Abstract P2-06-18: The Role of GREB1 in Breast Cancer: Identifying GREB1 Binding Proteins and Functional Pathways

Gene regulated in breast cancer 1 (GREB1) was initially discovered in breast cancers as an estrogen-regulated gene that mediates estrogen-stimulated cell proliferation and is a candidate clinical marker for response to endocrine therapy. However, little is known of the functional role of GREB1 protein in breast cancers, its interactive protein partners or the critical cellular pathways where GREB1 is involved. Our team employed a unique two-hybrid system to begin identifying GREB1 binding proteins from normal and cancerous breast cells as well as elucidating the molecular pathways with GREB1 involvement. The Matchmaker Gold Yeast Two-Hybrid System (Clontech Laboratories Inc., Mountain View, CA) was used to identify GREB1 binding proteins from immortalized human mammary epithelial cells (HMECs) and breast cancer cell lines. GREB1 served as a “bait” protein (pGBKT7-GREB1) to screen “prey” proteins generated from cDNA libraries created from immortalized HMECs, GREB1+ (MCF7, T47D) and GREB1- (SKBR3, MDA-464) breast cancer cells lines in yeast (Y187 MATα strain). Protein interactions were stringently screened using GAL4 activation and binding domainmediated reporters including aureobasidin A antibiotic resistance, two nutritional reporters (Histidine and Adenine) and a colorimetric reporter. Prey plasmid DNA, containing cDNA for GREB1 interactive protein candidates, was isolated from selected yeast colonies and sequenced to identify potential GREB1 binding proteins. These individual prey plasmids were subsequently re-introduced to the Y2H system to verify GREB1 interaction and measure the strength of this interaction. Confirmed GREB1 binding protein candidates were further screened using the Matchmaker Mammalian Two-Hybrid System, where interactions between GREB1 and a candidate GREB1-binding protein are verified in mammalian cell background (HEK 293 cells) using a quantitative secreted alkaline phosphatase (SEAP) reporter. This second screen in the mammalian background, unique to this Y2H system, ensures the conformation of GREB1 and the prey protein is similar to their natural state in breast cells and reflects interactions between proteins with greater authenticity than can be achieved in yeast alone. Finally, commercial monoclonal antibodies for GREB1 binding proteins identified using these two-hybrid systems were used to perform confirmatory co-immunoprecipitation experiments with the novel GREB1 monoclonal antibody developed in our laboratory and breast cell line lysates. Several GREB1 binding proteins, including PTMS, CRABP2, DHTKD1 and SEC23B, were confirmed by yeast and mammalian two-hybrid systems using cDNAs from GREB1+ breast cancer cell lines as well as co-immunoprecipitation assays. Subsequent pathway analysis using public databases (ex: GeneGo, Ingenuity) with GREB1 and these interactive partners suggest that GREB1 may be involved in regulation of gene transcription and cell cycle progression. These preliminary findings substantiate a critical role for GREB1 in breast cancer cell proliferation. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-06-18.