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

Abstract

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.