SEARCH

SEARCH BY CITATION

Keywords:

  • Apoptosis;
  • Cancer stem cells;
  • Cancer;
  • Glioma

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Glioblastoma multiforme (GBM) ranks among the deadliest types of cancer and given these new therapies are urgently needed. To identify molecular targets, we queried a microarray profiling 467 human GBMs and discovered that polo-like kinase 1 (PLK1) was highly expressed in these tumors and that it clustered with the proliferative subtype. Patients with PLK1-high tumors were more likely to die from their disease suggesting that current therapies are inactive against such tumors. This prompted us to examine its expression in brain tumor initiating cells (BTICs) given their association with treatment failure. BTICs isolated from patients expressed 110-470 times more PLK1 than normal human astrocytes. Moreover, BTICs rely on PLK1 for survival because the PLK1 inhibitor BI2536 inhibited their growth in tumorsphere cultures. PLK1 inhibition suppressed growth, caused G2/M arrest, induced apoptosis, and reduced the expression of SOX2, a marker of neural stem cells, in SF188 cells. Consistent with SOX2 inhibition, the loss of PLK1 activity caused the cells to differentiate based on elevated levels of glial fibrillary acidic protein and changes in cellular morphology. We then knocked glial fibrillary acidic protein (GFAP) down SOX2 with siRNA and showed that it too inhibited cell growth and induced cell death. Likewise, in U251 cells, PLK1 inhibition suppressed cell growth, downregulated SOX2, and induced cell death. Furthermore, BI2536 delayed tumor growth of U251 cells in an orthotopic brain tumor model, demonstrating that the drug is active against GBM. In conclusion, PLK1 level is elevated in GBM and its inhibition restricts the growth of brain cancer cells. STEM CELLS2012;30:1064–1075


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Glioblastoma multiforme (GBM) is among the most aggressive human tumors, and despite recent treatment advances, remains refractory to current therapies. There are three molecular subtypes of GBM—proliferative, proneural, and mesenchymal according to the molecular characterization of high-grade gliomas by Phillips et al. [1]. Patients with the proliferative subtype of the tumors are particularly prone to die from the disease. Although surgery is the most effective way for debulking tumors, complete resection is often difficult to achieve due to the location and infiltrative nature of GBM. In addition, adjuvant chemotherapy and radiation therapy often fail to provide long-term disease control, and disease relapses are common in GBM patients [2].

The cancer stem cell hypothesis postulates a hierarchical organization in tumors in which only a small subset of cells are capable of tumor formation. Similar to “stem cells,” tumor initiating cells (TICs) exhibit self-renewal and multilineage differentiation capabilities; moreover, TICs form tumors upon serial transplantation in immuno-compromised mice [3–6]. Brain tumor initiating cells (BTICs) have been found to be resistant to both chemo- and radiation therapies due to elevated expression of drug efflux proteins, enhanced DNA repair activity, and evasion of apoptosis [7–11]. Therefore, BTICs may play a role in tumor relapse.

Our lab has a long-standing interest in identifying molecular targets for the treatment of brain cancers [12–14]. We have recently performed a large-scale, genome-wide siRNA library screen of 691 human kinases, aiming to identify novel molecular targets for the treatment of childhood malignancies, including pediatric brain cancers. Results from the siRNA screen indicated that polo-like kinase 1 (PLK1) was one of the lead targets, which when inhibited, resulted in 80%-90% growth suppression of a panel of pediatric cancer cells including GBM in 72 hours [15]. PLK1 is a serine/threonine kinase that plays important roles in centrosome maturation, bipolar spindle formation [16–22], mitotic entry [23], metaphase-to-anaphase transition [24–26] and cytokinesis [27–30] in M phase of the cell cycle. This disease-relevant kinase is believed to be a promising therapeutic target for cancer treatments. The fact that PLK1 is differentially expressed in cancer and normal cells [31–36] and that malignant cells show exclusive dependence on PLK1 for growth and survival [37–40] suggest that a therapeutic window may exist for targeting this protein.

BI2536 is the first-in-class dihydropteridinone derivative, an ATP-competitive inhibitor that exhibits >1000× specificity to PLK1 compared to a panel of 63 kinases examined and has excellent in vivo antitumor activity [41]. The cytotoxic effect of PLK1 inhibition can be attributed to its antimitotic effects. Cells treated with PLK1 inhibitor are noted for the dumbbell-like chromatin structure, suggestive of 4N DNA cells arrested in pro-metaphase [42]. Disruption of bipolar spindle formation and chronic spindle checkpoint activation ultimately results in mitotic catastrophe [41, 43]. The remarkable cytotoxic activity of PLK1 inhibitors is a highly desirable feature in cancer treatments.

The promising results we have obtained from our initial kinase siRNA library screen prompted us to examine the functional role of PLK1 in brain cancers. Specifically, we questioned whether PLK1 would be a possible molecular target for the treatment of GBM. Therefore, in this study, we characterized the effects of PLK1 inhibition on adult and pediatric GBM cell lines as well as TICs isolated from patient GBM tumor specimens, as these cells are known to escape current therapy and may account for tumor relapse and recurrence. Here, we show that biological and pharmacological inhibition of this kinase leads to remarkable growth suppression, accompanied by apoptosis and loss of SOX2 expression in adult and/or pediatric GBM cells. In particular, treatment with the PLK1 small molecule inhibitor BI2536 impairs the ability of BTICs to self-renew and form tumorspheres in vitro. Furthermore, compared to the control, mice with intracranial GBM tumor survived significantly longer when treated with the PLK1 inhibitor. Together, these results suggest the potential therapeutic value of PLK1 inhibitor in the treatment of GBM.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Bioinformatic Analysis

Publicly available gene expression data for a series of pediatric [44] and adult [45, 46] primary GBM samples profiled on Affymetrix U133 platforms were downloaded and integrated in R 2.11 (http://www.r-project.org/). CEL files were read using the Affymetrix package of BioConductor 2.6, and normalized for each study using the robust multichip average (rma) algorithm [47]. Common probesets across the respective studies and platforms were extracted, and reduced from probeset to gene-level data (taking the maximum value across all samples), with unannotated probesets excluded. The data were row (gene)- and column (sample)-centered [48, 49], giving a working dataset of 467 samples and 13,169 unique known genes. Samples were clustered into “proneural,” “proliferative,” or “mesenchymal” subclasses based upon the relative expression of 323 overlapping genes from the Phillips 2006 classifier [1] using Ward's hierarchical clustering. PLK1 levels were investigated for subclass-specific expression correlations using ANOVA (Allocated Necrofeliac of Virtual Agency) and visualized by boxplots. Association with overall survival was performed using the log-rank test. Highly correlated genes were determined by calculating Pearson's correlation coefficients.

