BMI-1, also known as a stem cell factor, is frequently upregulated in several malignancies. Elevated expression of BMI-1 correlates with poor prognosis and is therefore considered a viable therapeutic target in a number of malignancies including ovarian cancer. Realizing the immense pathologic significance of BMI-1, small-molecule inhibitors against BMI-1 are recently being developed. In this study, we functionally characterize PTC-028, an orally bioavailable compound that decreases BMI-1 levels by posttranslational modification. We report that PTC-028 treatment selectively inhibits cancer cells in clonal growth and viability assays, whereas normal cells remain unaffected. Mechanistically, hyperphosphorylation-mediated depletion of cellular BMI-1 by PTC-028 coupled with a concurrent temporal decrease in ATP and a compromised mitochondrial redox balance potentiates caspase-dependent apoptosis. In vivo, orally administered PTC-028, as a single agent, exhibits significant antitumor activity comparable with the standard cisplatin/paclitaxel therapy in an orthotopic mouse model of ovarian cancer. Thus, PTC-028 has the potential to be used as an effective therapeutic agent in patients with epithelial ovarian cancer, where treatment options are limited. Mol Cancer Ther; 17(1); 39–49. ©2017 AACR.

BMI1/BMI-1 proto-oncogene, polycomb ring finger, a member of the Polycomb Repressor Complex 1 (PRC1) that mediates gene silencing by regulating chromatin structure is frequently upregulated in several types of cancer where its expression correlates with poor prognosis (1–5). We previously demonstrated that BMI-1 is overexpressed in epithelial ovarian cancer patient samples (3) and targeting BMI-1 sensitizes a variety of cancer cells to chemotherapeutics (3, 5–7). BMI-1 thus has been implicated in the propagation of several cancers with a role in self-renewal of cancer-initiating cells of glioblastoma and colorectal cancer (1, 2). Realizing this potential, Kreso and colleagues first described PTC-209, a small-molecule that decreased translation of BMI-1 mRNA (1), and inhibited self-renewal of cancer-initiating cells causing irreversible impairment in primary colorectal tumor growth when administered intratumorally. We subsequently reported that in ovarian cancer cells, PTC-209 potentiated autophagy-mediated necroptosis (8) and others reported Cyclin G2–mediated induction of autophagy in leukemia cells (9). Here, we investigated the biological and therapeutic activity of PTC-028, a novel compound with superior pharmaceutical properties that depletes BMI-1 at the protein level.

We report that PTC-028 significantly impacts clonal growth and viability of ovarian cancer cells by specifically decreasing BMI-1 through hyperphosphorylation-mediated degradation while normal cells, with minimal expression of BMI-1 are unaffected. Compared with PTC-209 (200 nmol/L), PTC-028 (100 nmol/L) depletes steady-state BMI-1 protein levels faster and induces depletion of ATP to potentiate caspase-dependent apoptosis through generation of mitochondrial reactive oxygen species (ROS). Importantly, orally administered PTC-028 exhibits significant single-agent antitumor activity in the orthotopic mouse model of ovarian cancer similar to that of the standard-of-care cisplatin/paclitaxel administered intraperitonealy. Therefore, PTC-028 could potentially be used as an effective therapeutic tool in several malignancies that are characterized by overexpression of BMI-1 including ovarian cancer.

Cell culture and chemicals

SV40-transformed primary normal ovarian epithelial cell line (OSE tsT/hTERT, henceforth OSE; ref. 10) was a kind gift from Dr. V. Shridhar (Mayo Clinic, Rochester, MN). SV40-transformed primary normal fallopian tube epithelial cells (henceforth FTE187 and FTE188; ref. 11) were kindly provided by Dr. Jinsong Liu (MD Anderson Cancer Center, Houston, TX). CP20 cell line was a kind gift from Dr. Anil K. Sood (MD Anderson Cancer Center, Houston, TX) and was authenticated through STR profiling facility at MD Anderson Cancer Center. OV90 and OVCAR4 cell lines were purchased from ATCC and NCI, respectively. OSE cells were routinely cultured in 1:1 MCDB 105 and Medium 199 (Corning) + 15% FBS (Gibco); FTE187 and FTE188 were cultured in 1:1 MCDB 105 and Medium 199 + 15% FBS + 0.01 μg/mL EGF; CP20, OV90, and OVCAR4 were routinely cultured in RPMI + 10% FBS. All the cells were cultured with 1× penicillin–streptomycin (Gibco) in a 5% CO2 humidified atmosphere and tested for mycoplasma contamination prior to any experiment. PTC-028 was obtained from PTC Therapeutics. PTC-209 (SML1143) was obtained from Sigma-Aldrich. FLAG-empty vector (FLAG-EV) or FLAG-BMI-1 was a kind gift from Dr. Damu Tang (McMaster University, Hamilton, Ontario, Canada). Gene silencing was performed using Hiperfect (Qiagen) and 10 picomoles siRNA (scrambled control D-001206-13-20, Dharmacon; BMI1 siRNA SASI-HS01-00175765 from Sigma in OPTIMEM (Invitrogen)

