Fig 2: FAK activation via ITGAvB3 induces AUY922 resistance in KRAS-mutated NSCLC cells. (A) Western blot analysis of the ITGB3 and ITGAv proteins in the acquired AUY922-resistant NSCLC cell lines (A549R and H1944R). (B) Real-time PCR analysis of miR-150 and miR-142 expression in the acquired AUY922-resistant NSCLC cell lines. (C) Western blotting analysis of ITGB3, pFAK, pRSK, RSK, pERK, AKT, pAKT, pMEK, and HSP90 expression in A549, A549R, H1944, and H1944R cell lines following incubation with 1 μM of AUY922. β-Actin was included as a loading control. FAK, focal adhesion kinase; ITGAv, integrin Av; ITGB3, integrin β3; NSCLC, non–small cell lung cancer; PCR, polymerase chain reaction.

Fig 3: FAK activation via miR-150 and miR-142 downregulation induce AUY922 resistance. (A) Real-time PCR analysis of ectopic miR-150 expression in A549R/miR-150 and H1944/miR-150 stable cells. (B) Effect of ectopic miR-150 expression on ITGB3, pFAK, TSPAN7 and N-cadherin in miR-control and miR-150 transfected resistant cells, revealed by western blot analysis. (C, D) Ectopic miR-150 expression renders A549R and H1944R cells sensitive to AUY922. Cell viability assays were performed at 72 hours after AUY922 treatment of the indicated cells at different concentrations. Data represent the mean±SD of triplicate experiments relative to untreated cells. (E) Real-time PCR analysis of ectopic miR-142 expression in A549R/miR-142 and H1944/miR-142 stable cells. (F) Effect of ectopic miR-142 expression on ITGB3, pFAK in miR-control and miR-142 transfected resistant cells, revealed by western blot analysis. (G, H) Ectopic miR-142 expression renders A549R and H1944R cells sensitive to AUY922. Cell viability assays were performed at 72 hours after AUY922 treatment of the indicated cells at different concentrations. Data represent the mean±SD of triplicate experiments relative to untreated cells. FAK, focal adhesion kinase; ITGAv, integrin Av; ITGB3, integrin β3; PCR, polymerase chain reaction; SD, standard deviation; TSPAN, tetraspanin 7.

Fig 4: Overexpression of ITGAv and ITGB3 induces AUY922 resistance in KRAS-mutated non–small cell lung cancer (NSCLC) cells. (A) Western blot analysis of ITGB3, ITGAv, and pFAK proteins in stably ITGAv and ITGB3 expressing NSCLC cell lines. (B, C) Effect of ITGAv and ITGB3 on AUY922 sensitivity of two KRAS-mutant NSCLC cell lines, A549/ITGAvB3 and H1944/ITGAvB3. CellTiter-Glo Luminescent Cell Viability Assays were performed 72 hours after drug treatment. Data represent the mean±SD of triplicate measurements relative to untreated cells. (D) IC50 values are reported in the table. (E) Anti-apoptotic effect of TAE226 in A549/ITGAvB3 and H1944/ITGAvB3 cell lines. CellTiter-Glo Luminescent Cell Viability Assays were performed 72 hours after drug treatment. (F) Western blot analysis of ITGB3, ITGAv, and pFAK proteins in stably ITGAv and ITGB3 knock-downed AUY922-resistant NSCLC cell lines. FAK, focal adhesion kinase; ITGAv, integrin Av; ITGB3, integrin β3; NSCLC, non–small cell lung cancer; SD, standard deviation.