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A GLI1-p53 inhibitory loop controls neural stem cell and tumour cell numbers

2009; Springer Nature; Volume: 28; Issue: 6 Linguagem: Inglês

10.1038/emboj.2009.16

1460-2075

Barbara Stecca, Ariel Ruiz i Altaba,

Genetics and Neurodevelopmental Disorders

Article12 February 2009Open Access A GLI1-p53 inhibitory loop controls neural stem cell and tumour cell numbers Barbara Stecca Barbara Stecca Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, SwitzerlandPresent address: Laboratory of Tumor Cell Biology, Core Research Laboratory—Istituto Toscano Tumori (CRL-ITT), Viale Morgagni 50, 50134 Florence, Italy Search for more papers by this author Ariel Ruiz i Altaba Corresponding Author Ariel Ruiz i Altaba Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland Search for more papers by this author Barbara Stecca Barbara Stecca Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, SwitzerlandPresent address: Laboratory of Tumor Cell Biology, Core Research Laboratory—Istituto Toscano Tumori (CRL-ITT), Viale Morgagni 50, 50134 Florence, Italy Search for more papers by this author Ariel Ruiz i Altaba Corresponding Author Ariel Ruiz i Altaba Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland Search for more papers by this author Author Information Barbara Stecca1 and Ariel Ruiz i Altaba 1 1Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland *Corresponding author. Department of Genetic Medicine and Development, University of Geneva Medical School, 1 rue Michel Servet, 8242 CMU, Geneva CH-1211, Switzerland. Tel.: +41 22 379 5646; Fax: +41 22 379 5962; E-mail: [email protected] The EMBO Journal (2009)28:663-676https://doi.org/10.1038/emboj.2009.16 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info How cell numbers are determined is not understood. Hedgehog-Gli activity is involved in precursor cell proliferation and stem cell self-renewal, and its deregulation sustains the growth of many human tumours. However, it is not known whether GLI1, the final mediator of Hh signals, controls stem cell numbers, and how its activity is restricted to curtail tumourigenesis. Here we have altered the levels of GLI1 and p53, the major tumour suppressor, in multiple systems. We show that GLI1 expression in Nestin+ neural progenitors increases precursor and clonogenic stem cell numbers in vivo and in vitro. In contrast, p53 inhibits GLI1-driven neural stem cell self-renewal, tumour growth and proliferation. Mechanistically, p53 inhibits the activity, nuclear localisation and levels of GLI1 and in turn, GLI1 represses p53, establishing an inhibitory loop. We also find that p53 regulates the phosphorylation of a novel N' truncated putative activator isoform of GLI1 in human cells. The balance of GLI1 and p53 functions, thus, determines cell numbers, and prevalence of p53 restricts GLI1-driven stem cell expansion and tumourigenesis. Introduction A key unresolved issue in biology is how cell numbers are determined during development, largely maintained in adulthood and deregulated in cancer. Hedgehog-Gli signalling is a key intercellular communication pathway involved in many aspects of development and cancer. During early brain development, it modulates precursor proliferation in different brain regions such as the neocortex, tectum and cerebellum (e.g., Dahmane and Ruiz i Altaba, 1999; Dahmane et al, 2001; Lien et al, 2006), while later on it regulates brain stem cell lineages (e.g., Lai et al, 2003; Palma and Ruiz i Altaba, 2004; Palma et al, 2005). It also drives rodent (Goodrich et al, 1997) and human (Dahmane et al, 2001) brain tumourigenesis and the self-renewal and survival of brain cancer stem cells (Clement et al, 2007). This dual involvement of Hh-Gli signalling in development and disease underlines its central role in controlling precursor cell numbers in the brain and other organs. It is not known, however, whether the level of GLI1 is critical to determine stem cell numbers and how GLI1 activity is restrained to prevent tumour formation. Here we have tested two hypotheses: (1) GLI1 is a central determinant of stem cell numbers; (2) p53, the major human tumour suppressor, which regulates stem cell and precursor numbers (Gil-Perotin et al, 2006; Meletis et al, 2006), does so by negatively modulating the activity of GLI1. Testing these two ideas is important, as GLI proteins can integrate patterning and proliferative inputs in addition to HH signals (Brewster et al, 2000; Palma and Ruiz i Altaba, 2004; Kasper et al, 2006; Riobó et al, 2006; Dennler et al, 2007), as well as those from oncogenic RAS-MEK/AKT (Stecca et al, 2007); and p53 suppresses whereas GLI1 promotes tumourigenesis. We report that (1) the level of GLI1 determines the number of neural stem cells in vivo, (2) GLI1 and p53 act in an inhibitory loop, (3) endogenous GLI1 isoforms exist, and (4) a novel N'Δ GLI1 isoform is subject to regulation by phosphorylation in a p53-dependent manner. Our present and previous data suggest a critical regulation of GLI1 function in stem cell lineages by oncogenes and tumour suppressors, which is