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Keywords:

  • Glioblastoma;
  • Cancer stem cells;
  • Hedgehog;
  • PTEN

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

The identification of brain tumor stem-like cells (BTSCs) has implicated a role of biological self-renewal mechanisms in clinical brain tumor initiation and propagation. The molecular mechanisms underlying the tumor-forming capacity of BTSCs, however, remain unknown. Here, we have generated molecular signatures of glioblastoma multiforme (GBM) using gene expression profiles of BTSCs and have identified both Sonic Hedgehog (SHH) signaling-dependent and -independent BTSCs and their respective glioblastoma surgical specimens. BTSC proliferation could be abrogated in a pathway-dependent fashion in vitro and in an intracranial tumor model in athymic mice. Both SHH-dependent and -independent brain tumor growth required phosphoinositide 3-kinase-mammalian target of rapamycin signaling. In human GBMs, the levels of SHH and PTCH1 expression were significantly higher in PTEN-expressing tumors than in PTEN-deficient tumors. In addition, we show that hyperactive SHH-GLI signaling in PTEN-coexpressing human GBM is associated with reduced survival time. Thus, distinct proliferation signaling dependence may underpin glioblastoma propagation by BTSCs. Modeling these BTSC proliferation mechanisms may provide a rationale for individualized glioblastoma treatment.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Author contributions: Q.X.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; X.Y. and G.L.: collection and/or assembly of data, data analysis and interpretation, final approval of manuscript; K.L.B.: financial support, provision of study material or patients, final approval of manuscript; J.S.Y.: conception and design, provision of study material or patients, data analysis and interpretation, manuscript writing, final approval of manuscript.

The cancer stem cell (CSC) hypothesis not only has provided a framework for understanding cancer heterogeneity, tumorigenesis, cancer progression, and cancer therapy but also has offered an alternative approach to modeling human cancer. Recent identification of cancer-initiating stem cells in brain tumor [1, 2], prostate cancer [3], colon cancer [4, 5], and breast cancer [6, 7] suggested that CSCs may play a central role in the propagation of several cancer types. CSCs have also been shown to be responsible for prevalent radioresistance and chemoresistance in glioma [8]. Compared with conventionally cultured human cancer cell lines, CSCs have been shown to recapitulate human brain tumors in phenotype and in cancer genetics and thus may more faithfully model mechanisms of tumorigenesis and tumor propagation [9].

Glioblastoma multiforme (GBM) is the most malignant form of human primary brain tumor and can be initiated from brain tumor stem-like cells (BTSCs) [8, 910]. The capability of BTSCs to sustain brain tumor growth apparently lies in their active self-renewal and/or suppressed cell differentiation [2]. Several major signaling pathways that are critical in brain development have also been implicated in tumorigenesis, including bone morphogenetic protein (BMP) [11], Notch [12], Sonic Hedgehog (SHH) [13, 14], epidermal growth factor receptor (EGFR) [15, 16], PTEN/ phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) [12, 15, 17], platelet-derived growth factor receptor (PDGFR) [18], and OLIG2 [19]. Recently, a gene expression profiling of gliomas has shown that SHH signaling is active in a subset of gliomas [20]. This study further showed that SHH signaling is essential for glioma CSC self-renewal and CSC-initiated brain tumor growth [20]. It is postulated that the relatively homogeneous population of CSCs, 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.

Here, we performed gene expression profiling of BTSCs and unveiled salient signaling pathway signatures. We identified both SHH signaling-dependent and -independent BTSCs that can initiate brain tumors retaining their respective characteristics of signaling dependence. BTSC proliferation could be abrogated in a pathway-dependent fashion in vitro and in an intracranial tumor model in SCID mice. Furthermore, hyperactive SHH-GLI signaling in PTEN-coexpressing tumors was associated with reduced survival times in glioblastoma patients.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Primary Brain Tumor Cell Culture

Primary brain tumor spheres were cultured as previously described [1]. Briefly, brain tumor stem-like cells were grown in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium (F12) supplemented with B-27 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 20 ng/ml basic fibroblast growth factor (bFGF), and 20 ng/ml epidermal growth factor (EGF) (Peprotech, Rocky Hill, NJ, http://www.peprotech.com). Alternatively, dispersed brain tumor stem-like cells were grown on laminin-coated surface in the same medium as described above. Primary human fetal neural stem cells were derived from primary cells obtained from Cambrex (East Rutherford, NJ, http://www.cambrex.com). The GBM cell line and adherent primary glioma cells were cultured in DMEM/F12 containing 10% fetal bovine serum. Some frozen primary GBM tissues were used to compare gene expression profiles of BTSCs and their parental tumors.

