Abstract
Glioblastoma (GBM) is a highly lethal brain tumour presenting as one of two subtypes with distinct clinical histories and molecular profiles. The primary GBM subtype presents acutely as a high-grade disease that typically harbours mutations in EGFR, PTEN and INK4A/ARF (also known as CDKN2A), and the secondary GBM subtype evolves from the slow progression of a low-grade disease that classically possesses PDGF and TP53 events1, 2, 3. Here we show that concomitant central nervous system (CNS)-specific deletion of p53 and Pten in the mouse CNS generates a penetrant acute-onset high-grade malignant glioma phenotype with notable clinical, pathological and molecular resemblance to primary GBM in humans. This genetic observation prompted TP53 and PTEN mutational analysis in human primary GBM, demonstrating unexpectedly frequent inactivating mutations of TP53 as well as the expected PTEN mutations. Integrated transcriptomic profiling, in silico promoter analysis and functional studies of murine neural stem cells (NSCs) established that dual, but not singular, inactivation of p53 and Pten promotes an undifferentiated state with high renewal potential and drives increased Myc protein levels and its associated signature. Functional studies validated increased Myc activity as a potent contributor to the impaired differentiation and enhanced renewal of NSCs doubly null for p53 and Pten (p53-/- Pten-/-) as well as tumour neurospheres (TNSs) derived from this model. Myc also serves to maintain robust tumorigenic potential of p53-/- Pten-/- TNSs. These murine modelling studies, together with confirmatory transcriptomic/promoter studies in human primary GBM, validate a pathogenetic role of a common tumour suppressor mutation profile in human primary GBM and establish Myc as an important target for cooperative actions of p53 and Pten in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal and tumorigenic potential.
High-grade malignant glioma, the most common intrinsic brain tumour, is uniformly fatal despite maximum treatment3. A wealth of molecular genetic studies has established central roles of the RTK-PI3K-PTEN, ARF-MDM2-p53 and INK4a-RB pathways in gliomagenesis3, 4. To explore the role of p53 and Pten in glioma, we used the hGFAP-Cre transgene5, 6 to delete p53 alone or in combination with Pten in all CNS lineages using conditional p53 (ref. 7) and Pten alleles (Supplementary Figs 1 and 2a–c). Because broad CNS deletion of Pten results in lethal hydrocephalus in early postnatal life (data not shown), modelling efforts henceforth emphasized the Ptenlox/+ genotype.
Clinically, between 15 to 40 weeks of ages, 42 out of 57 (73%) of the hGFAP-Cre+;p53lox/lox;Ptenlox/+ mice presented with acute-onset neurological symptoms—seizure, ataxia and/or paralysis (Fig. 1a). Histopathologically, all 42 neurologically symptomatic mice harboured malignant gliomas that were classified on the basis of WHO (World Health Organization) criteria8 as anaplastic astrocytomas (WHO III, n = 28, 66%) or GBM (WHO IV, n = 14, 34%; Fig. 1b). These GBMs had classical features of pseudopalisading necrosis, marked cellular pleomorphism, and highly infiltrative spread including perineuronal and perivascular satellitosis as well as subpial spread in the cerebral cortex (Supplementary Fig. 3a). Occasionally tumours had abnormal vessels suggestive of microvascular proliferation. All tumours showed increased mitoses (Ki67 staining) and expression of the classical human glioma markers Gfap and Nestin (Fig. 1c). Necropsy of 15 neurologically asymptomatic mice showed no cases of incipient low-grade glioma disease but 8 had high-grade pathology including very small lesions with anaplastic features of nuclear atypia, multinucleated tumour cells and/or high cellularity (Supplementary Fig. 3b). For the remaining genotypes, 4 out of 23 hGFAP-Cre+;p53lox/lox mice developed anaplastic astrocytoma (WHO III); conversely 19 out of 23 hGFAP-Cre+;p53lox/lox mice, 12 out of 12 hGFAP-Cre+;p53lox/+;Ptenlox/+ mice and 10 out of 10 hGFAP-Cre+;p53lox/+ mice had no CNS pathology and developed only non-CNS tumours (data not shown).
Figure 1: p53 and Pten inactivation cooperate to induce high-grade malignant gliomas.
a, Kaplan–Meier tumour-free survival curves for mice of indicated genotypes as a function of weeks. '+' designates the wild-type allele, 'L' denotes the conditional allele. b, Graph shows frequency and grade of gliomas versus non-CNS malignancies observed in end-stage of indicated mice from a. Asy* indicates neurological asymptomatic hGFAP-Cre+;p53lox/lox;Ptenlox/+ mice (n = 15) killed for non-CNS malignancies. c, Haematoxylin and eosin (H&E) histology and immunohistochemical staining of sections of WHO grade III and IV malignant gliomas from hGFAP-Cre+;p53lox/lox;Ptenlox/+ mice with antibodies against Ki67, Gfap and Nestin. Scale bars, 50 
m.
