異位ATP合酶刺激細胞外囊泡的分泌並促進癌細胞的免疫抑制

Abstract

異位的ATP合酶（eATP合酶）是指位於細胞表面的血漿膜上、朝向外部的腺苷三磷酸合酶（ATP合酶），已在多種細胞類型中觀察到，並被視為癌症治療的潛在靶點。先前的研究指出，在A549、SK-N-BE(2)C和T47D細胞中，饑餓期後eATP合酶的表達增加。然而，在癌細胞在饑餓狀態下，細胞表面的eATP合酶的確切功能仍不清楚。 本研究利用定量蛋白質組學對飢餓狀態A549細胞進行分析，我們觀察到在305個已定量的蛋白質中具有56個顯著差異表達蛋白，進一步的基因本體論(Gene ontology)分析揭示在饑餓壓力下，癌細胞表現較高的eATP合成酶，並增強胞外囊泡（EVs）的產生。後續研究結果顯示，在A549、SK-N-BE(2)C和T47D細胞中，eATP合成酶生成胞外ATP，通過增強P2X7受體介導的Ca2+流入來刺激EVs的分泌。EVs在腫瘤微環境中是重要的調節因子為了更深入地研究增加EV釋放的作用，我們對饑餓A549細胞衍生的EV進行了定量蛋白體分析，令人驚訝的是，我們發現eATP合成酶也位於腫瘤分泌的EVs表面。人體蛋白質微陣列顯示T細胞的細胞膜上表達的蛋白質Fyn與ATP合酶亞單位beta具有相互作用，實驗證實，EVs表面的eATP合成酶通過與Fyn結合，增加了對Jurkat T細胞中腫瘤分泌的EVs的攝取。eATP合成酶包被的EVs攝取隨後抑制了Jurkat T細胞的增殖和細胞因子分泌。 總結而言，這項研究不僅闡明了eATP合酶與EV分泌之間的關係，還揭示了癌細胞利用EV的一種新的通訊機制。這些發現對於開發針對eATP合酶的癌症治療和其參與EV介導的細胞間通訊具有潛在的意義。

The ectopic ATP synthase (eATP synthase) refers to plasma membrane-localized, outward-facing ATP synthase observed in various cancer cell types, recognized as a potential target for cancer therapy. Previous studies have indicated increased expression of eATP synthase post-starvation in A549, SK-N-BE(2)C, and T47D cells. However, the precise function of cell surface eATP synthase in cancer cells under starvation conditions remains unclear. In this study, quantitative proteomics analysis of starvation-stressed A549 cells revealed significant differential expression of 56 proteins out of 305 quantified proteins. Subsequent Gene Ontology (GO) analysis unveiled higher expression of eATP synthase in cancer cells under starvation stress, enhancing the production of extracellular vesicles (EVs). Further investigations demonstrated that eATP synthase generates extracellular ATP in A549, SK-N-BE(2)C, and T47D cells, stimulating EV secretion by enhancing P2X7 receptor-mediated Ca2+ influx. EVs are crucial regulators within the tumor microenvironment. To explore the role of increased EV release in depth, quantitative proteomic analysis of EVs derived from starved A549 cells was conducted. Surprisingly, eATP synthase was found on the surface of tumor-secreted EVs. Human protein microarray analysis revealed interaction between the plasma membrane protein Fyn on T cells and the ATP synthase subunit beta. Experimental validation confirmed that eATP synthase on EV surfaces enhances uptake by Jurkat T cells through association with Fyn, subsequently suppressing proliferation and cytokine secretion in Jurkat T cells. In summary, this study elucidates the relationship between eATP synthase and EV secretion, revealing a novel communication mechanism utilized by cancer cells through EVs. These findings hold potential implications for developing cancer therapies targeting eATP synthase and its involvement in EV-mediated intercellular communication.