Ovarian cancer is the leading cause of gynecologic malignancy deaths in the United States. In particular, ovarian carcinomas appears to be the most lethal tumor of the female genital tract and continues to be the major cause of mortality in female cancer patients. Metastases from the ovary are common by the time the ovarian enlargement leads to patient symptoms (at advanced stages III-IV). The biological factors affecting the ability of ovarian tumor cells to migrate and implant at distant locations within the peritoneal cavity remain unclear. In the proposed research, we plan to test the hypothesis that the CD44 isoform-p185HER2 interaction with the cytoskeleton plays an important role in promoting oncogenic signals required for tumor cell adhesion, motility, proliferation and growth during human ovarian cancer progression. This hypothesis clearly has important implications for the spread and progression of human ovarian cancers. Specifically, we plan to use a variety of biochemical, immunological and molecular biological techniques to elucidate the structural and functional association between the CD44s-p185HER2 complex and the cytoskeleton. We will also employ in vitro mutagenesis to construct CD44s deletion (or site- directed) mutants that lack certain portions (or replace certain amino acids) of the cytoplasmic domain required for the binding of the cytoskeleton. These mutants polypeptides will be expressed in ovarian tumor cells and their structural changes will be correlated with CD44s- p185HER2-mediated oncogenic properties (e.g. cell adhesion, motility, cell proliferation and growth). In the second part of the proposal, we plan to identify CD44 variant (CD44v) isoforms associated with human ovarian cancers using RT-PCR, DNA sequence analyses and in situ hybridization. We will clone the unique variant exons into standard CD44 cDNA and express these constructs in CD44v-negative (p185HER2- positive) ovarian epithelial cells. These CD44v transfectants will then be analyzed for their functional properties (e.g. CD44v-p185HER2 interaction, adhesion, tumor cell motility, proliferation and growth). We also plan to downregulate CD44v-p185HER2-mediated oncogenic signals by using dominant-negative mutants of Shc and/or Grb2. These mutant polypeptides will be expressed in ovarian tumor cells and their structural changes will be correlated with various oncogenic properties. Finally, specific antibodies against the """"""""fusion"""""""" proteins containing CD44 variant exons (related to ovarian cancer cells) will be prepared. The co-expression of certain CD44v isoforms with p185HER2 in ovarian carcinomas will be analyzed during ovarian cancer progression. We believe that the new information obtained from this proposal may establish the CD44v-p185HER2 complex as an important tumor marker for early detection and evaluation of oncogenic potential, as well as allow the development of new drug targets to inhibit tumor cell motility and proliferation/growth.
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