Subsphere Formation Assays

Subsphere formation assays were described before [1]. Briefly, cells in single-cell suspension were diluted and plated at a density of three to five cells per well. After plating, the cells were observed, and only wells containing a single cell were considered. Cells were fed by changing half of the medium every 2 days. The wells were scored for sphere formation after 14 days.

Reverse Transcription-Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction Analysis

Total RNAs were isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). cDNAs were synthesized by oligo(dT)-priming methods. Real-time polymerase chain reaction (PCR) was performed using the SYBR Green Supermix (Qiagen) according to the manufacturer's instructions (primers used are listed in supplemental online Table 1). Expression levels of β-actin or glyceraldehyde-3-phosphate dehydrogenase were used for normalization and quantification of gene expression levels.

Immunoblotting

Cells were lysed and homogenized in RIPA lysis buffer containing fresh protease inhibitors by standard procedures. Protein concentrations were quantified with the BCA protein assay kit (Pierce, Rockford, IL, http://www.piercenet.com), and 30 μg of proteins were separated in 4%–12% SDS-polyacrylamide gel electrophoresis gels, transferred to polyvinylidene difluoride membranes, and hybridized with a SUFU antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) by standard procedures. Signals were detected by chemiluminescence using ECL detection reagents (GE Health, Piscataway, NJ, http://www.gelifesciences.com).

Tissue Array and Immunohistochemistry

Glioblastoma (grade IV) tissue arrays were obtained from US Biomax (Rockville, MD, http://www.biomax.us). Paraffin-embedded tissue sections were dried for 2 hours at 60°C, dewaxed in xylene, and rehydrated with distilled water. Incubation with anti-human GLI1 (1:500; Millipore, Billerica, MA, http://www.millipore.com), anti-phospho-AKT (1:100; Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com), and anti-PTEN (1:200; Santa Cruz Biotechnology) was performed overnight at 4°C. Immunodetection was performed using the Elite Vector Stain ABC System (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). The expression levels of PTEN and nuclear GLI1 were detected by immunohistochemistry and blindly scored on a scale of 1–10. Samples were separated into two halves on the basis of phospho-AKT scores, and average GLI scores of the two groups were compared.

Cell Proliferation, 5-Bromo-2′-Deoxyuridine Labeling, and Apoptosis Assays

For cell proliferation assays, brain tumor stem-like cells were seeded in growth medium at a density of 5 × 103 cells per 96-well plate with or without inhibitors. At the indicated time of culture, the number of cells was determined using WST-1 colorimetric assay (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com). For the 5-bromo-2′-deoxyuridine (BrdU) labeling assays, cells were cultured for 48 hours after transfection with a pulse of BrdU (10 μM) for the last 3 hours. The cells were then fixed and stained with anti-BrdU antibody. The proportion of BrdU(+) cells was determined by fluorescence-activated cell sorting analysis performed in triplicate, in at least two independent experiments. Apoptosis assays were performed using the annexin V apoptosis kit (Roche Diagnostics).

RNA Interference and Cell Transfection

Short interfering RNA (siRNA) (5′-CUCCACAGGCAUACAGGAUUU-3′) against human GLI1 [21], with or without fluorescence-labeled Alexa Fluor 488 at the 3′ end, was obtained by Qiagen custom synthesis. Pretested control siRNA was obtained from Qiagen. Brain tumor stem-like cells were transfected using HiPerfect (Qiagen) following the manufacturer's instructions. Briefly, brain tumor spheres were triturated into small aggregates and cultured for 2 hours in the growth medium without heparin. For every 105 cells, 0.5 μg of control siRNA or GLI1 siRNA was diluted and mixed with 24 μl of HiPerfect reagent. After mixing and incubation for 10 minutes, the transfection mixture was added to the cells. Cells were examined for transfection efficiency at 8–16 hours and were used for functional assays 24 hours after transfection.

Intracranial Cell Transplantation into Nude Mice

Athymic nude mice (nu/nu; 6–8 weeks old; Charles River Laboratories, Wilmington, MA, http://www.criver.com) were anesthetized with i.p. ketamine and medetomidine and were stereotactically implanted with glioblastoma sphere cells (50,000 per mouse) in the right striatum. The experiment was repeated once under identical conditions. The implanted mice were euthanized at 6–12 weeks, followed by intracardiac perfusion-fixation with 4% paraformaldehyde. Brain tissues were retrieved for frozen section and analysis. For BrdU incorporation assays, mice were injected i.p. with 10 mg/ml BrdU (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) at 50 mg/kg once per day for 2 days before sacrifice. BrdU-positive cells were detected by immunofluorescence using anti-BrdU antibody (1:500; BD Biosciences, San Diego, http://www.bdbiosciences.com). All animals used were experimented on in strict accordance with the Institutional Animal Care and Use Committee guidelines enforced at the Cedars-Sinai Medical Center.

