Telomerase Inhibition as a Novel Therapy for Pediatric Ependymoma

Abstract

Ependymomas are the third most common pediatric brain tumor with an overall survival of ∼50%. Recently, we showed that telomerase [human telomerase reverse transcriptase (hTERT)] expression is a predictor of poor outcome in pediatric ependymoma. Thus, we hypothesized that ependymomas with functional telomerase may behave more aggressively and that these patients may benefit from anti‐telomerase therapy. To address our hypothesis, we investigated the effect of telomerase inhibition on primary ependymoma cells harvested at the time of surgery, as no animal models or established cell lines are readily available for this tumor. The cells were characterized for glial fibrillary acidic protein (GFAP) and hTERT expression, initial telomere length and telomerase activity. They were then subjected to telomerase inhibition (MST‐312, 1 µM) and tested for effects on cell viability (MTT assay), proliferation (MIB‐1), apoptosis (cleaved caspase 3) and DNA damage (γH2AX). After 72 h of telomerase inhibition, primary ependymoma cells showed a significant decrease in cell number (P < 0.001), accompanied by increased DNA damage (γH2AX expression) (P < 0.01) and decreased proliferative index (MIB‐1) (P < 0.01). Half showed an increase in apoptosis (cleaved caspase 3). These data suggest that telomerase inhibition may be an effective adjuvant therapy in pediatric ependymoma, potentially inducing tumor growth arrest in the short term, independent of telomere shortening.

INTRODUCTION

Central nervous system (CNS) neoplasms constitute the most common solid tumors of childhood. Although there has been great improvement in the treatment and prognosis in many pediatric tumor types, this has not occurred in most brain tumors, which are currently the major cause of mortality, as well as long‐term morbidity in pediatric oncology. Ependymomas are gliomas that arise from ependymal cells within the CNS. They constitute approximately 5% of adult intracranial gliomas and up to 10% of childhood CNS tumors, ranking them as the third most common CNS tumor of childhood 3, 22. The overall survival rate for pediatric ependymomas is approximately 50%, and has not changed substantially over the past several decades. The importance of a gross total resection has been well established (11), and current therapeutic trials are primarily aimed at determining optimal radiation dose, volume and fractionation. The role of chemotherapy has thus far been limited, and there have been no trials of targeted agents as have been undertaken in high‐grade astrocytic tumors (13).

Although recent attempts at improving the consistency of histological grading of ependymomas may help (21), there has been a general lack of consensus regarding grading of ependymomas which has likely contributed to the variable relationship between histology and outcome (22). This has led to confusion as to the interpretation of histological findings in clinical practice. Further, the lack of firm prognostic indicators has impeded consensus as to what constitutes optimum management.

Expression of human telomerase reverse transcriptase (hTERT), the enzymatic subunit of telomerase, has been shown to be a useful prognostic marker for many types of cancer (reviewed in (2)). Telomeres are unique structures located at the terminal ends of each chromosome. In vertebrates, telomeres are guanine‐rich tandem repeats of TTAGGG. They function as a cap that protects the ends of the DNA double helix, preventing DNA damage, and therefore promoting genomic stability. Because of the mechanism of DNA replication, a portion of each telomere is lost after each mitotic division. In normal somatic cells, the continual telomere shortening eventually results in a growth‐arrested state known as cellular senescence (10). In stem cells, germ line cells and cells in renewable tissue, a ribonucleoprotein called telomerase, is expressed (8). Telomerase consists of two essential subunits: the 127 kDa catalytic subunit, hTERT, and an RNA template for the telomere, hTR. Using the hTR as a template for the synthesis of the telomeric repeats, telomerase maintains the telomere length in these cells, thus increasing the proliferative potential. High telomerase activity has been observed in over 85% of human cancer cells (17), and this contributes to the limitless replicative potential characteristics of cancer. Interference with hTERT activity using telomerase inhibitors 7, 14, 15 or silencing RNA approaches (16) has been shown to induce cellular senescence, growth arrest, apoptosis, as well as telomeric erosion in various types of cancers.

