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.
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