Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2019 Jul 1.
Published in final edited form as: Mol Cancer Ther. 2018 May 1;17(7):1554–1565. doi: 10.1158/1535-7163.MCT-17-0945

Plk1 inhibition enhances the efficacy of BET epigenetic reader blockade in castration-resistant prostate cancer

Fengyi Mao 1,2,ǂ, Jie Li 1,ǂ, Qian Luo 1, Ruixin Wang 1, Yifan Kong 1,2, Colin Carlock 1, Zian Liu 1, Bennet D Elzey 3, Xiaoqi Liu 1,4,*
PMCID: PMC6030429  NIHMSID: NIHMS964934  PMID: 29716963

Abstract

Polo-like kinase 1 (Plk1), a crucial regulator of cell cycle progression, is overexpressed in multiple types of cancers, and has been proven to be a potent and promising target for cancer treatment. In case of prostate cancer, we once showed that anti-neoplastic activity of Plk1 inhibitor is largely due to inhibition of androgen receptor (AR) signaling. However, we also discovered that Plk1 inhibition causes activation of the β-catenin pathway and increased expression of c-Myc, eventually resulting in resistance to Plk1 inhibition. JQ1, a selective small molecule inhibitor targeting the amino-terminal bromodomains of BRD4, has been shown to dramatically inhibit c-Myc expression and AR signaling, exhibiting anti-proliferative effects in a range of cancers. Since c-Myc and AR signaling are essential for prostate cancer initiation and progression, we aim to test whether targeting Plk1 and BRD4 at the same time is an effective approach to treat prostate cancer. Herein, we show that a combination of Plk1 inhibitor GSK461364A and BRD4 inhibitor JQ1 had a strong synergistic effect on castration-resistant prostate cancer (CRPC) cell lines, as well as in CRPC xenograft tumors. Mechanistically, the synergistic effect is likely due to two reasons: 1) Plk1 inhibition results in the accumulation of β-catenin in the nucleus, thus elevation of c-Myc expression, whereas JQ1 treatment directly suppresses c-Myc transcription. 2) Plk1 and BRD4 dual inhibition acts synergistically in inhibition of AR signaling.

Keywords: Plk1, BRD4, JQ1, combination therapy, castration-resistant prostate cancer

Introduction

Prostate cancer (PCa) is among the most frequently diagnosed cancers in men and is one of the leading causes of cancer-associated death worldwide (1). The American Cancer Society estimates that there will be 161,360 newly diagnosed cases and 26,730 deaths due to PCa in the United States in 2017 (2). Androgen receptor (AR) signaling plays a critical role in PCa development. Consequently, androgen deprivation therapy (ADT) can manage the development of PCa initially and is the most widely used and effective systematic therapy to treat PCa patients with metastatic diseases. However, most patients eventually become resistant to ADT treatment, and the disease enters a stage called castration-resistant prostate cancer (CRPC), an uncurable disease at this moment (3,4). Therefore, the novel therapies for CRPC patients are urgently needed.

One potential target for therapy would be polo-like kinase 1 (Plk1), a serine-theronine kinase that plays a key role in mitosis (5), and has been demonstrated to be required in centrosome maturation and establishment of a bipolar spindle (6). Plk1 is overexpressed in multiple types of cancers, whereas Plk1 expression is low in surrounding normal, non-dividing tissue (5,7). It has been demonstrated that Plk1 is a potent and promising target for cancer treatment due to its critical role in cell proliferation and that Plk1 inhibitors are currently under heavy investigation to determine their efficacy and safety profiles in diverse tumor types (8). Among these inhibitors is GSK461364A (thiophene derivative)(9–13), which has been shown to potently inhibit the proliferation of various tumor cell lines (10).

BRD4, the most extensively investigated member of bromodomain and extraterminal domain (BET) family, functions as an epigenetic reader and regulates transcription (14). It has been discovered that BRD4 can recruit RNA polymerase II during the transcription, consequently not only regulating the transcriptional levels of FOS, Jun, and c-Myc (15), but importantly for this study, also physically interacting with AR to help localize AR to its target loci (16). JQ1, a small molecule inhibitor that is highly specific for BRD4 (17,18), can bind competitively to acetyl-lysine recognition motifs of BRD4 and disrupt its regulation, eventually effectively suppressing tumor growth (18).

Herein, we show a strong synergistic effect using a combinatorial treatment strategy with the Plk1 inhibitor GSK461364A and the BRD4 inhibitor JQ1 in 22RV1 and C4-2 cells, two very aggressive human CRPC cell lines, as well as in CRPC xenograft tumors. The combination treatment led to G2/M arrest, inhibition of cell growth, a massive increase in apoptosis, and a concordant drop in glycolysis. We have previously shown that Plk1 phosphorylation of Axin2, a member of β-catenin pathway, stabilizes the binding between GSK3β and β-catenin in the cytoplasm, resulting in increased degradation of β-catenin. Conversely, Plk1 inhibition would induce the accumulation of β-catenin in the nucleus, resulting in activation of c-Myc (19–21), while Plk1 inhibition can also inhibit AR signaling (22,23). As a BRD4 inhibitor, JQ1 could effectively antagonize AR signaling and repress c-Myc transcription (16). We thus propose that the observed synergistic effect for GSK461364A and JQ1 is due to their effects on c-Myc and AR signaling.

