Stress-induced Upregulation Of Mitochondrial ROS And Anterior Gradient 2 Promotes Gastric Cancer Progression.

A number of cellular stress conditions play active roles in the carcinogenesis and cancer progression. Sustained Helicobacter pylori infection and chronic inflammation has been associated with the development of gastric cancer(GC). Studies on how GC cells develop the potential of proliferating under stress will improve our understandings of tumorigenesis and progression of GC, thus provide possibilities to GC therapies.Although Toll-like receptor 4(TLR4) and reactive oxygen species(ROS) are showed to engage in the inflammation-associated carcinogenesis, their detailed mechanisms in gastric tumorigenesis are still not fully elucidated. Using immunohistochemical staining and immunoblotting, we found TLR4 was highly expressed in tumor cells of GC tissues and was associated with the aggressiveness of GC. Lipopolysaccharide(LPS) enhanced proliferation of GC cell lines through TLR4 signaling, which was abrogated by TLR4 neutralizing Ab. Increased ROS and mitochondrial ROS(mROS) production was observed when fluorescent probes were incubated with GC cells after activation of TLR4 signaling. Using antioxidants and oxidase inhibitors to intervene ROS and mROS production respectively, we found the blocking of mROS production rather than ROS neutralization resulted in cell cycle arrest and the loss of mitochondrial potential. Furthermore, the increased mROS owing to TLR4 signaling resulted in the activation of AKT phosphorylation and NF-κB p65 nuclear translocation, leading to enhanced proliferation of GC cells.Anterior gradient 2(AGR2) is showed to be induced when endoplasmic reticulum(ER) homeostasis was broken under stress, yet its role in GC progression remains unknown. We performed immunofluorescent staining on the slides of GC tissues and adjacent tissues, and found ER stress marker GRP78 was elevated in GC, which showed higher ER stress in GC tissues. The expression of GRP78 was positively correlated with that of AGR2. AGR2 was upregulated in GC tissues and sera of GC patients, especially diffused type and poorly differentiated GC. Hypoxia and serum deprivation or tunicamycin(TUN) treatment enhanced ER stress-induced AGR2 expression in GC cell lines. AGR2-overexpressed stable cell lines were established using vectors. We found AGR2 overexpression promoted proliferation, migration and invasion of GC cells, as well as tumor growth in nude mice. ERK and AKT phosphorylation accounted for the cancer progression driven by AGR2. AGR2 silencing by shRNA led to cell cycle arrest and reduced proliferation of GC cells. Silencing of AGR2 obviously sensitized GC cells to ER stress-induced apoptosis through caspase dependent mithchondrial pathway. Knockdown of unfolded protein response sensors partially abrogated ER stress-induced AGR2 expression.Collectively, here we showed two pathways involving in GC progression under cellular stress such as chronic inflammation. On the one hand, TLR4 signaling promoted GC cell proliferation through elevated mROS production. On the other hand, stress-induced AGR2 expression led to resistance to apoptosis, enhanced proliferation, migration and invasion. We also elucidated the underlying mechanisms. mROS promoted AKT phosphorylation and NF-κB p65 nuclear translocation, and AGR2 enhanced GC progression through ERK and AKT phosphorylation. These results suggest that blocking mROS and AGR2 production, as well as intervention of their pathway may inhibit GC progression under stress.