The androgen receptor (AR) plays a critical role in the development

The androgen receptor (AR) plays a critical role in the development and progression of prostate cancer (PC) and is a major therapeutic target in this disease. activity. Exogenous expression of ELF3 represses AR transcriptional activity when assessed using reporter-based transfection assays or when evaluated on endogenous AR target genes. Conversely ELF3 knockdown increases the AR transcriptional activity. Biochemical dissection of this activity indicates that it results from the physical interaction between ELF3 and AR and that this interaction inhibits the recruitment of AR to specific androgen response elements within target gene promoters. Significantly we observed that depletion of ELF3 expression in LNCaP cells promotes cell migration while increased ELF3 expression severely inhibits tumor growth and in a mouse xenograft model. Taken together these results suggest that modulation of ELF3 expression and/or AR/ELF3 interaction may have utility in the treatment of PC. and are found in a majority of advanced PCs25 26 It was of interest therefore that we recently identified ELF3 as a potential AR-interacting protein27. Following up on this finding we determined that ELF3 is indeed a coregulator functioning as a negative modulator of AR transcriptional activity. Furthermore manipulation of ELF3 inhibits androgen-dependent growth and migration of PC cells. These findings highlight the biological significance of the AR/ELF3 interaction in PC biology and suggest that this complex may be a target for novel therapeutic interventions. RESULTS Interaction between ELF3 and the androgen receptor Previously we reported the results of a high-throughput protein-protein interaction screen that employed T7 phage display technology to identify proteins that interacted either directly or indirectly with AR in the presence of different ligands. Over 300 interacting proteins were identified in this manner the functional significance of which remain under investigation27. Among the proteins that were identified in this manner was ELF3 a member of the ETS family of transcription factors a class of proteins that have been implicated in the pathology of solid tumors17. Furthermore we utilized paired-sample data from “type”:”entrez-geo” attrs :”text”:”GSE21034″ term_id :”21034″GSE2103428 to demonstrate in a case-by case manner that the expression of ELF3 is lost in primary prostate tumors when compared to expression in normal adjacent sites (Fig. 1A). Consequently we proceeded to evaluate the possibility that ELF3 was a biologically relevant regulator of AR action in the prostate. As a first step in this process we performed a co-immunoprecipitation assay to demonstrate an interaction between endogenous ELF3 and AR in LNCaP cells. As shown in Fig. 1B ELF3 was detected in cell lysate immunoprecipitated with AR antibody. In addition AR was also detected in cell lysate subjected to immunoprecipitation with ELF3 antibody. Interestingly the interaction was Lincomycin hydrochloride significantly enhanced in the presence of R1881. Subsequently GST-pull-down assays were used to show that recombinant AR and ELF3 interact directly and to define the domains within each protein that were required for the observed interaction. To this end purified full Lincomycin hydrochloride length GST-ELF3 was incubated with either translated full length AR or fragments thereof encoding selected functional domains of the receptor as shown in Fig. 1C. The results of this analysis confirm that the two proteins can directly interact with each other albeit weakly and Lincomycin hydrochloride that both the amino- and carboxy-terminal domains of AR bind MGC102953 to ELF3. Furthermore it was demonstrated that Flag-ELF3 co-localizes with AR in the nuclei of LNCaP cells following the addition of R1881 (Fig. 1D). In summary the results of these protein-protein interaction assays performed and within intact cells imply the existence of a close physical association between ELF3 and AR a finding that encouraged us to explore the functional consequences of this association. Figure 1 ELF3 expression is down regulated in PC tissues and interacts with AR. Waterfall Lincomycin hydrochloride plot of ELF3 expression in paired-sample data. Whole-transcript data was downloaded from the Gene Expression Omnibus under accession “type”:”entrez-geo” attrs :”text”:”GSE21034″ term_id :”21034″ … ELF3 represses androgen receptor-dependent transcriptional activity Lincomycin hydrochloride To address the functional role of ELF3 in AR signaling we first evaluated the impact of its expression on AR transcriptional activity in androgen responsive reporter gene assays in HEPG2 and LNCaP.