Abstract 5757: Identification of breast cancer-associated changes to the Rab11 interactome

Abstract Breast cancer is the second leading cause of death for American women. Recent advances have improved patient outcomes, but metastatic disease continues to be devastating, and its mechanisms are not fully understood. We made the exciting discovery that Rab11b, a small GTPase that controls endosomal recycling, is required for breast cancer brain metastatic outgrowth, through control of the cell surface proteome. Rab11b interacts with numerous proteins, including GTPase activating proteins (GAPs), guanine exchange factors (GEFs) and motor proteins to facilitate cargo recycling and control the cell surface proteome. We found that Rab11-mediated recycling is dependent on the cellular context, and hypothesize that these changes are driven by dynamic, microenvironment-mediated changes to the Rab11 interactome. To elucidate these changes, we used a proximity labeling approach to identify the Rab11 interacting proteins (the interactome). We used the promiscuous biotin ligase, TurboID, for proteomic analysis of protein-protein interactions. TurboID confers the advantage of detecting highly dynamic changes characteristic of GTPases. To identify the full range of changes to the Rab11 interactome, we are using HEK-293 cells to identify the canonical interactome, immortalized normal breast epithelial cells and triple negative breast cancer cells to identify the breast-specific Rab11 interactome. We have successfully expressed wild-type, dominant negative and constitutively active Rab11a and Rab11b constructs, and shown that they co-localize with endogenous proteins and that biotinylation is indeed spatially restricted. Proteomics studies identified known Rab11 interacting proteins, confirming the reliability of our approach. Importantly, we also identified differences in the canonical and breast cancer-associated Rab11 interactomes, supporting our hypothesis that recycling is context dependent. Current work is focused on validating two such proteins found to be differentially interacting with Rab11 in the different cellular contexts, AnnexinA1 and AnnexinA2. The annexins are phospholipid binding proteins that have a range of functions dependent on cellular location including roles in inflammation and cell trafficking. They’ve been historically implicated in tumorigenesis, though their functions as tumor suppressors or promoters vary greatly depending on tumor type, subtype, and even stage. Future work will focus on further elucidating the interaction between AnnexinA1/A2 and Rab11 in distinct metastatic microenvironments. Citation Format: Jessica Contreras, Joseph Kainov, Yaneli Betancourt, Oly Khowash, Charlie Yang, Erin N. Howe. Identification of breast cancer-associated changes to the Rab11 interactome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5757.