Introduction
Glioblastoma (GBM) is the most common and devastating tumor in the central nervous system. Despite optimal treatment, the patients with this disease can expect a dismal prognosis, a mean survival of 12–15 months after initial diagnosis [
1]. Chemotherapy plays an important role in the treatment of the disease. Currently, temozolomide (TMZ)-based chemotherapy significantly improves prognosis, and is recommended as a standard care for GBM patients [
2]. The main effect of TMZ is to methylate the O
6 residues of guanine so as to prevent DNA duplication during cell proliferation and to induce cell death and apoptosis [
3]. However, a DNA repair enzyme, O
6-methylguanine DNA methyltransferase (MGMT), is able to reverse the anti-tumor effect of TMZ, and mainly contributes to the chemoresistance in a fraction of patients. Normally, high promoter methylation status which means low MGMT activity predicts good response to TMZ chemotherapy and results in a longer survival period in GBM patients while low promoter methylation status, which leads to high MGMT expression is linked to a remarkable chemoresistance and a shorter survival period [
4,
5]. Bases on previous studies, there are about 50% patients with high MGMT activity in primary GBM [
2], and an even higher percentage in recurrent ones [
6]. Therefore, it is of great significance to alleviate the chemoresistance of GBM with high MGMT expression.
Hedgehog (HH) signaling pathway plays an important role in the development of central nervous system during embryogenesis [
7,
8]. In mammals, the canonical HH signaling pathway starts with binding of one of the three extracellular ligands (Sonic HH, Indian HH and Desert HH) to the transmembrane receptor protein Patched (PTCH). Two PTCH receptors, PTCH1 and PTCH2, have been identified for the ligands. Binding of HH ligands to PTCH relieves another transmembrane protein, smoothened (SMO), from the inhibitory effect of PTCH, resulting in transcription of downstream target genes through the Gli transcription factors [
9]. The Gli protein family, which consists of Gli1, Gli2 and Gli3, serves as the mediators of the HH signaling pathway. Gli1 acts as a main transcriptional activator in HH signaling pathway [
10]. Aberrant activation of HH/Gli1 signaling pathway has been implicated in a fraction of GBM, and inhibiting HH/Gli1 signaling results in tumor growth suppression [
11]. Our previous study showed that Gli1 was a potential target to alleviate multidrug resistance of human glioma by regulating the transcription of a series of chemoresistance-associated genes [
12], but the relationship between HH/Gli1 signaling and MGMT expression in GBM is yet to be clarified.
In this study, we first evaluated the relationship between Gli1 activity and MGMT expression in primary GBM tissues. In vitro cultured GBM cells lines, we explored the effects and mechanism of regulating the HH/Gli1 signaling pathway on MGMT expression and chemoresistance to TMZ. Finally, we performed in vivo studies to verify whether the signaling pathway can serve as a potential target to overcome chemoresistance in GBM with high MGMT activity.
Methods
Immunohistochemistry
With the approval of the institutional ethics committee, forty-eight patients with primary GBM whom underwent tumor resection in the Neurosurgical Department of Shanghai Tenth People’s Hospital from Jan 2011 to Dec 2013 were given informed consent and enrolled in this study. The median age was 58 years (range, 40–75); 20 patients were male and 28 female. All patients received surgical treatment and specimens were fixed with 10% formalin, embedded in paraffin, and examined histopathologically. Immunohistochemistry was done as previously described [
13], and the specimens were not affected by the chemotherapy. Primary antibodies were used as follow: rabbit polyclonal anti-Gli1 antibody (1:100, ab49314, Abcam) and mouse monoclonal anti-MGMT antibody (1:100, ab39253, Abcam). The percentage of tumor cells with Gli1 nuclear staining to total cancer cells being > 10%, it was judged to be Gli1-positive. With the ratio of tumor cells stained with MGMT > 5%, it was judged to be MGMT-positive [
14]. Sections from the same tissues without application of the primary antibodies were used as negative controls.
