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Cancer Research 67, 5179-5185, June 1, 2007. doi: 10.1158/0008-5472.CAN-06-4177
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Apoptosis Suppression by Somatic Cell Transfer of Bcl-2 Promotes Sonic Hedgehog–Dependent Medulloblastoma Formation in Mice

Todd D. McCall, Carolyn A. Pedone and Daniel W. Fults

Department of Neurosurgery, University of Utah School of Medicine, Salt Lake City, Utah

Requests for reprints: Daniel W. Fults, Department of Neurosurgery, University of Utah School of Medicine, 175 North Medical Drive East, Salt Lake City, UT 84132. Phone: 801-581-6908; Fax: 801-581-4385; E-mail: daniel.fults{at}hsc.utah.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Medulloblastomas are malignant brain tumors that arise in the cerebellum in children. Aberrant activation of the Sonic hedgehog (Shh) signaling pathway, which normally stimulates proliferation of granule neuron precursors (GNP) during cerebellar development, induces tumors in mice that closely mimic human medulloblastomas. Shh-dependent medulloblastoma formation is enhanced by hyperactive insulin-like growth factor (IGF) signaling and ectopic expression of Myc oncogenes. This enhanced tumorigenesis stems from the sensitivity of GNPs to IGF and Myc levels in regulating proliferation. An emerging theme in cancer research is that oncogene-induced cell proliferation cannot initiate neoplastic transformation unless cellular programs that mediate apoptosis are disabled. Here, we report a high frequency of medulloblastoma formation in mice after postnatal overexpression of the antiapoptotic protein Bcl-2 in cooperation with Shh. Ectopic expression of Bcl-2 alone or in combination with N-Myc did not induce tumors, indicating that Shh has essential transforming functions in GNPs not supplied by the mitogenic stimulus of N-Myc combined with a strong antiapoptotic signal provided by Bcl-2. Expression of endogenous Bcl-2 was not up-regulated in Shh-induced tumors. Instead, elevated levels of phosphorylated Akt were found, suggesting that activated phosphatidylinositol 3-kinase signaling is one intrinsic mechanism for suppressing apoptosis in Shh-dependent medulloblastomas. Thus, blockade of apoptosis cooperates with Shh-stimulated proliferation to transform GNPs and induce aggressive medulloblastomas. These findings provide insights into the molecular signals that initiate medulloblastoma formation and they support the importance of blocking apoptosis in carcinogenesis. [Cancer Res 2007;67(11):5179–85]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Medulloblastomas are malignant brain tumors that arise in the cerebellum in children. A growing body of evidence indicates that medulloblastomas can arise by transformation of granule neuron precursors (GNP). In mice and humans, GNPs undergo rapid, postnatal proliferation in the external granule layer, a germinal zone on the cortical surface of the developing cerebellum. GNP proliferation requires Sonic hedgehog (Shh), a morphogen that governs many aspects of neural development (reviewed in ref. 1). In vivo models of medulloblastoma that use genetically engineered mice have shown that aberrant activation of the Shh signaling pathway in GNPs induces tumors that closely mimic human medulloblastomas (reviewed in ref. 2). Rapid postnatal proliferation of GNPs in response to Shh creates a vulnerable setting for secondary transforming events like activation of signaling molecules that promote cell cycle progression or cell survival. For example, hyperactive insulin-like growth factor (IGF) signaling causes cerebellar hyperplasia in mice (3) and enhances Shh-induced medulloblastoma formation (4). Moreover, Shh and IGF signaling pathways cooperate to promote GNP proliferation (5) and medulloblastoma formation (6) by converging on the oncogenic transcription factor N-Myc through a two-pronged mechanism, in which Shh stimulates N-myc gene transcription and IGF signaling stabilizes the N-Myc protein.

