Role of DOK family proteins in lung tumor suppression

描述（由申请人提供）：肺癌是全球最常见的癌症相关死亡原因。本研究的目的是分析人肺癌中受到干扰的信号通路，重点研究DOK介导的肺肿瘤抑制机制。DOK（“酪氨酸激酶下游”）家族蛋白是调节酪氨酸激酶信号传导的信号传导蛋白。我们最近发现DOK-1、DOK-2和DOK-3是肺肿瘤抑制因子，我们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单基因敲除、双基因敲除和三基因敲除小鼠发生肺腺癌，其潜伏期和潜伏期依赖于Dok等位基因丢失的数量。来自Dok敲除小鼠的肿瘤表现出Akt和Erk活化，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基因组频繁丢失，同时DOK-2 mRNA表达减少。引人注目的是，DOK-2的基因组缺失与EGFR突变状态密切相关。因此，我们假设DOK-2对抗由致癌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-2L 138 S）可能是肺癌易感性的基础。本研究的目的是确定DOK介导的肺肿瘤抑制维斯EGFR-RAS通路的机制，并确定DOK-2等位基因变体和非肺细胞区室与DOK-无效肺癌发病机制的相关性，具体目标如下：（1）确定DOK-2在EGFR和KRAS突变型肺癌中的作用。为此，我们将利用现有的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型EGFR或KRAS驱动的肺癌。在初步分析中，我们发现Dok-2确实对抗由KRASG 12 D引发的肺肿瘤发生。(2)探讨DOK-2L 138 S在肺癌易感性中的作用。DOK-2L 138 S等位基因既作为人肺癌中发现的体细胞突变存在，也作为自然发生的生殖系多态性存在。体外分析表明，DOK-2L 138 S是一种功能丧失型突变体，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，我们将进行人类肺癌的病例/对照研究，并同时产生Dok-2L 138 S基因敲入小鼠模型。(3)评价细胞自主和非细胞自主机制对Dok突变小鼠肿瘤形成的贡献。Dok TKO造血表型的相关性将使用野生型和TKO动物的相互骨髓移植来确定。此外，肺特异性条件性Dok-2敲除小鼠模型的产生将允许明确确定细胞自主机制对Dok-2敲除肺表型的贡献。 公共卫生相关性：肺癌仍然是每年与癌症相关的死亡人数最多的原因，在从不吸烟者中发现的肺癌比例越来越高。因此，必须解构人类肺癌中的分子遗传学和干扰途径。我们的工作将定义在DOK无效的情况下，在体细胞和遗传背景下肿瘤易感性，启动和进展的机制，并剖析DOK-2丢失与EGFR和RAS的关系。这项工作可以确定预防和治疗人类肺癌的其他靶点，并发现决定肿瘤表型，进展和治疗反应的遗传标准。

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. PUBLIC HEALTH RELEVANCE: Lung cancer remains responsible for the most cancer-related deaths every year, with a growing proportion of lung cancer found in never smokers. It is therefore imperative to deconstruct the molecular genetics and the pathways perturbed in human lung cancer. Our work will define the mechanism of tumor predisposition, initiation and progression in a DOK-null scenario, in both a somatic and inherited setting, and dissect the relationship of DOK-2 loss to EGFR and RAS. This work could identify additional targets for the prevention and treatment of human lung cancer and discover genetic criteria that determine tumor phenotype, progression, and response to therapy.