Abstract 4466: Adenomatous polyposis coli transcriptionally regulates epithelial membrane protein 2 to regulate apical-basal polarity
Authors: Alyssa Lesko, Carolyn Ahlers, Anna T. Lyons, Jocelyn D. Gaona, Jenifer R. Prosperi
Journal: Cancer Research (2018)

Abstract Adenomatous Polyposis Coli (APC) is a well-known negative regulator of the Wnt pathway; however, it also regulates polarity proteins. Loss of apical-basal polarity disrupts several cellular processes including epithelial structure and intracellular signaling, and is an early marker for tumor development. We previously demonstrated that APC knockdown (APCKD) in Madin-Darby Canine Kidney (MDCK) cells altered cyst size and inverted polarity in 3D culture. Through microarray analysis we made the novel observation that APC loss increased Epithelial Membrane Protein 2 (EMP2) expression. Interestingly, EMP2 knockdown in APCKD cells decreased cyst size and restored apical polarity. These data suggest a previously unknown role for EMP2 in regulating polarity; however, how EMP2 influences APC-mediated polarity remains unknown. Therefore we hypothesize that APC transcriptionally regulates EMP2 to influence apical-basal polarity. In this study, we investigate transcriptional activation of EMP2 using bioinformatics, transcription factor DNA/protein arrays, luciferase reporters, and ChIP assays. EMP2 promoter screens (ConTra v3) identified binding sites for signal transducer and activator of transcription 1 and 3 (STAT1 and 3), and E2F transcription factor 1 (E2F1). Interestingly, APCKD cells exhibited increased expression of STAT1 and E2F1 in DNA/protein arrays compared to controls. Additionally, STAT3 activation was increased in APCKD cells in reporter assays. These studies identified STAT3, STAT1, and E2F1 as possible APC-mediated transcriptional regulators of EMP2. Current studies utilizing luciferase reporter assays aim to determine if E2F1 and STAT1 are transcriptionally active upon APC loss. ChIP assays are being utilized to determine if these candidate transcription factors can bind the canine EMP2 promoter. As CREB is a known transcriptional regulator of EMP2, it was used as a positive control for EMP2 promoter binding. However, no changes in CREB binding are expected between APCKD and MDCK cells, as DNA/protein arrays and luciferase reporter assays did not reveal changes in CREB expression or activity upon APC loss. In preliminary studies, E2F1 was found to weakly bind the canine EMP2 promoter, though this binding was not regulated by APC. Current studies seek to determine if STAT1 and STAT3 bind the EMP2 promoter. Reporter assays using mutant EMP2 promoter constructs are being used to assess if the candidate transcription factors drive EMP2 expression upon APC loss. Future studies will determine if candidate transcription factors influence APC-mediated polarity. Transcription factors will be inhibited in APCKD cells and activated in MDCK cells and EMP2 expression, apical polarity and cyst size will be evaluated in 3D culture. Understanding the interaction of APC and EMP2 and the influence on polarity will identify key players in APC disease progression. Citation Format: Alyssa Lesko, Carolyn Ahlers, Anna T. Lyons, Jocelyn D. Gaona, Jenifer R. Prosperi. Adenomatous polyposis coli transcriptionally regulates epithelial membrane protein 2 to regulate apical-basal polarity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4466.