N-WASP Attenuates Cell Proliferation and Migration through ERK2-Dependent Enhanced Expression of TXNIP

SIMPLE SUMMARY: Neural Wiskott–Aldrich Syndrome Protein (N-WASP) regulates actin cytoskeleton remodeling and can, it has been suggested, suppress several cancers. In this study, HSC-5 cells, a mammalian cell line with reduced N-WASP expression, were used to generate control cells and HSC-5 cells with increased N-WASP expression that is comparable to that of normal keratinocytes. The two cell lines were used to elucidate the regulation of cell proliferation and migration by N-WASP. Our findings suggest that N-WASP increases ERK2-dependent phosphorylation of FOXO1 and increases TXNIP expression, which reduces cell proliferation and migration. This study is the first to propose an antiproliferative role of N-WASP, which is mediated via ERK2, and it suggests new avenues for cancer therapeutic research and treatment. ABSTRACT: Neural Wiskott–Aldrich Syndrome Protein (N-WASP) regulates actin cytoskeleton remodeling. It has been known that reduced N-WASP expression in breast and colorectal cancers is associated with poor prognosis. Here, we found reduced N-WASP expression in squamous cell carcinoma (SCC) patient samples. The SCC cell line HSC-5 with reduced N-WASP expression was used to generate HSC-5(CN) (control) and HSC-5(NW) (N-WASP overexpression) cells. HSC-5(NW) cells had reduced cell proliferation and migration compared to HSC-5(CN) cells. HSC-5(NW) cells had increased phospho-ERK2 (extracellular signal-regulated kinase 2), phosphorylated Forkhead box protein class O1 (FOXO1) and reduced nuclear FOXO1 staining compared to HSC-5(CN) cells. Proteasome inhibition stabilized total FOXO1, however, not nuclear staining, suggesting that FOXO1 could be degraded in the cytoplasm. Inhibition of ERK2 enhanced nuclear FOXO1 levels and restored cell proliferation and migration of HSC-5(NW) to those of HSC-5(CN) cells, suggesting that ERK2 regulates FOXO1 activity. The expression of thioredoxin-interacting protein (TXNIP), a FOXO1 target that inhibits thioredoxin and glucose uptake, was higher in HSC-5(NW) cells than in HSC-5(CN) cells. Knockdown of TXNIP in HSC-5(NW) cells restored cell proliferation and migration to those of HSC-5(CN) cells. Thus, we propose that N-WASP regulates cell proliferation and migration via an N-WASP-ERK2-FOXO1-TXNIP pathway.