Cell lysis, cell fractionation, SDS-PAGE, and Western blotting

Total cell lysate was prepared in RIPA (purchased from Boston Bioproducts). Measurement of protein concentration, independent of the extraction method, was performed using BCA assay kit from Pierce. SDS-PAGE and immunoblotting was performed using standard protocol. The cell lysates were separated on 10% or 15% glycine SDS-PAGE gel and transferred to PVDF membrane. Membranes were blocked in 5% nonfat dry milk in TBS with 0.1% TWEEN-20 (TBST) for 1 hour at room temperature followed by incubation with indicated primary antibodies in TBST with 5% BSA. Antibodies were purchased from following vendors. BMI-1 was from Invitrogen (37-5400), Bethyl Laboratories (IHC-00606), and Proteintech (66161); uH2A (8240), H2A (2578), RING1A (2820), LC3B (2775), β-actin (4970), PARP (9542), cleaved caspase-3 (9664), cleaved caspase-7 (8438), cleaved caspase-9 (7237), NFκB/p65 (4764) from Cell Signaling Technology; RIPK1 (374639) from Santa Cruz Biotechnology; XIAP (MAB822) from R&D Systems, and secondary antibodies conjugated with horseradish peroxidase IgG rabbit (A6154) and mouse (A4416) from Sigma-Aldrich. Primary antibodies were used in dilutions recommended by the manufacturer. Secondary antibodies were used at a concentration of 1:10,000.

Determination of apoptosis, cell viability, and clonal growth

Apoptosis was determined by using the ApoTox-Glo triplex assay kit (G6321) from Promega. Briefly, PTC-028- or PTC-209–treated and untreated cells were incubated simultaneously to measure two protease activities; one is a marker of cell viability, and the other is a marker of cytotoxic cell death. The live- and dead-cell proteases produce different products, AFC and R110, which have different excitation and emission spectra, allowing them to be detected concurrently. The second part of the assay utilizes a luminogenic caspase-3/caspase-7 substrate (the tetrapeptide sequence DEVD), in a reagent optimized for caspase activity. Luminescence was proportional to the amount of caspase activity present. For clonal growth assay, cells were seeded as single cells (200 cells/well) in 6-well plates for 24 hours, treated with or without PTC-028 and cells were cultured for additional 7 days before staining with crystal violet (0.75% crystal violet, 50% ethanol, 0.25% NaCl, 1.57% formaldehyde) and counted.

Measurement of ATP and mitochondrial ROS

ATP was measured using CellTiter-Glo 2.0 Assay (G7571) from Promega as per the manufacturer's instructions. Briefly, cells were treated with PTC-028 or PTC-209 and the luminescence was recorded at various time intervals upto 48 hours using CLARIOstar (BMG Labtech). Luminescence intensity was determined (ATP) and normalized with respective viable cells by ApoTox-Glo triplex assay kit (G6321) in each group, and then expressed relative to respective control. Mitochondrial ROS was measured in cells by MitoSOX (Invitrogen) staining (2.5 μmol/L for 10 minutes at 37°C). Data were acquired with a FACSCalibur (BD Biosciences) and analyzed with FlowJo analytical software.

Preclinical model of ovarian cancer

Female athymic nude mice (NCr-nu; 6 to 8 weeks old) were purchased from the Harlan Laboratory. All mice were housed and maintained under specific pathogen-free conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the U.S. Department of Agriculture, U.S. Department of Health and Human Services, and NIH. All studies were approved and supervised by the University of Oklahoma Animal Facility under the guidance of the IACUC #13-101-SSH. For the generation of orthotopic ovarian tumor models, OV90 cells (1 million in 100-μL PBS) were orthotopically implanted in both the ovaries of each mice. Mice were randomized and divided into 3 groups (N = 7–10 mice/group): (i) vehicle control (0.5% HPMC,1% Tween 80), (ii) BMI-1 inhibitor PTC-028, and (iii) cisplatin + paclitaxel. Treatment was initiated 1 week after implantation and continued for another 3–4 weeks. After the final treatment and assessing tumor growth/regression in these animals, mice were sacrificed by CO2 inhalation with tumors and tissue harvested for further analysis.

IHC

Tumor grafts or mouse tissues were embedded in paraffin and sectioned (4 μm). These sections were deparaffinized in xylene, rehydrated in graded alcohol, subjected to heat-induced antigen retrieval with Target Retrieval Solution, and blocked with Protein Block. IHC was performed according to standard protocols. Antigen retrieval was achieved by heating sections in 95°C citrate buffer for 10 minutes. Sections were incubated with specific antibodies overnight at 4°C. For BMI-1 (1:300) and Ki67 (1:100) staining, the dark brown signal was revealed after incubation with the ABC kit (Vector), followed by a diaminobenzidine (DAB) and hydrogen peroxide reaction using the DAB detection kit (Vector). Counterstaining was performed by incubating the slides in hematoxylin for 5 minutes. Images were taken using Nikon Eclipse Ni microscope. To detect the apoptosis in tissue, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) positivity was assessed with a TUNEL apoptosis detection kit (DeadEnd Fluorometric TUNEL system, Promega) according to the manufacturer's instructions. IHC analysis of mouse tissues was performed in a blinded fashion.

Data analysis and statistics

All the experiments unless otherwise stated were repeated independently three times. Data are expressed as means ± SD. ANOVA was performed to compare the mean among three or more groups and Student t test was performed to compare the mean between two groups. Statistical significance was set at P < 0.05, using GraphPad Prism 6 software.