Materials and Methods

Cell culture and drugs

22RV1 and C4-2 cells, purchased from ATCC in 2016, were cultured in RPMI1640 medium supplemented with 10% (v/v) FBS and 100 U/mL penicillin, 100 U/mL streptomycin at 37C in 5% CO2. TRAMP-C2 cell were also purchase from ATCC in 2016 and it was cultured in Dulbecco’s modified Eagle’s medium with 5% fetal bovine serum, 5% Nu-Serum IV, 100 U/mL penicillin, 100 U/mL streptomycin at 37C in 5% CO2. All the cells were within 50 passages and Mycoplasma were detected every 3 months using MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-705). GSK461364A, JQ1 and BI2536 (24) were purchased from Selleckchem.

Cell viability assay

22RV1 and C4-2 cells were seeded with 5 × 103–1 × 104 per well in 96-well plates, cultured for 12 hours and treated with different concentrations of the drugs. After 72 hours of incubation, 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 μL of DMSO, cells were subjected to a plate reader to measure the absorbance at 570 nm. The IC50 values were obtained from the average viability curves generated by four independent measurements of each condition.

Combination index

The combination index (CI) was calculated using the following equation (25,26): CI = (Am)50/(As)50 + (Bm)50/(Bs)50. (Am)50 is the concentration of GSK461364A which could achieve 50% inhibitory effect in the combination with half of the concentration of the JQ1 IC50; (As)50 is the IC50 of GSK461364A; (Bm)50 is the concentration of JQ1 that will produce a 50% inhibitory effect in the combination with half of the concentration of GSK461364A IC50; and (Bs)50 is the IC50 of JQ1. Antagonism is indicated when CI > 1, CI = 1 indicates an additive effect, and CI < 1 indicates synergy.

Antibodies

Antibody against Axin2 and Ki67 were purchased from Abcam, whereas antibodies against β-actin (A-5441), and c-Myc were obtained from Sigma. Antibody against CD45, CD8, Foxp3, PD-L1, CD4, CD25, CD11b, Ly6C and Ly6G are purchased from Biolegend. All other antibodies were purchased from Cell Signaling Technology.

Immunoblotting (IB)

Cells were washed by PBS after harvest and then resuspended with TBSN buffer with protease inhibitors and phosphatase inhibitors. After sonication, cell lysates were collected, and protein concentrations were measured by using Protein Assay Dye Reagent from Bio-Rad. Mix the proteins from each group with SDS-PAGE loading respectively and boil it for 5 min. Upon transferring to polyvinylidene difluoride membranes, proteins were probed with indicated antibodies (27).

Immunofluorescence (IF) staining

After murine or human paraffin-embedded slides were deparaffinized and rehydrated, antigens were retrieved in antigen unmasking solution (Vector Laboratories). Samples were then incubated with primary antibodies against Ki67, cleaved-caspase 3 and PD-L1, followed by incubation with secondary antibodies and DAPI.

Colony formation assay

Cells (500–1000/well) were seeded in 6-well plates and cultured in medium alone or containing different drugs for 14 days, with medium change every 2 days. After culturing, cells were fixed in 10% formalin and stained with 0.5% crystal violet for 30 minutes, followed by counting of colony numbers.

Annexin V-fluorescein/isothiocyanate propidium iodide

Cells (5 × 105/well) were seeded in 6-well plates, cultured in medium alone or containing different drugs, and subjected to the procedure using the Annexin V apoptosis kit (BioVision, K101-25), followed by analysis with FACS Express 5.

FACS analysis

Cells (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/mL propidium iodide (PI), and subjected to FACS analysis.

Patient-derived xenograft model

Mice carrying LuCaP35CR tumors were obtained from Dr. Robert Vessella at the University of Washington (28). Tumors were amplified by cutting the original tumors into 20–30 mm3 pieces, followed by implantation into pre-castrated NSG mice. When tumors reached the size of 250–300 mm3, mice were randomly separated into four groups for control, GSK461364A alone, JQ1 alone, and combination treatment, respectively. The experiment was approved by the Purdue Animal Care and Use Committee (PACUC).

22RV1-derived mouse xenograft model

22RV1 cells were transfected with Flag-Axin2-WT and Flag-Axin2-311A plasmids and selected with 500 μg/mL G418 for 4 weeks. Cells (2.5 × 105 cells/mouse) were mixed with an equal volume of Matrigel (Collaborative Biomedical Products) and inoculated into the right flank of NSG mice (Harlan Laboratories). One week later, animals were randomized into treatment and control groups with 4 mice each. JQ1 was delivered via gavage, twice a week. Tumor volumes were estimated from the formula: V = L × W2/2 [V is volume (mm3); L is length (mm); W is width (mm)]. The experiment was approved by the Purdue Animal Care and Use Committee (PACUC).

TRAMP-C2-derived mouse allograft model

TRAMP-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 mice (Envigo). One week later, animals were randomized for control, GSK461364A alone, JQ1 alone, combination treatment, and BI2536 alone, respectively. Tumor volumes were estimated from the formula: V = 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-L1+/CD45− cell, T regulatory cells (Treg, CD4+CD25+FoxP3+) and Myeloid-derived suppressor cells (MDSC, CD45+Gr-1+CD11b+) were analyzed by flow cytometry. The experiment was approved by the Purdue Animal Care and Use Committee (PACUC).

Serum prostate-specific antigen (PSA) measurement

After blood was collected from tumor-carrying mice twice per week, serum PSA levels were measured by using a PSA (human) ELISA kit (Abnova, KA0208).

