In postmenopausal women, the adipose fibroblast is the major site of aromatase P450 (P45Oarom) expression, which catalyzes the conversion of C19-steroids to estrogens. Local estrogen production in the breast has been implicated in the growth of breast cancers. Recent work from our laboratory revealed the following: P45Oarom transcript levels and fibroblast content in adipose tissue proximal to a breast tumor are significantly higher than in the distant sites. Media conditioned by breast cancer cells stimulated aromatase activity of glucocorticoid- treated adipose fibroblasts, which was potentiated by E2 pretreatment of the cancer cells. P45Oarom gene expression in breast adipose tissue and fibroblasts is under the control of 3 alternative promoters, namely, II, I.3 and I.4, each giving rise to transcripts with unique 5'-ends. Treatment of fibroblasts with various hormones and cytokines causes major shifts in the promoter usage. We also have shown that IL-6 and IL-11 expressed in breast tumors and fibroblasts are potent stimulators of P45Oarom expression directed by promoter I.4 in adipose fibroblasts, via a Jak/STAT signalling pathway. Therefore, it appears that elevated aromatase activity in peritumoral adipose tissue promotes tumor growth, and that tumor-derived factors in turn regulate adipose aromatase expression and possibly peritumoral fibroblast proliferation, i.e., desmoplastic reaction. In order to characterize the cellular and molecular mechanisms involved in this interaction, we propose the following strategies: We will determine the P45Oarom promoter responsible for increased transcript levels in peritumoral adipose fibroblasts. Initially, a novel, standardized quantification method employing rapid amplification of P450arom cDNA 5'-ends will be developed. Using this method, levels of promoter-specific P45Oarom transcripts will be determined in each breast quadrant. This technology also permits us to simultaneously quantify total P45Oarom transcript levels. We will therefore identify the promoter used for increased aromatase expression proximal to a breast tumor. This will enable us to identify and study expression of tumor-induced trans- activating factors that bind to defined regulatory elements upstream of this promoter. Reduction mammoplasty specimens will be used as disease- free controls. We also seek to establish the regulation of IL-6, IL-11 and their alpha-subunit receptor (R-alpha) expression in T47D and MCF7 cancer cells and adipose fibroblasts in culture using immunoprecipitation and quantitative RT-PCR. Internal standard cRNA sequences homologous to IL-6, IL-11, IL-6R-alpha and IL-11R-alpha transcripts will be generated using the """"""""looped oligo"""""""" method. These will be used as controls in quantitative RT-PCR assays for measuring transcript levels. We will determine the effects of steroids and various growth substances on cytokine expression. We also seek to characterize the direct effects of IL-6 and IL-11 on tumor growth and peritumoral fibroblast proliferation. Flow cytometry will be used to determine growth stimulatory effects of these cytokines on breast cancer cells and on adipose fibroblasts. Finally, transcript levels of IL- 6, IL-11 and their receptors in breast adipose and tumor samples will be determined to better understand the in vivo pathophysiologic significance of these cytokines in human breast cancer. We believe that these studies will provide new insights into the role of peritumoral adipose stroma in supporting tumor growth.
