The role of Vimentin in cell adhesion and lung cancer metastasis

项目摘要 非小细胞肺癌（NSCLC）是最常见的肺癌类型。由于诊断频繁， 当原发性肿瘤的转移已经发生时，在疾病的晚期阶段，NSCLC具有5- 年生存率仅为15%。一旦转移已经发生，疾病是非常困难的， 治疗，使转移的分子机制的识别在治疗方面的进展至关重要。的 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 一种动态蛋白，其组装和拆卸对于形成新的局灶性粘连至关重要， 调节细胞粘附。除了发挥结构性作用外，有证据表明波形蛋白还发挥着 在粘着斑处的信号作用。波形蛋白与细胞粘附调节的分子联系 仍然是一个未开发的领域。通过Y397磷酸化激活的粘着斑激酶（FAK）也 局限于局部粘连从我们的初步数据中可以明显看出，波形蛋白丝被定向到 磷酸化FAK位点，表明波形蛋白和FAK磷酸化之间的潜在分子联系。我们 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， 波形蛋白表达导致FAK表达和Y397磷酸化降低。此外，若干 已经在波形蛋白上鉴定了磷酸化位点，并且与波形蛋白的分解和组装有关。 病灶粘连处的突起。这种情况发生的分子机制尚未探索。在 我们推测波形蛋白与FAK相互作用，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， 本研究的目的有两个：1）明确波形蛋白调控细胞增殖的分子机制 2）确定波形蛋白是否是体内肺肿瘤转移所必需的。第一个目标，我们 将特别关注波形蛋白调节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），它们是 对波形蛋白组装和拆卸很重要，在FAK活化和细胞粘附中起作用。在 第二个目的，我们将通过将波形蛋白缺失小鼠（Vim -/-）与LKB-/-杂交来创建新的体内小鼠模型。 KrasG 12 D肺癌小鼠模型，以确定波形蛋白表达是否是肿瘤转移所必需的。 原发性肺癌这将是第一次创建这样的小鼠模型， 转移需要波形蛋白表达。了解波形蛋白表达如何调节细胞 在分子水平上的粘附将鉴定新的治疗靶点以抑制转移并极大地改善肿瘤的生物学特性。 NSCLC患者的预后。

PROJECT SUMMARY 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 role 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.