SRC kinase promotes survival and invasion of non-small cell lung cancer cells with epidermal growth factor receptor abnormalities and is a potential candidate for combination targeted therapy

Abstract

Epidermal growth factor receptor (EGFR) mutations are common in female non-smoking non-small cell lung cancer (NSCLC) patients in Hong Kong. Tyrosine kinase inhibitor (TKI) against EGFR is an effective therapy for tumors with EGFR activating mutations. However, drug resistance due to second EGFR mutations or unknown mechanisms is frequently observed. Although Src is a mutual activator of EGFR, the effect of Src kinase inhibition on NSCLC is not fully known. To study the potential usefulness of Src kinase inhibition in treating NSCLC, we examined the effects of a specific Src inhibitor 4-(4’-Phenoxyanilino)-6,7-dimethoxyquinazolinee (SKI-1), alone or combined with TKI treatment, on apoptosis and invasion as well as the activation profiles of Src, EGFR and their downstream mediators in NSCLC cells harboring different EGFR abnormalities. SKI-1 specificity was validated by Src siRNA knockdown. Phosphorylation studies showed higher basal Src activities (pY416) in mutant (MT)-EGFR NSCLC cells (exon 19 deletion in HCC827; L858R substitution in FA31; L858R+T790M double substitution in H1975; amplification in H1819) than wild-type (WT) cells (H358 and HKULC2). The results suggest that NSCLC cells harboring MT-EGFR are more sensitive to SKI due to enhancement of basal Src kinase activation by MT-EGFR. Immunohistochemical study of clinical cancers showed correlation of Src activation and EGFR expression. Analysis using Hoechst 33258 staining, TUNEL, flow cytometry and PARP cleavage showed SKI-1 significantly induced apoptosis in H1819 and HCC827 but not H358 cells in a dose dependent manner. Co-treatment with SKI-1 and gefitinib (Iressa) synergistically enhanced apoptosis in FA31 and H1819 cells, suggesting a new combination treatment regime for NSCLC. Furthermore, SKI-1 significantly inhibited invasion and migration in cells with EGFR alterations in a dose dependent manner using Matrigel chamber and wound healing assays. The inhibition-profile of EGFR phosphorylation implicates that induction of apoptosis and sensitivity of HCC827 and H1819 to SKI treatment is in part related to EGFR downstream targets including Stat 3/5, ERK1/2 and Akt pathways. Phosphorylation of FAK and p130Cas is involved in Src-mediated invasion in SKI-1 sensitive cells, suggesting the involvement of Src in lung cancer metastasis. Overall, the findings indicate that Src kinase pathway could be a useful target for combination molecular treatment of NSCLC. We further suggest EGFR DNA sequence alterations are not the only factors that determine SKI sensitivity; other forms of abnormalities in EGFR may also be important determinants.