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Paracrine interactions between adipose fibroblasts and malignant epithelial cells are essential for structural and hormonal support of breast tumors. Factors derived from malignant epithelial cells inhibit adipogenic differentiation of fibroblasts and upregulate expression of aromatase, which stimulates estrogen synthesis and creates a localized, growth-stimulatory environment. Here, we characterized the gene expression profile of breast adipose fibroblasts in an in vitro model of malignancy to identify other paracrine interactions that support tumor growth. Primary breast adipose fibroblasts from cancer-free women were treated with conditioned media from malignant breast epithelial cells or normal breast epithelial cells, and differences in gene expression were identified by microarray. A total of 79 differentially regulated genes encoding cytokines, enzymes, angiogenic factors, cytoskeletal proteins, extra-cellular matrix remodeling proteins, signal transduction proteins and cell surface receptors were identified, and 6 of these were verified by real-time PCR. Among these, the expression of aldo-keto reductase family 1, member C3 (AKR1C3) was upregulated. AKR1C3 has multiple enzymatic properties, including conversion of estrone to estradiol and androstenedione to testosterone. Immunoreactive AKR1C3 was detected in epithelial and stromal components of benign lesions and ductal carcinomas in situ, and in 59.8% of epithelial and 69.6% of stromal cells in invasive breast carcinomas. AKR1C3 expression was significantly higher in myoepithelial cells surrounding the neoplastic epithelium of ductal carcinoma in situ compared with those surrounding benign epithelial lesions. Importantly, AKR1C3 and aromatase mRNA levels correlated positively in 61 malignant breast tumors (R=0.3967, p=0.00156). Malignant epithelial cell-conditioned medium significantly increased formation of testosterone and estradiol from androstenedione in breast adipose fibroblasts. In conclusion, malignant epithelial cell-derived factors significantly upregulate the enzymes AKR1C3 and aromatase that catalyze a series of complementary reactions to convert the circulating precursor androstenedione to biologically active estradiol in vitro in the stromal fibroblasts, and in vivo, in stromal component of breast tumors.

In breast cancer, a dense layer of undifferentiated fibroblasts is formed around malignant breast epithelial cells and referred to as desmoplastic reaction. These cells provide structural and functional support for tumor growth. Aromatase, the key enzyme in the biosynthesis of estrogen, is overexpressed in these undifferentiated fibroblasts, producing large quantities of estrogen, which in turn influences the growth and progression of malignant epithelial cells. We previously demonstrated that malignant epithelial cells produce large amounts of TNFalpha, which inhibit the differentiation of breast fibroblasts. TNF action is mediated by its two receptors (TNFRs), TNFR1, which mediates inhibition of adipocyte differentiation, and TNFR2, which was linked to the proliferation of thymocytes. We present evidence here that estrogen modulates the synthesis of receptors for TNF in human adipose fibroblasts (HAFs) from breast tissue in a paracrine fashion, which may serve as a mechanism for the inhibition of adipocyte differentiation in breast cancer. Estradiol (E(2)) treatment increased TNFR1 mRNA and protein levels in primary HAFs in a dose- and time-dependent manner, which could be reversed by the estrogen antagonist ICI182,780. Interestingly, higher concentration of E(2) inhibited whereas lower concentrations stimulated TNFR2 mRNA levels in HAFs. To investigate the specific roles of TNFRs in adipocyte differentiation, we incubated breast HAFs with receptor selective muteins of TNF. TNFR1-selective mutein decreased mRNA levels of aP2, a marker for adipogenic differentiation. This antiadipogenic effect was enhanced by cotreatment with E(2). We conclude that high levels of estrogen found in breast tumors promote the antiadipogenic action of TNF on breast adipose fibroblasts by selectively up-regulating TNFR1, which may be a critical mechanism for desmoplastic reaction.

The aromatase gene encodes the key enzyme for estrogen formation. Aromatase enzyme inhibitors eliminate total body estrogen production and are highly effective therapeutics for postmenopausal breast cancer. A distal promoter (I.4) regulates low levels of aromatase expression in tumor-free breast adipose tissue. Two proximal promoters (I.3/II) strikingly induce in vivo aromatase expression in breast fibroblasts surrounding malignant cells. Treatment of breast fibroblasts with medium conditioned with malignant breast epithelial cells (MCM) or a surrogate hormonal mixture (dibutyryl (Bt2)cAMP plus phorbol diacetate (PDA)) induces promoters I.3/II. The mechanism of promoter-selective expression, however, is not clear. Here we reported that sodium butyrate profoundly decreased MCM- or Bt2cAMP + PDA-induced promoter I.3/II-specific aromatase mRNA. MCM, Bt2cAMP + PDA, or sodium butyrate regulated aromatase mRNA or activity only via promoters I.3/II but not promoters I.1 or I.4 in breast, ovarian, placental, and hepatic cells. Mechanistically, recruitment of phosphorylated ATF-2 by a CRE (-211/-199, promoter I.3/II) conferred inductions by MCM or Bt2cAMP + PDA. Chromatin immunoprecipitation-PCR and immunoprecipitation-immunoblotting assays indicated that MCM or Bt2cAMP + PDA stabilized a complex composed of phosphorylated ATF-2, C/EBPβ, and cAMP-response element-binding protein (CREB)-binding protein in the common regulatory region of promoters I.3/II. Overall, histone acetylation patterns of promoters 1.3/II did not correlate with sodium butyrate-dependent silencing of promoters I.3/II. Sodium butyrate, however, consistently disrupted the activating complex composed of phosphorylated ATF-2, C/EBPβ, and CREB-binding protein. This was mediated, in part, by decreased ATF-2 phosphorylation. Together, these findings represent a novel mechanism of sodium butyrate action and provide evidence that aromatase activity can be ablated in a signaling pathway- and cell-specific fashion. [ABSTRACT FROM AUTHOR]