plk1 inhibition enhances the efﬁcacy of bet epigenetic reader … · cancer biology and...

Cancer Biology and Translational Studies

Plk1 Inhibition Enhances the Efficacy of BETEpigenetic Reader Blockade in Castration-Resistant Prostate CancerFengyi Mao1,2, Jie Li1, Qian Luo1, Ruixin Wang1, Yifan Kong1,2, Colin Carlock1,Zian Liu1, Bennet D. Elzey3, and Xiaoqi Liu1,4

Abstract

Polo-like kinase 1 (Plk1), a crucial regulator of cell-cycleprogression, is overexpressed in multiple types of cancers andhas been proven to be a potent and promising target for cancertreatment. In case of prostate cancer, we once showed thatantineoplastic activity of Plk1 inhibitor is largely due toinhibition of androgen receptor (AR) signaling. However, wealso discovered that Plk1 inhibition causes activation of theb-catenin pathway and increased expression of c-MYC, even-tually resulting in resistance to Plk1 inhibition. JQ1, a selectivesmall-molecule inhibitor targeting the amino-terminal bro-modomains of BRD4, has been shown to dramatically inhibitc-MYC expression and AR signaling, exhibiting antiprolifera-tive effects in a range of cancers. Because c-MYC and AR

signaling are essential for prostate cancer initiation and pro-gression, we aim to test whether targeting Plk1 and BRD4 at thesame time is an effective approach to treat prostate cancer.Herein, we show that a combination of Plk1 inhibitorGSK461364A and BRD4 inhibitor JQ1 had a strong synergisticeffect on castration-resistant prostate cancer (CRPC) cell lines,as well as in CRPC xenograft tumors. Mechanistically, thesynergistic effect is likely due to two reasons: (i) Plk1 inhibi-tion results in the accumulation of b-catenin in the nucleus,thus elevation of c-MYC expression, whereas JQ1 treatmentdirectly suppresses c-MYC transcription; (ii) Plk1 and BRD4dual inhibition acts synergistically in inhibition of AR signal-ing. Mol Cancer Ther; 17(7); 1554–65. �2018 AACR.

IntroductionProstate cancer is among themost frequently diagnosed cancers

inmen and is one of the leading causes of cancer-associated deathworldwide (1). The American Cancer Society estimates that therewill be 161,360 newly diagnosed cases and 26,730 deaths due toprostate cancer in the United States in 2017 (2). Androgenreceptor (AR) signaling plays a critical role in prostate cancerdevelopment. Consequently, androgen deprivation therapy(ADT) can manage the development of prostate cancer initiallyand is the most widely used and effective systematic therapy totreat patients with prostate cancer with metastatic diseases. How-ever,most patients eventually become resistant to ADT treatment,and the disease enters a stage called castration-resistant prostatecancer (CRPC), an incurable disease at this moment (3, 4).

Therefore, novel therapies for patients with CRPC are urgentlyneeded.

One potential target for therapy would be polo-like kinase 1(Plk1), a serine–threonine kinase that plays a key role in mitosis(5) and has been demonstrated to be required in centrosomematuration and establishment of a bipolar spindle (6). Plk1 isoverexpressed in multiple types of cancers, whereas Plk1 expres-sion is low in surrounding normal, nondividing tissue (5, 7). Ithas been demonstrated that Plk1 is a potent and promising targetfor cancer treatment due to its critical role in cell proliferation andthat Plk1 inhibitors are currently under heavy investigation todetermine their efficacy and safety profiles in diverse tumor types(8). Among these inhibitors is GSK461364A (thiophene deriva-tive; refs. 9–13), which has been shown to potently inhibit theproliferation of various tumor cell lines (10).

BRD4, the most extensively investigated member of bromo-domain and extraterminal domain (BET) family, functions as anepigenetic reader and regulates transcription (14). It has beendiscovered that BRD4 can recruit RNA polymerase II during thetranscription, consequently not only regulating the transcription-al levels of FOS, Jun, and c-MYC (15), but importantly for thisstudy, also physically interacting with AR to help localize AR to itstarget loci (16). JQ1, a small-molecule inhibitor that is highlyspecific for BRD4 (17, 18), can bind competitively to acetyl-lysinerecognition motifs of BRD4 and disrupt its regulation, eventuallyeffectively suppressing tumor growth (18).

Herein, we show a strong synergistic effect using a combina-torial treatment strategy with the Plk1 inhibitor GSK461364Aand the BRD4 inhibitor JQ1 in 22RV1 and C4-2 cells, two veryaggressive human CRPC cell lines, as well as in CRPC xenograft

1Department of Biochemistry, Purdue University, West Lafayette, Indiana.2Department of Animal Sciences, Purdue University, West Lafayette, Indiana.3Department of Comparative Pathobiology, Purdue University, West Lafayette,Indiana. 4Center for Cancer Research, Purdue University, West Lafayette,Indiana.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

F. Mao and J. Li contributed equally to this article.

Corresponding Author: Xiaoqi Liu, Department of Biochemistry, Purdue Uni-versity, 175 S. University Street, West Lafayette, IN 47906. Phone: 765-496-3764; Fax: 765-494-7897; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0945

�2018 American Association for Cancer Research.

