URI knockdown induces autophagic flux in gastric cancer cells.

ABSTRACT

URI, a member of the prefoldin family of molecular chaperones, functions in the regulation of nutrient-sensitive, mTOR-dependent transcription signaling pathways. Previous studies of several tumor types demonstrated that URI exhibits characteristics similar to those of an oncoprotein. URI has been shown as a mitochondrial substrate of S6 kinase 1 (S6K1), which acts to integrate nutrient and growth factor signals to promote cell growth and survival. Notably, the Akt/mTOR/p70S6K signaling pathway constitutes major negative regulatory mechanism of autophagy. However, the role of URI in autophagy has not been explored. Here, we investigated the involvement of URI in autophagy by manipulating its expression in MGC-803 and HGC-27 cells using siRNA and transfection approaches. GFP-LC3 punctum aggregation was assessed by confocal microscopy, whereas formation of autophagic vesicles was assessed using transmission electron microscopy. NH4Cl was used to inhibit autophagosome-lysosome fusion and to monitor autophagic flux. Expression of LC3-I, LC3-II, beclin1, total and phosphorylated mTOR, and p70S6k was assessed by Western blotting. The results showed that knockdown of URI induced significant autophagic flux in gastric cancer cells. URI regulates the expression of beclin1, which is essential for initiation of conventional autophagy. Levels of p-mTOR (Ser2448) and p-p70S6K (Thr389) increased in URI-overexpressing cells treated with the mTOR inhibitor rapamycin but decreased in URI-silenced cells. The inhibitory effect of URI silencing on mTOR and p70S6K phosphorylation was antagonized by the autophagy inhibitor 3-methyladenine. These results suggest that URI knockdown-induced autophagy is associated with the mTOR/p70S6K signaling pathway, indicating the potential existence of a novel autophagy regulatory mechanism mediated by URI.