5 fold in cells expressing SUMO deficient PR versus WT Dorsomorphin BMP PR expressing cells. These PR gene signatures were uploaded into Oncomine Research Premium Edition soft ware and the database was searched for associated concepts. Results PR SUMOylation alters promoter selection in T47D breast cancer cells For unknown reasons, there is little overlap between PR regulated genes in normal, relative to neoplastic, breast tissues. One mechanism for the apparent divergence of PR functions may relate to early events in breast can cer development, such as altered signal transduction. Based in part on our prior studies, we predict that the balance between SUMOylated and phosphory lated PRs is frequently altered in breast cancer, resulting in changes in PR promoter selec tivity and altered patterns of gene expression.
In a screen of ten Inhibitors,Modulators,Libraries breast tumors clinically defined as PR, we detected a wide range of total PR mRNA and protein expression. Of the seven Inhibitors,Modulators,Libraries breast tumors that were confirmed to be PR by both RT qPCR and western blotting, at least five samples also clearly con tained some level of phospho Ser294 PR B. Remarkably, two of ten tumors contained abundant phospho Ser294 PR B. Notably, PR B, but not PR A, Ser294 is rapidly phosphorylated in response to either progestins or peptide growth factors that input to proline Inhibitors,Modulators,Libraries directed protein kinases, primarily within the MAPK and CDK families. Consistent with this finding, EGF blocked progestin induced PR B, but not PR A SUMOylation. The broad range of PR expression in clinical specimens suggests that PR dependent gene expression may provide a more accurate marker of PR contribution to breast cancer phenotypes.
To address the unique actions of phosphorylated and SUMO deficient PR B, we measured the transcriptional profiles of breast cancer cells stably expressing either wild type or SUMO deficient PR B molecules using Inhibitors,Modulators,Libraries whole genome expres sion profiling. We first engineered multiple clones of vector matched PR null T47D breast cancer cells expres sing either Inhibitors,Modulators,Libraries WT PR B or mutant K388R PR B that is unable to undergo SUMO modification at Lys388, this SUMO deficient receptor is a functional mimic for PR B that is persistently phosphorylated on Ser294. Phospho Ser294 and S294D receptors are hyperactive transcription factors that undergo rapid ligand depen dent downregulation relative to WT PRs.
Cells expressing either WT or KR PR B were then treated with the synthetic progestin, R5020, for six hours. Upon ligand binding, PR is globally phosphorylated at multiple sites, as indicated by a slight gel upshift. Consistent with our previous reports hyperactivated KR PR undergoes slightly more rapid ligand induced downregulation www.selleckchem.com/products/AG-014699.html relative to WT PR. Using these experimental conditions, global gene expression profiles were simultaneously measured using Illumina HT 12v4 whole genome gene expression bead arrays. Top regulated genes were orga nized by heat maps showing up or down regulated genes.
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