MolecularCancerTherapeutics

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tumors. The combination treatment led to G2–M arrest, inhibi-tion of cell growth, a massive increase in apoptosis, and aconcordant drop in glycolysis. We have previously shown thatPlk1 phosphorylation of Axin2, a member of b-catenin pathway,stabilizes the binding between GSK3b and b-catenin in thecytoplasm, resulting in increased degradation of b-catenin. Con-versely, Plk1 inhibition would induce the accumulation of b-cate-nin in the nucleus, resulting in activation of c-MYC (19–21),whilePlk1 inhibition can also inhibit AR signaling (22, 23). As a BRD4inhibitor, JQ1 could effectively antagonize AR signaling andrepress c-MYC transcription (16). We thus propose that theobserved synergistic effect for GSK461364A and JQ1 is due totheir effects on c-MYC and AR signaling.

Materials and MethodsCell culture and drugs

22RV1 and C4-2 cells, purchased from ATCC in 2016, werecultured in RPMI1640 medium supplemented with 10% (v/v)FBS and 100 U/mL penicillin, 100 U/mL streptomycin at37�C in 5% CO2. TRAMP-C2 cells were also purchased fromATCC in 2016, and they were cultured in DMEM with 5% FBS,5% Nu-Serum IV, 100 U/mL penicillin, and 100 U/mL strep-tomycin at 37�C in 5% CO2. All the cells were within 50passages and Mycoplasma was detected every 3 months usingMycoAlert PLUS Mycoplasma Detection Kit (Lonza, LT07-705). GSK461364A, JQ1, and BI2536 (24) were purchasedfrom Selleckchem.

Cell viability assay22RV1 and C4-2 cells were seeded with 5 � 103–1 � 104 per

well in 96-well plates, cultured for 12 hours, and treated withdifferent concentrations of the drugs. After 72 hours of incuba-tion, cells were treated with the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)for 4 hours. Finally, upon resolving the crystal with 100 mL ofDMSO, cells were subjected to a plate reader to measure theabsorbance at 570 nm. The IC50 values were obtained from theaverage viability curves generated by four independent measure-ments of each condition.

Combination indexThe combination index (CI) was calculated using the fol-

lowing equation (25, 26): CI ¼ (Am)50/(As)50 þ (Bm)50/(Bs)50. (Am)50 is the concentration of GSK461364A that couldachieve 50% inhibitory effect in the combination with half ofthe concentration of the JQ1 IC50; (As)50 is the IC50 ofGSK461364A; (Bm)50 is the concentration of JQ1 that willproduce a 50% inhibitory effect in the combination with half ofthe concentration of GSK461364A IC50; and (Bs)50 is the IC50

of JQ1. Antagonism is indicated when CI >1, CI ¼ 1 indicatesan additive effect, and CI <1 indicates synergy.

AntibodiesAntibodies against Axin2 and Ki67 were purchased from

Abcam, whereas antibodies against b-actin (A-5441) and c-Mycwere obtained from Sigma. Antibodies against CD45, CD8,Foxp3, PD-L1, CD4, CD25, CD11b, Ly6C, and Ly6G were pur-chased fromBioLegend. All other antibodieswere purchased fromCell Signaling Technology.

ImmunoblottingCells were washed by PBS after harvest and then resuspended

with TBSN buffer with protease inhibitors and phosphataseinhibitors. After sonication, cell lysates were collected, and pro-tein concentrations were measured by using Protein Assay DyeReagent from Bio-Rad. Mix the proteins from each group withSDS-PAGE loading, respectively, and boil them for 5 minutes.Upon transferring to polyvinylidene difluoride membranes, pro-teins were probed with indicated antibodies (27).

Immunofluorescence stainingAfter murine or human paraffin-embedded slides were depar-

affinized and rehydrated, antigens were retrieved in antigenunmasking solution (Vector Laboratories). Samples were thenincubated with primary antibodies against Ki67, cleaved Caspase3, and PD-L1, followed by incubation with secondary antibodiesand DAPI.

Colony formation assayCells (500–1,000/well) were seeded in 6-well plates and cul-

tured in medium alone or containing different drugs for 14 days,withmedium change every 2 days. After culturing, cells were fixedin 10% formalin and stained with 0.5% crystal violet for 30minutes, followed by counting of colony numbers.