Cell culture, reagents and immunofluorescence
The GBM cell lines, U87, A172, and U251, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were regularly verified for cell morphology and growth characteristics by microscopic analysis. All the cells were cultured in 10% FBS-supplemented DMEM and maintained at 37 °C in a humidified atmosphere of 5% CO
2 and 95% air. The procedures to obtain and stain glioma stem/progenitor (GSP) cells from U87 cells were as previously described [
15]. The growth factor EGF and bEGF were purchased from PeproTech; B27 was purchased from Gibco. Rabbit polyclonal anti-Gli1 antibody (1:100, ab49314, Abcam) and mouse monoclonal anti-MGMT antibody (1:100, ab39253, Abcam) were used to stain GSP cells for immunofluorescence assay. Slides were stained with DAPI (Beyotime Biotechnology) for 5 min prior to examination using a confocal microscope.
Western Blot analysis
Whole-cell extracts were prepared after lysis in buffer containing RIPA (Beyotime Biotechnology) and PMSF (Beyotime Biotechnology) (100:1). Equal amounts of protein (30 μg/sample) were separated by 10 or 5% SDS-PAGE gels. Proteins were transferred to polyvinylidene difluoride membranes and then blocked in 5% BSA dissolved in PBST for 1 h. Membranes were incubated with primary antibodies (Gli1: GTX124274, GeneTex; MGMT: SAB1406122, Sigma-Aldrich; β-actin: ab8226, Abcam) in 5% BSA at 4 °C overnight followed by secondary antibody for 1 h at room temperature. Bands were visualized on an Odyssey Infrared Imaging System (LI-COR Biosciences).
Real-time quantitative PCR and methylation-specific PCR
Total RNA was extracted from cells using Trizol reagent (Invitrogen). To detect the mRNAs of the different genes examined, the primers applied for real-time PCR are as follows: Gli1 forward: 5′-TTCCTACCAGAGTCCCAAGT-3′; reverse: 5′-CCCTATGTGAAGCCCTATTT-3′; MGMT forward: 5′-CCTGGCTGAATGCCTATTTC-3′; reverse: 5′-GATGAGGATGGGGACAGGATT and GAPDH forward: 5′-TGCACCACCAACTGCTTAGC-3′; reverse: 5′-GGCATGGACTGTGGTCATGAG-3′. Average level of GAPDH RNA was used as internal control. Genomic DNA was extracted and treated with sodium bisulfite for methylation-specific PCR (MSP) using the EpiTect Plus LyseAll Lysis, Plus DNA Bisulfite, MSP Kit (Qiagen). The primers used for methylated product were: MGMT-M forward: 5′-GTTTCGGGTTTAGCGTAGTC-3′; reverse: 5′-TATCACAAAAATAATCCGCG-3′. The unmethylated primers are: MGMT-U forward: 5′-GTTTTGGGTTTAGTGTAGTT-3′; reverse: 5′-TATCACAAAAATAATCCACA.
Construction of Gli1 expression vector
The RT-PCR amplification mixture contained the primers followed: Gli1 forward: AGGGAGACCCAAGCTGgctagcATGTTCAACTCGATGACCCCACCACCAA; reverse: TAAGCTTGGTACCTCAtctagaTTAGGCACTAGAGTTGAGGAATTCTG. GCTAGC and TCTAGA were recognition sequences of the restriction endonucleases, NheI and XbaI.
Cell transfection
Cells were transiently transfected using Lipofectamine 2000 (Invitrogen) with vector pcDNA3.1-Gli1 or the empty vector pcDNA3.1. Cells were used at 24, 48, 72, 96 and 120 after transfection.
Cells proliferation and apoptosis assay
Cells were plated in 96-well plates at a density of 1 × 10
3 cells per well and incubated in 10% FBS-supplemented DMEM with 100 μM TMZ (Enzo Life Sciences) and maintained at 37 °C under a humidified atmosphere of 5% CO
2 and 95% air for 120 h. Cyclopamine (Cayman chemical) at the concentration of 5 μM was added as indicated in the “
Results” section. Cell proliferation was detected by the cell counting kit-8 (CCK8, Dojindo) and the absorbance was measured by microplate reader at 450 nm.
Cells were cultured in 6-well plates at a density of 2 × 105 cells per well for the apoptosis assay. The apoptotic effects of treatments were determined by using Vybrant Apoptosis Assay Kit (Invitrogen), followed by fluorescence-activated cell sorter analysis using a FACScanto flow cytometry.