Cell Culture

SF188 and U251 cells were obtained from the American Tissue Culture Collection (ATCC Manassas, VA, USA, http://www.atcc. org/). GBM4, GBM8, and BT74 primary BTICs were characterized by Wakimoto et al. describing the ability of these cells to form tumorspheres upon serial passaging in vitro and secondary tumors in vivo. Furthermore, the cells are capable of multilineage differentiation upon induction [50, 51]. L0, L1, and L2 primary BTICs were isolated from GBM patients as previously described [52]. The cells grow as tumorspheres, express markers of NSCs, and retain high self-renewal and differentiation potential. Similar methods were used to isolate primary GBM BTICs BT241 [4]. Primary brain tumor cells were isolated from BT005 and BT011 according to the protocol previously described by us [53]. The cDNA of human neural stem cells (hNSCs; hNSC101 and hNSC167) was synthesized using RNA extracted from fetal brains (no genetic defects detected) obtained from two abortion cases. All the primary cells and BTICs were obtained through informed consent in abidance with the respective Institutional Review Board guidelines.

Transfection and Immunofluorescence Staining

PLK siRNA transfections were performed using Lipofectamine RNAiMAX (Invitrogen, http://www.invitrogen.com/site/us/en/home.html) as previously described [15] using control oligo (sequence: UUC UCC GAA CGU GUC ACG U, Qiagen, Valencia, CA, USA, http://www.qiagen.com/default.aspx) and PLK1 siRNA oligo (siRNA#1 sense sequence: CGG GCA AGA UUG UGC CUA A dTdT; siRNA#2 sense sequence: ACG GCA GCG UGC AGA UCA A dTdT, Dharmacon, Lafayette, CO, USA, http://dharmacon.com/Home.aspx?id=642&wm_crID=9780433&wm_lpID=45432640&wm_ctID=388&wm_kwID=27522254&wm_mtID=3&wm_conte nt=0&wm_g_crID=8837334748&wm_g_kw=dharmacon&wm_g_ p cmt=&wm_g_cnt=0&gclid=CLfI8MaT9K4CFWYZQgodTzZTKw& wm_kw=dharmacon&utm_source=google&utm_medium=cpc&utm_ term=dhar macon&utm_campaign=company+ads&wm_sd=1). SOX2 siRNA was purchased from Sigma (SASI_Hs01_00050572). Primary antibodies used for Western blotting studies include anti-PLK1 (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada, http://www.sigmaaldrich.com/canada-english.html), anti-P-H2AXS139 antibody (Abcam, Cambridge, MA, USA, http://www.abcam.com/), anti-caspase 3 (Cell Signaling Technology, Danvers, MA, USA, http://www.cellsignal.com/index.jsp), anti-poly(ADP-ribose) polymerase (Cell Signaling Technology), anti-P-CDC25CSer198 (Cell Signaling Technology), anti-SOX2 (Millipore, Billerica, MA, USA, http://www.millipore.com/index.do and Cell Signaling Technology), anti-musashi (Abcam) and anti-Bmi1 (Abcam), anti-vinculin (Millipore, Billerica, MA, USA, http://www.millipore.com/index.do), anti-tubuin (Cell Signaling Technology) and anti-pan-actin (Cell Signaling Technology). Immunofluorescence staining was performed on the BT74 tumorspheres according to the procedure previously described by us [14]. To examine PLK1 and neural stem cell expression in BT74 single cells, the tumorspheres were dissociated using solution A, B and C in the Neurocult Chemical Dissociation Kit (STEMCELL Technologies, Vancouver, BC, Canada, http://www.stemcell.com/), fixed in 1:1 mixture of cold acetone and methanol on microscope slides and immediately incubated in −20°C for 20 minutes which allowed attachment of the cells to the glass slides. The cells were subsequently washed two times with phosphate-buffered saline (PBS) and incubated with PLK1 (Sigma, 1:300), SOX2 (Cell Signaling Technology, 1:100), musashi (Abcam, 1:100), and Bmi1 (Abcam, 1:100) antibodies overnight at 4°C. The following day, the cells were washed three times with PBS and incubated with mouse and rabbit secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 546 at room temperature for 1-hour. After the secondary antibody incubation, the cells were washed three times, 5 minutes each and Hoechst dye (diluted to 1 μg/ml) was added before the cells were visualized under the confocal microscope Fluoview FV10i.

Tumorsphere Assay

SF188, patient-derived BTICs: GBM4, GBM8, BT74, L0, L1, and L2, primary brain tumor cells: BT241, BT005, and BT011 were dissociated into single cells and plated in neurobasal medium (1 × 104 cells per well in six-well plates) supplemented with 20 ng/ml human recombinant epidermal growth factor (EGF), 20 ng/ml human recombinant basic fibroblast growth factor (FGF; STEMCELL Technologies) and 2 μg/ml heparin on ultra low attachment, coated culture plates (Corning, Corning, NY, USA, http://www.corning.com/index.aspx). Tumorspheres >50 μm were quantified and photomicrographs were taken 6 days after culture.

