Tumor suppressor FOXO3 mediates signals from the EGF receptor to regulate proliferation of colonic cells

Abstract

Epithelial proliferation, critical for homeostasis, healing, and colon cancer progression, is in part controlled by epidermal growth factor receptor (EGFR). Proliferation of colonic epithelia can be induced by Citrobacter rodentium infection, and we have demonstrated that activity of tumor suppressor FOXO3 was attenuated after this infection. Thus the aim of this study was to determine the contribution of FOXO3 in EGFR-dependent proliferation of intestinal epithelia and colon cancer cell lines. In this study we show that, during infection with C. rodentium, EGFR was significantly phosphorylated in colonic mucosa and Foxo3 deficiency in this model lead to an increased number of bromodeoxyuridine-positive cells. In vitro, in human colon cancer cells, increased expression and activation of EGFR was associated with proliferation that leads to FOXO3 phosphorylation (inactivation). Following EGFR activation, FOXO3 was phosphorylated (via phosphatidylinositol 3-kinase/Akt) and translocated to the cytosol where it was degraded. Moreover, inhibition of proliferation by overexpressing FOXO3 was not reversed by the EGFR signaling, implicating FOXO3 as one of the regulators downstream of EGFR. FOXO3 binding to the promoter of the cell cycle inhibitor p27kip1 was decreased by EGFR signaling, suggesting its role in EGFR-dependent proliferation. In conclusion, we show that proliferation in colonic epithelia and colon cancer cells, stimulated by EGFR, is mediated via loss of FOXO3 activity and speculate that FOXO3 may serve as a target in the development of new pharmacological treatments of proliferative diseases.

FOXO3 Growth Inhibition of Colonic Cells Is Dependent on Intraepithelial Lipid Droplet Density

“…Chromatin Immunoprecipitation (ChIP) Assay-The cells, after cross-linkage with 1% formaldehyde, were incubated in lysis buffer, sonicated to shear DNA (200 to 1000 bp), and equal amounts of protein were incubated with anti-FOXO3 antibody (Cell Signaling Technology) as previously described (5).…”

“…During tumorigenesis, the loss of FOXO3 activity promotes tumor growth (8 -12). In human colon cancer cells, FOXO3 negatively regulates proliferation, and the inactivation of FOXO3 leads to a decrease of the cell cycle inhibitor p27kip1 and a loss of cell cycle arrest (2,5,13). Moreover, several critical regulators in colon cancer progression, such as p53 and members of the Wnt/␤-catenin pathway (14,15), alter FOXO3 activity, further revealing the significance of FOXO3 in colon cancer growth.…”

Type 3 Muscarinic Receptors Contribute to Clearance of Citrobacter rodentium

“…Delayed C. rodentium clearance from Chrm3 −/− mice, determined by fecal bacteria counts, was confirmed by demonstrating persistent adherence of C. rodentium to colonic mucosa as well as a greater crypt hyperproliferation 13 DPI when compared to C. rodentium -infected WT mice. Crypt hyperplasia is important to the clearance of C. rodentium and is attributed to the secretion of pathogen proteins that promote adherence to epithelial cells (46), epithelial growth factor receptor activation (47), and IFN-γ (48). Delayed clearance from Chrm3 −/− mice occurred despite an amplified upregulation of colonic IFN-γ as well as IL-17A, indicating that the mucosal immune system responds appropriately to C. rodentium infection in the absence of M3R.…”

Polyethylene glycol diminishes pathological effects ofCitrobacter rodentiuminfection by blocking bacterial attachment to the colonic epithelia

“…26,30 EPEC and C. rodentium induce the activation of membrane EGFR in colonic epithelia; 29,34 which is also activated by inflammatory stimuli. 35 This study shows that PEG inhibits C. rodentium-induced EGFR expression and activation.…”

“…We previously demonstrated that C. rodentium infection induces expression and activation of EGFR in mouse colonic mucosa. 29 Thus, we further assessed whether PEG inhibits C. rodentium-induced EGFR activation in colonic epithelia. Increased expression of 208 ± 23% (and phosphorylation) of EGFR in colonic mucosa induced by C. rodentium infection (CR) was reduced to 81 ± 14% in the presence of PEG (PEG-CR) (Fig.…”