Annexin V-FITC propidium iodideCells (5 � 105/well) were seeded in 6-well plates, cultured in

medium alone or containing different drugs, and subjected to theprocedure using the Annexin V Apoptosis Kit (BioVision, K101-25), followed by analysis with FACS Express 5.

FACS analysisCells (5 � 105/well) were seeded in 6-well plates, cultured in

medium alone or containing different drugs, and harvested.Cells were then fixed in 70% ethanol, stained with 50 mg/mLpropidium iodide, and subjected to FACS analysis.

Patient-derived xenograft modelMice carrying LuCaP35CR tumors were obtained from Dr.

Robert Vessella at the University of Washington (Seattle, WA;ref. 28). Tumors were amplified by cutting the original tumorsinto 20 to 30 mm3 pieces, followed by implantation into preca-strated NSG mice. When tumors reached the size of 250 to300 mm3, mice were randomly separated into four groups forcontrol, GSK461364A alone, JQ1 alone, and combination treat-ment, respectively. The experiment was approved by the PurdueAnimal Care and Use Committee (PACUC).

22RV1-derived mouse xenograft model22RV1 cells were transfected with Flag-Axin2-WT and Flag-

Axin2-311A plasmids and selected with 500 mg/mL G418 for 4weeks. Cells (2.5 � 105 cells/mouse) were mixed with an equalvolume of Matrigel (Collaborative Biomedical Products) andinoculated into the rightflankofNSGmice (Harlan Laboratories).One week later, animals were randomized into treatment andcontrol groups with 4 mice each. JQ1 was delivered via gavage,twice a week. Tumor volumes were estimated from the formula:V ¼ L �W2/2 [V is volume (mm3); L is length (mm);W is width(mm)]. The experiment was approved by the PACUC.

JQ1 in CRPC Treatment

www.aacrjournals.org Mol Cancer Ther; 17(7) July 2018 1555




TRAMP-C2–derived mouse allograft modelTRAMP-C2 cells (1 � 105 cells/mouse) were mixed with an

equal volume of Matrigel (Collaborative Biomedical Products)and inoculated into the right flank of C57BL/6 wild-type (WT)mice (Envigo). One week later, animals were randomized forcontrol, GSK461364A alone, JQ1 alone, combination treatment,and BI2536 alone, respectively. Tumor volumes were estimatedfrom the formula: V ¼ L �W2/2 [V is volume (mm3); L is length(mm); W is width (mm)]. After 10 days of treatment, mice(5 mice/group) were sacrificed and the tumors were obtained.The proportion of PD-L1þ/CD45� cell, T regulatory cells (Treg,CD4þCD25þFoxP3þ), and myeloid-derived suppressor cells(MDSC, CD45þGr-1þCD11bþ) was analyzed by flow cytometry.The experiment was approved by the PACUC.

Serum PSA measurementAfter blood was collected from tumor-carrying mice twice per

week, serum PSA levels were measured using a PSA (human)ELISA Kit (Abnova, KA0208).

Histology and H&E stainingXenograft tumors were fixed in 10% neutral-buffered formalin,

paraffin embedded, sectioned to 5 mm, and stained using con-ventional hematoxylin and eosin (H&E) staining.

Seahorse analysis22RV1 cells (2 � 104/well) were seeded in XFe24 cell culture

microplates in RPMI1640 medium (10% FBS with antibiotics).After 12 hours of incubation, cells were treated with corre-sponding drug(s) for 24 hours. Cartridges were hydrated incalibrant buffer in a non-CO2 incubator at 37�C for at least12 hours before analysis. Before being subjected to Seahorseanalysis, cells were washed with corresponding medium twiceand incubated in a non-CO2 incubator for 1 hour. For glycol-ysis stress test (GST), GST medium was prepared by supple-menting XF base Medium with 2 mmol/L glutamine, and pHwas adjusted to 7.4. For mitochondrial stress test (MST), MSTmedium was prepared by supplementing XF base Medium with2 mmol/L glutamine, 1 mmol/L pyruvate, and 10 mmol/Lglucose, and pH was adjusted to 7.4. The drugs from the XFGST Kit and MST Kit were diluted with corresponding mediuminto designed concentrations and then added into correspond-ing ports of cartridge. After calibration of the cartridge, cellswent through GST or MST programs. Data were analyzed byusing the Seahorse XF Cell GST Report Generator and SeahorseXF Cell MST Report Generator, respectively.

Statistical analysisThe statistical significance of the results was analyzed using

an unpaired Student t test (StatView I, Abacus Concepts Inc.).A P value of less than 0.05 indicates statistical significance.

