|Home | About | Journals | Submit | Contact Us | Français|
During development, proliferation of cerebellar granule neuron precursors (CGNPs), candidate cells-of-origin for the pediatric brain tumor medulloblastoma, requires signaling by Sonic hedgehog (Shh) and insulin-like growth factor (IGF), whose pathways are also implicated in medulloblastoma. One of the consequences of IGF signaling is inactivation of the mTOR-suppressing Tuberous Sclerosis Complex (TSC), comprised of TSC1 and TSC2, leading to increased mRNA translation. We show that mice in which TSC function is impaired display increased mTOR pathway activation, enhanced CGNP proliferation, GSK-3α/β inactivation, and cytoplasmic localization of the cyclin-dependent kinase (cdk) inhibitor p27Kip1, which has been proposed to cause its inactivation or gain of oncogenic functions. We observed the same characteristics in wild-type primary cultures of CGNPs in which TSC1 and/or TSC2 were knocked down, and in mouse medulloblastomas induced by ectopic Shh pathway activation. Moreover, Shh-induced mouse medulloblastomas manifested Akt-mediated TSC2 inactivation, and the mutant TSC2 allele synergized with aberrant Shh signaling to increase medulloblastoma incidence in mice. Driving exogenous TSC2 expression in Shh-induced medulloblastoma cells corrected p27Kip1 localization and reduced proliferation. GSK-3α/β inactivation in the tumors in vivo and in primary CGNP cultures was mTOR-dependent, whereas p27Kip1 cytoplasmic localization was regulated upstream of mTOR, by TSC2. These results indicate that a balance between Shh mitogenic signaling and TSC function regulating new protein synthesis and cdk inhibition is essential for normal development and prevention of tumor formation or expansion.
Medulloblastoma is the most common malignant solid pediatric tumor. These tumors arise in young children in the cerebellum, a dorsal brain structure. Current treatment regimens for medulloblastoma cause developmental, behavioral, and cognitive disturbances in long-term survivors. These devastating side effects underscore the need to understand the basic mechanisms underlying medulloblastoma initiation, expansion, and recurrence, so that novel treatments can be developed which specifically target tumor cells without damaging the developing brain.
Cerebellar granule neuron precursors (CGNPs), a proposed cell-of-origin for medulloblastomas (1), proliferate rapidly during a transient post-natal expansion phase in the cerebellar external granule layer a (EGLa). CGNPs become post-mitotic in the EGLb, then migrate inward and terminally differentiate in the internal granule layer (IGL). The CGNP expansion phase requires signaling by Shh and insulin-like growth factor (IGF) (2, 3). Aberrant Shh pathway activation is found in sporadic medulloblastomas and in Gorlin’s Syndrome, in which patients carry mutations or deletions in the Patched (Ptc) gene (4, 5), a key inhibitory component of the Shh receptor complex.
IGF pathway activity is also found in medulloblastomas in both mice and humans (6, 7). IGF signaling activates the kinase mammalian target of rapamycin (mTOR), which promotes mRNA translation. Growth factors activate mTOR by turning off the tuberous sclerosis complex (TSC), composed of TSC1 (hamartin) and TSC2 (tuberin), which normally keeps mTOR in check (8, 9). TSC1 stabilizes TSC2 protein; TSC2’s GTPase activating protein (GAP) function inhibits mTOR. TSC2 over-expression results in decreased mTOR activity, while abrogating TSC1 or TSC2 function results in increased mTOR activity and excessive cell growth. Conversely, TSC1 or TSC2 over-expression impedes cell cycle progression (10, 11). TSC2 positively regulates the cyclin dependent kinase inhibitor p27Kip1 by preventing its degradation (12) and promoting its nuclear localization (13). Akt-mediated TSC2 phosphorylation impairs its ability to inhibit mTOR/S6K, but does not affect TSC2-mediated control of p27Kip1, indicating that TSC2’s regulation of mTOR and p27Kip1 are separable functions.