An emerging theme in cancer research is that oncogene-induced cell proliferation cannot initiate neoplastic transformation unless cell programs that mediate apoptosis become disabled (7). That is because mechanisms that drive cell cycle progression, like overexpression of Myc oncoproteins, trigger or sensitize cells to undergo apoptosis. The fact that human tumors often have dysregulated expression of genes that govern apoptosis supports the idea that uncoupling proliferation from apoptosis is required for tumor formation. For example, human follicular B-cell lymphomas overexpress the antiapoptotic protein Bcl-2 consequent to a t(14;18) chromosomal translocation, which juxtaposes the BCL2 gene and enhancer sequences from the immunoglobulin heavy chain locus (reviewed in ref. 8). Similarly, mutant MYC alleles prevalent in human Burkitt's lymphomas stimulate cell cycle progression but fail to induce apoptosis, thus allowing unchecked expansion of proliferating clones of B lymphocytes (9).

The fact that human medulloblastomas frequently overexpress Bcl-2 (10) suggested to us that disabled apoptosis might cooperate with Shh-stimulated proliferation to transform GNPs and induce medulloblastomas. To test this hypothesis, we used a version of the RCAS/tv-a system that allows postnatal gene transfer to neural progenitor cells in the cerebellum of mice (11). The system uses a replication-competent, avian retroviral vector (RCAS), derived from the avian leukosis virus ALV (subgroup A), and a transgenic mouse line (Ntv-a) that produces TV-A (the receptor for ALV-A) under control of the Nestin gene promoter. Nestin is an intermediate filament protein expressed by neuronal and glial progenitors. When mammalian cells are transduced with RCAS vectors, viral replication does not occur. Instead, the RCAS provirus integrates into the host cell genome and the transferred gene is expressed as a spliced message under control of the constitutive retroviral promoter, long terminal repeat. This somatic cell gene transfer system is well suited to identifying genes that initiate medulloblastoma formation because it targets GNPs during their postnatal expansion phase when these cells are highly susceptible to transformation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Vector construction. The RCAS-Bcl-2 vector was constructed by ligating a PCR-generated cDNA corresponding to the entire coding sequence of the human BCL2 gene into the parent retroviral vector, RCASBP(A) (12). Construction and characterization of RCAS-Shh, which has six repeats of the influenza virus hemagglutinin epitope (YPYDVPDYA), and RCAS-N-MycT50A have been described previously (6). To produce live virus, we transfected plasmid versions of RCAS vectors into immortalized chicken fibroblasts (DF-1 cells) and allowed them to replicate in culture.

In vivo somatic cell transfer in transgenic mice. Production of the Ntv-a transgenic mouse line has been described previously (11). The mice used in these experiments were mixtures of the following strains: C57BL/6, BALB/C, FVB/N, and CD1. To transfer genes via RCAS vectors, we injected DF-1 producer cells (105 cells in 1–2 µL of PBS) into the lateral cerebellum from an entry point just posterior to the lambdoid suture of the skull. We injected mice within 72 h after birth because the pool of nestin-positive cells producing ALV-A receptors diminishes progressively afterward. The mice were sacrificed 12 weeks after injection or sooner if they showed signs of increased intracranial pressure. The brains were fixed in formalin, embedded in paraffin, and sectioned for immunohistochemical analysis by parallel incisions in the coronal plane. We estimated tumor size by tracing tumor circumference from digitized photomicrographs of H&E-stained brain sections and calculated cross-sectional area using Zeiss Axiovision image analysis software.

