NPV-LDE-225 (Erismodegib) inhibits epithelial mesenchymal transition and self-renewal of glioblastoma initiating cells by regulating miR-21, miR-128, and miR-200

Abstract

Background

Glioblastoma multiforme is the most common form of primary brain tumor, often characterized by poor survival. Glioblastoma initiating cells (GICs) regulate self-renewal, differentiation, and tumor initiation properties and are involved in tumor growth, recurrence, and resistance to conventional treatments. The sonic hedgehog (SHH) signaling pathway is essential for normal development and embryonic morphogenesis. The objectives of this study were to examine the molecular mechanisms by which GIC characteristics are regulated by NPV-LDE-225 (Smoothened inhibitor; (2,2′-[[dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N-dimethylbenzenamine).

Methods

Cell viability and apoptosis were measured by XTT and annexin V–propidium iodide assay, respectively. Gli translocation and transcriptional activities were measured by immunofluorescence and luciferase assay, respectively. Gene and protein expressions were measured by quantitative real-time PCR and Western blot analyses, respectively.

Results and conclusion

NPV-LDE-225 inhibited cell viability, neurosphere formation, and Gli transcriptional activity and induced apoptosis by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase. NPV-LDE-225 increased the expression of tumor necrosis factor–related apoptosis inducing ligand (TRAIL)–R1/DR4, TRAIL-R2/DR5, and Fas and decreased the expression of platelet derived growth factor receptor–α and Bcl2, and these effects were abrogated by Gli1 plus Gli2 short hairpin RNAs. NPV-LDE-225 enhanced the therapeutic potential of FasL and TRAIL by upregulating Fas and DR4/5, respectively. Interestingly, NPV-LDE-225 induced expression of programmed cell death 4 and apoptosis and inhibited cell viability by suppressing micro RNA (miR)–21. Furthermore, NPV-LDE-225 inhibited pluripotency-maintaining factors Nanog, Oct4, Sox2, and cMyc. The inhibition of Bmi1 by NPV-LDE-225 was regulated by induction of miR-128. Finally, NPV-LDE-225 suppressed epithelial-mesenchymal transition by upregulating E-cadherin and inhibiting N-cadherin, Snail, Slug, and Zeb1 through modulating the miR-200 family. Our data highlight the importance of the SHH pathway for self-renewal and early metastasis of GICs.

Brain cancers have shown a remarkable ability to build resistance not only to current drug therapies but also to apoptosis. Glioblastoma multiforme (GBM) ranks among the deadliest types of cancer, and therefore new therapies are urgently needed. GBM can be developed from glioblastoma initiating cells (GICs) of the brain.^1–5 The capability of GICs to sustain brain tumor growth apparently lies in their active self-renewal and/or suppressed cell differentiation.⁶ Several major signaling pathways that are critical in brain development have also been implicated in tumorigenesis, including bone morphogenetic protein,⁷ Notch,⁸ sonic hedgehog (SHH),^8,9 epidermal growth factor receptor,¹⁰ phosphatase and tensin homolog/phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR),^11–15 platelet derived growth factor receptor (PDGFR),¹⁶ and Olig2.¹⁷ Recently, a gene expression profiling of gliomas has shown that SHH signaling is active in a subset of gliomas.¹⁸ This study further showed that SHH signaling is essential for glioma GIC self-renewal and GIC-initiated brain tumor growth.¹⁸ It is postulated that the relatively homogeneous population of GICs, rather than the heterogeneous tumor cells, may reveal key mechanisms of tumor initiation and propagation of primary tumors and hence predict tumor prognosis, therapy, and drug response.

The SHH signaling pathway is a key regulatory mechanism in neural development, and abnormal SHH signaling has been implicated in tumorigenesis in brain, such as that of glioblastoma.^3,19–21 The normal function of the SHH ligand in the SHH pathway is to serve as a morphogen, inducing proper differentiation in embryogenesis.^22–24 Genomic alterations of the SHH pathway have been shown to lead to the development of brain cancers.²⁵ Aberrant activation of the SHH pathway leads to an increase in cell survival and metastasis in cancer cells. Such aberrant activity includes inactivating mutations of Patched1 or Sufu as well as activating mutations of Smoothened (Smo).^26–28 The binding of the SHH ligand to its receptor, Patched, leads to the activation of transcription factors Gli1 and Gli2, as well as the inactivation of transcriptional repressor Gli3.^21,25 Several studies have supported the presence of stemlike cells in brain tumor cultures that are CD133 positive, are capable of self-renewal, and give rise to all cell types found within the tumor, potentially perpetuating growth. CD133 is a widely accepted marker for glioma-derived GICs. CD133-positive glioma cells overexpress genes involved in the SHH pathway, which contributes to chemoresistance, and its antagonism leads to an additive effect when used in combination with temozolomide.^1,29 In addition, aberrant activation of the SHH pathway has been tied to Gorlin syndrome, a disease that predisposes individuals to basal cell carcinoma and medulloblastoma.³⁰

Recently, SHH-Gli signaling was also found to be essential for glioma stem cell self-renewal.³¹ Abnormal regulation of the Gli family of genes had been shown to lead to tumorigenesis. The expression of Gli2 in certain types of cancer cells leads to increased invasiveness and metastatic capabilities of those cells.¹ Activation of Gli has been correlated with the expression levels of the SHH pathway as a whole, which suggests that the Gli family of transcription factors may serve as an indicator of SHH pathway activity.¹ Further studies have suggested that the Gli family may serve as a therapeutic target for anticancer drugs, as these transcription factors have been shown to be involved with advanced proliferation and metastasis in cancer cells.^22,25,32 A selective antagonist of Smo is NVP-LDE-225 ((2,2′-[[dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N-dimethylbenzenamine; Erismodegib). Recently, NVP-LDE-225 has been used in topical creams for the treatment of basal cell carcinoma and has shown promise in its ability to effectively inhibit the SHH pathway.³³ The selective inhibition of Smo by NVP-LDE-225 will effectively reduce the expression of Gli1 and Gli2. Inhibition of the Gli family of transcription factors would then decrease the transcription of genes associated with cell survival and proliferation in GICs.

The process of epithelial-mesenchymal transition (EMT), characterized by loss of intercellular adhesion and polarity, cytoskeletal reorganization that enhances cell motility, and degradation of the basement membrane, has been associated with tumor progression and metastasis.^34,35 Diverse signaling pathways regulate EMT, including the SHH pathway.³⁶ Induction of EMT functions in particular through downregulation of the epithelial adhesion protein E-cadherin (Cdh1) and direct repression of Cdh1 have been shown to be under the control of transcriptional regulators Zeb1, Zeb2, Twist1, Snail, and Slug, which also regulate a large number of other epithelial-related genes.³⁷ Transcription factors of the Zeb protein family (Zeb1 and Zeb2) and several micro RNA (miR) species (predominantly miR-200 family members) form a double negative feedback loop, which controls EMT and mesenchymal-epithelial transition (MET) programs in both development and tumorigenesis. However, the molecular mechanism by which the SHH pathway regulates EMT is not well understood.

MiRs are small, noncoding RNAs that play a critical role in developmental, stem cell maintenance, and physiological processes and are implicated in the pathogenesis of several human diseases, including GBM.³⁸ MiRs also play a role in cancer by controlling the expression of certain oncogenes and tumor suppressor genes.³⁹ MiR profiling has revealed distinct expression signatures in various human cancers, including glioma.⁴⁰ The functional significance of most of these alterations remains unclear.

