Hedgehog (HH) signaling is an important regulator of embryogenesis that has been associated with the development of several types of cancer. HH signaling is characterized by Smoothened (SMO)-dependent activation of the GLI transcription factors, which regulate the expression of critical developmental genes. Neuroblastoma, an embryonal tumor of the sympathetic nervous system, was recently shown to express high levels of key molecules in this signaling cascade. Using compounds blocking SMO (cyclopamine and SANT1) or GLI1/GLI2 (GANT61) activity revealed that inhibition of HH signaling at the level of GLI was most effective in reducing neuroblastoma growth. GANT61 sensitivity positively correlated to GLI1 and negatively to MYCN expression in the neuroblastoma cell lines tested. GANT61 downregulated GLI1, c-MYC, MYCN and Cyclin D1 expression and induced apoptosis of neuroblastoma cells. The effects produced by GANT61 were mimicked by GLI knockdown but not by SMO knockdown. Furthermore, GANT61 enhanced the effects of chemotherapeutic drugs used in the treatment of neuroblastoma in an additive or synergistic manner and reduced the growth of established neuroblastoma xenografts in nude mice. Taken together this study suggests that inhibition of HH signaling is a highly relevant therapeutic target for high-risk neuroblastoma lacking MYCN amplification and should be considered for clinical testing.
Neuroblastoma is the most common and deadly extracranial tumor of childhood where new treatment options, especially for children with high-risk disease, are greatly needed. Neuroblastomas are most probably derived from precursor cells residing in the neural crest, a transient structure arising from the dorsal region of the closing neural tube during embryogenesis.1–3
The Hedgehog (HH) signaling pathway is fundamental for numerous processes during embryonic development including the regulation of proliferation, migration and differentiation of cells in the developing neural crest.4, 5 Activation of the canonical HH signaling cascade is initiated by the binding of HH ligands (Sonic, Indian or Desert Hedgehog) to the Patched 1 (PTCH1) receptor, thereby relieving PTCH1 from inhibiting the signaling protein Smoothened (SMO). SMO signaling triggers a cascade of events that eventually results in the activation of the glioma-associated oncogene (GLI) transcription factors, GLI1 and GLI2, and degradation of the repressor forms of GLI (primarily GLI3). Activation of the GLI proteins stimulates the transcription of HH pathway target genes, including GLI1, which further amplifies the initial HH signal, GLI2 and PTCH1.6 HH signaling also induces the expression of cell cycle genes including Cyclin D1, c-MYC and MYCN.7
Aberrant activation of HH signaling is involved in several malignancies, including basal cell carcinoma, medulloblastoma, rhabdomyosarcoma and colon cancer. The HH signal activity in tumors can be a consequence of mutations in components of the pathway, resulting in a constitutively active signaling.6–8 Moreover, cancers without genetic aberrations may still rely on HH signaling for disease progression.6, 7 Evidence for a crucial role of the HH pathway in putative stem cells of these cancers has been reported.6, 8, 9 These findings highlight that HH signaling is an attractive target for cancer therapy.