ResultsCotreatment of GSK461364A and JQ1 acts synergistically

To investigate whether GSK461364A and JQ1 act synergis-tically to inhibit the growth of CRPC cells, 22RV1 and C4-2cells were treated with GSK461364A, JQ1, or a combinationof GSK461364A and JQ1, and harvested for immunoblottinganalysis of the apoptotic marker, cleaved PARP (Fig. 1A and B).As indicated in Fig. 1A, low-dose JQ1 treatment (100 nmol/L,

lane 3) showed a very weak cellular apoptotic response in22RV1 cells after 48-hour drug treatment. In contrast, thecombination treatment of GSK461364A and JQ1 led to asignificantly increased cellular apoptotic response (lane 5)compared with JQ1 or GSK461364A alone (lanes 2 and 3). InC4-2 cells, we also detected the same trend as 22RV1 cells andfound that the combination of two drugs resulted in dramat-ically increased apoptosis compared with monotherapy (Fig.1B). Furthermore, the combination treatment of GSK461364Aand JQ1 showed a much stronger inhibitory effect on both cellproliferation and colony formation in 22RV1 and C4-2 cells(Fig. 1C–F; Supplementary Fig. S1A and S1B). Furthermore,FACS analysis was performed to monitor the cell-cycle defectsupon drug treatment in 22RV1 cells. Under the combinationtreatment, the percentage of cells at G2–M phase significantlyincreased, indicating that the presence of JQ1 increased theefficacy of GSK461364A in arresting the cell cycle at G2–Mphase, which potentiated JQ1-associated cell death in 22RV1cells (Fig. 1G). In agreement, the synergistic effect was alsoobserved through the detection of apoptotic marker Annexin V,which showed a significantly increased population of apoptoticcells under the combination treatment compared with themonotherapy (Fig. 1H; Supplementary Fig. S1C). To furtherconfirm the synergistic effect between GSK461364A and JQ1,we measured the CI of GSK461364A and JQ1 (SupplementaryTables S1 and S2). The IC50 values of 22RV1 and C4-2 cells weremeasured to be 400 and 300 nmol/L, respectively, under JQ1treatment for 72 hours. However, the IC50 value of JQ1 wasdramatically reduced to 50 and 75 nmol/L upon cotreatmentwith GSK461364A. Next, the CI was calculated to be 0.625 and0.750, respectively (Supplementary Tables S1 and S2), reveal-ing a strong synergistic effect between GSK461364A and JQ1. Inshort, we have demonstrated that JQ1 treatment could effec-tively increase the efficacy of Plk1 inhibition.

GSK461364A plus JQ1 synergistically inhibits growth ofLuCaP35CR tumors

To further confirm the synergistic effect of GSK461364A andJQ1, we next tested the efficacy of combinatorial treatment inthe patient-derived xenograft LuCaP35CR mouse model. Com-pared with the untreated group, there was no significant dif-ference for tumor volumes in single treatment groups ofGSK461364A and JQ1 (Fig. 2A). However, the combinationtherapy led to strong inhibition of tumor growth, resulting in aremarkable decrease in final tumor volumes (Fig. 2A and B).Besides, it also showed a dramatic reduction in tumor weightsafter the combination therapy (Fig. 2C). A remarkable increasein the number of apoptotic bodies with condensed cytoplasmand pyknotic nuclei was also observed after cotreatment ofGSK461364A and JQ1 (Fig. 3A). Consistent with these obser-vations was the detection of a significant increase in cleavedCaspase 3–positive cells, along with a reduction in Ki67-pos-itive cells after combination treatment, which together suggestsa strong induction of cell apoptosis and inhibition of cellproliferation (Fig. 3B). Above all, these results are consistentwith our previous observation based on cell experiments,confirming that GSK461364A and JQ1 can act synergisticallyboth in vivo and in vitro. Also, combination therapy is muchmore effective compared with the monotherapies in CRPCtreatment, providing a novel therapeutic strategy for patientswith CRPC.

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Mol Cancer Ther; 17(7) July 2018 Molecular Cancer Therapeutics1556




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Cotreatment of GSK461364A and JQ1 acts synergistically in CRPC cells. A and B, 22RV1 cells and C4-2 cells were seeded in 6-well plates for 24 hours and then treatedfor 48 hours with DMSO, JQ1, GSK461364A, or a combination of JQ1 plus GSK461364A, respectively, followed by anti-cleaved PARP immunoblotting (IB) tomeasure apoptosis. C and D, 22RV1 cells (0.5 � 103) and C4-2 cells (1 � 103) were seeded in 6-well plates for 24 hours, treated with DMSO, 25 nmol/L JQ1, 1 nmol/LGSK461364A, or a combination of the two drugs, respectively. After changing fresh media containing drug(s) every 3 days for 2 weeks, cells were fixed withformalin and colony formationwasmonitored by crystal violet staining. The experiments shown are representatives of 3 repeats. E and F, 22RV1 and C4-2 cells (5� 103)were seeded in 6-well plates for 24 hours and treated with indicated drugs, followed by measurement of cell numbers for 5 days. G, 22RV1 cells (2.5 � 105) wereseeded in 6-well plates for 24 hours and then treated with DMSO, 500 nmol/L JQ1, 10 nmol/L GSK461364A, or JQ1 plus GSK461364A, respectively. After 24 hours oftreatment, cells were collected, fixed with 70% ethanol, stained with propidium iodide (PI) for 30 minutes, and analyzed with flow cytometry. H, 22RV1 cells(2.5 � 105) were seeded in 6-well plates for 24 hours and then treated with DMSO, 500 nmol/L JQ1, 10 nmol/L GSK461364A, or JQ1 plus GSK461364A, respectively.After 48 hours of treatment, cells were collected, stained with Annexin V/PI for 30 minutes, and 2 � 104 cells were analyzed per sample by flow cytometry.






