Osteosarcoma (OS) is the most prevalent primary cancer of the bone. Metastasis and chemoresistance are the major obstacles to the improvement of OS prognosis, in which N⁶-methyladenosine (m⁶A) modification plays an important role, but the exact molecular mechanisms are still unclear.

MeRIP-seq and RNA-seq were conducted on OS and paired adjacent normal tissue samples, which determined CACNA1E as a key m⁶A-modified molecule. In vitro and in vivo models were established to evaluate the function of CACNA1E on OS growth, metastasis, and methotrexate (MTX) resistance, and to explore the upstream regulators and downstream effectors of CACNA1E.

CACNA1E exhibited notable m⁶A hypermethylation and upregulated expression in OS than adjacent normal tissues. CACNA1E knockdown effectively hindered OS growth, lung metastasis, and MTX resistance. METTL3, an m⁶A “writer” boosted the mRNA stability of CACNA1E through m⁶A modification, and this process was recognized and enhanced by IGF2BP2, an m⁶A “reader”. WNT7B was identified as a downstream molecule of CACNA1E. CACNA1E facilitated OS progression and MTX resistance by enhancing the non-canonical Wnt/Ca²⁺ signaling through transcriptionally activating WNT7B. Furthermore, a novel combination treatment of targeted inhibition of CACNA1E with MTX had a synergistic effect on suppressing OS progression.

Collectively, our findings uncover that METTL3-mediated m⁶A modification of CACNA1E contributes to OS progression and chemoresistance through enhancing WNT7B-mediated non-canonical Wnt/Ca²⁺ signaling. Targeted inhibition of CACNA1E in combination with MTX may be a promising alternative therapeutic strategy for patients with MTX-resistant OS.