METTL3-Mediated m6A Modification Regulates the Polycomb Repressive Complex 1 Components BMI1 and RNF2 in Hepatocellular Carcinoma Cells.

Methyltransferase-like 3 (METTL3) is a primary RNA methyltransferase that catalyzes N6-methyladenosine (m6A) modification. The current study aims to further delineate the effect and mechanism of METTL3 in hepatocellular carcinoma (HCC). By using a murine model of hepatocellular cancer development induced via hydrodynamic tail vein injection, we showed that METTL3 enhanced HCC development. In cultured human HCC cell lines (Huh7 and PLC/PRF/5), we observed that stable knockdown of METTL3 by short hairpin RNA significantly decreased tumor cell proliferation, colony formation, and invasion, in vitro. When Huh7 and PLC/PRF/5 cells with short hairpin RNA knockdown of METTL3 were inoculated into the livers of SCID mice, we found that METTL3 knockdown significantly inhibited the growth of HCC xenograft tumors. These findings establish METTL3 as an important oncogene in HCC. Through m6A sequencing, RNA sequencing, and subsequent validation studies, we identified BMI1 and RNF2, two key components of the polycomb repressive complex 1, as direct downstream targets of METTL3-mediated m6A modification in HCC cells. Our data indicated that METTL3 catalyzed m6A modification of BMI1 and RNF2 mRNAs which led to increased mRNA stability via the m6A reader proteins IGF2BP1/2/3. Furthermore, we showed that the METTL3 inhibitor, STM2457, significantly inhibited HCC cell growth in vitro and in mice. Collectively, this study provides novel evidence that METTL3 promotes HCC development and progression through m6A modification of BMI1 and RNF2. Our findings suggest that the METTL3-m6A-BMI1/RNF2 signaling axis may represent a new therapeutic target for the treatment of HCC. Implications: The METTL3-m6A-BMI1/RNF2 signaling axis promotes HCC development and progression.

Chen W ，Zhang J ，Ma W ，Liu N ，Wu T ... -  《-》

MAZ-mediated N6-methyladenosine modification of ZEB1 promotes hepatocellular carcinoma progression by regulating METTL3.

Hepatocellular carcinoma (HCC) has a hidden onset and high malignancy. Its high metastasis, high recurrence, and short survival time have always been a difficult and hot spot in clinical practice. Our previous study revealed that myc-associated zinc finger protein (MAZ) is highly upregulated in HCC tissues and may promote the proliferation and metastasis of HCC cells by inducing the epithelial-mesenchymal transformation (EMT) process. However, the specific regulatory mechanism by which MAZ functions as an oncogene in HCC has still not been fully elucidated. Immunohistochemical staining and bioinformatics analyses were conducted to measure the expression of MAZ, key m6A enzymes, and ZEB1 in HCC tissues. RNA sequencing (RNA-seq) of MAZ knockdown HCC cells and human mRNA m6A sequencing (m6A-seq) of HCC tissues were intersected to screen the downstream targets for both MAZ and m6A methylation. The correlations between MAZ and its targets were analyzed by dual-luciferase assays and cell rescue experiments. Here, we report for the first time that MAZ is involved in m6A methylation of HCC by targeting the transcriptional regulation of key m6A enzymes. MAZ expression was significantly correlated with the expression of key m6A enzymes in HCC tissues and cell lines. Moreover, MAZ could bind to the promoters of key m6A enzymes, and multivariate Cox regression analysis suggested that MAZ and METTL3 expression were independent risk factors for the survival of HCC patients. Through RNA-seq and m6A-seq, we screened out EMT regulators ZEB1 and TRIM50 as the downstream targets for both MAZ and m6A methylation. Mechanistically, m6A sites with high confidence in ZEB1 and TRIM50 mRNA were identified by SRAMP, and there were significant relationships between ZEB1 and METTL3 in HCC tissues and cells. A nomogram model was established to better display the combined effect of MAZ, METTL3, and ZEB1 on HCC prognosis. Our study revealed a promising clinical application of MAZ, METTL3, and ZEB1 in HCC prognosis, further suggesting that MAZ can be used as a potential molecular biomarker for HCC diagnosis and prognosis.

Li D ，Xu L ，Liu R ，Yao Z ，Zheng C ，Jin S ，Guo X ，Zhang Z ，Tan S ，Zhu X ... -  《-》

METTL3-mediated m6A modification of ZNF384 promotes hepatocellular carcinoma progression by transcriptionally activating ACSM1.

