Annexin A2 in tumor-derived extracellular vesicles: Molecular contributions in metastatic triple negative breast cancer

Purpose

Tumor-derived extracellular vesicles (TEV) are highly implicated in tissue-specific metastasis. Additionally, TEV interacts with the distant microenvironment to shape a pre-metastatic niche (PMN) for homing the tumor cells.

Annexin A2 (AnxA2) is a plasma and endosomal membrane-associated protein. Its high levels have been correlated with poor distant metastasis-free survival and poor overall survival in triple negative breast cancer (TNBC) patients. It is also abundantly present in TEV and recruits TEV-associated cargo such as proteins and microRNAs.

Our lab reported that in vivo education with AnxA2 depleted EV led to reduced TNBC metastasis to lungs and brain suggesting a key role in the formation of a PMN. While the presence of AnxA2 in EV has been reported, its contribution in the formation & development of PMN via EV is still unexplored. We aim to evaluate the implications of AnxA2 in EV & elucidate the mechanisms promoting TNBC metastasis.

Methods

We used shRNA- mediated gene silencing to stably downregulate AnxA2 in organotropic TNBC cell lines derived from the parent MDA MB 231 cells. Differential ultracentrifugation was used to isolate EV from cell culture supernatant & size analysis was done using NTA. Biological characterization was done in concordance with MISEV 2018 guidelines using immunoblotting. Additionally, the EVs will be subjected to quantitative proteomic and transcriptomic analysis to identify differentially expressed proteins and genes upon loss of AnxA2.

Results

Upon depletion of AnxA2 protein, we observed a significant effect of AnxA2 depletion on its physiological role in plasmin generation. We observed a size distribution of the isolated EV between 30-300 nm. 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, calnexin & cytochrome c. Interestingly, we observed a reduced yield of EV with AnxA2 depletion indicating a potential effect on EV biogenesis & release.

Conclusion

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.