Study Of Anti-breast Cancer Mechanism Of Hsp70 Inhibitor(JG-98) And Identifying Synergistic Drug Combinations

BACKGROUND AND OBJECTIVEHeat shock protein 70(Hsp70/HSPA1A)is a stress-inducible molecular chaperone that participates in protection of proteome from aggregation and promote refolding and degradation of damaged polypeptides.All members of the Hsp70 family have an N-terminal nucleotide/ATPase binding domain(NBD)and a C-terminal substrate-binding domain.Hsp70 levels are highly elevated in a variety of tumors,and expression of Hsp70 strongly correlates with tumor grade,metastasis,chemotherapy resistance and poor prognosis,suggesting that it plays a special and broad role in cancer.For example,high Hsp70 levels are associated with adverse outcomes in breast,endometrial,colorectal,prostate cancers as well as certain leukemia.Indeed,genetic ablation of Hsp70 suppresses cancer development in mouse models.As a dispensable protein for growth and development of cancer,Hsp70 could be used as a drug target for cancer treatment.There have been several attempts to develop inhibitors of Hsp70 for cancer treatment,including compounds that interact with the ATPase domain,such as VER155008,MAL3-101,and YK-5 or inhibitors of substrate binding,for example,aptamers or pifithrin-p,.The concern with Hsp70 inhibitors is that Hsp70 is involved in many normal pathways,so the safety of its inhibition remains uncertain.In our previous study,we found that some anti-cancer effects of Hsp70 does not result from its core chaperone,but result from its interaction with a co-chaperone Bag3.Hsp70-Bag3 module rather than Hsp70 chaperone itself plays roles in multiple signaling pathways that is related to cancer development,suggesting that the Hsp70-Bag3 interaction may be a safer and more effective target.Dr.Jason Gestwicki and his colleagues discovered an allosteric inhibitor that binds Hsp70,then could inhibits the Hsp70-Bag3 interaction.A first generation molecule from this series,YM-01,mimics the effects of Hsp70 depletion on cancer signaling pathways and tumor growth.But half-life of YM-01 is too short for clinical application.A second generation molecule JG-98 was identified with greater affinity for Hsp70 and longer half-life,which makes it a promising new anti-cancer drug.Our previous study shows that by binding to ATPase domain of Hsp70,JG-98 could interfere the formation of Hsp70-Bag3 module,and is proved to have strong anti-cancer effects in animal models.Though pathways regulated by JG-98 which are responsible for its anti-cancer effect are not clear,which could be a daunting challenge of its safety in clinical application.On the other hand,inhibition of Hsp90,another member of Heat Shock Protein family,could also achieve anti-cancer effect.Additionally,a lot of potent Hsp90 inhibitors have already been developed.For example,tanespimycin,also called 17-AAG,has already stepped into phase II clinical trial.It is reported that simultaneous depletion of two major Hsp70 family members,inducible Hsp70 and constitutive Hsc73,could cause similar physiological responses as inhibition of Hsp90.Which raises a possibility that the physiological effect of Hsp70 inhibitor could be similar as Hsp90 inhibitor.If so,it will significantly reduce potential importance of JG-98 development.Broad Institute is a genomics study community that brings together researchers in medicine,biology,chemistry from across MIT,Harvard,and Harvard-affiliated hospitals.The Connectivity Map(also known as Cmap)is a Broad Institute project built based on RNA microarray technology.It is a collection of genome-wide transcriptional expression data from cultured human cells treated with different drugs and bioactive small molecules.These information enables the discovery of functional connections between drugs,genes and diseases through the generation and analysis of gene expression signatures.The big advantage of this approach is that instead of measuring all～20,000 genes in the human genome,Cmap uses an assay called L1000 to measure a select subset of approximately 1,000 genes and uses these "landmark"gene measurements to computationally infer a large portion