In the developing cerebellum, p27Kip1 is expressed the EGLb, the molecular layer, and the IGL (14, 15). In adult cerebella, p27Kip1 is expressed in cells of the IGL. p27Kip1 heterozygous or null mice possess cerebella that are larger than those of wild-type mice (14). Importantly, loss of p27Kip1 in Patched +/− mice increased the incidence of medulloblastoma (16). CGNPs prepared from p27Kip1-deficient mice show enhanced proliferation compared with CGNPs from wild-type mice, (14). The Roussel and Eisenman groups have demonstrated an antagonistic relationship between p27Kip1 and the Shh target N-myc during cerebellar development (17, 18). N-myc and D-type cyclins are destabilized by GSK-3α/β activity (2, 19).
We used transgenic mice constitutively expressing a dominant negative TSC2 allele (20) to investigate whether TSC inhibition plays roles in CGNP expansion and medulloblastoma. These mice possess clusters of granule neurons that fail to migrate to the internal granule layer, and they have cerebellar development abnormalities consistent with enhanced CGNP proliferation. CGNPs from these mice had mTOR signaling pathway activity resembling that of mouse Shh-driven medulloblastomas, including enhanced rp S6 phosphorylation, inactivation of GSK-3α/β and cytoplasmically localized p27Kip1. In Shh-driven medulloblastoma cells, introduction of wild-type TSC2 caused p27Kip1 to go to the nucleus and reduced proliferation. Interestingly, p27Kip1 was not re-localized to the nucleus by treatment with mTOR inhibitors, indicating that its sub-cellular localization is regulated by TSC2 upstream of mTOR. Our results suggest that loss of TSC activity can synergize with Shh signaling to alter cerebellar development and enhance medulloblastoma expansion via combined effects on mTOR activation, GSK-3α/β inactivation and p27Kip1 cytoplasmic localization.
We wished to determine how the TSC complex may be regulated in the context of normal and oncogenic Shh signaling. However, TSC1-null and TSC2-null mice die in utero; TSC1+/− and TSC2+/− mice are not reported to have cerebellar abnormalities (21), suggesting that the remaining TSC alleles are sufficient to support TSC function in the developing brain in those mice. Therefore, we made use of mice transgenic for a TSC2 allele lacking Rheb-GAP function (20), termed TSC2-RGΔ. The cerebellum of these mice was reported to contain “rests”, which may represent failed pre-neoplastic lesions (22, 23), but they do not develop medulloblastomas (20), consistent with the observation that humans with TSC mutations are not predisposed to medulloblastoma.
We evaluated TSC2-RGΔ mice at three stages of cerebellar development: Post-natal day (PN) 7, PN 15, and adult. RT-PCR analysis confirmed expression of the dominant negative TSC2 transgene (Supplementary Figure 1A). TSC2-RGΔ mice have a significantly thicker EGL compared to wild-type controls in PN 7 (Figure 1A, arrow, quantification shown in Supplementary Figure 1B). At PN 15, when CGNPs from wild-type mice have left the EGL, TSC2-RGΔ mice still possessed an EGL several cells thick. “Rests” were observed at the cerebellar surface in TSC2-RGΔ adult cerebella as previously reported (20).
We focused on PN7, the stage of development at which CGNP proliferation is at its peak, to identify mechanisms through which TSC inactivation contributes to CGNP proliferation. The cdk inhibitor p27Kip1 is an essential negative regulator of CGNP proliferation (14). In cell lines, p27Kip1 has been reported to interact with TSC2, which retains p27Kip1 in the nucleus (10, 13). When we analyzed p27Kip1 protein in wild type and TSC2-RGΔ cerebella, we detected nuclear p27Kip1 in PN 7 wild type mice (Figure 1B) in the EGLb, where CGNPs are leaving the cell cycle. In contrast, p27Kip1 was distributed throughout the EGL of TSC2-RGΔ mice. This p27Kip1 was entirely located in the cytoplasm, where other studies have proposed that it becomes degraded or takes on inappropriate functions (24–26).
