Pharmaceutically inhibiting polo-like kinase 1 exerts a broad anti-tumour activity in retinoblastoma cell lines
ABSTRACT
Background: Retinoblastoma is the most common malignant cancer of the eye in children. While metastatic retinoblastoma is rare, cure rates for this advanced disease remain below 50%. High-level PLK1 expression in retinoblastomas has previously been shown to be correlated with adverse outcome parameters. PLK1 is a serine/threonine kinase involved in cell cycle regulation at the G2/M transition. PLK1 inhibition has been demonstrated to have anti-tumour effects in preclinical models of several paediatric tumours. Here we assessed its efficacy against retinoblastoma cell lines.Methods: Expression of PLK1 was determined in a panel of retinoblastoma cell lines by PCR and western blot analysis. We analysed viability (MTT assay), proliferation (BrdU ELISA), cell cycle progression (PI staining) and apoptosis (Cell death ELISA) in three retinoblastoma cell lines after treatment with two ATP-competitive PLK1 inhibitors, BI6727 or GSK461364. Activation of PLK1 downstream signalling components including TP53 were assessed.Results: Treatment of retinoblastoma cells with either BI6727 or GSK461364 reduced cell viability and proliferative capacity, and induced both cell cycle arrest and apoptosis. PLK1 inhibition also induced the p53 signalling pathway. Analysis of key players in cell cycle control revealed that low nanomolar concentrations of either PLK1 inhibitor upregulated CCNB1 and increased activated CDK1 (phosphorylated at Y15) in retinoblastoma cell lines.Conclusions: These preclinical data indicate that PLK1 inhibitors could be useful as components in rationally designed chemotherapy protocols to treat patients with metastasised retinoblastoma in early phase clinical trials.
INTRODUCTION
Retinoblastoma is the most common malignant eye tumour in childhood.1 It is characterised by a biallelic loss of RB1, and is the paradigm for a tumour induced by loss of tumour suppressor gene function.2 Due to early detection, overall survival of children with retinoblastoma is high in developed countries. Metastatic retinoblastoma is rare, but patients have a dismal prognosis despite aggressive multimodal therapy.3,4 Worldwide, fewer than 50% of patients diagnosed with metastatic retinoblastoma can be cured with current treatment protocols. Localised intraocular retinoblastoma is curable, but many children lose their eyesight or suffer severe late effects after aggressive eye-preserving treatment. These current challenges for paediatric oncology create the need for novel effective and efficient therapeutic options to treat these patients.Recently, high-level expression of the PLK1 serine/threonine kinase in retinoblastoma was correlated with poor tumour differentiation and histopathological high-risk features.5 PLK1 acts as an essential regulator of cell cycle progression by promoting entry into the M phase at the end of the G2 phase 6 and inducing G2 checkpoint recovery after DNA damage.7 Elevated PLK1 activity might stimulate tumour growth by activating mitotic transcriptional programs and avoiding the DNA- damage checkpoint.8,9 PLK1 is overexpressed in a wide variety of cancers, and different PLK1 inhibitors have been introduced and investigated in preclinical models and clinical trials. BI6727 and GSK461364 are potent and selective ATP-competitive kinase inhibitors, which have demonstrated efficacy against several cancer types in preclinical and clinical studies.10 Both inhibitors target the ATP-binding pocket of PLK1, binding to the hinge region between the amino-terminal and the carboxy- terminal lobes of the kinase domain via hydrogen bonds between the dihydropteridinone inhibitor core with the backbone amino and carbonyl groups of C133.
