KDM6B Counteracts EZH2-Mediated Suppressionof IGFBP5 to Confer Resistance to PI3K/AKTInhibitor Treatment in Breast CancerWenyu Wang1, Keng Gat Lim1, Min Feng1, Yi Bao1, Puay Leng Lee1, Yu Cai2,Yufeng Chen1,3, Hao Zhang4, Diego Marzese5, Dave S.B. Hoon5, and Qiang Yu1,2,6,7
Abstract
Despite showing promise against PIK3CA-mutant breastcancers in preclinical studies, PI3K/AKT pathway inhibitorsdemonstrate limited clinical efficacy as monotherapy. Here,we found that histone H3K27me3 demethylase KDM6B-targeted IGFBP5 expression provides a protective mecha-nism for PI3K/AKT inhibitor-induced apoptosis in breastcancer cells. We found that overexpression of KDM6B andIGFBP5 in luminal breast cancer are positively associatedwith poorer disease outcomes. Mechanistically, KDM6Bpromotes IGFBP5 expression by antagonizing EZH2-medi-ated repression, and pharmacologic inhibition of KDM6Baugments apoptotic response to PI3K/AKT inhibitor treat-ment. Moreover, the IGFBP5 expression is upregulated upon
acquired resistance to the PI3K inhibitor GDC-0941, whichis associated with an epigenetic switch from H3K27me3to H3K27Ac at the IGFBP5 gene promoter. Intriguingly,GDC-0941–resistant breast cancer cells remained sensitiveto KDM6B or IGFBP5 inhibition, indicating the dependencyon the KDM6B–IGFBP5 axis to confer the survival advan-tage in GDC-0941–resistant cells. Our study reveals anepigenetic mechanism associated with resistance to targetedtherapy and demonstrates that therapeutic targeting of KDM6B-mediated IGFBP5 expression may provide a useful approachto mitigate both intrinsic and acquired resistance to thePI3K inhibitor in breast cancer. Mol Cancer Ther; 17(9); 1973–83.�2018 AACR.
IntroductionPIK3CA is found to be the most frequently mutated gene (up
to 45%) in luminal breast cancer, resulting in aberrant activa-tion of the PI3K/AKT signaling pathway (1). Accordingly, small-molecular inhibitors of PI3K/AKT have been actively investigatedin Phase II/III clinical trials in luminal breast cancers with PIK3CAmutations. Despite promising results obtained from preclinicalstudies, the clinical efficacy of single-agent PI3K inhibitor islimited or sub-optimal (2), probably due to intrinsic and acquiredresistance. As such, multiple drugs combination strategies with
an aim to enhance the efficacy of PI3K inhibitors are being testedin various late-phase clinical trials (3).
Epigenetic alterations such as aberrant histone modificationsare implicated in cancer initiation, progression, and metastasis(4, 5). Among them, EZH2-mediated histone three lysine 27 tri-methylation (H3K27me3) has been shown to be oncogenic topromote cancer development by repressing tumor-suppressorgenes (6). EZH2 can also work as a tumor suppressor (7, 8), andloss of H3K27me3 due to deletion of EZH2 causes T-cell acutelymphoblastic leukemia (T-ALL; ref. 9). Global reduction ofH3K27me3 caused by a pointmutation atH3 also drives pediatricbrain cancer progressions (10–12). Moreover, high-grade breast,ovarian and pancreas cancers have been found to harbor lowglobal H3K27me3 which is correlated with increased recurrenceand poor survival (13–15). These findings suggest that both thegain and loss of H3K27me3 can contribute to tumorigenesis in acontext-dependent manner.
The level ofH3K27methylation is tightly regulated. Removal ofH3K27 methylation mark is catalyzed by two H3K27 demethy-lases, KDM6A and KDM6B and both of which have been impli-cated in cancer progression (16). The two enzymes may haveredundant functions in the regulation of stem cell identity andanimal development but also sometimes exhibits contrastingfunctions in oncogenesis such as T-ALL (17). In breast cancer,KDM6B and estrogen receptor (ER) coordinate to promotethe expression of anti-apoptotic BCL2 by inactivating EZH2-mediated H3K27me3 at its promoter to enhance cell survival(18), whereas KDM6A and MLL4 co-regulate some proliferativeand survival genes (19). KDM6B also promotes epithelial tomesenchymal transition that mediates breast tumor invasionand metastasis (20). These findings suggest that KDM6B or
1Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore,A�STAR (Agency for Science, Technology, and Research), Biopolis, Singapore.2School of Pharmacy and Cancer Research Institute, Jinan University, Guangz-hou, Guangdong, China. 3The sixth affiliated hospital, Sun Yat-Sen University,Guangzhou, Guangdong, China. 4School of Medicine, Jinan University, Guangz-hou, Guangdong, China. 5Department of Translational Molecular Medicine, JohnWayne Cancer Institute, Santa Monica, California, USA. 6Department of Phys-iology, Yong Loo Lin School of Medicine, National University of Singapore,Singapore. 7Cancer and Stem Cell Biology, Duke-National University of Singa-pore Medical School, Singapore.
Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).
W. Wang and K.G. Lim are co-first authors of the article.
Corresponding Author: Qiang Yu, Genome Institute of Singapore, 60 BiopolisStreet, Singapore 138672. Phone: 65-6478-8127; Fax: 65-6808-9003; E-mail:[email protected]
KDM6A-mediated demethylation of H3K27 may have a role inpromoting cancer development and therapeutic targeting thismolecular event may present new therapeutic opportunities.
The level of H3K27methylation can be regulated by oncogenicsignaling pathway, which is exemplified by PI3K/AKT-mediatedinhibitory phosphorylation of EZH2, resulting in inhibition ofH3K27me3 (21). Alternatively, the inhibition of H3K27me3mayalso rise from the upregulation of KDM6B/KDM6A demethylasein cancer. Here, we investigated a role of KDM6B in regulatingPI3K/AKT inhibitor response in breast cancer and identified anovel KDM6B target gene, IGFBP5, being crucial in affecting theapoptotic response of PI3K inhibitor. We demonstrated thattherapeutic targeting of KDM6B is effective in overcoming PI3Kinhibitor resistance.