The TSC2-RGΔ cerebella display widespread rp S6 phosphorylation in the mitotic region EGLa and differentiated region IGL (Supplementary Figure 1C), indicating up-regulation of mTOR activity. Consistent with impaired cell cycle exit, the EGL of TSC2-RGΔ mice had increased Ki67 levels (Figure 1C) at PN 7 and at PN 15. Next, we examined expression of cyclin D2, an effector of N-myc activity in the developing mouse cerebellum (27). The expanded EGL at PN7 and the retained EGL at PN 15 of TSC2-RGΔ cerebella have increased cyclin D2 expression (Figure 1D). These findings suggest that TSC inactivation leads to abnormal cerebellar development due to increased proliferation in the EGL, associated with mis-localized p27Kip1.
Addition of Shh to the medium permits proliferation of CGNP primary cultures (28–30) while exogenous IGF is required for their survival (31, 32). Shh treatment elicited a significantly higher level of proliferation in TSC2-RGΔ CGNPs as compared with wild-type CGNPs (Figure 2A) as determined by BrdU incorporation and histone H3 phosphorylation. In comparison with wild-type CGNPs, TSC2-RGΔ CGNPs have increased endogenous levels of N-myc and cyclin D2 (Figure 2B). These results indicate that TSC2-RGΔ CGNPs have an increased intrinsic capacity for proliferation and that TSC inhibition may cooperate with Shh signaling to further enhance CGNP proliferation.
Since Shh-mediated medulloblastomas and TSC2-RGΔ mice might have mutations in other pathways that result in p27Kip1 dys-regulation and GSK-3α/β inactivation, we wished to determine how acute shRNA-mediated disruption of TSC signaling in wild-type CGNPs affects p27Kip1 and GSK-3α/β. As shown in Figure 2C, infection with shRNA-carrying lentiviruses targeting TSC1 and/or TSC2 effectively reduced their protein levels in infected CGNPs. GFP knock-down in CGNPs derived from Math-GFP mice did not affect mTOR pathway activity (Figure 2C).
TSC knockdown resulted in increased rp S6 phosphorylation. Compared to control-infected CGNPs, there was an increase of phospho-p27Kip1 (Ser10) levels following TSC knockdown. Phosphorylation at this site promotes p27Kip1 export to the cytoplasm (33, 34). Treatment with mTOR inhibitor rapamycin blocked S6 phosphorylation, but only modestly changed phospho-p27Kip1 levels compared to control CGNPs, suggesting that regulation of p27Kip1 in CGNPs depends on the TSC, and is not downstream of mTOR. These results contrast with a recent study carried out in cell lines, wherein p27Kip1 localization was found to be regulated downstream of mTOR (35). We also observed increased GSK-3α/β phosphorylation following TSC knockdown, which was reduced by rapamycin treatment. Cyclin D2, a CGNP proliferation marker, and Ki67 immunostaining show that proliferation is increased by TSC knockdown (Figure 2C, D).
TSC2-RGΔ CGNPs showed increased phosphorylation of the serine/threonine kinase mTOR and rp S6(Figure 3A), consistent with maintained mTOR activation, in comparison with wild type CGNPs. Interestingly, the TSC2-RGΔ cerebella also had increased levels of phosphorylated GSK-3α/β and Ser 10 phosphorylated p27Kip1 (25, 33, 34). Likewise, we detected phosphorylated mTOR, rp S6, GSK-3α/β, and p27Kip1 (Ser10) in medulloblastomas from Ptc+/− mice and SmoA1 mice, which express an activated mutant allele of the Shh receptor component Smoothened (36). Thus, the signaling abnormalities in TSC2-RGΔ cerebella are also present in mouse medulloblastomas. Taken together, our results indicate that in TSC inactivation in TSC2-RGΔ mice and Shh-induced mouse medulloblastomas could promote cell cycle progression as a result of increased mTOR-mediated protein synthesis, mis-localized p27Kip1, and inactivated GSK-3α/β, which is predicted to result in N-myc and D-type cyclin stabilization (2, 19).