Telomerase activity and hTERT expression have also been shown to correlate with grade and proliferative rate in a number of brain tumors (5). Using tissue microarrays and a matching clinical database for a cohort of pediatric ependymoma patients, we showed that expression of hTERT correlates with a worse outcome in these patients (19). Thus, we hypothesized that ependymomas with functional telomerase may behave more aggressively and that these patients may benefit from anti‐telomerase therapy. As animal models and cell lines are unavailable for ependymoma, we investigated the effect of telomerase inhibition on primary ependymoma cells harvested from freshly resected pediatric tumors.

MATERIALS AND METHODS

Patient samples

Ependymoma samples were accrued over a 1.5‐year period (2007–2008) on consecutive patients presenting to our institution with a new diagnosis of non‐myxopapillary ependymoma. Consent for use of tumor tissue for research was obtained for all patients. Six of seven patients presenting during this time period had sufficient leftover material for tissue culture and were successfully cultured.

Primary cell culture

Surgical ependymoma specimens were minced with a sterile scalpel in a 10 cm dish in the absence of medium followed by an addition of 2 mL RPMI‐1640/10% FBS medium. The entire mixture was then transferred to a 15 mL conical tube. Extra medium was added to reach a final volume of 10 mL, and mixed by pipetting. The tube was then allowed to sit at room temperature for 1 minute. Supernatant containing cells was then removed and dropped onto coverslips. The cells were then incubated at 37°C overnight and allowed to attach. Fresh medium was added on the following day. Cells were in culture for 72 h before being replated for use in the experiments outlined below.

Drug treatment

MST‐312 (Calbiochem, Gibbstown, NJ) is a synthetic compound that has been shown to be a potent telomerase inhibitor (14). MST‐312 powder was reconstituted in 5 mg/mL dimethylsulfoxide to give stock solution, and further diluted to the desired concentration. Primary ependymoma cells were seeded in 96‐well plates at equal numbers in triplicates, and incubated overnight for attachment prior to the inhibitor addition. MST‐312 was added at concentrations varying from 0 to 4 µM. A cytotoxicity assay (MTT assay, Roche Diagnostics, Mississauga, ON, Canada) was used to measure cell viability 72 h after MST‐312 treatment according to the manufacturer's instructions.

Immunofluorescence

For short‐term analysis, cells were cultured on glass coverslips in the cell culture medium described above for 72 h in the absence or presence of MST‐312. Medium was aspirated followed by three phosphate‐buffered saline (PBS) washes. Cells were then fixed onto coverslips using acetone for 10 minutes at −20°C followed by three PBS washes. Next, cells were permeabilized in 0.25% Triton X/PBS for 7 minutes followed by three PBS washes. The corresponding serum (1:50) was used as blocking solution for 3 h at room temperature. The appropriate antibody was added and incubated overnight at 4°C [glial fibrillary acidic protein (GFAP) 1:50 (Dako, Mississauga, ON, Canada), Ki67 1:50 (Dako), cleaved caspase 3 1:1000 (Cell Signaling, Danvers, MA), hTERT 1:50 (Novacastra, Norwell, MA) γH2AX 1:1000 (Upstate Cell Signaling, Temecula, CA)]. H2AX is protein belonging to a member of the histone H2A family. It is phosphorylated (S139) by ataxia telangiectasia, mutated following DNA damage. Phosphorylation of H2AX (γH2AX) has been reported to be one of the earliest measurable events following telomere‐associated DNA damage (4). Cells were washed with bovine serum albumin (BSA)/PBS (0.65 g/L) three times followed by addition of the appropriate secondary antibody (1:200) for 30 minutes. After PBS/BSA washes, the slides were counterstained with 4′,6′‐diamidino‐2‐phenylindole dihydrochloride, and viewed under a Nikon Eclipse E400 fluorescent microscope with appropriate filters (Nikon Instruments, Toronto, ON, Canada). Digital images were captured with a Nikon DXM1200F camera, and analyzed using open source software, ImageJ (http://rsb.info.nih.gov/ij/).

