Caf-derived IL-33 Promotes Gastric Cancer Progression Via ERK Signaling Pathway

Background: Cancer-associated fibroblasts(CAFs)paly important role in tumor-stroma interactions and gastric cancer(GC)progression.However,the mechanisms by which these interactions and CAFs contribute to GC progression are poorly understood.The aim of this study is to explore the mechanisms by which CAFs express IL-33 and CAF-derived IL-33 promotes GC progression.Transducin(?)-like 1 X-linked receptor 1(TBL1XR1)is an important regulator in controlling the master switch of gene activation and gene repression.Elevated TBL1XR1 expression has been found in many types of cancers and is associated with tumor development and progression.This study is aimed to investigate the clinical significance and biological mechanisms of TBL1XR1 in GC development.Methods: Part?: The expression levels of IL-33 and ST2 L in 134 pairs of GC tissues were detected by IF,IHC and q RT-PCR.Primary CAFs were isolated from GC tissues by tissue block method.The expression of TNF-a in GC tissues and cells was measured by q RTPCR.CAFs were co-cultured with GC cells(SGC7901 and MKN45),different experimental groups were set as follows: CAFs,CAFs+TNF-a,CAFs+GC cells,CAFs+GC cells+anti-TNF-a neutralizing antibody,then the expression of IL-33 in CAFs was detected by q RT-PCR and ELISA.We next detected the expression level of TNF-a receptor 2(TNFR2)in CAFs by q RT-PCR.And then CAFs were treated with TNF-a or NF-?B inhibitor(PDTC).Interferon regulatory factor 1(IRF-1)expression plasmids were transfected into CAFs using Lipofectamine 2000.Doulble luciferase reporter assay was used to prove the binding effects of IRF-1 on IL-33 promoter regions.GC cells were co-cultured with CAFs,or CAFs-si RNA/IL-33,or CAFs + anti-IL-33 neutralizing antibody,or CAFs + ERK inhibitor(U0126),or stimulated with IL-33,then cell migration and invasion assays were performed by using transwell chambers.EMT and activation of ERK pathway in GC cells were detected by q RT-PCR and Western blot(WB).GC cells were treated with IL-33 or U0126,or co-cultured with CAFs.SP1 expressing palsmids were transfected into GC cells.The expression of SP1,ZEB2 and p ERK were detected by q RTPCR and WB.Doulble luciferase reporter assay was used to prove the binding effects of SP1 on ZEB2 promoter regions.Part ?: TBL1XR1 expression level in GC tissues and cell lines were detected by q RTPCR,IHC and IF.We used sh RNA plasmids to down-regulate the expression of TBL1XR1 in GC cells NCI-N87 and SGC7901 cells,and used TBL1XR1 expressing plasmids to up-regulate the expression of TBL1XR1 in GC cells MKN45 and BGC823.CCK-8 assay,flow cytometry,wound healing assay,transwell migration and invasion assays were used to detect the proliferation,apoptosis,migration and invasion of GC cells.We used IHC and WB to detect the expression of p ERK and EMT markers in GC tissues and cells.U0126 was used to block the phosphorylation of ERK in NCI-N87 and SGC7901 cells,and then the proliferation,migration,invasion and EMT of GC cells were detected.U0126 was used to block the phosphorylation of ERK in BGC823/TBL1XR1 and MKN45/TBL1XR1 cells with up-regulated expression of TBL1XR1,and then the proliferation,migration,invasion and EMT of GC cells were detected.GC cells NCIN87/sh TBL1XR1 and BGC823/TBL1XR1 were injected into nude mice to explore the ability of tumorigenicity and peritoneal dissemination induced by TBL1XR1.Results: Part?:IL-33 and ST2 L were highly expressed in GC tissues.IL-33 was mainly expressed in CAFs,ST2 L was mainly expressed in GC cells,and both of them were correlated with clinicopathological features such as depth of invasion and TNM stage,and were prognostic markers for poor survival of GC patients.The up-regulation of IL-33 in CAFs can be induced by co-culture with GC cells or TNF-a stimulation,while deprivation of TNF-a using its neutralizing antibody inhibited the expression of IL-33.TNF-a was highly expressed in GC tissues and cells,TNFR2 was highly expressed in CAFs,and PDTC can inhibit the expression of IRF-1 and IL-33 in CAFs induced by TNF-a.The upregulated expression of IRF-1 in CAFs can promote the expression of IL-33.IRF-1 can promote the expression of IL-33 by binding to its promoter regions.GC cells co-cultured with CAFs or stimulated with IL-33 showed enhanced ability of migration,invasion and EMT than GC cells alone.However,adding anti-IL-33 or anti-ST2 L neutralizing antibody or CAFs/si IL-33 conditioned medium or U0126 into the co-culture system significantly decreased the ability of migration,invasion and EMT of GC cells.The expression of SP1 and ZEB2 were increased in GC cells treated with IL-33 or co-cultured with CAFs,but decreased when treated with anti-IL-33 neutralizing antibody or U0126.The up-regulation of SP1 in GC cells resulted in the enhanced expression of ZEB2,but had no effects on the expression level of p ERK.SP1 can promote the expression of ZEB2 by binding to its promoter regions.Part?: TBL1XR1 was highly expressed in GC tissues and cells and closely correlated with the depth of tumor invasion,TNM stage and poor prognosis.Knockdown of TBL1XR1 significantly suppressed the proliferation,migration,invasion and EMT of GC cells(NCI-N87 and SGC7901).Overexpression of TBL1XR1 significantly promoted the proliferation,migration,invasion and EMT of GC cells(BGC823 and MKN45).Knockdown of TBL1XR1 could significantly inhibit the phosphorylation of ERK in NCIN87 and SGC7901 cells,and U0126 could significantly inhibit the proliferation,migration,invasion and EMT in NCI-N87 and SGC7901 cells.Intriguingly,U0126 could also reverse the proliferation,migration,invasion and EMT induced by TBL1XR1 overexpression in BGC823/TBL1XR1 and MKN45/TBL1XR1 cells.Conclusions:1.IL-33 and ST2 L were highly expressed in GC tissues,and both of them were correlated with clinicopathological features and poor prognosis.GC cells enhanced the expression of IL-33 in CAFs via TNF-?/TNFR2/NF-?B/IRF-1 signaling pathway,while CAF-derived IL-33 promoted the migration,invasion,and EMT changes of GC cell by activation of ERK/SP1/ZEB2 signaling pathway via ST2 L.2.TBL1XR1 was highly expressed in GC tissues and cells,and it was correlated with clinicopathological features and poor prognosis.TBL1XR1 can promote GC cells proliferation,migration,invasion and EMT via the ERK signaling pathway.