SPHK1 Regulated Autophagy In Peritoneal Mesothelial Cell Enhances Gastric Cancer Peritoneal Dissemination

Objective:Gastric cancer remains the fifth most frequent cancer and the third leading cause of cancer death in global cancer statistics.Tumor metastasis is one of the main causes of poor prognosis,in which peritoneal dissemination is the most common form of metastasis in patients with gastric cancer.The median survival time after peritoneal dissemination is only 3 to 6 months.However,the mechanisms underlying gastric cancer peritoneal dissemination are not entirely clear.Therefore,it is of great significance to study the mechanism of gastric cancer peritoneal dissemination and to find key biomarkers and therapeutic targets for the prevention and treatment of peritoneal dissemination.Peritoneal dissemination is the result of the interaction between tumor cells and peritoneal microenvironment.The peritoneal cavity exfoliated cancer cells adhere to the peritoneal mesothelial cells,with injury of mesothelial cells,whereafter they infiltrate the submesothelial tissue and change the peritoneal microenvironment to make it suitable for the colonization of metastatic cancer.Our previous studies have shown that gastric cancer cells secrete transforming growth factor?1?TGF-?1?to induce mesothelial cell apoptosis and fibrosis.TGF-?1 is a member of TGF-?family,which is a kind of cytokine with many functions.TGF-?1 plays an crucial role in embryonic development,physiological state and disease state,such as extracellular matrix deposition,cell cycle control and immune response.Recently,TGF-?1 has been reported to regulate cell autophagy.Autophagy is a process of degrading intracellular components through the formation of autophagosomes and the degradation of lysosomes,which is important for the normal proliferation and differentiation of cells.It is also an adaptive response to maintain the viability of cells under stress.Subcellular components such as damaged,denatured,senescent or non-functional proteins and organelles are transported to lysosomes for degradation and recycling in order to maintain the stability of cell structure,function and metabolism.Interestingly,it has been demonstrated that TGF-?1 differentially regulates autophagy;specifically,the growth factor promotes autophagy in vascular endothelial cells and tubular epithelial kidney cells,but inhibits this process in fibroblasts from patients with idiopathic pulmonary fibrosis.In the process of gastric cancer peritoneal dissemination,cancer cells change the peritoneal microenvironment by secreting TGF-?1,so that it can form a favorable place for tumor colonization.Whether TGF-?1 can regulate the autophagy of peritoneal mesothelial cells and the effect of autophagic mesothelial on gastric cancer peritoneal dissemination remains largely unclear.Sphingosine kinase 1?SPHK1?is a widely expressed evolutionary conservative enzyme that catalyzes the phosphorylation of sphingosine to sphingosine 1-phosphate?S1P?.SPHK1 has been shown to regulate a wide range of cell processes,including promoting cell proliferation,survival and movement.In particular,SPHK1 plays an oncogenic role in some tumors,including ovarian cancer,liver cancer,leukemia,gastrointestinal tumors and so on.The abnormal expression of SPHK1 in tumors promotes the proliferation,migration and drug resistance of tumor cells.However,little is known about the role of SPHK1 in tumor stroma cells.Emerging evidence has implicated SPHK1 in cellular autophagy such as neuron cells,renal tubular epithelial cells and tumor cells.Whether SPHK1 is involved in regulating autophagy of peritoneal mesothelial cells has not been reported.In the present study,we will explore the effect of TGF-?1 secreted by gastric cancer cells on mesothelial autophagy by a coculture system of gastric cancer cells and mesothelial cells.In addition,whether SPHK1 is involved in the regulation of TGF-?1on mesothelial autophagy.Moreover,the relationship between mesothelial autophagy and fibrosis was also discussed.Methods:1.Peritoneal tissue specimens from 120 patients undergoing radical surgery were selected.The expression of SPHK1 and autophagy associated protein LC3B in peritoneal mesothelial cells was detected by immunohistochemistry.The relationship between SPHK1 and clinicopathological factors and prognosis was analyzed.2.The expression of TGF-?1 in several gastric cancer cell lines was detected by Western blot and ELISA,and the high expression gastric cancer cell line was selected.Short hairpin RNA?shRNA?lentivirus