Polycomb protein Bmi1 is a key regulator of hematopoietic, neural stem cell, and GIC populations. The Bmi1 gene is implicated in the pathogenesis of brain tumors, including glioma,⁴¹ and is an important epigenetic regulator of fate determination and proliferation in stem cell populations.^42,43 Bmi1 is upregulated in several cancer types and is a positive regulator of stem cell renewal,^44–46 and studies in transgenic mice revealed a critical role for Bmi1 in driving glioma growth.⁴¹ Previous reports have suggested that there is a potential link between SHH signaling and Bmi1, thus highlighting a novel regulatory mechanism whereby an external signaling morphogen interacts with cell-intrinsic epigenetic pathways controlling cell fate programs.⁴² MiR-128 is downregulated in gliomas, so that its expression reduces glioma cell proliferation, and thus Bmi1 is a direct target of miR-128.⁴⁷ Here, we propose that inhibition of the SHH pathway by NVP-LDE-225 may suppress Bmi1 through upregulation of miR-128.

The purpose of this study was to examine the effects of NVP-LDE-225 (also referred as LDE-225) on GICs, with a particular focus on the drug's impact on the SHH pathway and, subsequently, cell proliferation, neurosphere formation, EMT, and apoptosis. Overall, our findings suggest that inhibition of the SHH signaling pathway is a potential therapeutic strategy for glioblastoma, and the combination of NVP-LDE-225 with FasL or tumor necrosis factor–related apoptosis inducing ligand (TRAIL) can sensitize GICs that are resistant to death receptor (DR) agonists.

Materials and Methods

Reagents

Antibodies against caspase-3, PARP, Gli1, Gli2, Patched1, and Patched2 were obtained from Cell Signaling Technology. Antibodies against Fas, TRAIL-R1/DR4, TRAIL-R2/DR5, and β-actin were purchased from Santa Cruz Biotechnology. FasL and TRAIL were from R & D Systems. Enhanced chemiluminescence Western blot detection reagents were from Amersham Life Sciences. NVP-LDE-225 was purchased from ChemieTek, Indianapolis, IN. All other chemicals used were of analytical grade and were purchased from Fisher Scientific and Sigma-Aldrich. Lentiviral expression constructs of anti–miR-128, pre–miR-21, anti–miR-200a, anti–miR-200b, and anti–miR-200c were purchased from System Biosciences.

Primary Brain Tumor Cell Culture

Human GICs (CD133⁺) from human primary tumors were cultured on ultralow attachment culture dishes (Corning) in stem cell growth medium (Celprogen) supplemented with 1% N2 (Invitrogen), 2% B27 (Invitrogen), 20 ng/mL human basic fibroblast growth factor (Invitrogen), 100 ng/mL epidermal growth factor (Invitrogen), and 1% antibiotic-antimycotic (Invitrogen) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The population of CD133-positive GICs ranged from 3% to 5% from batch to batch. GICs were isolated from 5 primary tumors.

Lentiviral Particle Production and Gli1 and Gli2 shRNA Transduction

Gli1 shRNA (5′-GCCTGAATCTGTGTATGAA-3′; 5′-GTTTGAATCTGAATGCTAT-3′; 5′-AGCTAGAGTCCAGAGGTTC-3′; 5′-CCGGAGTGCAGTCAAGTTG-3′ and 5′-GGCTGGACCAGCTACATCA-3′) and Gli2 shRNA (5′-CCGAGAAGCAAGAAGCCAA-3′; 5′-CACAGCATGCTCTACTACT-3′; 5′-TCGCTAGTGGCCTACATCA-3′; 5′-TCCGAGAAGCAAGAAGCCA-3′ and 5′-CCAGACGACGTGGTGCAGT-3′) were obtained from Open Biosystems and cloned into TRIPZ vector. Lentivirus was produced by triple transfection of human embryonic kidney 293T cells. Packaging 293T cells were plated in 10-cm plates at a cell density of 5 × 10⁶ a day prior to transfection in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum without antibiotics. Transfection of packaging cells and infection of brain GICs were carried out using standard protocols³⁴ with some modifications. In brief, 293T cells were transfected with 4 µg of plasmid and 4 µg of lentiviral vector using lipid transfection (Lipofectamine-2000/Plus reagent, Invitrogen) according to the manufacturer's protocol. Viral supernatants were collected and concentrated by adding PEG-it virus precipitation solution (SBI System Biosciences) to produce virus stocks with titers of 1 × 10⁸ to 1 × 10⁹ infectious units per milliliter. Viral supernatant was collected for 3 days by ultracentrifugation and concentrated 100-fold. Titers were determined on 293T cells. Brain GICs were transduced with lentivirus expressing scrambled shRNA (control), Gli1 shRNA, or Gli2 shRNA. Brain GICs simultaneously expressing both Gli1 plus Gli2 shRNA were also generated. Following transduction, the GICs were washed 3 times with 1× phosphate buffered saline (PBS) and allowed to grow for 3 passages before screening for gene expression. Once decreased expression of the targeted gene was confirmed, the cells were used for experiments. Stable expression of Gli1 shRNA or Gli2 shRNA was ensured by culturing cells in the presence of a selection antibiotic puromycin (6.0 µg/mL), whereas induction of both Gli1 shRNA and Gli2 shRNA was performed in the presence of doxycycline (2.0 µg/mL). The transduced GICs were washed 3 times with PBS (without Ca⁺⁺ or Mg⁺⁺) and used for experiments.

Cell Viability and Apoptosis Assays

Cells (1.5 × 10⁴) were incubated with 0, 1, 5, and 10 µM of NVP-LDE-225 in 250 µL of culture medium in 96-well plates for 48 and 72 h before cell viability determination. Cell viability was determined by assay by XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium). In brief, a freshly prepared XTT–phenozine methosulfate labeling mixture (50 µL) was added to the cell culture. Absorbance was measured at 450 nm, with correction at 650 nm. Cell viability was expressed in terms of optical density (OD) (OD₄₅₀–OD₆₅₀). Apoptosis was determined by fluorescence-activated cell sorting analysis of PI-stained cells. In brief, cells were trypsinized, washed with PBS, and resuspended in 200 µL PBS with 10 µL RNAase (10 mg/mL) and incubated at 37°C for 30 min. After incubation, 50 µL PI solution (25 µg/mL) was added and cells were analyzed for apoptosis using flow cytometry. Apoptosis was also measured by annexin/PI staining as per manufacturer's instructions (BD Biosciences).

Motility Assay

Scratch migration assay was used to study the horizontal movement of cells. A confluent monolayer of cells was established and then a scratch was made through the monolayer, using a standard (1–200 μL) plastic pipette tip, which gave rise to an in vitro wound, then the monolayer was washed twice with PBS and replaced in media with or without NVP-LDE-225. GICs migrated into the scratch area as single cells from the confluent sides. The width of the scratch gap was viewed under the microscope in 4 separate areas each day until the gap was completely filled in the untreated control wells. Three replicate wells from a 6-well plate were used for each experimental condition.

