CXCL5 Promotes Gastric Cancer Metastasis By Inducing Epithelial-Mesenchymal Transition And Activating Neutrophils

Objective:Chemokines play important roles in the development of various malignant diseases.The dysregulated expression of CXCL5 is reported in a series of cancers and affects cancer progress.However,the biological roles of CXCL5 in gastric cancer remain uncertain.The present study was designed to detect the expression of CXCL5 in gastric cancer cells and tissues,serum from gastric cancer patients,and to analyze the correlation between its expression level and clinicopathological data.Moreover,we studied the biological functions and the EMT in gastric cancer cells and the activation of neutrophils after stimulation by CXCL5,to investigate the possible mechanism and provide new strategies for gastric cancer treatment.Materials and methods:(1)QRT-PCR was performed to detect the gene expression level of CXCL5 in GC cell lines and GC tissues.The correlation between the expression level of CXCL5 in GC tissues and the clinicopathological features(age,sex,tumor size,differentiation,lymph node metastasis,invasion and TNM stage)was analyzed.(2)ELISA assay was performed to measure the protein expression level of CXCL5 in serum and culture supernatants from GC tissues and GC cells.(3)GC cell HGC-27,BGC-23 and neutrophils were stimulated by rhCXCL5 and GC tissue culture supernatants.The migration and invasion abilities in GC cells and the chemotactic ability in neutrophils were then observed.QRT-PCR was performed to determine the expression levels of EMT related markers in GC cells and inflammatory factors in neutrophils.Western Blot analysis was used to detect the expression levels of EMT related markers and signaling protein in GC cells and neutrophils.(4)Conditioned medium of pre-treated neutrophils was used to stimulate GC cells.The migration and invasion abilities in GC cells were then observed.The expression levels of EMT related markers and signaling protein were tested by Western Blot analysis.Neutralization experiments were then performed to further verify thatneutrophil-derived IL-6 and IL-23 enhance the migration and invasion abilities and the EMT in GC cells.(5)Metastatic abdominal tumor model was established to observe the number of colonized and metastatic nodules.And the expression levels of EMT related markers were detected by immunohistochemistry,QRT-PCR and Western Blot.Results:(1)QRT-PCR results showed that the expression level of CXCL5 in GC cells and GC tissues was significantly higher than that in control groups.And the upregulation of CXCL5 was significantly correlated with lymph node metastasis and TNM stage.(2)ELISA results showed that the protein level of CXCL5 in serum from gastric cancer patients and culture supernatants from GC tissues and GC cells was higher than that in control groups.(3)GC cell HGC-27 and BGC-23 that stimulated by rhCXCL5 and GC tissue culture supernatants exhibited enhanced migration and invasion abilities and showed increased expression levels of EMT markers such as N-cadherin while decreased expression level of E-cadherin.Neutrophils migrating to rhCXCL5 were significantly increased and the pre-treated neutrophils showed increased expression levels of inflammatory factors such as IL-6,IL-8,IL-17,IL-23.Western Blot results showed higher expression level of phosphorylated ERK in pre-treated GC cells and higher expression levels of phosphorylated ERK and p38 in pre-treated neutrophils.(4)GC cells cultured with conditioned medium of neutrophils displayed enhanced migration and invasion abilities and increased expression levels of EMT markers such as N-cadherin while decreased expression level of E-cadherin.Blockades of IL-6 and IL-23 reversed those phenomena above.(5)Animal model showed more peritoneal colonized tumors and liver metastatic nodules in rhCXCL5 and neutrophil conditioned medium treated groups.IHC,QRT-PCR and Western Blot results showed increased expression levels of EMT markers such as N-cadherin while decreased expression level of E-cadherin in the tissues from metastatic nodules.Conclusion:The chemokine CXCL5 is upregulated in GC cells and GC tissues,and serum form gastric cancer patients.The upregulation of CXCL5 in GC tissues was associated with lymph node metastasis and TNM stage.On the one hand,CXCL5 activates ERK signaling pathway and induces EMT in GC cells;On the other hand,CXCL5 activates ERK and p38 signaling pathways and increases the expression levels of IL-6 and IL-23 in neutrophils,which subsequently induces EMT in GC cells.In conclusion,the direct and indirect functions of CXCL5 synergistically promote GC metastasis.Our study provides new evidence for revealing the mechanism of CXCL5 in GC development and new therapeutic targets for diagnosis and treatment of GC.