Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
Neuroendocrinology. 2012;96(3):228-37. doi: 10.1159/000337257. Epub 2012 Apr 27.
Epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) are crucial targets in cancer therapy. Combined inhibition of both targets yielded synergistic effects in vitro and in vivo in several cancer entities. However, the impact of EGFR and mTOR expression and combined inhibition in neuroendocrine lung tumors other than small-cell lung cancer remains unclear.
Expression and activation of EGFR/AKT/mTOR pathway constituents were investigated in typical and atypical bronchial carcinoid (AC) tumors and large-cell neuroendocrine lung carcinomas (LCNEC) by immunohistochemistry in 110 tumor samples, and correlated with clinicopathological parameters and patient survival. Cytotoxicity of mTOR inhibitor everolimus and EGFR inhibitor erlotinib alone and in combination was assessed using growth inhibition assay in NCI-H720 AC and SHP-77 LCNEC cells. Cell cycle phase distribution was determined by FACS. Apoptosis-associated activation of caspase-3/7 was measured by Caspase-Glo® assay. Activity status of EGFR and mTOR pathway components was analyzed by immunoblotting.
Activation of the EGFR/AKT/mTOR axis could be demonstrated in all entities and was significantly increased in higher grade tumors. Neoadjuvant chemotherapy correlated significantly with p-AKT expression and p-ERK loss. Erlotinib combined with everolimus exerted synergistic combination effects in AC and LCNEC cells by induction of apoptosis, while cell cycle phase distribution remained unaffected. These effects could be explained by synergistic downregulation of phospho-mTOR, phospho-p70S6 kinase and phospho-AKT expression by everolimus and erlotinib.
Our study indicates that EGFR and mTOR are clinically important targets in bronchial neuroendocrine tumors, and further in vivo and clinical exploration of combined inhibition is warranted.
表皮生长因子受体（EGFR）和哺乳动物雷帕霉素靶蛋白（mTOR）是癌症治疗的重要靶点。在几种癌症实体中，同时抑制这两个靶点在体外和体内产生协同作用。然而，除小细胞肺癌以外的神经内分泌肺肿瘤中 EGFR 和 mTOR 表达及其联合抑制的影响仍不清楚。
通过免疫组织化学方法在 110 个肿瘤样本中研究了典型和非典型支气管类癌（AC）肿瘤和大细胞神经内分泌肺癌（LCNEC）中 EGFR/AKT/mTOR 通路成分的表达和激活，并与临床病理参数和患者生存相关联。使用 NCI-H720 AC 和 SHP-77 LCNEC 细胞中的生长抑制测定法评估 mTOR 抑制剂依维莫司和 EGFR 抑制剂厄洛替尼单独和联合的细胞毒性。通过 FACS 确定细胞周期相分布。通过 Caspase-Glo®测定法测量与细胞凋亡相关的 caspase-3/7 的激活。通过免疫印迹分析 EGFR 和 mTOR 通路成分的活性状态。
所有实体中均能证明 EGFR/AKT/mTOR 轴的激活，并在高级别肿瘤中显著增加。新辅助化疗与 p-AKT 表达和 p-ERK 丧失显著相关。厄洛替尼联合依维莫司在 AC 和 LCNEC 细胞中通过诱导细胞凋亡产生协同的组合效应，而细胞周期相分布保持不变。依维莫司和厄洛替尼的协同下调磷酸化-mTOR、磷酸化-p70S6 激酶和磷酸化-AKT 表达可以解释这些作用。
我们的研究表明，EGFR 和 mTOR 是支气管神经内分泌肿瘤中临床重要的靶点，因此有必要进一步进行体内和临床联合抑制的探索。
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