The overall aim of my research is to understand the mechanism of prolactin (PRL) action in concert with estrogen (E), progesterone (P) and growth factors during growth, development, differentiation and tumorigenesis in the mammary gland. We have investigated two unique, interacting areas 1) delineating the role of PRL in the regulation of proliferation during normal mammary gland development and 2) defining the fundamental role of the PRL autocrine/paracrine system in human breast cancer. While past work has emphasized the first area, our ongoing and future research will concentrate more on the second area. To this aim we have initiated several collaborations with clinical researchers in the CCR. 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. The NMuMg-Msx2 cells constitutively over-express cyclin D1 and cyclin E and form multiple large colonies when grown in soft agar. 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 Clontech's Cancer Profiling Array, an increase in MSX-2 mRNA expression was observed in human breast cancer, but it did not reach statistical significance. In collaboration with Drs. Satoh and Shimosegawa in Sendai, Japan, we also showed that MSX-2 is over-expressed in pancreatic cancer where it appears to be involved in epithelial to mesenchymal transition. 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 are examining the effects of over-expression of BP-1 on tumor growth in nude mice and its expression in normal tissues from African American and Caucasian women. In addition, we are studying the mouse homolog, DLX4/7, in the mouse mammary gland where it is differentially expressed with developmental stage. We also have found that it is expressed in several hormonally responsive mouse mammary cancer cell lines. Factors regulating its expression are being investigated. Our continued studies of the changes in the vascular network that facilitates lactogenesis and tumorigenesis in the mammary gland analyzed the VEGF promoter for hormonally regulated transcriptional elements. Treatment of HC11 mouse mammary epithelial and Nb2 rat lymphoma cells with PRL induced VEGF expression. This induction was PRLR and JAK2 dependent. Deletion analysis of the VEGF promoter was utilized to identify the specific regulatory elements responsible for the interactive effects of PRL. Activation of both the mouse and the human VEGF promoters was achieved and the deletion analysis identified a PRL-inducible GC-rich region in the proximal region of the promoter. Using electrophoretic mobility shift analysis we showed that in addition to constitutive binding of SP1 to this region, PRL induced an additional complex identified as Egr-1.
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