Both inhibitors, BI6727 and GSK461364, are as monotherapy in clinical trials for patients with solid tumors.12–14 BI6727 is currently being tested in combination with conventional chemotherapy in several phase II trials for patients with acute myeloid leukemia and solid tumours.11,15 PLK1 inhibitors have also demonstrated their efficacy against paediatric solid tumours, particularly neuroblastomas and medulloblastomas.16, 17 PLK1 downregulation has been shown to induce apoptosis in medulloblastoma cells and sensitise them to ionising radiation in in vitro assays.17 PLK1 is a possible therapeutic target for retinoblastoma, and both BI6727 and GSK461364 are good candidates for inhibiting PLK1 as a part of tumour therapy.Here we investigated the effect of BI6727 and GSK461364 on cell viability, cell cycle progression, apoptosis and PLK1 signalling in three cell lines derived from retinoblastomas with variable genetic backgrounds.BI6727, N-[4-[4-(cyclopropylmethyl)piperazin-1-yl]cyclohexyl]-4-[[(7R)-7-ethyl-5- methyl-6-oxo-8-propan-2-yl-7H-pteridin-2-yl]amino]-3-methoxybenzamide, and GSK461364, (R)-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3- (1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxamide, were purchased from Selleckchem (Munich, Germany). Inhibitors were dissolved in DMSO at 10 mmol/l for stock solutions, and stored at -80°C.The human retinoblastoma cell lines, Y79, WERI-Rb1, RBL-13, RBL-15, RBL-30, RB383, RB3823, RB522, were cultivated in suspension in RPMI-1640 medium (Invitrogen, Waltham, MA, USA) supplemented with 10% fetal calf serum (Invitrogen), 100 U/ml penicillin/streptomycin (Invitrogen) and 2.5 mg/l amphotericin B (PAA Laboratories GmbH, Cölbe, Germany) in a humidified atmosphere of 5% CO2 at 37°C. The RB355 retinoblastoma cell line and the SAOS2 osteosarcoma cell line were cultivated as monolayers in the same medium and under the same conditions. The WERI-Rb1 and Y79 cell lines were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). RB355, RB383, RB3823, RB522 were kindly provided by Brenda Gallie (Department of Ophthalmology and Vision Sciences, Hospital for Sick Children, Toronto). RBL-13, RBL-15, RBL-30 were obtained from the Institute of Cell Biology, University hospital Essen, Germany. The human dermal fibroblast cell line (NHDF)used in control experiments was cultivated in DMEM (Invitrogen) with the supplements described above and under the same conditions. The identity of all cell lines was validated by analyses of RB1 mutational status and short tandem repeat (STR) analyses.
Retinoblastoma cell lines were seeded into 96-well plates in quadruplicate and incubated for 24 hours before treatment with inhibitors. Viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Carl Roth GmbH, Karlsruhe, Germany) following 72 hours of treatment with 1.25-1000 nM BI6727 or GSK461364.Apoptosis and proliferation were assessed using the Cell Death DetectionPLUS ELISA and the Cell Proliferation ELISA, BrdU (both from Roche GmbH, Karlsruhe, Germany), respectively, in WERI-Rb1, Y79 and RB355 cells following 72 hours of treatment with BI6727, GSK461364 or DMSO as a control. All assays were performed according to the manufacturer’s protocols and at least in triplicate.WERI-Rb1, Y79 and RB355 cells (250,000 cells/well in 12-well plates) were cultivated with 10 nM, 100 nM, 500 nM or 1000 nM of BI 6727, GSK461364 or DMSO for 72 hours in duplicate for cell cycle analyses. Cells from each treatment were fixed in ethanol at least 1 hour, washed three times with phosphate-buffered saline (PBS, Gibco, Carlsbad, CA, USA), digested with RNase A (Carl Roth GmbH) and the DNA stained with 1% propidium iodide (Carl Roth GmbH) in PBS. Cells were analysed on a FC500 flow cytometer (Beckman Coulter GmbH, Krefeld, Germany) and data were processed using the corresponding analysis software to identify the proportions of each cell population in each cell phase. Experiments were independently repeated at least three times.RNA was isolated from cells using the High Pure RNA isolation Kit (Roche GmbH), and cDNA was synthesised from 500 ng RNA using the Transcription First Strand cDNA Synthesis Kit (Roche GmbH) then diluted 1:20 for semi-quantitative analysis of BAX, CCNB1, E2F3, MDM2, MDM4, CDKN1A and TP53 using the polymerase chain reaction (PCR). GAPDH was co-amplified as a loading control in every semi- quantitative PCR. Real-time PCRs for PLK1 were also performed with a 1:20 dilution of cDNA and monitored using SYBR green fluorescence on a StepOnePlus Real-Time PCR system (Life Technologies, Carlsbad, CA, USA). Target gene expression was calculated using the delta Ct method with GAPDH as internal reference. Primer sequences and PCR conditions are provided in Supplementary Table 1.
