Role of DOK family proteins in lung tumor suppression

描述（由申请人提供）：肺癌是全球癌症相关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通路，重点探讨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蛋白。我们最近发现了DOK-1， DOK-2和DOK-3作为肺肿瘤抑制因子，我们进一步假设它们是致癌EGFR-RAS信号的负调控因子。在我们的初步分析中，我们发现Dok-1、Dok-2和Dok-3单、双和三重基因敲除小鼠发生肺腺癌，其外显率和潜伏期取决于丢失的Dok等位基因的数量。Dok缺失小鼠的肿瘤表现出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，我们观察到频繁的DOK-2基因组缺失，同时伴有DOK-2 mRNA表达的降低。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反对由致癌的EGFR和RAS引发的肺肿瘤发生。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。本研究旨在通过EGFR- ras通路确定DOK-2介导的肺肿瘤抑制机制，并确定DOK-2变异等位基因和非肺细胞区室与dok -无突变肺癌发病机制的相关性，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，我们将利用已经存在的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确实反对KRASG12D引发的肺肿瘤发生。(2)明确DOK-2L138S在肺癌易感性中的作用。DOK-2L138S等位基因既存在于人类肺癌中的体细胞突变，也存在于自然发生的种系多态性中。体外分析表明，DOK-2L138S是一个功能缺失突变体，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与肺癌易感性有关，我们将对人类肺癌进行病例/对照研究，同时建立一个Dok-2L138S敲入小鼠模型。(3)评估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通过使用野生型和TKO动物进行互惠骨髓移植来确定。此外，产生肺特异性条件Dok-2敲除小鼠模型将允许明确确定细胞自主机制对Dok-2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. 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.