TGF-β1 facilitates gallbladder carcinoma metastasis by regulating FOXA1 translation efficiency through m⁶A modification

TGF-beta 1 plays a pivotal role in the metastatic cascade of malignant neoplasms. N6-methyladenosine (m(6)A) stands as one of the most abundant modifications on the mRNA transcriptome. However, in the metastasis of gallbladder carcinoma (GBC), the effect of TGF-beta 1 with mRNA m(6)A modification, especially the effect of mRNA translation efficiency associated with m(6)A modification, remains poorly elucidated. Here we demonstrated a negative correlation between FOXA1 and TGF-beta 1 expression in GBC. Overexpression of FOXA1 inhibited TGF-beta 1-induced migration and epithelial-mesenchymal transition (EMT) in GBC cells. Mechanistically, we confirmed that TGF-beta 1 suppressed the translation efficiency of FOXA1 mRNA through polysome profiling analysis. Importantly, both in vivo and in vitro experiments showed that TGF-beta 1 promoted m(6)A modification on the coding sequence (CDS) region of FOXA1 mRNA, which was responsible for the inhibition of FOXA1 mRNA translation by TGF-beta 1. We demonstrated through MeRIP and RIP assays, dual-luciferase reporter assays and site-directed mutagenesis that ALKBH5 promoted FOXA1 protein expression by inhibiting m(6)A modification on the CDS region of FOXA1 mRNA. Moreover, TGF-beta 1 inhibited the binding capacity of ALKBH5 to the FOXA1 CDS region. Lastly, our study confirmed that overexpression of FOXA1 suppressed lung metastasis and EMT in a nude mice lung metastasis model. In summary, our research findings underscore the role of TGF-beta 1 in regulating TGF-beta 1/FOXA1-induced GBC EMT and metastasis by inhibiting FOXA1 translation efficiency through m(6)A modification.