Glucocorticoid modulatory element-binding protein 1 (GMEB1) interacts with the de-ubiquitinase USP40 to stabilize CFLAR_L and inhibit apoptosis in human non-small cell lung cancer cells.

BACKGROUND:GMEB1 was originally identified via its interaction with GMEB2, which binds to the promoter region of the tyrosine aminotransferase (TAT) gene and modulates transactivation of the glucocorticoid receptor gene. In the cytosol, GMEB1 interacts with and inhibits CASP8, but the molecular mechanism is currently unknown. METHODS:Human non-small cell lung cancer cells and 293FT cells were used to investigate the function of GMEB1/USP40/CFLAR_L complex by WB, GST Pull-Down Assay, Immunoprecipitation, Immunofluorescence and Flow cytometry analysis. A549 cells overexpressing green fluorescent protein and GMEB1 shRNA were used for tumor xenograft using female athymic nu/nu 4-week-old mice. RESULTS:We found GMEB1 interacted with CFLAR_L (also known as c-FLIP_L) in the cytosol and promoted its stability. USP40 targeted CFLAR_L for K48-linked de-ubiquitination. GMEB1 promoted the binding of USP40 to CFLAR_L. USP40 knockdown did not increase CFLAR_L protein level despite GMEB1 overexpression, suggesting GMEB1 promotes CFLAR_L stability via USP40. Additionally, GMEB1 inhibited the activation of pro-caspase 8 and apoptosis in non-small cell lung cancer (NSCLC) cell via CFLAR_L stabilization. Also, GMEB1 inhibited the formation of DISC upon TRAIL activation. CFLAR_L enhanced the binding of GMEB1 and CASP8. Downregulation of GMEB1 inhibited A549 xenograft tumor growth in vivo. CONCLUSIONS:Our findings show the de-ubiquitinase USP40 regulates the ubiquitination and degradation of CFLAR_L; and GMEB1 acts as a bridge protein for USP40 and CFLAR_L. Mechanistically, we found GMEB1 inhibits the activation of CASP8 by modulating ubiquitination and degradation of CFLAR_L. These findings suggest a novel strategy to induce apoptosis through CFLAR_L targeting in human NSCLC cells.

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