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. 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. However, in matched normal and cancerous tissue from the same patient, 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 be involved in epithelial to mesenchymal transition (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. BP-1 expression in normal tissues from African American and Caucasian women is being examined. In addition, we are studying the mouse homolog, DLX4/7, in the mouse mammary gland where it is differentially expressed with developmental stage. 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. These results indicate that PRL induces VEGF expression through Egr-1 and implicates VEGF as an intermediary of PRL-regulated angiogenesis. In addition, we found that the delta 3 splice variant of estrogen receptor alpha (ERdelta3) that is prevalent in advanced human breast cancer, activates VEGF expression 5 fold compared to a 2 fold increase by the wild type receptor when bound with estradiol. This ER variant lacks the DNA binding domain and therefore is unable to act through the classical ERE pathway. Our studies have revealed that ERdelta3acts through an essential SP1 site on the VEGF promoter thus implicating this hormone and receptor variant in local control of angiogenesis in ER positive breast cancers.
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