Immunocytochemistry. To analyze protein expression in tissue sections, we used an immunoperoxidase staining method detailed previously (13). Briefly, tissue sections (4 µm) were deparaffinized, rehydrated, and then autoclaved in a citrate-based antigen retrieval solution (Vector Laboratories) for 30 min before application of primary antibody. Immunoreactive staining was visualized using an avidin-biotin complex technique with diaminobenzidine as the chromogenic substrate (reddish brown color) and toluidine blue as a nuclear counterstain. We used the following monoclonal antibodies (and dilutions) from the indicated commercial sources: Mab100 (1:100)—human Bcl-2 (Santa Cruz Biotechnology); MabC2 (1:100)—mouse and human Bcl-2 (Santa Cruz Biotechnology); F7 (1:50)—hemagglutinin (Santa Cruz Biotechnology); 2F11 (1:100)—70-kDa neurofilament protein (DAKO); TuJ1 (1:400)—ßIII tubulin (Research Diagnostics); Mab377 (1:100)—NeuN (Chemicon); Mab9277 (1:50)—pS473Akt (Cell Signaling Technology); and DO-1 (1:50)—wild-type and mutant p53 (Santa Cruz Biotechnology). To detect Math1, we used a hybridoma developed by Dr. Jane Johnson (University of Texas Southwestern, Dallas, TX) and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Resources and maintained by The University of Iowa, Department of Biological Sciences (Iowa City, IA). We used polyclonal antibodies to detect expression of glial fibrillary acidic protein (1:1,000; DAKO), TrkC (1:25; R&D Systems), and TV-A (1:200; Dr. Andrew Leavitt, University of California at San Francisco, San Francisco, CA).

For double immunofluorescence staining, we incubated tissue sections sequentially with F7 antibody (against hemagglutinin-tagged Shh), biotinylated antimouse IgG, and FITC-conjugated avidin followed by Mab100 (against human Bcl-2), biotinylated antimouse IgG, and rhodamine-conjugated avidin. Washing and blocking reagents were applied according to the Vector M.O.M. basic kit (Vector Laboratories). Digital images were generated using an Olympus FV1000 confocal microscope.

Apoptosis and proliferation assays. Apoptosis was quantified by immunostaining formalin-fixed, paraffin-embedded tissue sections with an antibody against cleaved caspase-3 (Asp175) according to the manufacturer's protocol (Cell Signaling Technology). To calculate the apoptotic index, we counted caspase-3–positive cells in 6 to 14 contiguous x40 microscope fields (>1.3 x 103 cells counted) and averaged the percentage of positive cells from three to seven different tumors. The proliferation index was determined by the same method using a polyclonal antibody (1:1,000) against cell cycle protein Ki67 (Vector Laboratories).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Bcl-2 enhances Shh-dependent medulloblastoma formation. To investigate the effect of blocking apoptosis in Shh-induced medulloblastoma formation, we expressed Bcl-2 ectopically, alone, and in combination with Shh, in the postnatal mouse cerebellum. To do this, we generated an RCAS vector carrying the human BCL2 gene and then prepared retrovirus packaging cells producing RCAS-Bcl-2 and RCAS-Shh. A pellet containing 105 cells was injected into the lateral cerebellum of newborn mice expressing the tv-a transgene under control of the Nestin promoter. For experiments in which transfer of both Bcl-2 and Shh was the goal, the cell pellet was prepared by mixing equal numbers of both retrovirus-producing cells. The Nestin promoter directs expression of TV-A to central nervous system progenitor cells, thereby ensuring that RCAS retroviruses infect immature precursor cells in the injected region of the cerebellum. Because GNPs comprise the majority of cells in the postnatal cerebellum (14) and because the Nestin gene is expressed throughout the external granule layer (15), GNPs are expected to make up the majority of TV-A–expressing cells in the cerebellum when the injections took place. Figure 1G shows immunoreactive staining for TV-A in the external granule layer and the developing molecular layer of the cerebellum of Ntv-a mice on postnatal days 1 and 3—the time interval during which we carried out the injections. Another possible RCAS target in the postnatal cerebellum is a population of neural stem cells located in the white matter and expressing both nestin and prominin (14). Targeting ectopic Shh to these cells is unlikely to induce tumors because, unlike GNPs, they do not proliferate in response to Shh (14). RCAS retroviruses could also infect nestin-expressing Bergmann glia. Nevertheless, RCAS-mediated overexpression of Shh is unlikely to transform these cells because Shh promotes astrocytic differentiation of Bergmann glia in the postnatal cerebellum (16).


