Abstract P1-07-01: Novel role of the tumor suppressor ductal epithelium associated ring chromosome 1 (DEAR1) in regulation of breast stem/progenitor cell properties

Abstract

Abstract Introduction: Breast cancer is the most commonly diagnosed cancer and third-leading cause of cancer-related deaths in women in America. A quarter of lesions diagnosed annually are ductal carcinoma in situ (DCIS), one of the earliest pre-invasive forms of invasive ductal carcinoma (IDC). Without therapeutic intervention, 30-50% of DCIS cases can progress to IDC. Understanding the mechanisms regulating progression from DCIS to IDC would help identify biomarkers to stratify DCIS patients at higher risk of progression or recurrence. Cumulative literature suggests the earliest phase of dissemination from the primary tumor is driven by the epithelial-mesenchymal transition (EMT) program. DEAR1 is a tumor suppressor gene which is mutated, undergoes loss of heterozygosity in breast cancer, and is downregulated in DCIS lesions. DEAR1 regulates acinar morphogenesis and cell polarity, and is a negative regulator of TGF-beta-driven EMT by binding to and ubiquitinating SMAD3, thereby limiting the amount of SMAD3 available to activate an EMT signature. Overexpression of EMT master regulators, or exposure to TGF-beta in immortalized human mammary epithelial cells (HMECs), results in mammosphere formation and breast stem cell markers, thus linking the EMT process to acquisition of stem cell characteristics. Methods: Stable lentiviral shRNA knockdown, in vitro mammosphere assay, cytospin, immunofluorescence, immunoprecipitation, western blot analysis, ubiquitination and real-time quantitative PCR were performed. Results: DEAR1 knockdown in immortal HMECs resulted in a significant enhancement of primary mammosphere formation and growth compared to controls, suggesting that DEAR1 may regulate stem/progenitor cell properties; this effect was greater when cells were exposed to TGF-beta. To determine if DEAR1 regulates stem cell properties through regulation of SMAD3 levels, DEAR1-SMAD3 double knockdown clones were examined for mammosphere formation. Results indicated fewer mammospheres in double knockdown clones, but only in the presence of TGF-beta, suggesting that the mammosphere phenotype is partially dependent on the TGF-beta-SMAD3 pathway. The observation that loss of DEAR1 alone results in a stem/progenitor cell phenotype indicates that DEAR1 may regulate mammosphere formation independent of its role in regulating the TGF-beta pathway. HMEC mammospheres express higher levels of stem cell marker aldehyde dehydrogenase (ALDH1) and co-express luminal and basal cytokeratins, suggesting bipotential capacity. We also observed upregulation of SNAI2 and ZEB1, master EMT and stem cell regulators, in DEAR1 knockdown HMECs. DEAR1 interacted with SNAI2 by co-immunoprecipitation analysis and also promoted the polyubiquitination of SNAI2. Conclusions: Loss of the tumor suppressor/polarity regulator DEAR1 promotes stem cell properties in part through the DEAR1-TGF-beta-SMAD3 axis. We demonstrate a DEAR1-SNAI2 axis that may partially regulate stem/progenitor cell properties in HMECs through the polyubiquitination of SNAI2, a master regulator of EMT and stemness. These mechanisms governing acquisition of the stem cell properties may contribute to understanding how DCIS progress to IDC. Citation Format: Le U, Chen N, Balasenthil S, Killary AM. Novel role of the tumor suppressor ductal epithelium associated ring chromosome 1 (DEAR1) in regulation of breast stem/progenitor cell properties [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-07-01.