Mechanism of resistance to 17-DMAG, an Hsp90 inhibitor, in lung cancer cell lines with ALK rearrangement

Abstract

Heat shock protein 90 (Hsp90) inhibitors have shown clinical activity against lung cancer cells with rearrangement of the anaplastic lymphoma kinase (ALK) gene. We investigated the mechanism of acquired resistance to 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a geldanamycin analog Hsp90 inhibitor, in H3122 and H2228 lung cancer cell lines with ALK rearrangement. Resistant cell lines (H3122/DR-1, H3122/DR-2, and H2228/DR) were established by repeated exposure to increasing concentrations of 17-DMAG. MTT assay, western blotting analysis and reverse transcription-polymerase chain reaction were done to assess cytotoxicity, relative proteins and mRNA expression. Flow cytometry after incubation with rhodamine 123 (Rho123) performed for determining the activity of P-glycoprotein (P-gp

ABCB1/ MDR1). The resistant cells showed no cross-resistance to AUY922 or ALK inhibitors, suggesting persistent ALK dependency of cells with acquired resistance to 17-DMAG. Interestingly, all resistant cells showed the induction of P-gp at the protein and RNA levels, which was associated with increased efflux of the P-gp substrate Rho123. Transfection with siRNA directed against P-gp or treatment with verapamil, an inhibitor of P-gp, restored sensitivity to the drug in all cells with acquired resistance to 17-DMAG. Furthermore, we observed that the growth-inhibitory effect of 17-DMAG decreased in A549/PR and H460/PR cells generated to overexpress P-gp by long-term exposure to paclitaxel and that these cell lines recovered the sensitivity to 17-DMAG through inhibition of P-gp. Although the expression of NAD(P)H:quinone oxidoreductase 1 (NQO1), previously known as one of the factors associated with 17-DMAG resistance, decreased in H3122/DR-1 and H3122/DR-2 cells, NQO1 inhibition by dicumarol did not affect the response of H3122 cells to 17-DMAG. In summary, P-gp overexpression is one of the possible mechanisms of acquired resistance to 17-DMAG in lung cancer cells with ALK rearrangement.