Colony-Forming Cell Assay

Reagents specific for this assay were purchased from STEMCELL Technologies and the assay was performed according to manufacturer's instructions. Briefly, normal CD34+ cells were obtained from residual cells of bone marrow transplant and used following informed consent. BI2536 was diluted (0.1, 1, 2, 10, and 100 nM) and added to six separate tubes of MethoCult (STEMCELL Technologies), a methylcellulose matrix containing recombinant human cytokines stem cell factor, granulocyte macrophage colony-stimulating factor, interleukin-3, granulocyte colony stimulating factor, and erythropoietin. Following the addition of CD34+ cells at a final concentration of 5 × 103 cells per tube, the mixtures were vortexed and allowed to stand for 5 minutes. Dimethyl sulfoxide (DMSO) was used as vehicle control for BI2536. MethoCult mixtures were then dispensed into 35 mm dishes using 5 ml syringes and blunt end needles at a volume of 1.1 ml per dish. The medium was evenly distributed across the surface of each dish by gentle tilting and rotation. The dishes were then placed in a 150 mm tissue culture dish along with a 35 mm dish containing sterile water to maintain humidity. The tissue culture dish was placed in a 37°C humidified incubator containing 5% CO2 for 12 days. The number of myeloid and erythroid derived colonies in both the treated and control dishes was counted as described by the assay protocol.

Cell Cycle Analysis

Cells were harvested by trypsinization, washed once with cold PBS, and fixed in 70% ethanol overnight. The cells were washed once with cold PBS prior to the addition of staining buffer which was composed of 40 μg/ml propidium iodide and 200 μg/ml RNase A in cold PBS. The cells were incubated at room temperature, in the dark, for 30 minutes and 100 μl of cold PBS was added directly to the cell suspension when the cells were ready to be analyzed by flow cytometry.

Annexin V Staining and Quantification of Cell Growth by Hoechst Staining

SF188 cells were treated with 5 nM BI2536 for 48 hours and stained with Annexin V (Promega, Madison, WI, USA, http://www.promega.com) as previously described [54]. To evaluate the effect of PLK1 inhibition on cell growth, SF188 cells were plated (5,000 cell per well) in 96-well plates, treated with BI2536 or PLK1 siRNA for 72 hours and stained with Hoechst dye (1 μg/ml) in 100 μl of PBS containing 2% paraformaldehyde. The stained cells were kept at room temperature, in the dark, on a rocking platform for 30 minutes. The plates were analyzed and the images were taken on the ArrayScan VTI Reader (Cellomics, Pittsburgh, PA, USA, http://www.cellomics.com/).

BI2536 Efficacy Test in Orthotopic Xenograft Model

U251 human glioma cells were obtained from ATCC. Intracranial tumors were established using the protocol we previously published [55]. In brief, on day 0, 7.5 × 104 cells were implanted intracerebrally using a stereotaxic injection frame. After the orthotopic tumors established, BI2536 (50 mg/ml diluted in 0.1 N HCl) was delivered intravenously into the Rag2M mice (7-10 weeks old, eight mice per treatment group) once a week for 4 weeks. The health status and body weight of animals were monitored closely during the course of the experiment in accordance with the protocol approved by the British Columbia Cancer Research Centre.

Statistical Analysis

All quantitative data presented were analyzed as mean value ± standard error. For the microarray and animal studies, log-rank analysis was performed on the Kaplan-Meier curve to determine statistical significance of the results. The number of samples used and the respective p values are listed in the figure legends. The level of significance for the in vitro cell growth/death data was determined by Student's two-tailed t test (*p value <.05; **p value <.01).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

To establish the rationale for targeting PLK1 in GBM, we investigated its expression in primary tumors and more specifically in BTICs. Initially, we questioned whether PLK1 was highly expressed in primary GBM by evaluating publicly available expression microarray data from 467 cases. In this cohort, 400 cases were adult and 67 were pediatric GBM. PLK1 was notably expressed at high levels in the proliferative subclass (Fig. 1A, 1B) which has previously been associated with more aggressive disease and poor patient survival [1]. Consistent with these results, PLK1 was coexpressed with several genes associated with cell cycle progression, cytokinesis, and DNA replication (Fig. 1A and Supporting Information Table 1). In these patients, PLK1 expression was associated with less favorable overall survival (log-rank test, p = .042, data not shown). To independently confirm this trend, we analyzed PLK1 mRNA in 343 cases of gliomas from the UCSC database. PLK1 was upregulated in 49% of the patients (171/343) where high levels were associated with poor survival (Fig. 1C, log-rank test, p = 6.47 × 10−5).

thumbnail image

Figure 1. PLK1 is highly expressed in primary glioblastoma multiforme (GBM) where it is associated with the proliferative subtype and poor survival. (A): Heatmap shows the relative expression values for the most highly correlated genes with PLK1 in 467 pediatric and adult GBM, classified into subgroups according to the schema of Phillips et al. 2006 [1] (red = high expression and blue = low expression). Gene expression patterns across GBM subtypes indicate that PLK1 is coexpressed along with several genes involved in cell proliferation and division. Genes (horizontal axis) are listed in descending order of Pearson's correlation coefficient. (B): Boxplots (dark red = proliferative subgroup, dark blue = proneural subgroup, and dark green = mesenchymal subgroup) show mRNA expression values for PLK1 in 467 pediatric and adult GBM classified into subgroups according to the schema of Phillips et al. 2006 [1]. PLK1 is significantly overexpressed in the proliferative subclass when compared with the other two (p < .001, Allocated Necrofeliac of Virtual Agency (ANOVA)). (C): Kaplan-Meier curve shows the overall survival of patients with glioma (blue line) from the University of California Santa Cruz (UCSC) online database for which clinical outcome data was available (n = 343). PLK1 is highly expressed in 171/343 (49%) of the cases. Patients with high expression (red line) have significantly shorter survival than those with intermediate (yellow line) or low (green line) level of PLK1 (log-rank test, p = 6.7 × 10−5). Abbreviation: PLK1, polo-like kinase 1.

