Molecular Mechanism Of Synergistic Treatment With BET Inhibitor JQ1 And Autophagy Inhibitor In Vitro And In Vivo

BackgroundEpithelial ovarian cancer(EOC)is the leading cause of gynaecological cancer-associated death worldwide,partly because it is detected often at an advanced stage.Although carboplatin and paclitaxel chemotherapies are first-line treatment and the initial response rate is high(- 80%),most patients eventually recur,and mortality occurs within 5-years,and patients with high-grade serous carcinoma(HGSC)have shorter survival.BET bromodomain protein BRD4 has recently emerged as an exciting new class of target for the treatment of cancer,as BRD4 overexpression can enhance transcription of critical oncogenes,for example,MYC,Consequently,BET bromodomain inhibitors(BETi)were developed.Recent studies have shown that inhibition of BRD4 could reduce the expression of onco-genes and result in tumour regression.Therefore,clinical trials of BETi are in progress for many cancers.Although BETi showed great promise as cancer therapeutics,the anti-cancer effects of BETi were quite variable.Besides,emerging evidence showed that cancer cells acquire resistance to BETi,indicating single agent targeting of BRD4 may not produce ideal therapeutic response.JQ1,a selective BET inhibitor that mimics the acetyl moiety and occludes the acetyl-lysine binding pocket,thus replacing BET proteins from chromatin and shown to be highly effective against epithelial ovarian cancer.Recently JQ1 was shown to induce cell cycle arrest and apoptosis in EOC cells.Thus,JQ1 appears to be a promising therapeutic option for targeting EOC.However,EOC cell lines exhibit different proliferative and apoptotic responses to JQ1.The resistance of some EOC cell lines to JQ1 cannot be explained by differences in basal expression of BRD4 or changes in the levels of c-Myc and BRD4.These findings suggested that primary resistance to JQ1 may not describe its inability to suppress c-Myc or different expression levels of basalBRD4,but could be due to compensatory mechanism striggered by c-Myc inhibition..However,the mechanisms underlying different sensitivities of EOC cell lines to JQ1 remain elusive.A better understanding of the mechanisms involved in resistance of EOC cell lines to JQ1 is needed to combat BET inhibitor resistance in EOC.Autophagy was defined initially as a self-digestion process by which cytoplasmic contents were sequestered in autopha-gosomes and delivered to lysosomes for degradation.Autophagy plays a dual role both as pro-death or pro-survivalin malignant tumour treatment,largely depends on the tumour type and treatment characteristics.Many cellular pathways like the Akt/mTOR pathway and AMPK/ULK1 pathway have been reported to be involved in autophagy initiation.Autophagy protects MDR(multi-drug resistant)cancer cells from apoptosis and promotes resistance to chemotherapy treatment.Inhibition of autophagy may sensitise MDR cells to anti cancer drugs.It represents a new battle-line in the fight against drug resistance.Accordingly,it is vital to have a better understanding of the roles and mechanisms of autophagy and key signalling pathways involved in cancer resistance for targeting autophagy as an approach to overcome drug-resistance.Purposes:To explore whether ovarian cancer has developed resistance to the BET inhibitor JQ1,and to explore the mechanism of drug resistance and ways to reduce or even reverse drug resistanceMethods:1.To detect the activity of ovarian cancer cell line after treatment of JQ1:MTT and PI/annexin-v flow cytometry were used to detect the effect of BET inhibitor JQ1 on proliferation and apoptosis of ovarian cancer cell lines2.To detect the Effect of JQ1 on autophagy level of ovarian cancer cells:Changes in autophagy related protein expression levels after the action of JQ1 were detected by western-blot immunofluorescence assay to detect endogenous LC3B,and autophagy lysosomes were detected by AO staining3.To investigate the effect of autophagy inhibitor combined with JQ1 on the biological behavior of ovarian cancer cells:After JQ1 was combined with autophagy inhibitor 3-ma or CQ,MTT plate cloning assay and flow cytometry were used to detect the effect of combined drugs on the activity of ovarian cancer cells4.To detect the effect of JQ1 on AKT/mTOR autophagy pathway related proteins:Western Blot assay was used to detect the expression changes of AKT/mTOR pathway related proteins such as AKT,phosphorylated AKT(p-akt),phosphorylated mTOR(p-mtor),and phosphorylated P70S6K(p-p70s6k)in cells after the effect of