Cell surface integrin α5ß1 clustering negatively regulates receptor tyrosine kinase signaling in colorectal cancer cells via glycogen synthase kinase 3.

Abstract

As a key process within the tissue microenvironment, integrin signaling can influence cell functional responses to growth factor stimuli. We show here that clustering of integrin α5ß1 at the plasma membrane of colorectal cancer-derived epithelial cells modulates their ability to respond to stimulation by receptor tyrosine kinase (RTK)-activating growth factors EGF, NRG and HGF, through GSK3-mediated suppression of Akt pathway. We observed that integrin α5ß1 is lost from the membrane of poorly organized human colorectal tumors and that treatment with the integrin-clustering antibody P4G11 is sufficient to induce polarity in a mouse tumor xenograft model. While adding RTK growth factors (EGF, NRG and HGF) to polarized colorectal cancer cells induced invasion and loss of monolayer formation in 2D and 3D, this pathological behavior could be blocked by P4G11. Phosphorylation of ErbB family members as well as MET following EGF, NRG and HGF treatment was diminished in cells pretreated with P4G11. Focusing on EGFR, we found that blockade of integrin α5ß1 increased EGFR phosphorylation. Since activity of multiple downstream kinase pathways were altered by these various treatments, we employed computational machine learning techniques to ascertain the most important effects. Partial least-squares discriminant analysis identified GSK3 as a major regulator of EGFR pathway activities influenced by integrin α5ß1. Moreover, we used partial correlation analysis to examine signaling pathway crosstalk downstream of EGF stimulation and found that integrin α5ß1 acts as a negative regulator of the AKT signaling cascade downstream of EGFR, with GSK3 acting as a key mediator. We experimentally validated these computational inferences by confirming that blockade of GSK3 activity is sufficient to induce loss of polarity and increase of oncogenic signaling in the colonic epithelial cells.