Treatment of Brain Tumors Using siRNA and Rapamycin

Brain tumor stem-like cells were transfected with control siRNA or GLI1 siRNA (1.0 μg per 2 × 105 cells) at 2 hours prior to transplantation. For rapamycin treatment, cells were incubated with rapamycin (100 nM) for 2 hours prior to transplantation. At days 7 and 14 postoperation, animals were given siRNA (1.0 μg), rapamycin (200 ng), or the control, intratumorally.

GBM Patient Sample Analysis

Frozen tissues were obtained from 55 GBM patients (age range, 29–75) who were treated at Cedars-Sinai Medical Center, Los Angeles, CA. Tumor specimens were obtained according to a protocol approved by the Institutional Review Board of Cedars-Sinai Medical Center. The mRNA expression of PTEN, SHH, PTCH1, and GLI1 in 55 GBM tissues was determined by real-time PCR assays as described above. The expression levels of SHH, PTCH1, and GLI1 in PTEN-expressing (1%–100% of the maximal value) and PTEN-deficient (less than 1% of the maximal value) GBM tissues were analyzed. There was no significant difference in age between groups (PTCH1: 50 ± 11 vs. 51 ± 11, p = .78; SHH: 49 ± 10 vs. 52 ± 1, p = .60; GLI1: 54 ± 9 vs. 47 ± 11, p = .19). For the survival function analysis, the PTEN-expressing samples were grouped into two groups, greater than or less than the median values of SHH, PTCH1, and GLI1 expression, respectively, and the survival function was analyzed between the two groups for SHH, PTCH1, and GLI1 using Kaplan-Meier analysis. Survival was measured from the date of surgery of first diagnosis. All patients were diagnosed with de novo glioblastoma and underwent treatment with external beam radiation therapy to 60 Gy and chemotherapy.

Statistical Methods

To test whether variables differed across two groups, we used Student's t test (unpaired, two-tailed). The Kaplan-Meier (KM) analysis of survival distributions was performed using SPSS software (SPSS, Chicago, http://www.spss.com).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Pathway Gene Expression Profiles for Brain Tumor Stem-Like Cells

Previous studies in murine models have shown that the SHH signaling pathway plays a key role in the neural development and in neural stem cell self-renewal. We show that primary human fetal neurospheres grown in the EGF- and bFGF-supplemented medium express neural stem cell (NSC) markers CD133 and nestin (supplemental online Fig. 1). When the human NSCs were incubated with the SHH signaling inhibitor cyclopamine, cyclopamine dose-dependently inhibited cell proliferation. Furthermore, overexpression of GLI1 in human NSCs, using an adenoviral vector, enhanced human NSC proliferation, suggesting that SHH signaling is essential for human NSC proliferation (supplemental online Fig. 1). Although both NSCs and BTSCs express CD133 and form spheres in culture, it is unclear whether the requirement for SHH signaling in NSC proliferation is shared by BTSCs.

Previously we had isolated BTSCs from human GBM tissues and demonstrated that BTSCs, as opposed to the differentiated brain tumor cell counterparts, can generate tumors in immune-deficient mice [1]. It had been shown that BTSCs derived from glioblastoma manifest gene expression patterns different from those of corresponding serum-cultured cell lines and more closely recapitulate the phenotypes and biology of human glioblastoma [9]. Using real-time PCR analysis, we examined the expression of selected genes involved in major signaling pathways in BTSCs and in comparison with matched tumor cells and NSCs. As an example shown in Figure 1A, BTSC1 is different from both tumor cells (GBM1) and NSC in their gene expression patterns. For instance, the SHH pathway genes had the highest expression levels in BTSC1, whereas NSCs expressed the highest level of BMI1. Although the gene expression patterns of GBM1 and BTSC1 both showed high levels of expression of GLI1, suggesting active SHH signaling in these cells, only the BTSC1 profile revealed stronger and more consistent gene expression levels of the ligand (SHH), the receptor (PTCH1), and the downstream effectors (N-Myc and Cyclin D2). Therefore, BTSCs manifest gene expression profiles different from that of total tumor cells.

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Figure Figure 1.. Gene expression profiling of different BTSCs revealed distinct genetic programs and signaling pathway activities in brain tumor cells. (A): A molecular signature of BTSCs compared with that of human NSCs and their parent brain tumor cells (without culture). Both BTSCs and NSCs were grown in identical defined media. Frozen primary GBM tissues were used to compare gene expression profiles of BTSCs and their parental tumors. The mRNA expression levels for selected genes were quantified by real-time polymerase chain reaction (PCR). (B): Heat map image showing gene expression profiling of five different BTSCs. mRNA expression levels were quantified using real-time PCR and were normalized to glyceraldehyde-3-phosphate dehydrogenase expression. For each gene, the expression values were assigned a color using the minimum/maximum method. Red and green colors represent high and low mRNA expression levels, respectively. BTSC1–BTSC5 indicate five different BTSCs. (C): Detection of GLI1 protein expression and subcellular localization using immunocytochemistry assays. Panels 1–5 indicate BTSC1–BTSC5, respectively. Panel 6 shows an LN18 cell as a positive control. Abbreviations: BTSC, brain tumor stem-like cell; GBM, glioblastoma multiforme; hNSC, human fetal neural stem cell; NSC, fetal neural stem cell; SHH, Sonic Hedgehog.

