The Studies Of The Anti-HCC Effect And Underlying Molecular Mechanism By Targeting BRD4

Hepatocellular carcinoma(HCC)is one of the most aggressive malignant tumors,and it is also one of the most common malignant tumors in China.It has a high mortality rate and a poor prognosis.At present,the effective treatments for HCC include liver transplantation,surgical resection and local ablation,all of which can significantly improve the survival of patients with early HCC.However,these treatment methods are only suitable for a very small proportion of early HCC patients,and most patients are already in advanced stage at diagnosis and often require chemotherapy.Traditional chemotherapy is frequently ineffective in patients with late stage of HCC.Therefore,it is necessary to explore new treatment methods for HCC.The BET(Bromodomain and extra-terminal)proteins are epigenetic readers of acetylated histones,and play an important role in the transcription of genes that involve in cell cycle regulation,cell proliferation,and apoptosis.Bromodomaincontaining Protein 4(BRD4)is a member of BET family proteins.BRD4 has important epigenetic regulatory functions,and is the most widely studied member of the BET family of proteins in the cancer research field.Targeting BET proteins using small molecule BET inhibitors has become a novel cancer treatment strategy.Some studies have shown that BRD4 is highly expressed in a variety of tumors.As a transcriptional coactivator,the high expression of BRD4 promotes the abnormal expression of a variety of tumor-associated genes.The tumor-associated genesinvolved in BRD4 include: cell cycle regulatory genes such as cMyc,CDKN1A(p21)and CDKN1B(p27);apoptosis-regulating genes such as Myeloid Cell Leukemia Sequence 1(Mcl-1),B-cell lymphoma-extra large(Bcl-xL)and Bcl-2.Studies have also shown that the high expression of BRD4 is an important mediator of various tumorigenesis,and is closely related to the formation of a series of malignant biological behaviors such as non-controlled proliferation,apoptosis resistance,invasion and metastasis of some leukemia and solid tumors..Therefore,BRD4 plays an important role in gene transcription of cell cycle regulation and apoptosis,tumor-targeted therapeutic research by inhibiting the expression or function of BRD4 has drawn increasing attention in the field of anti-tumor research.Measures to inhibit the expression or function of BRD4 mainly include: genetic methods such as small RNA interference,gene knockout;small molecule inhibitors such as JQ1,OTX015 and HJB-97.A series of previous studies have shown that in the acute myeloid leukemia(AML),breast cancer,prostate cancer,colon cancer,osteosarcoma and other malignant tumors,inhibition of BRD4 can induce significant anticancer effect in vivo and in vitro experiments.Related compounds such as the small molecule inhibitor JQ1 have entered the clinical research stage of human cancer treatment and have shown certain anticancer effects.These evidences suggest that targeted therapies for BRD4 have a good anti-cancer prospect.In conclusion,BRD4 is an epigenetic regulator that has been found to be associated with up-regulation of multiple carcinogenic drivers in tumors.Targeting BRD4 with drug inhibitors such as JQ1 has become a novel approach to tumor therapy.However,whether targeting BRD4 has anticancer potential in HCC has not been studied in detail.To address this important issue,this study intends to investigate the expression of BRD4 in HCC and the biological activity by targeting BRD4 in HCC from the following four aspects.Part 1: The expression and significance of BRD4 in HCC.Part 2: The study of the anticancer effect and underlying molecular mechanism of small molecule BRD4 inhibitor JQ1 in HCC.Part 3: Upregulation of Mcl-1 expression inhibits JQ1-triggered anticanceractivity in HCC cells.Part 4: Oridonin synergistically enhances JQ1-triggered apoptosis in HCC cells through mitochondrial pathway.Part 1 The expression and significance of BRD4 in HCC ObjectiveTo study the expression of BRD4 in HCC cell lines,and the role of BRD4 in HCC cell proliferation.To