Melanoma cells release extracellular vesicle which contain H1° linker histone as well as RNA-binding proteins which bind to the H1° mRNA

We previously demonstrated that G26/24 oligodendroglioma cells release EVs that contain proteins, such as FasL and TRAIL, which induce apoptosis in rat cortical neurons [1] and astrocytes [2]. We also reported that cancer cells use EVs for transferring, into the environment [3], proteins such as extracellular matrix remodelling proteases [4], and H1°, a differentiation-specific histone [5]. In particular, by releasing H1°, cells could escape differentiation cues [5]. To verify the role of EVs in releasing specific proteins and mRNAs, in this study we used as a model A375 melanoma cells. METHODS EVs were purified from cell culture media as previously reported [1, 2]. T1 RNase-protection assays were performed on total cell lysates and EVs, as described elsewhere [6]. RNA-binding proteins (RBPs) were isolated by using a biotinylated H1° RNA as a bait. RESULTS We found that also melanoma cells synthesize H1° and secrete it via EVs. Interestingly, H1° histone sorted to vesicles is probably sumoylated. By T1 RNase-protection assay, we evidenced in EVs three main H1° RNA-protein complexes, the most abundant of which has a molecular mass of around 65 kDa. By using as a bait biotinylated H1° RNA, we isolated a few proteins, then analyzed by mass spectrometry. The most abundant protein was myelin expression factor-2 (MYEF2), which has a molecular mass of about 60 kDa. Finally, we confirmed MYEF2 presence in EVs by western blot. CONCLUSIONS We demonstrated that EVs released from melanoma cells contain the H1° linker histone, which is probably sumoylated before sorting to the vesicles. In addition, EVs contain H1° RNA-binding proteins, one of which seems to be MYEF2. [1] D’Agostino et al. 2006, Int J Oncol 29:1075-85. [2] Lo Cicero A et al. 2011, Int J Oncol 39:1353-7 [3] Di Liegro et al. 2015, Biomed Res Int, in press [4] Lo Cicero A et al. 2012, Matrix Biol 31:229-33 [5] Schiera G et al. 2013, Int J Oncol 43:1771-6 [6] Scaturro et al. 1998, J Biol Chem 273:22788-9