of the remainder.In our study,we used the Broad Institute Connectivity Map Platform to compare the physiological effects of JG-98 and Hsp90 inhibitors on cancer cells.Our research found that the effects of JG-98 are dissimilar from effects of Hsp90 inhibitor,thus justifying value of further development of JG-98.Additionally,we identified pathways modulated by JG-98 through inhibition of Hsp70 and tested the effect of Bag3 in modulation of these pathways by JG-98.Furthermore,using pooled shRNA genetic screen,we established gene sets that modulate the response of cancer cells to JG-98.Then by analyzing pathways from these genes or combining these information with Cmap,we developed approaches to predict potent drugs which could synergize with anti-cancer effects of JG-98,such as proteasome,RNApol II,AKT and RTK inhibitors.We also validated these predictions by in vitro and in vivo experiment,providing information in defining potential drug combination for clinical use of JG-98.METHODS1.Five different human cancer cell lines,including human breast cancer cell line MCF7,human prostate cancer cell line PC3,human liver cancer cell line HepG2,human colon cancer cell line HT29 and human acute T cell leukemia cell line Jurkat,were treated with five concentrations of JG-98 or Hsp90 inhibitor tanespimycin(starting from 5μM with sequential three-fold dilution)for 24 hours.RNA was isolated and run over the Broad Institute platform to compare the physiological effects of these two different drugs.Through Connectivity Map,effects of JG98 and tanespimycin on gene expression were compared with effects of other drugs in the database.2.Extracted RNA from MCF7 cells after JG-98 treatment and performed microarray analysis.Microarray data were analyzed using the Broad Institute software package for gene set enrichment analysis(GSEA)which allows deducing pathways up-and downregulated by JG98.We also profiled major signaling pathways using the protein phosphoralation level analysis service which provides information about phosphorylation states,levels or cleavage of more than 50 signaling factors that cover more than 20 major signaling pathways.3.To address the role of Hsp70-Bag3 interaction in cellular response to JG-98,we knock out Bag3 in breast cancer cell lines and then treated them with JG-98,collect and measure the protein level of MAPK,AKT and c-myc pathways related proteins.Using Inter Q method to test the interaction between Bag3 and ERK in MAPK pathway,as long as the effect of JG-98 on this interaction.Next,we tested if JG-98 inhibits dephosphorylation of ERK1/2.Collect JG-98 treated and untreated cells at different time point after stopping further ERK 1/2 phosphorylation by the upstream kinases,test the level of pho-ERK and evaluate its dephosphorylation rate in cells.4.Performed pooled shRNA screen in breast cancer cell line,the library covers about 20%of human ORFs.MCF7 cells were infected with the lentiviral shRNA library at low MOI.Two days post-infection,puromycin was added to select cells expressing shRNAs.After selection,divide cells into JG-98 treatment group and control group.Cells in JG-98 treatment group were incubated with 3μM JG-98 for 48 hours.Under these conditions,about 75%of cell died.Then collect control cells and cells survived JG-98 treatment for genomic DNA isolation.Pooled shRNA barcodes were amplified from the genomic DNA mix,sequenced by Ion Torrent,and frequencies of shRNA species presented in treated survivor cells versus un-treated control cells were established.ShRNAs whose frequencies signigicantly differed in treated and control samples above the established threshold were further analyzed.5.Sorted those genes that significantly affect sensitivity to JG-98 according to biological pathways.Information obtained was used to predict drugs which may potentiate effect of JG-98 on cancer cell killing.Predictions were validated in breast cancer cell lines(MDA-MB-231,BT474)by MTT,and also validated in vivo of mouse breast cancer tumor models.Combine the power of Cmap analysis with information obtained in our genetic screen to search for drugs that may sifnificantly increase the potency of