Insulin Growth Factor Binding Protein 2 Is Highly Expressed And Up-regulates VEGFA Expression By Activating EGFR-STAT3 Signaling Pathway In Esophageal Cancer

BackgroundInsulin-like growth factor-binding protein 2(IGFBP2)is one of the six homologous proteins of the insulin-like growth factor-binding protein family.IGFBP2 can prolong the half-life of insulin-like growth factor(IGF)and alter the interaction between IGF and its cell surface receptors.IGFBP2 has been shown to inhibit or promote cell growth by inhibiting or stimulating IGF.Resent studies has shown that IGFBP2 participates in the proliferation,migration,invasion and angiogenesis in a variety of human cancers.In our previous work,through high-throughput chip analysis,we found that serum IGFBP2 was up-regulated in patients with esophageal squamous cell carcinoma(ESCC),suggesting that serum IGFBP2 may be a biomarker for ESCC diagnosis.However,is the expression of IGFBP2 elevated in ESCC? What role does it play in the development of esophageal cancer? What is the molecular mechanism? These issues remain unclear.Therefore,this study aimed to explore the above scientific issues and provide useful experimental data for further understanding the role of IGFBP2 in ESCC.MethodsFirstly,the IGFBP2 mRNA expression data in ESCC were obtained from high-throughput GEO expression profile chip database(GSE7040 and GSE161533).The correlation between IGFBP2 mRNA expression and prognosis was analyzed according to GSE121932.Then,188 pairs of ESCC tissues with corresponding paracancer normal tissues and 51 cases of ESCC tissues collected from Shantou Central Hospital were divided into a test group(118 ESCC tissues and their paired paracancer normal tissues)and a validation group(70 pairs of ESCC tissues and paracancer normal tissues and 51 cases of ESCC tissues).The expression of IGFBP2 protein was detected by immunohistochemistry,and t-test and Kaplan-Meier survival analysis were used to explore the association between the IGFBP2 expression and prognosis of patients.Secondly,we evaluated the expression of IGFBP2 in different esophageal cancer cell lines by western blot,and constructed IGFBP2 stable overexpression cell lines by plasmid DNA transfection experiment.Thirdly,the biological functions of IGFBP2 in esophageal cancer cells were tested by cell proliferation assay,cell clonogenesis assay,cell scratch assay and transwell assay.Finally,the downstream oncogenic molecules of IGFBP2 were screened by differential gene enrichment analysis based on public data sets,and were validated by experiments including immunofluorescence assay,pathway inhibitor treatment and western blotting.Results1)In GEO expression profile chip database,IGFBP2 mRNA expression was up-regulated in ESCC tissues compared with normal adjacent tissues,and its high expression was associated with lower overall survival of ESCC patients.2)In the test set,compared with corresponding paracancer normal tissues,IGFBP2 was up-expressed in ESCC tissues.And its high expression was associated with lower overall survival time(p =0.039)and lower disease-free survival(p=0.0192).Moveover,COX multivariate analysis indicated that IGFBP2 was an independent prognostic factor for ESCC.Similar results were observed in the validation set,further confirming that IGFBP2 was highly expressed in ESCC tissues and was a prognostic biomarker.3)IGFBP2 expression levels were different in ESCC cell lines,with relatively lower expression in KYSE510 and KYSE30 cells.After stable over-expression of IGFBP2 in KYSE30 and KYSE510 cells,the abilities of cell proliferation and cloning were enhanced,as well as cell migration and invasion.In GEO dataset GSE69925,according to the differential expression of IGFBP2 mRNA in ESCC tissues,we analyzed the genes with differential expression and found that VEGFA mRNA was positively correlated with IGFBP2 mRNA expression.Then,related pathways were identified by western blotting assay,and the results suggested that the expression of p-EGFR and p-STAT3 and VEGFA in ESCC cells increased after the over-expression of IGFBP2.The same results were obtained after stimulation with exogenous IGFBP2 recombinant proteins.In addition,immunofluorescence assay showed that fluorescence intensity of p-EGFR,p-STAT3 and VEGFA could be enhanced when the IGFBP2 was over-expressed in KYSE30 cells,and they exhibited co-localization with IGFBP2.Western blotting assay indicated the expression of p-EGFR,p-STAT3 and VEGFA decreased when EGFR inhibitors erlotinib or gefitinib were added in IGFBP2-overexpressed KYSE30 and KYSE510 cells.ConclusionThis study demonstrated that IGFBP2 expression was up-regulated in ESCC tissues,and its high expression was associated with poor prognosis.Over-expression of IGFBP2 may enhance VEGFA expression by activating the EGFR-STAT3 signaling pathway,thereby promoting the malignant phenotype of esophageal cancer cells.These foundings suggest that IGFBP2 is a potential prognostic biomarker and therapeutic target for esophageal cancer.