Depletion of BMI-1 by PTC-028 inhibits ovarian cancer cell viability and clonal growth

The chemical structure of PTC-028 is shown in Fig. 1A and its synthesis is described in Supplementary Fig. S1. BMI-1 levels were determined in the immortalized nonmalignant ovarian surface epithelial cells (OSE) and fallopian tube epithelial cells (FTE 187 and 188), as well as in the malignant cisplatin-resistant CP20, OVCAR4, and OV90 epithelial ovarian cancer cells using immunoblotting. Compared with the normal OSE and FTE, BMI-1 levels were higher in the ovarian cancer cells (Fig. 1B). Using the MTS assay, we next evaluated the effect of PTC-028 on cell viability. In normal OSE and FTE cells, upto 500 nmol/L treatment with PTC-028 for 48 hours had minimal effect (∼18%–30% decrease; Fig. 1C). However, OVCAR4, OV90, and CP20 cells demonstrated significant dose-dependent decrease in cell viability with a half-maximum inhibitory concentration (IC50) of approximately 100 nmol/L and approximately 95% decrease at 500 nmol/L (Fig. 1D). The lack of effect in normal cells that express minimal BMI-1 supports specificity of PTC-028 and was further confirmed later. Because targeting BMI-1 in various systems inhibits self-renewal and clonal growth, we next evaluated the effect of PTC-028 in clonal growth assays. A significant dose-dependent decrease in clonal growth was observed in all the ovarian cancer cell lines tested (Fig. 1E). These results confirm that PTC-028 effectively inhibits ovarian cancer cell viability and clonal growth. Using immunoblotting, we then determined that treatment with PTC-028 significantly reduced levels of BMI-1 and its functional readout, ubiquitinated histone 2A (uH2A) in ovarian cancer cells (Fig. 1F). However RING 1A, a polycomb repressor complex 1 (PRC1) partner of BMI-1 or total H2A levels remained unchanged (Fig. 1F) indicating specificity of PTC-028 toward BMI-1. These results together confirm that compared with nonmalignant cells, PTC-028 selectively inhibits cancer cells that express relatively more BMI-1.

Figure 1.

PTC-028 depletes BMI-1 and decreases ovarian cancer cell viability and clonal growth. A, The chemical structure of PTC-028. B, Expression of BMI-1 in normal and malignant ovarian cells. Immortalized ovarian surface epithelium (OSE) or the fallopian tube epithelium (FTE) cells (C) and ovarian cancer cells (D) were treated with PTC-028 at the indicated concentrations for 48 hours and cellular viability was assessed using the MTS assay. Vehicle-treated control cells were set to 100%. Data are the mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing with respective control by a two-way ANOVA. E, Ovarian cancer cells were treated with PTC-028 at the indicated concentrations for up to 7 days for CP20 and 10 days for OV90 and OVCAR4, respectively, and clonal growth determined by counting crystal violet–stained colonies. Vehicle-treated control cells were set to 100%. Data are mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing with respective control by a two-way ANOVA. F, Ovarian cancer cells were treated with PTC-028 at the indicated concentrations for 48 hours. Expression of RING1A, BMI-1, uH2A, and total H2A was determined by immunoblotting.

Figure 1.

PTC-028 depletes BMI-1 and decreases ovarian cancer cell viability and clonal growth. A, The chemical structure of PTC-028. B, Expression of BMI-1 in normal and malignant ovarian cells. Immortalized ovarian surface epithelium (OSE) or the fallopian tube epithelium (FTE) cells (C) and ovarian cancer cells (D) were treated with PTC-028 at the indicated concentrations for 48 hours and cellular viability was assessed using the MTS assay. Vehicle-treated control cells were set to 100%. Data are the mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing with respective control by a two-way ANOVA. E, Ovarian cancer cells were treated with PTC-028 at the indicated concentrations for up to 7 days for CP20 and 10 days for OV90 and OVCAR4, respectively, and clonal growth determined by counting crystal violet–stained colonies. Vehicle-treated control cells were set to 100%. Data are mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing with respective control by a two-way ANOVA. F, Ovarian cancer cells were treated with PTC-028 at the indicated concentrations for 48 hours. Expression of RING1A, BMI-1, uH2A, and total H2A was determined by immunoblotting.

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PTC-028 induces hyperphosphorylation and decreases BMI-1 function

To determine temporal differences in depletion of BMI-1 by PTC-209 and PTC-028, we performed immunoblotting after treating with different concentrations of the compounds for 0–12 hours. A time-dependent increase in the phosphorylated BMI-1 species and subsequent reduction in the biochemical functional readout, uH2A was observed up to 12 hours with PTC-028 (100 nmol/L) in both CP20 (Fig. 2A and B) and OV90 cells (Fig. 2C and D) while total H2A levels remained unchanged. Accordingly, basal BMI-1 levels were reduced in CP20 and OV90 cells by approximately 53% and approximately 54%, respectively, at 12 hours with PTC-028 (Fig. 2B and D), suggesting depletion of BMI-1 following its hyperphosphorylation. At 200 nmol/L, PTC-209, however, did not affect BMI-1 levels up to 12 hours in either cell line. That PTC-028–mediated depletion of BMI-1 was due to hyperphosphorylation was confirmed by treating CP20 and OV90 cells with PTC-028 for 12 hours. Subsequently, the cellular lysates were treated with or without lambda phosphatase (λ-phosphatase) and subjected to immunoblotting. Disappearance of the lower mobility hyperphospho bands and accumulation of basal BMI-1 in the phosphatase-treated samples (Fig. 2E) confirmed induction of phosphorylation by PTC-028. Significant reduction of phosphorylated AKT in the PTC-028 and λ-phosphatase, dual treated samples confirmed efficiency of phosphatase action (Fig. 2E). These results clearly indicate a more potent depletion of BMI-1 by PTC-028 at a lower concentration than PTC-209 by 12 hours.