The first known inhibitor of the HH pathway, cyclopamine, a teratogenic alkaloid, binds and inhibits SMO.10 Since cyclopamine and other natural SMO inhibitors are of low affinity, have poor oral bioavailability and suboptimal pharmacokinetics, more potent and/or soluble SMO inhibitors have been developed, e.g. SANT1.6, 11, 12 Several small molecule antagonists targeting SMO have currently entered clinical trials in both adult and pediatric cancer.8 GANT61 was recently identified as a small molecule antagonist of GLI in a cell-based screening.13 GANT61 was shown to reduce the transcriptional activity of GLI1 and GLI2 and interfere with GLI1 DNA binding in the nucleus. In contrast to SMO inhibitors, GLI inhibitors suppress the activity of critical components further downstream in the pathway and, thus, have the advantage of functioning irrespective of the mode of HH signaling activation.13 GANT61 is spontaneously and rapidly metabolized to the GANT61-diamine, which inhibits GLI1 and GLI2 to the same extent.14
Recent studies demonstrate that primary neuroblastoma and cell lines express high levels of proteins involved in HH signaling.15, 16 Additionally, activation of the HH pathway was found to induce the expression of MYCN through both transcriptional and protein stabilization mechanisms.4, 7, 17 MYCN is widely expressed in the developing neural crest, where it orchestrates proliferation and differentiation of the neuroblasts.18MYCN amplification, which is found in 30–40% of high-risk neuroblastoma, is still the most reliable prognostic factor for aggressive neuroblastoma, although recent data have shown that high-risk patients without MYCN amplification have similar mortality rates.19
On the basis of these findings, we evaluated the effects of targeting HH signaling at the level of GLI1 in neuroblastoma. Moreover, we dissected the role of MYCN amplification in relation to aberrant HH signaling and provide evidence that specific inhibition of this pathway at the level of GLI may be a novel treatment option for children with high-risk neuroblastoma lacking MYCN amplification.
Seven neuroblastoma cell lines, with different stage and MYCN genetic characteristics, were grown and maintained as described.20 The identities of the cell lines were verified by short tandem repeat genetic profiling using the AmpFlSTR® IdentifilerTM PCR Amplification Kit (Applied Biosystems) in September 2010 and all cell lines were used in passages below 25. All experiments were executed in Opti-MEM (GIBCO) supplemented with glutamine, streptomycin and penicillin (HyClone ThermoScientific), except transfection experiments, which were performed without antibiotics.
Cyclopamine was purchased from LC Laboratories, SANT1 from Sigma-Aldrich, and GANT61 was a kind gift from Professor Rune Toftgård, Karolinska Institutet, Sweden. The drugs were dissolved in DMSO (Sigma-Aldrich) and further diluted with Opti-MEM or PBS. The DMSO concentration did not exceed 1% v/v in any experiment. For the in vivo studies, GANT61 was dissolved in ethanol and further diluted in glucose solution (50 mg/mL; Apoteket AB). Cisplatin, doxorubicin, irinotecan and vincristine (Apoteket AB) were dissolved according to guidelines from the manufacturer and further diluted in PBS.
Cell survival analysis
For cytotoxic evaluation, we used the fluorometric microculture cytotoxicity assay (FMCA), described in detail previously.21 Cells were seeded into drug-prepared 96- or 384-well microplates (0.055 × 106 cells/mL, except SK-N-BE(2): 0.028 × 106 cells/mL) and incubated for 72 hr. The cells were washed, fluorescein diacetate was added and after 40 min incubation, fluorescence was measured. Cell survival is presented as survival index (SI, %).
The effects of GANT61 in combination with chemotherapeutics were investigated using FMCA. The studies were designed as suggested in the CalcuSyn software manual, using a fixed ratio of the drugs across a concentration gradient.
To determine colony formation 200 cells/well were seeded in 6-well plates (Sarstedt), allowed to attach before drug exposure for 48 hr. After 8–12 days of incubation in drug-free medium, cells were washed, fixed, stained with Giemsa (Sigma-Aldrich) and colonies (>100 cells) with 50% plate efficiency were counted.22
Luciferase reporter assay
Cells were seeded in 24-well plates, left to attach and transfected with GLI reporter plasmid 12xGLIBS-Luc (400 ng)23 together with Renilla-Luc plasmid (40 ng) using Lipofectamine 2000 (Invitrogen). Twenty-four hr after transfection cells were drug treated. A Dual Luciferase Assay Kit (Promega) and a luminometer (Perklin Elmer) were used to measure luminescence. The values were normalized to the Renilla reporter before calculating relative levels.