Materials and MethodsReagents and antibodies
GDC-0941 (ref. 22; catalog number: Axon-1377) andMK2206(ref. 23; catalog number: Axon-1684) was purchased from AxonMedchem. GSKJ4 (ref. 24; catalog number: 12073-5) was pur-chased from Cayman Chemicals.
The following primary antibodies were used for Westernanalysis: total PARP (catalogue number: 9542), cleaved PARP(catalogue number: 9541), EZH2 (catalogue number: 3147),phospho-ERK 1/2 (catalogue number: 9101), total ERK 1/2(catalogue number: 9102), phospho-AKT (S473; catalogue num-ber: 9271), phospho-AKT (T308; catalogue number: 2965), H3(catalogue number: 9175 and 3638), H3K27me3 (cataloguenumber: 9733), survivin (catalogue number: 2802), and tubulin(catalogue number: 2146) were purchased from Cell SignalingTechnology. KDM6B (catalog number: 38113) and phospho-EZH2 S21 (catalog number: 84989)were purchased fromAbcam.KDM6A (catalog number: ABE409) was purchased from MerckMillipore. Actin (catalog number: A5441) was purchased fromSigma-Aldrich. Bim (catalog number: 559685) was purchasedfrom BD Biosciences. ChemiDoc MP Imaging Systems and ImageLab software (Bio-Rad) were used to detect chemiluminescenceintensity.
Western blotsSamples were prepared by using Blue Loading Buffer Pack
(Cell Signaling Technology, catalog number: 7722), and weredenatured at 95�C for 5 minutes. Equal amounts of proteinextract were loaded and run on SDS-polyacrylamide gels thentransferred to polyvinylidene fluoride (PVDF) membranes bysemi-dry transfer. Membranes were blocked for 1 hours at roomtemperature in TBST supplemented with 5% non-fat milk, andincubated overnight at 4�C with primary antibody diluted inthe same blocking buffer. After three washing in TBST, mem-branes were incubated for 1 hour at room temperature withhorseradish peroxidase (HRP)–conjugated secondary antibo-dies. After a further three times washing with TBST, membraneswere incubated with Pierce ECL2 Western Blotting Substrate(Thermo) and exposure with ChemiDoc MP Imaging Systemsand Image Lab software (Bio-Rad).
Breast cancer molecular subtype and survival analysisCancer subtype–specific gene expression analyses were per-
formed using data on UCSC cancer browser, https://genome-cancer.ucsc.edu/proj/site/hgHeatmap/ (TCGA RNAseq RPKM
level 3 value, N ¼ 1,215). Correlation between gene expressionsand overall survival of 1,789 patients was performed using theGOBO algorithm (http://co.bmc.lu.se/gobo/).
Cell cultureAll breast cancer cell lines were obtained by the ATCC. MCF-7,
T-47D, MDA-MB-361, MDA-MB-415, MDA-MB-231, BT-549,Hs 578T, and MDA-MB-157 breast cancer cell lines were grownin DMEM medium supplemented with 10% FBS, and theiridentities have been authenticated. SKBR3 cells were grown inMcCoy's 5A medium. HCC1806, HCC70, and HCC1937 weregrown in RPMI medium supplemented with 10% FBS. HMECand MCF10A normal breast epithelial cell line were grown inDMEM/F12 supplemented with 5% horse serum, 20 ng/mLepidermal growth factor, 0.5 mg/mL hydrocortisone, 100 ng/mLcholera toxin and 10 mg/mL insulin. All media were alsosupplemented with 5,000U/mL penicillin/streptomycin. Allcells were grown at 37 �C in a 5% CO2 atmosphere.
siRNAs and transfectionKDM6B siRNAs, IGFBP5 siRNAs, and EZH2 siRNAs listed
below were purchased from IDT (Integrated DNA Technologies,Singapore). For transient knockdown experiments, siRNAs weretransfected into cells using Lipofectamine RNAimax (ThermoFisher Scientific) following manufacturer's instructions. Forty-eight hours after transfection, cells were used for further experi-ments.
To generate the IGFBP5-overexpressing plasmid, complemen-tary DNAs of human IGFBP5 were amplified from pcDNA3-IGFBP5-V5 (Addgene plasmid: 11608) by PCR and inserted intothe GFP-based PMN retroviral expression vector (a gift from Dr.Linda Penn, University of Toronto, Canada).
Cell viability assays and flow cytometryCells were plated at a density of 1,000 cells/well in a 96-well
optical bottom plate (Corning) and left to incubate overnightbefore small interfering RNA or inhibitor treatment. Cell pro-liferation or viability was determined using CellTiter-Glo(Promega) following the manufacturer's instruction. Quanti-tation of DNA content using flow cytometry was used to ana-lyze cell cycle and quantify the sub-G1 population, which isreflective of the number of hypo-diploid cells undergoing a latestage of apoptosis following endonucleases activity. Briefly,treated cells were fixed with 70% ethanol for one hour at 4�Cand stained with propidium iodide (50 mg/mL) for 30 minutesin the dark. To verify cell apoptosis independently, treated cellswere also stained with JC-1 dye to analyze mitochondrialpermeability using the BD MitoScreen (catalog number:551302) following manufacturer's instruction. Briefly, cellswere harvested and collected by Centrifuge at 400 � g for 5
Wang et al.
Mol Cancer Ther; 17(9) September 2018 Molecular Cancer Therapeutics1974
minutes at room temperature. After removing the supernatant,cells were gently resuspended in 0.5 mL of freshly prepared JC-1Working Solution. Cells were incubated the in JC-1 WorkingSolution for 10 to 15 minutes at 37�C in a CO2 incubator. Thencells were washed twice with 1� Assay Buffer and at 400� g for5 minutes. The stained cells were analyzed using FACScaliburand CellQuest software (BD Biosciences) after resuspending in0.5 mL of 1 � Assay Buffer.