Figure 1
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  		Figure 1. Histopathology of medulloblastomas induced by Shh + Bcl-2. A, transaxial brain section showing tumor (Tu) in the dorsolateral cerebellum (H&E). B, microscopic appearance of medulloblastoma induced by Shh + Bcl-2, showing homogeneous sheets of tumor cells with hyperchromatic nuclei and scant cytoplasm (H&E). C and D, immunoperoxidase staining of medulloblastoma induced by RCAS-mediated transfer of hemagglutinin-tagged Shh and untagged Bcl-2. F7 antibody against the hemagglutinin epitope detects expression of retroviral Shh (C) and Mab100 antibody, specific for human Bcl-2, detects expression of retroviral Bcl-2 (D) in the cytoplasm of tumor cells. There is no immunoreactive staining in the molecular layer (Mo), Purkinje cell layer (Pu), or internal granule layer (IGL) of the adjacent cerebellum. E, double immunofluorescence staining of medulloblastoma induced by Shh + Bcl-2. FITC-labeled F7 antibody and rhodamine-labeled Mab100 detect expression of retroviral Shh and Bcl-2. The merged image shows expression of Shh (green), Bcl-2 (red), and both proteins (yellow; arrows) in individual tumor cells. F, immunoperoxidase staining of Shh-induced medulloblastoma with Math1-specific antibody shows nuclear immunoreactivity in tumor cells. G, immunoreactive staining for TV-A in the external granule layer (EGL) and the developing molecular layer of the cerebellum of Ntv-a mice on postnatal days 1 and 3. Bar, 1 mm (A), 25 µm (B–D), 16 µm (E and F), and 22 µm (G).

 
Animals showing increased head circumference (a sign of internal hydrocephalus) or debilitation were sacrificed and analyzed immediately. All remaining mice were sacrificed for analysis 12 weeks after injection. To assess tumor formation, the brains were dissected, sectioned, and stained with H&E. Medulloblastomas were detected in 34% of mice injected with RCAS-Shh alone (Table 1 ), consistent with the ability of constitutive Shh pathway activation in the developing cerebellum to induce medulloblastomas (2). As shown in Table 1, tumor incidence increased dramatically in mice injected with RCAS-Shh and RCAS-Bcl-2 together (78%; P = 0.0001 by {chi}2 contingency test). No tumors developed in mice that were injected with RCAS-Bcl-2 alone.


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  		Table 1. Medulloblastoma induction by RCAS-mediated transfer in Ntv-a mice

 
Brain specimens were scored as positive if they contained tumors large enough to show a clear cytologic pattern of medulloblastoma. We estimated tumor size by measuring cross-sectional area from transaxial sections through the cerebellum. As shown in Table 1, the median size of tumors induced by Shh + Bcl-2 (7.2 mm2) was larger than that of Shh-induced tumors (6.2 mm2; P = 0.01 by t test). Tumors induced by Shh + Bcl-2 ranged from 0.3 to 50.3 mm2, a size range corresponding to 1% to 182% of the average cross-sectional area of the brain stem and cerebellum of 3-month-old mice. Tumor latency was equivalent (48 days for Shh + Bcl-2 and 47 days for Shh). This suggests that Bcl-2 overexpression confers a growth advantage, perhaps by allowing expansion of Shh-stimulated cells that would otherwise be eliminated by compensatory activation of intrinsic cell death programs.