Total RNA was prepared from cells with the RNeasy kit (Qiagen) followed by cDNA synthesis with the High capacity RNA-to-cDNA kit (Applied Biosystems). PCR was performed with Power SYBR Green (Applied Biosystems) on a 7500 Real-Time PCR system (Applied Biosystems) with primers (New England Biolabs, Supporting Information Table S1). Relative expression was calculated with the 2−ΔΔCt method.
Protein extraction and western blotting were as earlier described.20 Membranes (PVDF, Millipore) were incubated with antibodies against cleaved Caspase-3 (1:1,000), PARP (1:1,000), Cyclin D1 (1:1,000), c-MYC (1:1,000), MYCN (1:500), β-actin (1:5,000) (all from Cell Signaling) and GLI1 (1:1,000) (Abcam). Anti-rabbit IgG, conjugated with horseradish peroxidase (Cell Signaling) was used for secondary detection and Pierce Super Signal (Pierce) for chemiluminescent visualization.
Cell cycle analyses
Cells were seeded in flasks, left to attach and drug treated. Cells were pulsed with Bromodeoxyuridine (BrdU 10 μM, 15 min), stained with 40,6-diamidino-2-phenylindole (DAPI) and Anti-BrdU monoclonal, phycoerythrin labeled antibody (Biosite) and subjected to cell cycle sorting using BD LSR II flow cytometry and analyses with the FACS Diva software (BD Biosciences).
Small interfering RNA (siRNA) analysis
SK-N-AS cells were seeded in 24-well dishes, left to attach and transfected using Lipofectamine 2000 with 20 pmol predesigned siRNAs targeting human SMO, GLI1, GLI2 and GLI3 (SiGenome SMART pools, Dharmacon) and nontargeting siRNA (fluorescent and nonfluorescent, Cell Signaling) as controls. After 48 hr, cell numbers were counted and the effectiveness of gene knockdowns was investigated by real-time RT-PCR.
In vivo xenograft studies
Immunodeficient nude mice (female 5- to 6-week old Sca:NMRI-nu/nu, Scanbur) were used for xenograft studies. Animals were maintained at a maximum of five per cage and given sterile water and food ad libitum.
Each mouse was injected subcutaneously on the flank with 20 × 106 cells of SK-N-AS under general anesthesia. When the tumor volumes were greater than 0.15 mL (mean 0.18 mL), the mice were randomized to receive either GANT61 (50 mg/kg, n = 10) or vehicle (n = 10); treatments were given orally through gastric feeding daily for 12 days. Tumors were measured daily, and tumor volume was calculated as (width)2 × length × 0.44. The animals were monitored for signs of toxicity including weight loss. At Day 12, the animals were sacrificed, tumors were dissected in smaller parts and either frozen or fixed in formaldehyde. Tumor volume index (TVI) was calculated as the measured tumor volume each day divided by the starting volume (Day 0).
All animal experiments were approved by the regional ethics committee for animal research (N304/08), appointed and under the control of the Swedish Board of Agriculture and the Swedish Court. The animal experiments presented herein were in accordance with national regulations (SFS 1988:534, SFS 1988:539 and SFS 1988:541).
The IC50 values (inhibitory concentration 50%) were determined from log concentrations-effect curves in GraphPad Prism (GraphPad Software) using nonlinear regression analysis. Comparison between two groups was made with t-test, and for comparison of three or more groups one-way ANOVA with Bonferroni multiple comparison test was used. Correlations were assessed with Spearman nonparametric test. All tests were two-sided and carried out in GraphPad Prism.
To interpret the drug combination data, the median-effect method of Chou and Talalay24 using the software CalcuSyn (Biosoft) was used. Analyses was done as previously described25 and combination index (CI) at different effect levels were reported. CI at 70% effect was chosen for presentation (IC70). Values of CI equal to 1, less than 0.8 and greater than 1.2 indicate additive, synergistic and antagonistic interactions, respectively. One sample t-test was used to determine if the CI significantly differed from 0.8 to 1.2.
