TNF-α drives bladder cancer metastasis via METTL3-mediated m6A modification to promote CLASP2/IQGAP1-dependent cytoskeleton remodeling.

BACKGROUND

Bladder cancer (BCa) metastasis is a multi-step process triggered by cytoskeleton reorganization. However, the regulation mechanism of cytoskeleton reorganization in BCa remains ambiguous. This study elucidated the influence of tumor necrosis factor-alpha (TNF-α) in cytoskeleton remodeling during BCa metastasis and its possible mechanisms.

METHODS

Colony formation, scratch, transwell, and the nude mouse model were adopted to evaluate the growth and metastasis. Molecular expression was assessed by immunohistochemical staining, quantitative real-time PCR (qRT-PCR), and Western blotting. The N6-methyladenosine (m6A) level was detected by methylated RNA immunoprecipitation (MeRIP). Protein interaction was validated by Co-immunoprecipitation (Co-IP). Immunofluorescence staining was used to identify rearrangement of actin filament fibers (F-actin) and protein colocalization.

RESULTS

TNF-α facilitated cytoplasmic linker associated protein 2 (CLASP2) and methyltransferase like 3 (METTL3) expression in a dose (10-50 ng/mL)-dependent manner in BCa. CLASP2 high expression suggested a shorter overall survival of BCa patients. CLASP2 deficiency suppressed BCa cell proliferation, migration, and invasion via disrupting F-actin cytoskeleton. Mechanistically, TNF-α promoted METTL3-mediated m6A modification of CLASP2 to enhance CLASP2 mRNA stability. Moreover, CLASP2 directly interplayed with IQ motif containing GTPase activating protein 1 (IQGAP1) to regulate F-actin cytoskeleton remodeling. In vivo data demonstrated that inhibition of METTL3/CLASP2 axis delayed lung metastasis in nude mice.

CONCLUSION

TNF-α favors BCa cell metastasis, which involves METTL3-mediated m6A modification of CLASP2 that interacts with IQGAP1, thus leading to F-actin cytoskeleton remodeling. Our findings suggest targeting TNF-α/METTL3/CLASP2/IQGAP1 axis as a potential avenue for promising treatment for BCa.