Tumors induced by Shh + Bcl-2 arise from neuronal precursors and resemble human medulloblastomas. The tumors arose in the dorsolateral cerebellum at the injection sites (Fig. 1A). Microscopically, the tumors induced by Shh and Shh + Bcl-2 resembled the most common histologic pattern found in human medulloblastomas, the classic subtype, which is characterized by densely packed sheets of cells with hyperchromatic nuclei and scant cytoplasm (Fig. 1B). To verify in vivo expression of genes we transferred via RCAS vectors, we showed specific staining of tumor cells with antibodies directed against the encoded proteins. To detect expression of retroviral Shh, we used an antibody against a hemagglutinin epitope appended to the carboxy terminus of Shh (Fig. 1C). For retroviral Bcl-2, we used an antibody (Mab100) that specifically detects human Bcl-2 and does not cross-react with endogenous mouse Bcl-2 (Fig. 1D). We found that >80% of tumor cells expressed retroviral Bcl-2, but only 30% had detectable levels of retroviral Shh. Double immunofluorescence staining showed that 5% to 10% of tumor cells coexpressed Shh and Bcl-2 (Fig. 1E). Thus, the induced tumors are mixtures of clones originating from cells infected with Shh alone or in combination with Bcl-2. Consistent with our previous reports on medulloblastomas induced by Shh and Myc oncoproteins (6, 13), these results suggest that Shh, a secreted protein, exerts a paracrine effect on neighboring cells.

To assess the differentiation status of the tumor cells, we carried out an immunocytochemical analysis with markers characteristic of immature neurons, mature neurons, and medulloblastoma. All tumors induced by Shh + Bcl-2 showed abundant ßIII tubulin and NeuN, markers of early neuronal differentiation, as well as the high-affinity neurotrophin receptor TrkC and the neurosecretory protein synaptophysin. We did not detect expression of neurofilament protein, a marker of terminally differentiated neurons, in any tumor. Immunoreactive staining for the astrocytic marker glial fibrillary acidic protein was visible only in processes of entrapped astrocytes and was not seen in tumor cells. The preferential expression of neuronal markers supports an origin of the induced tumors from neuronal precursors. We reported this same profile of neuronal differentiation markers in other mouse models of Shh-dependent medulloblastoma (4, 6, 13). Furthermore, the tumor cell nuclei showed positive immunoreactive staining for Math1 (mouse atonal homologue), a transcription factor that is specifically expressed in GNPs during normal cerebellar development (ref. 17; Fig. 1F).

It is possible that ectopic expression of Shh drives nestin+ cerebellar progenitors away from a glial fate toward a neuronal lineage. Studies from the developmental neurobiology literature, however, suggest that this is unlikely. In the cerebellum, Shh promotes postnatal differentiation of an already committed astrocytic lineage, Bergmann glia (16). In the developing spinal cord, Shh drives expression of early oligodendrocyte markers (18). Ectopic expression of Bcl-2, although not sufficient to induce tumors in the absence of Shh, could have perturbed cerebellar development, at least near the injection sites. In support of this, we observed focal areas of malformed cerebellar cortex containing cells expressing retroviral Bcl-2 (Supplementary Data). Alternatively, injection-related trauma could have been the cause.

Apoptosis suppression in Shh-dependent medulloblastomas—a role for phosphatidylinositol 3-kinase signaling. Bcl-2 protects cells from a wide variety of apoptosis-inducing stimuli that lead to mitochondrial outer membrane permeability (reviewed in ref. 19). Nevertheless, Bcl-2 cannot block all causes of apoptotic death, like activation of tumor necrosis factor receptors. To assess the effect of RCAS-mediated Bcl-2 overexpression on apoptosis, we carried out immunoperoxidase staining of tumor-bearing brain sections using an antibody that specifically detects the active proteolytic fragment of caspase-3 (cleaved caspase-3). Immunoreactive staining for this effector caspase is a reliable indicator of both mitochondria-mediated (intrinsic) and death receptor–mediated (extrinsic) apoptosis. The percentage of cells positive for cleaved caspase-3 (apoptotic index) in mouse medulloblastomas induced by Shh was 1.4 ± 0.9%, which is comparable with that reported in human medulloblastomas (20). As shown in Fig. 2A , the apoptotic index was suppressed in tumors induced by Shh + Bcl-2 to 0.3 ± 0.03% (P < 0.0001), thus confirming that the retroviral Bcl-2 was functional in vivo.