JG-98 actin.RESULTS1.Major pathways and genes modulated by JG-98 and Hsp90 inhibitors are very different,JG-98 represents an entirely novel pharmacological entity and could not be replaced by Hsp90 inhibitors.Gene expression changes in cancer cells stimulated by Hsp90 inhibitor(tanespimycin)are different from that stimulated by JG-98.Result from Cmap showed that effects of tanespimycin on gene expression were similar to effects of other Hsp90 inhibitors,including geldanomycin,NVP-AUY922,BIIB021.These inbinitors demonstrated high connectivity scores,up to 0.95.In contrast,Hsp90 inhibitors were not found among drugs that showed reasonable connectivity scores with JG-98.Additionally,the best connectivity score with JG-98 was relatively low,0.65,reflecting limited similarities in transcription effects of known drugs and bioactive compounds with JG-98.2.Gene set enrichment analysis(GSEA)showed 176 pathways were significantly downregulated by JG-98 and 19 pathways were significantly upregulated by JG-98.After analysis and integration of these results,we summarized them into several major pathways.Among downregulated functional gene families,we identified these major pathways(1)the cell cycle,(2)RNA processing and splicing,(3)proteasome,heat shock proteins and CCT chaperones,(4)cellular energetics,including respiratory chain,ATP synthetase,Crebs cycle and glycolysis,and(5)sterol biosynthesis.Among upregulated gene families we identified(1)unfolded protein response genes,(2)circadian rhythm,and(3)p53/DNA damage response genes.3.These results were partly confirmed by data from protein phosphoralation level analysis in MCF7 cells treated by JG-98 for 24 hours.Cell cycle progression related genes were suppressed by JG-98(upregulation of p21 and p27,and reduction of Rb phosphorylation),DNA damage response was activated(increased phosphorylation of histone H2Ax and Chk2)and UPR are activated(increased level of calnexin and PDI).4.JG-98 upregulated the level of activated ERK in breast cancer cells,upregulated MAPK pathway,and Hsp70-Bag3 module is related to this regulation effect.Validated by Western Blot,the phosphorylation level of ERK1/2 increased in MCF7 cells after JG-98 treatment than control cells,and the upregulation level increased with the drug concentration.Additionally,after knock out Bag3 by shRNA in MCF7 cells,the phosphorylation level of ERK1/2 also increased.Indicating inhibition of Hsp70-Bage3 module might paly role in activation of ERK1/2 by JG-98.Previous research suggested that Hsp70-Bag3 module could directly bind with ERK1/2 and then promote the dephosphorylation of ERK1/2 by MKP3 kinase.Proved by Inter Q that ERK1/2 readily forms complexes with Bag3 and JG-98 could reduce their interaction.Q-PCR showed that after treatment by 2μM JG-98 for 4 hours,the interaction between Bag3 and ERK1/2 decreases by 38.88%in MCF7 cells.Real-time testing of dephophrylation rate of ERK1/2 showed that in MCF7 cells treated by JG-98,the dephosphorylation rate of activated pho-ERK significantly decreased in compared to control cells.5.JG-98 downregulates AKT pathway and c-myc pathway,leads to decrease of pho-AKT and c-myc in breast cancer cells,and these modulations are independent of Bag3.Validated by Western Blot,the level of pho-AKT and c-myc decreased in MCF7 cells after incubation of JG-98.Otherwise,knock out of Bag3 by shRNA did not show influence on pho-AKT and c-myc levels.Additionally,JG-98 could cause enhanced decrease of pho-AKT and c-myc in Bag3 knock out MCF7 cells.6.Uncover functional pathways that are critical for JG-98-mediated killing breast cancer cells by pooled shRNA screens in MCF7 cells using shRNA library.The most under-represented shRNAs species in survivor cells were those targeting components of the proteasome pathway.Knock down proteasome related genes could increase the sensitivity of breast cnacer cells to JG-98.Other highly represented pathways include RNApol Ⅱ pathway,knock down genes encoding its components significantly sensitized breast cancer cell to JG-98.Golgi transport related pathway,knock down ARF1 or ARFGAP1 could increase breast cancer cell resistance to JG-98,to the