The role of Vimentin in cell adhesion and lung cancer metastasis

描述（申请人提供）：非小细胞肺癌（NSCLC）是最常见的肺癌类型。由于经常在疾病的晚期诊断，而此时原发肿瘤已经发生转移，NSCLC的5年生存率仅为15%。一旦发生转移，这种疾病就极难治疗，因此确定转移的分子机制对治疗进展至关重要。在迁移细胞中，中间丝蛋白，静脉蛋白被上调，表明在转移中起作用。Vimentin是一种动态蛋白，其组装和拆卸对于形成调控细胞粘附的新病灶粘附至关重要。除了发挥结构作用外，有证据表明vimentin在局灶粘连中也起信号作用。波形蛋白与细胞粘附调节之间的分子联系仍然是一个未开发的领域。局灶黏附激酶（Focal adhesion kinase， FAK）在Y397位点磷酸化激活，也定位于局灶黏附。从我们的初步数据中可以看出，vimentin细丝被引导到磷酸化的FAK位点，这表明vimentin和FAK磷酸化之间存在潜在的分子联系。我们对vimentin缺失细胞和对照细胞裂解物的初步western blot数据也显示，vimentin表达缺失导致FAK表达减少和Y397磷酸化。此外，在vimentin上发现了几个磷酸化位点，并与局灶黏附的拆卸和组装过程有关。这种情况发生的分子机制尚未被探索。在本研究中，我们假设vimentin与FAK相互作用，调节FAK的激活和表达，从而增加细胞粘附，促进原发性肺癌肿瘤的转移。为了验证这一点，我们将解决两个具体目标：1)确定vimentin调节细胞粘附的分子机制2)确定vimentin在体内是否是肺肿瘤转移所必需的。在第一个目标中，我们将专门研究vimentin调节FAK表达和激活的分子机制。我们还将测试三个特定的波形蛋白磷酸化位点（丝氨酸38、56和72）是否在FAK激活和细胞粘附中发挥作用，它们对波形蛋白的组装和拆卸很重要。在第二个目标中，我们将通过将vimentin缺失小鼠（vim -/-）与LKB-/- KrasG12D肺癌小鼠模型杂交，建立一种新的体内小鼠模型，以确定vimentin的表达是否需要原发性肺癌肿瘤的转移。这将是第一次建立这样的小鼠模型来观察vimentin表达对转移的要求。了解vimentin表达如何在分子水平上调控细胞粘附，将发现新的治疗靶点，抑制转移，极大地改善NSCLC患者的预后。

DESCRIPTION (provided by applicant): Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Due to frequent diagnosis during advanced stages of the disease when metastasis of the primary tumor has already occurred, NSCLC has a 5- year survival rate of only 15%. Once metastasis has already occurred, the disease is extremely difficult to treat, making identification of the molecular mechanism of metastasis crucial for advances in treatment. The intermediate filament, vimentin, is upregulated in migrating cells, indicating a rol in metastasis. Vimentin is a dynamic protein whose assembly and disassembly is critical for the formation of new focal adhesions that regulate cell adhesion. In addition to playing a structural role, evidence suggests that vimentin also plays a signaling role at the focal adhesions. The molecular link between vimentin and the regulation of cell adhesion remains an unexplored field. Focal adhesion kinase (FAK), which is activated by phosphorylation at Y397, also localizes to the focal adhesions. As evident from our preliminary data, vimentin filaments are directed into the phosphorylated FAK sites indicating a potential molecular link between vimentin and FAK phosphorylation. Our preliminary western blot data of lysates from vimentin depleted and control cells also shows that a loss of vimentin expression results in reduced FAK expression and phosphorylation at Y397. Additionally, several phosphorylation sites have been identified on vimentin and have been linked to the disassembly and assembly processes at focal adhesions. The molecular mechanism by which this occurs has not been explored yet. In this proposal we hypothesize that vimentin interacts with FAK to regulate activation and expression of FAK thereby increasing cell adhesion to facilitate metastasis of the primary lung cancer tumor. To test this we will address two specific aims: 1) To determine the molecular mechanism by which vimentin regulates cell adhesion 2) To determine whether vimentin is necessary for lung tumor metastasis in vivo. In the first aim, we will specifically look at the molecular mechanisms by which vimentin regulates FAK expression and activation. We will also test whether three specific vimentin phosphorylation sites (Serines 38, 56 and 72), which are important for vimentin assembly and disassembly, play a role in FAK activation and cell adhesion. In the second aim we will create a novel in vivo mouse model by crossing a vimentin null mouse (vim -/-) to the LKB-/- KrasG12D lung cancer mouse model to determine whether vimentin expression is required for metastasis of the primary lung cancer tumor. This will be the first time such a mouse model will be created to look at the requirement of vimentin expression for metastasis. Understanding how vimentin expression regulates cell adhesion at a molecular level will identify novel therapeutic targets to inhibit metastasis and greatly improve the prognosis of NSCLC patients. PUBLIC HEALTH RELEVANCE: Lung cancer, which is frequently diagnosed during late-stages of the disease when metastasis has already occurred, is the leading cause of death due to cancer in the United States and has a 5-year survival rate of only 15-25%. This proposal seeks to understand the molecular mechanisms of metastasis with the hope of identifying future therapeutic targets that could lead to a significant improvement in the prognosis for lung cancer patients. Specifically, we will look at the role of vimentin, which is overexpressed in invasive cancer tumors, in cell adhesion to the extracellular matrix.