We next introduced the TSC2-RGΔ transgene into mice with a heterozygous null mutation for Ptc and the NeuroD2-SmoA1 mice. Dominant negative TSC2 increased medulloblastoma incidence in both Ptc +/− (53.8% vs. 25%) and SmoA1 (72% vs. 52.8%) mice (Figure 3B). Also, TSC2 inactivation in SmoA1 tumor-bearing mice decreases survival latency (Figure 3C). Introducing the TSC2-RGΔ transgene increased phosphorylation of rp-S6, GSK-3α/β, and p27Kip1 (Figure 3D) in the tumors. Taken together, these results suggest that Shh signaling and TSC inactivation can cooperate to enhance medulloblastoma formation.
In order to determine whether TSC inactivation and p27Kip1 dys-regulation are related, we turned to a Shh-associated medulloblastoma cell line model, the Pzp53med cell line, derived from a Ptc+/−p53−/− mouse medulloblastoma (37). Recently the Scott group characterized Gli expression and sensitivity to sterol synthesis inhibitors in these cells (38). We found robust levels of phospho-rp S6 in Pzp53med cells (Figure 4A, left panel). Consistent with mTOR activation lying downstream of Akt, which phosphorylates and inactivates TSC2 (13, 39), treatment of the Pzp53med cells with the PI-3 kinase inhibitor wortmannin eliminated rp-S6 phosphorylation (Figure 4A, middle panel). Rapamycin treatment also eliminated rp-S6 phosphorylation (Figure 4A, right panel). Moreover, total TSC2 and p27Kip1 levels were low (Figure 4B, left panel) in the Pzp53med cells. Wortmannin treatment increased TSC2 protein levels and returned p27Kip1 to the nucleus, whereas rapamycin did not, suggesting that TSC2 inactivation and p27Kip1 stabilization are regulated upstream of mTOR (Figure 4B, middle and right panel) in the Pzp53med cells, as in CGNPs. To determine whether p27Kip1 localization was dependent on TSC in these cells, we introduced exogenous TSC2 by transient transfection. Cells expressing TSC2 had nuclear p27Kip1 (Figure 4C, left and middle panel). Indeed, nearly 90% of TSC2-positive cells had nuclear p27Kip1, as compared to only 10% of TSC2-negative cells (Figure 4C, right panel). These results suggest that low levels of TSC2 may be associated with increased p27Kip1 turnover in the medulloblastoma cells. To determine whether reconstituting TSC activity impedes cell cycle progression in Pzp53med cells, we co-immunostained for TSC2 and BrdU. We observed an inverse correlation between TSC2-positive cells and BrdU-positive cells: over-expression of TSC2 reduced proliferation by ~4 fold (Figure 4D). Finally, TSC2 and p27Kip1 are rapidly degraded in the cytoplasm of Pzp53 medulloblastoma cells (Figure 4B and Supplementary Figure 2, left panel). TSC2 accumulates in Pzp53med cells treated with the proteasome inhibitor lactacystin (Supplementary Figure 2, middle panel), and its localization to the nucleus is associated with increased nuclear p27Kip1 (Supplementary Figure 2, right panel). Together, our results show that p27Kip1 localization depends on TSC2 expression and function.