Histology and H&E staining

Xenograft tumors were fixed in 10% neutral buffered formalin, paraffin embedded, sectioned to 5 mm, and stained using conventional hematoxylin and eosin (H&E) staining.

Seahorse analysis

22RV1 cells (2 × 104/Well) were seeded in XFe24 cell culture microplates in RPMI 1640 medium (10% FBS with antibiotics). After 12 hours of incubation, cells were treated with corresponding drug(s) for 24 hours. Cartridges were hydrated in calibrant buffer in a non-CO2 incubator at 37°C for at least 12 hours before analysis. Before being subjected to seahorse analysis, cells were washed with corresponding medium for 2 times and incubated in a non-CO2 incubator for 1 hour. For glycolysis stress test (GST), GST medium was prepared by supplementing XF base Medium with 2 mM glutamine, and pH was adjusted to 7.4. For mitochondrial stress test (MST), MST medium was prepared by supplementing XF base Medium with 2 mM glutamine, 1 mM pyruvate and 10 mM glucose, and pH was adjusted to 7.4. The drugs from the XF GST kit and MST kit were diluted with corresponding medium into designed concentrations and then added into corresponding ports of cartridge. After calibration of the cartridge, cells went through GST or MST programs. Data were analyzed by using the Seahorse XF Cell GST Report Generator and Seahorse XF Cell MST Report Generator, respectively.

Statistical analysis

The 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.

Results

Co-treatment of GSK461364A and JQ1 acts synergistically

To investigate whether GSK461364A and JQ1 act synergistically to inhibit the growth of CRPC cells, 22RV1 and C4-2 cells were treated with GSK461364A, JQ1, or a combination of GSK461364A and JQ1, and harvested for IB analysis of the apoptotic marker, cleaved-PARP (Fig. 1A and 1B). As indicated in Fig. 1A, low-dose JQ1 treatment (100 nmol/L, lane 3) showed a very weak cellular apoptotic response in 22RV1 cells after 48 hours drug treatment. In contrast, the combination treatment of GSK461364A and JQ1 led to a significantly increased cellular apoptotic response (lane 5) compared with JQ1 or GSK461364A alone (lanes 2 and 3). In C4-2 cells, we also detected the same trend as 22RV1 cells and found that the combination of two drugs resulted in dramatically increased apoptosis compared to monotherapy (Fig. 1B). Further, the combination treatment of GSK461364A and JQ1 showed a much stronger inhibitory effect on both cell proliferation and colony formation in 22RV1 and C4-2 cells (Fig. 1C–1F; Supplementary Fig. S1A, and S1B). Furthermore, FACS analysis was performed to monitor the cell-cycle defects upon drug treatment in 22RV1 cells. Under the combination treatment, the percentage of cells at G2/M phase significantly increased, indicating that the presence of JQ1 increased the efficacy of GSK461364A in arresting the cell cycle at G2/M phase, which potentiated JQ1-associated cell death in 22RV1 cells (Fig. 1G). In agreement, the synergistic effect was also observed through the detection of apoptotic marker Annexin V, which showed a significantly increased population of apoptotic cells under the combination treatment compared to the monotherapy (Fig. 1H; Supplementary Fig. S1C). To further confirm the synergistic effect between GSK461364A and JQ1, we measured the Combination Index (CI) of GSK461364A and JQ1 (Supplementary Table S1 and S2). The IC50 values of 22RV1 and C4-2 cells were measured to be 400 nmol/L and 300 nmol/L respectively under JQ1 treatment for 72 hours. However, the IC50 value of JQ1 was dramatically reduced to 50 nmol/L and 75 nmol/L upon co-treatment with GSK461364A. Next, the CI was calculated to be 0.625 and 0.750 respectively (Supplementary Tables S1 and S2), revealing a strong synergistic effect between GSK461364A and JQ1. In short, we have demonstrated that JQ1 treatment could effectively increase the efficacy of Plk1 inhibition.

Figure 1. Co-treatment of GSK461364A and JQ1 acts synergistically in CRPC cells.

Figure 1

Open in a new tab

(A–B) 22RV1 cells and C4-2 cells were seeded in 6-well plates for 24 hours and then treated for 48 hours with DMSO, JQ1, GSK461364A, or a combination of JQ1 plus GSK461364A, respectively, followed by anti-cleaved PARP IB to measure apoptosis. (C–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 nM JQ1, 1 nM GSK461364A or a combination of the two drugs, respectively. After changing fresh media containing drug(s) every 3 days for two weeks, cells were fixed with formalin and colony formation was monitored by crystal violet staining. The experiments shown are representatives of 3 repeats. (E–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 five days. (G) 22RV1 cells (2.5 × 105) were seeded in 6-well plates for 24 hours and then treated with DMSO, 500 nM JQ1, 10 nM GSK461364A, or JQ1 plus GSK461364A, respectively. After 24 hours of treatment, cells were collected, fixed with 70% ethanol, stained with 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, 500nM JQ1, 10 nM GSK461364A, or JQ1 plus GSK461364A, respectively. After 48 hours of treatment, cells were collected, stained with Annexin V/PI for 30 minutes and 2,0000 cells were analyzed per sample by flow cytometry.