Immunohistochemistry

Five‐micron‐thick sections were dewaxed in xylene followed by rehydration in alcohol series. Slides were allowed to air dry. Antigen retrieval was done using a standard citrate buffer/pressure cooker method for 22 minutes. Methanol/H₂O₂ solutions were used to block endogenous peroxidase activity followed by avidin/biotin block. The appropriate serum block was used prior to antibody addition. Overnight incubation at 4°C was followed by wash in PBS/BSA solution. The appropriate horseradish peroxidase‐conjugated secondary antibody was added and incubated at room temperature for 30 minutes. Slides were washed in PBS/BSA. Avidin/biotin complex using the Vectastain ABC system (Vector Laboratories, Burlingame, CA) was added onto slides for an incubation of 40 minutes. After washing in PBS/BSA, the slides were rinsed in Tris buffer (pH 7.6), and color was developed using the 3,3′‐diaminobenzidine‐tetrachloride chromagen. Hematoxylin was used as a counterstain, and coverslips were added with mounting medium.

Telomere length assay

Telomere lengths were determined by a Terminal Restriction Fragment (TRF) kit (Roche Diagnostics, Mannheim, Germany) as described previously 18, 23. Briefly, the tumor DNA was restriction digested and run on 0.8% agarose. A biotinylated gamma DNA molecular weight marker was used as a DNA length standard. The DNA samples were depurinated in 0.25 mol/L of HCl, denatured in 0.4 mol/L of NaOH/3 mol/L of NaCl and transferred to a positively charged nylon membrane Hybond‐N (Amersham Pharmacia Biotech, Little Chalfont, England) and hybridized with a (TTAGGG)³telomere probe. Chemiluminescent detection was performed according to the detection kit (Roche Diagnostics, Basel, Switzerland). Detection was done on an X‐ray Hyperfilm ECL (Amersham/ GE Healthcare, Baie d'Urfe, Quebec, Canada). The X‐ray exposed film was analyzed using open software ImageJ (http://rsb.info.nih.gov/ij/) as described in Tabori et al (19).

Statistics

Student's t‐test using Microsoft Excel was used when comparing two samples. anova was used when comparing three or more samples. P < 0.05 was considered a significant difference.

RESULTS

Characterization of ependymomas and primary ependymoma cells

A total of six ependymoma cases were used in the current study. Clinical data of the corresponding patients are summarized in Supporting Information Table S1. Hematoxylin and eosin staining, as well as GFAP, MIB‐1 (Ki67) and hTERT immunostaining of the original tumors, was performed (Figure 1) with all the tumors expressing GFAP and hTERT. The diagnosis of ependymoma was confirmed by two neuropathologists.

Figure 1.

Histological and immunohistochemical characterization of ependymomas. Representative hematoxylin and eosin‐stained sections are shown in the first column. The following columns show immunohistochemical staining for glial fibrillary acidic protein (GFAP), MIB‐1 and human telomerase reverse transcriptase (hTERT), respectively. All tumors expressed GFAP and hTERT. The MIB‐1 proliferative indices ranged from ∼3 to 20%. EP1‐6 refers to the ependymoma specimen number.

Cells were harvested directly from the surgically resected tumor specimens. Once established in culture, the cells were assessed for GFAP and hTERT expression, and, where sufficient cells were available, for ultrastructural features of ependymal cells. Immunofluorescence showed that all harvested cells were GFAP and hTERT positive (Figure 2A). It is possible that successful tissue culture was biased toward hTERT‐positive samples and/or cells, with the result that all six of our successfully cultured samples were hTERT positive. The initial telomere lengths were measured in the five cases where sufficient material was available, and ranged from 5.9 to 7.1 kbp (Figure 2B,C).

Figure 2.

Characterization of primary ependymoma cells. A. Immunofluorescence shows that the harvested cells are human telomerase reverse transcriptase (red, left panel) and glial fibrillary acidic protein (green, right panel) positive. Cells were counterstained with 4′,6′‐diamidino‐2‐phenylindole dihydrochloride, and images were captured at 400× magnification. Figures are representative of all six tumor samples. B. Five of the six tumor samples were subjected to telomere length assay. C. Quantified results of Southern blot shows the telomere length ranges from 5.9 to 7.1 kbp.

