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.