vector was used to stabilize the silencing of gastric cancer cell line TGF-?1.The coculture system of gastric cancer cells and mesothelial cells was established by transell chamber.Western blot was used to detect the effect of TGF-?1 secreted by gastric cancer cells on the expression of mesothelial autophagy related protein and SPHK1,and fluorescence confocal microscopy was used to detect the formation of mesothelial autophagosomes.We used shRNA to target silenced mesothelial cells SPHK1.Western blot,fluorescence confocal microscopy and transmission electron microscopy were used to detect the effect of mesenchymal SPHK1 expression on the autophagy induced by gastric cancer cells.3.The effects of SPHK1-regulated mesothelial cell autophagy on the adhesion and invasion of gastric cancer cells were detected by gastric cancer cell-mesothelial cell adhesion test and transell invasion test in vitro,respectively.Gastric cancer cells and mesenchymal cells were mixed and intraperitoneally injected into nude mice to detect the effect of mesenchymal SPHK1 expression on the tumorigenic ability of gastric cancer peritoneum in vivo.4.Finally,we detected the effect of TGF-?1/SPHK1 pathway on mesothelial fibrosis in the coculture system by Western blot.The expression of fibrosis-related proteins in mesothelial cells were tested after treated with autophagy inhibitor 3-methylpurine.Results:1.The results of immunohistochemistry showed that the expression of SPHK1in peritoneal mesothelial cells was positively correlated with the expression of LC3B?Pearson test,r=0.456,P<0.001?.The high expression of SPHK1 was significantly correlated with tumor size,depth of tumor invasion,lymph node metastasis,TNM stage,high expression of LC3B and peritoneal recurrence?P<0.05?.Kaplan-Meier analysis showed that the patients with high expression of SPHK1 and LC3B had a poor overall survival,respectively(P_SPHK1<0.001,P_LC3B=0.009).COX multivariate analysis further confirmed that SPHK1 was an independent prognostic factor?HR=1.826,95%CI:1.057-3.155,P=0.031?.2.The expression of TGF-?1 in gastric cancer cell lines MKN45,MKN28,SGC-7901,MGC-803 and BGC-823 was detected by Western blot and ELISA.The results showed that the level of TGF-?1 protein in SGC-7901 cell line was the highest.Thus,SGC-7901 cells were selected for use in the coculture model for subsequent experiments.Then SGC-7901 cells were transfected with shRNAs to targeted silence TGF-?1,and shCtrl/shTGF-?1 SGC-7901 cells were cocultured with mesothelial cells,respectively.The autophagy level of mesothelial cells in TGF-?1 silent group was weakened.Similarly,the induction of mesothelial cell autophagy by SGC-7901 cells was inhibited by adding TGF-?1 receptor inhibitor into the coculture system.3.After silencing TGF-?1 in SGC-7901 cells or adding TGF-?1 receptor inhibitor to block the effect of TGF-?1 on mesothelial cells,the expression of SPHK1 in mesothelial cells decreased.When interfering with the expression of SPHK1 in mesothelial cells,the activation of autophagy in mesothelial cells was inhibited by TGF-?1 secreted by SGC-7901 cells.4.It was found that mesothelial autophagy promoted the adhesion and invasion of gastric cancer cells SGC-7901 and MGC-803 in vitro.When targeting silenced mesothelial cells SPHK1,the attachment and invasion of SGC-7901 and MGC-803cells to shSPHK1 mesothelial cell monolayers was significantly inhibited.Results of xenograft model of cancer cells mixed with stromal cells demonstrated that SGC-7901cells coinjected with shSPHK1 HPMCs exhibited reduced macroscopic nodules during peritoneal cavity dissemination5.Further experiments showed that SPHK1 expression in mesothelial cell also regulated mesothelial fibrosis induced by gastric cancer cells.Overexpression of SPHK1 activated both autophagy and fibrosis of mesothelial cells.The autophagy inhibitor 3-methylpurine inhibited the autophagy of mesothelial cells and reversed the fibrogenesis phenotype.Conclusion:The expression of SPHK1 in peritoneal mesothelial cells is related to peritoneal dissemination and prognosis in patients with gastric cancer.Gastric cancer cells up-regulate mesothelial cell SPHK1 by secreting TGF-?1 to activate mesothelial autophagy.SPHK1-induced mesothelial autophagy is beneficial to maintain its fibrotic phenotype and promote adhesion,invasion and peritoneal metastasis of gastric cancer cells.Taken together,our results provided new insights for understanding the mechanisms of gastric cancer peritoneal dissemination and established SPHK1 as a potential therapeutic target.