Aromatic fatty acids, of which phenylacetate (PA) is a prototype, constitute a new class of low toxicity drugs with demonstrated antitumor activity in experimental models and in humans. We and others have shown that PA inhibits the growth of human breast cancer cells. Our data indicates the ER+ cells are markedly more sensitive to PA than are ER- cells suggesting there is """"""""cross-talk"""""""" between the PA and estrogen signaling pathways. We have now confirmed this hypothesis by obtaining data showing that PA can inhibit gene transcription mediated by the """"""""classical"""""""" estrogen response element located in the vitellogenin gene promoter (EREv). The overall objective of this proposal is to delineate the precise mechanism(s) by which PA derivatives modulate estrogen-mediated gene regulation in human breast cancer cells.
Specific aim #1 is to determine the method by which PA and some of its more potent derivatives inhibit estrogen signaling through EREv. To do this, we will characterize the nuclear protein(s) that are induced by the PA compounds to bind EREv in breast cancer cells, and the gene and cell type specificity of these effects. Our preliminary studies have shown that these compounds also downregulate transcription of cyclin D1, an important estrogen-responsive gene that is overexpressed in roughly 40 percent of all breast tumors, but does not contain a canoncal ERE.
Specific aim #2 is to establish the mechanism of this activity. Using transient transfections of MCF-7 breast cancer calls with cyclin D1-promoter-luciferase constructs, the precise boundaries and critical nucleotide positions within the cyclin D1 promoter will be determined by delineating the effects of specific mutations on promoter function. Biochemical analysis using gel retardation and footprinting will be performed to correlate function of critical PA-responsive sequences with specific DNA-protein binding.
Specific aim #3 is to evaluate the therapeutic consequences of the PA blockage of estrogen responsive genes in an aromatase transgenic mouse model. Using these mice, we have already determined that aromatase/estrogen-induced preneoplastic/neoplastic changes following lactation can be completely abrogated by systemic administration of the PA derivative 4-chlorophenylacetate. Using a variety of treatment protocols, we will now assess the full potential of this and other PA compounds to act as chemoprotective agents against estrogen-induced carcinogenesis both without and with the addition of environmental carcinogens like DMBA. Taken together, the information gained from these studies will provide a basic framework for the rationale use of PA analogs or similar acting compounds in the clinic.
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