Figure 2
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  		Figure 2. Analysis of apoptosis and proliferation in medulloblastomas induced by Shh + Bcl-2. Columns and representative photomicrographs, percentage of tumor cells with positive immunoreactive staining for cleaved caspase-3 (apoptotic index; A) and Ki67 (proliferation index; B) in medulloblastomas induced by Shh alone or in combinations with Bcl-2, IGF-II, and Akt. A, overexpression of Bcl-2 or activation of PI3K signaling by IGF-II and Akt decreased tumor cell apoptosis in Shh-induced medulloblastomas. B, proliferation was comparable among tumors induced by Shh, Shh + Bcl-2, and Shh + IGF-II, and Shh + Akt. Columns, mean; bars, SE. Bar, 25 µm.

 
We showed previously that activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway by IGF-II or its downstream effector Akt synergizes with Shh to promote aggressive medulloblastoma growth in vivo (4). Because PI3K signaling has overlapping positive effects on cell cycle progression and survival in many cell types, it was unclear whether IGF was enhancing medulloblastoma growth by supplementing Shh-stimulated proliferation of GNPs (21) or by independently blocking apoptosis. We found that apoptosis was suppressed in mouse medulloblastomas induced by Shh + IGF-II and Shh + Akt to levels approaching that found in tumors induced by Shh + Bcl-2 (Fig. 2A). We then measured the percentage of tumor cells advancing through the cell cycle by immunostaining for Ki67, a protein expressed during cell cycle phases G1-M. Figure 2B shows that the proliferation index was comparable among tumors induced by Shh (17%), Shh + Bcl-2 (21%), Shh + IGF-II (21%), and Shh + Akt (24%). These results suggest that the prosurvival component of PI3K signaling is a more significant factor in the ability of this pathway to enhance Shh-induced medulloblastoma formation than its proproliferative component.

The hypothesis that uncoupling proliferation from apoptosis is required for neoplastic transformation (7) predicts that medulloblastomas induced by Shh alone should not be able to mount an effective apoptotic response. One mechanism whereby Shh might disable apoptosis is by up-regulating Bcl-2 expression. In the developing chick neural tube, Shh signaling promotes proliferation and inhibits apoptosis of neuroepithelial cells (22). Furthermore, Gli transcriptional activation, a downstream Shh response, increases Bcl-2 expression (22). Tight correlation between Bcl-2 and Gli1 mRNA levels in human medulloblastoma specimens, coupled with the observation that inhibiting Shh signaling in established cell lines by cyclopamine treatment can lower Bcl-2 levels and stimulate apoptosis, further supports the idea that Bcl-2 is a Shh transcriptional target (23). To determine whether expression of endogenous Bcl-2 was up-regulated in medulloblastomas induced by Shh in our mouse model, we probed tumor sections with an antibody (MAbC2) against mouse Bcl-2. Figure 3A shows immunoreactive staining in normal mouse spleen. Tumors induced by Shh + Bcl-2 showed intense staining because of cross-reactivity with human Bcl-2 expressed at high levels by the integrated RCAS retrovirus (Fig. 2B). MAbC2 did not detect endogenous Bcl-2, however, in mouse medulloblastomas induced by RCAS transfer of Shh alone (Fig. 3C) or in combination with N-Myc, IGF-II, or Akt—molecules that enhance Shh-dependent medulloblastoma formation. Figure 3D shows the absence of Bcl-2 immunoreactivity in a tumor induced by Shh + N-Myc.


Figure 3
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  		Figure 3. Immunocytochemical analysis of endogenous Bcl-2 and activated Akt in Shh-dependent medulloblastomas. Monoclonal antibody MabC2 detects endogenous Bcl-2 in normal mouse spleen (A) and retrovirally expressed (human) Bcl-2 in tumors induced by Shh + Bcl-2 (B). MabC2 does not detect endogenous Bcl-2 in mouse medulloblastoma induced by Shh (C) or Shh + N-Myc (D). Mab9277 antibody detects expression of Akt, activated by phosphorylation of amino acid residue S473, in the nucleus (arrowheads) and cytoplasm (arrows) of tumor cells in medulloblastomas induced by Shh (E) and Shh + IGF-II (F). Bar, 25 µm (A–D), 16 µm (E and F).