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Historically, TP53 inactivation has been considered a classical lesion in low-grade astrocytomas and secondary GBM but infrequently in primary GBM1, 9. The remarkable clinical and histological resemblance of this model to the primary GBM subtype in humans prompted TP53 and PTEN resequencing in human primary GBM. Of the 35 clinically annotated human primary GBM samples, 10 (29%) tumours registered prototypical TP53 mutations and 14 (40%) tumours had PTEN missense mutations, insertions, deletions or splicing mutations (Supplementary Table 1). Moreover, six out of ten tumours with TP53 mutations harboured concomitant PTEN mutations or homozygous deletion. Encouragingly, our mutational data agrees with The Cancer Genome Atlas data reporting TP53 and PTEN as the two most commonly mutated tumour suppressor genes (http://tcga-data.nci.nih.gov/tcga/findArchives.htm). These results, together with recent population-based studies10, 11, indicate that TP53 is a key tumour suppressor for both GBM subtypes.
Consistent with frequent PTEN loss of heterozygosity (LOH; 60–70%) in human high-grade glioma3, 16 out of 16 mouse high-grade gliomas showed no Pten expression in tumour cells but a robust signal in surrounding non-malignant cells and intratumoral vessels (Fig. 2a). Polymerase chain reaction (PCR) genotyping indicated that six out of seven tested tumours sustained loss of the wild-type Pten allele (Fig. 2b). The Pten reduction to homozygosity and the documented Cre-mediated deletion of both p53 floxed alleles indicate that the inactivation of both genes is required for gliomagenesis. Loss of Pten expression correlated with activation of key PI3K signalling surrogates: Akt and ribosomal protein S6 kinase (Fig. 2c). In accordance with human high-grade disease, eight out of eight malignant murine gliomas expressed high Vegf levels relative to normal brain tissue (Fig. 2c). Co-activation of multiple receptor tyrosine kinases in human primary GBM12 was also evident in the murine tumours with robust Pdgf
expression overlapping with strong regional activation of Egfr (Supplementary Fig. 4a–d).
Figure 2: hGFAP-Cre+;p53lox/lox;Ptenlox/+ gliomas mirror key features of human malignant gliomas.
a, Pten expression is completely extinguished in tumour cells. Sections of three independent malignant gliomas were stained with haematoxylin and eosin (H&E) and an anti-Pten antibody. 'N' indicates the adjacent normal regions of the tumour cells; the arrows point to Pten-positive vascular cells embedded in the tumour. b, The wild-type Pten allele is lost in glioma cells. Genomic DNA isolated from liver tissues and brain tumour cells was subjected to PCR-based assays for genotyping Pten and p53 alleles. '+' designates the Pten wild-type allele, 'L' denotes the conditional allele, and 'D' denotes the inactivated form of the conditional allele after Cre-mediated recombination. c, Immunohistochemical staining of mouse normal brain or glioma sections with antibodies against activated phosphorylated Akt (pAkt), phosphorylated ribosomal protein S6 kinase (pS6) and Vegf. d, TNS lines isolated from independent malignant gliomas were cultured in NSC medium or differentiation medium (1% fetal bovine serum (FBS)) and immunostained for Nestin, Gfap and Tuj1 as indicated. Scale bars, 50 m (a, c); original magnification in d 
400.
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A classical feature of human high-grade malignant glioma is a significant degree of intertumoral and intratumoral morphological and lineage heterogeneity, hence the moniker glioblastoma 'mutliforme'. This characteristic plasticity was evident in the hGFAP-Cre+;p53lox/lox;Ptenlox/+ gliomas in which occasional tumours (5 out of 50) presented with both astrocytic and oligodendroglial histopathological features (Supplementary Fig. 5). The basis for morphological variability is not known and may relate, among many possibilities, to the acquisition of an immature developmental state with multipotency and/or differentiative plasticity. Consistent with this notion, all murine tumours express stem or lineage progenitor markers (including Nestin, Gfap and Olig2) similar to human glioma profiles13 but are negative for mature neuronal and oligodendrocyte markers (NeuN and Mbp; Supplementary Fig. 6a, b). This stem/progenitor marker profile is in accord with the ability of all murine tumours tested to readily generate TNSs with (i) strong tumour-initiating potential with secondary tumours faithfully retaining the primary tumour's histological features (Supplementary Fig. 7); (ii) robust NSC marker Nestin expression; and (iii) limited capacity to differentiate into astrocytic and neuronal lineages after exposure to differentiation agents (Fig. 2d). As NSC/progenitor cells have been proposed to be the preferred cell-of-origin for GBM6, the immature marker profile and varied morphological presentation of our murine tumours prompted us to posit that Pten and p53 deficiencies might contribute to gliomagenesis by affecting NSC self-renewal and differentiation potential.