Download figure to PowerPoint

To investigate this further in patient samples, we obtained patient-derived BTICs from adult and pediatric brain tumor specimens and examined the transcript level of PLK1 by real-time polymerase chain reaction (RT-PCR). Primary adult BTIC lines—GBM4, GBM8, BT74, L0, L1, and L2—were well-characterized for their stem cell properties in vitro and vivo [50, 52]. Interestingly, PLK1 mRNA was expressed 110-470 times higher than normal human astrocytes (HAs). Likewise, PLK1 was highly expressed in BT241 which was derived from an adult GBM specimen, BT005 which are primary pediatric GBM isolates and SF188, which is a cell line established from a 8-year-old child with GBM [56] (Fig. 2A). Furthermore, we compared PLK1 level in BTICs and normal human neural stem cells isolated from fetal brains with no genetic defects. PLK1 was found to be 2.37-fold to 10.23-fold more abundant in GBM4, GBM8, and BT74 compared to hNSCs isolated from the two different samples (Supporting Information Fig. 2A, 2B) and its expression was downregulated significantly (∼59-fold to 83-fold) after linage differentiation, as evidenced by the low level of PLK1 in HAs compared to hNSC101 and hNSC167 (Supporting Information Fig. 2C, 2D). To confirm that PLK1 is expressed in tumorspheres formed by BTICs, we immunostained BT74 with PLK1 along with known neural stem cell markers SOX2 [57, 58], nestin [59, 60], and musashi1 [61] and found that they were coordinately expressed (Fig. 2B). BT74 tumorspheres were also dissociated into single cells, which were plated in monolayer and immunostained with PLK1, SOX2, musashi, and Bmi1 antibodies. We were able to confirm that PLK1 and these stem cell markers were coexpressed in single cells (Supporting Information Fig. 1).

thumbnail image

Figure 2. PLK1 is overexpressed in brain tumor initiating cells (BTICs) and its inhibition suppresses the self-renewal of these cells. (A): Real-time polymerase chain reaction was performed on the transcripts isolated from a panel of BTIC cultures—GBM4, GBM8, BT74, L0, L1, and L2, primary brain cancer cells—BT241 and BT005 (isolated from adult and pediatric malignant glioma, respectively), brain cancer cell line—SF188, and HA cell line to compare the level of PLK1. PLK1 transcript is between 110 and 470 folds more abundant in primary BTICs and cancer cell line than in the immortalized human astrocytes. (B): Whole-mount immunofluorescence staining of BT74 tumorspheres was performed to characterize PLK1 along with neural stem cell markers nestin, SOX2, and musashi. Z-stack images (×60 magnification) of the tumorspheres were taken on Fluoview FV10i (Olympus, Japan). (C): Primary BTICs: GBM4, GBM8, and BT74 (1 × 104 cells per well in six-well plates) were treated with 5 or 10 nM BI2536 for 6 days. The total number of tumorspheres (>50 μm) in each well was counted and photomicrographs of the spheres were taken (scale bar = 500 μm). The treatments were performed in duplicates on three separate occasions. (D): Self-renewal capability of BT74 was examined in tumorsphere assays. BT74 cells (1 × 104 cells per well in six-well plates) were treated with 10 nM BI2536 for 6 days. The tumorspheres formed were dissociated and replated in fresh medium containing the inhibitor for additional 6 days. The procedure was repeated until replating was unachievable due to low cell number. P0, P1, and P2 indicate primary, secondary, and tertiary tumorsphere formation, respectively. Scale bar = 500 μm. (E): Single cells were isolated from postsurgical pediatric malignant glioma BT005 according to the protocol we previously established [4] and cultured in neurosphere-supportive culture condition which allowed the cells to form tumorspheres in a week. The cells were subsequently dissociated and treated with 10 nM BI2536. The number of tumorspheres was enumerated and photomicrographs (scale bar = 200 μm) were taken after 6 days in culture. (F): Analysis of the effect of BI2536 on in vitro hematopoietic colony formation was performed on normal bone marrow derived CD34+ cells isolated from one patient. The cells were incubated with DMSO or increasing concentrations of BI2536 in methylcellulose cultures that contained cytokines to stimulate hematopoiesis. After 12 days in culture, myeloid and erythroid colonies were enumerated by counting under an inverted microscope based on morphology. Data presented here are from one of the two independent experiments. Abbreviations: DAPI, 4′,6-diamidino-2-pheylindole; DMSO, dimethyl sulfoxide; GBM, glioblastoma multiforme; HA, human astrocyte; and PLK1, polo-like kinase 1.

Download figure to PowerPoint

We then questioned whether PLK1 was essential for sustaining the growth of BTICs to provide further support for targeting this kinase in patients. Primary adult BTIC lines—GBM4, GBM8, BT74, L0, L1, and L2—were treated with PLK1 small molecule inhibitor BI2536 (5 or 10 nM) which is currently in several clinical trials [62] for other malignancies but not brain tumors. PLK1 inhibition blocked tumorsphere formation in all the BTIC lines examined (Fig. 2C and Supporting Information Fig. 3A). BT74 which was not very responsive to PLK1 inhibition initially (Fig. 2C) showed a decrease in secondary and tertiary sphere formation, indicated as P1 and P2, upon serial passaging (Fig. 2D). Of note, primary sphere formation was completely abolished in GBM4, GBM8, L0, and L2 because these BTICs were not viable after drug treatment (trypan blue staining, data not shown). In addition, we obtained a primary pediatric malignant glioma tumor specimen from the British Columbia Children's Hospital referred to as BT005. It too expressed PLK1 182 times higher than normal human astrocytes (Fig. 2A). These cells were characterized for their stem cell properties by RT-PCR and showed elevated levels of SOX2, musashi, and Bmi1 compared to normal human astrocytes (Supporting Information Fig. 3B). In addition, these cells responded very well to BI2536 in that the drug inhibited tumorsphere growth by >90% (Fig. 2E). On the contrary, the cells isolated from primary pediatric GBM tumor BT011, which curiously expressed negligible level of PLK1 (Supporting Information Fig. 3D), were insensitive to PLK1 inhibition (Supporting Information Fig. 3E), suggesting the importance of target specificity to effectively eliminate these cells. The control and BI2536-treated BT011 cells were dissociated and replated in fresh drug-containing medium every 6 days and no suppression in tumorsphere formation was observed even after the second serial passaging, as shown in Supporting Information Figure 3E. Together, these data suggest that children with GBM may benefit from being treated with PLK1 inhibitor; however, the potential for side effects to normal stem cells needs to be considered. In this realm, we performed a colony-forming assay on primary hematopoietic stem cells from two pediatric patients and assessed cellular growth and differentiation. The effect of BI2536 on the formation of multilineage colonies from CD34+ bone marrow cells was insignificant at doses that inhibited the cancer stem cells: that is, 5-10 nM (Fig. 2F and Supporting Information Fig. 3F).

