Malignant breast epithelial cells stimulate aromatase expression via promoter II in human adipose fibroblasts: an epithelial-stromal interaction in breast tumors mediated by CCAAT/enhancer binding protein beta.

Abstract

Expression of aromatase P450 (P450arom), which catalyzes the formation of estrogens, is aberrantly increased in adipose fibroblasts surrounding breast carcinomas, giving rise to proliferation of malignant cells. Aromatase in human adipose tissue is primarily expressed in undifferentiated fibroblasts under the control of several distinct and alternatively used P450arom promoters. In tumor-free breast adipose tissue, P450arom is usually expressed at low levels via a distal promoter (I.4), whereas in the breast adipose tissue bearing a tumor, P450arom is increased through the activation of two proximal promoters, II and I.3. Because the in vivo activation of P450arom promoter II is a key event responsible for aberrantly high P450arom expression in breast tumors, we studied the molecular basis for the enhancement of P450arom promoter II using human adipose fibroblasts (HAFs) in primary culture treated with T47D breast cancer cell-conditioned medium (TCM) as a model system. Upon treatment with TCM, HAFs displayed a striking induction of P450arom mRNA levels via promoter II usage. This effect appeared to be specific for malignant breast epithelial cells, because conditioned media from breast cancer cell lines T47D and MCF-7 induced promoter II activity, whereas normal breast epithelial cells or liver or prostate cancer cell lines did not produce such an effect. Although treatment with a cyclic AMP analogue also caused a switch in the promoter use from I.4 to II in cultured HAFs, TCM-induced promoter II use was found to be mediated via a cyclic AMP-independent pathway. Use of serial deletion mutants of the promoter II 5'-flanking sequence revealed the presence of critical cis-acting elements in the -517/-278 bp region, which regulate the baseline activity. TCM caused a 5.7-fold induction of the -517-bp promoter II construct, whereas site-directed mutagenesis of a CCAAT/enhancer binding protein (C/EBP) binding site (-317/-304 bp) abolished both baseline and TCM-induced activities. Ectopic expressions of C/EBPalpha and C/EBPbeta, but not C/EBPdelta, significantly induced promoter II activity. Moreover, we demonstrated the presence of both C/EBPbeta and C/EBPdelta but not C/EBPalpha in a DNA-protein complex formed by the nuclear extract from TCM-treated HAFs and a probe containing this critical C/EBP binding element (-317/-304 bp). Finally, treatment of HAFs with TCM strikingly induced C/EBPbeta expression, whereas this did not affect the levels of C/EBPalpha or C/EBPdelta transcripts. In conclusion, malignant breast epithelial cells secrete factors, which induce aromatase expression in adipose fibroblasts via promoter II. This is, at least in part, mediated by a TCM-induced up-regulation and enhanced binding of C/EBPbeta to a promoter II regulatory element.