The Function And Mechanism Of BRD4 Regulates RCC2 Transcription In Esophageal Squamous Cell Carcinoma

Background and research purposeEsophageal carcinoma is one of the most common cancers in China,which has two subtypes,esophageal squamous cell carcinoma and esophageal adenocarcinoma.Esophageal squamous cell carcinoma,which has been estimated to result in 508,585 deaths in 2018 worldwide,is the primary subtype of esophageal cancer.The 5-year survival rate for ESCC is less than 20% due to recurrence,acquired drug resistance,and lack of actionable targets.Therefore,investigations focusing upon the elucidation of critical molecular mechanisms governing ESCC tumor progression and the development of effective therapeutic strategies are urgently needed.Genetic mutations are closely related to ESCC carcinogenesis and progression.TP53 has been identified as the most prevalent gene mutation in ESCC,with a mutation frequency of 80.5% in patient tumor tissues.Despite the prevalence of TP53 mutations,targeted therapies have yet to be developed due to technical limitations.The other identified gene mutations are distributed among ESCC tumors at a rate less than 20%and similarly lack targeting drugs.Besides genetic mutations,frequently occurring copy number alterations have also been reported in ESCC.Amplification or mutation of EGFR was found to occur in 19 % of ESCC patients;however,phase III clinical trials testing the efficacy of the EGFR inhibitors gefitinib and panitumumab in the context of ESCC produced marginal results.Epigenomic dysregulation also affects the development of cancer.Epigenetic regulation is accomplished through the concerted effort of histone writers,readers,and erasers.Histone writers and erasers are responsible for adding and removing functional modifiers from histones,respectively,while histone readers typically function as scaffolds that recognize and often recruit additional proteins to modified histones.Importantly,inhibitors targeting epigenetic modulators have been approved for use in treating several cancers.SAHA and FK228,inhibitors targeting histone deacetylase,have been approved as treatments for lymphoma.Azacytidine,an inhibitor that targets DNA methyltransferases(DNMT),has been utilized to treat acute non-lymphocytic leukemia,colon cancer,breast cancer,and melanoma.Similarly,Tazemetostat,an inhibitor target,that targets EZH2,was approved for therapeutic use in patients with advanced epithelioid sarcoma.Thus,targeting chromatin regulators is emerging as a viable anti-cancer strategy.However,the study on the function of chromatin regulators and their inhibitor in ESCC is not clear.The role of BRD4(Bromodomain containing protein 4)in cancer has increasingly come under scrutiny in recent years.BRD4 is a "chromatin reader" protein that plays a critical role in regulating gene expression through mediating interactions between the transcription regulatory machinery at acetylated lysines 5 and 8 of nucleosomal histone H4(H4K5ac/K8ac)interspersed throughout the genome.Specifically,BRD4 is a transcription activator and regulates gene expression through interacting with specific transcription factors.BRD4 has been shown to facilitate the recruitment of P-TEFb to promoters,thereby enhancing RNA polymerase II activity.The primary mechanism of BRD4 in promoting tumor progression is through regulation of c-Myc expression and function.However,the role of BRD4 in cancer is complex as BRD4 has been shown to regulate variable targets across different tissue types.Importantly,BRD4 inhibitors have shown potent anti-proliferative effects in hematological cancers and some solid tumors.In this study,we will investigate the expression and function of BRD4 in ESCC and elucidate the oncogenic mechanism for BRD4 in the context of ESCC.We explored whether BRD4,a chromatin reader protein,could be a promising anti-cancer target against ESCC.Methods(1)Investigate the expression and function of BRD4 in ESCCTo investigate the importance of BRD4 in esophageal cancer,BRD4 expression were analyzed using two ESCC microarray datasets(GSE44021,GSE23400),which downloaded from the GEO(Gene Expression Omnibus).Both microarray datasets compare m RNA expression patient-derived ESCC tumor tissues to paired adjacent esophageal tissue.Next,the expression levels of BRD4 transcripts were analyzed in patient-derived samples based upon RNA-seq data provided by the TCGA database.To further verify BRD4 expression in the context of ESCC,BRD4 protein