Targeting BRD4 And Relative Mechanism On Invasion And Chemo-resistance Of Gastric Cancer

Gastric cancer(GC),as a malignant neoplasm with high incidence whose incidence has declined in the past 10 years with the popularization of helicobacter pylori standard radical surgery.However,GC is still the most common gastrointestinal tumor with the poor prognosis in China.While targeted therapy and immunol drugs has prevalently applicated in clinic,the five-year survival rates for GC patients have indeed climbed in recent years.Moreover,patients with distant metastasis at the time of diagnosis and drug resistance after treatment still lack of efficient therapy which causes the tumor progress and results in death ultimately.Due to the insidious onset of gastric cancer and the absence of specific symptoms in the early stage,some patients had been accompanied with distant metastasis at the time of clinical diagnosis,so metastasis is viewed as the first lethal factor for GC.Accounting for this characteristic,more accurate and efficient therapies are required to target intricated alteration of tumor phenotypes.Chemotherapy is one kind of auxiliary therapy to treat with GC patients as its substantial efficacy in clinical usage.However,resistance is prevalently existence which notes us that novel targeted therapy to sensitize GC cells which is combining with traditional therapy is worth exploring.The dynamic regulation of epigenetic modification is widespread and participates in a variety of tumor biology behaviors.So epigenetic-targeted therapy is one of the neoteric anti-tumor treatment to solve the problem of GC metastasis and drug resistance and improve the long-term survival rate of GC patients.Bromodomain and extra-terminal protein family(BETs)has been well known for its part in identifying acetylated histone like acetyl histone H3(K27)and non-histone proteins such as snail.BRD4,a member of BETs,plays as epigenomic reader which could be enriched and recruit mediator complex and Pol Ⅱ among active enhancers and super enhancers through its docking site-acetylated H3(K27),which result in transcriptional activation of specific genes.Our previous research found that aberrant expression of BRD4 identified acetylated snail which is related to epithelial-mesenchymal transition(EMT)and suppressed its ubiquitylated degradation,sustained its protein level and promoted GC metastasis ultimately.We also demonstrated that specific BRD4 inhibitor JQ1 restrained invasion and migration of GC cells by hindering the binding between BRD4 and snail which resulted in the degradation of snail.But little we know about the outcome when JQ1 treated in metastasis-associated mouse model.So we aim to dig out the conversion point to clinic whether targeting BRD4-snail axis can prevent GC patients from metastasis.Moreover,it’s still unclear whether BRD4 plays a part in regulating GC chemo-resistance and the potential mechanism needs to be illustrated.Therefore,in the second conception of this paper,which contains three parts will be shown as below.The role of BRD4-SOX9 axis in cisplatin resistance of GC cells,the inhibition effect of BETs inhibitor JQ1 and the primary mechanism exploration.To sum up,this article was based on "epigenome reader" BRD4 and its specific inhibitor JQ1,expounding the mechanism that BRD4 promotes GC progress in transcriptional and the non-transcriptional manner,meanwhile,we found that targeting BRD4 could inhibit GC cells metastasis and cisplatin-resistance in vivo.The evidences above remind us a fresh way to treat drug-resistant and metastasizing GC patients in future.MethodsExplore the effect of JQ1 on BRD4-snail axis and GC cell migrationIn this part of research,with transwell invasion and migration assay,as well as Peritoneal metastasis mouse model,the effect of JQ1 on BRD4-snail and GC cell migration was tested.Explore the role of BRD4-SOX9 axis in chemo-resistance of GC cellsIn this part,TCGA database,RNA-seq and IHC assay were used to analyze the correlation between BRD4 and SOX9;WB and RT-PCR were tested for variance analysis between WT and resistant GC cells,also tested to explore the regulation role of BRD4 on SOX9 that based on siBETs;the role of SOX9 in GC chemo-resistance were measured in CCK8,soft agar formation assay and colony formation assay.Identify the effect of JQ1 in sensitizing GC cells to cisplatin through targeting BRD4-SOX9 axisIn this section,with WB and RT-PCR,the effect of JQ1 to SOX9 and downstream genes expression was tested;CCK8,soft agar formation assay and colony formation assay were measured to clarify the combination effect of JQ1 and cisplatin in GC chemo-resistant cells;apoptosis stimulated by drug combination was tested by WB;Subcutaneous xenograft mouse model was used to explore the effect of drug combination in vivo.Explore the epigenetic mechanism of chemo-resistance based on BRD4-SOX9 axisIn this chapter,IHC and ChIP-seq were tested to found the correlation between SOX9 and H3K27Ac and the aberrant signal of H3K27Ac in SOX9 loci.ResultsJQ1 targets BRD4-snail axis and inhibit GC cell migration1.Overexpressed snail could eliminate the effect of JQ1 on suppressing invasion and migration of GC cells.2.Peritoneal metastasis mouse model demonstrated JQ1 could repress GC metastasis.BRD4-SOX9 axis promotes the chemo-resistance of GC cells1.BETs regulated the expression of SOX91.1 RNA-seq showed the high correlation between BRD4 and SOX9;1.2 Based on the databases analysis,high correlation appeared between BRD4 and SOX91.3 IHC showed the correlation between SOX9 and H3K27Ac.1.4 The mRNA and protein levels of SOX9 were inhibited after siRNA-BETs;1.5 The mRNA and protein levels of SOX9 were stimulated after overexpressing BETs;2.The aberrant expression of BRD4 and SOX9 in cisplatin resistant GC cells2.1 BETs and SOX9 were upregulated in cisplatin resistant GC cells;2.2 The expression of SOX9 was inhibited after siRNA-BETs in cisplatin resistant GC cells;3.SOX9 promotes chemo-resistance in GC cells3.1 With the cisplatin treatment,knockdown SOX9 suppressed cell viability and proliferation more than WT tumor cells.3.2 With the cisplatin treatment,overexpressing SOX9 stimulated cell viability and proliferation more than WT tumor cells.JQ1 sensitize GC cells to cisplatin through targeting BRD4-SOX9 axis1.Combined JQ1 with cisplatin treatment dramatically repressed tumor cells viability and proliferation.2.Combined JQ1 with cisplatin treatment dramatically simulated cell apoptosis.3.In subcutaneous xenograft mouse model,combined JQ1 with cisplatin treatment minimized tumor volume.4.In subcutaneous xenograft mouse model,combined JQ1 with cisplatin treatment inhibited tumor cell proliferation.The epigenetic mechanism of chemo-resistance based on BRD4-SOX9 axis1.IHC showed correlation between SOX9 and H3K27Ac;2.ChIP-seq showed the higher peak of H3K27Ac on SOX9 loci in cisplatin resistant GC cells compared with wide type;Conclusion1.JQ1 suppressed tumor metastasis by reducing snail protein stability.2.BRD4 showed correlation with SOX9,and regulated its expression which were upregulated in chemo-resistant GC cells.3.Combining JQ1 sensitized GC cells to cisplatin that displayed valuable clinical prospect.4.H3K27Ac peak recruited on SOX9 loci in chemo-resistant GC cells.