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Next, the gene expression profiles of five BTSCs from different GBM patients were compared and analyzed. All five BTSCs had a distinct gene expression profile (Fig. 1B). Furthermore, these BTSCs can be separated into two groups on the basis of their gene expression patterns. BTSCs 1, 2, and 3 form one group characterized by high expression levels and activities in SHH, Notch, and platelet-derived growth factor signaling pathways. The other group, including BTSCs 4 and 5, showed low expression of genes in these signaling pathways. Instead, these BTSCs seemed to have lost expression of one or more key tumor suppressor genes, such as PTEN, p21CIP, or BMP receptor. To confirm that SHH signaling is active only in BTSCs 1–3, the expression of GLI1 protein was examined in each BTSC. As shown in Figure 1C, strong nuclear immunostaining of GLI1 was present only in BTSCs 1–3 and not in BTSCs 4 and 5 (supplemental online Fig. 2). Thus, the tumor-initiating potential of BTSCs is associated with distinct molecular programs.

Identification of SHH Pathway-Dependent and -Independent BTSCs

The robust expression of the SHH-GLI1 pathway components in a group of BTSCs implied that hyperactive SHH-GLI1 signaling may play a role in cell proliferation of these BTSCs. We sought to inhibit this signaling by knocking down the downstream transcription factor GLI1 using RNA interference to determine the effect of signal inhibition on BTSC proliferation and self-renewal. The expression of GLI1 in tumor cells was suppressed by 90% using GLI1-targeting siRNA (supplemental online Fig. 3). siRNA of the same sequence had previously been used to effectively silence GLI1 expression [21]. Knockdown of GLI1 significantly inhibited BTSC1 cell proliferation but had no effect on BTSC4 (Fig. 2A). When both siRNA against GLI1 and siRNA against GLI2 were combined, the inhibiting effects on cell proliferation were similar (supplemental online Fig. 4). We also showed that GLI1 siRNA could inhibit cell proliferation in BTSC3, which has a SHH signaling signature, but not in SHH nonexpressing BTSC5 (supplemental online Fig. 5). To determine whether SHH signaling may be important for BTSC self-renewal, we performed subsphere formation assays. As shown in Figure 2A, the percentage of subsphere-forming BTSC1 cells, but not BTSC4 cells, was significantly reduced in GLI1 knockdown cells relative to control cells, suggesting that SHH signaling may promote BTSC1 cell proliferation through enhanced cell self-renewal.

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Figure Figure 2.. Sonic hedgehog-GLI1 signaling is required for the cell proliferation of some BTSCs. (A): BTSC1 (upper panel) and BTSC4 (middle panel) were transiently transfected with short interfering RNA (siRNA) (1.67 μM) targeting human GLI1 gene expression (▪) or control siRNA (♦), followed by cell proliferation assays at the indicated time points. *, p = .04 by Student's t test. Bottom panel: subsphere formation assay results showing percentages of dissociated BTSCs treated with GLI1 siRNA (open bars) or control siRNA (filled bars) forming secondary spheres. Results are from three independent experiments. **, p = .0002 by Student's t test. (B): GLI1 knockdown in BTSCs did not increase apoptotic cell death. Percentage of apoptotic cell death with (filled bars) or without (open bars) GLI1 knockdown were measured using an annexin V/PI staining kit. (C): BrdU incorporation assays in BTSCs without (upper panel) or with (lower panel) GLI1 knockdown. Results are representative of two experiments. (D): Misexpression of human SUFU in BTSCs as shown in Western blots. BTSC1 (lanes 1 and 2) and BTSC4 (lanes 3 and 4) were transiently transfected with hSUFU-expressing plasmid (lanes 2 and 4) or control plasmid (lanes 1 and 3). Cell lysates were analyzed at 48 h. (E): Effect of SUFU expression on BTSC proliferation. Cells were transfected with SUFU vector (filled bars) or empty vector (open bars) for 24 h, followed by WST-1 assays at the indicated times. Misexpression of SUFU in BTSC1 (upper panel), but not in BTSC4 (lower panel), inhibited cell proliferation. Results are from two independent experiments. **, p = .001 (72 h) and p = .003 (96 h) by Student's t test. Abbreviations: BrdU, 5-bromo-2′-deoxyuridine; BTSC, brain tumor stem-like cell; h, hours; PI, propidium iodide.

