Acetylation Modification Reader BRD4,an Underlying Therapeutic Target In Esophageal Squamous Cell Carcinoma

Background and objectiveBromodomain Containing 4(BRD4)is a member of bromodomain and extraterminal domain(BET)family,and contains two bromodomains and one extraterminal domain.In cell cycle,it combines with acetylated histones and non-histones via bromodomain,and recruits transcriptional regulatory factors such as Mediator,positive transcription elongation factor b(P-TEFb),thus playing a key role in gene transcription regulation,cell cycle progression,inflammation and tumor progression.Recent reports have indicated the disorder and dysfunction of BRD4 bear some relation to tumor genesis and progress in melanoma,breast cancer,colorectal cancer and acute myeloid leukemia,and BRD4 shRNA or BET inhibitor may induce tumor cell arrest,apoptosis,differentiation and inhibit metastasis.It seems BRD4 may be an underlying therapeutic target in tumors mentioned above or not.We observed the expression profiles of BRD4 esophageal squamous cell carcinoma issues to explore the relation between BRD4 expression and clinicopathologic features and prognosis in esophageal squamous cell carcinoma.In vitro cultured human esophageal squamous cell carcinoma cells,we applied BRD4 inhibitor JQ-1,then observed cell proliferation,analyzed cell apoptosis and the expression of related proteins.Experiment methodThe expression profiles of BRD4 esophageal squamous cell carcinoma issues and its clinical meaning.We detected BRD4 expression in 80 samples of esophageal squamous cell carcinoma tissue and the paired para-carcinoma tissue by immunohistochemical staining.Samples were from the thoracic department of Cixi city people's hospital,gained in operations from 2008 to 2010.We scored the results of immunohistochemical staining by intensity(negative,low,medium,and strong positive,0-3)and the percentage of positive staining cells(?10%,11%-30%,31%-50%and>50%,0-3),then added the two parts together,if total score>3,BRD4 was considered as high expression,score<3 as low expression.We analyzed the relation between BRD4 expression and clinicopathologic features by chi-square test,and investigate its effect on patients'prognosis by Kaplan-Meier univariate analysis.Whether BRD4 could be an underlying therapeutic target in esophageal squamous cell carcinoma.Human esophageal squamous cell carcinoma cell lines EC 109,TE-1 and TE-13 were cultured in vitro.The effect of cell proliferation by JQ-1 in different concentrations for 24,48,72 hours was analyzed by MTT assay.And in colony forming assay,we investigated the proliferation capacity of EC 109,TE-1 and TE-13 cells after JQ-1 treated for 2 weeks.Treated with JQ-1 for 24 hours,the apoptosis rates of TE-1 and TE-13 cells were analyzed by Annexin V-FITC/PI double staining,and the expression of apoptosis-related proteins,cleaved caspase 3 and cleaved PARP,was explored by Western blot,to further investigate whether BRD4 could be an underlying therapeutic target in esophageal squamous cell carcinoma.ResultIn BRD4 high expression group,we observed brown and dark brown granules in cellular cytoplasm and nucleus.Among cancer tissues,the percentage of BRD4 high expression was 42.5%(34/80)and it was 20.0%among para-carcinoma tissues(16/80),with significant difference(p=0.002).And BRD4 expression showed significant correlation with the differentiation,lymphatic metastasis and pathological stage of esophageal squamous cancer(p=0.009,p=0.008,p=0.003).In BRD4 high expression group,patients' 1 year cumulative survival rate was 73.5%,3 years cumulative survival rate was 11.8%,and 8.8%in 5 years.While in BRD4 low expression group cumulative survival rates were 78.3%,50.0%and 34.8%respectively,with five-year survival rate significantly higher than that of BRD4 high expression group(P=0.001).In Kaplan-Meier univariate analysis,we found that the level of BRD4 expression,lymphatic metastasis,T stage and pathological stage were prognostic factors of esophageal squamous cell carcinoma.The cell proliferation of esophageal squamous cell carcinoma cells EC 109,TE-1 and TE-13 was inhibited by BIX-01294 in a dose-dependent manner.JQ-1 treated for 24 hours in different concentrations(20,40,80 and 120 ?M),the survival rates in EC 109 were 101.5%± 6.4%,87.2%± 5.2%,42.3%±4.3%,5.6%± 2.2%;96.2%±5.8%,83.7%±4.4%,50.8%±3.2%,12.4%±2.6%in TE-1;105.2%±7.3%,74.8%±5.4%,37.2%±4.2%,7.2%±2.3%in TE-13.In colony forming assay,after JQ-1 treated for 2 weeks in EC109,there were 148.7±35.2 colony forming units in control group,22.5± 15.9,28.7± 12.4 and 19.6±8.7 units in 40,80 and 120 ?M concentration JQ-1 groups,the colony forming in EC109 was significantly inhibited(p<0.05),and similar results seen in TE-13,203.41±52.6 units in control group,27.5± 16.8,28.6±23.0 and 19.3±12.9 units in 40,80 and 120 ?M groups(p<0.05).Annexin V-FITC/PI double staining showed that,after JQ-1(40,80 and 120 ?M)treated for 24 hours,EC019 cell apoptosis rates respectively 8.1%±5.2%,49.7%±6,3%and 99.7%± 1.0%,higher than control group(2.6%±4,5%,p<0.05).And in TE-13,apoptosis rates respectively were 12.8%±7.3%,52.8%±4.9%and 97.3%± 1.8%,higher than control group(5.8%±3.7%,p<0.05).After JQ-1 24 hours treatment(40,80 and 120 ?M),the expression of apoptosis related proteins,cleaved caspase3 and cleaved PARP significantly upregulated with drug concentration increased.ConclusionBRD4 expression was obviously related to the disease progression of esophageal squamous cell carcinoma,and might be employed as an underlying prognostic marker.Endogenous BRD4 inhibition suppressed the cell proliferation of esophageal squamous cell carcinoma by inducing apoptosis,thus,BRD4 could be a therapeutic target for esophageal squamous cell carcinoma.