Small extracellular vesicles secreted from senescent cells promote cancer cell proliferation through EphA2.

Cellular senescence prevents the proliferation of cells at risk for neoplastic transformation. However, the altered secretome of senescent cells can promote the growth of the surrounding cancer cells. Although extracellular vesicles (EVs) have emerged as new players in intercellular communication, their role in the function of senescent cell secretome has been largely unexplored. Here, we show that exosome-like small EVs (sEVs) are important mediators of the pro-tumorigenic function of senescent cells. sEV-associated EphA2 secreted from senescent cells binds to ephrin-A1 that is highly expressed in several types of cancer cells and promotes cell proliferation through EphA2/ephrin-A1 reverse signalling. sEV sorting of EphA2 is increased in senescent cells due to its enhanced phosphorylation resulting from oxidative inactivation of PTP1B phosphatase. Our results demonstrate a novel mechanism of reactive oxygen species (ROS)-regulated cargo sorting into sEVs, which is critical for the potentially deleterious growth-promoting effect of the senescent cell secretome. To investigate the function of sEV-associated EphA2, we cultured breast cancer cell line MCF7 in the conditioned medium (CM) prepared from doxorubicin-induced senescent RPE1 expressing control or two different EphA2 shRNA. The effects of sEV-associated EphA2 on the gene expression profile of MCF7 were investigated using microarray. Microarray experiments were conducted using SurePrint G3 Human GE microarray 8 x 60K ver 2.0 (Agilent). Total RNA was isolated by Trizol Reagent. cDNA labelling, hybridizations, and scanning were performed by DNA ChiP Research Inc. (Japan), an Agilent-certified service provider. Data were then normalized to 75th percentile for inter-array comparison.