Chromatin Immunoprecipitation (ChIP) analysis
The ChIP analysis was conducted in the U251 cell line, using the EpQuik™ Chromatin Immunoprecipitation Kit (p-2002-2, epigentek) according to the manufacturer’s instructions. 1 μg mouse IgG and 1 μg anti-RNA polymerase II served as a negative and positive control respectively. For immunoprecipitation, 2 μg antibody against Gli1 (NB600-600, Novus Biologocals) was added to each well. The primers applied for ChIP analysis were as follows: Homo MGMT1 for site 1 (169 bp): forward: 5′-CCCCATCTCCAAATAAGGTC-3′; reverse: 5′-TAGACACTGCCAGAGCCTGA-3′; Homo MGMT2 for site 2 (210 bp): forward: 5′-GACGGCATCGCCCACCACA-3′; reverse: 5′-GCCCGAGTGGTCCTGAAAGC-3′; Homo MGMT3 for site 3 (276 bp): forward: 5′-TCAGGCGGAAGCTGGGAAGG-3′; reverse: 5′-CCGAGGACCTGAGAAAAGCAAGAG-3′. Primers for RegIV: forward: 5′-CTCGGAAGGTTTCTAATC-3′; reverse: 5′-TTCAACATGCGTGAGTTT-3′.
In vivo study
The Committee on Ethical Use of Animals of Shanghai Tenth People’s Hospital approved the in vivo study. The animal studies complied with the ARRIVE guidelines. Female nude mice of 4 weeks old were obtained from the Changzhou Cavens Laboratory Animal Co. Ltd. U251 cells in log-phase growth were suspended in PBS at 5 × 107/mL and then subcutaneously injected 200 μL per mouse into the right back of nude mice. When the tumor diameter reached about 5 mm, the mice were randomly divided into four groups with 5 mice per group and treated by the following agents: (a) the control: ethanol (1 mL/kg, days 1–2, p.o.) plus DMSO (1 mL/kg, days 1–5, p.o.); (b) TMZ alone: TMZ dissolved in equivalent dose of DMSO (42 mg/kg, days 1–5, p.o.) plus ethanol (1 mL/kg, days 1–2, p.o.); (c) cyclopamine alone: cyclopamine dissolved in equivalent dose of ethanol (25 mg/kg, days 1–2, p.o.) plus DMSO (1 mL/kg, days 1–5, p.o.); (d) TMZ plus cyclopamine: TMZ plus cyclopamine at the equivalent doses. Mouse body weight and the tumor size were measured every 5 days. The approximate volume of the tumor was calculated using the formula (length × width2)/2. All mice were sacrificed at day 30 and tumor tissues was stained by immunohistochemistry for Gli1 and MGMT expression.
Statistical analysis
All statistical analyses were performed with SPSS 16.0 for windows. Data was presented as the mean ± SD, and were compared by two-tailed unpaired Student’s t test or Person Chi Square test. Difference was considered significant when p value was below 0.05.
Discussion
To date, GBM remains the worst malignant human glioma despite of optimal therapeutic strategy. TMZ is currently recommended as the first-line agent to treat the disease [
19]. However, tumor’s resistance to TMZ or tumor recurrence are the main cause of treatment failure. Thus far, few approaches targeting chemoresistance in GBM have been introduced. O
6-Benzylguanine was used to restore TMZ sensitivity in patients with TMZ-resistant human glioma, but failed to restore TMZ sensitivity in GBM [
20]. Interferon-beta inactivates MGMT via p53 gene induction and enhances the therapeutic efficacy to TMZ [
21], showing benefits when combined with TMZ to treat newly diagnosed primary GBM [
22]. Our study reveals that HH/Gli1 signaling pathway may regulate MGMT expression and chemoresistance to TMZ in GBM.
