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》

Nicotine enhances the stemness and tumorigenicity in intestinal stem cells via Hippo-YAP/TAZ and Notch signal pathway.

Cigarette smoking is a well-known risk factor inducing the development and progression of various diseases. Nicotine (NIC) is the major constituent of cigarette smoke. However, knowledge of the mechanism underlying the NIC-regulated stem cell functions is limited. In this study, we demonstrate that NIC increases the abundance and proliferative activity of murine intestinal stem cells (ISCs) in vivo and ex vivo. Moreover, NIC induces Yes-associated protein (YAP) /Transcriptional coactivator with PDZ-binding motif (TAZ) and Notch signaling in ISCs via α7-nicotinic acetylcholine receptor (nAchR) and protein kinase C (PKC) activation; this effect was not detected in Paneth cells. The inhibition of Notch signaling by dibenzazepine (DBZ) nullified the effects of NIC on ISCs. NIC enhances in vivo tumor formation from ISCs after loss of the tumor suppressor gene Apc, DBZ inhibited NIC-induced tumor growth. Hence, this study identifies a NIC-triggered pathway regulating the stemness and tumorigenicity of ISCs and suggests the use of DBZ as a potential therapeutic strategy for treating intestinal tumors.

Isotani R ，Igarashi M ，Miura M ，Naruse K ，Kuranami S ，Katoh M ，Nomura S ，Yamauchi T ... -  《eLife》

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 ... -  《-》

Mechanism of METTL3 in the proliferation, invasion, and migration of intrahepatic cholangiocarcinoma cells via m6A modification.

Intrahepatic cholangiocarcinoma (ICC) is a primary invasive malignant tumor. This study was conducted to explore the role of methyltransferase-like 3 (METTL3)-mediated m6A modification in ICC cells and provide novel targets for ICC treatment. Levels of METTL3/YTH N6-methyladenosine RNA binding protein 2 (YTHDF2)/Nedd4 family interacting protein 1 (NDFIP1) in cells were determined. Cell viability, proliferation, invasion, and migration were evaluated. The enrichments of METTL3, YTHDF2, and m6A on NDFIP1 mRNA were analyzed. The mRNA stability was determined. Inhibition of YTHDF2 or NDFIP1 was combined with si-METTL3 to confirm the mechanism. The role of METTL3 in vivo was verified. METTL3 was overexpressed in ICC cells. METTL3 silencing suppressed ICC cell malignant behaviors, which were reversed by METTL3 overexpression. METTL3 increased m6A modification on NDFIP1 mRNA, facilitated YTHDF2 recognition of m6A, and promoted NDFIP1 mRNA degradation, thereby suppressing NDFIP1 expression. YTHDF2 inhibition increased NDFIP1 mRNA levels. NDFIP1 downregulation partially reversed the inhibitory effects of si-METTL3 on ICC cell behaviors, while NDFIP1 overexpression partially reversed the promotive effects of METTL3 on ICC cell behaviors. METTL3 downregulation suppressed ICC growth by increasing NDFIP1 expression. In conclusion, METTL3 aggravates ICC cell proliferation, invasion, and migration by promoting the degradation of NDFIP1 mRNA in a YTHDF2-dependent manner.

Jiang X ，Tan H 《-》

USP43 drives cervical carcinoma progression through regulation of the Hippo/TAZ pathway.

Cervical carcinoma (CC) poses significant health challenges, with its pathogenesis not fully understood. While ubiquitin specific peptidase 43 (USP43) is implicated in various cancers, its role in CC and regulation of the Hippo/Transcriptional Co-Activator With PDZ-Binding Motif (TAZ) pathway remain unexplored. This study examines USP43's impact on CC progression and its interaction with TAZ. USP43 expression levels in CC tissues and cell lines were assessed using reverse transcription real-time polymerase chain reaction (RT-qPCR) and Western blot. The effects of USP43 silencing on cell proliferation, migration, and invasion were evaluated through cell counting Kit-8 (CCK-8), 5-ethynyl-2'deoxyuridine (EdU), colony formation, and transwell assays. Immunofluorescence staining and co-immunoprecipitation (Co-IP) assays were used to explore the interaction between USP43 and TAZ. Polyubiquitination assays were performed to evaluate ubiquitination and stability of TAZ, and cycloheximide (CHX) chase experiments determined the half-life of TAZ. In vivo studies using BALB/c nude mice examined the impact of USP43 knockdown on tumor growth and metastasis. USP43 was overexpressed in CC tissues and cell lines. Silencing of USP43 reduced cell proliferation, migration, invasion, and the epithelial-mesenchymal transition (EMT) process. Co-IP and ubiquitination assays revealed that USP43 interacted with and stabilized TAZ by inhibiting TAZ ubiquitination. CHX chase experiments confirmed that USP43 prolonged TAZ protein stability. In vivo, USP43 knockdown led to reduced tumor growth and lung metastasis. Overexpression of TAZ reversed the inhibitory effects of USP43 silencing on CC cell proliferation, migration, invasion and EMT CONCLUSION: USP43 promotes CC cell proliferation, migration, invasion, and epithelial-to-mesenchymal transition (EMT) by activating the Hippo/TAZ pathway. These findings enhance our understanding of USP43's role in CC progression and highlight potential therapeutic targets for the treatment of CC.

Zhang Y ，Liu Y ，Deng Q ，Liu L ，Zhu W ... -  《-》