Ubenimex Enhances Brd4 Inhibition By Suppressing HEXIM1 Autophagic Degradation In Glioma Cells

Part 1:HEXIM1 expression plays an important role in BRD4 inhibitor’s sensitivity in glioma cellsBackgroundGlioma is the most common and lethality malignant brain tumor.Malignant glioma showed high invasion ability,leading to a high challenge of complete surgical radical resection.At the same time,the classic radiotherapy often leads to radiation damage on normal brain tissues as side effect.Meanwhile,a lot of glioma cases showed high incidence of chemotherapy resistance.Because of these,clinical application of the classic surgery + radiotherapy + chemotherapy showed limitation in treating this disease.New treatment methods are urgently needed to break the bottleneck of malignant glioma treatment.In recent years,with the development of tumor targeted therapy,there is an increasing potential using of BRD4 inhibitor in cancer treatment.BRD4 belongs to BET(bromodomain and extraterminal domain)subfamily of bromodomain-containing proteins comprised of Brd2,Brd3,Brd4,and Brdt carry out diverse functions as transcriptional regulators.Brd4 recruits the p-TEFb protein complex or Mediator to active promoters and super-enhancers on regulating target gene expression,cell cycle arrest,apoptosis and differentiation,showing great potential in treating glioma cancers.Brd4 inhibition by JQ1 treatment showed potential in glioma treatment.However,some cases showed low sensitivity of JQ1,besides,pre-clinical analysis showed its limitation by demonstrating transient treatment by JQ1 leads to aggressive development of tumor and therefore,accelerating death,emphasizing the importance of duration for clinical use.Thus,an improved understanding of the mechanisms underlying JQ1 sensitivity is urgently required to design strategies to improve its efficiency as well as overcome its limitation.Recently,research on BRD4i sensitivity were focus on P-TEFb pathway and its regulators such as HEXIMl.Brd4 recruits the p-TEFb protein complex that is comprised of cyclin-dependent kinase 9(Cdk9)and its regulatory partner cyclin T1 to active promoters and super-enhancers through its interaction with acetylated histones Through this recruitment,Brd4 positively regulates the transcription elongation carried out by RNA Polymerase Ⅱ,as well as the expression of a number of other genes involved in oncogenesis including Bcl2 and c-Myc.In an opposing interaction,the p-TEFb complex binds to a negative regulatory protein,hexamethylene bisacetamide-inducible protein 1(HEXIM1)that inhibits p-TEFb activity.It is the equilibrium between the positive regulation of pTEFb by Brd4 and its negative regulation by HEXIM1 that determines the extent of activation of the BET pathway.From this we can make a hypothesis:BRD4i sensitivity is related with HEXIM1 expression,by regulating level of HEXIM1 expression,we can develop a way to improve the efficiency of JQ1 treatment on glioma.Ubenimex(Bestatin)is a low-molecular-weight dipeptide molecule that enhances the function of immunocompetent cells and has diverse effects on the production of cytokines.It shows benefits in Glioma treatment by inhibiting 5-LO-LTA4 hydrolase pathway[Our previous study demonstrated its function as an Akt inhibitor and a regulator of autophagy in RCC cells and prostate cancer cells.Can ubenimex suppress Akt pathway and autophgy in glioma cell lines as well as in other cancers?Do Akt pathway and autophagy in glioma cells contributes to sensitivity to BET inhibitors and bring a new synergistic treatment of JQ1 on glioma?Aiming on those questions,we conduct this study.Objective:(1)To verify the relationship between HEXIMI expression and JQ1 sensitivity.(2)To verify whether Ubenimex has some coordination effects on BRD4i treatment,further more,to study its mechanism behind this effect such as down-regulating HEXIM1 expression in glioma cell lines.Materials and methods:(1)To investigate the effects of JQ1 on glioma cell proliferation,the WST-1 assay were employed to identify the effects of JQlon glioma cell lines.3 cell lines of interest were subject into test(U87M,T98G and U251);(2)Testing HEXIM1 expression in 3 cell lines by western blots;(3)By down-regulating HEXIM1 expression by using HEXIM1SiRNA in U87M and T98G(basal HEXIM1 expression in U251 is very low).After verified by Western Blot,HEXIM1 downregulated cells(Si-HEXIM1),empty vectors(Si-control)and control cells were subjected into WST-1 assay.(4)Test ubenimex pre-treated cells in the same way to identify whether HEXIM1 knock down has same effect on ubenimex enhanced JQ1 sensitivity.(5)All 3 groups(HEXIM1 SiRNA,empty vector and control cells)were pre-treated with ubenimex(0.5mg/ml,48 hours),then they were treated with JQ1 at varying concentrations(from lOnM to 5uM)for 72 hours.WST-1 were conduct to show whether down-regulating HEXIM1 could attenuate ubenimex enhanced JQ1 sensitivity.