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SHH signaling seemed to enhance BTSC proliferation through increased cell cycle progression, rather than promoting cell survival, as GLI1 knockdown in BTSC1 decreased BrdU incorporation rate from 32% to 19% (Fig. 2B). GLI1 knockdown in BTSC1 did not increase cell apoptosis, but rather decreased it (Fig. 2C). To further test whether SHH signaling is indeed critical for BTSC1 proliferation, cells were transiently transfected with a vector expressing SUFU, a negative regulator of SHH signaling (Fig. 2D). Overexpression of SUFU in BTSC1 significantly decreased cell proliferation at both 72 and 96 hours but had little effect on BTSC4 cells (Fig. 2E). Thus, we have identified both SHH signaling-dependent BTSCs and SHH signaling-independent BTSCs.

Inhibition of Tumor Growth from SHH Pathway-Dependent BTSCs by Targeting GLI1

These BTSCs had previously been shown to initiate tumor growth in immune-compromised mice [1]. To test whether tumor growth from BTSCs can be inhibited by targeting GLI1, BTSC1 (SHH-dependent) and BTSC4 (SHH-independent) were each implanted into nude mice after transfection with GLI1 siRNA or control siRNA, followed by intratumor injection of GLI1 siRNA or control. Animals implanted with BTSC1 or BTSC4 carried brain tumors in less than 12 weeks (Fig. 3A). Although BTSC4-initiated tumors were more malignant and invasive, the BTSC1 tumors were more confined (Fig. 3A). GLI1 siRNA treatment significantly reduced BTSC1 tumor volume (Fig. 3B) but had no effect on the BTSC4 tumor size. Characteristically, BTSC1-initiated brain tumors, but not BTSC4 tumors, contained a broad layer of GLI1-positive cells along the outer layer of the tumors, which were greatly reduced in GLI1 siRNA-treated tumors (Fig. 3C). Consistent with the tumor volume reduction, GLI1 siRNA treatment significantly reduced GLI1-positive cells and proliferating cells in BTSC1 tumors, as indicated by BrdU incorporation detection (Fig. 3D). Therefore, targeting GLI1 significantly inhibited SHH-GLI1 signaling-dependent brain tumor growth.

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Figure Figure 3.. Suppression of GLI1 expression as a potential therapy for sonic hedgehog signaling-active brain tumors. (A): BTSC1 and BTSC4 were implanted into the brains of nude mice with or without GLI1 siRNA treatment. Brain tissues were prepared and analyzed with H&E staining at 12 weeks. (B): GLI1 knockdown inhibited BTSC1 tumor growth as indicated by the tumor volume reduction (n = 5). **, p = .01 by Student's t test. (C): Brain tumors derived from BTSC1, but not those derived from BTSC4, manifested strong GLI1 expression, particularly in the tumor border regions. Brain tumors from BTSC1 with GLI1 siRNA treatment showed significantly reduced GLI1 expression. Note the nuclear staining of GLI1 in the GLI1-positive cells (insets). (D): GLI1 siRNA inhibited BTSC1 proliferation in vivo. At 12 weeks postimplantation, tumor cell proliferation without or with GLI1 siRNA treatment was assessed by BrdU incorporation assays. Abbreviations: BrdU, 5-bromo-2′-deoxyuridine; BTSC, brain tumor stem-like cell; siRNA, short interfering RNA.

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PI3K-mTOR Signaling Is Critical for the Growth of Brain Tumors Derived from BTSCs

The identification of SHH signaling-independent BTSCs and brain tumors prompted us to examine alternative signaling pathways. The PTEN loss-of-function and active PI3K-AKT pathway has been shown to be prevalent and critical in malignant brain tumors [12, 15, 17]. PI3K-AKT signaling is required in both SHH-dependent and -independent BTSC proliferation, as several PI3K-AKT pathway inhibitors, including LY294002 and AKT inhibitor VIII, inhibited cell proliferation in both BTSC1 and BTSC4 (supplemental online Fig. 6). A key downstream effector of PI3K-AKT signaling is mTOR [22]. Previous studies had implicated mTOR signaling in the progression of a wide range of cancers, including kidney [23], pancreatic [24], breast [25, 2627], prostate [28], lung [29], and glioma [17]. To test whether mTOR signaling is important in BTSC proliferation, we incubated BTSC1 and BTSC4 with or without rapamycin, an inhibitor of mTOR. As shown in Figure 4A, rapamycin dose-dependently inhibited the cell proliferation of both BTSC1 and BTSC4. Therefore, PI3K-mTOR signaling is critical for the cell proliferation of both SHH-dependent and -independent BTSCs.