expression levels were assessed in ESCC tissues by immunohistochemical(IHC)staining using 3normal esophageal tissue samples,13 adjacent tissue samples,and 16 ESCC tumor tissue samples.Moreover,to estimate the frequency of BRD4 up-regulation in ESCC,our analysis was extended to include an additional 81 paired ESCC tumor and adjacent tissues.Next BRD4 protein expression levels were determined in a normal human immortalized esophageal epithelial cell line(Shantou human embryonic esophageal cell line,SHEE)and several human ESCC cell lines by Western blot.MTT and soft agar colony formation assays were used to assess the extent that cell proliferation and colony formation are affected by BRD4 expression.To determine whether BRD4 could contribute to ESCC tumor growth in vivo,KYSE450 or KYSE510 cells depleted of BRD4 by knockdown were subcutaneously injected into nude mice.(2)Detect potential targets regulated by BRD4To further assess the oncogenic contribution of BRD4 in the context of ESCC,RNA-sequencing was performed using transcripts derived from KYSE450 cells depleted of BRD4 by either knockdown or inhibitors OTX015 and(+)-JQ1 treatment.To identify genes regulated at the protein expression levels by BRD4,proteomic profiling was performed using lysates derived from KYSE450 cells depleted of BRD4 by either knockdown or OTX015 and(+)-JQ1 inhibitor treatment.To detect potential targets regulated by BRD4 binding,our data was compared with previously published BRD4 Ch IP-Seq results.q PCR and Western blot were performed to assess the correlation between BRD4 and RCC2 expression at the protein levels.Next,the Ch IP assay was conducted to determine if BRD4 protein is distributed within the RCC2 promoter.(3)Uncover a potential regulatory mechanism facilitated by BRD4 upon RCC2To uncover a potential regulatory mechanism facilitated by BRD4 upon RCC2 promoter binding,the RCC2 promoter sequence was queried against known transcription factor consensus sequences,provided by the JASPAR database.To validate the in silico prediction provided by JASPAR,chromatin immunoprecipitationpolymerase chain reaction was performed in KYSE450 cells.We hypothesized that BRD4 may bind and was recruited by TP73 to the RCC2 promoter region and thereby modulate RCC2 expression.To test this hypothesis,a Co-IP assay was performed against BRD4 and TP73.To validate whether TP73-BRD4 interact facilitated the binding of BRD4 to the promoter region of RCC2,BRD4 protein expression levels were checked by western blot and occupation of BRD4 within the RCC2 promoter by Ch IP-PCR experiments upon TP73 depletion.TP73 protein expression levels were checked by western blot and occupation of TP73 within the RCC2 promoter by Ch IPPCR experiments upon BRD4 depletion.To determine whether TP73 facilitates RCC2 expression,TP73 was depleted in KYSE450 cells by knockdown and subsequently determined RCC2 expression by Western blot.To confirm whether BRD4 and TP73 regulate RCC2 promoter activity,the RCC2 promoter sequences were cloned into a luciferase vector and transfected in HEK293 T and KYSE450 cells.(4)Investigate the expression and function of RCC2 in ESCCTo investigate RCC2 expression in esophageal cancer,an ESCC microarray dataset(GSE23400)was downloaded from the Gene Expression Omnibus.Additionally,RCC2 transcript expression was analyzed in the TCGA-ESCA patient cohort.Additionally,RCC2 transcript expression was analyzed in the TCGA-ESCA patient cohort.To validate the results obtained from the GEO and TCGA datasets,proteins were extracted from 6 paired tumor and non-tumor ESCC patient tissues and RCC2 protein expression levels were measured by Western blot.To clarify if BRD4 induces cell proliferation via over-expression of RCC2,MTT and soft agar colony formation assays were performed using KYSE450 and KYSE510 cells depleted of RCC2 by knockdown.To evaluate the role of RCC2 on tumor growth in vivo,cellderived xenografts were established in nude mice using KYSE450 and KYSE510 cells depleted of RCC2 by knockdown.To determine whether BRD4 promotes ESCC proliferation by upregulation of RCC2,rescue experiments were performed in ESCC cell lines.As expected,exogenous FLAG-RCC2 expression in BRD4-ablated ESCC cells significantly attenuated the growth inhibition observed upon knockdown of BRD4.