3-knockdown HCC cells (Fig 2G and H) and sorafenib-resistant HepG-2 cells (Fig 2I) to sorafenib treatment. Consistently, the results of CCK8 cell growth assay in HCCs with METTL3 knockdown (Appendix Fig S2R and S) or sorafenib resistance (Appendix Fig S2T) showed a similar rescuing tendency, as well as a cell survival assay in sorafenib-resistant HepG-2 cells (Fig 2J), indicating the important role of the m6A modification mediated by METTL3 in sorafenib resistance in HCC. To further determine how METTL3 controls sorafenib resistance in HCC, we determined the influence of METTL3 on the tube-forming ability of human umbilical vein endothelial cells (HUVECs). Our results revealed that the tumor-conditioned medium (TCM) from HCC cells in the METTL3-knockdown groups showed increased capacity to stimulate HUVEC tube formation compared to the control groups (Fig 2K and K-1). Moreover, knockdown of METTL3 up-regulated RNA expression of some biomarkers of angiogenesis such as FGF, PDGF-B, STAT3, and VEGF-A in HCC cells under hypoxia (Fig 2L and M, Appendix Fig S2U). We further confirmed the induced protein levels of VEGF-A and PDGF-B by Western blot (Fig 2N and O). Taken together, our results demonstrate that METTL3 deletion enhanced sorafenib resistance in HCC. Autophagy aberration mediated METTL3-dependent sorafenib resistance in HCC Previous reports have shown that autophagy was associated with chemo-resistance in human cancers (Levine & Kroemer, 2008; Doherty & Baehrecke, 2018). To determine whether m6A regulation played a role in autophagy to mediate sorafenib resistance in HCC, transmission electron microscopy (TEM) was used to observe the morphological changes in SMMC-7721 cells with or without METTL3 depletion under hypoxia. Our results showed that knockdown of METTL3 increased the number of autophagosomes (Fig 3A), sugg