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Figure Figure 4.. Mammalian target of rapamycin (mTOR) activity was required for brain tumor growth. (A): Rapamycin, an mTOR inhibitor, dose-dependently inhibited BTSC1 (upper panel) and BTSC4 (lower panel) cell proliferation. BTSCs were incubated with Rap10, Rap100, or Rap2k for specified periods of time. Cell proliferation was determined using WST-1 assays. (B): BTSC4 formed invasive brain tumors that had little PTEN expression compared with BTSC1-derived brain tumors. Rapamycin-treated tumor showed a less invasive morphology. (C): Rapamycin treatment on animals bearing BTSC4 brain tumors had a survival benefit over the control group. p = .004 by Kaplan-Meier analysis (n = 10). Rapamycin treatment was administered immediately before surgery and again intratumor at days 7 and 14 postoperation. Abbreviations: BTSC, brain tumor stem-like cell; DMSO, dimethyl sulfoxide; h, hours; Rap10, 10 nM rapamycin; Rap100, 100 nM rapamycin; Rap2k, 2 μM rapamycin.

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To test whether suppressing mTOR signaling with rapamycin can inhibit BTSC-initiated brain tumor growth, BTSC4 was transplanted intracranially into nude mice, with or without rapamycin treatment, immediately before surgery and again at day 7 and day 14 postoperation. Whereas mock-treated animals developed highly invasive brain tumors, the rapamycin-treated tumors had a more defined boundary (Fig. 4B). Furthermore, rapamycin treatment increased animal survival (p = .004) (Fig. 4C). Consistent with previous data showing that BTSC4, but not BTSC1, had lost PTEN expression, there was little PTEN expression in the BTSC4-initiated brain tumors compared with the BTSC1 brain tumors (Fig. 4B). These data suggested that mTOR signaling is critical for SHH-signaling-independent brain tumor growth.

Differential SHH-GLI Signaling Activities in PTEN-Expressing and PTEN-Deficient GBMs

To survey the general status of SHH signaling activity and its relation to PTEN expression in human GBM tissues, we examined the expression of GLI1 and PTEN using tissue arrays with a panel of 40 GBM tissues. There were wide ranges of expression levels of GLI1 and PTEN, but high GLI1 expression was present in most GBM tissues (Fig. 5A). Interestingly, the GLI1 expression level in PTEN-expressing GBM tissues was significantly higher than that in PTEN-deficient GBM tissues (Fig. 5B). To further test the association of SHH signaling activity with PTEN expression status, we determined the gene expression levels of SHH, PTCH1, and GLI1 in tumor tissues from another 55 GBM patients. These samples were separated into a PTEN-expressing group and a PTEN-deficient group on the basis of their PTEN expression levels. As shown in Figure 5B, PTEN-deficient tumors manifested a significantly higher level of PTCH1 gene expression than PTEN-expressing tumors. However, the expression levels of SHH and GLI1 were significantly higher in PTEN-expressing cells than in PTEN-deficient cells (Fig. 5B). Therefore, there is a correlation between SHH-GLI signaling activity and PTEN activity in GBM tissues; SHH-GLI signaling activity appears to be higher in PTEN-expressing tumors than in PTEN-deficient tumors.

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Figure Figure 5.. SHH signaling activities in PTEN-positive brain tumors correlated with patient survival. (A): Variable GLI1 expression in human GBM tissues. Forty GBM tissues and normal brain tissues in a GBM tissue array were stained with GLI1 and PTEN antibodies and were scored. Representative pictures show GLI1 staining results for GBM tissues with different scores. Right: the average GLI1 expression level in the PTEN-expressing GBM tissues (open bar) was significantly higher than that in the PTEN-deficient GBM tissues (filled bar). *, p = .048. (B): The expression levels of SHH, PTCH1, and GLI1 in PTEN-expressing and PTEN-deficient GBM tissues. The mRNA expressions of PTEN, SHH, PTCH1, and GLI1 in 55 GBM tissues (ages 29–75) were determined by real-time polymerase chain reaction and were grouped (described in Materials and Methods). The gene expression levels in PTEN-expressing (open bars) and PTEN-deficient (closed bars) samples were compared. *, p = .006 (PTCH1), p = .008 (SHH), and p = .004 (GLI1) by Student's t test. (C): Effect of SHH-GLI signaling on the survival of PTEN-expressing GBM patients. GBM patient samples (age-matched) were grouped into lower expression (solid line) and higher expression (dotted line) on the basis of the expression levels of SHH (left panel), PTCH1 (middle panel), and GLI1 (right panel). The survival curves were generated using Kaplan-Meier analysis method. Abbreviations: GBM, glioblastoma multiforme; SHH, Sonic Hedgehog.

