The role of Vimentin in cell adhesion and lung cancer metastasis

描述（由申请人提供）：非小细胞肺癌（NSCLC）是最常见的肺癌类型。由于在原发性肿瘤已经发生转移的疾病晚期频繁诊断，NSCLC的5年生存率仅为15%。一旦转移已经发生，这种疾病是非常难以治疗的，使得转移的分子机制的鉴定对于治疗的进展至关重要。中间丝，波形蛋白，在迁移细胞中上调，表明在转移中的作用。波形蛋白是一种动态蛋白质，其组装和拆卸对于形成调节细胞粘附的新的粘着斑至关重要。除了发挥结构性作用外，有证据表明波形蛋白还在粘着斑中发挥信号传导作用。波形蛋白和细胞粘附调节之间的分子联系仍然是一个未探索的领域。通过Y397磷酸化激活的粘着斑激酶（FAK）也定位于粘着斑。从我们的初步数据中可以明显看出，波形蛋白丝直接进入磷酸化的FAK位点，表明波形蛋白和FAK磷酸化之间存在潜在的分子联系。我们对来自波形蛋白耗尽的细胞和对照细胞的裂解物的初步蛋白质印迹数据也显示，波形蛋白表达的丧失导致FAK表达和Y397处的磷酸化降低。此外，已经在波形蛋白上鉴定了几个磷酸化位点，并将其与粘着斑的分解和组装过程联系起来。这种情况发生的分子机制尚未探索。在这个提议中，我们假设波形蛋白与FAK相互作用以调节FAK的活化和表达，从而增加细胞粘附以促进原发性肺癌肿瘤的转移。为了验证这一点，我们将提出两个具体的目标：1）确定波形蛋白调节细胞粘附的分子机制2）确定波形蛋白是否是体内肺肿瘤转移所必需的。在第一个目标中，我们将专门研究波形蛋白调节FAK表达和激活的分子机制。我们还将测试是否三个特定的波形蛋白磷酸化位点（丝氨酸38，56和72），这是重要的波形蛋白的组装和拆卸，发挥FAK激活和细胞粘附的作用。在第二个目标中，我们将通过将波形蛋白缺失小鼠（Vim -/-）与LKB-/-KrasG 12 D肺癌小鼠模型杂交来创建新的体内小鼠模型，以确定原发性肺癌肿瘤的转移是否需要波形蛋白表达。这将是第一次建立这样的小鼠模型来研究转移对波形蛋白表达的要求。了解波形蛋白表达如何在分子水平上调节细胞粘附，将确定新的治疗靶点，以抑制转移，大大改善NSCLC患者的预后。 公共卫生相关性：在美国，肺癌是癌症死亡的主要原因，5年生存率仅为15- 25%。该提案旨在了解转移的分子机制，希望确定未来的治疗靶点，从而显著改善肺癌患者的预后。具体来说，我们将研究在侵袭性肿瘤中过度表达的波形蛋白在细胞粘附于细胞外基质中的作用。

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.