Specific GLI inhibition is more effective against neuroblastoma growth than SMO targeting
The cytotoxicity of the GLI inhibitor GANT61, and the two SMO inhibitors SANT1 and cyclopamine was evaluated on a panel of seven human neuroblastoma cell lines with different MYCN status using FMCA. GANT61 demonstrated the most effective inhibition of cell growth of the three HH signaling inhibitors tested (ANOVA, p < 0.001, Bonferroni multiple comparison test p < 0.001 in all cell lines except IMR-32 where GANT61 vs. SANT1 p < 0.01, Fig. 1a). GANT61 demonstrated concentration-dependent decrease in cell viability after 72 hr of treatment, with IC50 values ranging between 5.82 and 12.4 μM (Fig. 1b). The non-MYCN amplified neuroblastoma cell line SK-N-AS was most sensitive to GANT61 treatment while the MYCN amplified cell lines, SK-N-DZ, IMR-32 and SK-N-BE(2) the least sensitive to GANT61 treatment.
Three cell lines were selected for further studies, the SK-N-AS, SH-SY5Y and SK-N-BE(2), representing lower, medium and higher GANT61 resistance. Clonogenic assay further supported the growth inhibiting effects of GANT61 (Fig. 1c). GANT61 showed a dose-dependent inhibition of colony formation, the IC50 values ranged between 0.14 and 1.5 μM, with the same sensitivity pattern previously shown.
GANT61 potentiates standard neuroblastoma chemotherapeutics in vitro
Cancer treatment often consists of combinations of different drugs that target cell proliferation, and several clinical trials currently evaluate HH signaling inhibitors in combination with chemotherapeutic drugs. The effect of GANT61 in combination with cisplatin, doxorubicin, irinotecan and vincristine was evaluated in vitro on SK-N-AS, SH-SY5Y and SK-N-BE(2) cells. Interestingly, we demonstrated that by adding GANT61 to these standard drugs synergistic or additive effects were induced at IC70 with all tested cell lines (Fig. 1d). Synergism was observed when GANT61 was given in combination with doxorubicin (all three cell lines) and vincristine (two of three cell lines). Combinations with cisplatin and irinotecan were assessed as additive (Fig. 1d).
GANT61 reduces GLI activity and GLI1 mRNA expression correlates with GANT61 sensitivity
We hypothesized that the GANT61 cytotoxicity was dependent on the GLI factors. Thus, to identify the specific GLI molecular target, the log IC50 values were compared with the GLI1, GLI2 and GLI3 mRNAs. The GLI1 mRNA significantly correlated to the log IC50 for GANT61 (Spearman correlation: r = 0.96 and p = 0.0028; Fig. 2a), whereas no correlation was found to either GLI2 or GLI3 expression (GLI2: R2 = 0.083, GLI3 R2 = 0.034, data not shown). Interestingly, we found that the higher the expression of GLI1 in the neuroblastoma cells, the lower the amount of GANT61 was required to inhibit neuroblastoma growth (Fig. 2a).
To confirm these data, the ability of GANT61 to inhibit GLI transcriptional activity was studied using transient transfection with a GLI-dependent luciferase reporter plasmid in SK-N-AS and SK-N-BE(2) cells. The GLI-dependent luciferase activity and, consequently, the transcriptional activation in SK-N-AS, was almost completely suppressed (80%) after 48 hr of 10 μM GANT61 treatment compared with the control (ANOVA, p < 0.001, Fig. 2b). However, in the MYCN amplified cell line SK-N-BE(2) a less profound decrease (36%) of GLI activity was observed (ANOVA, p = 0.022, Fig. 2b).