Microarray gene expression and quantitative PCR analysisTotal RNA was isolated and purified using the RNeasy Mini Kit
(Qiagen). Human HT-12 V4.0 expression Beadchip (Illumina)was used for microarray hybridization and data were analyzedusing GeneSpring software (Agilent Technologies). The differen-tial gene expression was determined by 1.5-fold cutoff and sta-tistical analysis (P < 0.05) using student t test. After analysisdifferentially expressed gene sets were uploaded to IngenuityPathway Analysis (IPA; www.ingenuity.com) for gene ontologyanalysis. Allmicroarray data have been deposited at the depositedat the National Centre for Biotechnology Information's GeneExpression Omnibus (accession no. GSE74655). Reverse-tran-scription and quantitative PCR assays were performed usingHigh Capacity cDNA Archive kit and KAPA SyBr Fast qPCR kit(KAPA Biosystems). PCR reactions were analyzed in 96-wellformat using the Applied Biosystems PRISM 7500 Fast Real-Time PCR system. For quantification of mRNA levels, GAPDHwas used as internal controls. Real-time primer sequences are asfollows: KDM6B (Forward primer CGAGTGGAATGAGGT-GAAGA, and reverse primer GCAGCAGGCAGAACTTGAT);EZH2 (Forward Primer TGGTCTCCCCTACAGCAGA andreverse primer TCCTGATCTAAAACTTCATCTCCCA); IGFBP5(Forward primer GGTTTGCCTCAACGAAAAGA and reverseprimer CGGTCCTTCTTCACTGCTTC); KDM6A (Forward prim-er TTTGTCAATTAGGTCACTTCAACCTC and reverse primerAAAAAGGCAGCATTCTTCCAGTAGTC).
Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) assay was performed
as described previously (25). KDM6B (catalog number: ab38113)and H3K27ac (catalog number: ab4729) from Abcam, EZH2(catalog number: 39876) from Active Motif, H3K27me3 (catalognumber: 9733) from Cell Signaling Technology and IgG (catalognumber: sc2027) from Santa Cruz Biotechnology were used. TheimmunoprecipitatedDNAwas analyzed by real-time quantitativePCR and enrichments of target proteins on examined DNAregions were quantitated relative to the input DNA. The followingprimers were used to amplify the indicated regions surroundingthe transcription start sites of IGFBP5: P1(forward AGATCAG-GATCTGGGGGTGT and reverse TCTTGCTCGTTCAGTTCAGG);P2(forward TCATTGTGTTCACCCTGCTC and reverse GGAATG-TAAGAAAGGGGCAAG); P3(forward AGTGTGGGCTTTTTCC-CTTT and reverse AAACCCCAAACCCTAACACC); P4(forwardACCTGCTCTACCTGCCAGAA and reverse AGCGAGAGTG-CAGGGATAAA); P5(forward AACACCCCACATCCTTGGTA andreverse CCACAGGCAATCATCTTCAA).
Statistical analysisAll experiments were repeated at least three times unless
stated otherwise, and data are expressed as mean � SEM(standard error of the mean). Statistical analyses were per-formed using GraphPad PRISM6. The Student t test and
ANOVA followed by Tukey's, or Dunnett's tests were used tocalculate the P values (�, P < 0.05; ��, P < 0.01; ���, P < 0.001;����, P < 0.0001).
ResultsKDM6B is oncogenic in luminal breast cancer
Because the global level of H3K27me3 has been observed to bedownregulated in high-grade solid tumors, including breast can-cer (13, 14, 26), we hypothesized that a skewed balance betweenthe expression/activity of EZH2 andKDM6B/KDM6A could resultin the downregulation of H3K27me3. Our analysis using thebreast cancer online database of GOBO (http://co.bmc.lu.se/gobo) shows that luminal A and luminal B tumors have higherexpression of KDM6B and KDM6A but lower EZH2 levels com-pared with basal-like tumors (Fig. 1A; Supplementary Fig. S1A).Similarly, in breast cancer cell lines, KDM6B and KDM6A alsoshowed higher expression in luminal lines compared with basallines (Supplementary Fig. S1B). Moreover, western blot analysisalso showed that KDM6B is highly expressed in luminal breastcancer cell lines, especially in T-47D, MCF-7 and MDA-MB-361cells (Supplementary Fig. S1C). Analysis of the breast cancersurvival rates using GOBO (http://co.bmc.lu.se/gobo/gobo.pl)shows that KDM6B, but not KDM6A, is positively correlated withthe poor overall survival in luminal A and luminal B but not inbasal tumors (Fig. 1B; Supplementary Fig. S2). Given the previousreports showing that a low level of H3K27me3 is correlated withpoor survival in breast cancer (13–15), we hypothesized thatKDM6B is a key regulator of H3K27me3 in breast cancer.
Given a potential oncogenic role of KDM6B in luminal breastcancer, we sought to investigate whether pharmacologic inhibi-tion of KDM6B could provide therapeutic benefits. We treated apanel of breast cancer cells consist of varying PIK3CA mutationswith small-molecule GSKJ4, which has been shown to be aneffective inhibitor of H3K27me3 demethylase KDM6B/KDM6A(24). Strikingly, GSKJ4 preferentially induced cell death in lumi-nal breast cancer cell lines (MCF-7, T-47D, and MDA-MB-361)that carry activating PIK3CA mutations such as E545K andH1047R (Fig. 1C), with significantly lower IC50s, compared withPIK3CAwild-type cells (Fig. 1D). A time-course analysis shows theeffect of GSKJ4 onMCF-7, T-47D, andMDA-MB-361 cells but noton PIK3CA wildtype luminal MDA-MB-415 or non-cancerousMCF10A cells (Fig. 1E). A dose–response analysis shows thatGSKJ4 induced upregulation of H3K27me3 and increased PARPcleavage in MCF-7, T-47D and MDA-MB-361 cells but not inMDA-MB-415 and MCF10A cells (Fig. 1F), indicating a preferen-tial effect of GSKJ4 on PIK3CA mutant cancer cells. Similar toGSKJ4 treatment, knockdown of KDM6B induced H3K27me3 inall MCF-7, T-47D, MDA-MB-361 cells, SKBR3, MDA-MB-415,BT20 and SUM159 cells but only resulted in obvious PARPcleavage in PIK3CA-mutant luminal breast cancer cells: MCF-7,T-47D and MDA-MB-361 cells (Fig. 1G; Supplementary Fig. S3),which were accompanied by a deficiency in cell proliferation andcolony formation (Fig. 1H). These findings suggest that KDM6Bhas a role in conferring a survival advantage in PIK3CA-mutantluminal breast cancer.
