Tubulin microtentacles in detached mammary epithelial cells

 描述（由申请方提供）：已知细胞骨架改变会影响循环肿瘤细胞（CTC）的转移成功。然而，对肿瘤细胞骨架的研究几乎完全集中在附着于表面或细胞外基质的细胞上，留下了与转移相关的显著知识空白。PI研究小组最近的工作表明，循环肿瘤细胞的细胞骨架与附着在细胞外基质上的细胞反应非常不同。具体来说，自由漂浮的肿瘤细胞产生独特的 微管蛋白为基础的微触角（McTN），促进CTC再附着到内皮细胞层。活体动物的成像显示，CTC通过与McTN匹配的细胞骨架机制重新附着到血管壁上。来自PI实验室的新数据现在表明，在手术期间从乳腺癌患者中分离出来的几个小时内，肿瘤细胞中可以检测到McTNs。此外，患者的肿瘤细胞药物反应可以在几分钟内测量。由于McTNs由微管蛋白支持，因此常见的乳腺癌药物紫杉醇（Taxol）实际上增强了McTNs并加速了肿瘤细胞的重新附着。开发旨在通过靶向肌动蛋白细胞骨架来抑制附着的肿瘤细胞运动的癌症药物也增强了McTNs。这些结果强调了确定针对细胞分裂或附着的肿瘤细胞的运动性的癌症药物是否会无意中增加转移的重要性。由于手术和新辅助化疗都可以显著增加CTC的水平，因此了解哪些分子靶点会降低CTC的转移效率以及哪些分子靶点可能增加转移风险是很重要的。近90%的人类实体瘤是由上皮细胞产生的癌，其大的尺寸和刚性通常导致其在狭窄的毛细血管中破碎。这种对转移效率的限制可能会对循环癌细胞施加选择性压力，以发展重新附着到血管壁并逃避碎片的机制。PI实验室最近的工作已经确定，当质膜下的肌动蛋白皮质通过遗传改变或化学处理被破坏时，McTNs会增加。这项研究将测试的假设，遗传或治疗失调的肌动蛋白皮质诱导微管蛋白McTN，促进转移性再附着的循环肿瘤细胞。该假设的预测将在以下具体ais中进行检验：1）定义PTEN缺失促进McTN的分子机制。2)确定Src信号传导在McTN延伸和肿瘤转移中的作用。3)检查靶向Rho/ROCK介导的肌动蛋白收缩性对McTN产生的作用。该项目的长期目标是确定控制肌动蛋白破坏以促进循环肿瘤细胞中McTNs的分子机制。扩展我们对McTN结构和分子调控的理解，以及检查来自人类乳腺癌患者的肿瘤细胞中的McTN发生率和功能，将确定新的治疗靶点，并揭示当前癌症药物的潜在转移风险。

 DESCRIPTION (provided by applicant): Cytoskeletal alterations are known to influence the metastatic success of circulating tumor cells (CTCs). However, studies of the tumor cell cytoskeleton are almost exclusively focused on cells attached to surfaces or extracellular matrix leaving a significant knowledge gap relevant to metastasis. Recent work from the PI's research group has demonstrated that the cytoskeleton of circulating tumor cells responds very differently from cells attached to extracellular matrix. Specifically, free-floating tumor cells produce unique tubulin-based microtentacles (McTNs) that promote the reattachment of CTCs to endothelial cell layers. Imaging in live animals has shown that CTCs reattach to blood vessel walls via a cytoskeletal mechanism that matches McTNs. New data from the PI's lab now demonstrate that McTNs are detectable in tumor cells within hours of their isolation from breast cancer patients during surgery. Moreover, patient tumor cell drug responses can be gauged within minutes. Since McTNs are supported by tubulin, the common breast cancer drug, Paclitaxel (Taxol), actually strengthens McTNs and speeds tumor cell reattachment. Developing cancer drugs aimed at inhibiting attached tumor cell motility by targeting the actin cytoskeleton also enhance McTNs. These results emphasize the importance of determining whether cancer drugs aimed at cell division or the motility of attached tumor cells could inadvertently increase metastasis. Sinc both surgery and neoadjuvant chemotherapy can strongly increase the levels of CTCs, it is important to understand which molecular targets will reduce the metastatic efficiency of CTCs and which may increase metastatic risk. Nearly 90% of human solid tumors arise as carcinomas from epithelial cells, whose large size and rigidity often lead to their fragmentation in narrow capillaries. This restraint on metastatic efficiency could impose a selective pressure for circulating carcinoma cells to develop mechanisms to reattach to blood vessel walls and escape fragmentation. Recent work in the PI's lab has identified that McTNs are increased when the actin cortex beneath the plasma membrane is disrupted via either genetic alterations or chemical treatments. This study will test the hypothesis that genetic or therapeutic dysregulation of the actin cortex induces tubulin McTNs that promote the metastatic reattachment of circulating tumor cells. Predictions of this hypothesis will be tested in the following specific ais: 1) Define the molecular mechanism by which PTEN loss promotes McTNs. 2) Identify the role of Src signaling in McTN extension and tumor metastasis. 3) Examine the role of targeting Rho/ROCK mediated actin contractility on McTN generation. The long-term goals of this project are to define the molecular mechanisms that govern actin disruption to promote McTNs in circulating tumor cells. Extending our understanding of McTN structure and molecular regulation as well as examining McTN incidence and function in tumor cells derived from human breast cancer patients will both identify novel therapeutic targets and reveal potential metastatic risks of current cancer drugs.