Nuclear staining of Gli1 is a reliable indicator of HH pathway activity in tumor cells [
17]. Here, we investigate the relationship between Gli1 nuclear staining and MGMT expression in primary GMB tissues. The results showed that MGMT-positive tissues have a significantly higher rate of Gli1 nuclear staining than MGMT-negative ones, which implies there is a link between HH/Gli1 signaling activity and MGMT expression. Our following experiments in GBM cell lines revealed the transcriptional regulation of MGMT expression by Gli1 binding to its promoter region, demonstrating MGMT as a downstream target gene of HH/Gli1 signaling pathway. However, it is noteworthy that 5 of 26 (19.2%) MGMT-positive GBM tissues did not show positive nuclear Gli1 staining and 10 of 31 (32.2%) Gli1-positive GBM tissues also did not demonstrate positive MGMT expression. Comparatively, the well-known MGMT promoter methylation status predicts MGMT level more precisely in comparison to our Gli1 staining. We won’t say that Gli1 is a better predictive factor than MGMT promoter methylation status, but as our work shows, we can generate the MGMT expression and sensitivity to the chemotherapy by altering the HH/Gli1 pathway independent from the promoter status. There is a high possibility that 22 of 29 (75.9%) MGMT promoter unmethylated patients have an active HH/Gli1 pathway. We can take advantage here from our work, which could be a potential target that can help overcome the unmethylated patients’ resistance to TMZ. Interestingly, Xu et al. [
23] recently found that haplotype has an impact on transcription factor binding in the MGMT promoter/enhancer region consequently regulating MGMT expression. This finding may help our work on Gli1 to fit better on evaluating MGMT activity. Future work can explore the relationship between MGMT promoter and HH/Gli1 pathway statement. With the recruitment of MGMT promoter haplotypes analysis and more IDH-mutant patients, we may be able to establish a better evaluation system of MGMT activity and chemoresistance.
Although several studies have shown that HH/Gli1 signaling pathway is involved in regulation of chemosensitivity in a variety of organ neoplasms, including pancreatic cancers, prostate cancer, and breast caners [
24‐
26], few studies have focused on its influence on chemosensitivity in the central nervous system neoplasm. Activating HH signaling by Gli1 overexpression in A172 cells, resulted in synchronous increase of MGMT expression and enhanced chemoresistance to TMZ. Moreover, blocking the pathway by cyclopamine in U251 cells led to synchronous decrease of MGMT and diminished chemoresistance to TMZ. As the main transcriptional activator, Gli1 enhanced a number of downstream target genes of HH signaling pathway [
27]. Our ChIP analysis identified the potential binding site of Gli1 protein on the MGMT promoter region. Therefore, our study proved the involvement of HH/Gli1 signaling in the regulation of TMZ chemosensitivity in GBM.
There is a small subpopulation of tumor cells with stem-like phenotypes in human GBM. Expressing high levels of MGMT, these cells have been shown to be involved in chemotherapy resistance and responsible for tumor recurrence [
28]. Moreover, previous studies have shown that these stem-like cancer cells harbor aberrantly activated HH/Gli1 signaling pathway [
16,
29]. Furthermore, our study in the U87 GSP cells illustrated that inhibition of the HH/Gli1 signaling pathway by cyclopamine reduced MGMT expression. That indicates the existence of regulatory mechanism at the level of stem/progenitor cells and offers a new angle to mitigate chemoresistance in stem-like cancer cells.
O
6-Methylguanine DNA methyltransferase expression predicts the response to TMZ in GBM cells [
30]. TMZ alone did not effectively inhibit tumor growth because the GBM xenografts originated from the U251 cells that had a high MTMT activity. However, the tumor growth was significantly blocked by cyclopamine possibly due to an activated HH/Gli1 signaling in these cancer cells. The xenografts tissues after cyclopamine treatment showed low levels of Gli1 and MGMT measured by immunohistochemistry, indicating effective inhibition of the HH/Gli1 signaling pathway by cyclopamine. This finding is consistent with the previous study that targeted inhibition of HH signaling resulted in tumor growth repression in malignant glioma xenografts with active HH signaling [
31]. In the group of nude mice treated by cyclopamine plus TMZ, tumor growth inhibition was significant at 15 days after the treatment when compared with the control group, which was much earlier than the cyclopamine alone group (25 days). Moreover, the combination group showed significantly smaller tumor volumes than both the cyclopamine group and the TMZ group, demonstrating a synergistic effect on tumor growth inhibition.
In summary, this study shows that HH/Gli1 signaling pathway regulates MGMT expression and chemoresistance to TMZ in human GBM independent from MGMT promoter methylation status, which offers a potential target to restore chemosensitivity to TMZ in a fraction of GBM with high MGMT expression. As complex signaling networks regulating MGMT expression exist at different cellular levels including epigenetics, transcription, and posttranscription [
32‐
34], future studies will shed light on new methods to overcome chemoresistance in human GBM.
Authors’ contributions
KW conceived and designed the experiments. DJ performed most of the experiments. ZQ analyzed the data. LG and ML helped in sample collection. DJ wrote the manuscript and KW helped to draft the manuscript. KW, LG and DC provided the financial support and supervised the whole experimental work. All authors read and approved the final manuscript.