Abstract
One hallmark of cancer cells is sustaining proliferative signaling that leads to uncontrolled cell proliferation. Both the Forkhead box (FOX) M1 transcription factor and the Epidermal Growth Factor (EGF) receptor Pathway Substrate 8 (EPS8) are known to be activated by mitogenic signaling and their levels upregulated in cancer. Well-known to regulate Rac-mediated actin remodeling at the cell cortex, EPS8 carries a nuclear localization signal but its possible nuclear role remains unclear. Here, we demonstrated interaction of FOXM1 with EPS8 in yeast two-hybrid and immunoprecipitation assays. Immunostaining revealed co-localization of the two proteins during G2/M phase of the cell cycle. EPS8 became nuclear localized when CRM1/Exportin 1-dependent nuclear export was inhibited by Leptomycin B, and a functional nuclear export signal could be identified within EPS8 using EGFP-tagging and site-directed mutagenesis. Downregulation of EPS8 using shRNAs suppressed expression of FOXM1 and the FOXM1-target CCNB1, and slowed down G2/M transition in cervical cancer cells. Chromatin immunoprecipitation analysis indicated recruitment of EPS8 to the CCNB1 and CDC25B promoters. Taken together, our findings support a novel partnering role of EPS8 with FOXM1 in the regulation of cancer cell proliferation and provides interesting insight into future design of therapeutic strategy to inhibit cancer cell proliferation.
Highlights
Sustaining proliferative signaling in cancer cells can result from overproduction of growth factors in an autocrine manner or or constitutive activation of growth factor receptors by mutations [1]
EPS8 interacted with both the b and c isoforms of FOXM1 but the strength of interaction was stronger for FOXM1c, suggesting that exon Va that is differentially spliced out in FOXM1b contains part of the interaction interface
Our findings support that EPS8 is a critical partnering factor of FOXM1 in the regulation of mitotic gene expression and cell cycle progression. It is well-documented that the proliferation-associated Forkhead box transcription factor FOXM1 is activated by mitogenic signals
Summary
Sustaining proliferative signaling in cancer cells can result from overproduction of growth factors in an autocrine manner or or constitutive activation of growth factor receptors by mutations [1]. Binding of RTKs by growth factors such as Epidermal Growth Factor (EGF) leads to stimulation of their intrinsic intracellular protein-tyrosine kinase activity and the autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of RTKs. The resulting docking sites lead to recruitment and activation of specific SH2-domain-containing signal transduction proteins that can initiate several signal transduction cascades, in particular the Rat Sarcoma (RAS)/Mitogen-Activated Protein Kinase (MAPK) pathway that stimulates cell proliferation. RAS/MAPK signaling affects gene transcription by regulating the activity of transcription factors encoded by immediate early genes and the Forkhead box transcription factor FOXM1 [2]. With the expression highly associated with cell proliferation, EPS8 Is FOXM1-Cooperative Factor
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