Use of alternative promoters to express the aromatase cytochrome P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients.

Estrogen biosynthesis in adipose tissue has assumed great significance in terms of a number of estrogen-related diseases. Recent evidence suggests that estrogen synthesized locally in the breast is of singular significance in the development of breast cancer in elderly women. The biosynthesis of estrogen from C19 steroids is catalyzed by a specific form of cytochrome P450, namely aromatase cytochrome P450 (P450arom; the product of the CYP19 gene). The human CYP19 gene comprises nine coding exons, II-X, and its transcripts are expressed in the ovary, placenta, testes, adipose tissue, and brain. Tissue-specific expression of the CYP19 gene is determined, at least in part, by the use of tissue-specific promoters, which give rise to transcripts with unique 5'-noncoding termini. Transcripts in adipose tissue contain 5'-termini derived from specific untranslated exons, corresponding to expression derived from the proximal promoter II and its splice variant I.3, as well as a distal promoter, I.4. The object of the present study was to determine the distribution of these various exon-specific transcripts in breast adipose tissues from cancer-free women undergoing reduction mammoplasty and from patients with breast cancer, because this would provide important clues as to the nature of the factors regulating aromatase expression in these sites. To achieve this, we employed competitive RT-PCR, utilizing an internal standard for each exon-specific transcript of the CYP19 gene, as well as for the coding region, to evaluate total CYP19 gene transcripts. In cancer patients (n = 18), total CYP19 gene transcript levels were significantly higher in adipose tissue proximal to a tumor in comparison with adipose tissue distal to a tumor, in agreement with previous findings. Moreover, total transcript levels were higher in breast adipose tissue of cancer patients in comparison with those of cancer-free individuals (n = 9), even when the adipose tissue from the cancer patient was taken from a quadrant with no detectable tumor. We observed that exon I.4-specific transcripts were predominant in breast adipose obtained from cancer-free women. In this tissue, promoter-II-specific and exon I.3-specific transcripts were present in low copy number. On the other hand, in breast cancer patients, CYP19 gene transcripts from breast adipose tissue had primarily promoter-II-specific and exon I.3-specific sequence, whereas comparatively few transcripts had exon I.4-specific sequence at the 5'-terminus. We conclude that CYP19 gene transcription in breast adipose tissue of cancer-free individuals uses preferably promoter I.4, implicating a role of glucocorticoids and members of the IL-6 cytokine family in the regulation of this expression. On the other hand, the increased expression in breast adipose tissue bearing a carcinoma results from expression from promoters II and I.3, which are regulated by unknown factors acting via increased cAMP formation, which are presumably secreted by the tumor or associated cells.