Effects of Cancer-Associated EPHA3 Mutations on Lung Cancer
Effects of Cancer-Associated EPHA3 Mutations on Lung Cancer
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DOI：
10.1093/jnci/djs297
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发表时间：
2012-08-01
影响因子：
10.3
通讯作者：
Chen, Jin
中科院分区：
文献类型：
--
作者：
Zhuang, Guanglei;Song, Wenqiang;Chen, Jin
癌症基因组测序工作最近发现，编码EPHA3受体酪氨酸激酶的EPHA3是肺癌中最常见的突变基因之一。虽然受体酪氨酸激酶突变经常驱动致癌转化和肿瘤发生，但EPHA3突变在肺癌中的致癌潜力尚不清楚。方法采用免疫沉淀法、western blotting法和激酶法检测突变体EPHA3受体的活性和信号转导。从一个大型数据集生成突变相关基因签名，映射到另一个具有生存信息的训练数据集，并在第三个独立数据集中进行测试。通过配对正常肿瘤临床标本的定量逆转录聚合酶链反应和人肺癌组织微阵列的免疫组织化学检测EPHA3的表达水平。我们使用A549和H1299人肺癌细胞异种移植在小鼠体内评估肿瘤生长情况（每组n = 7-8只小鼠）。用溴脱氧尿苷掺入法测定肿瘤细胞增殖，用多种方法测定肿瘤细胞凋亡。所有P值均来自双侧检验。结果至少有两种与癌症相关的EPHA3体细胞突变是正常（野生型）EPHA3蛋白的显性抑制剂。发现EPHA3突变相关基因标记与患者生存不良相关。此外，EPHA3基因拷贝数和/或表达水平在来自大型肺癌患者队列的肿瘤中降低（例如，该基因在371例原发性肺腺癌中的157例（42%）中缺失）。在体外实验中，野生型EPHA3在人肺癌细胞系中重表达通过抑制AKT激活而增加细胞凋亡，并抑制肿瘤移植物的生长(例如，对于H1299细胞，野生型EPHA3的平均肿瘤体积= 437.4 mm（3)，而对照组= 774.7 mm(3), P < 0.001）。在肺癌患者中发现的显性阴性EPHA3体细胞突变可以在反式中覆盖野生型EPHA3的肿瘤抑制作用。结论肿瘤相关的EPHA3突变减弱了正常EPHA3在肺癌中的抑瘤作用。
Background Cancer genome sequencing efforts recently identified EPHA3, which encodes the EPHA3 receptor tyrosine kinase, as one of the most frequently mutated genes in lung cancer. Although receptor tyrosine kinase mutations often drive oncogenic conversion and tumorigenesis, the oncogenic potential of the EPHA3 mutations in lung cancer remains unknown.Methods We used immunoprecipitation, western blotting, and kinase assays to determine the activity and signaling of mutant EPHA3 receptors. A mutation-associated gene signature was generated from one large dataset, mapped to another training dataset with survival information, and tested in a third independent dataset. EPHA3 expression levels were determined by quantitative reverse transcription-polymerase chain reaction in paired normal-tumor clinical specimens and by immunohistochemistry in human lung cancer tissue microarrays. We assessed tumor growth in vivo using A549 and H1299 human lung carcinoma cell xenografts in mice (n = 7-8 mice per group). Tumor cell proliferation was measured by bromodeoxyuridine incorporation and apoptosis by multiple assays. All P values are from two-sided tests.Results At least two cancer-associated EPHA3 somatic mutations functioned as dominant inhibitors of the normal (wild type) EPHA3 protein. An EPHA3 mutation-associated gene signature that was associated with poor patient survival was identified. Moreover, EPHA3 gene copy numbers and/or expression levels were decreased in tumors from large cohorts of patients with lung cancer (eg, the gene was deleted in 157 of 371 [42%] primary lung adenocarcinomas). Reexpression of wild-type EPHA3 in human lung cancer lines increased apoptosis by suppression of AKT activation in vitro and inhibited the growth of tumor xenografts (eg, for H1299 cells, mean tumor volume with wild-type EPHA3 = 437.4 mm(3) vs control = 774.7 mm(3), P < .001). Tumor-suppressive effects of wild-type EPHA3 could be overridden in trans by dominant negative EPHA3 somatic mutations discovered in patients with lung cancer.Conclusion Cancer-associated EPHA3 mutations attenuate the tumor-suppressive effects of normal EPHA3 in lung cancer.