Hepatocellular carcinoma (HCC) is a lethal disease with a high mortality rate, and its development is influenced by various molecular mechanisms. Zinc finger protein 384 (ZNF384) has been reported to be involved in the progression of several cancers; however, its role in HCC remains elusive. mRNA expression levels were analyzed by quantitative real-time polymerase chain reaction, while western blotting and immunohistochemistry were performed to validate protein expression. Cell proliferation, apoptosis, and metabolic activities were examined using clonogenicity, flow cytometry, and specific assay kits. A xenograft mouse model was employed to assess the impact of acyl-CoA synthetase medium-chain family member 1 (ACSM1) depletion on HCC cell malignancy in vivo. Chromatin immunoprecipitation assay and dual-luciferase reporter assay were conducted to explore the association between ZNF384 and ACSM1. We found that ACSM1 and ZNF384 were significantly upregulated in HCC tissues and cells when compared with normal liver tissues and human liver immortalized cells. Knockdown of ACSM1 inhibited HCC cell proliferation and glucose metabolism and induced cell apoptosis. Furthermore, ACSM1 depletion suppressed the malignant progression of HCC cells in vivo. Our data indicated that ZNF384 transcriptionally activated ACSM1 in HCC cells. Overexpression of ACSM1 reversed the inhibitory effect of ZNF384 depletion on HCC cell malignancy. Further, methyltransferase-like 3 (METTL3) stabilized ZNF384 mRNA through m6A methylation. METTL3-mediated m6A modification of ZNF384 contributed to the progression of HCC by transcriptionally activating ACSM1. This finding suggests potential therapeutic targets for this devastating disease.

Zhang L ，Wang J ，Gui F ，Peng F ，Deng W ，Zhu Q ... -  《-》

Notch-Driven Cholangiocarcinogenesis Involves the Hippo Pathway Effector TAZ via METTL3-m6A-YTHDF1.

Notch and TAZ are implicated in cholangiocarcinogenesis, but whether and how these oncogenic molecules interact remain unknown. The development of cholangiocarcinoma (CCA) was induced by hydrodynamic tail vein injection of oncogenes (Notch1 intracellular domain [NICD]/AKT) to the FVB/NJ mice. CCA xenograft was developed by inoculation of human CCA cells into the livers of SCID mice. Tissues and cells were analyzed using quantitative reverse transcription polymerase chain reaction, Western blotting analyses, immunohistochemistry, chromatin immunoprecipitation-quantitative polymerase chain reaction and WST-1 cell proliferation assay. Our experimental findings show that TAZ is indispensable in NICD-driven cholangiocarcinogenesis. Notch activation induces the expression of methyltransferase like-3 (METTL3), which catalyzes N6-methyladenosine modification of TAZ mRNA and that this mechanism plays a central role in the crosstalk between Notch and TAZ in CCA cells. Mechanistically, Notch regulates the expression of METTL3 through the binding of NICD to its downstream transcription factor CSL in the promoter region of METTL3. METTL3 in turn mediates N6-methyladenosine modification of TAZ mRNA, which is recognized by the m6A reader YTHDF1 to enhance TAZ protein translation. We observed that inhibition of Notch signaling decreased the protein levels of both MELLT3 and TAZ. Depletion of METTL3 by short hairpin RNAs or by the next generation GapmeR antisense oligonucleotides decreased the level of TAZ protein and inhibited the growth of human CCA cells in vitro and in mice. This study describes a novel Notch-METTL3-TAZ signaling cascade, which is important in CCA development and progression. Our experimental results provide new insight into how the Notch pathway cooperates with TAZ signaling in CCA, and the findings may have important therapeutic implications.

Ma W ，Zhang J ，Chen W ，Liu N ，Wu T ... -  《Cellular and Molecular Gastroenterology and Hepatology》

N6-Methyladenosine modification activates the serine synthesis pathway to mediate therapeutic resistance in liver cancer.

Metabolic adaptation serves as a significant driving force for cancer growth and poses a substantial obstacle for cancer therapies. Herein, we unraveled the role of m6A-mediated serine synthesis pathway (SSP) regulation in both hepatocellular carcinoma (HCC) development and therapeutic resistance. We demonstrated that treatment of highly specific m6A inhibitor (STM2457) effectively inhibited HCC cell line growth and suppressed spontaneous HCC formation in mice driven by liver-specific Tp53 knockout and Myc overexpression. Using GLORI-seq, we delineated a single-base-resolution m6A landscape in human HCC cell lines. Interestingly, we identified three core enzymes in the SSP (PHGDH, PSAT1, and PSPH) as novel targets of METTL3-mediated m6A modification. In these SSP genes, m6A modification recruited m6A reader IGF2BP3 to stabilize their mRNA transcripts, thereby enhancing their mRNA and protein expression in HCC cells. Most importantly, our GLORI-seq data revealed that sorafenib-resistant HCC cells elevated m6A modification in SSP genes to promote protein expression and antioxidant production. STM2457 treatment attenuated the serine synthesis pathway, induced oxidative stress, and sensitized HCC cells to sorafenib and lenvatinib treatments. In conclusion, our findings suggest that targeting m6A could be a potential therapeutic strategy for HCC treatment.

Chan FF ，Kwan KK ，Seoung DH ，Chin DW ，Ng IO ，Wong CC ，Wong CM ... -  《-》