Basic research remains critical for our interpretation and improvement of ongoing chemo-endocrine breast cancer protocols. Our prototype model of tamoxifen-5fluorouracil (TAM-FUra) modulation remains the only in vitro example of chemo-endocrine synergy reported to date, and this project will continue to explore oncogenes (c-myc, c-fos, c-erbB-2) and estrogen receptors (ER) as targets for RNA-directed chemo-endocrine therapy. Human breast cancer cell lines will be used to i) complete studies comparing antiestrogen effects on c-myc and c-fos expression, ii) establish the therapeutic impact of inhibiting endogenous c-erbB-2 expression by transfecting antisense-erbB-2 genes, and iii) identify and characterize the small RNA species (RNAt) isolated from in vitro bound complexes of ER with its cognate DNA response element, ERE. Northern blot hybridization, nuclear run-on and in vitro transcript splicing assays will differentiate effects on c-myc and c-fos mRNA metabolism resulting from anti-estrogen therapy; and modulation of protein kinase C activity will help to determine if this signal transduction pathway is involved in these antiestrogen effects. Transfection of a constitutively promoted 5'-antisense-erbB-2 vector appears to inhibit growth of only those breast cancer cells showing deregulated endogenous c-erbB-2 overexpression; and further analysis of sense vs. antisense transfected subclones is necessary to document changes induced in c-erbB-2 transcript rates, mRNA and protein (pl85erbB-2) levels. Antisense-erbB-2 genes driven by inducible promoters (MMTV, hsp-70) will also be constructed to deliver the antisense response in combination with drug or endocrine therapy. Lastly, preliminary studies support a new model of ER gene activation involving a 4S RNA species (RNAt) that is necessary for and contained within in vitro ER-ERE complexes. Gel-shift will be used to purify RNAt, and to characterize its binding with ER and ERE. A therapeutic role for RNAt will be looked for by comparing drug and endocrine effects on ER-ERE complexes formed either in vitro or in vivo. These studies will hopefully provide new rationale and direction for improved chemo-endocrine therapies of breast cancer.
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