Dual inhibition of αV integrins and Src kinase activity in colon cancer cells.

e14609 Background: Integrins are commonly upregulated in tumor cells and are important regulators of invasion and metastasis. Integrin signaling is initiated upon engagement of ECM and requires Src as well as various other proteins including ILK, FAK, and paxillin. METHODS: Presence of αv integrins on CRC cell lines was established by flow cytometry. CNTO 95 (10μg/ml, a monoclonal anti-αv integrin antibody; Ortho Biotech), and dasatinib (<200nM; BMS, src small molecule inhibitor) were used to study the effects of combined integrin and src inhibition on proliferation and serum-induced migration of colon cancer cell lines in vitro. Downstream signaling changes were assessed by immunoblotting for phosphorylated forms of src, FAK (Y397, Y576/577 and Y925), paxillin, GSK3β and AKT (S473) in HT29, HCT116 and RKO. RESULTS: Proliferation was inhibited by dasatinib in HT29, HCT116, DLD1 and HCT15 in a dose-dependent manner, while RKO and SW48 were resistant. Combining CNTO 95 with dasatinib further inhibited proliferation in all dasatinib-sensitive cells, yet resistant cells were unaffected. Migration was blocked by both CNTO 95 and dasatinib in all cell lines, and combining the two drugs produced augmented effect. Src activation and src- dependent FAK phosphorylation at sites 576 and 925 were blocked by dasatinib; CNTO 95 had little effect alone, but potentiated the effect of dasatinib. Paxillin phosphorylation was modestly blocked by both compounds, but the combination produced significantly augmented inhibition in the three cell lines tested. The phosphorylation status of AKT and GSK-3β, which are downstream of ILK, was inhibited by both drugs as single agents. The combination of dasatinib and CNTO 95 produced further inhibition. CONCLUSIONS: Dual inhibition of Src by dasatinib and αv integrins by CNTO 95 produced additive to synergistic inhibition of proliferation in dasatinib sensitive CRC cell lines, and inhibition of migration in all cell lines tested. Decreased phospho-paxillin levels may be responsible for the pronounced inhibition of migration observed after dual treatment with dasatinib and CNTO 95 in these CRC cell lines. These data support the rationale for combined Src/integrin inhibition in colon cancer, and further suggest approaches to patient selection strategies. [Table: see text].

Duke Scholars

Author Andrew Benjamin Nixon Medicine, Medical Oncology