Role of DOK family proteins in lung tumor suppression

描述(申请人提供)：肺癌是世界上与癌症相关的死亡人数最多的疾病。本研究旨在剖析人肺癌中受干扰的信号转导途径，重点研究DOK介导的肺癌抑制机制。DOK(“酪氨酸激酶下游”)家族蛋白是调节酪氨酸激酶信号的信号蛋白。我们最近发现DOK-1、DOK-2和DOK-3是肺肿瘤抑制因子，并进一步假设它们是致癌EGFR-RAS信号的负调节因子。在我们的初步分析中，我们发现DOK-1、DOK-2和DOK-3单基因、双基因和三基因敲除小鼠发生肺腺癌的外显性和潜伏期取决于丢失的DOK等位基因的数量。Dok Null小鼠的肿瘤表现出Akt和Erk的激活，类似于EGFR或KRAS驱动的肿瘤发生模型。在人类非小细胞肺癌(NSCLC)中，我们观察到DOK-2基因频繁丢失，并伴随着DOK-2mRNA表达的降低。值得注意的是，DOK-2的基因组丢失与EGFR突变状态密切相关。因此，我们假设DOK-2反对由致癌的EGFR和RAS启动的肺肿瘤发生。此外，我们进一步假设DOK-2的变异等位基因(如DOK-2L138S)可能是肺癌易感性的基础。本研究旨在探讨DOK介导的肺肿瘤抑制机制与EGFR-RAS途径的关系，以及DOK-2变异等位基因和非肺细胞室与DOK缺失肺癌发病机制的关系。具体目的如下：(1)明确DOK-2在EGFR和KRAS突变肺癌中的作用。为此，我们将利用已有的EGFR和KRAS双转基因和Dok-2-/-小鼠模型来确定DOK-2是否在体内对抗突变的EGFR或KRAS驱动的肺癌。在初步分析中，我们发现DOK-2确实对抗KrasG12D启动的肺肿瘤形成。(2)明确DOK-2L138S在肺癌易感性中的作用。DOK-2L138S等位基因既是人类肺癌中发现的一种体细胞突变，也是一种自然发生的生殖系多态。体外分析表明，DOK-2L138S是一个功能缺失的突变体，在抑制EGF诱导的RAS和ERK活性方面存在缺陷。为了确定该等位基因是否有助于肺癌易感性，我们将对人类肺癌进行病例/对照研究，并同时产生DOK-2L138S敲入小鼠模型。(3)评价细胞自主和非细胞自主机制在DOK突变小鼠肿瘤形成中的作用。DOK TKO造血表型的相关性将通过使用野生型和TKO动物的相互骨髓移植来确定。此外，肺特异性条件性DOK-2基因敲除小鼠模型的建立将允许明确地确定细胞自主机制对DOK-2基因敲除肺表型的贡献。

DESCRIPTION (provided by applicant): Lung cancer is responsible for the most cancer-related deaths worldwide. This proposal aims at dissecting the signaling pathways perturbed in human lung cancer, with a focus on the mechanisms of DOK-mediated lung tumor suppression. DOK ("downstream of tyrosine kinase") family proteins are signaling proteins that modulate tyrosine kinase signaling. We recently identified DOK-1, DOK-2, and DOK-3 as lung tumor suppressors, and we further hypothesize that they are negative regulators of oncogenic EGFR-RAS signaling. In our preliminary analysis, we find that Dok-1, Dok-2, and Dok-3 single, double, and triple knockout mice develop lung adenocarcinoma with a penetrance and latency dependent on the number of lost Dok alleles. Tumors from Dok null mice exhibit Akt and Erk activation, similar to models of EGFR- or KRAS-driven tumorigenesis. In human non-small cell lung cancer (NSCLC), we observe frequent genomic loss of DOK-2 with a concomitant reduction of DOK-2 mRNA expression. Strikingly, genomic loss of DOK-2 is strongly associated with EGFR mutation status. We therefore hypothesize that DOK-2 opposes lung tumorigenesis initiated by oncogenic EGFR and RAS. Moreover, we further hypothesize that variant alleles of DOK-2 (such as DOK-2L138S) could underlie lung cancer susceptibility. The purpose of this proposal is to determine the mechanisms of DOK-mediated lung tumor suppression vis-¿-vis the EGFR-RAS pathway and to ascertain the relevance of variant DOK-2 alleles and non-pulmonary cellular compartments to the pathogenesis of DOK-null lung cancer with the following specific aims: (1) To define the role of DOK-2 in EGFR- and KRAS-mutant lung cancers. To this end, we will utilize already existing EGFR and KRAS bitransgenic and Dok-2-/- mouse models to determine if Dok-2 opposes mutant EGFR or KRAS-driven lung cancer in vivo. In a preliminary analysis, we find that Dok-2 does indeed oppose lung tumorigenesis initiated by KRASG12D. (2) To define the role of DOK-2L138S in lung cancer susceptibility. The DOK-2L138S allele is present both as a somatic mutation found in human lung cancer and as a naturally occurring germline polymorphism. In vitro analysis indicates that DOK-2L138S is a loss-of-function mutant that is defective at suppressing EGF-induced RAS and ERK activity. To determine if this allele contributes to lung cancer susceptibility, we will perform a case/control study of human lung cancer, and, in parallel, generate a Dok-2L138S knock-in mouse model. (3) Evaluate the contributions of cell autonomous and non-cell autonomous mechanisms to tumor formation in Dok mutant mice. The relevance of the Dok TKO hematopoietic phenotype will be determined using a reciprocal bone marrow transplant using wild-type and TKO animals. Furthermore, generation of a lung-specific conditional Dok-2 knockout mouse model will allow unequivocal determination of the contribution of cell autonomous mechanisms to the Dok-2 knockout lung phenotype.