(6)Annexin V-PI were employed to test apoptosis.Results:(1)The WST-1 assay demonstrated a dose-dependent decrease in all 3 cell’ s viability following treatment with JQ1.The IC50 values were 65 nM for U87M,480 nM forT98G and 3010 nM for U251.After 72 hours of JQ1 treatment,U87M showed the most sensitivity to JQ1,while U251 showed least response to JQ1 treatment.(2)WB showed U87M have highest HEXIM1 expression while U251 showed lowest,indicating HEXIM1 plays an important role in basal JQ1 sensitivity.(3)There is no significant difference between control(no treatment)and mock(empty vector transfected)groups.However,after downregulating HEXIM1 expression,all these 2 cell lines showed less sensitivity of JQ1 treatment compare to control group.The IC50 values shift to 110 nM for U87M HEXIM1 SiRNA,750nM for T98G HEXIM1 SiRNA.These results indicating HEXIM1 plays an important role in JQ1 sensitivity.(4)The WST-1 assay showed there is no cytoxicity of ubenimex pre-treatment alone on these 3 cell lines,however,data showed an increase of JQ1 sensitivity.The IC50 values shift to 50 nM for U87M,235 nM forT98G and 830 nM for U251.5.The results of WST-1 assay showed down-regulating HEXIM1 could attenuate ubenimex enhanced JQ1 sensitivity.(5)Compare to ubenimex pre-treated cells,the HEXIM1 knockdown groups,IC50 of JQ1 shift to 70 nM for U87M HEXIM1 SiRNA,380 nM for T98G HEXIM1 SiRNA.6.HEXIM1 could attenuate ubenimex enhanced JQ1 induced apoptosis in U87 cell line.(6)Ubenimex has some coordination effects on BRD4i treatment in every cell lines.Conclusion:1.HEXIM1 plays an important role in JQ1 sensitivity.HEXIM1 regulation could enhance JQ1 sensitivity.2.Ubenimex enhance the sensitivity of JQ1 induced growth inhibition by regulating HEXIM1 expression.3.HEXIM1 plays an important role in JQ1 induced apoptosis,ubenimex could enhance this effect.Part 2:Ubenimex improve the JQ1 treatment sensitivity of glioma by increasing HEXIM1 expression via regulating autophagyBackground:Autophagy is a catabolic pathway that directs excess or damaged cytoplasmic constituents to lysosomes for degradation and recycling for anabolic processes.Recent study demonstrate autophagy plays an important role in drug resistance of cancer treatments.By regulating autophagy could improve JQ1 treatment efficiency via regulating protein degradation and several related cell signal pathways.APN inhibitor ubenimex has been used as a anti-tumor drugs for several years,however,the pharmacological mechanism of this drug still remains unclear.It shows benefits in Glioma treatment by inhibiting 5-LO-LTA4 hydrolase pathway.Our previous study demonstrated its function as an Akt inhibitor and a regulator of autophagy in RCC cells and prostate cancer cells’ proliferation and migration suppression,indicating its role in regulating autophagy and its penitential in assisting cancer chemo-treatment or target treatment.It is the equilibrium between the positive regulation of pTEFb by Brd4 and its negative regulation by HEXIM1 that determines the extent of activation of the BET pathway.Besides,latest research demonstrated that HEXIM1 are degraded through the process of autophagy in a number of acute leukemia cell lines,inhibition of autophagy substantially increases the expression of HEXIM1,their results suggest that the activation of autophagy confer sensitivity to BET inhibitors.Our previous study verified that JQ1 sensitivity of glioma is related with the level of HEXIM1 expression.Regulating HEXIM1 expression could improve JQ1 sensitivity of glioma cells.We already verified ubenimex could improve JQ1 treatment efficient,further study need to be conduct to clarify whether this effect is associated withautophagy regulation.Objective:1.To verify the role of HEXIM1 autophagic degradation in JQ1 sensitivity of glioma by in vitro and in vivo experiments.2.Explore the role of autophagy in ubenimex enhanced JQ1 sensitivity in gliomas.Materials and methods:(1)To investigate the effects of ubenimex on JQ1 sensitivity,we pre-treat cells with ubenimex(0.5mg/ml)for 48 hours before JQ1 treatment.Test HEXIM1 expression in these 3 cells by Western Blot,as well as expression of autophagy marker such as p62 and LC3;(2)Autophagosomes was observed by using electron microscope;(3)Test HEXIM1 expression by using cell IHC after treatment by JQ1 or combined with ubenimex;(4)Test level of HEXIM1 expression by using WB after up-regulating autophagy by using rapamycin;(5)In order to verify whether ubenimex could improve JQ1 sensitivity in less sensitive gloma cells,T98G,U251 xenografts model was subject into in vivo study;(6)Tumor tissue from U251 xenografts were subjected into IHC staining to detect HEXIM1 expression;(7)In order to test whether ubenimex or JQ1 treatment could improve aggressive of glioma,dissection were conducted after U251 xenografts were sacrificed,metastasis to lung,liver and bone were observed.Results:(1)The