| Background and objective:Esophageal cancer(EC)is the fourth leading cause of cancer mortality in our country,ranking sixth for incidence rate,and about of 90%of which are esophageal squamous cell carcinoma(ESCC).In China,the distribution of ESCC shows significant regional characteristics,as the majority of the incidence rate occurred in Taihang Mountain,such as Lizhou(Henan),Shexian and Cixian(Hebei).Although the therapeutic strategies have been improved for decades,the efficacy is far from expectation.Therefore,it is of great value to elucidate the pathogenesis,find tumor marker,evaluate target therapy for ESCC,which could benefit to improve prognosis and survival rate of patients.Histone acetylation,one of the epigenetic modifications,has been demonstrated that regulated gene expression by chromatin remodeling.The dynamic equilibrium of histone acetylation is co-regulated by two sets of enzymes-histone deacetylase(HDAC)and histone acetyltransferase(HAT).There have been studies revealed that the dysregulated histone acetylation were more frequent than genomic alterations in ESCC patients,one of which over-expressed HDAC4 promoted ESCC proliferation and indicated poor prognosis.HDAC inhibitor(HDACi)have been detected effective for cancer treatment by targeting dysregulated HDAC and reorganizing the structural of chromatin,resulting in induction of apoptosis,cell cycle arrest,anti-angiogenesis,and differentiation.The selective HDACi LMK-235,targeting HDAC4 and HDAC5,has been detected significant cytotoxicity and lesser side-effects against several cancer cells,including ESCC cells.At present,it remains unclear about the anti-tumor mechanism of LMK-235 in ESCC.Tensin-3(TNS3),one of the tensins family,is characterized by SH2(Src homology 2)domain and phosphotyrosine-binding(PTB)domain at its C-terminus.These structures enable cells to communicating with their outside environment and cellular signaling pathways,leading to various cellular activities,such as cell proliferation,migration,and tumorigenesis.TNS3 have been demonstrated as an oncogene in advanced lung cancer,breast cancer,and melanoma.Super-enhancers(SEs),cis-regulatory elements with large clusters of enhancers,are characterized by a high level of transcriptional factors,cofactors,and epigenetic modifications(H3K27ac and H3K4me1)enrichment.The activation of critical oncogenes driven by SEs are usually expressed in tumor tissues,including ESCC,which indicates that SEs could serve as a hallmark of tumorigenesis.The underlying mechanism of TNS3 and its associated SEs in the treatment with LMK-235 in ESCC,however,have not been fully elucidated.And thus we will focus on this fields from the following four parts,contributing to the progression of the related clinical research,providing new evidence for targeted therapy.Part Ⅰ:Transcriptome analysis of LMK-235 inhibitory effects on ESCC cell proliferationObjectiveTo investigate the effects of LMK-235 on proliferation and transcriptome of ESCC cells.Methods(1)Evaluation the effect of LMK-235 in ESCC cells and it’s IC50 by CCK-8 assay.(2)Detection proliferation,cell cycle,and apoptosis of ESCC cells after treatment by LMK-235 by EdU incorporation and flow cytometry,respectively.(3)Measurement of the expression of histone acetylation(H3K27ac,H3K14ac&H3K9ac),HDAC4,and HDAC5 in ESCC cells treated with LMK-235 by Western Blot.(4)Transcriptome analysis of KYSE150 in response to the treatment of LMK-235.The underlying mechanism of the anti-proliferative effect by LMK-235 was analyzed by GSEA and KEGG pathway enrichment analysis.Results(1)There was a dose-dependent impaired variability of ESCC cells mediated by LMK-235,one of those KYSE150 was detected as the relative sensitive cells(IC50=0.824μm).(2)LMK-235 significantly inhibited proliferation and cell cycle progression,while increased the apoptosis rate of KYSE150.Compared with the vehicle control,EdU positive rate decreased 41.54%(t=-10.51,p=8.94e-03),the proportion of cells in G1 phase was about double(t=8.75,p=1.3e-02),and annexin V positive cells increased by 5.39 times(t=7.12,p=1.9e-02).(3)The histone acetylation protein(H3K27ac,H3K14ac&H3K9ac)expression of ESCC cells were significantly up-regulated by LMK-235,while HDAC4 and HDAC5 were down-regulated.And H3K27ac,one of the most distinctly regulated,increased by 6.38 times(t=11.65,p=2.69e-06)compared with the vehicle control.(4)Transcriptional landscape of KYSE150 was significantly regulated by LMK235,including 752 down-regulated and 981 up-regulated genes.GSEA and KEGG pathways analysis showed that these genes were involved in proliferation related pathways.Summary(1)LMK-235 inhibits ESCC proliferation,blocks Gl/S transition,and induces apoptosis.