Transwell Migration Assay

For transwell migration assays, 1 × 10⁵ brain GICs were plated in the top chamber onto the noncoated membrane (24-well insert; pore size, 8 μm; Corning Costar) and allowed to migrate toward serum-containing medium in the lower chamber. Cells were fixed after 24 h of incubation with methanol and stained with Diff-Quick Fixative Solutions (Dade Behring).

Transwell Invasion Assay

For invasion assay, 1 × 10⁵ cells were plated in the top chamber onto the Matrigel-coated membrane (24-well insert; pore size, 8 μm; Corning Costar). Each well was coated freshly with Matrigel (60 μg; BD Bioscience) before the invasion assay. Brain GICs were plated in medium without serum or growth factors, and medium supplemented with serum was used as a chemoattractant in the lower chamber. After 48 h, Matrigel-coated inserts were fixed and stained with Diff-Quick Fixative Solutions (Dade Behring). The number of cells invading through the membrane was calculated under a light microscope (40×, three random fields per well).

Neurosphere Assay

For neurosphere assay, cells were plated in 6-well ultralow attachment plates (Corning) at a density of 1000 cells/mL in Dulbecco's modified Eagle's medium supplemented with 1% N2 Supplement (Invitrogen), 2% B27 Supplement (Invitrogen), 20 ng/mL human platelet growth factor (Sigma-Aldrich), 100 ng/mL epidermal growth factor (Invitrogen), and 1% antibiotic-antimycotic (Invitrogen) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Neurospheres were collected after 7 days and dissociated with Accutase (Innovative Cell Technologies). The GICs obtained from dissociation were counted by Coulter counter using trypan blue dye.

Immunoblotting

GICs were lysed and homogenized in radioimmunoprecipitation lysis buffer containing fresh protease inhibitors by standard procedures. Protein concentrations were quantified with a bicinchoninic acid protein assay kit (Pierce; http://www.piercenet.com), and 40 µg of proteins were separated in 4%–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels, transferred to polyvinylidene difluoride membranes, and hybridized with primary by standard procedures. Signals were detected by enhanced chemiluminescence reagents (GE Health; http://www.gelifesciences.com).

RNA Isolation and mRNA Expression Analysis

Total RNAs were isolated using the RNeasy Mini Kit (Qiagen). Synthesis of cDNAs was performed by oligo(dT)-priming methods. RT-PCR was performed using SYBR Green Supermix (Qiagen) according to the manufacturer's instructions. Primers specific for each of the signaling molecules were designed using the National Center of Biotechnology Information/Primer–Basic Local Alignment Search Tool and were used to generate the PCR products. Expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPD) were used for normalization and quantification of gene expression levels. For the quantification of gene amplification, RT-PCR was performed using an Applied Biosystems 7300 Sequence Detection System in the presence of SYBR Green. The following gene-specific primers were used:

• Smo (5′-TCG CTA CCC TGC TGT TAT TC-3′, 5′-GAC GCA GGA CAG AGT CTC AT-3′)

• Patched1 (5′-TGA CCT AGT CAG GCT GGA AG-3′, 5′-GAA GGA GAT TAT CCC CCT GA-3′)

• Patched2 (5′-AGG AGC TGC ATT ACA CCA AG-3′, 5′-CCC AGG ACT TCC CAT AGA GT-3′)

• Gli1 (5′-CTG GAT CGG ATA GGT GGT CT -3′, 5′-CAG AGG TTG GGA GGT AAG GA-3′)

• Gli2 (5′-GCC CTT CCT GAA AAG AAG AC -3′, 5′-CAT TGG AGA AAC AGG ATT GG-3′)

• Myc (5′-CGA CGA GAC CTT CAT CAA AA-3′, 5′-TGC TGT CGT TGA GAG GGT AG-3′

• Nanog (5′-ACC TAC CTA CCC CAG CCT TT-3′, 5′-CAT GCA GGA CTG CAG AGA TT-3′)

• Sox2 (5′-AAC CCC AAG ATG CAC AAC TC-3′, 5′-GCT TAG CCT CGT CGA TGA AC-3′)

• Oct4 (5′-GGA CCA GTG TCC TTT CCT CT-3′, 5′-CCA GGT TTT CTT TCC CTA GC-3′)

• Snail (5′ACC CCA CAT CCT TCT CAC TG-3′, 5′-TAC AAA AAC CCA CGC AGA CA-3′)

• Slug (5′ACA CAC ACA CAC CCA CAG AG-3′, 5′-AAA TGA TTT GGC AGC AAT GT-3′)

• Zeb1 (5′-GCA CAA CCA AGT GCA GAA GA-3′, 5′-CAT TTG CAG ATT GAG GCT GA-3′)

• E-cadherin (5′-TGC TCT TGC TGT TTC TTC GG-3′, 5′-TGC CCC ATT CGT TCA AGT AG-3′)

• N-cadherin (5′-TGG ATG GAC CTT ATG TTG CT-3′, 5′- AAC ACC TGT CTT GGG ATC AA-3′)

• PDCD4 (5′-TTT GTA AGC GAA GGA GATGG-3′, 5′-ATG CCT TGT ACC CAA AAC AA-3′)

• Bmi1 (5′-AGC AGA AAT GCA TCG AAC AA-3′, 5′-CCT AAC CAG ATG AAG TTG CTGA-3′)

• Housekeeping-GAPD (5′-GAG TCA ACG GAT TTG GTC GT-3′, 5′-TTG ATT TTG GAG GGA TCT CG-3′)

Evaluation of Micro RNAs by qRT-PCR

Quantitative RT-PCR of miRs was performed using TaqMan microRNA assays (Applied Biosystems) in an Applied Biosystems 7300 Sequence Detection System. Ten nanograms of total RNA were reverse transcribed using a TaqMan MicroRNA Reverse Transcription kit from Applied Biosystems. Each reverse-transcribed reaction contained 1× stem-loop reverse-transcribed–specific primer, 1× reaction buffer, 0.25 mM each of deoxyribonucleodide triphosphate, 3.33 U/μL Multiscribe Reverse Transcription enzyme, and 0.25 U/μL RNAase inhibitor. The 15-μL reactions were incubated for 30 min at 16°C, for 30 min at 42°C, and for 5 min at 85°C and then held at 4°C. The PCR reaction was performed using standard protocol for the TaqMan PCR kit (Applied Biosystems). Briefly, following the reverse transcription step, 1.33 μL of the reverse-transcribed reaction was combined with 1 μL of a TaqMan microRNA assay (20×; forward primer, reverse primer, and probe) and 17.67 μL of TaqMan Universal PCR Master Mix, No AmpErase uracil N-glycosylase in a 20-μL final volume. The reactions were incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The expression of miR-34a was normalized against the expression of another small RNA, RNU48, as endogenous normalization control. All assays were performed in triplicate and were calculated on the basis of the ΔΔCt method. The n-fold change in miR expression was determined according to the 2^–ΔΔCT method.

Gli Reporter Assay (p-GreenFire1 Lenti-Reporter)

Gli reporter activity was measured as we described elsewhere.³² In brief, cop-GFP (control plasmid green fluorescent protein) and luciferase genes were cloned downstream of the Gli-response element, containing 4 Gli binding motifs (pGreen Fire1–4xGli-mCMV-EF1-Neo; System Biosciences). Brain GICs were transduced with lentiviral particles, and stable cells were selected. For transcription assay, GICs (5–10 000 cells per well) were seeded in 12-well plates and treated with or without NVP-LDE-225 (0–10 µM) for up to 48 h. After incubation, GICs were harvested and analyzed for luciferase reporter activity (Promega).

Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assay was performed as described elsewhere.³² In brief, Gli probes were end-labeled with [γ-³²P] dATP by incubating oligodeoxyribonucleotide strands with 5× reaction buffer and 10 U T4 polynucleotide kinase for 1 h at 37°C. Then labeled oligonucleotides were allowed to anneal at room temperature for 10 min, and 20 mg of protein from each sample was used in 25-mL binding reactions, which consisted of 1 mg poly dI-dC, in 5× binding buffer (50 mM Tris HCl; pH 8.0, 750 mM KCl, 2.5 mM EDTA, 0.5% Triton X-100, 62.5% glycerol [volume/volume] and 1 mM dithiothreitol). To determine specificity of DNA binding, samples were incubated with or without 20 ng of unlabeled competitor DNA for 10 min at room temperature. Then 0.1 ng of labeled probe of Gli was added and samples were further incubated for 20 min at room temperature. Samples were separated on a 5% nondenaturing polyacrylamide gel in 0.5% Tris-borate-EDTA and visualized by autoradiography.

Immunocytochemistry

Brain GICs were grown on fibronectin-coated coverslips (Becton Dickinson) in the presence or absence of NVP-LDE-225 (10 µM). Subsequently, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 in 1× PBS, washed, and blocked in 10% normal goat serum. After washing with PBS, cells were stained with Gli1 and Gli2 primary antibodies (1:100) for 16 h at 4°C and washed with PBS. Afterward, cells were incubated with fluorescently labeled secondary antibody (1:200) along with 4′,6 diamidino-2-phenylindole (DAPI; 1 mg/mL) for 1 h at room temperature. Finally, coverslips were washed and mounted using Vectashield (Vector Laboratories). Isotype-specific negative controls were included with each staining. Stained cells were mounted and visualized under a fluorescent microscope. Immunohistochemistry of brain tumor tissues was performed as described elsewhere.³⁵

Statistical Analysis

The mean and SD were calculated for each experimental group. Differences among groups were analyzed by a 1- or 2-way ANOVA, followed by Bonferroni multiple comparison tests using Prism statistical analysis software (GrafPad Software). Significant differences among groups were calculated at P < .05.

Results

NVP-LDE-225 Inhibits Cell Viability and Spheroid Formation and Induces Apoptosis in Brain GICs

We first confirmed the expression of a stem cell marker in GICs isolated from primary tumors. As shown in Fig. 1A, GICs express cell surface marker CD133. These cells were also positive for Abcg2. The SHH pathway is constitutively active in GBM.⁵ We therefore next sought to inhibit this pathway by NVP-LDE-225, a Smo inhibitor, and examine its effects on apoptosis and cell viability in neurospheres. We measured the effects of NVP-LDE-225 on apoptosis in brain GICs by 2 assays: annexin V/propidium iodide (PI) and PI staining (sub-G₀ cells). NVP-LDE-225 induced apoptosis in a dose-dependent manner, as measured by both of the assays (Fig. 1B and C). We next examined the effects of NVP-LDE-225 on cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). As shown in Fig. 1C, treatment of brain GICs resulted in an increased expression of cleaved caspase-3 and PARP, both markers of apoptosis. NVP-LDE-225 inhibited neurosphere formation in a dose-dependent manner (Fig. 1E). Similarly, NVP-LDE-225 inhibited cell viability in primary, secondary, and tertiary neurospheres in a dose-dependent manner (Fig. 1F). These data suggest that NVP-LDE-225 inhibits cell viability in neurospheres and neurosphere formation and induces apoptosis in a dose-dependent manner, and thus can be used for the treatment of brain cancer by targeting GICs.

Fig. 1.

Effects of NVP-LDE-225 on apoptosis, caspase-3 and PARP cleavage, spheroid formation, and cell viability in GIC neurospheres. (A) Isolation and characterization of GICs from human primary tumors. GICs were isolated using anti-CD133 antibody by flow cytometry. CD133⁺ GICs also express Abcg2. (B) GICs were treated with NVP-LDE-225 (0, 1, 5, and 10 µM) for 48 h. At the end of incubation period, apoptosis was measured by annexin/PI staining. Data are representative of 3 independent experiments. LDE-225 = NVP-LDE-225. (C) GICs were treated with NVP-LDE-225 (0, 1, 5, and 10 µM) for 48 h. At the end of incubation period, apoptosis was measured by PI staining. Data are representative of 3 independent experiments. (D) Expression of cleaved caspase-3 and PARP. GICs were treated with NVP-LDE-225 for 48 h, and the expression of cleaved caspase-3 and PARP was determined by Western blot analysis. (E) Effects of NVP-LDE-225 on neurosphere formation. GICs were seeded in suspension and treated with NVP-LDE-225 (0–10 µM) for 7 days. At the end of incubation period, neurospheres were photographed. Data are representative of 3 independent experiments. (F) Cell viability in neurospheres. GICs were seeded in suspension and treated with NVP-LDE-225 (0–10 µM) for 7 days. At the end of incubation period, neurospheres were collected, and dissociated with Accutase (Innovative Cell Technologies). For secondary and tertiary neurospheres, cells were reseeded and treated with NVP-LDE-225 for an additional 7 days. Cell viability was measured by trypan blue assay. Data represent mean ± SD. Data are representative of 3 independent experiments. *, #, %, &, and ^ indicate significant difference from control, P < .05. Abbreviations: APC, allophycocyanin; PE, phycoerythrin.

NVP-LDE-225 Inhibits the Components of the SHH Pathway, Gli Transcriptional Activity, and Gli Nuclear Translocation in Brain GICs

Since NVP-LDE-225 inhibited cell viability and induced apoptosis in brain GICs, we next sought to examine its effects on various components of the SHH pathway in brain GICs by quantitative real-time (qRT) PCR analysis (Fig. 2A). NVP-LDE-225 inhibited the expressions of effectors (Gli1 and Gli2) and receptors (Patched1 and Patched2) of the SHH pathway in brain GICs (Fig. 2A). We next confirmed the effects of LDE-225 on the expression of Gli1, Gli2, Patched1, and Patched2 by Western blot analysis. As shown in Fig. 2B, LDE-225 inhibited the expression of Gli1, Gli2, Patched1, and Patched2 in GICs. These data suggest that NVP-LDE-225 can regulate GIC characteristics by inhibiting the various components of the SHH pathway.

Fig. 2.

