Epidermal Growth Factor Substrate 8 is an essential partnering factor of the Forkhead box transcription factor FOXM1 in the regulation of cell proliferation in cervical cancer cells

Abstract

The proliferation-associated Forkhead box transcription factor FOXM1 (named Molecule of the Year in 2010) is increasingly recognized as marker and therapeutic target in cancers of multiple tissues of origin. FOXM1 plays critical role in maintaining the proliferation of cancer cells and its function is activated by mitogenic signals. In a yeast two-hybrid screen for FOXM1-interacting proteins, we identified Epidermal Growth Factor (EGF) Substrate 8 (EPS8), the phosphorylation of which is stimulated by EGF signaling. FOXM1 co-localized with EPS8 at the G2/M phase of the cell cycle and both proteins can be reciprocally immunoprecipitated from lysates prepared from cervical cancer cells. Interestingly, the nuclear translocation of EPS8 was dramatically enhanced by treatment with the exportin 1 inhibitor leptomycin B, suggesting that EPS8 is actively regulated by nuclear export. Site-directed mutagenesis defined a functional nuclear export signal within EPS8. To test the relevance of FOXM1-EPS8 interaction in the regulation of FOXM1 function, we depleted EPS8 expression in cervical cancer cells by RNA interference. Importantly, depletion of EPS8 led to G2/M arrest and suppression of expression of FOXM1 and its known target genes including CCNB1 and CDK1. Moreover, chromatin immunoprecipitation analysis revealed recruitment of both FOXM1 and EPS8 to the promoters of CCNB1 and CDK1, consistent with EPS8 being a partnering factor of FOXM1 in transcriptional regulation. Taken together, our findings provide the first evidence to support a novel role of EPS8 in modulating the function of FOXM1, which is relevant to the development of cancer diagnostic strategy and targeted therapy.