Role And The Underlying Mechanism Of Epigenetic Reader BRD4 Contributing To Gastric Cancer Cell Invasion And Metastasis

With the inovation of sequencing and proteomics technology,limited progression has been achieved during the past decades.However,it is self-evident that the traditional central dogma is too circumscribed to elucidate the complexity of cancer malignant biological properties.Epigenomics dysregulation,which has been proved to exert the vital role in cancer progression,was consider as the replenishment for the canonical conception.Accordingly,targeting the master epigenetic events in cancer development and exploring the new anti-cancer targets,named Epigenetic Therapeutics,was the new strategy for the next generation of anti-cancer drug development and research.Bromodomain and extra terminal protein（BET）family,which includes BRD2,BRD3,BRD4 and BRDT（only expressed in testis）,is one the most concerned potential targets,thought BRD4 was consider as gem for cancer targeted therapy.Regarding as the epigenetic reader,BRD4 directly recognizes and binds to the acetylated lysine of histone,thus recruiting series of pivotal transcriptional complex such as P-TEFb and JMJD6 to coordinate the transcription of panels of oncogenes such as MYC and BCL2,resulting in the boost of cancer cells proliferation.However,whether BRD4 may control the other cancer malignant bioprocess was unknown.Mechanically,whether BRD4 regulate the related biological process independent on the transcriptional regulation also needs to be elucidated.In 2011,two newly launched selective small molecular inhibitor JQ1 and I-BET against bromodomain of BRD4 has been highly recommended,because of their distinctly effective for cancer proliferation restrain.JQ1 competitively binds to the two bromodomains of BRD4,thus restricting its ability as the epigenetic reader.The bromodomain-blocked BRD4 could not activate the transcription of the oncogenes like MYC and BCL2,resulting in tumor survival suppression.To date,JQ1 had been underwent into clinical trial（Clinical Trials.gov identifiers NCT01987362、NCT01587703、NCT01949883、NCT01713582 and NCT01943851）,which demonstrates the potential application in clinical work in the near future.Nevertheless,the mechanism of BRD4 in JQ1 regulating the malignant biological process in gastrointestinal carcinomas,such as gastric cancer and colorectal cancer is unclear.Therefore,regarding the gastric cancer as the disease model,taking advantage of BRD4 as the potential therapeutic target,demonstrates the higher novelty and the potential application value for clinical translation.Snail family transcriptional repressor 1,Snail,is the key transcriptional factor in epithelial to mesenchymal transition（EMT）process,that serve as the transcriptional suppressor of CDH1 gene,resulting in E-cadherin inhibition and EMT activation,thus stimulating the cancer malignancies such as cancer cells invasion,metastasis,self-renewal and chemoresistance.Meanwhile,a range of research exhibit that Snail is the freestanding oncogene independent on the EMT process.Mechanically,Snail is a labile protein with a relative short half-time 2-3 hours,which is regulated by ubiquitin-proteosome system（UPS）.A cohort of E3 ubiquitin ligases,such asβ-TRCP1,FBXL14,FBXO11 and A20,were participated in the regulation of Snail protein stability.Nonetheless,the mechanism antagonizing the degradation-related ubiquitination for Snail is ambiguous.Gastric cancer（GC）is one of the prevalent carcinomas around word,especially in China,which means the second leading of morbidity and mortality after lung cancer,though the incidence rate was relatively decreased because of the qualitative life improvement and the popularity of Helicobacter Pylori eradication.GC usually occults in onset with no special symptom in early stage,contributing to progressive statue when the patients were diagnosed.However,the understanding of the pathogenesis of GC is limited,giving rise to the lack of the molecular subtyping guiding for clinical work.Metastasis is the leading cause of death for GC patients.The limited and unmatured therapeutic strategy for this cohort of patients results in the unfavourable clinical outcomes.Plenty of researches try to target the potential oncogenic factors such as HER2,VEGF and EGFR,which were testified the pleasant clinical benefit in other types of malignancies,while both of them could not extent the median survival time over one year,although some of them did reached the profit in statistic level.It is the urge need for the research aiming at the driving mechanism and the treatment for metastatic GC.After the accomplishment of Human Genome Project（HGP）,series of items based on high throughput sequencing reveal the importance of homeostasis disorder in epigenetics.Therefore,by focusing on the metastatic gastric cancer patients,it is feasible and transformable that explored the mechanism driving the malignant behavior for this cohort,which is valuable for the research and development of new targeted drug.Accordingly,this article lay emphasis on the epigenetic reader BRD4 and its uncanonical mechanism,which recognizing and binding to the Snail in an acetylation dependent manner,contributing to the Snail stability and cancer invasion.Utilizing the bromodomain inhibitors JQ1 and I-BET,the new epigenetic targeted drugs,is