AP-2ß Defines a Mammary Epithelial Subpopulation with a potential Role in Lobular Carcinoma Initiation

Summary: Background: Invasive Lobular Carcinoma (ILC) is the most common special histological subtype of breast cancer, comprising ~15% of all breast cancer cases and characterized by a loss of E-cadherin (CDH1). Although ILC differs clinically and molecularly from NST, it receives similar treatment and has worse long-term outcomes. This highlights the need to better understand the molecular drivers of ILC. Our group has recently identified TFAP2B (AP-2ß) as one of the most hypomethylated genes in ILC. Non-invasive lesions such as Lobular Carcinoma in Situ have strong expression of AP-2ß, while NST tumors display TFAP2B hypermethylation and minimal protein expression. Prior studies show that AP-2ß is almost entirely co-expressed with luminal markers GATA3 and cytokeratins 8/18 and partially co-expressed with hormone-receptors estrogen and androgen receptors in normal mammary epithelium. This suggests that AP-2ß may serve as a lineage marker during progression from normal epithelium to precancerous lesions and ultimately ILC. Hypothesi: I hypothesize that AP-2ß marks a distinct mammary epithelial subpopulation capable of surviving loss of CDH1, an event that usually results in apoptosis. I also hypothesize that this accomplished through AP-2ß regulating transcription of survival pathways which then also play a role in transition from LCIS to ILC. Methods: We examined AP-2ß expression in normal mammary epithelium from human, mouse, and rats, as well as mouse-derived organoids (MDOs) from C57Bl/6 Crefl/fl, using immunoblotting, immunohistochemistry (IHC), immunofluorescence (IF). We also examined Ap-2ß expression in publicly available single-cell RNA-seq datasets. Results: IHC across human, mouse, and rat tissues revealed sporadic AP-2ß expression in normal epithelium and strong, uniform expression in ILC tumors. IF revealed co-expression of AP-2ß with key luminal markers including FOXA1, GATA3, CK8/18, ER, and AR. Single-cell RNA-seq analysis of normal human breast showed that the expression of AP-2b was limited to hormone responsive (HR) epithelial cells while it was absent in hormone secretory cells. HR cells also overexpressed FOXA1, GATA3, ER, AR and CK8/18, and AR signaling and ROS were the top enriched pathways. In CDH1-deficient MDOs, cells exhibited proliferation arrest and apoptosis; ongoing studies are investigating whether AP-2ß overexpression can rescue these cells from CDH1 loss-induced apoptosis. Conclusion: Our findings support a model in which AP-2ß marks a unique epithelial cell population in the normal breast. We have successfully established models and techniques to now define its functional role in cell survival following loss of CDH1, and its contribution to the molecular events driving the transition from normal epithelium to LCIS and finally ILC. Hunter Waltermire, Abdalla Wedn, Chelsea Chen, Jagmohan Hooda, Rohit Bhargava, Adrian Lee, and Steffi Oesterreich