GSK461364A plus JQ1 synergistically inhibits growth of LuCaP35CR tumors

To further confirm the synergistic effect of GSK461364A and JQ1, we next tested the efficacy of combinatorial treatment in the patient-derived xenograft (PDX) LuCaP35CR mouse model. Compared with the untreated group, there was no significant difference for tumor volumes in single treatment groups of GSK461364A and JQ1 (Fig. 2A). However, the combination therapy led to strong inhibition of tumor growth, resulting in a remarkable decrease in final tumor volumes (Fig. 2A and 2B). Besides, it also showed a dramatic reduction in tumor weights after the combination therapy (Fig. 2C). A remarkable increase in the number of apoptotic bodies with condensed cytoplasm and pyknotic nuclei was also observed after co-treatment of GSK461364A and JQ1 (Fig. 3A). Consistent with these observations was the detection of a significant increase in cleaved-caspase 3 positive cells, along with a reduction in Ki67 positive cells after combination treatment, which together suggests a strong induction of cell apoptosis and inhibition of cell proliferation (Fig. 3B). Above all, these results are consistent with our previous observation based on cell experiments, confirming that GSK461364A and JQ1 can act synergistically both in vivo and in vitro. Also, combination therapy is much more effective compared to the monotherapies in CRPC treatment, providing a novel therapeutic strategy for CRPC patients.

Figure 2. GSK461364A plus JQ1 synergistically inhibits growth of LuCaP35CR tumors.

Figure 2

Open in a new tab

(A) Growth curves of LuCaP35CR tumors. Pre-castrated NSG mice were inoculated with LuCaP35CR tumors, administered with GSK461364A (12 mg/kg body weight, intravenous injection, twice a week), JQ1 (25 mg/kg body weight, oral gavage, twice a week) or a combination of both drugs. The sizes of the tumors in each group were measured every 3 days (mean ± SEM; n = 4 mice from each experiment group). **, P < 0.01 compared with the monotherapy group or the untreated group at the end of the study. (B) Representative images of the tumors at the end of the study. (C) Tumor weight measurement after being freshly removed from the bodies. **, P < 0.01 compared with monotherapy group at the end of the study.

Figure 3. Histologic analysis of LuCaP35CR-derived xenograft tumors.

Figure 3

Open in a new tab

(A) Representative images of H&E staining on formaldehyde-fixed, paraffin-embedded tumor sections from different treatment groups. (B) Top: Representative images of IF staining for Ki67 and cleaved caspase-3 with tumors as 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 within each field (x 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).

The synergistic effect of GSK461364A plus JQ1 is due to suppression of AR signaling and c-Myc

To investigate the mechanism underlying this synergistic effect, 22RV1 and C4-2 cells were treated singly or in combination for 48 hours, and then harvested for western blotting. The expression levels of AR and c-Myc, two proteins critical to the development of CRPCs, were analyzed (Fig. 4A and 4B). We found that combinatorial treatment was remarkably more effective at reducing the expression levels of c-Myc, AR full length and AR variants than either single treatment in 22RV1 cells. Further, in C4-2 cells, we showed that the combination treatment significantly decreased the expression levels of c-Myc, AR and PSA compared to monotherapy (Fig. 4B). Previous studies done by our lab indicate that Plk1 could phosphorylate Axin2 at S311 and that Plk1 phosphorylation of Axin2 promotes β-catenin phosphorylation, thus its degradation. As such, treatment of CRPC cells with Plk1 inhibitor results in activation of the β-catenin pathway, eventually increasing of the level of c-Myc protein (19). Because JQ1 directly inhibits c-Myc transcription, we compared response to JQ1 in cells expressing different forms of Axin2 (WT or S311A). Of note, Axin2-S311A mutant was used to mimic Plk1 inhibition 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 expressing Axin2-S311A upon JQ1 treatment, indicating that inhibition of Axin2-S311 phosphorylation could render cells more sensitive to JQ1 (Fig. 4C and 4D). Consistent with this observation, JQ1 treatment also led to dramatic decrease of the levels of AR, for both full length and variants, and of c-Myc in cells expressing Axin2-S311A (Fig. 4D and 4E). In summary, these data support our hypothesis that JQ1 could enhance the efficacy of Plk1 inhibition through suppression of AR and c-Myc signaling.

Figure 4. The synergistic effect of GSK461364A plus JQ1 in 22RV1 and C4-2 cells is due to suppression of AR signaling and c-Myc.

Figure 4

Open in a new tab

(A) 22RV1 cells (5 × 105) were seeded in 6-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 AR and 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 with JQ1 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 nM 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 nM JQ1 for 48 hours, and subjected for IB to measure the levels of AR and c-Myc.

22RV1-derived tumors expressing Axin2-S311A are more sensitive to JQ1

To further confirm our hypothesis, we next transfected 22RV1 cells with Flag-Axin2 plasmids (WT or S311A) and tested the efficacy of JQ1 treatment in xenograft tumors derived from these cells. As expected, compared with tumors derived from cells expressing Axin2-WT, tumors derived from cells expressing Axin2-S311A were much more aggressive and grew much faster (Fig. 5A), consistent with our previous finding (19). By contrast, low-dose JQ1 treatment (6.25 mg/kg body weight) showed a strong inhibitory effect on tumors derived from cells expressing Axin2-S311A and a significant reduction of tumor volumes (Fig. 5B). Serum PSA concentrations followed a similar trend, with JQ1 treatment reducing the serum PSA levels of the Axin2-311A-expressing group to near WT (Fig. 5C). Morphologically, there was a dramatic increase in the number of apoptotic bodies with condensed cytoplasm and pyknotic nuclei after treatment of JQ1 when compared with the non-treated group (Fig. 5D). It was also 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 a significant inhibition of cell growth along with an induction of apoptosis following JQ1 treatment. In summary, it confirms our hypothesis that JQ1 could enhance the efficiency of Plk1 inhibition through overcoming the side effects of Plk1 inhibitor that causes accumulation of nuclear β-catenin.