NVP-LDE-225 downregulates the SHH signaling pathway in GICs. (A) GICs were treated with NVP-LDE-225 (10 μM) for 36 h. At the end of incubation period, RNA was extracted and expressions of Gli1, Gli2, Patched1 (Ptch1), and Patched2 (Ptch2) were measured by qRT-PCR. Data represent mean ± SD (n = 4). * = significant difference from control (P < .05). (B) Protein expressions of Gli1, Gli2, Patched1, and Patched2. GICs were treated with NVP-LDE-225 for 48 h, and expressions of Gli1, Gli2, Ptch1, and Ptch2 were determined by Western blot analysis. (C) GICs were treated with LDE-225 (0–10 μM) for 24 h. Nuclear extracts were prepared and electrophoretic mobility shift assay was performed as described in Materials and Methods. Lane 1 = control (without LDE-225); lanes 2, 3, and 4 = LDE-225–treated samples (1, 5, and 10 µM, respectively). (D) NVP-LDE-225 inhibits Gli transcriptional activity. GICs were transduced with lentiviral particles expressing Gli-dependent luciferase reporter and were treated with NVP-LDE-225 (0, 5, and 10 µM) for 24 h. Lysates were prepared, and luciferase activity was measured as described in Materials and Methods. Normalized luciferase activity is presented as mean ± SD. * = significant difference from control (P < .05). (E) NVP-LDE-225 inhibits expression of Gli1 and Gli2 in GICs. The cells were seeded on fibronectin-coated coverslips and treated with NVP-LDE-225 (5 µM) for 48 h. Subsequently, cells were fixed with 4% paraformaldehyde, blocked in 5% normal goat serum, and stained with Gli1 and Gli2 primary antibodies (1:100) for 16 h at 4°C and washed with PBS. Afterward, cells were incubated with fluorescently labeled secondary antibody (1:200) along with DAPI (1 mg/mL) for 1 h at room temperature and cells were mounted and visualized under a fluorescent microscope. The color of DAPI was shifted to red to enhance the red and green overlap to produce yellow.

We next examined the effects of LDE-225 on Gli–DNA interaction by electrophoretic mobility shift assay in GICs (Fig. 2C). Treatment of GICs with LDE-225 (0–10 µM) at 24 h resulted in decreased Gli–DNA binding activity. Since LDE-225 inhibited Gli–DNA binding, we went on to examine the effects of NVP-LDE-225 on Gli transcriptional activity. Brain GICs were transduced with a Gli-dependent luciferase reporter construct and treated with NVP-LDE-225 for 24 h (Fig. 2D). NVP-LDE-225 inhibited Gli-dependent luciferase reporter activity in a dose-dependent manner. Hence, these data suggest that inhibition of the SHH pathway by NVP-LDE-225 can reduce transcriptional activity of the Gli target gene.

We next employed an immunofluorescence technique to examine the effect of NVP-LDE-225 on the expression of Gli1 and Gli2 to nuclei (Fig. 2E). Brain GICs were treated with NVP-LDE-225, and the expression/translocation of Gli1 and Gli2 was observed under a fluorescent microscope. NVP-LDE-225 inhibited expression/translocation of Gli1 and Gli2 to the nuclei. Overall, these results suggest that NVP-LDE-225 can inhibit components of the SHH pathway, Gli–DNA binding and transcriptional activities, and Gli nuclear translocation.

NVP-LDE-225 Inhibits the Expression of Genes Involved in Maintaining Pluripotency

Since NVP-LDE-225 inhibited the SHH pathway, we next examined the expression of genes that play roles in maintaining pluripotency. Brain GICs were exposed to NVP-LDE-225 (1, 5, and 10 µM) for 24 h, and the expression levels of cMyc, Nanog, Sox2, and Oct4 were measured by qRT-PCR. NVP-LDE-225 inhibited the expressions of cMyc, Nanog, Sox2, and Oct4 in brain GICs in a dose-dependent manner (Fig. 3A). We confirmed the effects of NVP-LDE-225 on the expressions of cMyc, Nanog, Sox2, and Oct4 in neurospheres by immunocytochemistry. As shown in Fig. 2B, NVP-LDE-225 inhibited the expressions of cMyc, Nanog, Sox2, and Oct4 in brain GIC' neurospheres. These data suggest that inhibition of the SHH pathway can suppress the self-renewal capacity of GICs by inhibiting the factors required for maintaining pluripotency.

Fig. 3.

NVP-LDE-225 differentially regulates genes involved in self-renewal and pluripotency. (A) GICs were treated with NVP-LDE-225 (0–10 µM) for 36 h, and expressions of cMyc, Sox2, Nanog, and Oct4 were measured by qRT-PCR. HK-GAPD was used as the endogenous normalization control. Data represent mean ± SD. * = significant difference from control, P < .05. (B) Immunohistochemical examination of Nanog, cMyc, Sox2, and Oct4. GBM GICs were grown in suspension and treated with NVP-LDE-225 (5 µM) for 48 h. At end of incubation period, neurospheres were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked in 5% normal goat serum. After blocking, neurospheres were stained with primary antibody (1:100) overnight at 4°C, washed twice with PBS, and incubated with fluorescently labeled secondary antibody (1:200) along with DAPI (1 mg/mL) for 1 h at room temperature. Finally, neurospheres were mounted and visualized under a fluorescent microscope. (C) Top left, GICs were treated with NVP-LDE-225 (0–10 µM) for 36 h, and expression of Bmi1 was measured by qRT-PCR. Data represent mean ± SD. * = significant difference from control, P < .05. Top right, GICs were treated with NVP-LDE-225 (0–10 µM) for 36 h, and expression of miR-128 was measured by qRT-PCR. Data represent mean ± SD. * = significant difference from control, P < .05. Bottom left, GICs were transduced with either control or anti–miR-128 viral particles. Expression of miR-128 was measured by qRT-PCR. Data represent mean ± SD. * = significant difference from control, P < .05. Bottom right, GICs were transduced with either control (scrambled) or anti–miR-128 viral particles and treated with NVP-LDE-225 (0–10 µM) for 36 h. At end of incubation period, the expression of Bmi1 was measured by qRT-PCR. Data represent mean ± SD. * = significant difference from control, P < .05.

The polycomb group gene Bmi1 is overexpressed in glioblastoma GICs and pediatric medulloblastoma.^6,48 The downregulation of Bmi1 resulted in inhibition of clonogenic ability in vitro and brain tumor formation in vivo.⁴⁹ Bmi1 is required for spontaneous de novo development of the brain tumor and is considered a key factor required for SHH pathway–driven tumorigenesis.⁶ We therefore examined whether NVP-LDE-225 regulates the expression of Bmi1 in brain GICs. As shown in Fig. 3C, LDE-225 inhibited the expression of Bmi1 in a dose-dependent manner, suggesting the requirement of Bmi1 for cell survival.

We next examined whether miR-128 mediates the inhibitory effects of NVP-LDE-225 on Bmi1 expression in GICs. NVP-LDE-225 induced the expression of miR-128 in GICs (Fig. 3C). Transduction of GICs with anti–miR-128 inhibited the expression of miR-128. Furthermore, transduction of GICs with anti–miR-128 blocked the inhibitory effects of NVP-LDE-225 on Bmi1 expression. These data suggest that NVP-LDE-225 inhibits Bmi1 expression by inducing the expression of miR-128.

NVP-LDE-225 Differentially Regulates Genes Involved in Cell Survival and Cell Death

Since NVP-LDE-225 inhibited the SHH pathway, we next examined the expression of genes that play roles in cell survival and apoptosis. Brain GICs were exposed to NVP-LDE-225 (1, 5, and 10 µM) for 24 h, and the expression levels of Bcl2 family members, inhibitors of apoptosis (IAps), PDGFRα, and programmed cell death 4 (PdCD4) were measured by qRT-PCR. Treatment of brain GICs with NVP-LDE-225 increased the expressions of Bak and Bax (Fig. 4A). NVP-LDE-225 inhibited the expressions of Bcl2, Bcl-X_L, X-linked IAp, survivin, cellular (c)IAp1, and cIAp2 (Fig. 4B). Expression of PDGFRα was decreased and that of PdCD4 was increased following NVP-LDE-225 treatment (Fig. 4C and D).

