p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation - PubMed (original) (raw)

. 2008 Oct 23;455(7216):1129-33.

doi: 10.1038/nature07443.

Haoqiang Ying, Haiyan Yan, Alec C Kimmelman, David J Hiller, An-Jou Chen, Samuel R Perry, Giovanni Tonon, Gerald C Chu, Zhihu Ding, Jayne M Stommel, Katherine L Dunn, Ruprecht Wiedemeyer, Mingjian J You, Cameron Brennan, Y Alan Wang, Keith L Ligon, Wing H Wong, Lynda Chin, Ronald A DePinho

Affiliations

p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation

Hongwu Zheng et al. Nature. 2008.

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 events. 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.

PubMed Disclaimer

Figures

Figure 1

Figure 1. p53 and Pten inactivation cooperate to induce high-grade malignant gliomas

a, Kaplan-Meier tumor-free survival curves for mice of indicated genotypes as a function of weeks. b, Graph shows frequency and grade of gliomas vs. non-CNS malignancies observed in end-stage of indicated mice from a. Asy* indicates neurological asymptomatic hGFAP-Cre;P53lox/lox;Ptenlox/+ mice (n=15) sacrificed for non-CNS malignancies. c, H&E histology and immunohistochemical staining of sections of WHO grade III and grade IV malignant gliomas from hGFAP-Cre;P53lox/lox;Ptenlox/+ mice with antibodies against Ki67, GFAP, and Nestin. Scale bar, 50 µm.

Figure 2

Figure 2. hGFAP-Cre;P53lox/lox;Ptenlox/+ gliomas mirror key features of human malignant gliomas

a, Pten expression is completely extinguished in tumor cells. Sections of three independent malignant gliomas were stained with H&E, and an anti-Pten antibody. Note “N” indicates the adjacent normal regions of the tumor cells; the arrows point to Pten-positive vascular cells embedded in the tumor. b, Wild-type Pten allele is lost in glioma cells. Genomic DNA isolated from liver tissues and brain tumor cells were subjected to PCR-based assays for genotyping Pten and p53 alleles. Note “+” designates the Pten wild-type allele; “_L_” the conditional allele; and “_D_” 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 p-AKT, p-S6 kinase, and VEGF. d, TNS lines isolated from independent malignant gliomas were cultured in NSC medium or differentiation medium and immuno-stained for Nestin, GFAP and Tuj1 as indicated. Scale bar, 50 µm.

Figure 3

Figure 3. p53 and Pten coordinately regulate c-Myc protein level as well as NSC self-renewal and differentiation

a, The number of neurospheres formed by p53/Pten double-null NSCs in culture is significantly increased as compared to wild-type or singly null NSCs (*P < 0.001; n=3). Values represent mean ± s.d. from three experiments. b, The multi-lineage differentiation potential was impaired in double-null NSCs. Note “WF” designates for white-field; N for Nestin (red); G for GFAP (green); T for Tuj-1 (red); O4 (red); D for DAPI (blue). c, Combined inactivation of p53 and Pten in NSCs stimulates c-Myc protein expression. d, Knockdown of c-Myc expression restores p53/Pten double-null NSCs differentiation capacity. Lower panel, western blot of double-null NSC c-Myc protein expression after infected with indicated lenti-shRNA. Note c-Myc expression in shMyc #2 and #3 infected double-null cells is comparable to that in _p53_-null cells, and shMyc #1 as a control shows no knockdown.

Figure 4

Figure 4. Attenuated c-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 (5 µM) for 7 days before being subjected to immuno-staining with antibodies against Nestin (red), GFAP (Green) and Tuj1 (red). b, Inhibition of the AKT pathway in TNS cells with triciribine attenuates their cellular c-Myc expression. c, Knockdown of c-Myc expression in TNS cells reduces their self-renewal potential assessed by sphere formation (*P < 0.001, n=3). Values represent mean ± s.d. from three experiments. d, ShRNA-mediated reduction of c-Myc expression in TNS cells sensitizes cells to differentiation stimuli. Cells infected with control and indicated shRNA were incubated with differentiation medium before subjected to indicated lineage marker analysis. e, ShRNA-mediated reduction of c-Myc expression represses TNS tumorigenic potency in orthotopically transplanted SCID mice.

Similar articles

Cited by

References

    1. Kleihues P, Ohgaki H. Primary and secondary glioblastomas: from concept to clinical diagnosis. Neuro Oncol. 1999;1:44–51. - PMC - PubMed
    1. Zhu Y, Parada LF. The molecular and genetic basis of neurological tumours. Nat Rev Cancer. 2002;2:616–626. - PubMed
    1. Furnari FB, et al. Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev. 2007;21:2683–2710. - PubMed
    1. Wiedemeyer R, et al. Feedback circuit among INK4 tumor suppressors constrains human glioblastoma development. Cancer Cell. 2008;13:355–364. - PMC - PubMed
    1. Zhuo L, et al. hGFAP-cre transgenic mice for manipulation of glial and neuronal function in vivo. Genesis. 2001;31:85–94. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources