Specific cross-talk between epidermal growth factor receptor and integrin alphavbeta5 promotes carcinoma cell invasion and metastasis

Abstract

Tyrosine kinase receptors and integrins play essential roles in tumor cell invasion and metastasis. Previously, we showed that epidermal growth factor (EGF) stimulation of pancreatic carcinoma cells led to invasion and metastasis that was blocked by antagonists of integrin alpha(v)beta(5). Here, we show that EGF stimulates metastasis of carcinoma cells via a Src-dependent phosphorylation of p130 CAS leading to activation of Rap1, a small GTPase involved in integrin activation. Specifically, EGF receptor (EGFR)-induced Src activity leads to phosphorylation of a region within the CAS substrate domain, which is essential for Rap1 and alpha(v)beta(5) activation. This pathway induces alpha(v)beta(5)-mediated invasion and metastasis in vivo yet does not influence primary tumor growth or activation of other integrins on these cells. These findings show cross-talk between a tyrosine kinase receptor and an integrin involved in carcinoma cell invasion and metastasis and may explain in part how inhibitors of EGFR affect malignant disease.

Fig. 1. (A) EGF induces avβ5-mediated Rap1 activation and cell metastasis

(A) EGF treatment increased activity of Rac1 and Rap1 in FG cells plated on anti-β5, but not anti-β1, integrin antibodies. (B) Rap1 knockdown blocked EGF-induced cell migration on vitronectin but not fibronectin. (C) Knockdown of β5 expression blocked EGF-induced pulmonary metastasis, but not primary tumor weight, in the chick CAM model. Mean±SEM, n≥6, *p≤0.05.

Fig. 2. Src kinase is necessary for EGF induced β5-mediated migration and metastasis

(A)Pretreatment with a Src inhibitor (SKI-606, 500nM) blocked EGF-induced migration on vitronectin. (B) Expression of CSK, but not CSK kinase dead (CSK-KD), blocked EGF-induced migration on vitronectin. Blockade of Src kinase activity and expression of CSK and CSK-KD was confirmed by immunoblotting. **p≤0.01 (C) Expression of CSK blocked EGF-induced pulmonary metastasis, but not primary tumor growth, in the chick CAM model. mean±SEM, n≥6, **p≤0.01.

Fig. 3. Src kinase is sufficient for β5-mediated migration and metastasis

(A) Expression of active Src (SrcA) selectively induced migration on vitronectin which could be blocked with anti-avβ5. (B) Intravenous injection with 100μg of a function-blocking antibody to β5 (P1F6), but not a non-function blocking β5 antibody (11D1), blocked pulmonary metastasis but not primary tumor growth in the chick CAM model. mean±SEM, n≥6, **p≤0.01. (C) Pretreatment with a Src inhibitor (SKI-606, 500nM) blocked EGF-induced migration of MCF-7 breast carcinoma cells on vitronectin. (D) EGF-induced migration of MCF-7 cells on vitronectin was blocked by treatment with an anti-β5 function-blocking antibody.

Fig. 4. FAK and CAS are substrates of Src in FG pancreatic carcinoma cells

(A) Src inhibition (SKI-606, 500nM) blocked EGF-induced tyrosine phosphorylation of 120-140kD proteins (arrow). (B) Treatment with a FAK inhibitor (PF228, 1mM) blocked FAK autophosphorylation on Y397, EGF-induced FAK pY861, and EGF-induced migration on vitronectin and fibronectin. **p≤0.01. (C) The Src inhibitor (SKI-606, 500nM) blocked EGF-induced CAS phosphorylation in FG cells, and constitutive CAS activity in FG cells expressing active Src (SrcA).

Fig. 5. The first 9 YXXP tyrosine residues in the CAS substrate domain are required for αvβ5-mediated migration and metastasis

(A) Expression of the F1-15 or F1-9 CAS mutants blocked EGF-induced migration toward vitronectin. The Src inhibitor (SKI-606, 500nM) blocked EGF-induced migration of FG cells expressing WT CAS or the F10-15 CAS mutant. (B) Expression of the F1-9 (but not F10-15) CAS mutant blocked EGF-induced Rap1 activity, but did not impact Rac, Rho, or cdc42. Vertical lines separate samples from a single gel. (C) Expression of the F1-9 CAS mutant blocked EGF-induced pulmonary metastasis but not primary tumor growth in the chick CAM model. mean±SEM, n=7, *p≤0.05. (D) EGF stimulates EGFR to recruit and activate Src kinase and β5 integrin, leading to activation of CAS and Rap1, which facilitate actin remodeling to enable cell invasion and metastasis.