The synergistic effect of GSK461364A plus JQ1 is due tosuppression of AR signaling and c-MYC

To investigate themechanism underlying this synergistic effect,22RV1 andC4-2 cells were treated singly or in combination for 48hours, and then harvested for Western blotting. The expressionlevels of AR and c-MYC, two proteins critical to the developmentof CRPCs, were analyzed (Fig. 4A and B). We found that combi-natorial treatment was remarkably more effective at reducing theexpression levels of c-MYC, AR full length, and AR variants thaneither single treatment in 22RV1 cells. Furthermore, in C4-2 cells,we showed that the combination treatment significantlydecreased the expression levels of c-MYC, AR, and PSA comparedwith monotherapy (Fig. 4B). Previous studies done by our lab-oratory indicate that Plk1 could phosphorylate Axin2 at S311 andthat Plk1 phosphorylation of Axin2 promotes b-catenin phos-phorylation, thus its degradation. As such, treatment of CRPCcells with Plk1 inhibitor results in activation of the b-cateninpathway, eventually increasing the level of c-MYC protein (19).Because JQ1 directly inhibits c-MYC transcription, we comparedresponsewith JQ1 in cells expressing different forms of Axin2 (WTor S311A). Of note, Axin2-S311Amutant was used tomimic Plk1inhibition as S311A cannot be phosphorylated by Plk1 anymore.Accordingly, 22RV1 and C4-2 cells were transfected with Flag-Axin2 constructs (WT or S311A), treated with JQ1, and harvested.The cleaved PARP signal was clearly elevated in cells expressingAxin2-S311A upon JQ1 treatment, indicating that inhibition of

Axin2-S311 phosphorylation could render cells more sensitive toJQ1 (Fig. 4C and D). Consistent with this observation, JQ1treatment also led to dramatic decrease of the levels of AR, forboth full length and variants, and of c-MYC in cells expressingAxin2-S311A (Fig. 4D and E). In summary, these data support ourhypothesis that JQ1 could enhance the efficacy of Plk1 inhibitionthrough suppression of AR and c-MYC signaling.

22RV1-derived tumors expressing Axin2-S311A are moresensitive to JQ1

To further confirm our hypothesis, we next transfected 22RV1cells with Flag-Axin2 plasmids (WT or S311A) and tested theefficacy of JQ1 treatment in xenograft tumors derived from thesecells. As expected, compared with tumors derived from cellsexpressing Axin2-WT, tumors derived from cells expressingAxin2-S311A were much more aggressive and grew much faster(Fig. 5A), consistent with our previous finding (19). In contrast,low-dose JQ1 treatment (6.25 mg/kg bodyweight) showed astrong inhibitory effect on tumors derived from cells expressingAxin2-S311A and a significant reduction of tumor volumes (Fig.5B). SerumPSA concentrations followed a similar trend, with JQ1treatment reducing the serum PSA levels of the Axin2-311A–expressing group to near WT (Fig. 5C). Morphologically, therewas a dramatic increase in the number of apoptotic bodies withcondensed cytoplasm and pyknotic nuclei after treatment of JQ1when compared with the nontreated group (Fig. 5D). It was also

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GSK461364A plus JQ1 synergisticallyinhibits growth of LuCaP35CR tumors.A, Growth curves of LuCaP35CRtumors. Precastrated NSG mice wereinoculated with LuCaP35CR tumors,administeredwith GSK461364A (12mg/kg bodyweight, intravenous injection,twice a week), JQ1 (25 mg/kgbodyweight, oral gavage, twice aweek),or a combination of both drugs. Thesizes of the tumors in each group weremeasured every 3 days (mean � SEM;n ¼ 4 mice from each experimentgroup). �� , P < 0.01 compared with themonotherapy group or the untreatedgroup at the end of the study. B,Representative images of the tumors atthe end of the study. C, Tumor weightmeasurement after being freshlyremoved from the bodies. �� , P < 0.01compared with monotherapy group atthe end of the study.