(2)Transcriptome change mediated by LMK-235 inhibited KYSE150 cell proliferation.Part Ⅱ:Identification of target genes in ESCC treated by LMK-235ObjectiveTo screen the proliferation related target genes by LMK-235,and evaluate the correlations between this gene and the clinical characteristics of ESCC patients.Methods(1)The differentially expressed genes of ESCC were identified based on five microarray data from GEO database,including GSE20347,GSE23400,GSE44021,GSE38129 and GSE75241.(2)The differentially expressed genes of ESCC were intersected with genes significantly regulated by LMK-235,which were used for identifying putative oncogenes and tumor suppressor genes.(3)Small interfering RNA(siRNA)library was employed to detect the candidate genes related with proliferation based on high content screening platform.(4)Detection the expression of TNS3 in ESCC tissues,corresponding para-cancerous tissues,and corresponding normal esophageal mucosa based on GSE161533.(5)Detection the expression of TNS3 in esophageal cancer cells,including ESCC and EAC,treated with the selective HDACi MS-275,AZA,or combined based on GSE57130.(6)Exploration of the correlation between TNS3 expression and ESCC patients’characteristics based on TCGA database.(7)Measurement of TNS3 expression in ESCC cell treated with LMK-235 and SAHA compared with Het-1A cell,respectively.And cell immunofluorescence(IF)was conducted to detect the localization and expression of TNS3 in response to the treatment of LMK-235 in KYSE150 and TE-1.Results(1)1301 differentially expressed genes were identified in ESCC based on the above-mentioned five microarray data,including 634 up-regulated and 667 down-regulated genes.(2)Combined with 1733 genes significantly regulated by LMK-235,61 putative oncogenes and 84 putative tumor suppressor genes were identified from the 1301 differentially expressed genes,respectively.(3)Six proliferation related genes were detected from the candidate genes by siRNA library,one of which TNS3 was the most significantly influenced.The EdU positive rate was decreased by 37.67%conducted by silencing TNS3 compared with the negative control(t=-8.21,p=1.57e-03).(4)The expression of TNS3 was significantly up-regulated in ESCC tissues compared with the related normal esophageal mucosa and para-cancerous tissues(χ2=20.79,p=3.07e-05).(5)The expression of TNS3 was significantly down-regulated by MS-275 or combined with AZA in OE21(χFrideman2=8.20,p=4e-02),while not in OE33(χFrideman2=5.85,p=0.12).(6)The expression of TNS3 was positively correlated with the clinical TNM stage except for stage Ⅳ(p trend=2.79e-03),and exhibited a possible trend to significance(F=2.75,p=0.07)with advanced histopathological grade.(7)The expression of TNS3 was significantly down-regulated by LMK-235 or SAHA in five ESCC cells,including KYSE150,KYSE510,TE-1,TE-7,and EC109,compared with Het-1A.TNS3 mainly localized at the cytoplasm in KYSE150 and TE-1 cells,while their expressions were also down-regulated by LMK-235.Summary(1)TNS3 is highly expressed in ESCC tissues,which is positively correlated with malignant characteristics of patients.(2)LMK-235 inhibited ESCC proliferation by targeting TNS3.Part Ⅲ:The study on the mechanism of TNS3 promotes ESCC proliferationObjectiveTo explore the underlying mechanism of oncogene TNS3 in the regulation of proliferation.Methods(1)The correlations between TNS3 and the proliferation cell nuclear antigen Ki-67(MKI67)were examined based on 33 cancers data from TCGA database,including ESCC and EAC.(2)Detection the expression of TNS3 in ESCC tissues and its correlation with ESCC patients’ characteristics by immunohistochemistry(IHC).(3)Measurement the effect of TNS3 in the proliferation of ESCC cells by EdU incorporation assay.(4)Verification the pro-proliferative effect of TNS3 in vivo by cell line-derived xenograft(CDX)animal experiment.Results(1)TNS3 was positively correlated with MKI67 among 15 cancers,one of which ESCC showed the most relevant(r=0.36,p=7.53e-04),while not in EAC.(2)TNS3 was highly expressed in ESCC tissues(LogeWWilcoxon=8.24, p=1.25e-11),and positively correlated with cTNM stage(Kendall’s τ-b=0.40,p=2.22e-08)and histopathological grade(Kendall’s τ-b=0.42,p=3.14e-09)of ESCC patients.Besides,the higher expression of TNS3 indicated poor prognosis(χ2=7.10,p=7.0e-03)(3)The knockdown efficiency of TNS3 was about 70%at least by two siTNS3(#1),one of which siTNS3(#2)decreased TNS3 expression up to 79.84%(t=-61.59,p=2.69e-12)and 72.20%(t=-44.59,p=3.53e-11)in KYSE150 and TE-1 cells,respectively.