We wished to determine whether mTOR inhibition affects tumor growth and p27 localization in SmoA1 medulloblastoma in vivo. We treated wild-type adult mice with the mTOR inhibitor CCI-779 for 9–10 days using intra-peritoneal injection of the drug. Data shown are representative of results from 5 treated mice and 5 untreated mice. Before treatment, phosphorylation of rp-S6 is restricted to Purkinje cells of normal adult cerebella. Following treatment, phosphorylated rp-S6 was virtually undetectable (Figure 5A). As shown in Figure 3 and Figure 5A, B, SmoA1 medulloblastomas possessed Bmi1(40, 41), a progenitor cell marker, and phosphorylated rp S6, indicating mTOR pathway activation. The phospho-rp S6 signal was not found in GFAP-positive cells (ie astrocytes) present in the tumor (Figure 5A). Both phospho-GSK-3α/β and Akt-mediated phosphorylation of TSC2 were found in tumor cells (Figure 5B, top row). Since we observe that TSC2 is phosphorylated on inactivating sites in SmoA1 medulloblastomas, we asked whether p27Kip1 was mis-localized in these tumors. As shown in Figure 5B (top row), p27Kip1 was found in the cytoplasm of the tumor cells. Indeed, no cells were observed with nuclear p27Kip1. We also detected only cytoplasmic p27Kip1 in Ptc+/− medulloblastomas (data not shown).
Recent reports (35, 42) have indicated that GSK-3α/β can be phosphorylated downstream of mTOR, and that p27Kip1 localization can be regulated downstream of mTOR. Following CCI-779 treatment, rp-S6 phosphorylation was eliminated in NeuroD2-SmoA1 medulloblastomas (Figure 5B, bottom row). GSK-3α/β phosphorylation was also down-regulated, suggesting that as in primary CGNPs (Figure 2 and Figure 3), GSK-3α/β phosphorylation is dependent on mTOR under TSC-inactive conditions (Figure 5B, bottom row). The loss of GSK-3α/β phosphorylation was not due to effects of CCI-779 on Akt, as TSC2 phosphorylation on the Akt-targeted site T1462 was not affected by CCI-779 treatment (Figure 5B, bottom row). Lastly, CCI-779 treatment did not promote p27Kip1 re-localization to the nucleus, indicating that in medulloblastomas, p27Kip1 subcellular localization is regulated upstream of mTOR (Figure 5B, bottom row). Quantification of histone H3 phosphorylation (Figure 5C) revealed a significant reduction in proliferation in treated medulloblastomas, as did western blotting for N-myc and cyclin D2 (Figure 5D). We also observed decreased levels of vascular endothelial growth factor (VEGF) protein following CCI-779 treatment (Figure 5D). Previous reports have shown that mTOR induces VEGF production, which is vital for tumor growth through angiogenesis (43).
Somatic mutations in TSC1 or TSC2 cause tuberous sclerosis (TS) (39, 44). Although TS patients themselves are not predisposed to develop medulloblastoma, the TSC can be inactivated by phosphorylation downstream of growth factors associated with medulloblastoma, such as PDGF and IGF, and a small study identified a point mutation in TSC2 in a subset of medulloblastomas (45). Here we have asked whether loss of TSC activity may contribute to proliferation during normal cerebellar development and to incidence or growth of medulloblastomas.
We found that mouse Shh-mediated medulloblastomas had high levels of mTOR activity, and biochemically resembled CGNPs derived from mice constitutively expressing a dominant negative TSC2 allele (TSC2-RGΔ mice). When we analyzed cerebellar development in these mice, we found that their CGNPs had an extended proliferative capacity in vivo, and in vitro they exhibited Shh-independent proliferation. When the TSC2-RGΔ allele was introduced into mice with aberrantly activated Shh signaling, the incidence of medulloblastoma was markedly increased, with reduced latency to tumor formation. These lines of evidence suggest that inactivation of the TSC complex can contribute to medulloblastoma formation, perhaps as a “second hit”. Indeed, when we recently analyzed a human tumor collection for TSC-inactivating mutations, we identified TSC1 deletions in a significant number of tumors, and these tumors belonged to the Shh-associated subclass of medulloblastomas (Supplementary Figure 3). The only known function of TSC1 is to stabilize TSC2 (46), therefore loss of TSC1 in tumors results in loss of TSC2 activity. We also explored the expression analysis of TSC2 and CDKN1B (p27Kip1) in human Shh-subgroup medulloblastomas and found moderate decreases compared to adult cerebellum (Supplementary Figure 4). TSC2 and p27 are not classic tumor suppressors, as they are rarely mutated or deleted in cancer, but they are often deregulated in cancer by post-translational modifications, as supported by our study and previous reports (47–49).