To further our understanding of the potential of PLK inhibitors to block the growth of pediatric GBM, we treated SF188 cells with siRNA as well as BI2536. Following PLK1 knockdown, the growth of SF188 cells was suppressed by ∼75-80% in 72 hours (crystal violet stained cells and bar graph in Fig. 3A; Supporting Information Fig. 4A). Likewise, BI2536 suppressed the growth of these cells in a dose-dependent manner (crystal violet stained cells and growth curve in Fig. 3B). The IC90 after 72 hours was ∼5 nM (Fig. 3B). BI2536 treatment reduced the phosphorylation of CDC25CSer198, a known PLK1 substrate (Fig. 3B) and this corresponded with a large increase in G2 fraction, suggesting of a G2/M arrest (Fig. 3C). The loss of PLK1 expression via siRNA or kinase activity by way of BI2536 treatment led to apoptosis as evidenced by increased Annexin V staining (Fig. 3D), poly (ADP-ribose) polymerase (PARP) cleavage cleavage and phosphorylation of histone H2AX (Fig. 3E). PLK1 inhibition by siRNA or small molecule inhibitor also suppressed the growth of Gli36 (Supporting Information Fig. 4B), another GBM cell line (provided by Dr. David Louis) that expresses abundant human epidermal growth factor receptor. The necessity for the PLK1 pathway was specific for cancer cells as its blockade with siRNA or BI2536 had no effect on the growth of normal human astrocytes (Fig. 3F).

thumbnail image

Figure 3. PLK1 inhibition suppresses cell growth and induces apoptosis in brain cancer cells SF188. (A): SF188 cells were treated with 5 nM PLK1 siRNA for 72 hours, stained with Hoechst, and quantified. The growth of the cells was suppressed by ∼75%-80% after PLK1 siRNA treatment, a result confirmed by crystal violet staining of the cells. PLK1 knockdown by siRNA decreased its protein level, as shown by immunoblotting. (B): SF188 cells were treated with BI2536 (0.5-100 nM) for 24, 48, and 72 hours. At the end of each time point, the cells were stained with Hoechst and quantified. BI2536 inhibited the growth of the cells in 72 hours. The cells were additionally stained with crystal violet at 72 hours and growth suppression was confirmed. The experiment was performed in triplicates on two separate occasions. (C): SF188 cells were treated with 5 nM BI2536 for 24 hours, fixed in cold 70% ethanol, stained with propidium iodide, and subjected to flow cytometry for analysis of cell cycle profile. G2/M cell cycle arrest was observed after PLK1 inhibition. (D): SF188 cells were treated with 5 nM BI2536 for 48 hours. Apoptosis was measured by Annexin V-PE/7AAD staining. Induction of apoptosis in PLK1-inhibited cells was demonstrated by an increase in the percentage of Annexin V-PE-positive cells. (E): SF188 cells were treated with 5 nM PLK1 siRNA or inhibitor and the total proteins were extracted for immunoblotting. Apoptosis was confirmed at the molecular level by PARP cleavage and phosphorylation of H2AX at Ser139. (F): Immortalized human astrocytes HA were treated with either PLK1 siRNA (5-20 nM) or BI2536 (2.5-10 nM) and cell growth in monolayer was assessed 72 hours after treatments. Abbreviations: AAD, amino-actinomycin D; DMSO, dimethyl sulfoxide; PARP, poly (ADP-ribose) polymerase; PLK1, polo-like kinase 1; and V-PE, Annexin V-PE, Annexin V-phycoerythrin.

Download figure to PowerPoint

Having demonstrated that PLK1 inhibition suppressed cell growth and induced apoptosis of SF188 cells in monolayer, next we questioned whether it may also affect the expansion of putative cancer stem-like cells in neurosphere cultures. Preliminary results from us indicated that SF188 cells plated at low cell seeding density were capable of proliferating clonally to form tumorspheres >50 μm within 1 week in neurosphere culture condition (data not shown). This suggests to us that there might exist a population of cancer stem-like cells in this cell line. Similar to the results we obtained from the patient-derived primary BTICs, the putative cancer stem-like cells in SF188 were susceptible to PLK1 inhibition as the tumorsphere formation was significantly inhibited upon BI2536 or siRNA treatment. The inhibitor and siRNA reduced the total number as well as the size of the tumorspheres formed (Fig. 4A and Supporting Information Fig. 3C). Consistent with the inhibition of stem cell properties, PLK1 knockdown (Fig. 4B) or BI2536 treatment (Fig. 4C) reduced the mRNA and protein expression of SOX2 and musashi. In addition, PLK1 knockdown with two different PLK1-targeting siRNAs consistently decreased SOX2 expression (Supporting Information Fig. 5A). Furthermore, corresponding to the reduction in the expression of stem cell markers, SF188 cells that survived 6 days after siRNA or BI2536 treatment intriguingly underwent dramatic cellular morphological changes that rendered these cells to assume a stellate appearance, bearing structural similarity to astrocytes (Fig. 4D and Supporting Information Fig. 5B for additional images) and this was accompanied by an increase in the transcript level of glial fibrillary acidic protein (GFAP) (Fig. 4E). Next, we questioned whether the loss of cell proliferation, survival and stem-like properties of PLK1-inhibited cells was associated with the decrease in SOX2. Therefore, we silenced the expression of SOX2 and examined the biological effects on GBM cells. SOX2 knockdown reduced cell growth (Fig. 4F) and induced cell death (Fig. 4G) in SF188 cells in 72 hours. Phosphorylation of H2AX, an early marker of apoptosis, was observed at 48 hours (Fig. 4H) while cleavage of caspase 3 (Fig. 4I) occurred at 72 hours in siSOX2 cells.

