HGF Regulated Vesicle Trafficking and Tumor Invasion

描述（由申请人提供）：调节肿瘤侵袭和转移的机制在很大程度上仍然未知，这些过程是常见的危及生命的癌症并发症。临床和基础研究表明，细胞因子HGF及其受体c-Met在调节导致大多数人类癌症的肿瘤侵袭的细胞内信号通路中起重要作用。初步结果表明，HGF激活前列腺癌细胞系DU 145中的Ras、PI 3-激酶和MAPK途径，导致细胞分散，这是由E-钙粘蛋白和其他粘附蛋白连接（AJ）蛋白的内吞作用引起的。随后是侵袭增加，这是由组织蛋白酶B阳性溶酶体沿微管沿着的极化胞吐作用引起的。膜运输的调节在癌症进展中起作用，但对相关机制知之甚少。我们推测，HGF激活Ras信号通路，刺激AJ蛋白的巨胞饮内化，并诱导非激酶依赖性溶酶体胞吐，导致组织蛋白酶B的极化分泌，并增加侵袭和转移。在目标#1中，将采用生物化学和显微镜方法来确定巨胞饮作用是否在AJ摄取中起重要作用。显微镜和分子遗传学方法也将用于确定AJ蛋白积累的细胞内区室的性质。最后，我们将确定Ras，Racl和PI 3-激酶相互作用介导HGF诱导的AJ蛋白内化的顺序。在目标#2中，将探索HGF诱导的溶酶体胞吐中涉及的机制。这将涉及使用显微镜和生物化学方法来确定HGF是否诱导溶酶体沿沿着运动。小G蛋白（RhoG，RhoA等）和分子马达（驱动蛋白，肌球蛋白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用于确定通过灭活选择的靶蛋白来阻断溶酶体再分布是否会防止或减少侵袭。在目标#3中，将使用动物模型来确定失活显示调节HGF诱导的顺行溶酶体转运的蛋白质是否会预防或减少侵袭和转移。该项目的完成将增加我们关于囊泡运输在肿瘤侵袭中的作用的知识，并将确定潜在的治疗靶点以减缓或预防癌症扩散。

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.