contrary,knock down ARFGEF2 could increase cell sensitivity to JG-98.TGF-β pathway,knock down its positive regulatory factor TGFBP1,FNTA,SMAD2,AXIN and Usp9 could increase cell resistance to JG-98,knock down its negative regulatory factor STRAP could increase cell sensitivity to JG-98.And also Wnt pathway,knock down FXD3,FZD4,FZD7,FZD8,FGF8 and TCF7L1 increase sensivity,knock down AXIN increase resistance.7.The proteasome inhibitor was validated to increase breast cancer cell sensitivity to JG-98 by in vitro cell viability experiment and in vivo experiment using xenograft breast cancer model.Also demonstrates that shRNA library screen could be useful tool to predict drug combination.MTT experiment showed that proteasome inhibitor(MG132)could significantly potentiate cell killing by JG-98.More breast cancer cells(MDA-MB-231,BT474)died when being treated by JG-98 combined with MG132 than being treated by either drug alone.We next validated this finding by in vivo experiment using MDA-MB-231 xenograft cancer models.For these texts,we change to an FDA-approved proteasome inhibitor,bortezomib.Results showed that bortezomib and JG-98 caused suppression of tumor growth even when injected alone.Importantly,their combination caused a significantly stronger suppression of tumor growth,demonstration that this combination is more potent than the individual drugs alone.8.RNApol II inbibitor a-amanitin could synergize with JG-98 and increase breast cancer cell sensitivity to JG-98.In vitro MTT experiment demonstrated that a-amanitin could increase anti-cancer effect of JG-98 in breast cancer cell lines(HCC1937,BT549).9.Applying shRNA library screen result to connectivity map showed that AKT pathway inhibitor(LY294002)and tyrosine kinase inhibitor(sunitinib)could enhance breast cancer cell sensitivity to JG-98.In vitro experiment validated both drugs could potentiate JG-98 in killing cancer cells.CONCLUSIONS1.JG-98 triggers unique and novel physiological response in diverse cancer cells.Physiological effects of JG-98 are distinct from those of Hsp90 inhibitors.Hsp70 inhibitor series,such as JG-98 represent an entirely novel pharmacological entity and could be a promising anti-cancer target.2.Gene sets downregulated by JG-98 in breast cancer cells include(1)the cell cycle,(2)RNA processing and splicing,(3)proteasome,heat shock proteins and CCT chaperones,(4)cellular energetics,including respiratory chain,ATP synthetase,Crebs cycle and glycolysis,and(5)sterol biosynthesis.Gene sets upregulated by JG-98 in breast cancer cell include(1)unfolded protein response genes,(2)circadian rhythm,and(3)p53/DNA damage response genes.3.We indicated the mechanism of JG-98 in upregulate MAPK pathway in breast cancer cells.Upregulation of ERK1/2 by JG-98 is related to Hsp70-Bag3 module.Through inhibiting formation of Hsp70-Bag3 complex,JG-98 interrupt interaction between Bag3 and ERK1/2,decrease dephosphorylation of ERK1/2,thus increasing ERK1/2 activity.4.JG-98 has downregulation effect on AKT and c-myc pathway,and the downregulation effect is independent of Bag3.5.Either shRNA screen combined with pathway analysis or combined with Connectivity Map could be a useful approach to predict anti-cancer mechanism of drugs and to predict potential drug combination therapy.6.Proteasome inhibitor(MG132/bortzomib),RNApol II inhibitor(α-amanitin),AKT pathway inhibitor(LY294002),tyrosine kinase inhibitor(sunitinib)could enhance sensitivity of breast cancer cells to JG-98,could be candidates for clinical drug combination with JG-98.SIGNIFICANCE1.Demonstrate the difference between physiological effect of JG-98 and Hsp90 inhibitors using gene expression analysis based on the Connectivity Map Platform developed by Broad Institute.2.Firstly identified pathways regulated by JG-98 in anti-breast cancer effect,and then assessed the role of disruption of Hsp70-Bag3 interaction in pathway regulation by JG-98.3.Developed new approaches to design potent combination of known drugs with JG-98.First approach is straightforward pathway analysis,second approach is a combination of genetic screen data(shRNA library screen)and gene expression data(Connectivity Map).