Abstract:

Objective  To explore how mTOR signaling may help 17-DMAG overcome acquired resistance to alectinib in H3122 lung cancer cells positive for the EML4-ALK fusion gene. Methods H3122 cells were induced to become resistant to alectinib by adding transforming growth factor alpha（TGF-α） or epidermal growth factor （EGF） at 100 ng/ml，then the ability of 17-DMAG to overcome this resistance was checked after 72 h using the CCK-8 assay and AnnexinⅤ-PE apoptosis assay. Western blotting was used to detect expression of ALK，EGFR and phosphorylated protein，as well as determine the expression and activation levels of key proteins in the mTOR signaling pathway. Results Alectinib inhibited the viability of H3122 cells over 72 h in a dose-dependent manner （IC₅₀=0.042 μmol/L）. After treatment with TGF-α or EGF，the cells were resistant to alectinib （IC₅₀>10 μmol/L），and 17-DMAG reduced their viability in a dose-dependent manner （IC₅₀=0.245 μmol/L），even in the presence of TGF-α（IC₅₀=0.251 μmol/L） or EGF（IC₅₀=0.301 μmol/L）. The apoptosis rate of H3122 cells was （30.01±0.92）% after treatment with 0.05 μmol/L alectinib for 72 h，which was significantly higher than the rate of （6.36±0.14）% after the combination of alectinib and TGF-α，and （6.13±0.21）% after the combination of alectinib and EGF（both P<0.001）. Apoptosis rate was similar after 72　h treatment with 0.03 μmol/L 17-DMAG alone （28.37±1.75）% or with 17-DMAG combined with TGF-α （26.69±1.2）% or EGF （26.62±0.72）% （P>0.05）. Alectinib inhibited p-ALK and p-mTOR，as well as activation of key proteins up-and downstream of the mTOR pathway. EGF significantly increased expression of p-EGFR，p-mTOR and activation of key proteins up-and downstream of the mTOR pathway in cells. Although alectinib inhibited p-ALK，it did not inhibit EGF-induced up-regulation of p-mTOR or activation of key proteins up-and downstream in the mTOR pathway. 17-DMAG inhibited expression of ALK，EGFR，mTOR and their activated forms，regardless of whether EGF was present or absent. Conclusions It is possible to activate signaling pathways that bypass acquired resistance to alectinib in H3122 lung cancer cells positive for the EML4-ALK fusion gene. In the case of 17-DMAG，this bypass involves the mTOR signaling pathway.

Key words: Lung neoplasms, EML4-ALK fusion gene, H3122 cell line, Alectinib, 17-DMAG, mTOR signaling pathway