The main objective of this application is to determine epithelial-stromal interactions, which increase local estrogen biosynthesis and inhibit adipocyte differentiation in malignant breast tumors. We hypothesize that malignant epithelial cells inhibit the differentiation of surrounding adipose fibroblasts to mature adipocytes. This results in the formation of a dense fibroblastic layer around epithelial cells. These fibroblasts provide structural and estrogenic support to epithelial cells. Epithelial factors also upregulate aromatase expression in these undifferentiated fibroblasts by activating aberrant promoter regions of the CYP19 (aromatase) gene. Our preliminary data are as following: (i) Expression of aromatase, the rate-limiting enzyme in estrogen biosynthesis, is regulated by multiple cell-specific promoters distributed within the 90-kb 5'-flanking region of the CYP19 gene via alternative splicing. (ii) Malignant epithelial cells secrete factors that aberrantly activate promoter II in adjacent adipose tissue fibroblasts. This increases the tissue levels of aromatase mRNA originating from promoter II but containing an identical coding region and thus giving rise to an identical functional protein regardless of the promoter use. (iii) The switch from the physiological adipose tissue promoter (I.4) to the pathologically used cancer tissue promoter (II) in adipose fibroblasts is regulated by differential binding of coactivator or corepressor complexes to these regions in a reciprocal fashion. (iv) In response to estrogen, malignant epithelial cells secrete large quantities of TNF and IL-11 that inhibit the differentiation of adipose fibroblasts to mature adipocytes, and thus, increase the number of aromatase-expressing fibroblasts adjacent to malignant cells. (v) We recently cloned a novel promoter region of the CYP19 (aromatase) gene, which is upregulated in breast cancer tissue compared with breast fat distal to cancer. In order to define molecular and cellular mechanisms responsible for aromatase overexpression and inhibition of adipocyte differentiation in breast cancer tissue, we are proposing the following aims: 1) to characterize paracrine and autocrine mechanisms responsible for estrogen-mediated upregulation of TNF, IL-11 and their receptors in breast tumor tissue; 2) to identify common transcriptional enhancers and associated signaling pathways that activate the CYP19 gene and other estrogenic genes in adipose tissue fibroblasts in response to malignant epithelial cell-derived factors; 3) to determine the cell-specific distribution of a novel CYP19 (aromatase) gene promoter and its biological roles in breast cancer and disease-free tissues.
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