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Receptor ligand-triggered resistance to alectinib and its circumvention by Hsp90 inhibition in EML4-ALK lung cancer cells.
Tanimoto A
,
Yamada T
,
Nanjo S
,
Takeuchi S
,
Ebi H
,
Kita K
,
Matsumoto K
,
Yano S
.
Abstract
Alectinib is a new generation ALK inhibitor with activity against the gatekeeper L1196M mutation that showed remarkable activity in a phase I/II study with echinoderm microtubule associated protein-like 4 (EML4)--anaplastic lymphoma kinase (ALK) non-small cell lung cancer (NSCLC) patients. However, alectinib resistance may eventually develop. Here, we found that EGFR ligands and HGF, a ligand of the MET receptor, activate EGFR and MET, respectively, as alternative pathways, and thereby induce resistance to alectinib. Additionally, the heat shock protein 90 (Hsp90) inhibitor suppressed protein expression of ALK, MET, EGFR, and AKT, and thereby induced apoptosis in EML4-ALK NSCLC cells, even in the presence of EGFR ligands or HGF. These results suggest that Hsp90 inhibitors may overcome ligand-triggered resistance to new generation ALK inhibitors and may result in more successful treatment of NSCLC patients with EML4-ALK.
Figure 1. 17-DMAG suppresses the growth of EML4-ALK NSCLC cells in the presence of HGF and EGFR ligandsThe EML4-ALK lung cancer cell lines human H2228 and human H3122 were treated with increasing concentrations of alectinib or 17-DMAG, with or without HGF (50 ng/mL), EGF (100 ng/mL), HB-EGF (10 ng/mL), and TGF-α (100 ng/mL), and cell viability was determined after 72 h by MTT assay. Data shown are representative of at least 3 independent experiments. Error bars indicate standard deviation (SD) of triplicate cultures.
Figure 2. HGF-gene transfection resulted in reducing susceptibility of EML4-ALK NSCLC cells to alectinib but not 17-DMAG H2228/Vec(A) or H2228/HGF (B) cells were treated with increasing concentrations of alectinib or 17-DMAG, and cell viability was determined after 72 h by MTT assay. Data shown are representative of at least 3 independent experiments. Error bars indicate SD of triplicate cultures.
Figure 3. 17-DMAG reduced MET protein expression and inhibited downstream pathways, even in the presence of HGFH2228/Vec or H2228/HGF cells were treated with or without alectinib (0.3 μmol/L) for 2 h or 17-DMAG (0.3 μmol/L) for 24 h and then stimulated with or without HGF (50 ng/mL) for 10 minutes. The resultant cells were lysed, and the indicated proteins were detected by immunoblotting. Data shown are representative of at least 3 independent experiments.
Figure 4. 17-DMAG reduced MET protein expression and inhibited downstream pathways, even in the presence of EGFR ligandsH2228 cells were treated with or without alectinib (0.3 μmol/L) for 2 h or 17-DMAG (0.3 μmol/L) for 24 h, and then stimulated with or without EGF (100 ng/mL), HB-EGF (10 ng/mL), and TGF-α (100 ng/mL) for 10 min. The resultant cells were lysed, and the indicated proteins were detected by immunoblotting. Data shown are representative of at least 3 independent experiments.
Figure 5. 17-DMAG induced apoptosis of EML4-ALK NSCLC cells, even in the presence of HGFA. Apoptotic cells were evaluated by the 7-AAD cell viability assay, as described in the Materials and Methods. B. Quantification of apoptotic cells.
Figure 6. 17-DMAG reduced viability of EML4-ALK NSCLC cells, even in the presence of both HGF and TGF-αH2228 and H3122 cells were incubated with or without alectinib (0.1 μmol/L), crizotinib (0.1 μmol/L), and/or HGF (50 ng/mL) and TGF-α (100 ng/mL), and cell viability was determined after 72 h by MTT assay. The percentage of cell viability is shown relative to controls without HGF or TGF-α treatment. *, P < 0.001 (one-way ANOVA). NS, not significant. Data shown are representative of at least 3 independent experiments. Error bars indicate SD of triplicate cultures.
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