Uptake and tumor-suppressive pathways of exosome-associated GKN1 protein in gastric epithelial cells

Background Gastrokine 1 (GKN1) is a stomach-specific tumor suppressor that is secreted into extracellular space as an exosomal cargo protein. The objective of this study was to investigate the uptake and tumor-suppressive pathways of exosome-associated GKN1 protein in gastric epithelial cells. Methods Immunofluorescent and Western blot analysis were used to investigate gastric-specific uptake of HFE-145-derived exosomes. Binding affinity of HFE-145 derived exosomes with integrin proteins was examined using protein microarray chip. Tumor suppressor activities of exosome-carrying GKN1 protein were analyzed using transwell co-culture, MTT assay, BrdU incorporation, immunoprecipitation, and Western blot analysis. Results HFE-145-derived exosomes were internalized only into HFE-145 gastric epithelial cells and gastric cancer cells. Gastric-specific uptake of stomach-derived exosomes required integrin alpha 6 and alpha X proteins. Clathrin and macropinocytosis increased the uptake of exosomes into gastric epithelial cells, whereas caveolin inhibited the uptake of exosomes. Transwell co-culture of AGS cells with HFE-145 cells markedly inhibited viability and proliferation of AGS cells. Following uptake of HFE-145-derived exosomes in recipient cells, GKN1 protein bound to HRas and inhibited the binding of HRas to b-Raf and c-Raf which subsequently downregulated HRas/Raf/MEK/ERK signaling pathways in AGS, MKN1 cells, and MKN1-derived xenograft tumor tissues. In addition, exosomal GKN1 protein suppressed both migration and invasion of gastric cancer cells by inhibiting epithelial-mesenchymal transition. Conclusions Gastric-specific uptake of exosomes derived from gastric epithelial cells requires integrin alpha 6 and alpha X proteins in both gastric epithelial cells and exosomes. Exosomal GKN1 protein inhibits gastric carcinogenesis by downregulating HRas/Raf/MEK/ERK signaling pathways.