Cells were washed with cold PBS and lysed in RIPA buffer (50 mM TRIS, 150 mM NaCl, 1 mM EDTA, 1% NP40, 1 % sodium deoxycholate, and 0.11% SDS, pH7.4) containing Roche cOmplete™ protease inhibitor and PhosSTOP™ phosphatase inhibitor cocktails (Roche GmbH). Gel electrophoresis, transfer to nitrocellulose membranes, blotting and visualisation were performed as described 18, and membranes were probed with antibodies against PLK1 (1:1000; #4535,Cell Signaling, Danvers, MA, USA), TP53 (1:500; sc71817, Santa Cruz Biotechnology Inc., Dallas, TX, USA), CCNB1 (1:500; H00000891-M01, Abnova, Taipei City, Taiwan), CDK1 (1:500; MA5-11472, Milipore, Billerica, MA, USA,), CDK1 phosphorylated at Y15 (1:1000; #9111, Cell Signaling), MDM2 (1:500, sc5304, Santa Cruz Biotechnology Inc.), MDM4 (1:500, a300-287a, Bethyl Laboratories; Montgomery, TX, USA) and GAPDH (1:1000, MAB374, Millipore).Graph Pad Prism 5.0 (San Diego, CA, USA) was used to calculate IC50 concentrations and conduct Student’s two-sided t-tests to compare all interval variables. Error bars express +/− standard error of the mean (SEM).
RESULTS
PLK1 expression was examined at both the transcript and protein levels in the Y79, WERI-Rb1, RBL-13, RBL-15, RBL-30, RB355, RB383, RB3823, and RB522 cell lines.Several controls were included to estimate normal expression levels. Transcript levels were compared with those in the normal human dermal fibroblast (NHDF) cell line, healthy retinal tissue adjacent to the tumour and the SAOS2 cell line derived from an osteosarcoma, which is an embryonal tumour of the bone and a frequent secondary tumour arising in patients who received curative treatment for retinoblastoma. Protein levels were only compared with those from NHDF and SAOS2 cells. All retinoblastoma cell lines investigated expressed higher PLK1 levels compared with healthy retinal tissue, NHDF and SAOS2 cells (Fig. 1A). Elevated PLK1 expression in retinoblastoma cell lines was confirmed at the protein level compared to SAOS2 or NHDF cells (Fig. 1B). Our data indicate a general elevation of PLK1 expression in pre-clinical cellular models of retinoblastoma.We selected the well-characterised retinoblastoma cell lines, WERI-Rb1, Y79 and RB355, to investigate the molecular effects downstream of PLK1 inhibition in functional in vitro analyses. To estimate effects of inhibitors on non-tumorigenic cells we used NHDF cells. PLK1 inhibitor concentrations causing a 50% reduction of cell viability after 72 hours of treatment (IC50) were determined using MTT assays. IC50 values were in the nanomolar concentration range for both BI6727 and GSK461364 in all three retinoblastoma cell lines (Fig. 2A).
Even treatment of NHDF cells with 1000 nM BI6727 or GSK461364 did not reduce cell viability below 50% (69.51% or 89.44%, respectively) confirming an inhibitory effect specific for tumour cells. Results in MTT assays were confirmed using BrdU assays, which showed that 100 – 1000 nM concentrations of either inhibitor reduced proliferation significantly to 0.4 – 40.8% in all three cell lines, although proliferation was more completely suppressed in Y79 and WERI-Rb1 cells than in RB355 cells (Fig. 2B). PLK1 inhibition also significantly increased the fraction of apoptotic cells in the cell death ELISA (Fig. 2C). The strongest induction occurred in RB355 cells, in which 10 or 100 nM GSK461364 produced the same effect and increased the apoptotic fraction by 3-fold, and in Y79 cells, in which 100 nM of either inhibitor induced the apoptotic fraction by 4-fold. We next examined the effect of treatment with BI6727 or GSK461364 on both the apoptotic cell fraction (subG1) and cell cycle progression using flow cytometry. PLK1 inhibition significantly increased the fractions of Y79 and RB355 cells in subG1 (Fig. 2D), confirming the strong apoptotic effect we observed in the cell death ELISA (Fig. 2C). PLK1 inhibition increased the Y79 subG1 fraction in a concentration-dependent fashion, peaking at 45.4% (500 nM BI6727) and 50.5% (1000 nM GSK461364) in subG1. Treatment of RB355 cells with 10 nM concentrations of BI6727 or GSK461364 resulted in 45.5% and 30.5% of cells in subG1, respectively. With increasing doses of PLK1 inhibitors, we observed a significant accumulation of RB355 and WERI-Rb1 cells in the G2 phase, indicating that PLK1 inhibition arrests a proportion of retinoblastoma cells at the G2 restriction point. PLK1 inhibitor treatment also increased the fraction of WERI-Rb1 cells in subG1, but to a lesser extent even at higher doses. We have previously demonstrated that GSK461364 downregulates E2F3, which encodes a transcription factor involved in cell cycle progression and apoptosis, in neuroblastoma cell lines (unpublished data).
