The major goal of this proposal is to provide direct evidence (causal relationship) that organ-derived paracrine growth factors regulate the site-specific growth of malignant human colon carcinoma (HCC) cells that possess the appropriate receptors. Recent data from our laboratory demonstrates that the ability of HCC cells to produce hepatic metastasis directly correlates with expression of epidermal growth factor receptor (EGF-R). Highly metastatic HCC cells also respond to specific mitogenic signals produced by tissue undergoing repair (hepatectomy followed by liver regeneration) demonstrating that physiological signals can be utilized by neoplastic cells.
Four specific aims will be pursued. EGF-R, hepatocyte growth factor (c- met), and insulin-like growth factor-1 receptor (IGF-1-R) will be functionally characterized (alone and in combination) in an isogeneic HCC cell system using highly metastatic versus non- and low-metastatic cells growing in vitro, in co-culture with human hepatocytes, and orthotopically, ectopically, or at the metastatic site in athymic nude mice using standard molecular, biochemical and tumor biological techniques. Secondly, the production and putative upregulation of the ligands for these receptors (transforming growth factor alpha (TGF-alpha), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1)) will be assessed in host tissue adjacent to the HCC growing at orthotopic, ectopic, and metastatic sites in athymic nude mice using in situ mRNA hybridization (ISH), immunohistochemistry, and reverse transcriptase- polymerase chain reaction (RT-PCR) analyses. Thirdly, to determine if these receptors are directly involved (causal relationship), or alternatively, act as markers for the metastasis and growth of HCC cells in the liver environment, methodologies designed to disrupt the function or decrease the levels of the receptors will be invoked, i.e., antisense RNA, protein tyrosine kinase (PTK) inhibition, and expression of dominant negative mutant receptors. Lastly, fresh and archival primary and metastatic colon carcinoma surgical specimens will be analyzed for expression of EGF-R, c-met, and IGF-1-R by the HCC and production of their corresponding ligands by the adjacent host tissue using mRNA, protein ISH, and immunohistochemical techniques for the relevance of the in vitro and nude mouse findings to clinical reality. The knowledge gained from this research should extend our understanding of host:tumor interactions and provide a therapeutic basis for interfering with hepatic metastases produced by HCC by downregulation of PTK receptor number and function.
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