Figure 2.

Temporal depletion of BMI-1 by PTC-028 and PTC-209. A, CP20 cells were treated with PTC-028 at 100 nmol/L or with PTC-209 at 200 nmol/L for the indicated times. Expression of BMI-1, uH2A, total H2A, and β-actin was determined by immunoblotting. B, Top, represents NIH ImageJ quantitation of the hyperphosphorylated (pBMI-1) bands (high exposure) as indicated in A. Bottom, similar representation of the basal BMI-1 (bBMI-1; low exposure) as indicated in A. Data represent mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing with respective control by a one-way ANOVA. C, OV90 cells were treated with PTC-028 at 100 nmol/L or with PTC-209 at 200 nmol/L for the indicated times. Expression of BMI-1, uH2A, total H2A, and β-actin was determined by immunoblotting. D, Top, represents NIH ImageJ quantitation of the hyperphosphorylated bands as indicated in C. Bottom, shows similar representation of the basal BMI-1 as indicated in C. Data represent mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing with respective control by a one-way ANOVA. E, CP20 or OV90 cells were treated with PTC-028 at 100 nmol/L for 12 hours with or without treatment with λ-phosphatase. The expression of BMI-1, pAKT (S473), and β-actin was determined by immunoblotting.

Figure 2.

Temporal depletion of BMI-1 by PTC-028 and PTC-209. A, CP20 cells were treated with PTC-028 at 100 nmol/L or with PTC-209 at 200 nmol/L for the indicated times. Expression of BMI-1, uH2A, total H2A, and β-actin was determined by immunoblotting. B, Top, represents NIH ImageJ quantitation of the hyperphosphorylated (pBMI-1) bands (high exposure) as indicated in A. Bottom, similar representation of the basal BMI-1 (bBMI-1; low exposure) as indicated in A. Data represent mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing with respective control by a one-way ANOVA. C, OV90 cells were treated with PTC-028 at 100 nmol/L or with PTC-209 at 200 nmol/L for the indicated times. Expression of BMI-1, uH2A, total H2A, and β-actin was determined by immunoblotting. D, Top, represents NIH ImageJ quantitation of the hyperphosphorylated bands as indicated in C. Bottom, shows similar representation of the basal BMI-1 as indicated in C. Data represent mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing with respective control by a one-way ANOVA. E, CP20 or OV90 cells were treated with PTC-028 at 100 nmol/L for 12 hours with or without treatment with λ-phosphatase. The expression of BMI-1, pAKT (S473), and β-actin was determined by immunoblotting.

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Depletion of BMI-1 by PTC-028 induces apoptosis

To determine the mechanism by which PTC-028 inhibits cancer cell growth, CP20, OV90, and OVCAR4 cells were treated with increasing concentrations of PTC-028 for 48 hours followed by the ApoTox-Glo Triplex assay that simultaneously determines cell viability, cell death, and caspase 3/7 activity by utilizing two different protease markers and a luminogenic caspase substrate, respectively (8). A dose-dependent decrease in cell viability and simultaneous increase in caspase-3/7 activity was observed, without any effect on cytotoxic cell death (Fig. 3A). We previously confirmed that a cytotoxic cell death signal is generated in this assay only by a disruption of the cellular membrane (Triton-X) but not by an apoptotic stimuli, such as cisplatin (8). Recent evidences suggest a fallopian tube origin of ovarian cancer (12, 13) rendering the FTE188 cells a suitable model for normal ovarian cells. In FTE188 cells that express minimal BMI-1 (Fig. 1B), PTC-028 had no effect (Fig. 1C); however, forced expression of BMI-1 (Supplementary Fig. S2A) sensitized FTE188 cells to PTC-028 demonstrating specificity toward BMI-1 (Supplementary Fig. S2B). Furthermore, silencing BMI-1 in CP20 or OV90 cells rendered them insensitive to PTC-028 confirming specificity (Supplementary Fig. S2C). To determine whether decreased cellular viability was due to apoptosis, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) positivity was evaluated in the ovarian cancer cells that were treated with PTC-028 (100 nmol/L) for 48 hours. Compared with the untreated control, significant TUNEL positivity was observed in the PTC-028 treated CP20 (∼32%), OV90 (∼34%), and OVCAR4 (∼35%) cells, respectively (Fig. 3B). Having demonstrated that OVCAR4 cells responded similarly to PTC-028 with respect to cellular viability and apoptosis, henceforth, further experiments were performed using CP20 and OV90 cell lines. In contrast to PTC-028, PTC-209 potentiates autophagy leading to RIPK-mediated necroptosis in ovarian cancer cells (8). To assess potential differences in signaling, we treated ovarian cancer cells with PTC-209 (200 nmol/L) or PTC-028 (100 nmol/L) for 48 hours followed by immunoblotting. Appreciable depletion of BMI-1 was observed by both the compounds (Fig. 3C). Consistent with our prior report (8), PTC-209 induced the expression of LC3B-II and X-linked inhibitor of apoptosis (XIAP) while RIPK1 levels remained unchanged (Fig. 3C). PTC-028 however decreased the expression of XIAP and RIPK1 while LC3B levels remained unchanged compared with that of the control (Fig. 3C). In addition, significant cleavage of caspase-7, caspase-9, and PARP was observed in PTC-028, but not in PTC-209–treated ovarian cancer cells (Fig. 3C). These results clearly suggest that depletion of BMI-1 by PTC-028 induces caspase-mediated apoptosis.