To explore this hypothesis, we characterized primary murine embryonic day (E)13.5 NSC cultures singly or doubly null for p53 and Pten. Compared to NSCs null for either Pten or p53, which show only modestly increased proliferation and self-renewal reflected by neurosphere formation capacity14, 15, 16, NSCs null for both p53 and Pten showed significant proliferation and self-renewal activity (Fig. 3a and Supplementary Fig. 8a). This effect on NSC renewal, coupled with the aforementioned varied tumour histology, suggests that combined p53 and Pten loss might cooperate in tumorigenesis by impairing NSC differentiation potential. When NSCs were continuously cultured in NSC medium, all genotypes showed a similar robust expression of NSC/progenitor markers (for example Nestin) and minimal expression of differentiated lineage markers (Supplementary Fig. 8b). After exposure to differentiation-inducing medium, wild-type and single-null NSC cultures differentiated into Gfap-positive astrocytes, Tuj1-positive neurons, or O4-positive oligodendrocytes. In contrast, p53-/- Pten-/- NSCs failed to respond to these differentiation cues and retained stem-cell-like morphology and lineage marker (Nestin) expression (Fig. 3b and Supplementary Fig. 9a). Similar differentiation defect and abnormal self-renewal potential were also observed in adult NSCs that were deleted for Pten and p53 postnatally (Supplementary Fig. 9b, c). The contribution of Pten deficiency in maintaining impaired differentiation was further verified by the ability of the Akt inhibitor triciribine17 to enable differentiation of NSCs null for both Pten and p53 (Supplementary Fig. 10a, b).
Figure 3: p53 and Pten coordinately regulate Myc protein level as well as NSC self-renewal and differentiation.
a, The number of neurospheres formed by p53-/- Pten-/- NSCs in culture is significantly increased as compared to wild-type or singly null NSCs; asterisk, P < 0.001; n = 3. Values represent mean 
s.d. from three experiments. b, The multilineage differentiation potential was impaired in p53-/- Pten-/- NSCs. D, DAPI (blue); G, Gfap (green); N, Nestin (red); O4 (red); WF, white field; T, Tuj-1 (red). c, Combined inactivation of p53 and Pten in NSCs stimulates Myc protein expression. d, Knockdown of Myc expression restores p53-/- Pten-/- NSC differentiation capacity. Lower panel, western blot of double-null NSC Myc protein expression after infected with indicated lentiviral shRNA. Note Myc expression in shMyc2- and shMyc3-infected p53-/- Pten-/- cells is comparable to that in p53-/- cells, and shMyc1 as a control shows no knockdown. Original magnification used for b and d: 200.
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To understand the molecular basis of impaired differentiation capacity, we performed transcriptome comparisons of murine p53-/- NSCs with p53-/- Pten-/- NSCs at 1 day after exposure to the differentiation inducer. Among the 410 genes showing significant differential expression (Supplementary Table 2) promoter analysis identified E2F and Myc motifs as two of the most enriched promoter binding elements (1.7 and 1.4, respectively; P < 10-4). Notably, promoter analysis using 69 pretreatment human primary GBM cases in the TCGA database showed strong enrichment of MYC binding elements: 10 p53-/- Pten-/- double-mutant tumours versus the 59 remaining tumours (1.40, P = 2.20 10-3) or versus the 12 p53-/- single-mutant tumours (1.46, P = 1.54 10-3).
MYC is well-known for its roles in cell cycle progression and apoptosis18 as well as in stem cell self-renewal and differentiation during development and oncogenic processes19, 20, 21, 22. It is also notable that both p53 and Pten/PI3K pathways can directly regulate MYC with p53 repressing MYC transcription by directly binding to the MYC promoter23, whereas downstream PI3K pathway arms can modulate MYC translation and protein degradation24, 25. In agreement, Myc protein levels were substantially increased in the murine double-null NSCs, but only marginally elevated in p53-/- or Pten-/- NSCs when compared to wild-type controls (Fig. 3c), raising the possibility that p53 and Pten cooperate to regulate Myc levels which in turn could control NSC self-renewal and differentiation.
