Mutant EGF Receptor-Dependent Lung Cancer in Human Cell Lines and Transgenic Mice

描述(申请人提供)：在本奖项的前三年，我们表征了由多西环素诱导的转基因编码在相应类型的人类癌症中发现的两种常见形式的突变EGFR的肺腺癌小鼠模型。这些小鼠会发展出与人类癌症难以区分的肿瘤；当癌基因被去诱导或患者使用的药物抑制突变激酶时，肿瘤会退化，并且在某些情况下会产生抗药性，在某些情况下，这是由于在大约一半的患者耐药肿瘤中也发现了EGFR的二次突变。我们现在提议通过几种方法扩展我们对这些小鼠模型的工作，目标是(I)评估可能影响EGFR启动的肺肿瘤发生的基因和蛋白(包括肿瘤抑制基因、EGFR家族成员和含磷酸酪氨酸的蛋白)；(Ii)发现更多导致肿瘤形成、进展或耐药的基因；(Iii)评估不同基因类型的肿瘤在体外培养中转移并成为成熟细胞系的可能性；以及(Iv)研究在这些小鼠肿瘤中观察到的炎症细胞的来源和功能。为了实现这些目标，我们将利用几个有条件的小鼠突变，我们自己的蛋白质组学调查结果，睡美人转座系统，以及免疫细胞的表征方法。我们希望我们的发现能为EGFR介导的肺癌发生、肿瘤进展、继发性耐药以及免疫细胞在实体瘤中的作用提供新的见解。此外，我们可能会产生新的小鼠肿瘤细胞系用于实验，并识别对肺癌的诊断、分类和治疗有用的基因。 公共卫生相关性：肺癌是美国和世界上癌症死亡的主要原因，而腺癌是最常见的肺癌形式。我们的实验室是发现美国大约10%的肺腺癌患者对一系列新药-抑制一种名为蛋白酪氨酸激酶的酶的药物--有反应的人之一，因为他们的癌症是由影响生长因子受体EGFR的两种特征性突变之一引起的。不幸的是，对这些药物的有益反应是短暂的；在大约一半的病例中，获得性耐药性是由于我们几年前发现的EGFR基因的第二个突变。为了比在人类患者身上更深入地研究这种类型的肺癌，我们在小鼠身上操纵了基因，使动物患上了与带有EGFR突变的人类肺癌非常相似的肺癌；正如我们在这项拨款的第一阶段所展示的那样，癌症对相同的药物产生反应，产生类似的耐药性，如果突变基因被关闭，这些癌症就会消失。我们现在建议使用这些强大的模型来研究肺癌的一些相关方面。首先，我们将研究突变的EGFR导致肺癌时，其他基因可能扮演的角色。这些其他基因包括众所周知的肿瘤抑制基因，以及我们通过研究肺癌细胞中被EGFR或相关酶修饰的蛋白质而确定的基因。在这项工作的过程中，我们还将询问需要什么才能使肺癌细胞转移到其他部位，并试图从转移的地方发展小鼠肺癌细胞系。为了寻找可能有助于肺癌加速生长或耐药发展的新基因，我们将利用一个可移动的DNA单元来识别这些基因。最后，我们将探索免疫细胞在小鼠肺肿瘤中的惊人表现，以努力了解这些细胞是如何到达肺组织的，以及它们具有哪些促进或延缓肿瘤发展的功能。

DESCRIPTION (provided by applicant): During the first three years of this award, we have characterized mouse models of lung adenocarcinoma initiated by doxycycline-inducible transgenes encoding the two common forms of mutant EGFR found in the corresponding type of human cancer. These mice develop tumors indistinguishable from the human cancers; the tumors regress when the oncogene is de-induced or when the mutant kinase is inhibited with drugs used in patients, and they develop drug resistance under certain conditions, in some cases as a result of a secondary mutation in EGFR also found in about half of drug-resistant tumors in patients. We now propose to extend our work with these mouse models in several ways with the goals of (i) evaluating genes and proteins (including tumor suppressor genes, members of the EGFR family, and phosphotyrosine-containing proteins) that might influence EGFR-initiated lung tumorigenesis; (ii) discovering additional genes that contribute to tumor formation, progression or drug resistance; (iii) assessing the potential of tumors with various genotypes to metastasize and become established cell lines in culture; and (iv) studying the origins and functions of inflammatory cells observed in these mouse tumors. To pursue these goals, we will take advantage of several conditional mouse mutants, results from our own proteomics surveys, the Sleeping Beauty transposition system, and methods for characterization of immune cells. We expect our findings to offer new insights into EGFR-mediated lung carcinogenesis, tumor progression, secondary drug resistance, and the role of immune cells in solid tumors. In addition, we may generate new mouse tumor cell lines for experimental use and identify genes that are useful for diagnosis, classification, and treatment of lung cancers. PUBLIC HEALTH RELEVANCE: Lung cancer is the major cause of death from cancer in the United States and the world, and adenocarcinoma is the most common form of lung cancer. Our laboratory is among those who found that about ten percent of patients in the US with lung adenocarcinomas respond to a new set of drugs--- drugs that inhibit a type of enzyme called a protein-tyrosine kinase---because their cancers are caused by one of two characteristic mutations affecting a growth factor receptor called EGFR. Unfortunately, the beneficial response to these drugs is short-lived; in about half the cases, the acquired drug resistance is due to a second mutation in the EGFR gene that we discovered a few years ago. To study this type of lung cancer more thoroughly than is possible in human patients, we have manipulated genes in mice so that the animals develop lung cancers that are very similar to human lung cancers with EGFR mutations; as we have shown during the first phase of this grant, the cancers respond to the same drugs, develop similar drug resistance, and disappear if the mutant gene is turned off. We now propose to use these powerful models to study a number of related aspects of lung cancer. First, we will look at the roles that might be played by other genes when mutant EGFR causes lung cancer. These other genes include well-known tumor suppressor genes and genes identified through studies we have done to characterize proteins that are modified by EGFR or related enzymes in lung cancer cells. In the course of this work, we will also ask what is required to make lung cancer cells metastasize to other sites and attempt to develop mouse lung cancer cell lines from the metastases. To seek novel genes that might contribute to accelerated growth of a lung cancer or to the development of drug resistance, we will make use of a moveable DNA unit that can identify such genes. Finally, we will explore the striking appearance of immune cells in mouse lung tumors, in an effort to understand how such cells arrived in the lung tissue and what functions they serve to promote or retard tumor development.