Figure 3.

PTC-028 induces caspase-dependent apoptosis. A, CP20, OV90, and OVCAR4 cells were treated with increasing concentrations of PTC-028 for 48 hours and cell viability, cytotoxic cell death, and caspase-3/7 activity was evaluated using the ApoTox-Glo Triplex assay. Data are mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing with respective vehicle-treated control by a two-way ANOVA. B, CP20, OV90, and OVCAR4 cells were treated with 100 nmol/L PTC-028 for 48 hours, cells were subjected to the TUNEL assay and analyzed by fluorescence microscopy. The number of TUNEL-positive nuclei were counted from approximately 300 cells per treatment group. Data represent the mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing with respective control by Student t test. C, CP20 or OV90 cells were treated with 100 nmol/L PTC-028 or 200 nmol/L PTC-209 for 48 hours. Expression of BMI-1, RIPK1, XIAP, LC3B, cleaved caspase-7, cleaved caspase-9, PARP, and β-actin was determined by immunoblotting. D, CP20 cells were treated with PTC-028 at 100 nmol/L or with PTC-209 at 200 nmol/L for the indicated times. Expression of BMI-1, PARP, and β-actin was determined by immunoblotting (left panel). The quantitation of basal, residual BMI-1 by NIH Image J is shown in the right panel. E, OV90 cells were treated with PTC-028 at 100 nmol/L or with PTC-209 at 200 nm for the indicated times. Expression of BMI-1, PARP and β-actin was determined by immunoblotting (left). NIH ImageJ quantitation of the basal, residual BMI-1 is shown in the right. Data represent mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing between each group at respective time point by a two-way ANOVA.

Figure 3.

PTC-028 induces caspase-dependent apoptosis. A, CP20, OV90, and OVCAR4 cells were treated with increasing concentrations of PTC-028 for 48 hours and cell viability, cytotoxic cell death, and caspase-3/7 activity was evaluated using the ApoTox-Glo Triplex assay. Data are mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing with respective vehicle-treated control by a two-way ANOVA. B, CP20, OV90, and OVCAR4 cells were treated with 100 nmol/L PTC-028 for 48 hours, cells were subjected to the TUNEL assay and analyzed by fluorescence microscopy. The number of TUNEL-positive nuclei were counted from approximately 300 cells per treatment group. Data represent the mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing with respective control by Student t test. C, CP20 or OV90 cells were treated with 100 nmol/L PTC-028 or 200 nmol/L PTC-209 for 48 hours. Expression of BMI-1, RIPK1, XIAP, LC3B, cleaved caspase-7, cleaved caspase-9, PARP, and β-actin was determined by immunoblotting. D, CP20 cells were treated with PTC-028 at 100 nmol/L or with PTC-209 at 200 nmol/L for the indicated times. Expression of BMI-1, PARP, and β-actin was determined by immunoblotting (left panel). The quantitation of basal, residual BMI-1 by NIH Image J is shown in the right panel. E, OV90 cells were treated with PTC-028 at 100 nmol/L or with PTC-209 at 200 nm for the indicated times. Expression of BMI-1, PARP and β-actin was determined by immunoblotting (left). NIH ImageJ quantitation of the basal, residual BMI-1 is shown in the right. Data represent mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing between each group at respective time point by a two-way ANOVA.

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To further appreciate the temporal link between depletion of cellular steady-state BMI-1 levels and apoptosis, we treated CP20 and OV90 cells with 100 nmol/L PTC-028 or 200 nmol/L PTC-209 for 0–48 hours. A gradual depletion of cellular BMI-1 levels was induced by PTC-028; however, maximal depletion by PTC-209 was observed between 36 and 48 hours (Fig. 3D and E). At 48 hours, however, both the compounds significantly inhibited BMI-1 to similar levels. Interestingly, while there was no PARP cleavage in PTC-209–treated cells, earliest PARP cleavage could be observed as early as 12 hours after treatment with PTC-028 (Fig. 3D and E) suggesting activation of apoptosis.

To establish that PTC-028–mediated decrease in cell viability was due to apoptosis, we treated the cells with the pan-caspase inhibitor z-VAD-fmk (10 μmol/L) for 3 hours with or without PTC-028 (100 nmol/L) for 48 hours and analyzed cell viability using the MTS assay. Compared with PTC-028 only, dual treatment with z-VAD-fmk significantly increased cell viability by approximately 30% in CP20 and approximately 41% in OV90 cells, respectively (Supplementary Fig. S3A). To further confirm the involvement of the caspase cascade in PTC-028–mediated apoptosis, z-VAD-fmk pretreated cells with or without PTC-028 were subjected to immunoblotting. Compared with PTC-028 only, dual treatment with z-VAD-fmk partially restored RIPK1, XIAP, and NFκB/p65 levels (Supplementary Fig. S3B). Prior reports indicate that caspase-mediated depletion of RIPK1 potentiates apoptosis if levels of antiapoptotic NFκB or XIAP are low; furthermore, such apoptosis can be blocked by treatment with z-VAD-fmk (14). Together, these results establish that PTC-028 –mediated apoptotic cell death is specific toward high BMI-1–expressing cells and is caspase-dependent.

