THE AUTOPHAGY-RELATED-PROTEIN VMP1 IS SECRETED IN EXOSOMES FROM PANCREATIC TUMOR CELLS

PAULA SEPÚLVEDA ACUÑA; MARIA INES VACCARO; MARIA NOE GARCIA; TAMARA ORQUERA; FELIPE ARIEL RENNA

San Diego

Congreso; Digestive Disease Week 2019; 2019

American Gastrological Asociation (AGA)

Different from degradative autophagy, secretory autophagy is thus a newly recognized mechanism of increasing relevance to explain the secretion of some molecules of critical biologicalimportance. VMP1 is an autophagy-related transmembrane protein essential for autophagosome biogenesis. VMP1 is induced in pancreas by pancreatic diseases such as pancreatitisand pancreatic ductal adenocarcinoma. Exosomes are small vesicles (40-120 nm) which arereleased to extracellular medium by secretory autophagy. Here we demonstrate that VMP1is involved in secretory autophagy and it is released from pancreatic cells as a membraneprotein of the exosomes. Setting up two exosome purification protocols, by ultracentrifugationand by isolation with magnetic beads fussed to anti-VMP1 antibodies, we identified thepresence of VMP1-exosomes, labeled with CD63, in supernatant of different pancreatic cancercell lines. We confirmed the secretion of VMP1-exosomes by electron microscopy, westernblot and flow cytometry and using other markers of exosomes, such as CD81, CD9 andAlix. By means of the induction of autophagy with starvation, rapamycin treatment oroverexpression of VMP1 and the autophagy inhibition by shATG5, shVMP1, 3-MA andchloroquine, we confirmed that VMP1-exosome secretion depends on autophagosome formation. Using VMP1-exosome fraction secreted by cell lines expressing CD63-GFP, we demonstrated that VMP1-exosomes are able to be up-taken by other cell lines, suggesting thatVMP1-exosomes might be able to mediate remote communication between cells. Finally,using three different antibodies, we detected VMP1 in human serum from donors (N=20)and we purified exosomes from human serum using magnetic beads fussed to anti-VMP1antibodies. In conclusion, we detected for the first time, an autophagic protein secreted tothe cellular environment, which is able to be up-taken by other cells. Further, the demonstration of VMP1-exosomes in human serum is of high potential as a relevant molecule fordiagnosis, treatment and monitoring diseases such as pancreatitis and pancreatic ductal adenocarcinoma