study the expression of BRD4 in clinical HCC tissues,adjacent non-tumor liver tissues,normal tissues,and its relationship with clinicopathological features,and explore the clinical significance of BRD4.MethodsSeven human HCC cell lines,such as Hep3 B,HCCLM3,HuH7,HepG2,SMMC7721,BEL7402 and MHCC97 H,and normal liver cell line LO2 were used for the study.Western blotting was used to analyze the expression of BRD4 in HCC cells and LO2 cells;siRNA inhibited the expression of BRD4 in HCC cells and detected the effect on the proliferation of HCC cells.Tumor tissues and corresponding adjacent non-tumor liver tissues of 72 HCC patients were collected,and liver tissues from three healthy normal humans were used as controls.The expression levels of BRD4 in HCC tissues,adjacent non-tumor liver tissues and normal liver tissues were examined by tissue microarray,immunohistochemistry(IHC),western blotting and trypan blue staining test.The clinical and pathological data were used for statistical analysis.Results1.BRD4 is highly expressed in HCC cell lines.We first examined the expression of BRD4 in normal liver cell line LO2 and seven human HCC cell lines,such as Hep3 B,HCCLM3,HuH7,HepG2,SMMC7721,BEL7402 and MHCC97 H.Western blotting showed that BRD4 was highly expressed in all HCC cell lines compared to normal liver cell line LO2.2.Knockdown of BRD4 by siRNA transfection significantly inhibits the growth of HCC cells.To investigate the role of BRD4 in HCC cell proliferation,we used two different siRNAs to knock down BRD4 expression in Hep3 B and HCCLM3 cells.Western blotting was used to determine the efficacy of transfection,and trypan blue test was used to detect the number of transfected cells on the 4th day after transfection.It was found that knockdown of BRD4 significantly inhibited the growth of HCC cells compared with the siCON group(P < 0.01).These results suggested that BRD4 played an important role in the proliferation of HCC cells.3.BRD4 is highly expressed in HCC tumor tissues.The expression of BRD4 was detected by IHC staining in tumor tissues of 72 HCC patients and corresponding tissue cores adjacent to non-tumor liver tissues,and liver tissues from three healthy normal humans were used as controls.IHC results showed that BRD4 was mainly expressed in the nucleus,and the expression of BRD4 in HCC tissues was significantly higher than that in non-tumor liver tissues(P<0.01).These results demonstrated that BRD4 was highly expressed in HCC tissues.Western blotting analysis of 5 pairs of HCC tissues and corresponding adjacent non-tumor liver tissues further confirmed that the expression of BRD4 in tumor tissues was significantly higher than that in adjacent non-tumor liver tissues.4.The clinical significance of BRD4 expression in HCC tissues.IHC staining intensity analysis showed that the average scores of AJCC stage I,II,III and IV were 1.7±0.2,2.1±0.1,2.5±0.1 and 2.6±0.2,respectively.The expression of BRD4 in HCC tumor tissues and AJCC cancer stage was positively correlated(P<0.05).We further determined the relationship between the expression of BRD4 in HCC tissues and the age,sex,serum AFP,lymph node metastasis,AJCC staging and vascular invasion.After statistical analysis of clinical data of 72 patients,we found that the expression of BRD4 was not associated with age,gender,AFP and lymph node metastasis,but was significantly associated with AJCC staging and vascular invasion(P < 0.01).Further analysis of clinical data revealed that the survival rate of patients with higher BRD4 expression was significantly lower than those with lower BRD4 expression(P < 0.05).These results indicated that BRD4 expression was highly up-regulated in HCC tissues and increased further as the disease progressed.Conclusions1.BRD4 is highly expressed in HCC cells,and significantly promotes the proliferation of HCC cells.2.The expression level of BRD4 in tumor tissues is significantly higher than that in adjacent non-tumor liver tissues,which is positively correlated with AJCC staging and vascular invasion of HCC.Part 2 The study of the anticancer effect and underlying molecular