CFTR regulates migration and EGFR signaling in fetal human colon cells

Abstract

Objective To investigate the pathogenesis of colon cancer by determining the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in regulating cell migration and epidermal growth factor receptor (EGFR) signaling. Background Individuals with cystic fibrosis (CF) over the age of 40 are 4–8 times more likely to get colon cancer than the general population. CF patients who have a history of distal intestinal obstruction syndrome and organ transplant have even higher risk of developing colon cancer. Better understanding the pathogenesis of colon cancer in CF patients may lead to improved treatments for this population. The mechanisms leading to increased colon cancer in CF patients is not fully understood, but dysfunctional Cl− transport is highly implicated. CFTR dysfunction in several tumor types has been linked to increased malignant features, such as decreased cell-to-cell adhesion and epithelial-to-mesenchymal transformation. CFTR dysfunction has also been linked to increased inflammation. We hypothesize that CFTR dysfunction in CF patients leads to colon cancer by promoting increased cell migration and altered epidermal growth factor receptor (EGFR) signaling. Methods Scratch assays were performed on fetal human colon epithelial cells (FHC) and the rate of cell migration was measured 2 days after treatment with either CFTR activating or inhibiting compounds. EGFR localization was analyzed using standard immunohistochemical labeling techniques and the fluorescence intensity of EGFR was analyzed using ImageJ. Results Colon cells treated with 500 µM IBMX and 5 µM forskolin (CFTR activators) had significantly decreased rates of cell migration (0.031 mm2/24 hours) compared to the control group (0.26 mm2/24 hours) n=6; p<0.05. Cells treated with CFTR siRNA had significantly increased rates of cell migration (0.23 mm2/24 hours) compared to the scramble siRNA control (0.08 mm2/24 hours) n=4; p<0.05. Immunohistochemistry analysis of control and colon cells treated with 500 µM IBMX and 5 µM forskolin showed low levels of EGFR labelling (restricted to cell periphery), whereas cells treated with 5 mM CFTR(inh)-172 (CFTR inhibitor) showed more robust EGFR signal throughout the cytoplasm in 3 independent observations. Likewise, CFTR siRNA significantly increased punctate EGFR labeling in colon cells. Significance This project provides mechanistic insight into a new and critically important trend recently identified in CF patients that have increased risk for colon cancer. Our results indicate that CFTR plays an important role in regulating cell growth and migration via EGFR signaling in colon epithelia. Future studies will be aimed towards identifying novel therapeutic targets for CF and colon cancer patients to improve health outcomes.