supposed to be the promising strategy for metastatic GC treatment by aiming at the BRD4/Snail axis.The main results and conclusion of this research are as follows:1.High expression of BRD4 indicates the tumor metastasis and poor prognosis（1）The mRNA expression of BRD4 is higher in GC tissues compared with the normal mucosa;（2）The protein expression of BRD4 is higher in GC tissues compared with the normal mucosa;The protein expression of BRD4 is higher in metastatic GC tissues compared with non-metastasis group;（3）Higher expression of BRD4 in GC tissues demonstrates the tumor metastasis and unpleasant prognosis.2.BRD4 promote the invasion and metastasis of GC cells independent the putative target genes MYC and BCL2（1）BRD4 promote the proliferation of GC cells through the MYC process;（2）BRD4/MYC axis induce the tumorigenesis of GC cells in vivo;（3）BRD4 depletion inhibit the migration and invasion of GC cells;（4）BRD4 knockout restrain the metastasis and dissemination of GC cells in vivo.3.BRD4 upregulates and stabilize the Snail protein in a post-transcriptional manner（1）Ectopic expression of BRD4 promotes the protein expression of Snail but not RNA expression;（2）Downregulation of BRD4 inhibits the protein expression of Snail but not RNA expression;（3）JQ1 treatment suppresses the protein expression of Snail but not RNA expression;（4）Ectopic expression of BRD4 promotes the protein stability of Snail,while BRD4depletion destabilizes the protein stability of Snail.4.The acetylation-dependent interaction between BRD4 and Snail inhibits the degradation-related polyubiquitination of Snail protein（1）Endogenous BRD4 was colocalized and interacted with Snail in GC cells;（2）JQ1 suppresses the interaction between BRD4 and Snail in an exogenous condition;（3）Depletion of BRD4 stimulates the polyubiquitination of Snail;（4）Ectopic expression of BRD4 inhibit the polyubiquitination of Snail;（5）JQ1 antagonizes the Snail polyubiquitination restrain by BRD4 accumulation;（6）BRD4 interacts with Snail in an acetylation-dependent manner;（7）Conserved 146 lysine（K146）and K187 in Snail are the key acetylation modification sites;（8）Acetylation deficiency mutation of K146 and K187 antagonizes the stability of Snail regulated by BRD4;（9）Acetylation deficiency mutation of K146 and K187 promote the polyubiquitination of Snail regulated by BRD4.5.BRD4 competes with the E3 ligases FBXL14 andβ-Trcp1 to bind Snail,thereby decreasing Snail polyubiquitination（1）Ectopic expression of BRD4 restrain the recognition of Snail by its E3 ubiquitin ligasesβ-TRCP1 and FBXL14;（2）The interaction between Snail and its E3 ubiquitin ligasesβ-TRCP1 and FBXL14inhibits the binding of BRD4 to Snail;（3）Depletion of BRD4 strengthens the recognition of Snail by its E3 ubiquitin ligasesβ-TRCP1 and FBXL14;（4）Acetylation deficiency mutation of Snail reinforces the recognition of Snail by its E3ubiquitin ligasesβ-TRCP1 and FBXL14;（5）BRD4 and FBXL14 own the overlapping binding site in Snail;（6）BRD4 restrains the Snail phosphorylation regulated by GSK-3β,thereby weakens the interaction between Snail andβ-TRCP1;（7）BRD4 antagonizes the FBXL14-regulated polyubiquitination of Snail,thus stimulate its expression;（8）BRD4 antagonizes theβ-TRCP1-regulated polyubiquitination of Snail,thus stimulate its expression.6.BRD4/Snail axis is the key signaling pathway in GC invasion and metastasis（1）Reactivation of Snail in BRD4^-/-recovery the migration and invasion ability in GC cells;（2）Overexpression of wild type but not the acetylation deficiency Snail promotes the migration and invasion ability in GC cells;（3）Ectopic expression of Snail in BRD4^-/-recovery the metastatic ability in vivo;（4）Reactivation of Snail in BRD4^-/-partially rescues the proliferation inhibition in GC cells;（5）The expression of BRD4 is in a positive correlation with Snail;（6）The expression of Snail is negatively correlated withβ-TRCP1 and FBXL14;（7）BRD4-Snail signaling controls subsets of metastatic signatures.In Conclusion,the main results of the research exhibit below:（1）BRD4 is higher expression in GC tissues especially in metastatic group;Higher expression of BRD4 is the independent prognostic factor,indicating the poor prognosis in GC cohort;（2）Higher expression of BRD4 binds to the acetylated Snail,excluding the recognition of Snail by the canonical E3 ubiquitin ligasesβ-TRCP1 and FBXL14,therefore restrain its degradation-related polyubiquitination and promote the Snail protein stability;（3）BRD4 promotes the gastric cancer migration and invasion both in vitro and in vivo,through stabilization of Snail;（4）Targeting the BRD4/Snail axis is the potential strategy for metastatic GC treatment;This research firstly demonstrated the regulation of BRD4 in GC cells invasion and metastasis both in vitro and in vivo,elucidating a new regulatory mechanism of BRD4independent on the classical transcriptional regulation that BRD4 specially competes with the E3 ligases FBXL14 andβ-Trcp1 to interact with Snail,thereby promoting Snail expression and GC malignancy.Our findings fill up the gap in the noncanonical role of BRD4,which was considered as the ideal target because its special anti-cancer effects both in proliferation and metastasis.By utilizing the bromodomain inhibitors to target the BRD4/Snail axis could be the new choice and direction for metastatic GC epigenetic therapeutics.