WTAP regulates TRAF6 expression and vascular plasticity-associated phenotypes in experimental cell models

Background Hepatocellular carcinoma (HCC) is a highly vascular malignancy characterized by marked tumor vascular plasticity. Vasculogenic mimicry (VM)-associated phenotypes have been linked to aggressive tumor behavior and reduced sensitivity to antiangiogenic therapy, but the epitranscriptomic mechanisms contributing to these phenotypes in HCC remain incompletely defined. This study examined whether Wilms tumor 1-associated protein (WTAP) regulates cell growth and vascular plasticity-associated phenotypes in experimental models through m6A-associated post-transcriptional regulation of TNF receptor-associated factor 6 (TRAF6). Methods WTAP and TRAF6 expression was measured in HepG2 and BEL-7402 cells and the hepatocyte comparator using RT-qPCR and Western blotting. Gain- and loss-of-function experiments were performed to assess cell viability, wound closure, invasion, the ability of the examined cells to support vascular-like network formation in tumor cell-HUVEC co-culture, and the expression of vascular plasticity- and EMT-associated proteins. MeRIP-qPCR was used to evaluate m6A enrichment of TRAF6 transcripts, and an Actinomycin D chase assay was performed to assess TRAF6 mRNA stability. WT/MUT reporter assays, YTHDF1 RNA immunoprecipitation followed by qPCR (RIP-qPCR), and YTHDF1 depletion were used to examine additional aspects of TRAF6 regulation. Subcutaneous xenograft models were used to evaluate tumor growth and PAS-positive/CD31-negative VM-like structures. Results WTAP and TRAF6 were upregulated in the examined cells. WTAP overexpression enhanced cell viability, wound closure, invasion, tumor cell-supported vascular-like network formation, and vascular plasticity/EMT-associated molecular changes. WTAP manipulation altered TRAF6 mRNA abundance, m6A enrichment, and decay; its effect was greater on the WT than the MUT reporter. WTAP overexpression also increased TRAF6 RNA recovery in YTHDF1 immunoprecipitates. YTHDF1 depletion reduced the associated TRAF6 protein increase despite persistently elevated mRNA. TRAF6 knockdown attenuated WTAP-driven phenotypes and tumor growth, while TRAF6 overexpression partially rescued the effects of WTAP knockdown. PAS/CD31 staining provided qualitative evidence of VM-like morphology. Conclusion WTAP contributes to the observed cellular and xenograft phenotypes, partly through TRAF6 post-transcriptional regulation associated with altered m6A enrichment and mRNA stability. YTHDF1 may regulate TRAF6 protein, but its role in mRNA stabilization remains unproven. The precise m6A sites and perfusion of VM-like structures remain unclear and warrant validation in clinically relevant HCC models and human specimens.