Novel nuclear partnering role of EPS8 with FOXM1 in regulating cell proliferation

Abstract

The forkhead box transcription factor M1 (FOXM1) is ubiquitously expressed in proliferating cells and regulates expression of genes controlling cell cycle progression. Its overexpression was reported in various human malignancies, indicating dependence of tumor cell growth on FOXM1. Recently studies associated FOXM1 with “epithelial-mesenchymal transition (EMT)”, an event closely related to cancer metastasis. These render FOXM1 a diagnostic marker and target for development of anti-cancer drugs as it is crucial for cancer cell proliferation and migration/invasion. Interestingly, previous screening studies in our lab identified Epidermal Growth Factor Receptor (EGFR) Pathway Substrate 8 (EPS8) as a specific interactor of FOXM1. Similar to FOXM1, EPS8 contributes to cell proliferation, and its overexpression and constitutive phosphorylation are observed in numerous cancers, suggesting that crosstalk may exist between them.

In this study, I first attempted to validate the interaction between FOXM1 and EPS8, using coimmunoprecipitation and yeast two-hybrid analyses. Coimmunoprecipitation using human cervical cancer cell lines confirmed that EPS8 is an interactor of FOXM1. Yeast two-hybrid studies suggested FOXM1c as a stronger interactor of EPS8 than FOXM1b, and the SH3 domain of EPS8 was not necessary for interaction. Indeed, two critical FOXM1-binding regions was identified.

Next, I investigated the correlation of EPS8 with the cell cycle. Synchronization of HeLa cells at G1/S phase and subsequent release into the cell cycle revealed that EPS8 exhibited nuclear staining at late S and G2/M phases, colocalizing with FOXM1. Knockdown of EPS8 expression in HeLa cells led to decreased expression of FOXM1 and its downstream target Cyclin B1, which controls G2/M progression. Consistently, DNA/PI analysis revealed that cell cycle progression at G2/M phase was decelerated upon EPS8 depletion.

As data suggested that EPS8 can enter the nucleus, similar to FOXM1, the mechanisms of its regulated nuclear entry was investigated. Treatment of HeLa, C33A and HEK293T cells with Leptomycin B (LMB) resulted in nuclear retention of EPS8, suggesting regulation by CRM1-dependent nuclear export. Subsequent analysis by overexpressing EGFP-EPS8 mutants in HEK293T cells confirmed the existence of a functional nuclear export sequence (NES) in EPS8. EPS8 belongs to a family of proteins, with EPS8L2 as the most closely related member with overlapping expression patterns. Interestingly, EPS8L2 was also detected in human cervical cancer cells, and was responsive to LMB treatment, similar to EPS8.

Recent studies reported association of nuclear cargo proteins with certain phosphorylated tyrosine residues on EPS8, but EGFP-EPS8 mutants carrying constitutively phosphorylated or non-phosphorylated mimics of the residues showed no difference in subcellular localization from wild-type EPS8 in this study.

In summary, this study provided evidence to support the notion that EPS8 has a novel nuclear partnering role with FOXM1, and its interaction with FOXM1 affects expression of FOXM1 downstream target genes that regulate cell cycle progression, especially G2/M-specific events. This provides important insight into the crosstalk of growth factor signaling pathways with FOXM1 in the regulation of cancer cell proliferation, which is of major biological interest and is useful for developing future strategies against cancer cell proliferation and migration/invasion.