E2F3 expression was significantly reduced by at least 48 hours of treatment with BI6727 in WERI-Rb1 and RB355 cells and by 48 hours of treatment with GSK461364 in WERI-Rb1 cells, indicating reduced cell cycle activity (Suppl. Fig. 1). GSK461364 treatment had no effect on E2F3 expression in RB355 and Y79 cells, and BI6727 treatment upregulated E2F3 expression Y79 cells (Suppl. Fig. 1). E2F3 has also been reported to be involved in the apoptotic pathway.19 Upregulation in Y79 cells is in line with an induction of apoptosis after PLK1 inhibition as demonstrated in flow cytometry.To confirm the on-target specificity of PLK1 inhibition and further elucidate the mechanism of PLK1 inhibition in retinoblastoma cell lines, we assessed Y15 phosphorylation in CDK1 and mRNA and protein expression of the CDK1 interaction partner, CCNB1, after 24, 48 and 72 hours of treatment with either BI6727 or GSK461364. CDC25C is known to be a direct downstream target of PLK1, and is the phosphatase responsible for dephosphorylating Y15 and T14 in CDK1 to activate the CDK1-CCNB1 complex and allow entry into the M phase of the cell cycle. BI6727 or GSK461364 increased levels of phosphorylated CDK1 in the three retinoblastoma cell lines examined by 24 hours of treatment (Fig. 3A). Our flow cytometry experiments showed that treatment of WERI-Rb1 and RB355 cells with BI6727 or GSK461364 enhanced the cell fraction in G2 (Fig. 2D), and these results are corroborated by the accumulation of CCNB1 after PLK1 inhibition (Fig. 3A, 3B). Our combined data from pre-clinical in vitro models for retinoblastoma show that low nanomolar concentrations of either BI6727 or GSK461364 reduced cell viability and proliferation and induced apoptosis. Cell cycle analysis and fine examination in time course showed that pharmaceutically inhibiting PLK1 arrested the cell cycle at the G2/M phase restriction point and induced apoptosis.
It is known that PLK1 depletion and inhibition of its downstream target, CDK1, triggers p53 signalling.20,21 We next investigated p53 involvement in the signalling cascade induced by pharmaceutical PLK1 inhibition. Expression of several p53 signalling components were investigated using western blotting and semi- quantitative PCR in Y79, RB355 and WERI-Rb1 after 24, 48 and 72 hours of BI6727 or GSK461364 treatment (Fig. 4). TP53 levels increased in all three cell lines in as little as 48 hours of treatment with either PLK1 inhibitor (Fig. 4A). Expression of the TP53 target gene, CDKN1A (formerly p21), in Y79 cells was approximately 2-fold higher after 48 hours of treatment with 100 nM BI6727 or GSK461364 (Fig. 4B). CDKNA1 was moderately upregulated in WERI-Rb1 and RB355 cells by BI6727 (1.3- and 1.1-fold, respectively) and GSK461364 (1.9- and 1.3-fold, respectively). BAX and MDM2 are target genes affected further downstream in p53 signalling. PLK inhibition only moderately increased their expression in Y79, WERI-Rb1 and RB355 cells in some cases (Fig. 4 C,D). GSK461364 induced a more pronounced activation of p53 signalling than BI6727 in all three retinoblastoma cell lines.TP53 protein level and mRNA levels of BAX, CDKNA1, MDM2 were higher in Y79, a cell line with high constitutive TP53 levels compared to RB355 and WERI-Rb1 cells,which show low constitutive levels (Suppl. Fig. 2). These in vitro data reveal that retinoblastoma cell lines respond to PLK1 inhibition via BI6727 or GSK461364 treatment by enhancing p53 signaling depending on constitutive TP53 expression levels of the cell lines.It is known, that TP53 expression levels are regulated by MDM2 and MDM4 and previously published expression data showed high levels of MDM2 and MDM4 in retinoblastoma cell lines. 22 In our study, MDM2 and MDM4 levels were higher in WERI-Rb1 and RB355 cells than in Y79 and this is in line with lower TP53 levels in WERI-Rb1 and RB355. (Suppl. Fig. 2).