Combined KDM6B and PI3K inhibition sensitize cancercells to apoptosis
We next tested whether a combination of GSKJ4 with a PI3Kinhibitor GDC-0941 or an AKT inhibitor MK2206 would
KDM6B-Induced IGFBP5 Confers Resistance to PI3K Inhibitor
www.aacrjournals.org Mol Cancer Ther; 17(9) September 2018 1975
produce more cell death. Indeed, the combination treatmentresulted in more cell death compared with single-agent treat-ment in MCF-7 and T-47D cells but not in SKBR3, BT20 andSUM159 cells as determined by propidium iodide DNA stain-ing of cells in Sub-G1 phase (Fig. 2A). An independent apo-ptosis assay, JC-1 that correlates mitochondrial health withapoptosis further confirmed the result (Fig. 2B). Western blotanalysis shows that, compared with single-agent treatment, thecombination treatments induced more PARP cleavage whichcorrelated with increased H3K27me3 (Fig. 2C). Of note, theinhibitory phosphorylation of EZH2 at serine 21 (p-EZH2 S21)was reduced by GDC-0941 or MK2206 treatment which wasmore evident in the combined treatment conditions (Fig. 2C).Consistent with pharmacologic inhibition, genetic depletion of
KDM6B also sensitized MCF-7 and T-47D cells but not SKBR3,BT20 and SUM159 cells to GDC-0941 (Fig. 2D). These datasuggest that co-targeting KDM6B and PI3K/AKT effectivelyinduced more up-regulation of H3K27me3, which was accom-panied by more robust apoptosis.
IGFBP5 downregulation contributes to apoptosis induced bycombined KDM6B and PI3K inhibition
Co-inhibition of PI3K and KDM6B may increase theH3K27me3 level by simultaneously relieving inhibitory EZH2phosphorylation at serine 21 while suppressing the KDM6Bdemethylase activity. This led us to hypothesize that an increasedH3K27me3may lead to inhibition of an active gene transcriptionprogram which might be important to coordinate or mediate the
H
D
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T-47D
siKDM6B: NC #1 #2 NC #1 #2
BT2
0H
CC
1806
HC
C70
HC
C19
37M
DA
-MB
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MD
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31S
UM
159
BT5
49H
s578
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-MB
-157
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3
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MCF-7 MCF10AT-47D MDA-MB-361 MDA-MB-415
Pro
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n(%
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mol
/L)
PI3K Mutant PI3K Wild-type
0 1 2 3 4 50
2 0 0
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6 0 0 0
0 .10 .512 .55
MC
F-7
T-47
DM
DA
-MB
-361
MD
A-M
B-4
15
E54
5KH
1047
RE
545K
H10
47R
H10
47L
NormalBasal-likeLuminal
AAll tumors : KDM6BHU subtypes P = <0.00001
357 152 482 289 257 344
2
Log2
Exp
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ion
All tumors : EZH2HU subtypes P = <0.00001
Bas
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- like
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-like
Unc
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0
-1
-2
-3
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0
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-6
357 152 482 289 257 344
(-3.19, -0.04): n = 86(0.04, -2.56): n = 103
Luminal A
Ove
rall
surv
ival
(%) KDM6B
Luminal B
Time (years) Time (years) Time (years)
B
P = 0.0005(-3.19, -0.04): n = 63(0.04, -2.56): n = 35
KDM6B overexpression is associated with poor clinical outcome and depletion of KDM6B triggers apoptosis. A, Expression levels of KDM6B and EZH2 mRNA indifferent subtypes of breast cancer from GOBO database. B, Kaplan–Meier analysis of overall survival of breast cancer patients with tumors expressing high (red) orlow (gray) levels of KDM6B using the GOBO database. Curves were compared by log-rank test. C, Cell death induced by histone demethylase inhibitorGSKJ4 (10 mmol/L) in a panel of breast cell lines with and without PIK3CA mutations. Data represent the percentage of cells in sub-G1 phase as measured by flowcytometry following propidium iodide staining. D, IC50 of GSJ4 in a panel of breast cell lines as measured by CellTiter-Glo Assay. E, Cell viability measured byCellTiter-Glo Assay of PI3K mutant cells (MCF-7, T-47D, MDA-MB-361) and PI3K wildtype cells (MDA-MB-415, MCF10A) treated with GSKJ4 at the indicatedconcentrations and time points up to 5 days. F, Western blot analysis of PI3K mutant cells (MCF-7, T-47D, MDA-MB-361) and PI3K wildtype cells (MDA-MB-415,MCF10A) treatedwith GSKJ4 for 3 days at the indicated concentrations.G, Effects of two independentKDM6B siRNAs onmRNA levels byQuantitative RT-PCR (top)and protein expression levels of indicated histone mark and apoptosis genes by Western blot analysis (bottom) in MCF-7, T-47D, and MDA-MB-361cell lines. H, Cell viability measured by CellTiter-Glo Assay (top) and representative colony formation images (bottom) of MCF-7, T-47D and MDA-MB-361cells depleted of KDM6B by two independent siRNAs. Patients and cell lines are stratified according to breast cancer subtype as indicated. � , P < 0.05, pairedtwo-tailed t test (C). Data represent mean � SEM of three replicates.
