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

Expression of aromatase P450 (P450arom), which catalyzes the formation of e strogens, 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 prom oters. In tumor-free breast adipose tissue, P450arom is usually expressed a t low levels via a distal promoter (I.4), whereas in the breast adipose tis sue bearing a tumor, P450arom is increased through the activation of two pr oximal promoters, II and I.3. Because the in vivo activation of P450arom pr omoter II is a key event responsible for aberrantly high P450arom expressio n in breast tumors, we studied the molecular basis for the enhancement of P 450arom promoter II using human adipose fibroblasts (HAFs) in primary cultu re treated with T47D breast cancer cell-conditioned medium (TCM) as a model system. Upon treatment with TCM, HAFs displayed a striking induction of P4 50arom mRNA levels via promoter II usage. This effect appeared to be specif ic fur malignant breast epithelial cells, because conditioned media from br east cancer cell lines T47D and MCF-7 induced promoter II activity, whereas normal breast epithelial cells or liver or prostate cancer fell lines did not produce such an effect. Although treatment with a cyclic AMP analogue a lso caused a switch in the promoter use from I.4 to II in cultured HAFs. TC M-induced promoter II use was found to be mediated via a cyclic AMP-indepen dent 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-fol d induction of the -517-bp promoter II construct, whereas site-directed mut agenesis of a CCAAT/enhancer binding protein (C/EBP) binding site (-317/-30 4 bp) abolished both baseline and TCM-induced activities, Ectopic expressio ns of C/EBP alpha and C/EBP beta, but not C/EBP delta, significantly induce d promoter II activity. Moreover, we demonstrated the presence of both C/EB P beta and C/EBP delta but not C/EBP alpha in a DNA-protein complex formed by the nuclear extract from TCM-treated HAFs and a probe containing this cr itical C/EBP binding element (-317/-304 bp), Finally, treatment of HAFs wit h TCM strikingly induced C/EBP beta expression, whereas this did not affect the levels of C/EBP alpha or C/EBP delta transcripts. In conclusion, malig nant breast epithelial cells secrete factors, which induce aromatase expres sion in adipose fibroblasts via promoter II. This is, at least in part, med iated by a TCM-induced up-regulation and enhanced binding of C/EBP beta to a promoter II regulatory element.