To further validate these effects on the transcriptional level, cells exposed to GANT61 (10 μM for 48 or 72 hr) were analyzed for GLI1 and GLI2 expression with real-time RT-PCR. In SK-N-AS cells the mRNA levels of both GLI1 and GLI2 were strongly downregulated with approximately 75% decreased expression after 48 hr (ANOVA, GLI1: p < 0.001, GLI2: p = 0.0029, Figs. 2c and 2d). In SH-SY5Y and SK-N-BE(2) cells the expression levels of GLI1 showed no change (Fig. 2c). However, expression levels of GLI2 were suppressed by nearly 50% in SH-SY5Y (ANOVA, p = 0.014, Fig. 2d) whereas no effect was observed in SK-N-BE(2) cells. Additionally, the inhibitory effect of GANT61 on GLI1 expression was further verified in SK-N-AS cells by western blotting (Fig. 2e).
MYCN mRNA levels negatively correlate with GANT61 sensitivity and GANT61 reduces MYCN and c-MYC expression
Given that MYCN amplification has a strong prognostic value in neuroblastoma, we investigated the expression of MYCN in relation to GANT61 sensitivity in the seven neuroblastoma cell lines. We found a negative correlation between the log IC50 values for GANT61 and MYCN gene expression (Spearman correlation: r = −0.89 and p = 0.012, Fig. 3a). Additionally, both MYCN and c-MYC are reported to be regulated by HH signaling. To analyze this in neuroblastoma, cells exposed to GANT61 (10 μM for 48 or 72 hr) were examined with real-time RT-PCR for MYCN expression. In SK-N-AS cells, the MYCN mRNA levels were distinctly reduced (ANOVA, p = 0.0017, Fig. 3b). Also in SH-SY5Y and the MYCN amplified cell line SK-N-BE(2) MYCN mRNA levels were suppressed to some extent (ANOVA, SH-SY5Y: p = 0.0014, SK-N-BE(2): p = 0.0024, Fig. 3b). To validate these results, protein levels of MYCN and c-MYC were examined. We found a reduced expression of c-MYC (in c-MYC positive cells: SK-N-AS and SH-SY5Y), respectively, MYCN [in MYCN amplified cell line SK-N-BE(2)] in cells treated with GANT61 (Fig. 3c).
Apoptosis follows GANT61 treatment in neuroblastoma cells
To further study the mechanisms of GANT61 on neuroblastoma growth, the effects on proliferation, cell cycle progression and apoptosis were evaluated. BrdU analyses revealed that SK-N-AS cells in S-phase were reduced from 40 to 24% following GANT61 treatment (10 μM, 72 hr) (t-test, p = 0.0084, Fig. 4a and Supporting Information Table S2). Moreover, a pronounced accumulation of SK-N-AS cells with hypodiploid DNA content, i.e. in sub-G1 phase, was observed after treatment with GANT61 (t-test, p = 0.0036, Fig. 4a and Supporting Information Table S2). Similar results were obtained in SH-SY5Y and SK-N-BE(2) cells but a higher GANT61 concentration (20 μM) was needed, as the 10 μM concentration was ineffective (SH-SY5Y, t-test, sub-G1: p = 0.0005, S: p = 0.002) and SK-N-BE(2); t-test, sub-G1: p = 0.041, S: p = 0.015; Figs. 4b and 4c, Supporting Information Table S2). To further validate the cell cycle effects, western blotting for Cyclin D1 was performed. A reduced protein expression was found after GANT61 treatment for 72 hr in SK-N-AS cells (Fig. 4d).
To clarify the mechanism of the GANT61 effects on neuroblastoma cells, expression of essential proteins in the apoptotic cascade was studied. Activation of caspase-3 and subsequent cleavage of poly (ADP)-ribose polymerase (PARP), its downstream substrate, was evident in SK-N-AS cells following 10 μM GANT61 treatment for 72 hr (Fig. 4e). Protein expression of active caspase-3 was also seen after 20 μM GANT61, 72 hr, in SH-SY5Y and SK-N-BE(2) cells (Fig. 4e).