mechanism of small molecule BRD4 inhibitor JQ1 in HCC ObjectiveTo explore anticancer potential of small molecule BRD4 inhibitor JQ1 in HCC using in vitro and in vivo models,and to elucidate the underlying molecular mechanism of small molecule BRD4 inhibitor JQ1 in HCC.MethodsSeven HCC cell lines,including Hep3 B,HCCLM3,HuH7,HepG2,SMMC7721,BEL7402 and MHCC97 H,were used for this study.Immunohistochemistry,Quantitative Real-time Polymerase Chain Reaction(qRT-PCR),siRNA transfection,CCK8,clone formation,primary culture,chromatin immunoprecipitation(ChIP),flow cytometry,western blotting,tumor-bearing experiments in nude mice and second-generation transcriptome sequencing techniques were also used.Results1.The small molecule BRD4 inhibitor JQ1 dramatically inhibits the proliferation of human HCC cells.In the first part of the experiment,our experimental results found that BRD4 knockdown by siRNA inhibited the growth of HCC cells.We next investigated theanti-HCC activity of targeting BRD4 by small molecule JQ1 in 7 HCC cell lines including Hep3 B,HCCLM3,HuH7,HepG2,SMMC7721,BEL7402 and MHCC97 H.CCK8 assay showed that JQ1 inhibited cell growth in a dose-dependent manner in 7HCC cell lines,Hep3 B and HCCLM3 were the most sensitive cell lines with IC50 values of 0.08 ?M and 0.14 ?M,respectively.The results of colony formation assay showed that treatment of Hep3 B and HCCLM3 cell lines by JQ1 for 14 days inhibited the formation of HCC cells in a dose-dependent manner.In the primary culture of freshly isolated primary HCC cells from surgically resected tumor tissues,the CCK8 assay also showed that JQ1 treatment for 5 days inhibited the growth of primary HCC cells.2.JQ1 induces cell cycle arrest and triggers apoptosis in HCC cells.We used flow cytometry to analyze cell cycle distribution and found that JQ1 treatment for 48 hours resulted in an increase in the percentage of HCC cells in G1 phase and a decrease in the percentage of S phase;western blotting revealed that caspase-3 and caspase-9 were activated and PARP was cleaved after JQ1 treatment.JQ1 treatment also induced the release of cytochrome C from the mitochondria into the cytoplasm.These results indicated that JQ1 activated apoptotic signals.3.The mitochondrial apoptotic pathway mediates JQ1-induced apoptosis in HCC cells.In order to determine whether caspase-9 is required for JQ1-induced anticancer activity,we pretreated with caspase-9 inhibitor Z-LEHD-FMK for 1 h,and examined cell death by trypan blue staining.The results showed that in Hep3 B and HCCLM3 cells,the killing effect of JQ1 in Z-LEHD.fmk pretreatment group was significantly weaker than that in un-pretreated group(P < 0.01).These results suggested that caspase-9-dependent mitochondrial apoptotic pathway mediated JQ1-induced anti-HCC cell activity.4.JQ1 inhibits the expression of cMyc in HCC cells.qRT-PCR and western blotting showed that JQ1 inhibited the expression of cMyc in a time-dependent manner.ChIP assay showed that BRD4 was enriched in MYC enhancer in both HCC cell lines.These results indicated that JQ1 inhibited the transcription of cMyc by reducing the binding of BRD4 to the cMyc enhancer.Western blotting further confirmed that JQ1 reduced the protein expression level of cMyc in time-and concentration-dependent manners in Hep3 B and HCCLM3.We also confirmed by RNA-Seq analysis that JQ1 down-regulated the expression of cMyc at the mRNA level.These results indicated that cMyc was a key mediator of JQ1-triggered anticancer effect.Western blotting results also found that JQ1 treatment led to increased expression of p27 in Hep3 B and HCCLM3 HCC cell lines,but had a strong inhibitory effect on p21 expression.This suggested that p27 might be a key downstream mediator of cMyc-induced cell proliferation inhibition.5.In the HCC cell line,inhibition of cMyc promoted JQ1-mediated anticancer activity.We used specific siRNA to inhibit the expression of cMyc in Hep3 B and HCCLM3 cells.Western blotting showed that siRNA inhibited cMyc and achieved good transfection.It also up-regulated the expression of Bim and P27 in Hep3 B and HCCLM3 cell lines.CCK8 test