Screening And Identification Of Protein Profile For Lung Cancer Early Diagnosis And Deregulation Of Chromatin Remodeling Of SWI/SNF In Lung Cancer

Objective:To identify a panel of tumor associated autoantibodies which can potentially be used as biomarkers for the early diagnosis of non-small cell lung cancer(NSCLC).Background:Lung cancer histologically consists of small cell lung cancer(SCLC)and NSCLC which accounts for about 80%cases of all lung cancer.Lung caner is the leading cause among cancer-related death worldwide,in part because most cases are in the late metastasis stage when diagnosed.Recent studies indicated that the autoantibodies to the lung cancer-associated specific antigens may be present in the patient peripheral blood and can potentially be used as a biomarker for early detection of lung cancer.Materials and Methods:We established a phage display library derived from 20 NSCLC tumor tissues(stage ?-?).We performed biopan enrichment and immunochemical analysis of this library with sera from patients and normal healthy controls.The presumptive phage expressed tumor-associated proteins were initially proposed based on the differential affinity to the sera antibodies of patients versus normal healthy controls.After the gene sequence confirmation,the in-frame phage expressed proteins were correlated with their respective gene expression profiles generated from microarray in NSCLC cell lines and tissue samples.The up-regulated gene encoded proteins were selected to develop phage protein enzyme-linked immunoadsorbent assays(ELISAs).We further tested the validity of these tumor-associated autoantibodies makers with serum samples from 40 NSCLC patients and 36 healthy matched controls.Logistic regression models and leave-one-out validation tests were used to evaluate prediction ability of a single marker versus combined markers,and the latter statistic method was also employed to analyze the data in association with the disease stage of lung cancer.Results:Of the 148 putative tumor-associated phage clones,35 were unique and in-frame expressed phage proteins.Based on the gene expression profiling,four proteins were selected for further study.Affymetrix microarray database showed that the mRNA transcripts of the proteins were significantly up-regulated in NSCLC cell lines when compared with normal HBECs(human bronchial epithelial cells)(p<0.05).Illumina microarray database showed that the gene expression level of 96-12 and BT9-3(protein code)was up-regulated in NSCLC cell lines and 83 NSCLC tissue samples in contrast to normal HBECs/HSAECs and paired non-malignant lung tissues,respectively(p<0.05).A logistic regression analysis achieved 47.5%sensitivity and 97.3%specificity in predicting NSCLC status,while leave-one-out validation achieved 66.7%sensitivity and 60.0%specificity.Both receiver operating characteristic curve analysis and leave-one-out method revealed that combined measurements of the four antibodies produced better predictive accuracies than any single marker did alone.Leave-one-out validation also showed that the sensitivity were 63.6%,62.5%,82.4%and 50%for detecting stage ?,?,? and stage ? NSCLC patients,respectively.Conclusion:our data presented here demonstrated that the serum antoantibody was existed in lung cancer patient sera.The identified four lung cancer-related proteins may be associated with cancer-specific autoantibody production at early stage of tumorigenesis.The panel of autoantibodies generated from the assays has a high potential for detecting early stage NSCLC.Objective:To study deregulation of the SWI/SNF chromatin remodeling complex in lung cancer tumors and cell lines with the emphasis on SMARCA2,SMARCA4 and ARID1A.Background:The DNA in each mammalian cell is compacted about 5000 fold into chromatin,and in the compacted state is unavailable for transcription.This compaction is controlled by three mechanisms:including the ATP-dependent chromatin remodeling complex SWI/SNF,consisting of 20 genes that code for about 12 protein subunits(some of which can substitute for each other).There are over 280 possible subunit permutations,influencing transcription,chromatin binding and remodeling,and tissue and gene specificity.Several subunit genes function as tumor suppressor genes(TSGs)including the interchangeable ATP catalytic components SMARCA2(protein = BRM)and SMARCA4(protein = BRG1)and the ARID1A(protein = BAF250a)accessory gene.The catalytic components are known to be inactivated in several cancers including lung,and ARID1 A in ovarian cancer.Materials and Methods:Total 101 cell lines and 83 non small cell lung cancer and paired non-malignant lung tissue samples arising in smokers and never smokers were studied.NextGen and Sanger sequencing of the SWI/SNF complex genes were performed on the cell lines.Western blot of nuclear extracts and methylation were also performed on cell lines.Genome wide gene copy number(by SNP analyses)and microarray expression studies were performed on the tumors,the control tissues and cell lines.Immunostaining(for BRG1 and BAF250a)were performed on lung cancer microarrays.Results:Mutations rates in NSCLC lines were:SMARCA4 28%(mainly homozygous deletions),SMARCA2 0%,ARID1A 11%(mainly heterozygous point mutations).However,a marked decrease in nuclear protein expression was frequently present in NSCLC lines:BRG1 32.5%,BRM 25%,BAF250a 23%.Loss of BRG1,BRM,or BAF250a was present in 41%of the NSCLC lines,and 16%had loss of two or three.Combined analyses with microarray expression and immunostaining studies of these three genes indicated frequent low expression in both NSCLC tumors and cell lines.In addition,DNA copy number by SNP analyses of NSCLC tumors indicated widespread loss of alleles of multiple members of the SWI/SNF complex.Evidence for epigenetic inactivation of one or more complex genes was present.Conclusions:Our data presente here indicate that there are frequent inactivation of one or more members of the SWI/SNF complex by a variety of mechanisms,including deletions,mutations,epigenetic,transcriptional and translational control.The predicted downstream effects on transcription and histone regulation are likely to be widespread and possibly demonstrating cell type and gene specific effects.Although highly complicated,elucidation of the precise chromatin complex abnormalities,mechanisms and downstream effects indicate the possibility of multiple new therapeutic targets for lung cancer.