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Hyperactive SHH-GLI Signaling in Brain Tumors with PTEN Coexpression Is Associated with Reduced Survival Time

Since SHH-GLI signaling activity is high in PTEN-expressing brain tumors and this signaling pathway is also essential for BTSC self-renewal and proliferation, we next studied the potential connection between SHH-GLI signaling activity and GBM patient survival time. For this purpose, the KM survival function analysis was performed within the PTEN-expressing group of GBM samples, using the gene expression levels of SHH, PTCH1, or GLI1 as the variables. As shown in Figure 5C, higher expression levels of SHH, PTCH1, and GLI1 were associated with reduced survival time. For the groups with high expression levels of SHH, PTCH1, and GLI1, the median survival times were 48, 52, and 53 weeks, respectively, compared with 70 weeks for the groups with lower SHH, PTCH1, and GLI1 expression. In particular, the survival benefits for the groups with lower expression of SHH and PTCH1 are statistically significant (Fig. 5C). Together, these data suggest that active SHH signaling is associated with poor prognosis for patients bearing PTEN-coexpressing glioblastoma.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

This study has generated the first molecular signatures of GBM cancer stem cell proliferation to identify both SHH signaling-dependent and -independent BTSCs and brain tumors. Previously, gene expression profiling using GBM cell lines of primary tumor cells has identified numerous, yet diverse, signature genes or gene sets [30, 3132]. Using genetically homogeneous BTSCs, tumor classifications and their molecular signatures can be revealed with relatively small sample sizes because of reduced noise levels. We also show that the growth of brain tumors initiated from SHH-dependent BTSCs can be inhibited by targeting GLI1, whereas both SHH-dependent and -independent brain tumor growth requires PI3K-mTOR signaling. Furthermore, we found that the expression levels of SHH and GLI1 are significantly higher in PTEN-expressing cells than in PTEN-deficient cells. Finally, we present evidence indicating that hyperactive SHH-GLI signaling in human brain tumors with PTEN-coexpression is associated with reduced survival time.

SHH signaling pathway is a key regulatory mechanism in neural development, and abnormal SHH signaling has been implicated in the tumorigenesis of pediatric brain tumors, such as medulloblastoma [33, 34, 3536]. Recently, SHH-GLI signaling was also found to be essential for glioma stem cell self-renewal [20]. Our study of signaling in GBM, however, demonstrated that there are both SHH signaling-dependent and -independent brain tumors and BTSCs. SHH signaling-dependent BTSCs, such as BTSC1, express high levels of SHH, PTCH1, and GLI1 and initiate brain tumors characterized by a cancer stem cell zone of GLI1-positive cells. Both overexpression of SUFU and knockdown of GLI1 expression inhibited BTSC1, but not BTSC4, cell proliferation. Unlike the study by Clement et al. [20], our study found that cyclopamine could not inhibit BTSC growth at normal dose levels (no more than 10 μM). This discrepancy could be due to experimental conditions, or it could reflect a difference in ligand dependence, cancer genetics, or drug resistance of the cancer cells. However, we could significantly inhibit SHH signaling by targeting GLI1 with siRNA both in vitro and in vivo. Because the duration of siRNA action is transient, the inhibition of BTSC1 by siRNA in culture also showed transient effects. Future work using stable inducible systems would provide further insight into this mechanism. Consistent with our data demonstrating a critical role for SHH signaling in some BTSCs, a recent study showed that inhibition of SHH signaling could deplete glioblastoma stem cells [37].

In our study, gene expression profiling indicated that active SHH signaling in the BTSCs is associated with high activities in the signaling pathways of Notch, PDGFR, and OLIG2, which are also known to be important in neural development and neural stem cell functions. Deregulated signaling of BMP [11], Notch [12], PDGFR [18], and OLIG2 [19] has been implicated in the development and progression of GBM. For instance, it has been shown that the BMP signaling pathway is suppressed in GBMs and that exogenous BMPs can arrest BTSC-initiated tumor growth by blocking cell differentiation [11]. These disparate signaling pathways may contribute to GBM development independently. Alternatively, SHH signaling may interact with each of these signaling pathways. For instance, it has been shown that activation of the SHH pathway could induce PDGFRα and OLIG2 expression during neural development [38, 39]. It remains to be seen whether PDGFR signaling and OLIG2 expression are regulated by SHH signaling in these BTSCs.

The identification of both SHH signaling-dependent and -independent brain tumors in this study suggested that there are molecularly distinct subclasses of GBMs that have an effect on progression and prognosis. These findings are reminiscent of a recent study in which high-grade gliomas were classified into a proneural subclass and a mesenchymal subclass, resembling two stages in neurogenesis [12]. In that study, Phillips et al. discovered a prognostic model using PTEN-AKT and Notch signatures to predict poor versus better glioma prognosis, respectively [12]. In our study, the SHH signaling-dependent brain tumors showed high activities in Notch and PDGFR pathways, whereas the SHH signaling-independent brain tumors showed PTEN deficiency and high activity in the PI3K-AKT pathway. Our study emphasized that BTSCs with distinct signaling patterns determined the different GBM phenotypes and progression. The presence of SHH-independent GBM may also partly explain the result in a recent study showing that SHH signaling is active in grade II–III gliomas but not GBMs [40].