thumbnail image

Figure 4. PLK1 inhibition downregulates the expression of SOX2, which is required for the growth and survival of glioblastoma multiforme cells. (A): SF188 cells (1 × 104 cells per well in six-well plates) were plated in neurobasal growth medium containing 5 or 10 nM BI2536 for 6 days. The total number of tumorspheres (>50 μm) in each well was counted and photomicrographs of the spheres were taken (scale bar = 500 μm). The treatments were performed in duplicates on three separate occasions. (B): The transcript and protein expression of neural stem cell markers SOX2, musashi, and Bmi1 was measured by real-time polymerase chain reaction (RT-PCR) and immunoblotting 36 and 48 hours, respectively, after PLK1 siRNA treatment in SF188 cells. (C): The transcript and protein expression of SOX2, musashi, and Bmi1 were measured by RT-PCR and immunoblotting 36 and 48 hours, respectively, after BI2536 treatment in SF188 cells. (D): SF188 cells were treated with 5 nM PLK1 siRNA or BI2536 for 6 days and photomicrographs were taken on the cells that remained after the treatment. Representative photomicrographs of the cells that underwent dramatic cellular morphological alterations are shown. Scale bar = 280 μm. (E): Total RNA from the cells treated with 5 nM PLK1 siRNA #1 and #2 for 36 hours was extracted and subjected to RT-PCR to quantify the transcripts of PLK1, SOX2, and GFAP. (F): SF188 cells were treated with 100 nM SOX2 siRNA for 72 hours. The cells were stained with Hoechst and quantified. The number of viable cells was enumerated and the relative fold difference in cell growth is shown in the bar graph. (G): The number of nonviable cells after 100 nM, 72 hours siSOX2 treatment was enumerated based on enhanced Hoechst staining due to chromatin condensation and the relative fold difference in cell death is shown in the bar chart. (H): Proteins extracted from the cells treated with 100 nM control or SOX2 siRNA for 48 hours were subjected to immunoblotting to examine the phosphorylation of H2AX at Ser139, which is a marker of early apoptosis. (I): SF188 cells were treated with 100 nM control or SOX2 siRNA for 72 hours and immunoblotting was performed on the total protein lysates. Increased caspase 3 cleavage was observed in the siSOX2-treated cells compared to the control cells. Abbreviations: DMSO, dimethyl sulfoxide; GFAP, glial fibrillary acidic protein; and PLK1, polo-like kinase 1.

Download figure to PowerPoint

Finally, we tested the in vivo efficacy of BI2536 in mice against the well-established U251 model that was previously characterized by our group [55]. First, we conducted a time course study to establish whether or not U251 (adult GBM) cells were sensitive to PLK1 inhibition. BI2536 blocked U251 growth in a dose-dependent manner after 72 hours in vitro (Fig. 5A). To further support these results, PLK1 was inhibited with siRNA where the loss of PLK1 expression led to 90% growth suppression (Fig. 5B). This was correlated with a loss of SOX2 expression (Fig. 5C). We therefore established that U251 cells were sensitive to PLK1 inhibition with siRNA as well as BI2536, warranting further examination in mice. U251 cells were introduced via intracranial injection and tumors were allowed to form before the animals were randomized into three groups (control, vehicle-treated, or BI2536-treated). The test group of mice were treated with BI2536 (50 mg/kg) once weekly for 4 weeks. BI2536 prolonged the survival of animals compared to those that were untreated or given vehicle control agent (Fig. 5D). Health status and body weight of the mice were monitored throughout the study. The PLK1 inhibitor did not cause adverse side effects to the animals as they appeared healthy during the course of the treatment (days 21-42) and maintained normal body weight (Fig. 5E). We concluded that BI2536 was well tolerated and that it had the desired effect of significantly delaying brain tumor progression.

thumbnail image

Figure 5. BI2536 suppresses the growth of glioblastoma multiforme (GBM) cell line U251 in vitro and tumor formation in vivo. (A): U251 cells were treated with increasing concentrations of BI2536 (0.5-100 nM) in a 72-hour time course study. BI2536 (5 nM) inhibited the growth of U251 cells in 72 hours. The experiment was performed in triplicates on two separate occasions. (B): U251 cells were treated with 5 nM PLK1 siRNA for 72 hours, stained with Hoechst, and quantified. PLK1 knockdown decreased U251 cell growth by ∼90%. The experiment was repeated on three separate occasions. (C): Total RNA was extracted from U251 cells treated with BI2536 for 36 hours. The transcripts were quantified by real-time polymerase chain reaction (RT-PCR). The RT-PCR experiment was performed in triplicates and the experiment was repeated on two separate occasions. (D): Orthotopic GBM tumors were established by intracranial injection of U251 cells into Rag2m mice (n = 8 per treatment group), which were subsequently treated with 50 mg/kg BI2536 i.v. weekly for four weeks. Log-rank test was performed on the Kaplan-Meier curves of the experimental animals. The difference in the survival of vehicle control and BI2536-treated mice was statistically significant with a p value of .012. (E): The body weight of the untreated, vehicle control-treated and BI2536-treated animals was measured regularly for 60-80 days. The mice that were treated with BI2536 showed significantly less body weight loss compared to the control mice during the course of the experiment. Abbreviations: DMSO, dimethyl sulfoxide; PLK1, polo-like kinase 1.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

In this study, we conducted a comprehensive investigation on the potential of PLK1 inhibitors in GBM. We approached this by integrating microarray data from 467 patients and identified PLK1 to be overexpressed in the proliferative subtype of GBM, which is associated with poor prognosis [1]. Subsequently, we examined the effect of PLK1 inhibition on GBM in vitro and in vivo. To address this question, we have used multiple models, including cell lines (SF188, U251, Gli36, and immortalized HAs), cancer stem-like cells derived from patients (GBM4, GBM8, BT74, L0, L1, and L2), primary cells isolated from adult and pediatric GBM specimens (BT241, BT005, and BT011), and hematopoietic stem cells derived from two patients. We reported that pharmacological or genetic inhibition of PLK1 remarkably suppressed growth, induced cell cycle arrest and apoptosis in these aggressive brain tumor cells, and shut down the growth of BTICs. These changes were associated with a loss of SOX2, the inhibition of which partially phenocopied the effects of growth suppression and apoptosis observed following PLK1 suppression.

