Moonlighting proteins in Extracellular Vesicles: do Hsp70 and Alpha-enolase share common localization and function?

Moonlighting proteins (MPs) exhibit more than one physiologically relevant function at distinct cellular and extracellular sites. A large number of MPs are cytoplasmic chaperones and enzymes that can play a different role on the cell surface. Among them Hsp70 and alpha-enolase, a chaperone and a glycolytic enzyme respectively, are both intracellular and surface MPs commonly overexpressed in different tumor types and present in extracellular vesicles (EVs). Surface alpha-enolase acts as receptor for plasminogen and this enolase activity has been directly linked to tumor invasion and metastasis [1, 2], likewise surface Hsp70 is a recognized marker of tumor cells [3]. In breast cancer cells, Hsp70 is a molecular partner of alpha-enolase and we have shown that the protein-protein interaction is involved in the surface localization of the enzyme, which lacks a canonical localization signal [4]. Moreover, in breast cancer cells both proteins are overexpressed in the plasma membrane in response to inflammatory stimuli, namely LPS and EGF [4,5], and are released in EVs when cells are exposed to cytokines, such as TGF-?1, CCL2, and TNF-? [5]. It has been shown that alphaenolase- exposing EVs enhance cancer cell migration and invasion [5]. On the other hand, Hsp70 on the surface of EVs elicits different immune responses, mainly immunosuppression, contributing to tumor immune escape. In particular, the interaction between cell toll-like receptor 2 (TLR2) and Hsp70 exposed on the tumor-derived small EVs or exosomes results in the activation of myeloidderived suppressive cells (MDSC) and the consequent inhibition of adaptive and innate immunity [6]. Similarly, EVs from microorganisms, like protozoa or bacteria, modulate host immune response [7]. Despite both alpha-enolase and Hsp70 surface localizations are promoted by factors associated with tumor progression, and their exteriorization contributes to cell invasion and immunoregulation, the functional relationship between these proteins in EVs is still unknown. We propose that Hsp70 and alpha-enolase at the surface of EVs may cooperate to modulate the activity of the immune system using overlapping mechanisms. To this end, we generated a cellular model ectopically expressing epitope-tagged alpha-enolase and Hsp70 and we verified the presence of these proteins in the EVs, than we performed preliminary experiments to investigate the localization of alphaenolase and Hsp70 in the vesicles lumen and/or surface. These data may contribute to elucidate the mechanisms by which EVs transfer materials or induce signaling in immune cells and to ascertain novel EV surface markers. References [1] Hsiao, K. C. et al. 2013. Surface ?-enolase promotes extracellular matrix degradation and tumor metastasis and represents a new therapeutic target. PLoS One. Jul 19; 8(7) [2] Principe M et al 2015. Targeting of surface alpha-enolase inhibits the invasiveness of pancreatic cancer cells. Oncotarget. May 10; 6(13) [3] Stangl, S. et al. 2011. Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody. Proc. Natl. Acad. Sci. U. S. A. 108, 733-8 [4] Perconti et al. 2017. Pro-invasive stimuli and the interacting protein Hsp70 favour the route of alpha-enolase to the cell surface. Sci Rep. Jun 19; 7(1) [5] Didiasova et al. 2015. STIM1/ORAI1-mediated Ca2+ Influx regulates Enolase-1 exteriorization. J Biol Chem. May 8; 290(19) [6] Jessica Gobbo et al. 2015. Restoring anticancer immune response by targeting tumor-derived exosomes with a HSP70 peptide aptamer. J Natl Cancer Inst. Nov 22; 108(3) [7] Li S. et al. 2018. Extracellular Vesicles secreted by Neospora caninum are recognized by Toll-Like Receptor 2 and modulate host cell innate immunity through the MAPK signaling pathway. Front Immunol. Jul 24; 9: