HGF Regulated Vesicle Trafficking and Tumor Invasion

描述（申请人提供）：调控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激活前列腺癌细胞DU145中的Ras、PI 3-激酶和MAPK通路，导致细胞散射，然后是E-cadherin和其他粘附连接（AJ）蛋白的内吞噬，随后是侵袭的增加，与组织蛋白酶b阳性溶酶体沿微管极化胞外分泌平行。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。我们假设HGF激活Ras信号通路刺激AJ蛋白的巨噬细胞内化和诱导激酶不依赖的溶酶体胞外分泌，导致组织蛋白酶B的极化分泌和侵袭和转移的增加。在Aim #1中，将采用生化和显微镜方法来确定巨噬细胞是否在AJ摄取中起重要作用。显微和分子遗传学方法也将用于确定AJ蛋白积累的细胞内腔室的性质。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Aim #2中，将探讨hgf诱导的溶酶体胞吐作用的机制。这将涉及使用显微镜和生化方法来确定HGF是否诱导溶酶体沿着微管运动。小G蛋白（RhoG， RhoA等）和分子马达（运动蛋白，肌凝蛋白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是否阻断溶酶体再分配通过失活选择的目标蛋白将阻止或减少入侵。在Aim #3中，将使用动物模型来确定失活调节hgf诱导的顺级酶体运输的蛋白质是否会阻止或减少入侵和转移。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认识，并将确定减缓或预防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.