results showed that cMyc knockdown inhibited the growth of HCC cells(P<0.01);however,the inhibition of cells by siCON group and siMYC group was significantly weaker than that induced by siCON+JQ1 group(P<0.01).These results indicated that inhibition of cMyc only had a partial role in JQ1-mediated anticancer activity;JQ1 might have other functions besides inhibiting cMyc.6.JQ1 can also induce up-regulation of BIM expression in HCC cells.RNA-Seq analysis found that the expression of cMyc gene was inhibited by JQ1 in HCCLM3 cells,and the expression of proapoptotic gene BCL2L11(BIM)was up-regulated;which was further verified by siRNA,qRT-PCR and western blotting.The trypan blue staining test further found that BIM knockdown significantly inhibited JQ1-induced cell death in HCC cell lines by more than 50%(P < 0.05);Western blotting results showed that JQ1-induced caspase-3 activation and PARP cleavage was attenuated after BIM knockdown.These results indicated that the upregulation of BIM expression played an important role in JQ1-triggered apoptosis in HCC cells.7.JQ1 inhibits tumor growth in the HCC xenograft model.By establishing an HCC xenograft model of HCCLM3 and Hep3 B,we found that the mean tumor volume in the JQ1 treatment group was smaller than the control group after treatment for 14 day(P < 0.05);western blotting analysis revealed that JQ1 treatment resulted in BIM accumulation,PARP cleavage and caspase-3activation,indicating the activation of apoptotic signaling in tumor tissues;IHC detection showed that the expression of cMyc and Ki67 in HCC tumor tissues was also inhibited by JQ1 treatment.These data indicated that JQ1 inhibits the expression of cell cycle regulators,activated the apoptotic signals,and promoted the antitumor activity in vivo.Conclusions1.Small molecule inhibitor JQ1 targeting BRD4 can inhibit the proliferation,and induce apoptosis in HCC cells and in HCC xenograft tumors.2.Inhibiting cMyc and increasing the expression of Bim can play important roles in JQ1-mediated anti-HCC activity.Part 3 Upregulation of Mcl-1 expression inhibits JQ1-triggered anticancer activity in HCC cells.ObjectiveBy analyzing the results of previous studies,we found that the expression of Bcl-2 family anti-apoptotic protein Mcl-1 was up-regulated after JQ1 treatment in HCC cells,and Mcl-1 might be a key molecule of HCC-resistant to JQ1.This part explored the negative role of Mcl-1 in JQ1-triggered anti-HCC activity.MethodsHCCLM3,BEL7402,MHCC-97 H,HepG2 and Huh7 cell lines were used in this study.RNA-Seq,siRNA transfection,flow cytometry,trypan blue assay,qRT-PCR and western blotting analysis were used to analyze the expression of Mcl-1 in JQ1 treated HCCLM3 and BEL7402 cell lines.The role of Mcl-1 in JQ1-mediated anti-HCC activity was investigated by RNA interference and flavopiridol,a CDKinhibitor and also a well-known Mcl-1 inhibitor.Results1.JQ1 induces up-regulation of Mcl-1 expression at mRNA levels in HCC cells.We analyzed the effect of JQ1 on the expression of Bcl-2 family members by RNA-Seq.The results showed that Bcl-xl was down-regulated,and Bim expression was up-regulated after 4 hours of JQ1 treatment.However,we also observed an increase in the expression level of the anti-apoptotic protein Mcl-1 in the HCCLM3 and BEL7402 cell lines after JQ1 treatment.The results of qRT-PCR further showed that the expression of Mcl-1 mRNA in the treatment group was significantly higher than that in the control group in HCCLM3 and BEL7402 cell lines(P<0.01).2.JQ1 induces up-regulation of Mcl-1 expression at the protein level in HCC cells.We further investigated the effect of JQ1 on the expression of Mcl-1 protein in HCC cells by western blotting analysis.The results showed that JQ1 increased the expression level of Mcl-1 in dose-and time-dependent manners in HCCLM3 and BEL7402 cell lines.We also validated this result in MHCC97 H,HepG2 and Huh7 cells.Our results suggested that up-regulation of Mcl-1 triggered by JQ1 might be widespread in HCC cells.3.Knockdown of Mcl-1 can enhance JQ1-mediated apoptosis of HCC cells.We first used two different siRNAs targeting