PTC-028–mediated apoptosis is dependent on mitochondrial ROS

While caspase activation is a manifestation of apoptotic cell death, reduced ATP levels and compromised redox balance have been attributed as causes (15). Therefore, we determined ATP levels in PTC-02-8 and PTC-209–treated cells (Fig. 4A). Similar to BMI-1 depletion (Fig. 3D and E), a gradual depletion in cellular ATP levels was observed in PTC-028–treated cells (Fig. 4A). However in PTC-209–treated cells, ATP levels remained steady up to 36 hours after which a significant drop was observed at 48 hours (Fig. 4A). Next using MitoSOX Red, we determined mitochondrial superoxide formation in PTC-028- or PTC-209–treated cells at 48 hours. A robust increase in mean fluorescence intensity of oxidized MitoSOX was evidenced after PTC-028, but not PTC-209 treatment (Fig. 4B). To evaluate whether mitochondrial ROS was required for the induction of apoptotic cell death, both OV90 and CP20 cells were pretreated with mitoquinone (MitoQ), a mitochondria-targeted antioxidant (16) followed by PTC-028 for 48 hours and cell viability determined by the MTS assay. Pretreatment with MitoQ at 10 μmol/L rescued PTC-028–mediated decrease in cell viability by approximately 28% in OV90 and by approximately 29% in CP20 cells, respectively (Fig. 4C). To determine the status of the apoptotic markers, MitoQ pretreated cells with or without PTC-028 were subjected to immunoblotting. Compared with PTC-028 only, dual treatment with MitoQ restored XIAP and PARP levels to near control while cleaved caspase-9 was absent from the dual MitoQ and PTC-028–treated cells (Fig. 4D). Together, these results confirm that PTC-028 induces mitochondria-mediated apoptosis in ovarian cancer cells.

Figure 4.

PTC-028–mediated apoptosis is dependent on cellular ATP depletion and mitochondrial ROS. A, CP20 or OV90 cells were treated with PTC-028 at 100 nmol/L or with PTC-209 at 200 nmol/L for the indicated times and intracellular ATP levels determined and normalized with respective number of viable cells in each group. Data represent mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing between each group at respective time point by a two-way ANOVA. B, OV90 or CP20 cells were treated with 200 nmol/L PTC-209 or 100 nmol/L PTC-028 for 48 hours followed by MitoSOX staining and analyzed by a FACSCalibur flow cytometer. A representative histogram depicting mean fluorescence intensity from three independent replicates is shown. C, OV90 or CP20 cells were pretreated with or without Mitoquinone (MQ) 10 μmol/L for 3 hours followed by PTC-028 at 100 nmol/L for 48 hours. Cell viability was assessed by the MTS assay. Vehicle-treated control cells were set to 100%. Data represent mean ± SD of three independent experiments performed in triplicate and *, P < 0.05 by a two-way ANOVA. D, OV90 or CP20 cells were pretreated with or without mitoquinone at 10 μmol/L for 3 hours followed by PTC-028 at 100 nmol/L for 48 hours. Expression of XIAP, PARP, cleaved caspase-9 and β-actin was determined by immunoblotting.

Figure 4.

PTC-028–mediated apoptosis is dependent on cellular ATP depletion and mitochondrial ROS. A, CP20 or OV90 cells were treated with PTC-028 at 100 nmol/L or with PTC-209 at 200 nmol/L for the indicated times and intracellular ATP levels determined and normalized with respective number of viable cells in each group. Data represent mean ± SD of three independent experiments performed in triplicate. *, P < 0.05 when comparing between each group at respective time point by a two-way ANOVA. B, OV90 or CP20 cells were treated with 200 nmol/L PTC-209 or 100 nmol/L PTC-028 for 48 hours followed by MitoSOX staining and analyzed by a FACSCalibur flow cytometer. A representative histogram depicting mean fluorescence intensity from three independent replicates is shown. C, OV90 or CP20 cells were pretreated with or without Mitoquinone (MQ) 10 μmol/L for 3 hours followed by PTC-028 at 100 nmol/L for 48 hours. Cell viability was assessed by the MTS assay. Vehicle-treated control cells were set to 100%. Data represent mean ± SD of three independent experiments performed in triplicate and *, P < 0.05 by a two-way ANOVA. D, OV90 or CP20 cells were pretreated with or without mitoquinone at 10 μmol/L for 3 hours followed by PTC-028 at 100 nmol/L for 48 hours. Expression of XIAP, PARP, cleaved caspase-9 and β-actin was determined by immunoblotting.

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Therapeutic activity of PTC-028 in vivo

