The Role Of S100A8 In The Metastasis And Prognosis Of Colorectal Cancer

Colorectal cancer(CRC)is the most common malignant disease in the digestive tract.According to statistics,CRC ranks the top three causes of cancer morbidity and mortality in both men and women in the United States.There are 331,000 new cases of CRC in China every year,and 159,000 people still die of this disease.Though the early diagnosis and treatment methods are improved in recent years,the morbidity and mortality of CRC are not decreased.Metastasis is the most critical cause of death.Now Chemotherapy is the major method for metastatic CRC.Although many molecular targeted drugs have been approved by the food and drug administration(FDA)for adjuvant treatment of metastatic colorectal cancer,the effects were in great discrepancy for the individual difference.The efforts in studying the mechanism of CRC metastasis and searching for potential molecular targets are needed to improve treatment and survival for CRC patients.Epithelial-mesenchymal transition(EMT)is a process that epithelial cells lose their cell polarity and cell-cell adhesion,with the observation by reduced epithelial marker like E-cadherin(E-cad),increased mesenchymal markers like vimentin and activated transcription factor like snail.EMT is regulated by the complex networks of intracellular and extracellular factors.Many growth factors and other ligands from tumor cells and stroma cells bind to the receptors on tumor cells,following activation of several pathways,then activate several transcription factors that regulate EMT markers.TGF-? is the key regulators and major inducer of EMT.TGF-? and other EMT inducers are not only origin from tumor cells but also come from the stroma surrounding tumor cells.Tumor microenvironment is the internal environment in which tumor cells produce and live,including not only tumor cells themselves,but also fibroblasts,immune and inflammatory cells,and other cells around them,as well as matrix,microvessels and biological molecules.The tumor microenvironment plays an important role in regulating EMT,but the specific regulatory mechanism is not clear.The invasive front is considered to be a hot spot of EMT when tumor cells infiltrate and metastasize to the deep.At the invasive margin,there is a unique type of tumor cells,which are usually detached from the main tumor body and appear as single cell or clusters of no more than five cells,called tumor buds.Tumor buds are considered as a typical morphology of EMT.In order to investigate the effects of the different tumor microenvironment on EMT and metastasis,we previously applied microdissection to capture tumor buds,stroma cells surrounding tumor buds,tumor cells,tumor stoma cells and normal epithelial cells and normal stroma cells.A series of differentially expressed genes were identified by microarray analysis and calculation.Among them,the expression of S100A8 gene in the normal stroma,tumor center stroma and tumor invasive front stroma gradually increased,suggesting that S100A8 may affect EMT and metastasis in CRC.S100A8 protein,also known as MRP8 or calgranulin A,is a low molecular weight(10.8 kDa)calcium binding protein and belongs to the S100 calcium binding protein family..The expression of S100A8 is up-regulated in many tumors such as breast cancer,pancreatic cancer,bladder cancer,gastric cancer,lung cancer,ovarian cancer and skin cancer.However,the differential expression of S100A8 in tumors and the dual effects of anti-tumor and pro-tumor effects lead to the complex relationship between S100A8 and tumor.At present,most studies focus on the function of S100A8 acting as a secretory protein in tumor cells,but its function in the tumor cells was not clear.In CRC,in which cells do S100A8 protein express?Which extracellular signals regulate S100A8 expression in tumor cells?What is the relationship between intracellular S100A8 expression and EMT?All these problems need to be clarified.The upstream transcription factor(USF),which has two variants USF1 and USF2,is widely expressed in eukaryotes as a transcription factor.USF2 is able to form a dimer that binds to E-box on the promoter of the target genes.However,the relationship between USF2 and EMT has not been reported.It has been reported that TGF-?secreted by tumor cells can induce T-cell immunoglobulin mucin 3(Tim 3)by upregulating USF2.In addition,inhibiting DNA binding ability of USF2 can significantly reduce TGF-?