Effect of telomerase inhibition on primary ependymoma cells

Primary ependymoma cells harvested from surgically removed tumors were treated with MST‐312 for 72 h. Because of limited cell number, only three samples were subjected to MTT assay. Of the three samples tested, all showed a significant dose‐dependent decrease in cell number compared to vehicle‐treated cells (P < 0.001) following 72 h of MST‐312 treatment (Figure 3).

Figure 3.

Short‐term telomerase inhibition with MST‐312 decreases cell viability. Three of the primary ependymoma cells (EP1, EP4 and EP6) were subjected to MST‐312 treatment for 72 h. MTT assay shows a significant (P < 0.05) dose‐dependent decrease in cell viability in all samples relative to control (vehicle‐treated) cells.

The observed cell number decrease may have been caused by a decrease in cell proliferation and/or an increase in cell death/apoptosis. To explore these possibilities, the proliferative index and apoptotic index of telomerase‐inhibited cells were assessed by immunofluorescence using anti‐Ki67/MIB‐1 antibodies and anti‐cleaved caspase 3 antibodies, respectively. Immunofluorescence staining showed an approximately 50% decrease (P < 0.05) in the MIB‐1 proliferative index (Figure 4A) following 72 h of 1 µM MST‐312 in all the samples tested. An increase in apoptosis was detected in three of the six samples tested (Figure 4B), and overall was not statistically significant (P > 0.05).

Figure 4.

Short‐term telomerase inhibition with MST‐312 activates H2AX and decreases MIB‐1 proliferative index. A. After 72 h of 1 µM MST‐312 treatment, there was a significant (P < 0.05) decrease in the MIB‐1 proliferative index relative to vehicle control in all ependymoma samples (n = 6). B. Increased apoptosis was detected in three out of six cases, but was not significant overall. C. There was a significant (P < 0.05) increase in γH2AX relative to no treatment controls in all ependymoma samples (n = 6). Error bars represent standard deviation of the mean of six samples.

The percent of cells showing γH2AX expression with and without MST‐312 treatment was assessed by immunofluorescence using an anti‐γH2AX antibody. The percent of cells expressing γH2AX was significantly (P < 0.05) higher in the treated cells compared to vehicle control (Figure 4C). This was observed in all the primary ependymoma cells tested. Taken together, these data show that telomerase inhibition using MST‐312 results in acute DNA damage, decreased proliferation and a decrease in cell number in primary ependymoma cells.

DISCUSSION

Our results demonstrate acute induction of DNA damage, decreased cell proliferation and decreased cell number within 72 h of exposure to the telomerase inhibitor MST‐312 (summarized in Supporting Information Table S1). This was independent of initial telomere length and telomere length maintenance.

Interestingly, our in vitro results are in keeping with our findings in a larger cohort of paraffin‐embedded ependymoma samples (20). Ependymomas which lacked hTERT showed more γH2AX expression (DNA damage) and lower MIB‐1 proliferation indices, similar to what we observed following telomerase inhibition in hTERT‐positive ependymomas in vitro.

The conventional view of anti‐telomerase therapies is that one must wait for the telomeres to erode to a critically short length before any cytotoxic effect can be observed. However, our results suggest that hTERT inhibition could lead to decreased proliferation and an elevation of DNA damage in ependymomas without having to wait for the telomeres to shorten first. Acute effects of telomerase inhibition on cancer cells have been reported in several other studies 6, 9, 12 with decreased proliferation observed as early as 48 h following treatment.

The mainstay of treatment in ependymoma is surgery aiming for maximal resection, as the extent of resection is currently the primary determinant of outcome (1). The acute response of primary ependymoma cells observed in this study, coupled with our previous observation of a better outcome in patients with hTERT‐negative ependymomas (18), suggests that hTERT‐positive ependymomas are telomerase dependent and may be sensitive to anti‐telomerase therapies. This potential needs to be explored further in clinical trials given the poor outcome of children with hTERT‐positive ependymomas. Several clinical trials using oligonucleotide and therapeutic vaccines are already under evaluation, and our data support the idea of extending these trials to include those children with hTERT‐positive ependymomas.

Supporting information

Table S1. Summary of patient characteristics and primary ependymoma cell response to telomerase inhibition.

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Supporting info item