DISCUSSION
A recent report has highlighted an association between high PLK1 expression and adverse histopathological risk factors, including poor tumour cell differentiation, in retinoblastoma.5 Here we show a massive upregulation of PLK1 on both the mRNA and protein levels in a panel of retinoblastoma cell lines in comparison with healthy retina tissue and NHDF cells. NHDF cells were chosen as a cell line with regular cell cycle function because no retinal cell lines are available. In fact, PLK1 expression in pre-clinical cellular models of retinoblastoma was much higher than in an osteosarcoma cell line, a frequent secondary tumour of retinoblastoma, for which PLK1 inhibition has a strong adverse effect in vitro.23 PLK1 governs several key processes in cell cycling, including the initiation of entry into mitosis, centrosome maturation and separation, metaphase to anaphase transition, mitotic exit and the onset of cell division.20 Therapy targeting cell cycle regulators with small molecule inhibitors is promising, and a number of small molecule inhibitors have recently entered clinical trials, including the PLK1 inhibitors, BI6727 and GSK461364, which are now under evaluation for treatment of acute myeloid leukemia and solid tumours, respectively.13,14,24 Inhibiting PLK1 function by treatment with BI6727 or GSK461364 induced a dose-dependent decrease in cell viability and proliferation in our in vitro models of retinoblastoma. This effect was mediated via cell cycle arrest in G2, apoptosis or both, and was dependent on the cellular background. IC50 values for BI6727 and GSK461364 ranged from 4 – 14 nM for retinoblastoma cell lines, which is at the lower end of the range of IC50 values (6 nM-135 nM) reported for BI6727 in cell lines derived from other paediatric cancers, including acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma, neuroblastoma, rhabdomyosarcoma and rhabdoid tumours.
Data from clinical trials for PLK1 inhibitors in paediatric cancers have yet to be published, but BI6727 administered by infusion has been reported to be well tolerated in adults, with mostly haematological adverse events that were manageable and reversible.10 Data for the intraocular concentration of PLK1 inhibitors after systemic application are currently not available, In general, delivering sufficient drug concentration to the posterior part of the eye remains a challenge due to anatomical barriers, but specialized local administration protocols for intravitreal injections have been developed for the treatment of retinoblastoma.26–28 We demonstrated here that BI6727 or GSK461364 treatment suppressed CDK1/CCNB1 complex activity via transient downregulation of active CDK1, which is phosphorylated at Y15, and increased CCNB1 levels, which also contributed to G2 arrest. Activation of CDC25, that phosphorylates CDK1 is complex and not regulated by PLK1 exclusively. As one example, CDC25 is also activated by PP1 due to dephosphorylation of Ser287.29The complexity of CDC25 activation may explain the transient effects of PLK1 inhibition on the active phosphorylated CDK1.The TP53 transcription factor functions as a guardian for genome integrity, and can induce either cell cycle arrest or apoptosis in response to DNA damage or stress. PLK1 downregulation has been shown to activate the p53 signalling cascade in HeLa cells.20 We have previously reported that inhibiting CDK1, a downstream target of PLK1 also activates p53 signalling in neuroblastoma cells.21 Here we show that PLK1 inhibition via treatment with BI6727 or GSK461364 activates the p53 signalling cascade in retinoblastoma cells. TP53 protein accumulated after PLK1 inhibition in all three retinoblastoma cell lines investigated, and expression of TP53 downstream targets, CDKN1A, BAX and MDM2, increased.
Transcriptional activation of target genes was most pronounced in Y79 cells, consistent with the high constitutive TP53 expression level in this cell line. TP53 downstream target gene upregulation was more moderate in the WERI-Rb1 and RB355 cell lines, which maintain lower constitutive TP53 levels. This could probably be due to upregulation of MDM2 and MDM4, which we could demonstrate in this study and which was previously reported by McEvoy et al.22 We hypothesize that p53 signalling induction by BI6727 or GSK461364 may be mediated by the inactivation of the PLK1 target, CDK1, which together with CCNB1 phosphorylates TP53 at S315 to destabilise TP53 activity.30 In 75 % of retinoblastoma, p53 signaling is disrupted due to overexpression of MDM2 and MDM4 31 so further studies are required to clarify the role of TP53 on the induced signaling pathway in retinoblastoma after PLK1 inhibition.Here we demonstrate that pharmaceutically inhibiting PLK1 using BI6727 or GSK461364 reduces viability and induces apoptosis and cell cycle arrest in cellular models of retinoblastoma. The clinical correlation between elevated PLK1 levels in primary retinoblastomas with parameters for poor patient outcome makes PLK1 a promising therapeutic target for patients with advanced stage, disseminated retinoblastoma, where systemic application of inhibitors could be combined with current multimodal treatment strategies.