Interestingly, in the TSC2-RGΔ cerebellar EGL and NeuroD2-SmoA1 medulloblastomas, we observed p27Kip1 in the cytosol in all cells, associated with increased levels of phosphorylation on Ser10. Phosphorylation on p27Kip1 Ser10 leads to its nuclear export and increased stability in the cytosol (33). When we transfected Pzp53med cells with exogenous TSC2, we found nuclear p27Kip1 in nearly 90% of TSC2-positive cells. A correlation between poor prognosis and cytoplasmic localization of p27Kip1 in human tumors has led to the hypothesis that p27Kip1 has an active tumor-promoting function in the cytoplasm (49). A recent study tested for CDK-independent functions of p27Kip1 by analyzing knock-in mice expressing a mutant p27 (p27CK−) that is unable to inhibit cyclin–CDK complexes (24). These mice developed various tumors, and the p27CK− mutant localizes to the cytoplasm, hinting that a cytoplasmic function might mediate oncogenic effects of p27Kip1.
Taken together, our findings suggest the model shown in Figure 6. Under normal developmental conditions, TSC modulates mTOR signaling, and stabilizes p27Kip1 (Figure 6A). Under conditions of TSC loss (Figure 6B), mTOR activity is increased, p27Kip1 is localized to the cytoplasm where it may be degraded, inactivated, or gain abnormal functions, and GSK-3α/β is phosphorylated (42), thus increasing N-myc protein levels and CGNP proliferation. In the setting of Shh-induced medulloblastoma (Figure 6C), signaling by growth factors such as PDGF (9) or IGF can lead to TSC inactivation via phosphorylation by Akt or Erk2; alternately the TS complex can be disrupted by mutation. Signaling by mTOR is activated, leading to increased translation of growth-associated proteins and potential inhibition of GSK-3α/β. These factors can all contribute to enhanced tumor growth or decreased latency.
Development of hedgehog pathway inhibitors such as cyclopamine and HhAntag (50) has raised the hope that targeting this pathway will be an effective anti-medulloblastoma strategy (50, 51). However, it has recently been shown that systemic treatment of young animals with hedgehog pathway inhibitors results in devastating, irreversible effects on bone growth (52). Gli1-transformed epitheloid cells require mTOR activity for their survival (53). Our observation that the mTOR pathway is activated in mouse and human medulloblastomas suggests that inhibitors of this pathway may be useful. Moreover, our demonstration that modulation of mTOR is not the only tumor suppressive function of the TSC in the developing brain and in brain tumors suggests that using drugs to block activation of TSC2-inhibiting pathways, or that can promote p27Kip1 nuclear localization may present even more effective future medulloblastoma therapies.
TSC2-RGΔ, Ptc+/−, and NeuroD2-SmoA1 mice were generated and maintained on C57/BL6 background. Mice were managed according to MSKCC IACUC policies described in AMK’s IACUC-approved protocol. NeuroD2-SmoA1 mice were provided by Dr. Jim Olson (Fred Hutchison Cancer Center). Math1-GFP mice were provided by Jane Johnson (UTSW).
For CGNP culture, post-natal day 4 or 5 mice were used as previously described (54). The CGNPs were treated with Sonic hedgehog (1 µg/mL, R&D Systems) to promote proliferation. Where indicated, rapamycin (Calbiochem) and wortmannin (Calbiochem) were used at 10 nM for 6 hours. Proteasome inhibitor lactacystin (Calbiochem) was used at 10 nM for 4 hrs.
