BACH1 Autoantibody Validation And Its Functional Study In Esophageal Squamous Cell Carcinoma

Esophageal carcinoma(EC)is the seventh common malignant cancer and the sixth leading cause of cancer-related death worldwide.Chinese annual incidence and mortality rate of EC occupied more than half of that in the world.Approximately 90%of the histologic types of EC are esophageal squamous cell carcinoma(ESCC).Due to its highly invasion and metastasis,10-30%of early stage ESCC cases especially for tumors with submucosal invasion can metastasize to regional lymph nodes and behave like advanced tumor.Accurate diagnose of early stage ESCC with lymph nodes metastasis(LNM)extremely impacts the selection of treatment protocols,and also associates with better overall survival rate of patients.Despite multimodal diagnostic methods,such as endoscopic ultrasound,computed tomography(CT),positron emission tomography(PET),endoscopy biopsy,are used for staging tumor depth and nodal status,the diagnostic accuracy of detecting LNM is not completely satisfactory.Thus,further research into the discovery and validation of effective and noninvasive blood-based biomarkers for ESCC lymph nodes metastasis is benefit for diagnosis,treatment and prognosis.In our previous study,we adopted the Human Proteome Micro-arrays containing 19,394 recombinant proteins to comprehensively discover the differentially expressed serum autoantibodies during healthy controls(HC)individuals as well as ESCC without lymph node metastasis patients(ESCC-nLNM)and ESCC with lymph node metastasis patients(ESCC-LNM).Compared with HC and ESCC-nLNM groups,twenty-eight IgG autoantibodies and nine IgM autoantibodies were found to be especially upregulated in ESCC-LNM group.Anti-BACH1(BTB domain and CNC homolog 1)IgG was found to be one of upregulated autoantibodies.In this study,we further evaluated Anti-BACH1 IgG in a larger independent ESCC validation cohort by the semi quantitative immunoblotting analysis.The results revealed that the levels of anti-BACH1 IgG in ESCC patients' serum were significantly upregulated compared with HC group(P<0.01),and the levels of anti-BACH1 IgG in patients' serum with ESCC-LNM were notablely increased compared with ESCC-nLNM group(P<0.01).Receiver operating characteristic(ROC)analysis was applied for evaluating the use of anti-BACH1 IgG as ESCC lymph node metastasis candidate markers,the sensitivity is 23.1%,specificity is 92%and area under the curve(AUC)was 0.672.The positive rate of anti-BACH1 IgG was significantly correlated with LNM(P=0.0145)and AJCC stage(P=0.0012),however it didn't have significant correlation with sex,histological grade,distal organ metastasis and other clinical feathers.Overall,those data suggested that anti-BACH1 IgG autoantibody could be used as a potential biomarker of ESCC with lymph node metastasis.The production of most tumor associated autoantibodies is due to loss of immune tolerance caused by abnormal expression of tumor associated antigens(TAA)in malignancy tumor.In this study,we detected the expression of BACH1 in ESCC tissues by immunohistochemistry(IHC),BACH1 protein was overexpressed in ESCC tissues compared with paracarcinoma epithelial tissues(P<0.01).Analysis of the Cancer Genome Atlas(TCGA)ESCC RNA-seq data set showed that the mRNA expression level of BACH1 was significantly increased in ESCC tissues(P=0.0344),Higher BACH1 mRNA levels were remarkably associated with decreased overall survival(OS,P=0.0385)and shorter disease-free survival(DFS,P=0.0112)rates in ESCC patients.Collectively,these data suggested that BACH1 might be a potential oncogene for the progression and metastasis of ESCC.Our study further explored the function and potential mechanism of BACH 1 in the progression of ESCC.We found that silencing BACH1 attenuated the migration,invasion of KYSE30 and KYSE170 cells in vitro and the growth of xenografts tumor in vivo.Conversely,BACH1 overexpression increased the migration,invasion of KYSE150 cells and growth of tumor xenografts in vivo.Further mechanism analysis suggested that BACH1 promoted the metastasia of ESCC by triggering epithelial-mesenchymal transition(EMT)via directly upregulating the expression of CDH2,SNAI2 and VIM.Luciferase assay indicated that BACH1 overexpression significantly enhanced CDH2 promoter activity,triggered the expression of N-Cadherin.High level of BACH1 protein showed a strong association with upregulated levels of N-Cadherin,Slug and vimentin in ESCC tissues.Moreover,BACH1 knockdown significantly decreased mRNA expression of vascular endothelial growth factor C.BACH1 promoted VEGFC expression by regulating its transcription.The analysis of GEO database and TCGA database also confirmed that the mRNA expression of VEGFC was significantly upregulated in ESCC tissues,and it was positively correlated with BACH1 expression.IHC of tumor xenografts tissues showed that N-Cad,Slug,Vimentin,VEGFC and CD31+vessel density were significantly downregulated in BACH1 knockdown group compared with control group.BACH1 might inhibit the proliferation of ESCC by inhibiting angiogenesis and EMT in vivo.This study found that Anti-BACH1 IgG autoantibody may be a potential biomarker for lymph node metastasis of ESCC.The high expression of BACH1 is significantly associated with poor prognosis.BACH1 plays a critical role in promoting the proliferation and metastasis of ESCC.This study enriched the biological function of BACH 1 in ESCC and provided a noval target for exploring biomarkers of ESCC.