C/EBPß LIP and c-Jun synergize to regulate expression of the murine progesterone receptor.

CCAAT/enhancer binding protein ß (C/EBPß) is required for murine mammary ductal morphogenesis and alveologenesis. Progesterone is critical for proliferation and alveologenesis in adult mammary glands, and there is a similar requirement for progesterone receptor isoform B (PRB) in alveologenesis. We examined C/EBPß regulation of PR expression. All three C/EBPß isoforms, including typically inhibitory LIP, transactivated the PR promoter. LIP, particularly, strongly synergized with c-Jun to drive PR transcription. Endogenous C/EBPß and c-Jun stimulated a PR promoter-reporter and these two factors showed promoter occupancy on the endogenous PR gene. Additionally, LIP overexpression elevated endogenous PR protein expression. In pregnancy, both PRB and the relative abundance of LIP among C/EBPß isoforms increase. Consistent with a role in PRB expression, in vivo C/EBPß and PR isoform A expression showed mutually exclusive localization in mammary epithelium, while C/EBPß and PRB largely co-localized. We suggest a critical role for C/EBPß, particularly LIP, in PRB expression.

Progesterone receptor A-regulated gene expression in mammary organoid cultures.

Progesterone, through the progesterone receptor (PR), promotes development of the normal mammary gland and is implicated in the etiology of breast cancer. We identified PRA-regulated genes by microarray analysis of cultured epithelial organoids derived from pubertal and adult mouse mammary glands, developmental stages with differing progesterone responsiveness. Microarray analysis showed significant progestin (R5020)-regulation of 162 genes in pubertal organoids and 104 genes in adult organoids, with 68 genes regulated at both developmental stages. Greater induction of receptor activator of NFkappaB ligand and calcitonin expression was observed in adult organoids, suggesting possible roles in the differential progesterone responsiveness of the adult and pubertal mammary glands. Analysis of the R5020-responsive transcriptome revealed several enriched biological processes including cell adhesion, immune response, and survival. R5020 both induced Agtr1 and potentiated angiotensin II-stimulated proliferation, highlighting the functional significance of the latter process. Striking up-regulation of genes involved in innate immunity processes included the leukocyte chemoattractants serum amyloid A1, 2 and 3 (Saa1, 2, 3). In vivo analysis revealed that progesterone treatment increased SAA1 protein expression and leukocyte density in mammary gland regions undergoing epithelial expansion. These studies reveal novel targets of PRA in mammary epithelial cells and novel linkages of progesterone action during mammary gland development.

Identification of an octamer-binding site controlling the activity of the small breast epithelial mucin gene promoter.

e human small breast epithelial mucin (SBEM) gene has been identified as being preferentially expressed in mammary epithelial cells and over-expressed in breast tumors. In this report, we have characterized the promoter of SBEM gene in order to identify sequences responsible for this strong mammary expression. A series of SBEM promoter/luciferase constructs were transiently transfected into both breast (MCF-7, BT-20) and non-breast (HeLa and HepG2) cell lines. In addition to the minimal promoter and to a repressor region, we have identified an 87-bp sequence (-357/-270) driving a strong breast-specific expression. Site-directed mutagenesis of a putative octamer-binding transcription factor binding site located within this latter region led to a strong decrease of the transcriptional activity of the SBEM promoter. Furthermore, transient over-expression of Oct1 and Oct2 not only increased SBEM promoter reporter activity, but also enhanced endogenous SBEM mRNA level. Overall, the data suggest that octamer-binding transcription factors participate in the strong expression of SBEM gene in breast tissues. Clarifying the SBEM gene regulation will help to dissect mechanisms underlying transcription of normal breast and breast cancer-associated genes.

Expression of an estrogen receptor variant lacking exon 3 in derivatives of MCF-7 cells with acquired estrogen independence or tamoxifen resistance.

The estrogen receptor (ER) plays important roles in the development and progression of breast cancer, and is a major target for tumor therapy. In this study, we investigated ER function in two derivatives of MCF-7 cells that were selected for their ability to proliferate in the absence of estrogen or in the presence of the antiestrogen, tamoxifen. Reporter gene assays indicated decreased ER activity in both cells lines, although the activity remaining retained responsiveness to both estrogen and tamoxifen. The decreased ER activity correlated with expression of a 61 kDa variant ER protein, and sequencing of RT-PCR products indicated that this variant was the product of an exon 3 deletion (ERDeltaE3). To study its effects on cell proliferation, ERDeltaE3 cDNA was stably transfected into both the MCF-7 cell line and its estrogen-independent/tamoxifen-sensitive derivative MCF-7/LCC1 (LCC1), and the phenotypes of transfectants were examined. Expression of ERDeltaE3 was not sustainable in MCF-7 cells, but was maintained for at least 17 passages in LCC1 cells. These results are in agreement with previous reports that ERDeltaE3 inhibits wild-type ER activity and negatively regulates proliferation of MCF-7 cells. They further suggest that the alteration that leads to estrogen independence in LCC1 cells allows for sustained expression of ERDeltaE3, and that additional changes are required to confer tamoxifen resistance to these cells.

Identification of a novel breast- and salivary gland-specific, mucin-like gene strongly expressed in normal and tumor human mammary epithelium.

Expression profiling using the public expressed sequence tag (EST) and serial analysis of gene expression (SAGE) databases resulted in the identification of a putative breast-specific mRNA that we have termed small breast epithelial mucin (SBEM). Hybridization analysis performed on 43 normal human tissues revealed that the SBEM gene was only expressed in mammary and salivary glands. Further reverse-transcription PCR analyses confirmed SBEM expression in most of established human breast epithelial cell lines analyzed (7 of 8) but not in cell lines of non-breast origin (0 of 6). SBEM mRNA expression was detected in >90% of invasive ductal carcinomas and correlated with the expression of a previously characterized breast-specific gene, mammaglobin-1 (n = 54; Spearman r = 0.34, P = 0.011). Interestingly, a higher SBEM:mammaglobin-1 ratio was observed in primary tumors with axillary lymph node metastasis than in node-negative tumors (n = 46; Mann-Whitney, P = 0.04). In a subset of 20 primary breast tumors and their matched axillary lymph nodes, a high concordance (Fisher's exact test, P < 0.001) was seen between PCR detection of SBEM mRNA in lymph node tissue and their histopathological status, indicating that SBEM mRNA expression is conserved in nodal metastasis. The SBEM gene is predicted to code for a putative low molecular weight, secreted sialoglycoprotein, potentially useful for the diagnosis of metastatic breast cancer.