Fig. 4.

NVP-LDE-225 regulates genes involved in apoptosis and cell survival. (A) GICs were treated with NVP-LDE-225 (0–10 µM) for 36 h, and expressions of Bak, Bax, Bcl2 and Bcl-X_L were measured by qRT-PCR. HK-GAPD was used as the endogenous normalization control. Data represent mean ± SD. * = significant difference from control, P < .05. (B) GICs were treated with NVP-LDE-225 (0–10 µM) for 36 h, and expressions of X-linked IAp, survivin, cIAp1, and cIAp2 were measured by qRT-PCR. Data represent mean ± SD. * = significant difference from control, P < .05. (C) GICs were treated with NVP-LDE-225 (0–10 µM) for 36 h, and expression of PDGFRα was measured by qRT-PCR. Data represent mean ± SD. * = significant difference from control, P < .05. (D) GICs were treated with NVP-LDE-225 (0–10 µM) for 36 h, and expression of PdCD4 was measured by qRT-PCR. Data represent mean ± SD. * = significant difference from control, P < .05. (E) GICs were treated with NVP-LDE-225 (0–10 µM) for 36 h, and expression of miR-21 was measured by qRT-PCR. Data represent mean ± SD. * = significant difference from control, P < .05. (F) NVP-LDE-225 induces PdCD4 and apoptosis and inhibits cell viability by suppressing miR-21. GICs were transduced with control (scrambled) or pre–miR-21 viral particles and treated with NVP-LDE-225 (0–10 µM) for 36 h. The expression of miR-21 and PdCD4 was measured by qRT-PCR. Data represent mean ± SD. * = significant difference from control, P < .05. Similarly, GICs were transduced with control (scrambled) or pre–miR-21 viral particles and treated with NVP-LDE-225 (0–10 µM) for 48 h. Cell viability and apoptosis were measured by XTT and assay by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), respectively. Data represent mean ± SD. * = significant difference from control, P < .05.

It has been shown that miR-21 is expressed at higher levels in primary glioblastoma-tissue and glioblastoma-derived cell lines than in normal brain tissue.⁵⁰ Since NVP-LDE-225 induced the expression of PdCD4, we hypothesized that this expression was regulated by miR-21. As shown in Fig. 4E, NVP-LDE-225 inhibited the expression of miR-21. We next assessed whether miR-21 mediated the biological functions of NVP-LDE-225 (Fig. 4F). Overexpression of pre–miR-21 enhanced the expression of miR-21 in GICs. Furthermore, overexpression of pre–miR-21 suppressed NVP-LDE-225–induced expression of PdCD4. Interestingly, overexpression of pre–miR-21 suppressed the inhibitory effects of NVP-LDE-225 on cell proliferation and stimulatory effects on apoptosis. Our data indicate that miR-21 mediates the effects of NVP-LDE-225 on the expression of PdCD4, which may regulate cell proliferation and apoptosis in brain GICs.

NVP-LDE-225 Sensitizes Brain GICs to FasL and TRAIL by Upregulating Fas and TRAIL-R1/DR4 and TRAIL-R2/DR5 Receptors, Respectively

Inhibition of the SHH pathway has been shown to upregulate the expressions of Fas, TRAIL-R1/DR4, and TRAIL-R2/DR5 in GICs.³² Fas and TRAIL have been shown to induce apoptosis in GBM with varying sensitivity.^51–55 We therefore first measured the expression levels of Fas, DR4, and DR5 in brain GICs treated with NVP-LDE-225. As shown in Fig. 5A, NVP-LDE-225 upregulated the expressions of Fas, DR4, and DR5 in brain GICs. We next examined the preclinical significance of upregulating Fas and DRs by treating brain GICs with the combination of NVP-LDE-225 with FasL or TRAIL. As shown, treatment of brain GICs with NVP-LDE-225 resulted in apoptosis. By comparison, FasL and TRAIL alone had no significant effect on apoptosis in brain GICs. Interestingly, pretreatment of GICs with NVP-LDE-225 sensitized GICs that were resistant to FasL or TRAIL alone. These data suggest that NVP-LDE-225 may be combined with FasL or TRAIL to obtain maximum therapeutic benefits in brain cancer.

Fig. 5.

NVP-LDE-225 enhances the apoptosis-inducing potential of FasL and TRAIL. (A) GICs were incubated with NVP-LDE-225 (0, 1, 5, and 10 µM) for 36 h. Surface expressions of Fas, DR4, and DR5 were measured by flow cytometry. Immunoglobulin (Ig)G was used as an isotype control. (B) GICs were pretreated with NVP-LDE-225 (5 µM) for 24 h, followed by treatment with FasL (100 nM) for another 24 h, and apoptosis was measured by TUNEL assay. (C) GICs were pretreated with NVP-LDE-225 (5 µM) for 36 h, followed by treatment with TRAIL (100 nM) for another 24 h, and apoptosis was measured by TUNEL assay.

Regulation of Downstream Targets of Gli by NVP-LDE-225

In order to examine the effects of Gli1 and Gli2 on downstream targets, we inhibited the expression of Gli1 and Gli2 by short hairpin (sh)RNA. As shown in Fig. 6A, lentiviral mediated expression of Gli1 and Gli2 shRNA inhibited the expression of Gli1 and Gli2 proteins in GICs. To genetically determine the effects of Gli1 plus Gli2, transduced GICs were treated with NVP-LDE-225 for 48 h, and Western blot analysis was performed to determine the expression of genes involved in the regulation of cell death (Fas, DR4/TRAIL-R1, and DR5/TRAIL-R2) and cell survival (PDGFRα and Bcl2). NVP-LDE-225 induced the expressions of Fas, DR4, and DR5, and inhibited the expressions of Bcl2 and PDGFRα in cancer stem cell/scrambled cells (Fig. 6B). By comparison, LDE-225 had no significant effects on expression of these downstream targets of the SHH signaling pathway in Gli1 shRNA + Gli2 shRNA GICs, supporting Gli-mediated cytotoxic effects of NVP-LDE-225 in GICs.

Fig. 6.

Regulation of downstream targets of the SHH pathway. (A) Knockout of Gli1 plus Gli2 by shRNA in GICs. GICs were transduced with lentiviral particles expressing either scrambled or Gli1 plus Gli2 shRNA. Western blot analysis was performed to measure expressions of Gli1 and Gli2. β-actin was used as the loading control. (B) Transduced GICs were treated with NVP-LDE-225 (0 and 10 µM) for 48 h, and lysates were extracted to determine expressions of Fas, TRAIL-R1/DR4, TRAIL-R2/DR5, Bcl2, and PDGFRα by Western blot analysis. β-actin was used as the loading control.