Figure 5. Xenograft tumors derived from 22RV1 cells expressing Axin2-S311A mutant are more sensitive to JQ1 treatment than those expressing WT Axin2.

Figure 5

Open in a new tab

(A) Growth curves of tumors derived from 22RV1 cells expressing different forms of Axin2 (WT or S311A). Pre-castrated NSG mice were inoculated with 22RV1 cells (2.5×105) expressing different forms of Axin2 (WT or S311A) for 22 days and administrated with JQ1 (6.25 mg/kg body weight) by oral gavage every 3 days, followed by measurement of tumor sizes (mean ± SEM; n = 4 mice from each experiment group). **, P < 0.01 compared with the monotherapy group or the untreated group at the end of the study. (B) Representative images of the tumors taken at the end of the study. (C) After the serum for each group was collected every 3 days, PSA levels were measured using a PSA Elisa Kit (mean ± SEM; n=4 mice from each experiment group). **, P < 0.01 compared with the monotherapy group or the untreated group at the end of the study. (D) Representative images of H&E staining and IF staining for Ki67 and cleaved caspase-3 on formaldehyde-fixed, paraffin-embedded tumor sections from different treatment groups as in (A).

Co-treatment with GSK461364A and JQ1 has a minor impact on PD-L1 expression

It has been reported recently that BRD4 inhibition can effectively suppress PD-L1 expression level (29). We, therefore, aimed to ask whether the combination therapy have any impact on PD-L1 expression. As indicated in Supplementary Fig. S2A and S2B, we failed to detect obvious expression of PD-L1 in 22RV1 and C4-2 cells, even under the induction of a high concentration of interferon-gamma (10 μg/mL), indicating that these two cell lines express low levels of PD-L1. We also showed that both monotherapy and dual-treatment had no significant impact on PD-L1 expression in C4-2 and 22RV1 cells. To better understand the effect of the combination treatment on PD-L1 expression, we then used TRAMP-C2, an aggressive mouse prostate cell line that expresses PD-L1 under induction of interferon-gamma (Supplementary Fig. S2C). In this model, both monotherapy and combination treatment slightly induced PD-L1 expression, but the two drugs did not show a synergistic effect. Moreover, we performed immunofluorescent (IF) staining of PD-L1 of LuCaP35CR and 22RV1 xenografts (Supplementary Fig. S3A and S3B). Consistently, we failed to detect high level of PD-L1 in these two xenograft models, even after the combination treatment. Furthermore, as TRAMP-C2 cells can express PD-L1, cells were implanted in C57/B6 wild type mice and treated with vehicle, GSK461364A, JQ1, combination of GSK461364A and JQ1, and BI2536. It has been reported previously that BI2536 can inhibit both Plk1 and BRD4 (30), so it was used as a positive control in this allograft experiment. Compared with the control group, there was no significant difference for tumor volumes in all the treated groups (Supplementary Fig. S4A). However, we detected an obvious reduction in tumor weights after the combination therapy (Supplementary Fig. S4B and S4C). After the tumors were harvested, they were digested into single cells and stained with corresponding antibodies to detect proportions of certain cell populations by flow cytometry. None of the drug treatments had a major impact on the levels of PD-L1 expression in tumor cells (Supplementary Fig. S4D), Myeloid-derived suppressor cells (Supplementary Fig. S4E) and T regulatory cells (Supplementary Fig. S4F), indicating that the combination therapy of GSK46164A and JQ1 may have limited effect on immune system under this experimental condition.

Co-treatment with GSK461364A and JQ1 dramatically inhibits cell glycolysis

Since both AR and c-Myc are crucial in CRPC development and involved in metabolism regulation, it is possible that combinatorial treatment of GSK461464A and JQ1 could lead to severe metabolic defects affecting energy production, eventually resulting in increased cell death. As aerobic glycolysis and mitochondrial oxidative phosphorylation are two major ways for cells to produce enough ATP for survival, we decided to test the glycolytic ability and mitochondrial function of cells undergoing this treatment. As indicated, 22RV1 cells were treated either singly or combination for 24 hours, after which we tested glycolytic ability and mitochondria function using GST and MST, respectively. For GST, cells were incubated without CO2 for 1 hour, then extracellular acidification rates (ECAR) were measured as graphed (Fig. 6A and 6B). Data were analyzed by using stress test report generator from Seahorse Bioscience. As expected, monotherapy treatments only slightly inhibited cell glycolysis, glycolytic capacity and glycolytic reserves after 24 hours of treatment. By comparison, the combinatorial therapy showed significant inhibition of glycolysis rate compared with single treatments, with strong reductions in glycolytic capacity and glycolytic reverse. Altogether this indicates that the combinatorial treatment likely directly interferes with the glycolytic pathway components. In addition, we tested whether combination of two drugs would affect mitochondrial function using MST. 22RV1 cells were treated as indicated for 24 hours, followed by measurement of oxygen consumption rates (OCR) as graphed (Fig. 6C and 6D). Interestingly, there were no appreciable differences in spare respiratory capacity and proton leakage between each group, indicating that both monotherapy and combination therapy could not influence mitochondrial function and oxidative phosphorylation. Taking the above all into consideration, the data indicates that GSK461364A and JQ1 could act synergistically in inhibition of glycolysis of CRPCs, while both drugs have no effect on the oxidative phosphorylation.

