mTOR 信号通路在 17-DMAG 克服 EML4-ALK 阳性肺癌细胞株H3122对alectinib耐药中的作用机制研究

目的 观察mTOR信号通路在17-DMAG克服旁路信号通路激活诱导棘皮动物微管相关蛋白样4与间变性淋巴瘤激酶（echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase，EML4-ALK）融合基因阳性肺癌细胞株H3122（以下简称“H3122细胞”）对alectinib耐药中的变化，并探讨其内在的调控机制。方法 加入100 ng/mL转化生长因子α（transforming growth factor-α，TGF-α）和100 ng/mL表皮生长因子（epidermal growth factor ，EGF）诱导H3122细胞对alectinib耐药，观察加入17-DMAG后上述耐药能否被克服。采用CCK-8法检测不同处理方式下H3122细胞增殖，流式细胞术检测细胞凋亡，蛋白质免疫印迹（Western blot）技术检测不同处理方式下细胞中ALK、EGFR及其磷酸化蛋白的表达，观察其下游mTOR信号通路关键蛋白及活化水平的表达情况。结果 alectinib作用72 h后H3122细胞增殖率随药物浓度升高而下降，IC₅₀为0.042 μmol/L；联合TGF-α或EGF后H3122细胞对alectinib耐药，IC₅₀ 远大于10 μmol/L；单药17-DMAG处理后H3122细胞增殖率随药物浓度升高而下降，IC₅₀为 0.245 μmol/L；联合TGF-α或EGF后H3122细胞增殖率亦被17-DMAG抑制，且呈剂量依赖性，IC₅₀分别为 0.251 μmol/L和0.301 μmol/L。经0.05 μmol/L alectinib作用72 h后H3122细胞凋亡率为（30.01±0.92）%，alectinib联合TGF-α或EGF后细胞凋亡率分别为（6.36±0.14）%和（6.13±0.21）%，显著低于alectinib单药处理（P＜0.001）；经0.3 μmol/L 17-DMAG单药或联合TGF-α、EGF作用72 h后H3122细胞凋亡率分别为（28.37±1.75）%、（26.69±1.2）%和（26.62±0.72）%，差异无统计学意义（P＞0.05）。alectinib可抑制H3122细胞中p-ALK、p-mTOR的表达，也可抑制mTOR上、下游关键蛋白的活化状态；EGF可明显增加细胞中p-EGFR、p-mTOR及mTOR上、下游关键蛋白的活化水平表达；alectinib可抑制p-ALK表达，但联合EGF后不能抑制mTOR及mTOR上、下游关键蛋白活化水平的表达；即使联合EGF后，17-DMAG亦能抑制H3122细胞中ALK、EGFR、mTOR信号通路蛋白及其活化状态蛋白的表达。结论 旁路信号通路激活介导EML4-ALK融合基因阳性肺癌细胞株H3122对alectinib耐药的方式具有可行性，mTOR信号通路在 17-DMAG克服旁路信号通路激活引起alectinib的获得性耐药过程中有一定作用。

Role of the mTOR signaling pathway in the ability of 17-DMAG to overcome acquired resistance to alectinib in the H3122 lung cancer cell line positive for the EML4-ALK fusion gene

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.