HGF Regulated Vesicle Trafficking and Tumor Invasion

描述(申请人提供)：调节肿瘤侵袭和转移的机制仍然很不清楚，这些过程是常见的和威胁生命的癌症并发症。临床和基础研究表明，细胞因子HGF及其受体c-Met在调节大多数人类肿瘤侵袭的细胞内信号通路中发挥重要作用。初步结果表明，HGF激活了前列腺癌DU145细胞中的RAS、PI 3-激酶和MAPK通路，导致细胞分散，在此之前是E-钙粘素和其他粘附素连接(AJ)蛋白的内吞，随后是侵袭力的增加，与之平行的是组织蛋白酶B阳性的溶酶体沿微管极化的胞吐。膜转运的调控在癌症的发展中起着一定的作用，但人们对相关的机制知之甚少。我们假设HGF激活RAS信号通路，刺激AJ蛋白的大胞内化和诱导不依赖于运动的溶酶体胞吐，导致组织蛋白酶B的极化分泌，增加侵袭和转移。在目标1中，将使用生化和显微方法来确定巨噬细胞吞噬是否在AJ摄取中起重要作用。显微和分子遗传学方法也将被用来确定AJ蛋白在细胞内积累的性质。最后，我们将确定Ras、racl和PI3-激酶相互作用的顺序，以介导HGF诱导的AJ蛋白内化。在目标#2中，将探索HGF诱导的溶酶体胞吐的机制。这将涉及使用显微镜和生化方法来确定HGF是否诱导溶酶体沿微管移动。小G蛋白(RhoG、RhoA等)和分子马达(Kinesin、Myosin V)在溶酶体运动中的作用将被确定。最后，分子遗传学方法也将被用来确定通过灭活特定的目标蛋白来阻止溶酶体重分布是否可以防止或减少入侵。在目标3中，动物模型将被用来确定失活调节HGF诱导的前向瘤小体运输的蛋白质是否可以防止或减少侵袭和转移。该项目的完成将增加我们对囊泡运输在肿瘤侵袭中的作用的了解，并将确定减缓或防止癌症扩散的潜在治疗靶点。

DESCRIPTION (provided by applicant): The mechanisms regulating tumor invasion and metastasis remain largely unknown and these processes are common and life-threatening complications of cancer. Clinical and basic research have demonstrated that the cytokine HGF and its receptor c-Met play an important role in regulating intracellular signaling pathways leading to tumor invasion of most human cancers. Preliminary results have indicated that HGF activates Ras, PI 3-kinase and the MAPK pathway in the prostate cancer cell-line DU145, leading to cell scattering, preceeded by endocytosis of E-cadherin and other adherins junction (AJ) proteins This is followed by increases in invasion that are paralleled by the polarized exocytosis of cathepsin B-positive lysosomes along microtubules. The regulation of membrane trafficking plays a role in cancer progression, yet very little is known about the pertinent mechanisms. We hypothesize that HGF activates the Ras signaling pathway to stimulate both macropinocytic internalization of AJ proteins and to induce kinesindependent lysosome exocytosis, leading to the polarized secretion of cathepsin B and increases in invasion and metastasis. In Aim #1, biochemical and microscopic approaches will be employed to determine if macropinocytosis plays an important role in AJ uptake. Microscopic and molecular genetic approaches will also be used to determine the nature of the intracellular compartment AJ proteins accumulate in. Finally, we will determine the order Ras, Racl, and PI 3-kinase interact to mediate HGF-induced AJ protein internalization. In Aim #2, the mechanisms involved in HGF-induced lysosome exocytosis will be explored. This will involve using microscopic and biochemical approaches to determine if HGF induces lysosome movement along microtubules. The role of small G proteins (RhoG, RhoA, etc) and molecular motors (kinesin, myosin V) in lysosome movement will be determined. Finally, molecular genetic approaches will also be used to determine if blocking lysosome redistribution by inactivating select target proteins will prevent or reduce invasion. In Aim #3, animal models will be used to determine if inactivating the proteins shown to regulate HGF-induced anterogradelysosome transport will prevent or reduce invasion and metastasis. Completion of this project will increase our knowledge concerning the role of vesicle trafficking in tumor invasion and will identify potential therapeutic targets to slow or prevent cancer spread.