NVP-LDE-225 Inhibits Motility, Invasion, and Migration of GICs

During cancer metastasis, the mobility and invasiveness of cancer cells increase.³⁴ To detach from neighboring cells and invade adjacent cell layers, carcinoma cells must lose cell–cell adhesion and acquire motility. The highly conserved EMT program has been implicated in dissemination of carcinoma cells from primary epithelial tumors.³⁴ Tumor progression is frequently associated with the downregulation of E-cadherin³⁴ and the upregulation of vimentin and several transcription factors, including Snail, Zeb1, and Slug.⁵⁶ GICs undergoing metastasis usually express low levels of epithelial markers and high levels of mesenchymal markers. Since GICs appear to play a significant role in early metastasis,⁵⁷ we sought to measure the effects of NVP-LDE-225 on motility, migration, and invasion of GICs (Fig. 7A and B). NVP-LDE-225 inhibited motility, migration, and invasion of brain GICs. These data suggest that NVP-LDE-225 can inhibit early metastasis of brain GICs. NVP-LDE-225–treated GICs changed their morphology from mesenchymal to epithelial, a feature similar to reversal of EMT (data not shown).

Fig. 7.

Regulation of cell motility, migration, invasion, and expression of EMT factors by NVP-LDE-225 in GICs. (A) Motility assay; photomicrographs demonstrating the results of the in vitro motility of GICs using the simple scratch technique. GICs were grown in monolayers, scratched, and treated with or without NVP-LDE-225 for 24 or 48 h. Data are representative of 3 independent experiments. (B) Transwell migration assay (left panel), GICs were plated in the top chamber of the transwell and treated with NVP-LDE-225 (0–10 µM) for 24 h. Cells migrated to the lower chamber were fixed with methanol, stained with crystal violet, and counted. Data represent mean ± SD. * = significant difference from respective controls, P < .05. Matrigel invasion assay (right panel), GICs were plated onto the Matrigel-coated membrane in the top chamber of the transwell and treated with NVP-LDE-225 (0–10 µM) for 24 h. Cells invaded to the lower chamber were fixed with methanol, stained with crystal violet, and counted. Data represent mean ± SD. * = significant difference from respective controls, P < .05. (C), Left panel, GICs were treated with NVP-LDE-225 (0–10 µM) for 36 h. At end of incubation period, expressions of Snail, Zeb1, and Slug were measured by qRT-PCR. Data represent mean ± SD. * = significant difference from respective controls, P < .05. Middle and right panels, GICs were treated with NVP-LDE-225 (0–10 µM) for 36 h. At end of incubation period, expressions of E-cadherin and N-cadherin were measured by qRT-PCR. Data represent mean ± SD. * = significant difference from respective controls, P < .05. (D) NVP-LDE-225 inhibits migration and invasion by upregulating miR-200a, miR-200b, and miR-200c. Left panel, GICs were treated with NVP-LDE-225 (5 µM) for 36 h, and expressions of miR-200a, miR-200b, and miR-200c were measured by qRT-PCR. Data represent mean ± SD. * = significant difference from control, P < .05. Middle panel, NVP-LDE-225 inhibits cell migration by upregulating miR-200a/b/c. GICs were transduced with control (scrambled) or anti–miR-200a/b/c viral particles and treated with NVP-LDE-225 (0–10 µM) for 24 h. Cell migration was measured as previously described. Data represent mean ± SD. * = significant difference from control, P < .05. Right panel, NVP-LDE-225 inhibits cell invasion by upregulating miR-200a/b/c. GICs were transduced with control (scrambled) or anti–miR-200a/b/c viral particles and treated with NVP-LDE-225 (0–10 µM) for 24 h. Cell invasion was measured as previously described. Data represent mean ± SD. * = significant difference from control, P < .05.

Since NVP-LDE-225 inhibited EMT, we next examined the regulation of EMT-inducing transcription factors Snail, Slug, and Zeb1 (Fig. 7C, left panel). NVP-LDE-225 inhibited the expression of Snail, Slug, and Zeb1 as measured by qRT-PCR. The expression of cadherins has been demonstrated to change during EMT.³² We therefore examined the effects of NVP-LDE-225 on the expression of E-cadherin and N-cadherin (Fig. 7C, middle and right panels). NVP-LDE-225 enhanced the expression of E-cadherin and inhibited the expression of N-cadherin, a phenomenon known as cadherin switch during EMT. These data suggest that NVP-LDE-225 can regulate early metastasis by modulating the expression of cadherins and EMT transcription factors.

EMT and MET represent a mechanistic basis for epithelial cell plasticity implicated in cancer.³⁴ Transcription factors of the Zeb protein family and several miR species (predominantly miR-200 family members) form a double negative feedback loop, which controls EMT and MET programs in both development and tumorigenesis. We therefore examined the role of the miR-200 family on the regulation of EMT by SHH pathway inhibition. NVP-LDE-225 induced the expression of miR-200a, miR-200b, and miR-200c in GICs (Fig. 7D, left panel). Transduction of GICs with anti–miR-200a/b/c blocked the inhibitory effects of NVP-LDE-225 on cell migration and invasion (Fig. 7, middle and right panels). These data suggest that NVP-LDE-225 inhibits EMT by upregulating the miR-200 family.

Discussion

Recent evidence suggests that GICs may be important for the initiation, propagation, and recurrence of glioblastoma, and hence glioblastoma GICs are now drawing attention as critical therapeutic targets.⁵⁸ An understanding of the molecular mechanisms involved in the regulation of glioblastoma GICs may thus be clinically significant. Here, we showed that NVP-LDE-225 inhibits GBM GIC characteristics in vitro by suppressing the SHH pathway.

Signaling pathways that control cell differentiation and proliferation, motility, cell survival, and apoptosis during embryonic development have been the subject of intensive studies for developing new therapeutic targets. Aberrant activation of the SHH pathway has been reported in several pediatric neural tumors (neuroblastomas, ependymomas, medulloblastomas, and primitive neuroectodermal tumors and GBM).⁴⁸ Inhibitors of the SHH pathway are therefore attractive targets for these tumors. Clinical trials of SHH-pathway antagonists are already under way in medulloblastoma.^59,60 These antagonists have also been shown to have encouraging, although not sustained, results in relapsed cases.² In malignant gliomas, cyclopamine, which specifically inhibits the membrane protein Smo, has been shown to deplete glioma GICs² and, in combination with temozolomide, has shown the ability to suppress GIC proliferation.⁹ In medulloblastoma, it has been demonstrated that addition of the PI3K inhibitor NVP-BKM120 or the dual PI3K–mTOR inhibitor NVP-BEZ235 to the initial treatment with the Smo antagonist markedly delayed the development of resistance of the SHH pathway.¹¹ In the present study, we found that these GICs consistently express various components of the SHH signaling pathway, including signaling the molecules Gli1, Gli2, Patched1, Patched2, Smo, and SHH, suggesting that the SHH pathway is one of the “core” signaling pathways or an autocrine mode of SHH signaling in these cells. NVP-LDE-225 inhibited EMT, which was associated with inhibition in Snail, Slug, Zeb1, and N-cadherin and with upregulation in E-cadherin.