Figure 6. Co-treatment with GSK461364A and JQ1 dramatically inhibits glycolysis.

Figure 6

Open in a new tab

(A) ECAR under single or dual treatment was measured by Seahorse XFe24 analyzer. 22RV1 cells were seeded in XFe24 cell culture microplates, treated with GSK461364A, JQ1 or both for 24 hours, and subjected to the protocol for GTT in which glucose, oligomycin and 2-deoxyglucose (2-DG) were added at the time points indicated. (B) Calculated glycolysis rates, glycolysis capacity and glycolytic reserve. The data was normalized by relative cell number of treated group compared to control group. (C) OCR under single or dual treatment was measurement by Seahorse XFe24 analyzer. 22RV1 cells were seeded in XFe24 cell culture microplates, treated with GSK461364A, 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 time points indicated. (D) Calculated basal respiratory rate, spare respiratory capacity, proton leak and ATP production. The data was normalized by relative cell number of treated group compared to control group.

Discussion

BRD4, a critical epigenetic reader, could directly interact with the positive transcription elongation factor complex b (P-TEFb) (31), through which it can mark select M/G1 genes in mitotic chromatin as transcriptional memory and direct post-mitotic transcription (32), and regulate c-Myc transcription as well. As c-Myc is the major oncogenes in multiple cancers, scientists are interested in targeting at BRD4 as an effective cancer therapy. JQ1, a highly specific small chemical inhibitor of BRD4, could trigger acute c-Myc repression, induce apoptosis, and inhibit growth in multiple types of cancer cells (16,33). Furthermore, it was shown that the WNT/β-catenin signaling is involved in both primary and acquired BET resistance (33). Herein, we presented a novel therapeutic strategy which can significantly increase the efficiency of JQ1 (Supplementary Fig. S5).

Beyond its function in regulating cell cycle, Plk1 is also over-expressed in multiple human tumors and interacts with other important cancer-associated pathways (8). Increasing evidence supports that Plk1 might be involved in acquisition of drug resistance, making it an effective target for novel cancer therapies. For example, phosphorylated Orc2 by Plk1 would induce continued DNA replication, contributing to gemcitabine resistance in treatment of pancreatic cancer (34). Besides, it has been reported by our lab, that Plk1 phosphorylation of two microtubule plus end-binding proteins, CLIP-170 and p150Glued, enhances the microtubule dynamics, resulting in resistance to docetaxel in prostate cancer (35). Furthermore, Plk1 inhibition could also enhance the efficiency of metformin and β-catenin inhibitor in CRPC (19,36). Therefore, Plk1 appears to be a promising target in CRPC therapy.

Herein, we investigated the efficacy of not only the monotherapy of Plk1 inhibitor GSK461364A and Brd4 inhibitor JQ1, also the combination therapy, in 22RV1 cells and C4-2 cells, which are androgen independent (37). We found that Plk1 inhibitor GSK461364A and BRD4 inhibitor JQ1 act synergistically in reduction of cell growth, induction of cell apoptosis and G2/M arrest (Fig. 1A–H and S1A–C). Meanwhile, the in vivo xenograft models of LuCaP35CR and 22RV1 also confirmed the in vitro observation that dual inhibition of Plk1 and BRD4 synergistically enhance the efficacy than monotherapy. Surprisingly, the combination therapy not only dramatically inhibited c-Myc expression, but also suppress expression and function of AR, both full length and variants. Further, considering that it has been reported previously that BET inhibitor can engage the host immune system and regulate expression of PD-L1(29), we tested efficacy of the combination treatment on immune system. As indicated in Supplementary Fig. S2 and S3, not only human prostate cancer cell lines used in this study, 22RV1 and C4-2, also LuCaP35CR and 22RV1-derived xenograft tumors, showed no significant expression of PD-L1. In addition, allograft model was performed with TRAMP-C2 cells, which can express PD-L1 upon interferon-gamma induction (Supplementary Fig. S2C), indicating that the combination therapy indeed had synergistic inhibition on tumor growth (Supplementary Fig. S4B), but limited effect on immune system, including the levels of PD-L1 expression in tumor cells (Supplementary Fig. S4D), Myeloid-derived suppressor cells (Supplementary Fig. S4E) and T regulatory cells (Supplementary Fig. S4F). However, more experimentations are required to further investigate the influence of GSK461364A and JQ1 combination therapy on immune system in other prostate cancer models.

In summary, our in vitro and in vivo data support a strong synergy of Plk1 and BRD4 inhibition in CRPC progression from two aspects: 1) JQ1 suppresses c-Myc expression, thus enhancing the efficacy of Plk1 inhibitor; and 2) Plk1 and BRD4 inhibitors act synergistically in inhibition of AR signaling. Thus, the novel combination strategy can be considered for clinical trials to reduce the resistance and increase the efficiency of JQ1.

Supplementary Material

1
2
3
4
5
6
7

Acknowledgments

We gratefully acknowledge Sandra Torregrosa-Allen and Melanie Currie for their help with animal study.

Footnotes

Disclosure of Potential Conflicts of Interest: The authors declare no potential conflicts of interest.