-induced plasminogen activator inhibitor-1 transcription(PAI-1).PAI-1 is an important molecule to promote renal fibrosis,and EMT is one of the important mechanisms of renal fibrosis.Therefore,whether TGF-? can affect EMT by regulating the transcriptional activity of USF2 remains to be studied.Could USF2 regulate S100A8 expression and promoting EMT?The expression of S100A8 in the stroma and tumor cells was counted by immunohistochemical method.In order to clarify the significance of S100A8 in the tumor microenvironment and tumor cells,the immunohistochemical results were separated between stroma cells and tumor cells.The density of S100A8 was analyzed with the survival of patients and EMT related indicators.After treatment of CRC cell lines with human recombinant protein S100A8,the changes in cell motility and protein markers were detected.In the CRC cells,S100A8 and USF2 were overexpressed and knocked-down respectively,to detect EMT markers and cell biological behavior.In vivo,the formation of lung metastases was detected after the S100A8-overexpressed tumor cells were injected into mice:The immunohistochemical results suggested that S100A8 were mainly expressed in neutrophils and monocytes in the tumor stroma.After counting the number of S100A8 positive cells in the stroma TC and TF,it was found that the more number of S100A8 in stroma TF,the longer survival patients had,while S100A8 in stroma TC was not significantly related to the survival of the patients.CRC cells(SW480 and DLD1)were administered by recombinant S100A8 protein in the concentration gradients.High concentrations of S100A8 reduced tumor cell migration and invasion.Western blot results showed that the expression of E-cadherin increased and vimentin decreased after S100A8 treatment.The expression of transcription factor snail and anti-apoptosis factor Bcl2 decreased.It indicated that the extracellular S100A8 prevented EMT and induced apoptosis.The number of tumor buds in TF was counted.Patients with higher tumor buds had shorter survival time,which was different from the prognosis of patients with more positive S100A8 cells in the stroma TF.Therefore,the new indicator SATB(S100A8+-associated Tumor bud)was obtained by combining S100A8 with tumor buds.The higher SATB score(the less S100A8 positive number and the more tumor buds number),the worse survival of patients.The results of multivariate proportional risk model analysis showed that SATB could independently predict the prognosis of patients.Following the biological significance of S100A8 positive cells in the tumor stroma,we try to determine the association of S100A8 expressing tumor cells and the patient prognosis.When S100A8 was positive in the tumor cells,the patients had more lymph node metastasis(p = 0.029)and distant organ metastasis(p = 0.021),higher TNM stage(p = 0.013)and lower survival rate(p = 0.039).That TGF-P induced EMT was accompanied by up-regulation of S100A8 in SW480 cells and DLD1 cells.Then S100A8 was overexpressed in SW480 and HCT8 cells,and knocked-down in DLD1 and HT29 cells respectively.In S100A8 overexpressed group,cell migration and invasion were enhanced,E-cadherin expression was decreased,vimentin expression was increased,and nuclear snail expression was increased;while in S100A8 knockdown group the results were opposite.It suggested S100A8 promoted EMT and metastasis.In vivo,the lung metastasis experiment confirmed S100A8 promoted metastasis.S100A8-knockdown DLD1 and HT29 cells were stimulated with TGF-?.TGF-? could not enhance cell migration and invasion in S100A8-knockdown cells compared with the control group.In order to clarify the regulating mechanism of S100A8,we use the online software to predict USF2 binding site and finding that USF2 could bind to the S100A8 promoter(aacacgtcc)sequence.ChIP(chromatin immunoprecipitation)experiments confirmed that USF2 could bind to this promoter sequence.Luciferase reporter gene results showed that USF2 could increase the promoter activity.The reporter gene signal was significantly reduced after mutation of the promoter sequence.After overexpression of USF2 in SW480 and HCT8 cells,S100A8 expression increased and EMT occurred.S100A8 expression decreased after knocking down USF2 in DLD1 and HT29,and EMT did not occur.TGF-? could not enhance cell migration and invasion in USF28-knockdown cells compared with the control group.Recombinant S100A8 protein treatment decreased the expression of nuclear USF2 in tumor cells,while TGF-? stimulation increased expression of nuclear USF2 in tumor cells.In order to explore whether USF2 is a node of intracellular and extracellular functional transformation,we stimulated with Recombinant protein S100A8 in the USF2 blank and overexpression groups,respectively,EMT in the USF2 overexpressed group was not inhibited by recombinant S100A8 protein,nor was apoptosis induced as compared with the control group.EMT could not be induced after TGF-? stimulation in USF2 knockdown group.These results suggested that USF2 plays an important regulatory role in the intracellular and extracellular functional transformation of S100A8.Based on the above data,we draw the following conclusions:The S100A8 positive cells in the stroma of invasive front predict good prognosis for colorectal cancer patients and inhibit EMT.In CRC cells,S100A8 induces EMT and promotes metastasis.The expression of S100A8 is regulated by TGF-?/USF2,which is an unfavorable factor for prognosis.USF2 plays an important regulatory role in the intracellular and extracellular functional transformation of S100A8.