The pivotal role of PTEN expression status in GBM development and progression is further supported in our study using BTSCs. PTEN expression is widely lost in GBM and other malignant tumors [7, 41, 42, 4344]. Coexpression of PTEN with active EGFR in GBM determines clinical cancer responses to an EGFR inhibitor [15]. PTEN loss and AKT activation are associated with more invasive and malignant cancer and poor prognosis for GBM patients [12]. Furthermore, a drug that potently inhibits GBM cell proliferation was found to be an inhibitor of both PI3Kα and mTOR [17]. We found that there are both PTEN-expressing and PTEN-deficient BTSCs and that all BTSCs tested are inhibited by PI3K and AKT inhibitors.

Furthermore, we found that proliferation of the BTSCs was most strongly inhibited by rapamycin, an mTOR inhibitor. Rapamycin also inhibited BTSC-initiated brain tumor and extended animal survival. mTOR is a critical effector downstream of growth factor (through PI3K-AKT) and nutrient signaling pathways [22]. mTOR has also emerged as a key target that is commonly deregulated in human cancers [22]. Recently, a combination therapy with rapamycin and an EGFR inhibitor inhibited the growth of both peripheral lung cancer and chemoresistant bronchial lung cancer [29]. Clinical trials with mTOR inhibitors in the treatment of GBM have yielded contradictory results [45, 4647]. The inadequate penetration of the drug across the blood-brain barrier into the tumor sites was cited as the reason for the failure [46]. We demonstrated that rapamycin effectively inhibited BTSC growth and that intratumor administration of rapamycin prolonged animal survival. In addition, combination therapy of an mTOR inhibitor and an EGFR/vascular endothelial growth factor receptor inhibitor had been reported to offer increased benefit in glioma treatment [48].

Our study presents evidence indicating genetic interaction between the SHH signaling pathway and PTEN in human glioblastoma. After analysis of a tissue array of GBM tumors and additional frozen GBM tissues, we found that the average SHH signaling activity is significantly higher in PTEN-expressing tumors than in PTEN-deficient tumors. Although the expression levels of SHH and GLI1 are significantly higher in PTEN-expressing tumors, the expression level of PTCH1, a SHH receptor, is significantly lower. PTCH1 is tumor suppressor and a negative regulator of SHH signaling in the absence of the ligands. These results suggest that hyperactive SHH-GLI signaling is critical for brain tumor cell proliferation only when the tumor suppression mechanism of PTEN pathway is intact. When PTEN expression is lost through genetic or epigenetic alteration, alternative signaling pathways, including the PI3K-AKT-mTOR pathway, are usually activated, leading to bypassing of the requirement for an active SHH-GLI signaling pathway. PTEN expression alone, however, was not a strong indicator of malignancy in our study, as there is no association between PTEN expression in GBM and patient survival. Finally, we have shown that higher SHH signaling in PTEN-expressing GBM is associated with reduced survival time, further supporting a critical role for SHH signaling in the PTEN-coexpressing subset of GBM tumors.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

In summary, we demonstrate that the distinct cell proliferation signaling dependence of glioblastoma can be uncovered using gene expression profiling with brain tumor stem-like cells. Activated SHH-GLI signaling is required for the growth of glioblastomas with a PTEN-coexpression signature, whereas the PI3K-AKT-mTOR signaling pathway is generally critical for glioblastoma growth. These findings imply that future development of glioblastoma treatment based on signaling pathway inhibitors may benefit from identification of tumor molecular signatures and consideration of the genetic context.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

We thank Gretchen Duvall and Akop Seksenyan for critical reading of the manuscript and helpful advice. We also thank Mario Castro for providing the adenoviral vectors. This work was funded in part by NIH grants NS048959 and NS048879 (to J.S.Y.).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information
FilenameFormatSizeDescription
SC-08-0459_Suppl_Fig_1.tif2533KSupplemental Figure 1
SC-08-0459_Suppl_Fig_2.tif784KSupplemental Figure 2
SC-08-0459_Suppl_Fig_3.tif1621KSupplemental Figure 3
SC-08-0459_Suppl_Fig_4.tif349KSupplemental Figure 4
SC-08-0459_Suppl_Fig_5.tif382KSupplemental Figure 5
SC-08-0459_Suppl_Fig_6.tif204KSupplemental Figure 6
SC-08-0459_Suppl_Tab_1.tif492KSupplemental Table 1

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