Financial information: NIH R01 CA157429 (X. Liu), NIH R01 CA192894 (X. Liu), NIH R01 CA196835 (X. Liu) and NIH R01 CA196634 (X. Liu)

References

  • 1.Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA: a cancer journal for clinicians. 2015;65(2):87–108. doi: 10.3322/caac.21262. [DOI] [PubMed] [Google Scholar]
  • 2.Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA: a cancer journal for clinicians. 2017;67(1):7–30. doi: 10.3322/caac.21387. [DOI] [PubMed] [Google Scholar]
  • 3.Harris WP, Mostaghel EA, Nelson PS, Montgomery B. Androgen deprivation therapy: progress in understanding mechanisms of resistance and optimizing androgen depletion. Nature clinical practice Urology. 2009;6(2):76–85. doi: 10.1038/ncpuro1296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Chandrasekar T, Yang JC, Gao AC, Evans CP. Mechanisms of resistance in castration-resistant prostate cancer (CRPC) Translational andrology and urology. 2015;4(3):365–80. doi: 10.3978/j.issn.2223-4683.2015.05.02. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Strebhardt K. Multifaceted polo-like kinases: drug targets and antitargets for cancer therapy. Nat Rev Drug Discov. 2010;9(8):643–U24. doi: 10.1038/nrd3184. [DOI] [PubMed] [Google Scholar]
  • 6.Combes G, Alharbi I, Braga LG, Elowe S. Playing polo during mitosis: PLK1 takes the lead. Oncogene. 2017;36(34):4819–27. doi: 10.1038/onc.2017.113. [DOI] [PubMed] [Google Scholar]
  • 7.Cholewa BD, Liu XQ, Ahmad N. The Role of Polo-like Kinase 1 in Carcinogenesis: Cause or Consequence? Cancer Res. 2013;73(23):6848–55. doi: 10.1158/0008-5472.CAN-13-2197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Liu X. Targeting Polo-Like Kinases: A Promising Therapeutic Approach for Cancer Treatment. Translational oncology. 2015;8(3):185–95. doi: 10.1016/j.tranon.2015.03.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Erskine S, Madden L, Hassler D, Smith G, Copeland R, Gontarek R. Biochemical characterization of GSK461364: A novel, potent, and selective inhibitor of Polo-like kinase-1 (Plk1) Cancer Res. 2007;67(9 Supplement):3257. [Google Scholar]
  • 10.Gilmartin AG, Bleam MR, Richter MC, Erskine SG, Kruger RG, Madden L, et al. Distinct concentration-dependent effects of the polo-like kinase 1-specific inhibitor GSK461364A, including differential effect on apoptosis. Cancer Res. 2009;69(17):6969–77. doi: 10.1158/0008-5472.CAN-09-0945. [DOI] [PubMed] [Google Scholar]
  • 11.Sutton D, Diamond M, Faucette L, Giardiniere M, Zhang S, Gilmartin A, et al. Efficacy of GSK461364, a selective Plk1 inhibitor, in human tumor xenograft models. Cancer Res. 2007;67(9 Supplement):5388. [Google Scholar]
  • 12.Laquerre S, Sung C-M, Gilmartin A, Courtney M, Ho M, Salovich J, et al. A potent and selective Polo-like kinase 1 (Plk1) Inhibitor (GSK461364) induces cell cycle arrest and growth inhibition of cancer cell. Cancer Res. 2007;67(9 Supplement):5389. [Google Scholar]
  • 13.Kuntz K, Salovich J, Mook R, Emmitte K, Chamberlain S, Rheault T, et al. Identification of GSK461364, a novel small molecule polo-like kinase 1 inhibitor for the treatment of cancer. Cancer Res. 2007;67(9 Supplement):4171. [Google Scholar]
  • 14.Devaiah BN, Gegonne A, Singer DS. Bromodomain 4: a cellular Swiss army knife. Journal of leukocyte biology. 2016;100(4):679–86. doi: 10.1189/jlb.2RI0616-250R. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Adelman K, Lis JT. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. Nature reviews Genetics. 2012;13(10):720–31. doi: 10.1038/nrg3293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Asangani IA, Dommeti VL, Wang X, Malik R, Cieslik M, Yang R, et al. Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer. Nature. 2014;510(7504):278–82. doi: 10.1038/nature13229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Ferri E, Petosa C, McKenna CE. Bromodomains: Structure, function and pharmacology of inhibition. Biochem Pharmacol. 2016;106:1–18. doi: 10.1016/j.bcp.2015.12.005. [DOI] [PubMed] [Google Scholar]
  • 18.Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O, et al. Selective inhibition of BET bromodomains. Nature. 2010;468(7327):1067–73. doi: 10.1038/nature09504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Li J, Karki A, Hodges KB, Ahmad N, Zoubeidi A, Strebhardt K, et al. Cotargeting Polo-Like Kinase 1 and the Wnt/beta-Catenin Signaling Pathway in Castration-Resistant Prostate Cancer. Molecular and cellular biology. 2015;35(24):4185–98. doi: 10.1128/MCB.00825-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Momota H, Shih AH, Edgar MA, Holland EC. c-Myc and beta-catenin cooperate with loss of p53 to generate multiple members of the primitive neuroectodermal tumor family in mice. Oncogene. 2008;27(32):4392–401. doi: 10.1038/onc.2008.81. [DOI] [PubMed] [Google Scholar]