To translate our in vitro results in vivo, we determined plasma concentration of PTC-028 after administration of single oral doses to the CD-1 mice. Total plasma AUC0–24h were 10.9 and 26.1 μg/h/mL at doses of 10 and 20 mg/kg, which suggests dose proportional pharmacokinetics. The Cmax for PTC-028 at 10 and 20 mg/kg was 0.79 and 1.49 μg/mL, respectively. The Cmax was reached at both dose levels 1-hour postdose after which plasma concentrations slowly reduced (Supplementary Fig. S4). Next, we implanted OV90 cells in the bilateral ovarian bursa of athymic female nude mice. A week after implantation, mice were randomized in to three different groups. PTC-028 was administered orally at a dose of 15 mg/kg twice weekly to one group. The second group received intraperitoneal injections of cisplatin at a dose of 3 mg/kg/weekly and paclitaxel at 15 mg/kg/weekly following the established standard-of-care for treatment of epithelial ovarian cancer (17, 18), while the third group received vehicle control orally and intraperitoneally. Three weeks after treatment the mice were euthanized and tumor tissues collected, weighed, and processed for further analysis by IHC. Compared with the control (average tumor weight, ∼3 g), single-agent PTC-028 and cisplatin+paclitaxel caused approximately 94% (0.169 g) and approximately 99% (0.025 g) reduction in tumor weight, respectively (Fig. 5A and B). No obvious toxicity was noted in the animals during therapy experiments as assessed by mean body weight (Fig. 5B). The tumor tissues were further analyzed by IHC and compared with the control, the expression of BMI-1 was reduced by 8-fold in the PTC-028 group and by 3.5-fold in the cisplatin/paclitaxel–treated group. Similarly, the proliferation marker Ki67 decreased by 2.6-fold and 3.6-fold in the PTC-028 and the cisplatin/paclitaxel–treated group, respectively (Fig. 5C). Consequently, a significant increase in TUNEL positivity was observed in the PTC-028 (8.7-fold) and cisplatin/paclitaxel (6.1-fold) treated groups compared with the control (Fig. 5C). These results confirm the specificity of PTC-028 toward BMI-1 in vivo and indicate that anti-BMI-1 strategies are comparable in their efficacy to standard therapy in the orthotopic OV90 model.

Figure 5.

Therapeutic efficacy of PTC-028 in the orthotopic OV90 model. A, OV90 cells were implanted in the bursa of bilateral ovaries in athymic female nude mice. One week later, mice were randomized into three groups of 7–10 each and treatment initiated. PTC-028 was administered orally at 15 mg/kg twice weekly. Cisplatin at 3 mg/kg/weekly and paclitaxel at 15 mg/kg/weekly were administered by intraperitoneal injections and vehicle control given orally and intraperitoneally. Three weeks after treatment, the mice were euthanized and tumor tissues collected, weighed, and processed. Representative images of the tumors within the ovary and after resection from each group are shown. B, Top, average and individual tumor weight ± SD from each group. *, P < 0.05 compared with the control group by a one-way ANOVA. Bottom, average body weight of the mice after treatment from each group. C, IHC staining of tumor xenografts for BMI-1 (top), Ki67 (middle), and TUNEL positivity (bottom). After quantification, fold changes with respect to the control are shown graphically on the right, scale bar represents 50 μm for BMI-1, Ki67 and 10 μm for TUNEL. *, P < 0.05 compared with the control group by one-way ANOVA.

Figure 5.

Therapeutic efficacy of PTC-028 in the orthotopic OV90 model. A, OV90 cells were implanted in the bursa of bilateral ovaries in athymic female nude mice. One week later, mice were randomized into three groups of 7–10 each and treatment initiated. PTC-028 was administered orally at 15 mg/kg twice weekly. Cisplatin at 3 mg/kg/weekly and paclitaxel at 15 mg/kg/weekly were administered by intraperitoneal injections and vehicle control given orally and intraperitoneally. Three weeks after treatment, the mice were euthanized and tumor tissues collected, weighed, and processed. Representative images of the tumors within the ovary and after resection from each group are shown. B, Top, average and individual tumor weight ± SD from each group. *, P < 0.05 compared with the control group by a one-way ANOVA. Bottom, average body weight of the mice after treatment from each group. C, IHC staining of tumor xenografts for BMI-1 (top), Ki67 (middle), and TUNEL positivity (bottom). After quantification, fold changes with respect to the control are shown graphically on the right, scale bar represents 50 μm for BMI-1, Ki67 and 10 μm for TUNEL. *, P < 0.05 compared with the control group by one-way ANOVA.

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Accumulating evidences have established BMI-1 as an important therapeutic target in several different malignancies including ovarian cancer (1, 3–5, 19). Consequently, PTC-209, a small molecule that inhibited BMI-1 reporter expression (1), potentiated autophagy leading to RIPK-mediated necroptotic cell death (8) in ovarian cancer and impaired primary colorectal tumor growth. Here we report for the first time that PTC-028, a second-generation inhibitor that depletes BMI-1 at the protein level, activates caspase-dependent apoptosis and significantly reduces ovarian tumor growth similar to the standard-of-care cisplatin/paclitaxel.

The specificity of PTC-028 toward BMI-1 was demonstrated by the fact that normal OSE and FTE cells that express low levels of BMI-1 were nonresponsive while ovarian cancer cells that express higher BMI-1 showed a dose-dependent decrease in cell viability upon treatment with PTC-028. Furthermore, forced expression of BMI-1 sensitized FTE188 cells to PTC-028, thus confirming specificity toward BMI-1.

Interestingly, although at 48 hours, relative depletion of cellular BMI-1 by either PTC-028 (100 nmol/L) or PTC-209 (200 nmol/L) was comparable, there was a distinct difference at earlier time points. A gradual depletion of cellular BMI-1 that reached approximately 50% by 12 hours was observed in PTC-028–treated cells. However, in PTC-209–treated cells a severe decline in BMI-1 levels was observed only between 36 and 48 hours of treatment. Similar to depletion of BMI-1, cellular ATP levels also gradually decreased over time with PTC-028 but drastically dropped between 36 and 48 hours in PTC-209–treated cells. Furthermore, at 48 hours, significant induction of mitochondrial ROS was observed with PTC-028 but not PTC-209 treatment. These results lead us to conclude that a gradual decrease in cellular BMI-1 levels is linked to the decrease in ATP and increase in mitochondrial ROS generation. Prior studies from our and other groups have demonstrated that BMI-1 supports mitochondrial complex activity, absence of which leads to decreased ATP production and enhanced ROS generation (8, 20). We therefore speculate that a gradual drop in ATP coupled with a compromised redox balance potentiates apoptosis (15) in PTC-028–treated cells. However, a swift, drastic drop in ATP coupled with induction of XIAP and lack of ROS prevents apoptosis in PTC-209–treated cells. Indeed, prior studies have demonstrated that intracellular ATP levels determine cell death fate (21, 22). A transient and moderate depletion of ATP in cells induces apoptosis while potent and prolonged depletion causes necrosis (23–27) because apoptosis is an energy requiring process while necrosis is not (21).