JQ15.Over-expression of AKT activates the AKT/mTOR pathway for rescue experiment:ovarian cancer cell lines were transient transfected AKT plasmid to construct AKT over-expression model,with negative control sequence(negative control,NC)cells as a control,using Western Blot experiments to detecct its expression efficiency and impact on AKT/mTOR pathway related proteins.Using Western Blot and immunofluorescence to detect the changes of level of autophagy after JQ1 treatment.Flow cytometry was used to detect the effect of JQ1 on the apoptosis level of AKT overexpressed ovarian cancer cell lines and NC cell lines6.To detect the effect of JQ1 combined with autophagy inhibitor CQ on xenograft tumor growth in nude mouse ovarian cancer:Use normal ovarian cancer A2780 cell lines to construct nude mice subcutaneously into tumor model,divided into blank group?JQ1 injection group?and combination group,every three days,intraperitoneal injection of drugs on measurement of tumor size,tumor volume calculation,draw the tumor growth curve,stripping tumors after weighing tumors had the volume and weight,further validation JQ1 and autophagy inhibitor CQ combination effects on transplanted tumor growthResults:1.Study on the inhibitory effect of JQ1 on the growth of ovarian cancer cell lines:After treatment with different concentrations of JQ1 for 48h,JQ1 significantly inhibited the proliferation of four ovarian cancer cell lines in a concentration-dependent manner.As shown in FIG.1A,the IC50 of four A2780 HO8910?SKOV-3?HEY cell lines was 6.963um 5.18um 1.503um 0.503um after 48h of action of JQ1.The results showed that JQ1 inhibited the proliferation of four ovarian cancer cell lines,and the difference was statistically significantEffect of JQ1 on apoptosis detected by flow cytometry PI/Annexin V staining was performed on the cells after 48h of effect of JQ1 at different concentrations.The results of apoptosis level detected by flow cytometry were shown in FIG.1B 1C:after 48h of effect of JQ1,the apoptosis levels of all four ovarian cancer cell lines were significantly increasedAs shown in FIG.1D 1E:after the action of JQ1,the levels of BRD4 protein and c-myc in four ovarian cancer cell lines were significantly down-regulated,and there was no significant difference in the basic expression levels of BRD4 and c-myc in four cell lines,indicating that A2780 HO8910 SKOV-3 The differences are not influenced by the expression level of basal BRD4 and the insensitivity of BRD4 protein,but by other underlying mechanisms2.Study on autophagy induced by JQ1 in drug resistance group:To explore the potential mechanism of the different reactivity of the two groups of cells to JQ1,as shown in figure 2A:after a certain concentration of JQ1(2.5um)was used for 48h,the LC3-II/LC3-I ratio in A2780 and ho-8910 cell lines significantly increased,but there was no change in sensitive groupAs Showed in figure 2B:Western Blot to detect the changes in levels of autophagy marker proteins after the action of JQ1:the expression levels of autophagy markers ATG5 Beclinl and LC3-? were significantly increased in the resistance group of ovarian cancer.SQSTM1 expression level decreased significantly,there was no change in the oarian cancer sensitive group.Cell immunofluorescence assay and AO staining further confirmed the increase of endogenous LC3 puncta density and the increase of acidic organelle(AVO)in the resistant group after the action of JQ1.The above results showed that the autophagy level of the drug-resistant group was significantly enhanced after the action of JQ1,while the autophagy level of the sensitive group was not significantly changedAs shown in FIG.3A,after using CQ to block autophagic lysosome degradation,the intracellular LC3-II/LC3-I ratio in the drug-resistant group still increased,indicating that JQ1-induced autophagy enhancement increased the formation of autophagy flow rather than hindered the degradation of autophagic lysosome.3.The combination of JQ1 and autophagy inhibitor significantly enhanced the anti-tumor effect of JQ1:As shown in figure 3A,LC3-II/LC3-I ratio was significantly down-regulated in the combined group of 3MA and JQ1 compared with the single group of JQ1,suggesting that 3-MA successfully blocked the occurrence of jq1-induced autophagy.As shown in figure 3C,the proliferation capacity of ovarian cancer cell lines was significantly reduced in the group treated with autophagy inhibitor compared with the group treated with JQ1 alone The plate cloning experiment was conducted with the same concentration of autophagy inhibitor.FIG.3B showed