up-regulation of HEXIM1 by ubenimex treatment in conjunction with markedly increased expression of p62 and decreased LC3B expression suggested the presence of autophagy down regulatin;(2)A reduced count of autophagosomes was observed in ubenimex treated groupby by using electron microscope;(3)JQ1 treatment alone has no effect on HEXIM1 expression,however,combined with ubenimex down-regulated HEXIM1 expression significantly;(4)After upregulating autophagy level by using rapamycin,HEXIM1 expression decreased dramatically;(5)Consistent with in vitro study,T98G showed better response to JQ1 treatment than U251 xenografts model;(6)Consistent with in vitro study,combined with ubenimex oral feeding,tumor volumes grows slower or shrinked much faster,indicating ubenimex improved JQ1 efficiency in controlling glioma tumor volume in vivo;(7)Tumor tissue from U251 xenografts were subjected into IHC staining to detect HEXIM1 expression,data showed higher HEXIM1 expression in ubenimex treated tumor tissues,consistent with in vitro study;(8)Combined with ubenimex oral feeding significantly reduced metastasis ratio compare to JQ1 treatment alone(0/20 vs3/20).However,no apparent difference between JQ1 treatment group and control group(DMSO group,4/20 vs 3/20).Conclusion:1.HEXIM1 expression were upregulated after ubenimex treatment via suppressing autophagic protein degradation.2.After upregulating autophagy level,HEXIM1 expression decreased dramatically,leading to decreased JQ1 sensitivity.3.Ubenimex improved JQ1 efficiency in controlling glioma tumor volume and invasion in vivo,which is related to higher HEXIM1 expression after ubenimex combine treatment.Part 3:Role of Akt pathway in Ubenimex enhanced JQ1 sensitivity and inhibiting invasion of glioma cellsBack ground:The autophgy level in cancer is regulated by several cell signal pathways.Recent study showed most related pathway-Akt,was verified to play an important role in JQ1 sensitivity.However,Role of Akt pathway in JQ1 sensitivity remains controversial.Cheng’s study indicates higher Akt expression rescuing c-Myc from JQ1 induced suppression in glioblastoma,whereas this study indicates the converse,where c-Myc down-regulation effects Akt down-regulation;His studly also demonstrated cells genetically engineered for Akt hyperactivation did not compromise JQ1 efficacy,suggesting that these frequently mutated signaling pathways may not confer resistance to JQ1.Pre-clinical analysis from Vishal etc.suggested JQ1 treatment can induce Akt suppression only at long time point compare to a shorter time point,indicating role of Akt pathway plays a context depended role in JQ1 treatment in gloma cells.Pre-clinical analysis showed its limitation by demonstrating transient treatment byJQ1 leads to aggressive development of tumor and therefore,accelerating death.Akt pathway palys an important role in regulating cell invasion.Our previous study verified ubenimex could inhibit RCC cell migration by inhibiting Akt pathway.As one of the potential risk of JQ1 treatment is aggressive development of tumor,an method to reduce glioma cell migration is essential in JQ1 treatment.Can ubenimex also inhibit glioma invasion and migration?Is this effect relate with Akt pathway?In order to clarify these question,Further study has been done to investigate Akt pathway in ubenimex enhanced JQ1 sensitivity and inhibiting invasion of glioma.Objective:To investigate the role of Akt pathway in ubenimex enhanced JQ1 sensitivity and inhibiting invasion of glioma.Materials and methods:(1)To investigate the effects of ubenimex on Akt signaling pathways(p-Akt-ser437)in glioma cell,we pre-treated cells with ubenimex(0.5mg/ml)for 48 hours before JQ1 treatment.Test the expression of phosphorylated proteins involved in the Akt signaling pathways in these 3 cells by Western Blot;(2)Using Akt agonist(10nM)regulats Akt signal level in glioma cells,to investigate the effects of Akt signaling pathway on the efficacy of JQ1 in glioma cells;(3)Transwell assays were performed to determine whether ubenimex affects the migration capacity of glioma U251 cell lines.Using Akt agonist regulats Akt signal level in glioma cells,then detect its role in suppressing glioma cell migration by combining ubenimex with JQ1.Results(1)Western blot showed Ubenimex treatment significantly reduced phosphorylated proteins involved in the Akt signaling pathways(p-Akt-ser437);(2)WST-1 assay showed treatment with an Akt agonist(10nM)compromise JQ1 efficacy;(3)The migration capacity of the glioma cells was significantly suppressed by combining ubenimex with JQ1;(4)Akt agonist could significantly attenuated this effect indicating its role in suppressing glioma cell migration by combining ubenimex with JQ1.Conclusion:1.Akt inhibition is one of the mechanisms of ubenimex enhanced JQ1 sensitivity.2.Combing with ubenimex inhibit cell invasion in an Akt dependent pathway.