WZ4002, a third-generation EGFR inhibitor, can overcome anoikis resistance in EGFR-mutant lung adenocarcinomas more efficiently than Src inhibitors.

Src has a role in the anoikis resistance in lung adenocarcinomas. We focused on two epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cell lines, HCC827 (E746-A750 deletion) and H1975 (L858R+T790M), in suspension to elucidate whether suspended lung adenocarcinoma cells are eradicated by long-term treatment with Src tyrosine kinase inhibitors (TKIs). We also examined metastasis-positive lymph nodes from 16 EGFR-mutant lung adenocarcinoma patients for immunohistochemical expression of mutant-specific EGFR. Almost all suspended HCC827 cells underwent apoptosis after 144 h of combination treatment with AZD0530, trichostatin A (TSA), and ABT-263, whereas many suspended H1975 cells survived the treatment. AZD0530 is a Src TKI, TSA is a histone deacetylase inhibitor, and ABT-263 is a Bcl-2 inhibitor. During the therapy, the phosphorylation of EGFR decreased in HCC827 cells and remained stable in H1975 cells. The phosphorylated EGFR of Src TKI-resistant H1975 cells, as well as HCC827 cells, was completely suppressed by the third generation EGFR TKI, WZ4002. Consequently, both the suspended cell lines were almost completely eradicated within 144 h, with the combined therapy of WZ4002, ABT-263, and TSA. Interestingly, treated suspended cells underwent apoptosis to a greater extent than did adherent cells. Intrasinus floating lung adenocarcinoma cells in the lymph nodes expressed a mutant-specific EGFR. These findings suggest that suspended EGFR-mutant lung adenocarcinoma cells depend significantly more on EGFR activation for survival than attached cells do. The tumor cells circulating in vessels, which express mutant-specific EGFR, would be highly susceptible to the combination therapy of WZ4002, ABT-263, and TSA.

ABT-263, a Bcl-2 inhibitor, enhances the susceptibility of lung adenocarcinoma cells treated with Src inhibitors to anoikis.

The tyrosine kinase Src plays an important role in the development of anoikis resistance in lung adenocarcinomas. Several suspension lung adenocarcinoma cell lines, which express phosphorylated Src, undergo apoptosis, or anoikis, in the presence of Src kinase inhibitors. However, lung adenocarcinoma cell lines vary in their sensitivity to Src inhibitors. We hypothesized that the addition of ABT-263, a potent Bcl-2 inhibitor, should significantly enhance the degree of anoikis in lung adenocarcinoma cells treated with Src inhibitors. In this study, we treated four suspension lung adenocarcinoma cell lines with ABT-263, an Src inhibitor (bosutinib or PP1), or a combination of both. In LC-KJ and HCC827 cells, combined treatment with ABT-263 and an Src inhibitor effectively induced anoikis, and the extent of anoikis was significantly greater than that induced by each agent alone; the synergy between the two drugs was apparent. Although we did not observe a marked increase in anoikis in LC-KJ and HCC827 cells treated with ABT-263 alone, H1650 and H1975 cells treated with ABT-263 (1 µM) upon detachment significantly underwent apoptosis, the levels of which were much greater than those observed upon attachment. However, the levels of anoikis induced by combination treatment were still greater than those by the individual agents in H1650 and H1975 cells. These findings provide a biological rationale to test combination therapy with ABT-263 and Src inhibitors in patients with lung adenocarcinoma.