 
Shh-driven hyperproliferation of GNPs could render these cells susceptible to secondary mutations that disabled their apoptotic response via molecular mechanisms that do not require Bcl-2. The complexity of apoptosis control programs makes a comprehensive analysis of Bcl-2–independent mechanisms very difficult in our in vivo model system. Nevertheless, one way for tumor cells to evade apoptotic death without raising Bcl-2 levels is by inactivating p53. In fact, loss of p53 increases medulloblastoma penetrance in mice with targeted deletions of genes governing DNA repair or cell cycle control (24, 25). We did not detect p53, however, in Shh-induced medulloblastomas by immunostaining tissue sections with an antibody (DO-1) that detects elevated levels of p53 protein—a characteristic signature of cells that have mutant (defective) p53 alleles (data not shown).

Another mechanism whereby Shh-stimulated cells could escape negative selection normally imposed by intrinsic cell death programs is through enhanced PI3K signaling, a reliable indicator of which is phosphorylation of Akt at its activating amino acid S473. We found that the majority of tumor cells showed immunoreactive staining for pS473Akt (in either the nucleus or the cytoplasm) in mouse medulloblastomas induced by Shh (Fig. 3E). The percentage of pS473Akt-positive cells was equivalent to that in medulloblastomas in which the PI3K pathway was activated directly by RCAS-mediated overexpression of IGF-II (Fig. 3F). Thus, activated PI3K signaling is one plausible, intrinsic mechanism for suppressing apoptosis in Shh-driven medulloblastomas.

Shh is essential for transformation of GNPs. In an earlier report, we showed that ectopic expression of N-Myc strongly enhanced Shh-induced medulloblastoma formation, whereas elevated levels of N-Myc alone were not sufficient for tumor induction (6). A possible explanation for the inability of N-Myc to transform GNPs is that Myc oncoproteins can coordinately stimulate cell proliferation and apoptosis (26). Therefore, we asked whether N-Myc could induce medulloblastomas if Myc-induced apoptosis were overcome by coexpression of Bcl-2. To address this question, we used an RCAS vector to drive expression of an N-myc allele encoding a stabilized protein (N-MycT50A) that is resistant to glycogen synthase kinase 3ß–mediated degradation (5). N-MycT50A stimulates GNP proliferation and enhances Shh-dependent medulloblastoma formation more potently than wild-type N-Myc (5, 6). Nevertheless, we could not generate tumors in mice by RCAS-mediated transfer of Bcl-2 + N-MycT50A (Table 1). These results indicate that Shh has essential transforming functions in GNPs not supplied by the mitogenic stimulus of N-Myc combined with a strong antiapoptotic signal provided by Bcl-2.

Using a somatic cell gene transfer model, we report here that blockade of apoptosis by Bcl-2 cooperates with Shh-stimulated proliferation to transform cerebellar progenitor cells and induce aggressive medulloblastomas. A clinical correlation study did not show a correlation between Bcl-2 expression and patient prognosis (10), suggesting that Bcl-2 might not be required for the maintenance of established tumors. Nevertheless, the findings presented here provide insights into the molecular signals that initiate medulloblastoma formation and they support the importance of blocking apoptosis in carcinogenesis.


   Acknowledgments
 
Grant support: NIH CA108622.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Chris Rodesch (Cell Imaging Facility, University of Utah Health Sciences Center) for assistance with confocal microscopy and Dr. Andrew Leavitt (University of California San Francisco) for the TV-A antibody.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 11/13/06. Revised 3/13/07. Accepted 3/22/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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