Regulated CYP19 aromatase transcription in breast stromal fibroblasts.

Extraglandular estrogen synthesis mediates the proliferation of estrogen-responsive breast cancer in postmenopausal women. Aromatase, the cytochrome P450 Cyp19 enzyme, catalyzes the rate-limiting step in estrogen biosynthesis. Activity is present in both normal and neoplastic breast tissue, and Cyp19 protein is localized by immunohistochemistry predominantly in breast stromal fibroblasts. In cultured breast stromal fibroblasts, both activity and Cyp19 messenger ribonucleic acid are increased to a substantial degree by hormonal and growth factor regulators of transcription. Transcriptional regulation of CYP19 is complex in breast tissues, in which exon switching in the usage of alternative first exons occurs from predominantly EI.4 in breast tissue from cancer-free women to predominantly EI.3 and PII in breast tumors and quadrants with or without tumor. The present study questioned whether the first exon switch occurs as a result of an inherent difference between fibroblasts in normal and tumor tissues or because of differences in local regulators between these tissues. To distinguish between these two possibilities, we examined fibroblasts cultured from breast tumor, benign breast, and reduction mammoplasty tissues for the ability of various CYP19 transcriptional regulators to modulate first exon EI.3, EI.4, and PII usage. A semiquantitative RT-PCR method was used to identify transcripts containing six of the nine known CYP19 first exons. Combinations of cAMP and Dex regulated transcription from first exons EI.3, EI.4, and PII in fibroblasts cultured from all tissues, but not in reduction mammoplasty epithelial cells. These results provide evidence that the fibroblasts from these breast tissues are not inherently different in transcriptional regulation of CYP19 first exon usage and that transcriptional regulatory molecules are likely to mediate the exon switch phenomenon.

Membrane association of cathepsin B can be induced by transfection of human breast epithelial cells with c-Ha-ras oncogene.

Alterations in trafficking and increases in expression of the lysosomal proteases cathepsins B, D and L have been observed in transformed cells and malignant tumors, including human breast carcinoma. ras and the related rab proteins participate in the vesicular transport processes required for normal trafficking of lysosomal enzymes. In addition, transfection of murine fibroblasts with the ras oncogene has been shown to increase the expression of cathepsins L and B. As human cancers are primarily epithelial in origin, we have investigated whether there are alterations in the trafficking and expression of cathepsin B in MCF-10 human breast epithelial cells transfected with wild-type and mutated ras. In all cells examined, i.e. mortal MCF-10M cells, immortal MCF-10A or MCF-10F cells, and transfected MCF-10A cells (transfected with the neomycin resistance gene (MCF-10Aneo) or cotransfected with wild-type proto-oncogenic ras (MCF-10AneoN) or mutated oncogenic ras (MCF-10AneoT)), levels of mRNA transcripts for cathepsin B were similar. However, alterations in trafficking of cathepsin B were observed in the cells transfected with oncogenic ras. In these cells there was an increased association of cathepsin B activity and cathepsin B protein with plasma membrane/endosomal fractions and a more peripheral distribution of immunofluorescent staining for cathepsin B. At the electron microscopic level, immunogold labeling for cathepsin B was localized to the cell membrane as well as to vesicles in the microvilli and adjacent to the cell membrane. In the parental MCF-10A cells, in contrast, cathepsin B was localized to vesicles in the perinuclear region. The cathepsin B associated with plasma membrane/endosomal fractions in the cells transfected with oncogenic ras was mature cathepsin B as demonstrated by immunoblot analysis. This was confirmed further by showing an absence of peripheral immunofluorescent staining in these cells using an antibody specific for the propeptide of cathepsin B. Thus, we have demonstrated by multiple techniques that transfection of human breast epithelial cells with oncogenic ras results in alterations in the trafficking of cathepsin B similar to those observed previously in human and animal tumors of both epithelial and mesenchymal origin.