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Histologic analysis of LuCaP35CR-derived xenograft tumors. A, Representative images of H&E staining on formaldehyde-fixed, paraffin-embedded tumorsections from different treatment groups. B, Top, representative images of immunofluorescence (IF) staining for Ki67 and cleaved Caspase 3 with tumorsas in A. Bottom, quantification of Ki67- or cleaved Caspase 3–positive cells within total number of cells. For quantification, at least 300 cells were scored withineach field (�20 fields, more than 3 sections at different tumor depths/mouse) as the percentages of Ki67- or cleaved Caspase 3–positive cells. � , P < 0.05;�� , P < 0.01 (two-tailed unpaired t test).






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The synergistic effect of GSK461364A plus JQ1 in 22RV1 and C4-2 cells is due to suppression of AR signaling and c-MYC. A, 22RV1 cells (5 � 105) were seeded in6-well plates for 24 hours and treated for 48 hours with DMSO, JQ1, GSK461364A, or combination of the two drugs, respectively, followed by IB to detect ARand c-MYC. B, C4-2 cells (3 � 105) were seeded in 6-well plates for 24 hours and treated for 48 hours with indicated drugs, followed by IB to detect AR, c-MYC,and PSA. C, 22RV1 cells (3 � 105) were seeded in 6-well plates for 24 hours, transfected with Flag-Axin2 plasmids (WT or S311A) for 48 hours, treated withJQ1 for 24 hours, and harvested for anti–cleaved-PARP IB. D, C4-2 cells (2 � 105) were seeded in 6-well plates for 24 hours, transfected with Flag-Axin2 plasmids(WT or S311A) for 48 hours, treated with 500 nmol/L JQ1 for 48 hours, and followed by IB to detect the levels of Flag, cleaved PARP, AR, c-MYC, and PSA.E, 22RV1 cells (3 � 105) were seeded in 6-well plates for 24 hours, transfected with Flag-Axin2 plasmids (WT or S311A) for 48 hours, treated with 500 nmol/LJQ1 for 48 hours, and subjected for IB to measure the levels of AR and c-MYC.

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Figure 5.

Xenograft tumors derived from22RV1 cells expressing Axin2-S311Amutant are more sensitive to JQ1treatment than those expressingWT Axin2. A, Growth curves oftumors derived from 22RV1 cellsexpressing different forms of Axin2(WT or S311A). Precastrated NSGmice were inoculated with 22RV1cells (2.5� 105) expressing differentforms of Axin2 (WT or S311A) for 22days and administrated with JQ1(6.25 mg/kg body weight) by oralgavage every 3 days, followed bymeasurement of tumor sizes (mean� SEM; n ¼ 4 mice from eachexperiment group). �� , P < 0.01compared with the monotherapygroup or the untreated group at theend of the study. B, Representativeimages of the tumors taken at theend of the study. C, After the serumfor each groupwas collected every 3days, PSA levels were measuredusing a PSA Elisa Kit (mean � SEM;n ¼ 4 mice from each experimentgroup). �� , P < 0.01 compared withthe monotherapy group or theuntreated group at the end of thestudy. D, Representative images ofH&E staining and IF staining for Ki67and cleaved Caspase 3 onformaldehyde-fixed, paraffin-embedded tumor sections fromdifferent treatment groups as in A.






observed that treatment of JQ1 reduced the population of Ki67-positive cells and increased the population of cleaved Caspase 3–positive cells in Axin2-311A tumors. This is taken to indicate asignificant inhibition of cell growth along with an induction ofapoptosis following JQ1 treatment. In summary, it confirms ourhypothesis that JQ1 could enhance the efficiency of Plk1 inhibi-tion through overcoming the side effects of Plk1 inhibitor thatcauses accumulation of nuclear b-catenin.