Pzp53med cells (BB and AMK) (kindly provided from Dr. Matt Scott, Stanford University) were grown in DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. Pzp53med cells were transfected with either pcDNA3.1-TSC2 or pRK-TSC2 (both plasmids kindly provided by Dr. Pier Paolo Pandolfi, Harvard Medical School) using Fugene 6. Cells were fixed after 48 hrs post-transfection.
TSC1, TSC2, and GFP shRNA lentiviral constructs were obtained from Sigma. Four constructs of each were transfected into a mouse macrophage cell line or GFP-expressing cells, and western blotting was used to determine which shRNAs effectively and specifically targeted TSC1, TSC2, and GFP (data not shown, Figure 2). Effective contructs were used to prepare lentiviruses.
Protein extraction and western blotting were carried out as previously described (54). Primary antibodies were: TSC1 (Cell Signaling), TSC2 (Cell Signaling), GSK-3α/β (Upstate Biotechnology), p-GSK-3α/βSer21/9 (Cell Signaling), cyclin D2 (Santa Cruz), N-myc (Santa Cruz), p27Kip1 (BD Transduction Laboratories), p-p27Ser10 (Santa Cruz), ribosomal protein S6 (Cell Signaling), p-rp S6Ser235/236 (Cell Signaling), p-mTORSer2481 (Cell Signaling), mTOR (Cell Signaling), PCNA (Calbiochem), GFP (Calbiochem), VEGF (Santa Cruz) and β-tubulin (Sigma). HRP conjugated secondary antibodies were: Goat anti-rabbit IgG (H+L) at 1:3000 (Pierce Labs) and Donkey anti-mouse IgG (H+L) at 1:5000 (Jackson Laboratories).
Post-natal day 7 brains were fixed in 4% paraformaldehyde (PFA) at 4°C, processed for paraffin-embedding and sectioned at 10 µm. Immunohistochemistry and RNA in situ hybridization were performed using standard methods. Immunohistochemistry for phosphorylated ribomsomal S6 protein and in situ hybridization for cyclin D2 was performed on sagittal sections. Detailed protocols available at http://www.mskcc.org/mskcc/html/77387.cfm.
CGNPs and Pzp53med cells were fixed in 4% PFA for 10 minutes. Post-natal day 7, 15, and adult brains were fixed in 4% paraformaldehyde (PFA) at 4°C, processed for paraffin-embedding and sectioned at 10 µm. Immunochemistry and BrdU incorprtaion detection were carried out as described (54). Primary antibodies are listed above, with the exception of BrdU (BD Transduction Laboratories), Ki67 (Vector Labs), p-H3Ser10 (Cell Signaling), GFAP (Cell Signaling), and Bmi1 (Upstate Biotechnology). Secondary antibodies were Alexa Fluor 555 goat anti-rabbit IgG (H+L) at 1:1000 (Invitrogen) and Alexa Fluor 488 goat anti-mouse IgG (H+L) at 1:1000 (Invitrogen). BrdU incorporation was measured using MetaMorph Imaging system.
We thank Jeffrey Miller, PhD, Andy Koff, PhD, Andrej Dlugosz, PhD, and Timothy Gershon, MD, PhD for their advice, helpful discussion, and expertise.
Financial support: The American Brain Tumor Association (BB, in memory of Davis Ferguson). The NINDS (R01NS061070, AMK), Children’s Brain Tumor Foundation (AMK), the Childhood Brain Tumor Foundation of Maryland (AMK), the Pediatric Brain Tumor Foundation (AMK), the Sontag Foundation (AMK, MDT), and the Brain Tumor Center of Memorial Sloan-Kettering Cancer Center (BB).
Conflicts of Interest: None