  • 21.Wang G, Wang J, Sadar MD. Crosstalk between the Androgen Receptor and beta-Catenin in Castrate-Resistant Prostate Cancer. Cancer Res. 2008;68(23):9918–27. doi: 10.1158/0008-5472.CAN-08-1718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Zhang Z, Chen L, Wang H, Ahmad N, Liu X. Inhibition of Plk1 represses androgen signaling pathway in castration-resistant prostate cancer. Cell cycle. 2015;14(13):2142–8. doi: 10.1080/15384101.2015.1041689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Zhang Z, Hou X, Shao C, Li J, Cheng JX, Kuang S, et al. Plk1 inhibition enhances the efficacy of androgen signaling blockade in castration-resistant prostate cancer. Cancer Res. 2014;74(22):6635–47. doi: 10.1158/0008-5472.CAN-14-1916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Steegmaier M, Hoffmann M, Baum A, Lenart P, Petronczki M, Krssak M, et al. BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo. Current biology : CB. 2007;17(4):316–22. doi: 10.1016/j.cub.2006.12.037. [DOI] [PubMed] [Google Scholar]
  • 25.Dai D, Holmes AM, Nguyen T, Davies S, Theele DP, Verschraegen C, et al. A potential synergistic anticancer effect of paclitaxel and amifostine on endometrial cancer. Cancer Res. 2005;65(20):9517–24. doi: 10.1158/0008-5472.CAN-05-1613. [DOI] [PubMed] [Google Scholar]
  • 26.Chou TC. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 2010;70(2):440–6. doi: 10.1158/0008-5472.CAN-09-1947. [DOI] [PubMed] [Google Scholar]
  • 27.Wu ZQ, Yang X, Weber G, Liu X. Plk1 phosphorylation of TRF1 is essential for its binding to telomeres. The Journal of biological chemistry. 2008;283(37):25503–13. doi: 10.1074/jbc.M803304200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Corey E, Quinn JE, Buhler KR, Nelson PS, Macoska JA, True LD, et al. LuCaP 35: a new model of prostate cancer progression to androgen independence. The Prostate. 2003;55(4):239–46. doi: 10.1002/pros.10198. [DOI] [PubMed] [Google Scholar]
  • 29.Hogg SJ, Vervoort SJ, Deswal S, Ott CJ, Li J, Cluse LA, et al. BET-Bromodomain Inhibitors Engage the Host Immune System and Regulate Expression of the Immune Checkpoint Ligand PD-L1. Cell reports. 2017;18(9):2162–74. doi: 10.1016/j.celrep.2017.02.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Ciceri P, Muller S, O’Mahony A, Fedorov O, Filippakopoulos P, Hunt JP, et al. Dual kinase-bromodomain inhibitors for rationally designed polypharmacology. Nature chemical biology. 2014;10(4):305–12. doi: 10.1038/nchembio.1471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Bisgrove DA, Mahmoudi T, Henklein P, Verdin E. Conserved P-TEFb-interacting domain of BRD4 inhibits HIV transcription. Proceedings of the National Academy of Sciences of the United States of America. 2007;104(34):13690–5. doi: 10.1073/pnas.0705053104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Dey A, Nishiyama A, Karpova T, McNally J, Ozato K. Brd4 marks select genes on mitotic chromatin and directs postmitotic transcription. Molecular biology of the cell. 2009;20(23):4899–909. doi: 10.1091/mbc.E09-05-0380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Rathert P, Roth M, Neumann T, Muerdter F, Roe JS, Muhar M, et al. Transcriptional plasticity promotes primary and acquired resistance to BET inhibition. Nature. 2015;525(7570):543–7. doi: 10.1038/nature14898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Song B, Liu XS, Rice SJ, Kuang S, Elzey BD, Konieczny SF, et al. Plk1 phosphorylation of orc2 and hbo1 contributes to gemcitabine resistance in pancreatic cancer. Molecular cancer therapeutics. 2013;12(1):58–68. doi: 10.1158/1535-7163.MCT-12-0632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Hou X, Li Z, Huang W, Li J, Staiger C, Kuang S, et al. Plk1-dependent microtubule dynamics promotes androgen receptor signaling in prostate cancer. The Prostate. 2013;73(12):1352–63. doi: 10.1002/pros.22683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Shao C, Ahmad N, Hodges K, Kuang S, Ratliff T, Liu X. Inhibition of polo-like kinase 1 (Plk1) enhances the antineoplastic activity of metformin in prostate cancer. The Journal of biological chemistry. 2015;290(4):2024–33. doi: 10.1074/jbc.M114.596817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Sramkoski RM, Pretlow TG, 2nd, Giaconia JM, Pretlow TP, Schwartz S, Sy MS, et al. A new human prostate carcinoma cell line, 22Rv1. In vitro cellular & developmental biology Animal. 1999;35(7):403–9. doi: 10.1007/s11626-999-0115-4. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS964934-supplement-1.docx (15.7KB, docx)
2
NIHMS964934-supplement-2.docx (15.5KB, docx)
3
NIHMS964934-supplement-3.pptx (145.7KB, pptx)
4
NIHMS964934-supplement-4.pptx (69.5KB, pptx)
5
NIHMS964934-supplement-5.pptx (5.8MB, pptx)
6
NIHMS964934-supplement-6.pptx (581.2KB, pptx)
7
NIHMS964934-supplement-7.pptx (21KB, pptx)

RESOURCES