Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
J Natl Cancer Inst. 2012 Aug 8;104(15):1182-97. doi: 10.1093/jnci/djs297. Epub 2012 Jul 24.
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.
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.
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.
Cancer-associated EPHA3 mutations attenuate the tumor-suppressive effects of normal EPHA3 in lung cancer.
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 是肺癌中突变最频繁的基因之一。虽然受体酪氨酸激酶突变通常驱动致癌转化和肿瘤发生，但肺癌中 EPHA3 突变的致癌潜力尚不清楚。
我们使用免疫沉淀、western blot 和激酶测定来确定突变的 EPHA3 受体的活性和信号转导。从一个大型数据集生成一个与突变相关的基因特征，将其映射到具有生存信息的另一个训练数据集，并在第三个独立数据集进行测试。通过定量逆转录聚合酶链反应在配对的正常-肿瘤临床标本中确定 EPHA3 表达水平，并通过免疫组织化学在人肺癌组织微阵列中进行。我们在小鼠中使用 A549 和 H1299 人肺癌细胞异种移植评估体内肿瘤生长（每组 7-8 只小鼠）。通过溴脱氧尿苷掺入测量肿瘤细胞增殖，通过多种测定法测量细胞凋亡。所有 P 值均来自双侧检验。
至少两种与癌症相关的 EPHA3 体细胞突变作为正常（野生型） EPHA3 蛋白的显性抑制剂起作用。确定了与患者生存不良相关的 EPHA3 突变相关基因特征。此外，在大量肺癌患者的肿瘤中，EPHA3 基因拷贝数和/或表达水平降低（例如，在 371 例原发性肺腺癌中，该基因缺失 157 例[42%]）。野生型 EPHA3 在人肺癌细胞系中的重新表达通过抑制 AKT 激活体外增加了细胞凋亡，并抑制了肿瘤异种移植物的生长（例如，对于 H1299 细胞，野生型 EPHA3 的平均肿瘤体积为 437.4mm3，而对照为 774.7mm3，P<0.001）。在肺癌患者中发现的显性负 EPHA3 体细胞突变可以在转染中克服野生型 EPHA3 的肿瘤抑制作用。
与癌症相关的 EPHA3 突变减弱了肺癌中正常 EPHA3 的肿瘤抑制作用。