Cotreatment with GSK461364A and JQ1 has aminor impact onPD-L1 expression

It has been reported recently that BRD4 inhibition can effec-tively suppress PD-L1 expression level (29). We, therefore, aimedto ask whether the combination therapy has any impact on PD-L1expression. As indicated in Supplementary Fig. S2A and S2B, wefailed to detect obvious expression of PD-L1 in 22RV1 and C4-2cells, even under the induction of a high concentration of IFNg(10 mg/mL), indicating that these two cell lines express low levelsof PD-L1. We also showed that both monotherapy and dual-treatment had no significant impact on PD-L1 expression in C4-2and 22RV1 cells. To better understand the effect of the combi-nation treatment on PD-L1 expression, we then used TRAMP-C2,an aggressive mouse prostate cell line that expresses PD-L1 underinduction of IFNg (Supplementary Fig. S2C). In this model, bothmonotherapy and combination treatment slightly induced PD-L1expression, but the two drugs did not show a synergistic effect.Moreover, we performed immunofluorescence staining of PD-L1of LuCaP35CR and 22RV1 xenografts (Supplementary Fig. S3Aand S3B). Consistently, we failed to detect high level of PD-L1 inthese two xenograft models, even after the combination treat-ment. Furthermore, as TRAMP-C2 cells can express PD-L1, cellswere implanted in C57/B6 WT mice and treated with vehicle,GSK461364A, JQ1, combination of GSK461364A and JQ1, andBI2536. It has been reported previously that BI2536 can inhibitboth Plk1 and BRD4 (30), so it was used as a positive control inthis allograft experiment. Compared with the control group, therewas no significant difference for tumor volumes in all the treatedgroups (Supplementary Fig. S4A). However, we detected anobvious reduction in tumor weights after the combination ther-apy (Supplementary Fig. S4B and S4C). After the tumors wereharvested, they were digested into single cells and stained withcorresponding antibodies to detect proportions of certain cellpopulations byflow cytometry.None of the drug treatments had amajor impact on the levels of PD-L1 expression in tumor cells(Supplementary Fig. S4D),MDSCs (Supplementary Fig. S4E), andTregs (Supplementary Fig. S4F), indicating that the combinationtherapy of GSK46164A and JQ1 may have limited effect onimmune system under this experimental condition.

Cotreatment with GSK461364A and JQ1 dramatically inhibitscell glycolysis

Because both AR and c-MYC are crucial in CRPC developmentand are involved in metabolism regulation, it is possible thatcombinatorial treatment of GSK461464A and JQ1 could lead tosevere metabolic defects affecting energy production, eventuallyresulting in increased cell death. As aerobic glycolysis and mito-chondrial oxidative phosphorylation are twomajor ways for cellsto produce enough ATP for survival, we decided to test theglycolytic ability and mitochondrial function of cells undergoingthis treatment. As indicated, 22RV1 cells were treated either singlyor combination for 24 hours, after which we tested glycolytic

ability and mitochondria function using GST and MST, respec-tively. For GST, cells were incubated without CO2 for 1 hour, andthen extracellular acidification rates were measured as graphed(Fig. 6A and B). Data were analyzed by using stress test reportgenerator from Seahorse Bioscience. As expected, monotherapytreatments only slightly inhibited cell glycolysis, glycolytic capac-ity, and glycolytic reserves after 24 hours of treatment. In com-parison, the combinatorial therapy showed significant inhibitionof glycolysis rate compared with single treatments, with strongreductions in glycolytic capacity and glycolytic reverse. Altogether,this indicates that the combinatorial treatment likely directlyinterferes with the glycolytic pathway components. In addition,we tested whether combination of two drugs would affectmitochondrial function using MST. 22RV1 cells were treatedas indicated for 24 hours, followed by measurement of oxygenconsumption rates as graphed (Fig. 6C and D). Interestingly,there were no appreciable differences in spare respiratorycapacity and proton leakage between each group, indicatingthat both monotherapy and combination therapy could notinfluence mitochondrial function and oxidative phosphoryla-tion. Taking all of the above into consideration, the dataindicate that GSK461364A and JQ1 could act synergisticallyin inhibition of glycolysis of CRPCs, whereas both drugs haveno effect on the oxidative phosphorylation.

DiscussionBRD4, a critical epigenetic reader, could directly interact with

the positive transcription elongation factor complex b (P-TEFb;ref. 31), through which it can mark select M–G1 genes in mitoticchromatin as transcriptional memory and direct postmitotictranscription (32), and regulate c-MYC transcription as well. Asc-MYC is the major oncogene in multiple cancers, scientists areinterested in targeting at BRD4 as an effective cancer therapy. JQ1,a highly specific small chemical inhibitor of BRD4, could triggeracute c-MYC repression, induce apoptosis, and inhibit growth inmultiple types of cancer cells (16, 33). Furthermore, it was shownthat theWNT/b-catenin signaling is involved in both primary andacquired BET resistance (33). Herein, we presented a noveltherapeutic strategy that can significantly increase the efficiencyof JQ1 (Supplementary Fig. S5).

Beyond its function in regulating the cell cycle, Plk1 is alsooverexpressed inmultiple human tumors and interacts with otherimportant cancer-associated pathways (8). Increasing evidencesupports that Plk1 might be involved in acquisition of drugresistance, making it an effective target for novel cancer therapies.For example, phosphorylated Orc2 by Plk1 would induce con-tinuedDNA replication, contributing to gemcitabine resistance intreatment of pancreatic cancer (34). Besides, it has been reportedby our laboratory, that Plk1 phosphorylation of twomicrotubuleplus end-binding proteins, CLIP-170 and p150Glued, enhances themicrotubule dynamics, resulting in resistance to docetaxel inprostate cancer (35). Furthermore, Plk1 inhibition could alsoenhance the efficiency of metformin and b-catenin inhibitor inCRPC (19, 36). Therefore, Plk1 appears to be a promising target inCRPC therapy.

