The Mechanism Of JWA Via CXCR4 And CCR5 Inhibits Gastric Cancer Metastasis

Gastric cancer is the 5^th most common cancer and the 2^nd largest cause of cancer death worldwide.Over the past decade,the prognosis of gastric cancer is still unsatisfactory although the development of surgery,radiation and chemotherapy technology.The prognosis of tumor is closely related to its metastasis.The 5-year survival decreases to 27%and 3%from 63%once the tumor has spread to regional lymph nodes or distant organ sites.The process of tumor metastasis is very complex and its molecular mechanism is not very clear.During metastatic dissemination,a cancer cell from a primary tumor executes the following sequence of steps:it locally invades the surrounding tissue,enters the microvasculature of the lymph and blood systems?intravasation?,survives and translocates largely through the bloodstream to microvessels of distant tissues,exits from the bloodstream?extravasation?,survives in the microenvironment of distant tissues,and finally adapts to the foreign microenvironment of these tissues in ways that facilitate cell proliferation and the formation of macroscopic secondary tumor.Therefore understanding of the molecular mechanism of tumor metastasis will provide new theoretical bases for the treatment of tumor.This will also become a key point of tumor metastasis.Since 1996,we have focused on the relationship between environment response gene-JWA with tumor development.Our previous data showed the melanoma cells with reduced expression of JWA indicated significant higher migration,invasion,adhesion ability than that with vector control.Retrospective cohort study on gastric cancer showed that JWA expression was significantly down-regulated in gastric cancer compared with normal gastric tissues.The lower expression of JWA in gastric cancer increased the risk of the lymph node metastasis and distant metastasis.JWA low expression was an unfavourable factor for the prognosis of gastric cancer patients.Previous studies also found that JWA can regulate the expression of CXCR4 and CCR5.The present study was designed to explore how JWA regulates the gastric cancer cell metastasis through cell chemokine receptors CXCR4 and CCR5.Methods:Scratches and transwell chambers migration experiments were used to observe the ability of gastric cancer cell migration after interfering or increasing the expression of JWA in human gastric cancer SGC7901 and HGC-27 cells.Then Western blotting and real time quantitative PCR methods were conducted to show the influence of JWA on CXCR4 and CCR5 expressions.Then we investigated the influence of JWA on p-AKT activation after inhibiting the the expression of CXCR4and CCR5.Western blot was used to test the expressions of JWA,CXCR4 and CCR5in fresh gastric cancer and the corresponding normal gastric mucosa tissues.Simultaneously,the retrospective gastric cancer set was studied,and immunohistochemisty was used to detect the expression of CXCR4 and CCR5 in the gastric cancer tissue microarrays?TMAs?.The association between CXCR4 and CCR5 expression and clinicopathological parameters was evaluated by Fisher's exact test.The significance of CXCR4 and CCR5 staining in primary tumors compared with their corresponding non-tumors was assessed by the paired Wilcoxon test?raw scores?.Probability of differences in OS as a function of time was ascertained by use of the Kaplan-Meier method,with a log-rank test probe for significance.Result:?1?JWA inhibits gastric cancer cell migration.The gastric cancer cells'migration ability was increased after interference the expression of JWA.On the contrary,the cells migrated slower after overexpression of JWA in the wound healing experiment.The similar result was observed in the transwell chamber migration experiment:after high expression of JWA,the scratch width of HGC-27 cells were increased from 50%to 80%after 24 hours;While in SGC7901 cells with low expression of JWA,the scratch width of were decreased from 90%to 65%after 48 hours.?2?JWA via ubiquitin proteasome pathway regulats CXCR4 and CCR5 protein expressions.In gastric cancer cells,JWA negatively regulated expressions of CXCR4and CCR5.Further study demonstrated that low expression of JWA could inhibit the degradation of CXCR4 and CCR5,and high expression of JWA could accelerate the degradation of CXCR4 and CCR5 in gastric cancer cells.Using ubiquitin proteasome inhibitor PS341 we blocked the regulation of JWA on CXCR4 and CCR5 protein expression.?3?JWA reduces p-AKT activity via inhibition of CXCR4 and CCR5.The activity of p-AKT was reduced after high expression of JWA or low expression of CXCR4 or CCR5.On the contrary,low expression of JWA or overexpression of CXCR4 or CCR5 could increase p-AKT activity.Interference of CXCR4 and CCR5 expression could reverse the increased activity of p-AKT caused by the low expression of JWA.P-AKT or CXCR4 and CCR5 inhibitors could reverse the enhanced migration ability of the gastric cancer cells caused by the low expression of JWA.?4?JWA expression was negatively correlated with expressions of CXCR4 and CCR5in gastric cancer tissues.The expression of JWA was reduced and the expression of CXCR4 and CCR5 was raised in seven fresh gastric cancer tissues compared with the paired normal tissues.The similar results were showed in gastric cancer tissue microarray chip of the retrospective cohort study.?5?JWA is negatively correlated with clinical pathologic characteristics in retrospective cohort study of gastric cancer:the expression of CXCR4 in gastric cancer tissues was correlated with lymph node metastasis?P=0.002?and TNM staging?P=0.043?,the expression of CCR5 in gastric cancer tissues was associated with the degree of differentiation.And the expression of CXCR4?P<0.001?or CCR5?P=0.044?indicated a negative correlation with JWA expression.High expression of CXCR4 and CCR5 patients showed poor survival outcomes in gastric cancer patients.In conjoint analysis with JWA,we found that gastric cancer patients with low expression of JWA and high expression of CXCR4 and CCR5 showed the worst survival outcomes.Conclusion:In the present study,we carried out series cell culture models and analysis of TMA combined with fresh gastric cancer tissue as well as clinical data base and confirmed an important role of JWA in the metastasis related processes of gastric cancer metastasis.In the mechanism study,we found that JWA eventually inhibited the gastric cancer metastasis by negative regulation of CXCR4 and CCR5through ubiquitin proteasome pathway.These finds would not only provide the theoretical basis for treatment strategies and methods of inhibiting gastric cancer metastasis targeted to JWA,but also put forward a new insight for the molecular mechanism of the gastric cancer metastasis treatment.