Wang et al.
Mol Cancer Ther; 17(9) September 2018 Molecular Cancer Therapeutics1976
apoptotic response to the combination treatment. As these threeinhibitors all induced H3K27me3, we sought to determine thecommon gene set which can be repressed by all the three inhi-bitors. Microarray gene expression analysis of MCF-7 cells treatedwith GSKJ4, GDC-0941, and MK2206, revealed a common set of35 genes whose expression were downregulated (using a 1.5-foldcutoff, P < 0.05) by all the three inhibitors (Fig. 3A). We hypoth-esized that among these 35 genes there are potential candidatesthat are co-regulated by both KDM6B and PI3K/AKT,whichmightbe responsible for the combinatorial killing effect. We first ana-lyzed 1,215 breast tumors fromTCGAand found that 14 out of 35genes were expressed significantly higher in luminal tumorscompared with basal tumors (Supplementary Fig. S4A). Furtheranalysis of gene expression and patient survival data (GOBO)identified IGFBP5 as the only gene (out of the above 14 genes)being both upregulated in luminal tumors and significantlyassociated with poor survival in luminal A breast cancer patients(Fig. 3B and C; Supplementary Fig. S4B). Consistent with themRNA level, secreted IGFBP5 proteins are alsomuch higher in theluminal breast cancers (Supplementary Fig. S4C). These in silicoanalyses of cancer genomic databases and clinical information
support IGFBP5 being a key candidate regulated by KDM6B andPI3K/AKT/EZH2 in luminal breast cancer.
RT-PCR and Western blotting further validated the micro-array data and showed that GSKJ4, MK2206 or GDC-0941treatment downregulated the IGFBP5 expression, which wasmore evident in combination treatments (Fig. 3D). KDM6Bknockdown was also able to reduce IGFBP5 expression in bothMCF-7 and T-47D cells (Fig. 3E), but KDM6A knockdown didnot affect the IGFBP5 expression (Supplementary Fig. S4D),excluding a role of KDM6A in regulating IGFBP5 expression.Furthermore, depletion of IGFBP5 in MCF-7 and T-47D cellsresulted in PARP cleavage, decreased survivin and p-ERK 1/2, aswell as increased Bim expression (Fig. 3F). Interestingly,although reduced IGFBP5 was also caused by KDM6B knock-down in MDA-MB-415 and BT20 cells (Supplementary Fig.S5A), neither KDM6B knockdown nor IGFBP5 knockdownresults in substantial cell death in SKBR3, MDA-MB-415, BT20and SUM159 cells (Supplementary Figs. S3 and S5B). Ectopicexpression of IGFBP5 partially rescued cell death induced bytreatment with GSKJ4 and GDC-0941(Fig. 3G). Together, thesedata indicate that downregulation of IGFBP5 is necessary for
D
0
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− + − + − +GSKJ4:− − + + − −− − − − + +
MK2206:GDC-0941:
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**
Apo
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is(%
)A
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(%)
C
H3
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H3K27me3
p-Akt (S473)
Actin
p-ERK 1/2
c-PARP
p-EZH2 (S21)
MCF-7
− + − + − +GSKJ4:− − + + − −− − − − + +
MK2206:GDC-0941:
1.0 16 25 42 22 31
1.0 2.7 2.2 2.8 2.3 3.4
1.0 0.7 0.5 0.3 0.2 0.2
1.0 0.8 0.4 0.3 0.4 0.5
1.0 0.8 0.5 0.2 0.4 0.2
MW(kDa)
37
50
75
2015
5037
20155037
100
100
SKBR3
PI staining
si.KDM6B#1: _ _ _ _+ +
GDC-0941: _ + _ +_ +
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0
si.NC: + + _ __ _
si.KDM6B#2: _ _ _ _ + +
BT20
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si.KDM6B#1: _ _ _ _+ +
GDC-0941: _ + _ +_ +
50
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si.NC: + + _ __ _
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PI staining
si.KDM6B#1: _ _ _ _+ +
GDC-0941: _ + _ +_ +
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T-47D
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si.KDM6B#1: _ _ _ _+ +
GDC-0941: _ + _ +_ +
50
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(%)
Sub
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(%)
Sub
-G1
(%)
Sub
-G1
(%)
si.KDM6B#1: _ _ _ _+ +
GDC-0941: _ + _ +_ +
100
80
60
40
20
0
si.NC: + + _ __ _
si.KDM6B#2: _ _ _ _ + +
BT2050
40
30
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10
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SKBR3
PI staining
50
40
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SUM159
GSKJ4:MK2206:
GDC-0941:
_ + _ + _ +_ _ _ _+ +
_ _ + +_ _
50
40
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Figure 2.
Combined inhibition of KDM6B and PI3K sensitizes cancer cells to apoptosis. A, Measurement of apoptotic cells in sub-G1 by propidium iodide followed by flowcytometry after MCF-7, T-47D, SKBR3, BT20 and SUM159 cells were treated with GSKJ4 (5 mmol/L), MK2206 (2 mmol/L) and GDC-0941 (1 mmol/L) or incombination for 3 days as indicated.B,Measurement of the percentage of apoptotic cells by JC1Mitochondrial Membrane Potential assayafterMCF-7 and T-47Dcellswere treated in the same condition as in (A). C, Western blot analysis in MCF-7 cells treated with the indicated inhibitors at the same concentrations as in(A) and (B) for 3 days. Western blot bands were quantified by Image lab and normalized to actin or their respective unmodified total protein, then comparedwith vehicle-treated controls. D, The percentage of apoptotic cells by propidium iodide staining of KDM6B-depleted MCF-7, T-47D, SKBR3, BT20, andSUM159 cells treated with GDC-0941 (1 mmol/L) for 3 days as indicated, respectively. P values (���� , P < 0.0001; ��� , P < 0.001; �� , P < 0.01; � , P < 0.05) in thisfigure denote ANOVA followed by Dunnett's test (A and B) and paired two-tailed t test (D). Data represent mean � SEM of three replicates.
