Tanizaki J et al. (2012), Combined effect of ALK and MEK inhibitors in EM...

ECB-ART-50135

Br J Cancer 2012 Feb 14;1064:763-7. doi: 10.1038/bjc.2011.586.

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Combined effect of ALK and MEK inhibitors in EML4-ALK-positive non-small-cell lung cancer cells.

Tanizaki J , Okamoto I , Takezawa K , Sakai K , Azuma K , Kuwata K , Yamaguchi H , Hatashita E , Nishio K , Janne PA , Nakagawa K .

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BACKGROUND: Although most non-small-cell lung cancer (NSCLC) patients with the echinoderm microtubule-associated protein-like 4 (EML4) - anaplastic lymphoma kinase (ALK) fusion gene - benefit from ALK tyrosine kinase inhibitors (ALK-TKIs), the efficacy of these drugs varies greatly among individuals. METHODS: The antitumour action of ALK-TKIs in EML4-ALK-positive NSCLC cell lines was evaluated from their effects on cell proliferation, signal transduction, and apoptosis. RESULTS: The ALK-TKI TAE684 inhibited cell proliferation and induced apoptosis, in association with inhibition of STAT3 and ERK phosphorylation, in EML4-ALK-positive H3122 cells. TAE684 inhibited STAT3 phosphorylation, but not ERK phosphorylation, and it showed little effect on cell proliferation or apoptosis, in EML4-ALK-positive H2228 cells. The combination of TAE684 and a MEK inhibitor-induced marked apoptosis accompanied by inhibition of STAT3 and ERK pathways in H2228 cells. Such dual interruption of STAT3 and ERK pathways induced downregulation of the antiapoptotic protein survivin and upregulation of the proapoptotic protein BIM. CONCLUSION: Our results indicate that interruption of both STAT3-survivin and ERK-BIM pathways is required for induction of apoptosis in NSCLC harbouring EML4-ALK, providing a rationale for combination therapy with ALK and MEK inhibitors in EML4-ALK-positive NSCLC patients for whom ALK inhibitors alone are ineffective.

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	Figure 1. Effects of ALK inhibitors on cell proliferation in lung cancer cell lines positive for EML4âALK. H3122 or H2228 cells were cultured for 72âh in complete culture medium-containing various concentrations of TAE684 (A) or crizotinib (B), after which cell viability was assessed. Data are expressed as percent survival and are meansÂ±s.e. of triplicates from an experiment that was repeated a total of three times with similar results.
	Figure 2. Effects of ALK inhibition on signal transduction in lung cancer cells positive for EML4âALK. (A) H3122 or H2228 cells were incubated with the indicated concentrations of TAE684 for 48âh, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p) or total forms of ALK, STAT3, ERK, or AKT as well as with those to BIM, to survivin, to PARP, or to Î²-actin (loading control). The positions of the bands for EML4âALK, BIMEL, and intact and cleaved forms of PARP are shown for the corresponding antibodies. (B) Cells were transfected with non-specific (Con) or ALK siRNAs for 48âh, after which cell lysates were prepared and subjected to immunoblot analysis as in (A).
	Figure 3. Effects of the combination of the MEK inhibitor AZD6244 with TAE684 on signal transduction and apoptosis in lung cancer cells positive for EML4âALK. (A) H2228 cells were incubated in the absence or presence of TAE684 (30ânM), AZD6244 (1âÎ¼M), or both agents for 48âh, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the indicated proteins. (B) H3122 or H2228 cells were incubated in the absence or presence of the indicated concentrations of TAE684, AZD6244, or both agents for 60âh, after which the proportion of apoptotic cells was determined by staining with annexin V and propidium iodide followed by flow cytometry. Data are meansÂ±s.e. from three independent experiments. P<0.05 for the indicated comparisons; NS, not significant. (C) Nude mice with tumour xenografts established by subcutaneous implantation of H2228 cells were treated for 26 days by daily oral gavage with vehicle (control), TAE684 (0.5âmgâkgâ1), AZD6244 (25âmgâkgâ1), or the combination of both drugs. Tumour volume was determined at the indicated times after the onset of treatment. Data are meansÂ±s.e. for six mice per group. P<0.05 for the combination of TAE684 and AZD6244 vs either drug alone. (D) Lysates prepared from tumour xenografts at the completion of the experiment in (C) were subjected to immunoblot analysis with antibodies to the indicated proteins.
	Figure 4. Effects of the combinations of the MEK inhibitor, AZD6244, with either STAT3 depletion or survivin depletion on signal transduction and apoptosis in lung cancer cells positive for EML4âALK. (A) H2228 cells were transfected with non-specific (â) or STAT3 siRNAs and incubated with or without AZD6244 (1âÎ¼M) for 48âh, after which cell lysates were subjected to immunoblot analysis with antibodies to the indicated proteins (left). Alternatively, the cells were transfected and treated with AZD6244 for 60âh, after which the proportion of apoptotic cells was determined by staining with annexin V and propidium iodide followed by flow cytometry (right). (B) H2228 cells were transfected with non-specific (â) or survivin siRNAs and incubated with or without AZD6244 (1âÎ¼M) for 48âh, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the indicated proteins (left). Alternatively, the cells were transfected and treated with AZD6244 for 60âh, after which the proportion of apoptotic cells was determined by staining with annexin V and propidium iodide followed by flow cytometry (right). All quantitative data are meansÂ±s.e. from three independent experiments. *P<0.05 for the indicated comparisons.

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