Growing evidence suggests that GICs have aberrant or constitutively active self-renewal pathways that are controlled by genetic or epigenetic mechanisms and that lead to unrestrained proliferation. The Myc oncoproteins are highly amplified or constitutively expressed in pediatric lymphomas, neuroblastomas, and medulloblastomas. Interestingly, overexpression of cMyc has been correlated with a higher histological grade in gliomas.^61,62 Relative to nonstem glioma cells, cMyc is highly expressed in glioma cancer stem cells⁶² and has been described as highly expressed in glioma GICs relative to the bulk of tumor cells, thereby suggesting a role in GIC proliferation and survival. Knockdown of cMyc using shRNAs showed reduced glioma GIC proliferation, increased apoptosis, and cell cycle arrest in the G₀/G₁ phase. Moreover, downregulation of cMyc in the GIC population resulted in the inability to form neurospheres or tumors in vivo.⁶² In the present study, NVP-LDE-225 inhibited the expressions of Oct4, Nanog, Sox2, and cMyc. These factors have been shown to be required for maintaining pluripotency in normal and tumor-initiating cells.

MiR-21 is expressed at higher levels in primary glioblastoma-tissue and glioblastoma-derived cell lines than in normal brain tissue. The downregulation of miR-21 in glioblastoma-derived cell lines results in increased expression of its target, PdCD4, a known tumor suppressor gene.⁵⁰ Furthermore, either downregulation of miR-21 or overexpression of its target, PdCD4, in glioblastoma-derived cell lines leads to decreased proliferation, increased apoptosis, and decreased colony formation in soft agar. Using a glioblastoma xenograft model in immune-deficient nude mice, in vivo downregulation of miR-21 levels or overexpression of PdCD4 resulted in decreased tumor formation and growth.⁵⁰ In the present study, miR-21 mediates the effects of NVP-LDE-225 on the expression of PdCD4, which may regulate cell proliferation and apoptosis in brain GICs. These findings demonstrate an important functional linkage between miR-21 and PdCD4 and further elucidate the molecular mechanisms by which the known high level of miR-21 expression in glioblastoma can attribute to tumorigenesis, that is, through inhibition of PDCD4 and its tumor-suppressive functions.

Polycomb group proteins regulate gene expression through modifications in chromatin structure. The polycomb group gene Bmi1 plays a role in proliferation of cerebellar precursor cells and was shown to be overexpressed in pediatric medulloblastoma.^6,48 More recently, Bmi1 was found to be highly enriched in glioblastoma GICs, and its downregulation resulted in inhibition of clonogenic ability in vitro and brain tumor formation in vivo.⁴⁹ Bmi1 is required for spontaneous de novo development of a solid tumor arising in the brain, suggesting a crucial role for Bmi1-dependent, nestin-expressing progenitor cells in medulloblastoma expansion and implicating Bmi1 as a key factor required for SHH pathway–driven tumorigenesis.⁶ In our study, NVP-LDE-225 inhibited the expression of Bmi1, which may contribute to self-renewal capacity of brain GICs. Furthermore, the inhibitory effects of LDE-225 on Bmi1 were exerted through upregulation of miR-128. In another study, using a panel of patient glioblastoma specimens, the upregulation of Bmi1 expression and downregulation of miR-128 compared with normal brain were demonstrated.⁴⁷ Bmi1 functions in epigenetic silencing of certain genes through epigenetic chromatin modification. In the same study, miR-128 expression caused a decrease in histone methylation (H3K27me(3)) and Akt phosphorylation and upregulation of p21/Cip1 levels, consistent with Bmi1 downregulation.⁴⁷

The SHH pathway regulates components of both cell-intrinsic and cell-extrinsic pathways of apoptosis. We have shown that NVP-LDE-225 inhibited prosurvival proteins Bcl2 and Bcl-X_L and pro-apoptotic proteins Bak and Bax in brain GICs. Furthermore, NVP-LDE-225 inhibited the expression of X-linked IAp, survivin, cIAp1, and cIAp2. Thus, our data demonstrate that inhibition of the SHH pathway by NVP-LDE-225 can regulate multiple genes involved in cell-intrinsic and cell-extrinsic pathways.

The components of the death receptor pathway are expressed in GBM. NVP-LDE-225 induced the expressions of Fas, DR4, and DR5, suggesting that the activation of these receptors with their respective ligands may induce apoptosis in brain GICs. Although neither Fas nor TRAIL alone was effective in inducing apoptosis in brain GICs, NVP-LDE-225 sensitized these GICs to ligand Fas and TRAIL. The inhibition of the SHH pathway by NVP-LDE-225 has significant clinical implications. Specifically, the combination of NVP-LDE-225 with Fas or TRAIL can be used for the management of GBM by targeting GICs.

EMT during embryogenesis, adult tissue homeostasis, and carcinogenesis is characterized by a class switch from E-cadherin to N-cadherin. Accumulating evidence suggests that EMT plays an important role during malignant tumor progression. During EMT, transformed epithelial cells can activate embryonic programs of epithelial plasticity and switch from a sessile, epithelial phenotype to a motile, mesenchymal phenotype. Induction of EMT can, therefore, lead to invasion of surrounding stroma, intravasation, dissemination, and colonization of distant sites. It is now clear that sustained metastatic growth requires the dissemination of GICs from the primary tumor, followed by their reestablishment in a secondary site. Thus, EMT can confer metastatic ability on carcinomas. Snai1 (Snail), Snai2 (Slug), Snai3, Zeb1, Zeb2 (Sip1), KLF (Kruppel-like factor)8, Twist1, and Twist2 are EMT regulators repressing the Cdh1 gene encoding E-cadherin. Hedgehog signals induce Jag2 upregulation for Notch-CSL (CBF [C promoter binding factor]1 suppressor of hairless, Lag-1)-mediated Snai1 upregulation and also induce transforming growth factor (TGF)β1 secretion for Zeb1 and Zeb2 upregulation via TGFβ receptor and nuclear factor–kappa B. Hedgehog signaling activation indirectly leads to EMT through fibroblast growth factor, Notch, TGFβ signaling cascades, and miR regulatory networks. Our results indicate a key and essential role of the SHH-Gli pathway in promoting brain GIC tumor growth, stem cell self-renewal, and metastatic behavior. NVP-LDE-225 inhibited EMT, as demonstrated by inhibition in cell motility, invasion, and migration. The inhibition of EMT was associated with suppression of EMT transcription factors (Zeb1, Snail, and Slug) and cadherin switch (upregulation of E-cadherin and downregulation of N-cadherin) in GICs, suggesting a potential role of NVP-LDE-225 in early metastasis. Targeting Gli1/2 is thus predicted to decrease tumor bulk and eradicate GICs and metastases.

In conclusion, we showed that a novel system operates in GBM and also demonstrated an undocumented role for the SHH pathway in GICs. The inhibition of Smo function by NVP-LDE-225 resulted in regulation of GIC proliferation, EMT, and apoptosis. Furthermore, NVP-LDE-225 inhibited GIC characteristics that were associated with inhibition of Gli1 and Gli2 and regulation of Bcl2 family members, IAps, and induction of death receptors (Fas, DR4, and DR5). In addition, the inhibition of EMT by NVP-LDE-225 was regulated by induction of the miR-200 family. Overall, our findings suggest that inhibition of the SHH signaling pathway is a potential therapeutic strategy for glioblastoma, and the combination of NVP-LDE-225 with FasL or TRAIL can sensitize GICs that are resistant to death receptor agonists.

Funding

This work was supported in part by grants from the National Institutes of Health (R01CA125262, RO1CA114469, and RO1CA125262-02S1) and the Kansas Bioscience Authority.