siRNA knockdown of GLI1, GLI2, GLI3, but not SMO reduces neuroblastoma growth
To investigate the role of SMO and the three GLI transcription factors for the proliferation of neuroblastoma cells, we performed siRNA experiments. SK-N-AS cells were transfected with siRNAs directed against GLI1, GLI2, GLI3 and SMO. When comparing cell numbers 48 hr after transfection, GLI1, GLI2 and GLI3 knockdown significantly reduced neuroblastoma cell growth compared with nonsilencing siRNA control (ANOVA p = 0.0003; GLI1: p < 0.001, GLI2: p < 0.01, GLI3: p < 0.01), while SMO knockdown had no significant effect (Fig. 5a). The mRNA expression levels were reduced to at least 60% of the nonsilencing control using the different siRNAs (Fig. 5b).
GANT61 reduces neuroblastoma growth in vivo
To examine if GANT61 was suitable for oral treatment in vivo a pharmacokinetic study was performed. It concluded that only the active metabolite GANT61-diamine could be measured by liquid chromatography tandem mass spectrometry in plasma 15 min after dosage initiation (data not shown).
Consequently, to investigate the therapeutic effect of GANT61 on neuroblastoma growth in vivo, nude mice carrying SK-N-AS xenografts were treated with 50 mg/kg GANT61 orally (by gavage). Tumor growth was significantly inhibited at Day 12 (t-test, p = 0.03, Fig. 6), as the tumor volume was reduced to 63% compared with controls. All mice gained in weight during the experiment and showed no signs of toxicity (data not shown).
Mutations and amplifications of genes involved in HH signaling are a characteristic of many human tumors of different origin. Additionally, introduction of the corresponding gain or loss of function mutations in mice results in the development of tumors closely resembling the human counterparts. Taken together these observations established an apparent contribution of HH signal transduction to oncogenesis.6, 17 Recent studies have also shown that several types of cancers, including neuroblastoma, exhibit upregulation of the HH ligands or key elements in the HH signaling cascade.15–17 The molecular mechanisms for this upregulation are currently elusive but increased secretion of HH ligands from stromal cells or downregulation of miRNAs targeting GLI1 and SMO has been suggested.17 The findings of aberrant HH signaling in cancer have initiated efforts on the discovery of natural, and the development of synthetic, pharmacologic compounds, which target key components in the HH pathway. Currently, all substances that have entered clinical trials act directly on SMO.6 Hence, these compounds will presumably, not affect molecular lesions located downstream of SMO and their use is, therefore, limited. In this study, we show that neuroblastoma cells are less sensitive to the SMO inhibitors cyclopamine and SANT1 compared with the GLI inhibitor GANT61 (Fig. 1a). The concentrations needed to inhibit neuroblastoma cell growth by SANT1 were much higher than the one reported to inhibit SMO.12 Additionally, siRNA knockdown of SMO expression in neuroblastoma cells had no effect on cell survival whereas siRNA directed against GLI1, GLI2 or GLI3 significantly reduced neuroblastoma cell proliferation (Fig. 5a). Furthermore, the mRNA levels of GLI1 expression significantly correlated with GANT61 sensitivity (Fig. 2a). These results suggest that direct targeting of the GLI transcription factors, rather than SMO, is more efficient in suppressing neuroblastoma cell growth. Similarly, inhibition of HH signaling at the level of GLI but not SMO has also been reported to be an effective treatment of rhabdomyosarcoma and colon cancer cell growth.26–28
Among the neuroblastoma cell lines tested in this study GANT61 induced cytotoxicity in all cells, with IC50 values ranging between 5.82 and 12.4 μM after 72 hr incubation (Figs. 1a and 1b). Interestingly, a negative correlation between the log IC50 values of GANT61 and MYCN mRNA expression was also detected. The SK-N-AS cell line, expressing very low levels of MYCN but high GLI1, was the most sensitive to GANT61 treatment whereas the MYCN amplified cell line SK-N-BE(2), expressing high levels of MYCN but low GLI1, was the least (Figs. 2a and 3a). However, in the neuroblastoma cell lines expressing very low to intermediate levels of MYCN (SK-N-AS and SH-SY5Y) and