Herein, we investigated the efficacy of not only the monother-apy of Plk1 inhibitor GSK461364A and Brd4 inhibitor JQ1, alsothe combination therapy, in 22RV1 cells andC4-2 cells, which areandrogen independent (37). We found that Plk1 inhibitorGSK461364A and BRD4 inhibitor JQ1 act synergistically in

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Cotreatment with GSK461364A and JQ1 dramatically inhibits glycolysis. A, Extracellular acidification rates (ECAR) under single or dual treatment weremeasured by Seahorse XFe24 analyzer. 22RV1 cells were seeded in XFe24 cell culturemicroplates, treatedwith GSK461364A, JQ1 or both for 24 hours, and subjectedto the protocol for GTT in which glucose, oligomycin, and 2-deoxyglucose (2-DG) were added at the time points indicated. B, Calculated glycolysis rates, glycolysiscapacity, and glycolytic reserve. The data were normalized by relative cell number of treated group compared with control group. C, Oxygen consumption rates(OCR) under single or dual treatment was measurement by Seahorse XFe24 analyzer. 22RV1 cells were seeded in XFe24 cell culture microplates, treated withGSK461364A, JQ1 or both for 24 hours, and subjected to the protocol for MTT in which oligomycin, FCCP, and Rotenone/antimycin A were added at the timepoints indicated. D, Calculated basal respiratory rate, spare respiratory capacity, proton leak, and ATP production. The data were normalized by relative cellnumber of treated group compared with control group.






reduction of cell growth, induction of cell apoptosis, and G2–Marrest (Fig. 1A–H; Supplementary Fig. S1A–S1C). Meanwhile, thein vivo xenograft models of LuCaP35CR and 22RV1 also con-firmed the in vitro observation that dual inhibition of Plk1 andBRD4 synergistically enhance the efficacy than monotherapy.Surprisingly, the combination therapy not only dramaticallyinhibited c-MYC expression, but also suppressed expression andfunction of AR, both full length and variants. Furthermore,considering that it has been reported previously that BET inhibitorcan engage the host immune system and regulate expression ofPD-L1 (29), we tested efficacy of the combination treatment onimmune system. As indicated in Supplementary Figs. S2 and S3,not only human prostate cancer cell lines used in this study,22RV1 and C4-2, also LuCaP35CR and 22RV1-derived xenografttumors, showed no significant expression of PD-L1. In addition,allograft model was performed with TRAMP-C2 cells, which canexpress PD-L1 upon IFNg induction (Supplementary Fig. S2C),indicating that the combination therapy indeed had synergisticinhibition on tumor growth (Supplementary Fig. S4B), but lim-ited effect on immune system, including the levels of PD-L1expression in tumor cells (Supplementary Fig. S4D), MDSCs(Supplementary Fig. S4E), and Tregs (Supplementary Fig. S4F).However, more experimentation is required to further investigatethe influence of GSK461364A and JQ1 combination therapy onimmune system in other prostate cancer models.

In summary, our in vitro and in vivo data support a strongsynergy of Plk1 and BRD4 inhibition in CRPC progression fromtwo aspects: (i) JQ1 suppresses c-MYC expression, thus enhancingthe efficacy of Plk1 inhibitor; and (ii) Plk1 and BRD4 inhibitorsact synergistically in inhibition of AR signaling. Thus, the novel

combination strategy canbe considered for clinical trials to reducethe resistance and increase the efficiency of JQ1.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: F. Mao, J. Li, X. LiuDevelopment of methodology: F. Mao, Q. LuoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Mao, J. Li, Q. Luo, R. Wang, Z. Liu, B.D. ElzeyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Mao, J. Li, Q. Luo, Y. Kong, C. Carlock, X. LiuWriting, review, and/or revision of the manuscript: F. Mao, J. Li, C. Carlock,X. LiuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Q. Luo, C. Carlock, B.D. Elzey, X. LiuStudy supervision: J. Li, B.D. Elzey, X. Liu

AcknowledgmentsWe gratefully acknowledge Sandra Torregrosa-Allen and Melanie Currie for

their help with animal study. This work was supported by NIH R01 CA157429(to X. Liu), NIH R01CA192894 (to X. Liu), NIH R01 CA196835 (to X. Liu), andNIH R01 CA196634 (to X. Liu).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 28, 2017; revised February 7, 2018; accepted April 25,2018; published first May 1, 2018.

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2018;17:1554-1565. Published OnlineFirst May 1, 2018.Mol Cancer Ther Fengyi Mao, Jie Li, Qian Luo, et al. Blockade in Castration-Resistant Prostate CancerPlk1 Inhibition Enhances the Efficacy of BET Epigenetic Reader

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