KDM6B-Induced IGFBP5 Confers Resistance to PI3K Inhibitor
www.aacrjournals.org Mol Cancer Ther; 17(9) September 2018 1977
the effective apoptosis induction by co-targeting KDM6B andPI3K/AKT.
The IGFBP5 expression is co-regulated by both KDM6B andPI3K/AKT/EZH2
To determine whether IGFBP5 is a direct target of KDM6B andEZH2 (whose activity is regulated by PI3K/AKT through inhibi-tory phosphorylation; ref. 21), we performed ChIP analysis inMCF-7 cells. Using known EZH2 targets CDKN1C and CDH1 aspositive controls, we show that both KDM6B and EZH2 wereenriched in the IGFBP5 gene promoter flanked byH3K27me3 andH3K27Ac marks (Fig. 4A). Depletion of EZH2 by two indepen-dent siRNAs resulted in decreased H3K27me3 and increased
IGFBP5 expression (Fig. 4B). Similarly, pharmacologic inhibitionof EZH2 byGSK126 also reduced H3K27me3 and augmented theIGFBP5 expression (Fig. 4C). However, this effect of GSK126 onboth H3K27me3 and IGFBP5 was reversed by co-treatment withGDC-0941(Fig. 4C), as it is known that inhibition of PI3K/AKTbyGDC-0941 can induce H3K27me3 by removal of AKT-inducedinhibitory phosphorylation of EZH2. Moreover, overexpressionof a constitutively activemutant of PIK3CA- PIK3CAE545K in 293Tcells upregulated IGFBP5 through inhibition of EZH2 activitywhereas re-introduction of EZH2 in these PIK3CAE545K-expres-sing cells partially reversed the IGFBP5 level (Fig. 4D). Thesefindings support a direct and counteractive regulation of IGFBP5by KDM6B and PI3K/AKT-regulated EZH2. Moreover, consistent
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Figure 3.
Combined KDM6B and PI3K inhibition targets IGFBP5 leading to cancer cell apoptosis. A, Venn diagram (top, not drawn to scale) showing the overlap of genesdownregulated by GSKJ4 (5 mmol/L), MK2206 (2 mmol/L) and GDC-0941 (1 mmol/L) in MCF-7 cells for 3 days. Bottom heatmap shows 35 commonlydownregulated genes by each inhibitor. B, Scatter plots showing the overall expression levels of IGFBP5 in breast cancers, analyzed from the GOBO databaseas indicated. C, Kaplan-Meier curves comparing overall survival of breast cancer patients with tumors expressing high (red) or low (gray) levels of IGFBP5using the GOBO database. Curves were compared by log-rank test. D, Measurement of IGFBP5 mRNA level by Quantitative RT-PCR (top) and IGFBP5 proteinslevels by Western blot (bottom) in MCF-7 cells treated with indicated inhibitors at the same concentration and duration as in (A). E, Measurement of IGFBP5mRNA level by Quantitative RT-PCR (top) IGFBP5 proteins levels by Western blot (bottom) in indicated cells depleted of KDM6B by siRNAs. F, Westernblot analysis of indicated pro-apoptotic proteins in MCF-7 and T-47D cells where IGFBP5 is depleted by two independent siRNA sequences. G, The percentage ofapoptotic cells by propidium iodide staining (left) in MCF-7 cells with ectopic IGFBP5 expression combined with GSKJ4 (5 mmol/L) and GDC-0941 (1 mmol/L)treatment for 3 days. IGFBP5 protein levels are shown in the right. P values (���� , P < 0.0001; ��� , P < 0.001; � , P < 0.05) in this figure denote ANOVAfollowed by Dunnett's test (D and E) and paired two-tailed t test (G). Data represent mean � SEM of three replicates.
Wang et al.
Mol Cancer Ther; 17(9) September 2018 Molecular Cancer Therapeutics1978
with the combined effect on IGFBP5 expression, co-treatment ofGSKJ4 with MK2206 or GDC-0941 resulted in increasedH3K27me3 but reduced H3K27Ac at the IGFBP5 promoter (Fig.4E). These data indicate that co-targeting of KDM6B and PI3K/AKT was able to downregulate IGFBP5 effectively through mod-ulatingH3K27me3 andH3K27Acmarkers, resulting in apoptosis.
Breast cancer cells acquiring resistance to PI3K inhibitorremain highly sensitive to KDM6B inhibition
To determine whether therapeutic targeting of KDM6B is rel-evant in breast cancer cells resistance to a PI3K inhibitor, wegenerated three PIK3CA mutant breast cancer cell lines (MCF-7,T-47D, and MDA-MB-361) that had acquired resistance to PI3Kinhibitor treatment following prolonged exposure to GDC-0941(Fig. 5A). Interestingly, although the three GDC-0941-resistantlines were refractory to GDC-0941 treatment compared with theparental lines, they remained sensitive to GSKJ4 treatment at a
level comparable to their corresponding parental lines (Fig. 5B).Similarly, depletion ofKDM6B reduced expression of IGFBP5 andinduced PARP cleavage and cell death equally effectively in bothparental and resistant cells (Fig. 5C). These data suggest thatalthough these cells become resistant to the PI3K inhibitor, theyremain sensitive to KDM6B inhibition.
Upregulationof IGFBP5due to epigenetic switch inGDC-0941–resistant cells confers a growth dependency
Strikingly, IGFBP5 expression was upregulated in GDC-0941-resistant cells compared with parental cells (Fig. 6A andB), which was however accompanied by decreased p-AKT andincreased H3K27me3 (Fig. 6B). This finding suggests that PIK3/AKT-mediated regulation on EZH2/H3K27me was not predom-inant in regulating IGFBP5 expression in GDC-0941–resistantcells. Examination of the IGFBP5 gene promoter by ChIPshowed an increase in H3K27Ac and a decrease in H3K27me3
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Figure 4.