different Mcl-1 gene sequences,to knock down Mcl-1 expression in HCCLM3 and BEL7402 cell lines.Western blotting analysis showed that siMcl-1 transfection significantly inhibited Mcl-1 expression,and also was very effective in inhibition of JQ1-induced upregulation of Mcl-1.We further treated cells transfected cells with 0.5 ?M JQ1.Western blotting revealed that JQ1 induced caspase-3 activation and PARP cleavage.Notably,the same concentration of JQ1 treatment resulted in almost compete cleavage of full-length PARP ion Mcl-1 knockdown HCCLM3 cells,whereas it only slightly reduced the level of full-length PARP in the control cells.The apoptosis rate of HCCLM3 cells with Mcl-1 knockdown treated by JQ1 was(64.7±5.2)% and(70.7±5.5)%,respectively,which were significantly higher than control cells(26.7±1.2)%(P < 0.05).BEL7402,MHCC97 H,HepG2 and Huh7pre-transfected with siRNA were treated with JQ1.Trypan blue staining showed that when Mcl-1 was knocked down in all four HCC cell lines,JQ1 elicited more death cells(P < 0.05);meanwhile,western blotting showed that JQ1 had stronger PARP cleavage activity in HCC cells transfected with siMcl-1.These results indicated that JQ1 activated a stronger apoptotic signal when Mcl-1 was inhibited in HCC cells.4.Flavopiridol enhances JQ1-triggered apoptotic signaling and cell death induction by inhibiting Mcl-1 in HCC cells.Flavopiridol is an FDA-approved CDK inhibitor for the treatment of human cancers,and has the effect of inhibiting Mcl-1 expression in tumor cells.To further verify the above results obtained by RNA interference,we treated the HCCLM3 and BEL7402 cell lines by JQ1(0.5 ?M),flavopiridol(0.5 ?M)or both for 48 hours.Trypan blue staining showed that a large number of cell deaths occurred in JQ1 combined with Flavopiridol treatment(60-80%),which was significantly higher than that in the control group and the monotherapy groups(P < 0.01).Western blotting analysis showed that 0.5?M flavopiridol significantly reduced the basal level of Mcl-1,and also inhibited the up-regulation of Mcl-1 to a large extent in HCC cells;the results also showed that the combination of JQ1 and flavopiridol induced a large amount of caspase-3 activation and PARP cleavage.These results indicated that flavopiridol enhanced JQ1-induced apoptosis by inhibiting Mcl-1 expression in HCC cells.Conclusions1.Up-regulation of Mcl-1 expression inhibits the anti-HCC effect mediated by the small molecule BRD4 inhibitor JQ1.2.Inhibition of Mcl-1 significantly enhances JQ1-triggered apoptosis in HCC cells.3.The combination of JQ1 and Mcl-1 inhibitors improves the treatment of HCC and provides a direction for the combined treatment of HCC.Part 4 Oridonin synergistically enhances JQ1-triggered apoptosis inHCC cells through mitochondrial pathway ObjectiveMany solid tumors are inherently resistant to BET protein inhibitors.Our previous studies found that HCC cells have some resistance to BRD4 inhibitor JQ1.In order to explore new methods for improving HCC treatment by BRD4 inhibitors,we studied the combined action of JQ1 and Oridonin in HCC cells.MethodsHCCLM3,BEL7402,MHCC97 H and SMMC7721 cell lines were used in this study,CCK8 assay,flow cytometry,trypan blue assay,cell fractionation,western blotting analysis and other methods were used in our study.We explored the mechanism of the apoptosis of HCC cells induced by oridonin combined with JQ1,and its effect on the spheroid formation activity of HCC.Results1.Oridonin inhibits Bcl-2,Mcl-1 and xIAP expression in HCC cells.Firstly,we examined the inhibitory effect of Oridonin alone on the cell viability in a panel of 4 HCC cell lines that include HCCLM3,BEL7402,MHCC-97 H and SMMC7721 cell lines,CCK8 assay showed that treatment with Oridonin at 2.5-10?M for 24 h partially inhibited cell viability in these HCC cell lines.We then treated HCC HCCLM3,BEL7402,MHCC-97 H and SMMC7721 cell lines with this concentration-range for 24 h,western blotting showed that Oridonin profoundly and dose-dependently reduced protein level of Bcl-2 and Mcl-1,as well as xIAP in all 4HCC cell lines.In contrast,Oridonin had minimal effect on the