(4)Silencing TNS3 distinctly inhibited ESCC cells proliferation.The EdU positive rate of KYSE150 and TE-1 was decreased 41.70%(t=-18.31,p=5.23e-05)and 32.02%(t=-5.59,p=5.0e-03)by siTNS3(#2)compared with NC,respectively.(5)Down-regulation of TNS3 by shTNS3(#2)significantly suppressed ESCC CDX tumor growth in vivo.The weight and volume of tumor were decreased 69.23%(t=-12.83,p=1.38e-05)and 80.60%(days=22;F=28.03,p=9.04e-4)compared with NC,respectively.(6)The protein expressions of TNS3,Ki-67,p-Akt(Ser473),and p-Akt(Thr308)were significantly down-regulated in CDX tumor blocks of shTNS3(#2)by IHC,compared with NC.Summary(1)TNS3 promotes ESCC cell proliferation and indicates poor prognosis,which demonstrates squamous cell carcinoma tissue specificity.(2)The highly expressed TNS3 promotes ESCC proliferation may mediated by activating PI3K/AKT signaling pathways.Part Ⅳ:Epigenetic mechanisms of TNS3-SE involved in the anti-proliferation effect of LMK-235ObjectiveTo illustrate the epigenetic mechanisms of TNS3-SE involved in the anti-proliferation effect of LMK-235 and provide evidence for clinical translation.Methods(1)Identification of TNS3-SE in esophageal carcinoma(ESCC&EAC)by ROSE algorithm based on GEO H3K27ac ChIP-Seq data,including GSE76859,GSE106433,GSE106563,GSE155183,GSE166231,and GSE132680.The enrichment of transcription factor,including p63,BRD4,CTCF,and SOX2,were detected within TNS3-SE.(2)Measurement of the histone acetylation landscape,SEs and its associated genes of KYSE150 treated with LMK-235 by H3K27ac ChIP-Seq.(3)Detection of the chromatin accessibility within TNS-SE and the correlation with TNS3 expression by Pearson coefficient based on TCGA esophageal carcinoma ATAC-Seq data,including 18 ESCC and 12 EAC samples.(4)Identification of the constituent enhancers of TNS3-SE based on the bioinformatics analysis and the narrowPeak data from H3K27ac ChIP-Seq of KYSE150.(5)Identification of the core constituent of TNS3-SE by dual-luciferase reporter assay.(6)Prediction of transcription factors enriched within TNS3-SE based on motif analysis of E3 enhancer and TNS3 promoter from JASPAR database,and H3K27ac ChIP-Seq of KYSE150 by Homer suite(De Novo).(7)Measurement the expression of TNS3 driven by TNS3-SE via CRISPR-Cas9.(8)Detection the effect of TNS3-SE on KYSE150 proliferation by EdU incorporation assay.Results(1)TNS3-SE was identified in ESCC cells,which was enriched by H3K4mel,SOX2,and CTCF,while not it EAC cells.(2)The expression of genes driven by SEs were generally higher and more sensitive than those mediated by TEs and non-enhancers(H=644.92,p<2.22e-16).The activity of TNS3-SE was inhibited by LMK-235 in ESCC cells.(3)TNS3-SE was the accessible chromatin in ESCC compared with EAC.(4)E3 was the core constituent enhancer of TNS3-SE,whose relative luciferase activity was 20.76 times higher than negative control(p=6.09e-10,Tukey HSD).(5)Combined with the results from JASPAR database,six transcription factors were identified enriched within TNS3-SE,including AR,CTCFL,NEUROG2,THAP1,ZNF768,and NR4A1.(6)Compared with the negative control,EdU positive rate of KYSE150 was decreased 45.54%(t=-17.24,p=1.32e-04)by TNS3-SE knock-down mediated by CRISPR-Cas9.Summary(1)TNS3-SE is specific to ESCC.(2)The anti-proliferation effect of LMK-235 is based on TNS3 down-regulation mediated by TNS3-SE inhibition.ConclusionsFirstly,we demonstrated that the proliferation of ESCC cells were significantly inhibited by LMK-235,The transcriptional profiles of KYSE150 was distinctly regulated by LMK-235,followed by GSEA and KEGG analysis.Combined with differentially expressed genes of ESCC based on GEO database,we initially identified 61 putative oncogenes and 84 putative tumor suppressor genes from the 1733 significantly regulated genes by LMK-235.Next,10 candidate genes from those were screened for proliferation based on high content screen platform,one of the most proliferation impaired gene TNS3 was selected.Bioinformatics analysis showed that TNS3 was positively correlated with the malignant phenotype of ESCC patients.The expression of TNS3 was down-regulated by LMK-235 compared with Het-1A.And then TNS3 was detected to indicate poor prognosis of ESCC patients by IHC.Finally,the anti-proliferation effect of LMK-235 is based on TNS3 down-regulation mediated by TNS3-SE inhibition,which was demonstrated by H3K27ac ChIP-Seq.The study illustrated the underlying epigenomic mechanism TNS3-SE involved in the anti-proliferation effect of LMK-235,which could promote the related clinical research,and provide new evidence for clinical translation. |
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