(5)Uncover the effect and mechanism of BRD4 inhibition attenuates tumor growth in the ESCC patient-derived xenograft(PDX)To evaluate the therapeutic potential of targeting BRD4 in vivo,BRD4 protein expression levels were detected in several ESCC PDX model cases.Next the BRD4 inhibitors,(+)-JQ1 and OTX015,were administered to assess their effects on ESCC tumor growth.Immunoblotting indicated whether BRD4 inhibitor-treated tumors also displayed lower RCC2 expression.Results(1)BRD4 is up-regulated in ESCCBRD4 exhibited increased expression in ESCC tumors compared with the paired adjacent tissues in two ESCC microarray datasets downloaded from the Gene Expression Omnibus(GSE44021,GSE23400).BRD4 transcript levels were significantly highly expressed in esophageal adenocarcinoma(EAC)and ESCC tissues compared with adjacent tissues in TCGA database.The IHC analysis confirmed that BRD4 protein expression levels were increased in the tumor tissue relative to normal and adjacent tissues.A general trend increased of BRD4 expression in the ESCC cell lines compared to the SHEE control.(2)Genetic and pharmacological inhibition of BRD4 suppress ESCC growth in vitro and in vivoThe cell growth and colony formation potential were significantly reduced in KYSE450 and KYSE510 cells with stable expression of BRD4 sh RNA or after BRD4 inhibitor treatment.The tumors generated from cells depleted of BRD4 were significantly smaller compared to the sh Scramble controls cells in the xenograft mouse model.(3)BRD4 up-regulates RCC2 expression31 genes were regulated(12 up and 19 down)at both the transcript and protein expression levels after BRD4 depletion by knockdown or inhibitor treatment.Compared with previously published BRD4 Ch IP-Seq results,11 genes(3 up-regulated and 8 down-regulated)of 31 differentially expressed genes identified in our combined transcriptomic and proteomic dataset were potentially regulated by BRD4.Among those genes,RCC2 was found to be potentially regulated by BRD4.RCC2 is expressed at reduced levels in KYSE450 and KYSE510 cells depleted of BRD4 by either knockdown or OTX015 and(+)-JQ1 inhibitor treatment.The Ch IP results show that BRD4 occupancy was enriched within the RCC2 promoter.(4)TP73-BRD4 interact facilitates the binding of BRD4 to the promoter region of RCC2TP73 binding sites located within the RCC2 promoter region.The results of the Ch IP-PCR experiments confirmed RCC2 promoter occupancy by BRD4 and TP73.TP73 was found to be associated with BRD4.TP73 depletion didn’t reduce BRD4 protein expression levels in KYSE450 and KYSE510 cells.But TP73 knockdown decreased the occupation of BRD4 within the RCC2 promoter region.BRD4 knockdown or inhibition also decreased the occupation of TP73 within the RCC2 promoter region.(5)BRD4 and TP73 regulates promoter activity of RCC2BRD4 and TP73 regulate RCC2 promoter activity.TP73-BRD4 interact facilitated the binding of BRD4 to the promoter region of RCC2 and thereby modulate RCC2 expression.(6)RCC2 is up-regulated in ESCCIncreased RCC2 expression in ESCC tumors compared to the paired adjacent tissues in the Gene Expression Omnibus(GSE23400).RCC2 transcript expression is highly expressed in ESCA and ESCC tissues compared to normal tissues in TCGAESCA patient-cohort.Western blotting analysis showed that RCC2 protein expression levels was increased in ESCC patient tumor tissue relative to matched adjacent tissue.(7)Inhibition of RCC2 suppresses ESCC growth in vitro and in vivoCell proliferation and colony formation were significantly inhibited upon stable knockdown of RCC2 in KYSE450 and KYSE510 cells using an MTT assay.A decreased tumor growth in the RCC2 knockdown cell-derived xenograft mouse model compared to the control.(8)BRD4 promotes ESCC proliferation by upregulation of RCC2Exogenous FLAG-RCC2 expression in BRD4-ablated ESCC cells significantly attenuated the growth inhibition observed upon knockdown of BRD4.(9)BRD4 inhibition attenuates tumor growth in the ESCC patient-derived xenograft(PDX)and down-regulates RCC2 in vivoBRD4 inhibitors,(+)-JQ1 and OTX015,to assess their effects on ESCC tumor growth.Moreover,we observed greater growth inhibition in tumors expressing higher levels of BRD4 upon treatment with(+)-JQ1 or OTX015.Immunoblotting indicated the BRD4 inhibitor-treated tumors also displayed lower RCC2 expression.ConclusionsIncreased BRD4 expression facilitates ESCC cell proliferation and tumor growth.TP73-BRD4 interact facilitates the binding of BRD4 to the promoter region of RCC2,thereby regulating RCC2 expression.Inhibition of BRD4 effectively suppresses ESCC proliferation in vitro as well as in vivo in PDX models.BRD4 can be a promising anticancer target against ESCC.