The essential roles of m 6 A RNA modification to stimulate ENO1-dependent glycolysis and tumorigenesis in lung adenocarcinoma

Background Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer. Patient prognosis is poor, and the existing therapeutic strategies for LUAD are far from satisfactory. Recently, targeting N6-methyladenosine (m 6 A) modification of RNA has been suggested as a potential strategy to impede tumor progression. However, the roles of m 6 A modification in LUAD tumorigenesis is unknown. Methods Global m 6 A levels and expressions of m 6 A writers, erasers and readers were evaluated by RNA methylation assay, dot blot, immunoblotting, immunohistochemistry and ELISA in human LUAD, mouse models and cell lines. Cell viability, 3D-spheroid generation, in vivo LUAD formation, experiments in cell- and patient-derived xenograft mice and survival analysis were conducted to explore the impact of m 6 A on LUAD. The RNA-protein interactions, translation, putative m 6 A sites and glycolysis were explored in the investigation of the mechanism underlying how m 6 A stimulates tumorigenesis. Results The elevation of global m 6 A level in most human LUAD specimens resulted from the combined upregulation of m 6 A writer methyltransferase 3 (METTL3) and downregulation of eraser alkB homolog 5 (ALKBH5). Elevated global m 6 A level was associated with a poor overall survival in LUAD patients. Reducing m 6 A levels by knocking out METTL3 and overexpressing ALKBH5 suppressed 3D-spheroid generation in LUAD cells and intra-pulmonary tumor formation in mice. Mechanistically, m 6 A-dependent stimulation of glycolysis and tumorigenesis occurred via enolase 1 (ENO1). ENO1 mRNA was m 6 A methylated at 359 A, which facilitated it’s binding with the m 6 A reader YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) and resulted in enhanced translation of ENO1. ENO1 positively correlated with METTL3 and global m 6 A levels, and negatively correlated with ALKBH5 in human LUAD. In addition, m 6 A-dependent elevation of ENO1 was associated with LUAD progression. In preclinical models, tumors with a higher global m 6 A level showed a more sensitive response to the inhibition of pan-methylation, glycolysis and ENO activity in LUAD. Conclusions The m 6 A-dependent stimulation of glycolysis and tumorigenesis in LUAD is at least partially orchestrated by the upregulation of METTL3, downregulation of ALKBH5, and stimulation of YTHDF1-mediated ENO1 translation. Blocking this mechanism may represent a potential treatment strategy for m 6 A-dependent LUAD.