levels of Bak and cIAP-1/2.These results suggested that Oridonin effectively inhibited the expression of multiple anti-apoptosis proteins in HCC cells.2.Oridonin enhances JQ1-mediated apoptosis in HCC cells.We treated HCC cell lines with Oridonin alone,JQ1 alone or their combination for 48 h,and then stained with Annexin V-FITC and propidium iodide(PI).Wefound that in all 4 HCC cell lines,treatment with Oridonin alone at 5 ?M did not dramatically increase the percentage of Annexin V-FITC(+)cell subpopulation,and treatment with JQ1 alone at 1 ?M increased the percentage of Annexin V-FITC(+)cell subpopulation by 26.2% and 16.5%,respectively,in HCCLM3 and BEL7402 cell lines.In striking contrast,their combination treatment resulted in 51.1% and 49.7%cells positively stained with Annexin V-FITC,respectively,in the two cell lines.These results suggested that Oridonin strongly enhanced JQ1-triggered apoptosis in HCC cells(P<0.01).3.Oridonin enhances JQ1-triggered apoptotic signaling in HCC cells.We examined the cell viability of HCCLM3,BEL7402,MHCC-97 H and SMMC7721 cell lines by trypan blue staining assay.The results showed that in four HCC cell lines,Oridonin combined with JQ1 significantly inhibited the viability of HCC cells(P < 0.01).We next examined the activation of apoptosis signaling in 4HCC cell lines treated by Oridonin alone,JQ1 alone or their combination.Western blotting analysis showed that treatment with 5 ?M Oridonin for 48 h did not trigger caspase-9 activation and PARP cleavage.Treatment with 1 ?M JQ1 for 48 h induced accumulation of cleaved caspases-9 and caspases-3.Consistently,JQ1 treatment resulted in modest cleavage of PARP accompanied by reduction of full-length of PARP level.In striking contrast,the combination not only induced a large amount of accumulation of cleaved caspases-9,caspases-3 and cleaved PARP,but also almost completely eliminated the level of full-length PARP.These results indicated that Oridonin enhanced JQ1-triggered apoptotic signaling in HCC cells.4.Mitochondrial apoptosis pathway mediates apoptosis by the combination of Oridonin with JQ1 in HCC cells.We treated 4 HCC cell lines with DMSO,Oridonin alone,JQ1 or both for 48 h,followed by cell fractionation.Western blotting showed that the combination treatment caused damage of mitochondrial membrane integrity,which led to proapoptogenic cytochrome c released into cytoplasm in HCC cells.To further investigate the role of mitochondrial pathway in the combination,we pretreated the cells with caspase-9 inhibitor Z-LEHD-FMK or caspase-8 inhibitor Z-IETD-FMK,followed by treatment of the combination.The trypan blue staining test showed thatthe Oridonin and JQ1 combination-induced cell death significantly was inhibited when the mitochondrial pathway initiator caspase-9 was suppressed as compared with the control group(P < 0.01).However,inhibition of caspase-8,the initiator caspase for death-receptor pathway had no significant effect on the activity of the combination.These experimental results indicated that the mitochondrial pathway was required for the induction of apoptosis.5.Oridonin and JQ1 acts synergistically in inhibition of cell viability in HCC cell lines.We treated the HCC cells with Oridonin alone,JQ1 alone or their combination for 5 days.CCK8 assays showed that either JQ1 or Oridonin alone dose-dependently inhibited cell viability,and achieved IC50 of 1.8,4.2 ?M,respectively,in HCCLM3 cell line.In striking contrast,their combination had IC50 of 0.17 ?M,reducing by10.6 and 24.7 folds as compared to the IC50 values of either JQ1 or Oridonin alone.Similarly,the two agents also had strong combination activity in BEL7402,SMMC7721 and MHCC-97 H cell lines.Combinational index values are 0.09,0.39,0.46,0.54 ?M for HCCLM3,MHCC-97 H,BEL7402,SMMC7721 cell lines,respectively.These results suggested that the two agents had synergistic effect in all these four HCC cell lines.Conclusions1.Oridonin can enhance the sensitivity of small molecule BRD4 inhibitor JQ1 in HCC cells.2.Oridonin synergistically enhances JQ1-triggered apoptosis in HCC cells through the mitochondrial pathway.