colony formation capacity was significantly reduced after JQ1 was combined with autophagy inhibitor.The synergistic effect of autophagy inhibitor and JQ1 is not the additive effect of autophagy inhibitor and JQ1Figure 3D showed Combined with autophagy inhibitor,JQ1 significantly enhanced the level of apoptosis.The above results indicated that JQ1 induces the production of cell-protective autophagy,and blocking autophagy with autophagy inhibitor can significantly improve the sensitivity of ovarian cancer cell lines to JQ1.Autophagy inhibitor and BET inhibitor JQ1 have synergistic effects on ovarian cancer cells4.Study on the molecular mechanism of autophagy induced by JQ1:To investigate whether the AKT/mTOR pathway is involved in the generation of autophagy induced by JQ1,western blot was used to detect the changes in the expression levels of AKT/mTOR pathway related proteins after the action of JQ1 at different concentrations.After the action of JQ1 the AKT/mTOR path-related protein p-akt p-mtor p-p70s6k in the JQ1 drug-resistant cell group was significantly down-regulated in a concentration dependent manner.The inactivation of AKT/mTOR pathway induced by JQ1 was demonstrated5.Study on the molecular mechanism of activating AKT/mTOR pathway to increase the sensitivity of cells to JQ1 in the drug-resistant group:Figure S1C showed that the expressions of p-akt p-mtor and p-p70s6k were up-regulated after the transfection of AKT plasmids,indicating that the AKT/mTOR pathway was activated.FIG.4C demonstrated the activation of AKT/mTOR pathway after the cell translocated AKT plasmid.It was demonstrated that the activation of AKT/mTOR pathway inhibited autophagy in the jq1-induced drug resistance group.Further AKT pathway inhibitor LY294002 was used to verify the involvement of autophagy induced by AKT/mTOR pathway.Cell immunofluorescence assay was performed to detect the endogenous LC3B puncta density.It is further confirmed that the AKT/mTOR pathway is involved in the generation of autophagy induced by JQ1 To further detect the effect of autophagy induced by inactivation of AKT/mTOR pathway on cell activity,a rescue experiment was conducted.The result is shown in figure 4E.The AKT1 plasmid group was compared to the blank plasmid group.The level of apoptosis was significantly up-regulated,further confirming that inactivation of AKT/mTOR pathway is involved in the generation of protective autophagy6.Study on the synergistic effect of JQ1 and autophagy inhibitor in transplanted tumor of nude mice:Subcutaneous xenograft model of ovarian cancer was constructed by subcutaneous inoculation of normal A2780 cell line in nude mice.The results showed that the growth inhibition was the most obvious in the combined drug group,and the tumor weight of the combined drug group was significantly lower than that of the control group.Tumor-forming experiments in nude mice have shown that JQ1 combined with autophagy inhibitor CQ can significantly improve the resistance of ovarian cancer to BET inhibitor JQ1 compared with JQ1 aloneConclusion:1.In drug-resistant cells JQ1 induces autophagy by inducing inactivation of AKT/mTOR pathway.Autophagy has a protective effect on ovarian cancer cells and is involved in the development of resistance to JQ1 in ovarian cancer cells.2.The combination of autophagy inhibitor and JQ1 can significantly enhance the anti-tumor effect of JQ1 and improve the sensitivity of ovarian cancer cells and xenograft tumors to JQ1.Innnovations and LimitationsInnovations:1.In this study,we studied the antitumor effect of BET inhibitor JQ1 on ovarian cancer cell lines.It was found that JQ1 induced autophagy is a potential mechanism of drug resistance of some ovarian cancer cell lines to JQ1..2.Vitro experiments confirmed that the inactivation of Akt/mTOR pathway was involved in JQ1 induced autophagy of ovarian cancer cell lines,and the "recovery experiment" confirmed that the inactivation of Akt/mTOR pathway induced autophagy was the potential mechanism of drug resistance of ovarian cancer cell lines to JQ1.3.Vivo and in vitro experiments showed that the combination of autophagy inhibitor 3-mA or CQ and BET inhibitor JQ1 had synergistic antitumor effect.Limitations:1.According to the sensitivity of four ovarian cancer cell lines to JQ1,we divided them into two groups:JQ1 sensitive group and JQ1 resistant group.No JQ1 resistant cell line was cultured and compared with the parent sensitive cell line.2.This paper pays less attention to JQ1 sensitive group,some experiments are not also verified in JQ1 sensitive group,so lack of logic.