We have examined the role that the Hox-related homeobox containing gene, Msx2, plays during branching morphogenesis where our studies in vivo and in vitro showed that P in the presence of E regulates its expression. The over-expression of Msx-2 in stable transfectants of the normal mouse mammary epithelial cell line, NMuMg, resulted in a highly branched phenotype compared to control cells transfected with the empty vector (EV) when grown in collagen gels. In the normal mammary gland, Msx2 is induced by progesterone acting through the A isoform of the progesterone receptor and the BMP2/4 signaling pathway. The NMuMg-Msx2 cells constitutively over-express cyclin D1 and cyclin E and form multiple large colonies when grown in soft agar. They undergo epithelial to mesenchymal transition (EMT) through a Cripto-1 activation pathway. When the NMuMg-Msx2 cells were implanted into nude mice either subcutaneously or in the mammary fat pad, rapidly-growing, poorly differentiate tumors arose within 15 weeks in 97% of the mice compared to small, slow-growing, well differentiated tumors in animals given the NMuMg-EV cells. By real time PCR and hybridization to Clontechs Cancer Profiling Array, an increase in MSX-2 mRNA expression was observed in human breast cancer, but it did not reach statistical significance. Mining of published databases found a strong correlation between elevated MSX2 expression and estrogen receptor (ER) positive breast cancer. Thus we utilized matched normal and cancerous tissue from the same patient and found a significant increase in expression of MSX2 was observed. In collaboration with Dr. Satoh in Sendai, Japan, we also showed that MSX-2 is over-expressed in human pancreatic cancer where it appears to induce EMT. Utilizing our knowledge of the MSX homeobox genes, we are examining the role of the homeobox-containing gene, BP-1, in normal development and in human breast cancer. In collaboration with Dr. Pat Berg of George Washington University who showed this gene to be more prevalent in breast cancer from African American women vs. Caucasians, and over-expressed in all ER negative breast cancers examined, we have shown that cells over-expressing BP-1 develop larger, more aggressive tumors in nude mice. Expression of BP-1 in breast cancer cells is regulated by PRL. We are examining the role of PRLR isoforms and autocrine/paracrine PRL in tumorigenesis and carcinogenic susceptibility. Using RT-PCR and the Clontech Cancer Profiling Array, we found that there was significantly higher expression of the PRLR mRNA in the cancerous vs. the normal tissue from the same patient. The various forms of the PRLR (LF, SF1a and SF1b) differ in their cytoplasmic domains due to alternate splicing. The ability of SF1b and SF1a to transduce PRLs mitogenic signal or to act as a dominant negative of the LF in transducing the mitogenic signal, is being assessed by siRNA knock-down experiments. We are examining the role that the PRL autocrine system may have in metastasis. To this end we have stably transfected MDA-MB4-35 cells with hPRL and noted an increase in cells growth, expression of PRL receptors, colony formation in soft agar and migration in culture. In addition, the MDA-MB-435PRL cells undergo EMT and overexpress MSX2 and BP-1.The effects of PRL in this system are through activation of the wnt pathway utilizing wnt16 as ligand. The MDA-MB4-35 cells develop larger and more aggressive tumors in nude mice. These data strongly suggest that the various isoforms of PRLR may be good therapeutic targets for a subclass of breast cancers. Autocrine PRL expression is primarily regulated by a distal promoter located 5.8 kb upstream to the pituitary promoter. As a result of alternative promoter usage, autocrine PRL is regulated by different signaling pathways and different hormones, cytokines or neuropeptides compared to regulation in the pituitary. We have shown that estrogen directly induces hPRL gene expression in T47D human breast cancer cells. We have identified a functional, non-canonical estrogen responsive element (ERE) and an AP1 site located in the hPRL distal promoter. Gel shift and chromatin immunoprecipitation assays demonstrated that both ER alpha and ER beta directly bind to the ERE. However, only ER alpha interacts with AP1 proteins that bind to the AP1 site in the hPRL distal promoter. Promoter-reporter gene studies demonstrate that both ERE and AP1 sites are required for full induction of the promoter activity by estradiol. Our studies suggest that the interactions between estrogens, ERs, the ERE and AP1 transcription factors in regulation of autocrine/paracrine PRL in the human breast may be critical for oncogenesis and may contribute to progression of breast cancer. I have established several collaborations with clinical researchers including Drs. Sheila Prindiville and Superna Wedam, to explore the role of the PRL autocrine system in breasts of at-risk women being treated with aromatase inhibitors and women with high breast density. Increased breast density is an independent risk factor for breast cancer and is believed to be largely due to the stromal content of the breast. Recent studies have indicated that autocrine PRL is made in the stromal cells of the normal human breast. Using normal tissue from organ donors and reduction mammoplasties we are exploring the level of expression of the PRL autocrine system in the epithelium vs. stroma (intralobular vs. interlobular) of the human breast
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