Co-regulation of IGFBP5 expression by KDM6B and PI3K/AKT/EZH2.A, Left showsH3K27me3 and EZH2 enrichments on promoters of two knownEZH2 targets. Topright diagram shows PCR primers location encompassing the IGFBP5 promoter. The bottom right panel shows Chromatin immunoprecipitation-quantitativeRT-PCR showing EZH2, KDM6B, H3K27me3, and H3K27Ac enrichments near the IGFBP5 promoter in MCF-7 cells. B, Quantitative RT-PCR for IGFBP5mRNA levels(top) and Western blot analysis of indicated proteins (bottom) in MCF-7 cells depleted of EZH2 by two independent siRNA sequences. C, Quantitative RT-PCR forIGFBP5 mRNA levels (top) and Western blot analysis of indicated proteins (bottom) in MCF-7 cells treated with GSK126 (2.5 mmol/L), GDC-0941 (1 mmol/L),or in combination for 3 days.D,Western blot analysis of indicated proteins in 293T cellswhere PIK3CAE545K or/and EZH2 is overexpressed after 48 hours as indicated.E, Chromatin immunoprecipitation-quantitative RT-PCR of H3K27me3 (top) and H3K27Ac (bottom) enrichments on IGFBP5 promoter in MCF-7 cells treatedwith GSKJ4 (5 mmol/L), MK2206 (2 mmol/L), GDC-0941 (1 mmol/L) or in combination as indicated for 48 hours. P values (���� , P < 0.0001; ��� , P < 0.001; �� , P < 0.01;� , P < 0.05) in this figure denote ANOVA followed by the Dunnett's test (B and E), Tukey's test (C).
KDM6B-Induced IGFBP5 Confers Resistance to PI3K Inhibitor
www.aacrjournals.org Mol Cancer Ther; 17(9) September 2018 1979
in GDC-0941–resistant cells as compared with the parentalcells (Fig. 6C), suggesting an epigenetic switch at the chromatinlevel leading to the upregulation of IGFBP5. Similar to KDM6Bknockdown, IGFBP5 knockdown equally or even more prom-inently induced PARP cleavage and cell death in GDC-0941–resistant lines compared with parental lines (Fig. 6D). Collec-tively, these findings suggest that IGFBP5 expression is pro-moted by both KDM6B and PI3K/AKT/EZH2 through suppres-sing H3K27me3 at the IGFBP5 promoter and KDM6B may playa major role in regulating IGFBP5 expression to confer survivaladvantage upon resistance to PI3K inhibitor (Fig. 6E).
DiscussionPIK3CA mutations occur in up to 40% of luminal breast
tumors, which provides a rationale for therapeutic targeting ofPI3K/AKT signaling in PIK3CA-mutant breast cancers (27, 28).However, inmultiple clinical trials, PI3Kor AKT inhibitors used asa single-agent have repeatedly demonstrated lower than expectedefficacy with partial tumor regression and long-term diseasestabilization only in a small number of patients (29). Here, weprovide evidence that inhibition of an epigenetic regulator, theH3K27demethylase KDM6B, can enhance the apoptotic responseto PI3K/AKT inhibitors in breast cancer cells.
We show that IGFBP5 is a direct target of KDM6B and thatIGFBP5 expression level is a crucial modulator of PI3K/AKTinhibitor response. IGFBP5 is also a target of EZH2 whose activitytowards H3K27me3 is negatively regulated by PI3K/AKT-medi-ated phosphorylation. Thus, we propose that epigenetic (KDM6Boverexpression) and oncogenic (PI3K activation) deregulationsmay converge on the chromatin level, resulting in a low enrich-ment of H3K27me3 at the IGFBP5 promoter, resulting in IGFBP5overexpression in luminal breast cancer. As such, combinedinhibition of both KDM6B and PI3K/AKT is necessary to inhibitIGFBP5 expression to trigger effective apoptosis. This strategydemonstrates that epigenetic approaches to increase H3K27me3and thus suppress some key pro-survival oncogenes might beexploited to enhance therapeutic response. Our study supports apotential use of GSKJ4 in combination with PI3K inhibitor inluminal breast cancers with PIK3CAmutation.Of note, GSKJ4 hasbeen shown to be effective in cervical cancer, acute lymphoblasticleukemia, and prostate cancer (17, 30, 31), and recent researchalso showed that GSKJ4 is an effective inhibitor targeting breastcancer stem cells (32). Our findings advocate the use of GSKJ4 ormore specific KDM6B inhibitors to treat luminal breast cancerwith PIK3CA mutation in future clinical trials.
Although both H3K27me3 demethylases KDM6B and KDM6Ashowed higher expression in luminal than basal tumors, only
Breast cancer cells acquiring resistance to PI3K inhibitor remain highly sensitive to KDM6B inhibition.A,Cell viability by CellTiter-Glo AssaymeasuringMCF-7, T-47D,and MDA-MB-361 parental cell lines compared with corresponding GDC-0941–resistant cell lines generated by exposing parental cell lines to gradually increasethe concentration of GDC-0941 until a target concentration of 1 mmol/L. B, Cell viability assay measuring three parental cell lines and correspondingGDC-0941–resistant cell lines. Cells were treated with DMSO, GSKJ4 (5 mmol/L) or GDC-0941 (1 mmol/L) at indicated time points for 6 days, respectively. C,Westernblot analysis of indicated proteins in MCF-7 and T-47D parental cell lines and corresponding GDC-0941–resistant lines (left), and percentage of cells inSub-G1 by propidium iodide staining (right) in indicated cell lines where KDM6B was depleted by two independent siRNAs.
Wang et al.
Mol Cancer Ther; 17(9) September 2018 Molecular Cancer Therapeutics1980
KDM6B is significantly associated with patient survival, suggest-ing an oncogenic role of KDM6B but not KDM6A in breast cancer.Consistent with this notion, we found that depletion of KDM6B,but not KDM6A, downregulated IGFBP5. The oncogenic role ofKDM6B in prognosis is consistent with previous findings that lowH3K27me3 predicts poor prognosis, though a low level of EZH2shows the opposite (13–15). These data suggest that KDM6Bcould be a more important regulator of H3K27me3 in luminalbreast tumors compared with EZH2 whose epigenetic activity isoften restricted by inhibitory phosphorylationmediated by onco-genic signaling such as AKT.