in the MYCN amplified SK-N-BE(2) cell line treatment with GANT61 downregulated MYCN and/or c-MYC expression (Figs. 3b and 3c). Additionally, a significant inverse correlation between GLI1 and MYCN was evident using the publicly available R2 microarray analysis data from 88 neuroblastoma tumors (available at (http://r2.amc.nl, data not shown),29 which confirms our experimental observations on the cell lines. These findings may reflect the transition from HH signaling dependency to MYCN driven proliferation and differentiation of neuroblasts during neural crest development, which is independent of HH signaling.18 In medulloblastoma, an embryonal tumor of the CNS containing several molecular signatures similar to neuroblastoma, the expansion of granular progenitor cells and medulloblastoma growth is dependent of the expression of MYCN even in the presence of active HH signaling.30 Also, in Ptch1+/- mouse models of medulloblastoma, induction of sustained MYCN expression radically changes the fate of preneoplastic cells by preventing differentiation and driving tumor progression. This MYCN driven process is independent of HH signaling making the resulting tumor resistant to HH antagonists.31 Given that MYCN is an oncogenic driver in neuroblastoma32 together with the fact that MYCN amplification is a powerful predictor of aggressive biological behavior and the most reliable indicator of poor clinical prognosis,1, 2 our findings suggest that GLI antagonists like GANT61, are compounds that will affect early events in tumor cell growth that are independent of MYCN expression.
Molecular analysis of the GANT61 effects on the growth of neuroblastoma cells revealed a combination of cell cycle arrest in early-S phase accompanied by reduced Cyclin D1 expression and cleavage of caspase-3 and PARP, indicating apoptosis (Figs. 4a–4e). Similar findings have been reported in HT29 colon cancer cells treated with GANT6126 suggesting the importance of GLI transcription factors in maintaining survival of tumor cells that may not necessarily be characterized by mutations in HH signaling components.
The combinational studies of GANT61 with clinically relevant chemotherapeutic drugs revealed promising results. Synergistic effects of GANT61 were observed in all tested cell lines with the topoisomerase II inhibitor doxorubicin and in two out of three tested cell lines with the mitotic inhibitor vincristine (Fig. 1d). Since the synergy was also evident in cell lines with low expression of GLI1 (SK-N-BE[2)] this indicate that the effects are mediated through a nontarget specific mechanism. It has been reported that another HH inhibitor, HhAntag691 (GDC-0449) inhibits multiple ATP-binding cassette transporters such as P-glycoprotein.33 As both vincristine and doxorubicin are substrates for P-glycoprotein this may be a possible mechanism for the synergy.
The GANT61 effects were also investigated in established neuroblastoma xenografts, revealing a significant inhibition of neuroblastoma growth (Fig. 6). For these in vivo experiments we chose to deliver GANT61 (50 mg/kg) by daily oral gavage. The only other study investigating the in vivo effect of GANT61 on tumor growth involved subcutaneously injections proximal to the growing tumor.13 This resulted in a more pronounced tumor inhibition effect compared with our data.13 However, this form of drug administration is not feasible in the treatment of neuroblastoma. Given the short half-life of GANT6114 and the fact that we did not observe any signs of toxicity, there is potential for more extensive effects by optimizing the GANT61 concentration, administration route and frequency.
Taken together this study suggests that inhibition of HH signaling at the level of the GLI transcription factors is an effective way to target high-risk neuroblastoma without MYCN amplification and should be further considered as a treatment option for neuroblastoma patients. Our findings also indicate that the full potential of compounds inhibiting GLI transcription factors should be further investigated since these molecules act downstream of the majority of the mutations present in tumors with aberrant HH signaling activation.
The work of Juan Castro is acknowledged and Professor Rune Toftgård is acknowledged for the kind gift of GANT61.