To test this hypothesis, we examined the effect of Myc knockdown on murine p53-/- Pten-/- NSC differentiation potential and observed that Myc short hairpin RNA (shRNA; shcMyc2 and shcMyc3), which reduced Myc levels to those in p53-/- NSCs, largely restored their differentiation capacity (Fig. 3d and Supplementary Fig. 11a). Conversely, enforced Myc expression in p53-/-NSCs repressed their differentiation and enabled retention of stem/progenitor marker expression (Nestin and Sox2; Supplementary Fig. 11b), indicating that the concomitant loss of p53 and Pten elevates Myc activity to impede NSC differentiation capacity.
The strong pleiotropic activities attributed to MYC demands tight control of its expression to avoid development of diverse human malignancies, including gliomas21, 26. Our finding that the concomitant loss of p53 and Pten compromises NSC differentiation capacity by means of elevated Myc levels prompted further assessment of its relevance in the so-called 'brain cancer stem cells' in our model. Using murine TNSs which are enriched for such tumour initiating cells (TICs), Akt inhibitor treatment strongly reduced Myc protein and promoted differentiation (Fig. 4a, b and Supplementary Fig. 12a). Correspondingly, Myc knockdown in TNS cells not only markedly reduced their proliferation and self-renewal capacity (Fig. 4c and Supplementary Fig. 12b, c) but also strongly sensitized them to differentiation induction (Fig. 4d and Supplementary Fig. 12d). Notably, although ten out of ten intracranial injections of vector-transduced murine TNSs resulted in lethal infiltrating gliomas within 1 month, nine out of ten mice injected with Myc knockdown TNSs survived for more than 3 months (Fig. 4e). Thus, Myc has a crucial involvement in maintaining the impaired differentiation and potent tumorigenic potential of p53- and Pten-inactive TNSs (Supplementary Fig. 13).
Figure 4: Attenuated Myc expression restores hGFAP-Cre+;p53lox/lox;Ptenlox/+ TNS differentiation potential and reduces tumorigenic potential.
a, Inhibition of the Akt pathway by triciribine induces TNS cell differentiation. Two independent TNS lines were cultured in 1% FBS in the absence or presence of triciribine (Tri, 5 M) for 7 days before being subjected to immunostaining with antibodies against Nestin (Nes, red), Gfap (Green) and Tuj1 (red). D, DAPI (blue). b, Inhibition of the Akt pathway in TNS cells with triciribine attenuates their cellular Myc expression. c, Knockdown of Myc expression in TNS cells reduces their self-renewal potential assessed by sphere formation; asterisk, P < 0.001; n = 3. Values represent mean s.d. from three experiments. d, shRNA-mediated reduction of Myc expression in TNS cells sensitizes cells to differentiation stimuli. Cells infected with control (shGfp) and the indicated shRNA were incubated with differentiation medium before being subjected to lineage marker analysis. e, shRNA-mediated reduction of Myc expression represses TNS tumorigenic potency in orthotopically transplanted SCID mice. Original magnification for a and d: 200.
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The identification of TICs with stem-like properties in diverse human cancers including GBM represents an important conceptual advance in cancer biology with therapeutic implications27, 28. These TICs seem to constitute a reservoir of self-sustaining cells with potent tumorigenic potential. However, unlike normal NSCs which readily differentiate along a developmental hierarchy into lineage-restricted differentiated progenies, TNSs derived from p53-/- Pten-/- malignant gliomas show resistance towards differentiation cues. The diminished tumorigenicity of these TICs on restoration of differentiation potential, along with recent reports supporting pro-differentiation as a potential strategy to inhibit GBM-derived TICs29, 30, encourages the identification and testing of agents targeting these differentiation pathways including MYC in the treatment of primary GBM in humans.
Methods Summary
Standard gene targeting and chimaera formation methods were used to generate the conditional PtenL allele in the mouse germ line in which Pten exon 5 is flanked by loxP sites; the conditional p53L mouse was generated by A. Berns; hGFAP-Cre mice were purchased from the Jackson Laboratory. All mice were maintained in pathogen-free facilities and followed for development of neurological deficits. After culling, tissues were collected and processed for histological, immunohistochemical, immunofluorescence or western blot analyses, as detailed in the Methods. For p53 and Pten mutation analysis, surgically resected human primary glioblastoma were flash-frozen and genomic DNA was prepared from frozen tumour samples. For microarray analysis, total RNAs from indicated early passage NSCs were amplified and labelled by standard methods and hybridized to Affymetrix 430 2.0 chips. For tumorigenic analysis, TNS cells isolated from hGFAP-Cre+;p53lox/lox;Ptenlox/+ murine malignant gliomas were infected with indicated lentivirus shRNA and orthotopically injected into the forebrain of SCID mice. Animals were observed daily for the development of neurological deficits and subjected to histological analysis once killed.
Full methods accompany this paper.
550 motifs in the TRANSFAC 12.1 database. Enrichment was measured against control regions at a comparable distance from the transcription start sites of random genes.
