Abstract 1314: Molecular contribution of Annexin A2 in triple negative breast cancer metastasis via tumor-derived extracellular vesicles

Abstract

Abstract Triple negative breast cancer (TNBC) is the most lethal subtype of all breast cancers leading to high mortality rates. Tumor-derived extracellular vesicles (TEV) along with their cell-type specific cargo are greatly implicated in tissue-specific metastasis. In addition to metastasis, TEV play a crucial role in interacting with the distant microenvironment & shaping a pre-metastatic niche (PMN) for tumor cells to home. Understanding the mechanisms by which TEV promote PMN development has great potential to improve the diagnosis, prognosis, & treatment of TNBC. Annexin A2 (AnxA2) is a plasma & endosomal membrane-associated protein. High levels of AnxA2 in TNBC patients have been correlated with poor distant metastasis-free survival and poor overall survival. It is abundantly present in the TEV & recruits cargo such as proteins and microRNAs. Our lab reported that in vivo education with AnxA2 depleted TNBC-derived extracellular vesicles (EVs) led to reduced metastasis in lungs & brain suggesting a key role in the formation of a PMN. While the presence of AnxA2 in TNBC-derived EVs has been reported, its molecular role in the formation & development of PMN via EVs is still unexplored. We aim to evaluate the implications of AnxA2 in EVs at distant sites to mediate tumor cell homing & elucidate mechanisms promoting TNBC metastasis. We have employed RNA interference-mediated gene silencing to stably downregulate AnxA2 in organotropic TNBC cell lines derived from the parent MDA MB 231 cells. We observed a significant effect of AnxA2 depletion on its physiological role in plasmin generation. Differential ultracentrifugation was used to isolate EVs from cell culture supernatant and the physical characterization was done using Nanoparticle Tracking Analyzer. Biological characterization was done in concordance with MISEV 2018 guidelines. Using immunoblotting we confirmed reduced levels of AnxA2 in EVs derived from AnxA2 depleted TNBC cells. We verified their purity using EV enriched markers - ESCRT, Heat shock proteins & tetraspanins such as CD81, CD9, CD63 & confirmed the absence of negative markers - GM130 & cytochrome c. Interestingly, a reduced yield of EV was observed with AnxA2 depletion indicating a potential effect on EV biogenesis and release. Additionally, the EVs will be subjected to quantitative proteomic analysis to identify differentially expressed proteins upon loss of AnxA2. We will further carry out transcriptome profiling of the AnxA2 depleted TNBC cells and -derived EVs to identify the differentially expressed genes. The role of AnxA2 in TEV biogenesis, release and selective cargo loading will lead to potential identification and understanding of the novel secretory and EV protein that may act as a functional regulator in promoting advanced metastasis in TNBC. [This research is supported by the NCI under Award Number R01CA220273 (JKV), awarded to Dr. Jamboor K. Vishwanatha.] Citation Format: Rucha Trivedi, Jamboor K. Vishwanatha. Molecular contribution of Annexin A2 in triple negative breast cancer metastasis via tumor-derived extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1314.