PTC-028–mediated apoptotic cell death was exemplified by the enhanced caspase activity, TUNEL positivity, and cleavage of PARP. Furthermore, decreased cell viability could be rescued by the pan-caspase inhibitor z-VAD-fmk. In addition, RIPK1 and XIAP levels were significantly decreased by PTC-028, but not by PTC-209. Complex roles of RIPK1 in regulating NFκB-mediated inflammatory signaling, FADD/caspase-8–mediated apoptotic signaling or RIPK1/RIPK3–mediated necroptotic signaling are beginning to be appreciated (14, 28). It is noteworthy that the RIPK1−/− mice display extensive apoptosis in their lymphoid and adipose tissue and die within 1–3 days of birth (14, 29). Also, RIPK1 is an important component of major cellular death pathways, such as those regulated by Fas/FADD, TRAIL-R, and TNFR signaling (14, 30–32). In all these pathways, caspase-8–mediated cleavage of RIPK1 potentiates apoptosis if levels of antiapoptotic NFκB or XIAP are low that can be blocked by treatment with z-VAD-fmk (Fig. 6; ref. 14). Our results are consistent with these observations in that z-VAD-fmk restores levels of RIPK1, XIAP, and NFκB/p65 in PTC-028–treated cells.

Figure 6.

Scheme of action of PTC-028. PTC-028 induces hyperphosphorylation and subsequent depletion of BMI-1 that induces reduction of ATP and induction of mitochondrial ROS along with inhibition in expression of RIPK1 and XIAP. Low XIAP and increased ROS then activate caspase-9 that is followed by caspase-3/7. RIPK1 can be targeted by caspases leading to downregulation of NFκB that reciprocally regulates XIAP. Together, these signaling ultimately lead to apoptosis. Dashed arrows represent published reports as elaborated in the Discussion section.

Figure 6.

Scheme of action of PTC-028. PTC-028 induces hyperphosphorylation and subsequent depletion of BMI-1 that induces reduction of ATP and induction of mitochondrial ROS along with inhibition in expression of RIPK1 and XIAP. Low XIAP and increased ROS then activate caspase-9 that is followed by caspase-3/7. RIPK1 can be targeted by caspases leading to downregulation of NFκB that reciprocally regulates XIAP. Together, these signaling ultimately lead to apoptosis. Dashed arrows represent published reports as elaborated in the Discussion section.

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Furthermore, in contrast to PTC-209 that was administered subcutaneously at 60 mg/kg/day for 10 days to inhibit subcutaneous xenograft tumors (1), PTC-028, administered orally at a dose of 15 mg/kg twice weekly, showed impressive single-agent activity similar to that of cisplatin/paclitaxel administered by intraperitoneal delivery in the orthotopic OV90 ovarian cancer model without any obvious toxicity.

In summary, PTC-028 induces hyperphosphorylation and gradual depletion of BMI-1 that results in depletion of ATP and induction of mitochondrial ROS with decreased expression of both RIPK1 (33) and XIAP (34) leading to activation of the caspase cascade. In addition, caspase targeting of RIPK1 can lead to downregulation of NFκB, which has a reciprocal feedback regulation with XIAP (35, 36). Together, the signaling ultimately leads to apoptotic cell death (Fig. 6). Therefore, PTC-028 could potentially be used as an effective therapeutic for management of patients with epithelial ovarian cancer, for whom treatment options are limited.

No potential conflicts of interest were disclosed.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conception and design: A. Dey, Y.-C. Moon, R. Bhattacharya

Development of methodology: A. Dey, X. Xiong, R. Baiazitov, T. Davis

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Dey, X. Xiong, S.K.D. Dwivedi, S.B. Mustafi, R. Baiazitov, T. Davis, R. Bhattacharya

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Dey, A. Crim, S.K.D. Dwivedi, R. Baiazitov, R. Bhattacharya

Writing, review, and/or revision of the manuscript: A. Dey, A. Crim, P. Mukherjee, L. Cao, R. Bhattacharya

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Dey, L. Cao, R. Baiazitov, M. Dumble, R. Bhattacharya

Study supervision: A. Dey, P. Mukherjee, L. Cao, R. Bhattacharya

Other (design and synthesis of novel BMI-1 inhibitors): N. Sydorenko

We thank an Institutional Development Award (IDeA) grant (P20 GM103639) from the National Institute of General Medical Sciences of the National Institutes of Health supporting the use of the Histology and Immunohistochemistry Core. We thank the Laboratory for Molecular Biology and Cytometry Research at OUHSC for the use of the Flow Cytometry and Imaging facility. This study was supported by the NIH CA 157481 (to R. Bhattacharya), Foundation for Women's Cancer (FWC), St. Louis Ovarian Cancer Awareness Research Grant (to A. Dey).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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