SOX2 [(sex determining region Y)-box 2] is a transcription factor that is required for the self-renewal of embryonic stem cells [63] as well as primitive neural cells [63–67]. Recent experimental evidence indicates that SOX2 is not only a marker of neural stem cells but is also functionally involved in the pathogenesis of brain cancers [68–71]. In addition, SOX2 was demonstrated to confer proliferative advantage and to maintain tumorigenicity of glioma-initiating cells [72, 73]. Together these results led us to question whether the growth suppression and apoptosis from PLK1 inhibition would be mediated in part through the downregulation of SOX2. For the first time, we showed that SOX2 expression is regulated by PLK1. In addition, SOX2 knockdown partially phenocopied the effect of PLK1 inhibition; however, the magnitude of growth suppression and cell death was not as significant as what was observed in PLK1 inhibition and this is likely due to the pivotal and pleiotropic roles that PLK1 plays in mitosis[16–30]. However, these results have nevertheless provided a rational and probable explanation for the loss of proliferative capacity and stemness properties of BTICs treated with BI2536 in tumorsphere assays. Research is currently underway to understand the mechanism by which PLK1 modulates SOX2 expression, the link between these two proteins and their functional cooperation in regulating the proliferation and survival of GBM cells. Finally, results from our in vivo xenograft experiment indicated that systemic administration of BI2536 prolonged the survival of animals with orthotopic GBM tumors. Together, these results suggest the potential of targeting PLK1 in brain cancer treatments.

PLK1 is an emerging new molecular target for therapy. This is recognized by the fact that several PLK1 inhibitors have been developed and are currently under investigations in phase I or II clinical trials. BI2536 passed phase I clinical trials and demonstrated measurable antitumor activity with favorable toxicity profiles in advanced solid tumors [74, 75]. BI6727 is a second-in-class, ATP-competitive PLK1 inhibitor tested in non-Hodgkin's lymphoma, acute myeloid leukemia, small-cell lung cancer, and solid tumors [76]. The result from phase I trial is promising showing disease stabilization in 32% of the patients with advanced or metastatic solid tumors [62]. Additional PLK1 inhibitors such as GSK461364 and ON01910 have been evaluated in phase I clinical trials and demonstrated therapeutic efficacy in patients with advanced solid tumors [77, 78]. PLK1 inhibitors may be an alternative treatment to tumors refractory to standard antimitotic agents due to mutations in tubulin proteins [79] or overexpression of drug efflux proteins such as p-glycoproteins [80]. In addition, unlike standard antimitotic agents such as vinca alkaloids and taxanes, PLK1 inhibition does not cause chemotherapy-induced peripheral neuropathy, a pathological condition associated with motor and sensory deficits. The major side effect and dose-limiting toxicity of PLK1 inhibitors is neutropenia [74] which is reversible and clinically manageable. Our finding has shown that at the concentration of 10 nM BI2536 for instance, where an inhibition of >70% of tumor cell growth was achieved, no significant inhibitory effect was observed on hematopoietic colony formation. In addition to testing PLK1 inhibitor on hematopoietic stem cells, we believe that it is equally important to evaluate the effects of BI2536 on normal hNSCs to assess the safety of the inhibitor. In our study, we have shown that PLK1 transcripts were 2.37-10.23 times more abundant in the BTICs compared to normal human NSCs. This result suggests to us that there may exist a therapeutic window for the clinical use of PLK1 inhibitors at a dose range that would show effective antitumor activity with minimal hematopoietic and neuro-toxicities. However, additional correlative and toxicity studies in animal models and early phase clinical trials are needed to confirm this observation.

Intriguingly, pediatric GBM cells SF188 treated with PLK1 siRNA or inhibitor may have undergone differentiation as evidenced by a dramatic alteration in cellular morphology and increased level of GFAP. It is believed that cancers may arise from aberrations in the lineage differentiation of stem and progenitor cells due to genetic or epigenetic abnormalities; therefore these cells are trapped in a perpetual state of self-renewal and proliferation that prevent them from becoming terminally differentiated [81]. In this study, we demonstrated that PLK1 inhibition not only induced apoptosis of pediatric GBM cells but may have also forced these cells down the differentiation path that will decelerate their growth and possibly render them sensitive to conventional anticancer therapies. Given the well-known difficulty in treating glial tumors, our results argued for the possibility of treating this patient population with PLK1 inhibitors. This could therefore shed light on the treatment of GBM, a rare type of brain tumor found in children where new therapies are desperately needed because unlike adults patients, children afflicted with malignant glioma do not respond well to Temozolomide (TMZ) [82–84], a front-line therapy for this disease.

Disease relapse is one of the major road-blocks to successful treatments in brain cancers. BTICs reportedly express elevated levels of MGMT, BCL-2, and BCL-xL transcripts [9] and have an enhanced ability to repair DNA following radiation [7]. Given these features, it is not surprising that these cells survive radiation and chemotherapy. The fact that BTICs are resistant to conventional anticancer therapies and are enriched in tumors of relapsed patients suggest that they play a clinically important role in disease recurrence [85]. We demonstrated that PLK1 inhibition affected not only the cell division of rapidly proliferating SF188 cells but also the self-renewal of putative cancer stem-like cells from patient samples. BTIC lines GBM4, GBM8, L0, and L2 could not be serially passaged because the cells died upon BI2536 treatment. In addition, even those that were capable of primary sphere formation such as BT74, self-renewal was greatly impaired when BI2536 treatment was continued. Recently, BT74 cells have been shown to be resistant to chemotherapy and molecular-targeting agents [86, 87]. Thus, our results are particularly exciting as it suggests that PLK1 inhibitor may potentially eliminate the cells that make up the bulk of the tumors as well as deplete the notoriously chemotherapy and radiation-resistant BTICs. Our results are consistent with a study recently published by Grinshtein et al. who demonstrated that PLK1 inhibition suppressed the expansion of TICs isolated from bone marrow metastases of neuroblastoma patients [40]. As a result, PLK1 is a unique molecular target because its inhibition kills a range of cancer cells.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

To conclude, we have identified PLK1 as a factor critical to the survival of brain cancer cells and BTICs. Thus, PLK1 is positioned as a promising molecular target that has the capacity to overcome therapy resistance imposed by BTICs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

