Breast cancer is the most common cancer among women living in western societies. Many breast tumors are estrogen (E2) responsive, but the precise role of E2 in carcinogenesis is unknown. We have established and characterized a highly novel system in which to identify the critical factors associated with E2-induced malignant progression, and the progression to E2-independence in human breast cancer. Sublines of the E2-dependent MCF-7 cell line form stable, invasive, E2-independent tumors in the mammary fat pads of ovariectomized athymic nude mice. We have clearly implicated the regulation of specific genes in E2-induced progression. Of 8 differentially expressed proteins we have identified, two exhibit substantial differential expression. HD-6 is present in E2-withdrawn MCF-7 cells and absent in E2-treated MCF-7 and E2- independent variants (plus/minus E2). We hypothesize that HD-6 may exhibit E2-induced suppressor-like activity. HI-10 is present in E2-treated MCF-7 cells, in E2-independent cell (plus/minus E2) but absent in E2-withdrawn MCF-7 cells. We hypothesize that HI-10 exhibits E2-induced progression-like activity. We also hypothesize that HD-6 and HI-10 contribute to the E2-induced progression observed in human breast cancer cells.
Our specific aims are to isolate and characterize the HD-6 and HI-10 proteins and cDNAs. Our first priority is to isolate and identify the HD-6 and HI-10 proteins. Partial N-terminal amino acid sequence will be obtained to facilitate cDNA cloning. Internal peptides will be generated to enable sequence analysis of N-terminal blocked proteins. The HD-6 and HI-10 cDNAs will be cloned either by PCR, or by screening appropriate lambda gt11 cDNA libraries with specific antisera. Antisera specific for HD-6 and HI-10 will be raised and used to determine expression of the HD-6 and HI-10 proteins in normal, benign and malignant human breast tissues by immunohistochemical analyses. The function of the HD-6 and HI-10 proteins will be investigated by transducing malignant or normal breast cells with the respective cDNA and investigating the phenotype of the transduced cells both in vitro and in vivo. The long term aims are to further extend and support our hypothesis by determining the relevance of HD-6 and HI-10 and other proteins to E2-induced progression, and their utility as both prognostic indicators and targets for therapeutic intervention.
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