Our study suggests a role of IGFBP5 in luminal breast cancer topromote survival. Although a tumor-suppressor role of IGFBP5has also been shown (33–35), our data are consistent with theprevious reports showing that IGFBP5 facilitates survival in breastcancer MCF-7 cells (33) and promotes resistance to IGF-IR inhib-
itor (36). Furthermore, high level of IGFBP5 has been found intumors and has the pro-metastatic capacity (37), and overexpres-sion of IGFBP5 was associated with advanced tumor stage, poorclinical outcomes in breast cancer patients and urothelial carci-noma patients (38, 39). As a critical mediator of PI3K/AKT andMAPK/ERK survival pathways, IGFBP5not only acts to inhibit IGFbinding to the insulin receptor to inhibits the downstream PI3Ksignaling output but also functions in the cytoplasm to promotegrowth in an IGF-independent manner (40–42). Therefore, onepossibility is that IGFBP5 has both tumor suppressor role and theoncogenic role and its fine regulation determine its main activityin a context-dependent manner. Interestingly, upon acquiringresistance of breast cancer cells to GDC-0941, IGFBP5 was upre-gulated, and its promoter underwent an epigenetic switch fromH3K27me3 to H3K27Ac. Importantly, breast cancer cells acquir-ing resistance to GDC-0941 showed remained dependency on
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IGFBP5 upregulation confers a growth advantage in PI3K inhibitor-resistant cells. A, Quantitative RT-PCR analysis of IGFBP5 mRNA in MCF-7, T-47D parental celllines, and corresponding GDC-0941–resistant cell lines. B, Western blot analysis of indicated protein levels in MCF-7, T-47D parental cell lines, and correspondingGDC-0941–resistant cell lines. C, Chromatin immunoprecipitation-quantitative RT-PCR of H3K27Ac (left) and H3K27me3 (right) enrichments on IGFBP5 promoter inMCF-7 parental cells compared with GDC-0941–resistant cells. D, Western blot analysis of indicated proteins in MCF-7 and T-47D parental cell lines andcorresponding GDC-0941–resistant lines (left), and percentage of cells in Sub-G1 by propidium iodide staining (right) in indicated cell lines where IGFBP5 wasdepleted by two independent siRNAs. E, Schematicmodel showing co-dependency of PI3K/AKT/EZH2 and KDM6B signaling pathways in luminal breast cancer withPIK3CA mutation that underlie the rationale for co-targeting these pathways to improve initial response and overcome resistance to PI3K inhibition.
KDM6B-Induced IGFBP5 Confers Resistance to PI3K Inhibitor
www.aacrjournals.org Mol Cancer Ther; 17(9) September 2018 1981
KDM6B and IGFBP5 for survival, suggesting when PI3K signalingis inhibited, the cellsmay still rely on IGFBP5 expression to confera survival advantage. These findings support the notion thatupfront KDM6B inhibition in luminal breast cancer might beable to sensitize PI3K inhibitor treatment as well as to forestallresistance to PI3K inhibitors.
Our study provides an example of how H3K27me3-mediatedrepression of an oncogene such as IGFBP5 can be targeted toachieve a therapeutic effect. In this scenario, increasingH3K27me3 by targeting H3K27me3 demethylase might be auseful approach to inhibit the expression of a crucial oncogene,though there is a concern that the global effect on H3K27me3might also restore the expression some tumor-suppressor genes tocounteract the benefit.
In summary, ourfindings demonstrate that targetingKDM6B incombination with PI3K inhibition represents a novel strategy toimprove initial response and overcome resistance to PI3K inhi-bitors. This combination treatment effectively reduces IGFBP5 insensitive and PI3K inhibitor-resistant cells to induce apoptosis.Given that IGFBP5 is overexpressed in luminal breast cancer andfurther upregulated upon acquiring resistance to PI3K inhibition,it warrants further study to determinewhether IGFBP5 level can beused as a predictive biomarker to monitor response to KDM6B/PI3K inhibition and development of drug resistance. Our strategyof combining KDM6B and PI3K inhibition represents a newparadigm in harnessing epigenetic therapeutics to overcome drugresistance. Further studies are thus warranted to evaluate thiscombination in a clinical setting.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: W. Wang, K.G. Lim, Q. YuDevelopment of methodology: W. Wang, K.G. Lim, M. FengAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W. Wang, K.G. Lim, Y. Bao, D.S.B. HoonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. Wang, K.G. Lim, M. Feng, Y. Bao, H. Zhang,D. Marzese, D.S.B. Hoon, Q. YuWriting, review, and/or revision of themanuscript:W.Wang, K.G. Lim, Y. Cai,D. Marzese, D.S.B. Hoon, Q. YuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K.G. Lim, M. Feng, P.L. Lee, Y. Chen, Q. YuStudy supervision: Q. Yu
AcknowledgmentsThis work was supported by the Agency for Science and Technology of
Singapore (A�STAR to Q.Yu); Margie Petersen Breast Cancer Program (JWCI; toD.S.B. Hoon) and National Medical Research Council (NMRC) of Singapore(OFIRG16may081; to Q. Yu).
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.
Received August 24, 2017; revised March 28, 2018; accepted June 14, 2018;published first June 20, 2018.
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www.aacrjournals.org Mol Cancer Ther; 17(9) September 2018 1983
KDM6B-Induced IGFBP5 Confers Resistance to PI3K Inhibitor
2018;17:1973-1983. Published OnlineFirst June 20, 2018.Mol Cancer Ther Wenyu Wang, Keng Gat Lim, Min Feng, et al. CancerConfer Resistance to PI3K/AKT Inhibitor Treatment in Breast
toIGFBP5KDM6B Counteracts EZH2-Mediated Suppression of
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