We identified genes that were upregulated 1. 5 fold by PR in a LD orLI manner and discovered gene expres sion overlap between cells expressing either KR or WT receptors, as well as subsets of uniquely regulated genes. We next validated the expression profiles for numerous PR target kinase inhibitor Brefeldin A genes from these classes using RT qPCR. Notably, RGS2 Inhibitors,Modulators,Libraries expression is over expressed in the basal myoepithelial compartment and substantially elevated in a majority of breast tumors. In contrast, BCL2L11 is a pro apoptotic mediator involved in ERBB MAPK dependent luminal cell clearing whose expression is primarily upregu lated by WT but not KR receptors. As these examples suggest, our gene array robustly identified diverse classes of PR target genes, and contains gene expression profiles indicative of mechanisms of PR mediated cellular prolif eration and survival.

These results essentially repeated in T47D cells engi neered to express either WT or KR PR from an induci ble vector system. In this model, inducible expression of PRs is solely dependent on the presence of a small molecule dimeri zer, AP21967, added to the cell culture medium, equal levels of either iWT or iKR were induced upon treat ment with AP21967 Inhibitors,Modulators,Libraries and these receptors were equally phosphorylated on Ser294 in response to progestin. Cells were treated with AP21967 and R5020 and assayed for changes in gene expression using the Affymetrix U133A 2. 0 microarray platform. Remarkably, PR dependent gene expression profiles obtained from T47D cells stably expressing PR were significantly similar to gene array data obtained from the same par ental cells inducibly expressing Inhibitors,Modulators,Libraries PR.

Together, our arrays identified a greater number of PR regulated genes than previous reports, microarray Inhibitors,Modulators,Libraries platforms now contain thou sands more reporters relative to earlier technologies. Notably, we identified 70% of the previously known PR target genes but also revealed hundreds of novel PR tar get genes. Phosphorylation of PR Ser294 drives Inhibitors,Modulators,Libraries SUMO deficient PR gene expression To investigate mechanisms of regulation of SUMO sen sitive PR target genes, we selected four genes from our microarray analysis for further study. These specific genes were dramatically upregulated apply for it in cells expressing KR, but not WT recep tors. A query of the Onco mine database demonstrated that all four genes are amplified in breast carcinomas relative to normal breast tissue and blood. To validate SUMO depen dent changes in PR target gene expression in an addi tional breast cancer model, we stably introduced vector control, WT or KR receptors into MCF 7 cells expres sing low levels of endogenous PR.