This work was supported by the Michael Smith Foundation for Health Research and Hannah's Heroes Foundation (SED and CL). It was also funded by the C17 Research Network (SED and AN). We are also grateful to STEMCELL Technologies for their kind support. CJ acknowledges NHS funding to the NIHR Biomedical Research Centre. We also thank our collaborators Dr. Brent Reynolds as well as Dr. Paul Steinbok, Dr. Doug Cochrane, Dr. Rod Rassekh, Dr. Vesna Popovska, and Dr. Glenda Hendson at the B.C. Children's Hospital for helping us obtain patient tumor specimens and for their contribution to the study.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
SC_11-1161_sm_supplFigure1.tif2931KSupplementary Figure 1. PLK1 and neural stem cell markers SOX2, musashi and Bmi1 are co-expressed in dissociated BT74 cells. BT74 tumourspheres were dissociated into single cells and fixed in 1:1 mixture of acetone and methanol directly on glass slides. The cells were stained with antibodies against PLK1, SOX2, musashi and Bmi and the images (60x, scale bar=100μm) were obtained by confocal microscopy on Fluoview FV10i (Olympus, Japan). PLK1 was co-expressed with SOX2, musashi and Bmi1 in the nuclei of the majority of the cells examined.
SC_11-1161_sm_supplFigure2.tif2931KSupplementary Figure 2. PLK1 level is elevated in BTICs compared to normal human neural stem cells and its expression is significantly down-regulated after lineage differentiation. (A) RT-PCR was performed to examine PLK1 level in hNSC167 and GBM4, GBM8 and BT74. The transcript level of PLK1 is 4.1-fold, 6.41- fold, 10.23-fold higher in GBM4, GBM8 and BT74, respectively, compared to hNSC167. (B) PLK1 level in hNSC101 and BTICs was examined by RT-PCR. The transcript level of PLK1 is 2.37-fold, 3.71-fold, 5.92-fold higher in GBM4, GBM8 and BT74, respectively, compared to hNSC101. (C) RT-PCR was performed to examine PLK1 level in hNSC167 and HA. The HA express ∼59-fold less PLK1 transcripts compared to the neural stem cells. (D) PLK1 transcript level in hNSC101 and HA was quantified by RT-PCR. PLK1 level is ∼83-fold lower in HA compared to the neural stem cells.
SC_11-1161_sm_supplFigure3.tif2931KSupplementary Figure 3. BI2536 suppresses tumoursphere formation of primary brain tumour cells but exerts a minimal effect on the differentiation of primary hematopoietic stem cells isolated from a patient. (A) Primary BTICs: L0, L1 and L2 (1x104 cells per well in 6-well plates) were treated with 5 or 10nM BI2536 for 6 days. The total number of tumourspheres (>50μm) in each well was counted and photomicrographs of the spheres were taken (scale bar=500μm). (B) Total RNA extraction was performed on the immortalized human astrocytes HA and tumourspheres of BT005, followed by reverse transcription and qPCR. The total cDNA level of neural stem cell markers SOX2, musashi and Bmi1 was quantified and compared (in relative fold difference) between the two cell cultures. (C) SF188 cells were transfected with 5nM PLK1 siRNA for 24hrs and re-plated in neurobasal medium supplemented with EGF and FGF, a condition that supports the growth and expansion of stem and progenitor cell population. The number of tumourspheres was enumerated 6 days after culturing and representative photomicrographs (scale bar=200μm) are shown to compare the colonies in the control and siPLK1 treatment. (D) Single cells were isolated from a post-surgical pediatric GBM specimen (referred to as BT011) and grown as tumourspheres for two weeks. The RNA from the tumourspheres was isolated and RT- PCR was performed to quantify the level of PLK1 in BT011 and HA. (E) BT011 cells were plated in BI2536-containing medium for 6 days and the cells were serially passaged two more times before the tumourspheres were quantified. PLK1 inhibitor did not suppress the self-renewal and/or proliferation of these primary brain tumour cells, which expressed negligible level of PLK1. (F) Analysis of the effect of BI2536 on in vitro hematopoietic colony formation was performed on normal bone marrow derived CD34+ cells isolated from a second patient. The cells from this patient were incubated with DMSO or increasing concentrations of BI2536 (0.1-100nM) in methylcellulose cultures that contained cytokines to stimulate hematopoiesis. After 12 days in culture, myeloid and erythroid colonies were enumerated by counting under an inverted microscope based on morphology.
SC_11-1161_sm_supplFigure4.tif2931KSupplementary Figure 4. PLK1 inhibition represses the cell growth of pediatric and adult GBM cell lines SF188 and Gli36. (A) SF188 cells were treated with two different siRNAs targeting PLK1. The growth of the cells was assessed in 72hrs by Hoechst staining and quantification on the Cellomics high-content screening instrument. PLK1 siRNA #1 and #2 inhibited cell growth to a similar extent in 72hrs. (B) The effect of PLK1 inhibition by siRNA or small molecular inhibitor was evaluated in Gli36 (adult GBM cell line) 72hrs after treatment. Gli36 cells were also very sensitive to PLK1 inhibition as 5nM of siRNA or BI2536 suppressed cell growth by ∼80-90%.
SC_11-1161_sm_supplFigure5.tif2931KSupplementary Figure 5. PLK1 knockdown by two targeting siRNAs decreases the transcript and protein levels of SOX2 and alters cellular morphology. (A) SF188 cells were treated with 5nM of PLK1 siRNA #1 or #2. Total RNA and proteins were extracted for RT-PCR and immunoblotting (36hrs and 48hrs respectively) to examine the expression of SOX2. (B) SF188 cells were treated with 5nM of PLK1 siRNA or BI2536 for 6 days and photomicrographs were taken on the cells that remained after the treatment. Additional representative photomicrographs of the cells that underwent dramatic cellular morphological alterations are shown (scale bar=280μm)
SC_11-1161_sm_supplTable1.tif2931KSupplementary Table 1. Correlation between PLK1 and similarly expressed genes expressed in primary GBM based on Affymetrix U133 microarrys. The genes that are most highly correlated with PLK1 by mRNA expression from Affymetrix U133 microarray profiling 467 pediatric and adult glioblastomas are listed in descending order according to their Pearson's correlation coefficients.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.