off-balance in disease. Results Elevation of GLI1 levels in neural progenitors results in a larger brain with expanded progenitor populations To enhance Gli1 activity in stem cell lineages in vivo, we developed doxycycline (dox) inducible bigenic mice using the Nestin intron II driver (Nestin->rtTA-IRES-LacZ driver; Supplementary Figure 1), which is strongly expressed in embryonic neural progenitors but only very weakly postnatally (Figure 1; Supplementary Figures 1, 2, 3, 4 and not shown). As a responder, we made a bidirectional GFP myc-GLI1 transgene by random insertion (one line) or by targeting into the hprt locus (three lines). All bigenic lines showed the same phenotypes, and we focused on perinatal phenotypes of the first line resulting from sustained embryonic dox administration (Supplementary Table 1; Supplementary Figure 1). Figure 1.Enhanced GLI1 activity induces a larger brain with hyperplasias. (A–D) DT+dox bigenic mice with strong phenotypes are perinatal lethal exhibiting a larger brain than DT−dox siblings, seen dorsally (A, B) or in sagittal sections after Xgal staining highlighting the expression of the Nestin->rtTA-LacZ driver (C, D) (see Supplementary Figure 1). (E–H) Phenotypes detected in cross section in the thalamus (Thal; E, F), cerebellum (Cb) and medulla (Md; G, H) showing LacZ (Xgal) labelling (E–H), BrdU incorporation (G, H), GLI1 and Ptch1 (F) or GFP (H) expression in DT−dox and DT+dox siblings as indicated. (I–K) HP1γ−/Nestin+/GFP+ precursors from the ventricle (v) invade the parenchyma through the cortical plate and expand near the pial (p) surface in the cingulate cortex (I, J). Invading GFP+ cells associate with CD34+ endothelial cells in vessels as seen in a Z-stack confocal reconstruction (K) and a confocal image detail (K inset). (L–P) Phenotypic analyses in cross sections of subventricular zone of the lateral ventricle (SVZ) (L) and thalamus (M–P) for the markers indicated. Myc-GLI1 was mostly cytoplasmic and Prox1 nuclear (O inset). L shows a confocal image. Tissue is counterstained with eosin (e.g., C, D, M) and nuclei with DAPI (e.g., I, L). Hip, hippocampus; Sc, spinal cord; Tct, tectum; vz, ventricular zone; Scale bar=1.5 mm (A, B), 1 mm (C, D), 0.5 mm (E–H), 100 μm (O inset, J, P), 200 μm (I), 70 μm (L; H inset), 14 μm (K), 10 μm (K inset), 0.6 mm (M, N, O), 150 μm (M inset). Download figure Download PowerPoint Double-transgenic (DT)-treated mice (DT+dox), but not untreated (DT−dox) siblings or treated single transgenics, expressed GLI1 and GFP in precursors and displayed larger brains with hyperplasias of progenitor zones throughout from forebrain to spinal cord (Figure 1A–D; Supplementary Figure 1 and not shown). The strength of the phenotype varied within a litter. DT+dox mice with a strong phenotype (Figure 1) were embryonic lethal or died shortly after birth. Surviving animals had milder alterations (Supplementary Figure 3A). Three brain regions with strong phenotypes (cortex, thalamus and cerebellum) and the spinal cord had high levels of LacZ, GFP and Nestin and of GLI1 and endogenous Ptch1 mRNAs in progenitor cells (Figure 1E–H, M and P; Supplementary Figures 2, 3, 4). The cerebellar external germinal layer, which has the highest levels of endogenous Gli1 (Dahmane and Ruiz i Altaba, 1999), was largely unaffected, whereas the ventricular zone (VZ) was hyperplastic (Figure 1G and H; Supplementary Figure 3A, B). The tectum (colliculi) was also larger but did not show convoluted tissue as the cerebellum, thalamus or medulla (Figure 1A–D; Supplementary Figure 1). Massive and highly invasive precursor hyperplasias were also detected in the spinal cord (Supplementary Figure 4). Transgenic GLI1 induced ∼4–13-fold the activity of the endogenous Hh-Gli pathway as assessed by the increased expression of Gli1 and Ptch1 (Figure 2A; Supplementary Figure 5). It also boosted 2–8-fold BrdU incorporation in a region-specific manner (Figure 1G, H and P; Supplementary Figure 2C, D, G, H). In the cortex and subventricular zone of the lateral ventricle (SVZ), we found high GFP+, LacZ+, Ptch1+, myc-GLI1+ groups of cells streaming from the VZ to the pial surface (Figure 1I and J; Supplementary Figure 2A–E, I). These cells were HP1γ−, a heterochromatin (neuronal and senescent cell) marker (Figure 1I); they expressed high levels of Nestin (Figure 1J; Supplementary Figure 2B, E), associated with CD34+ vessels (Figure 1K) similarly to human gliomas (Farin et al, 2006), and derived from Nestin+, GFAP− VZ cells (Figure 1L). Figure 2.Gene expression and cellular behaviour in neurospheres and stem cells. (A) RT–qPCR analyses of P0 dissected tissues from cortex (Ctx), thalamus (Thal) or cerebellum (Cb). qPCR values in all panels reflect ct values after normalisation with the geometrical mean of the values of Gapdh, βactin and EFIα and shown as the DT+dox/DT−dox value ratios. See Supplementary Figure 5. (B) Phase contrast and fluorescence images of DT+dox NS. (C–E). Histograms of the number of neurospheres (C, D) or BrdU+ cells (E) as indicated. Primary spheres were quantified per 250 000 brain cells. (F) The level of GLI1 determines NS numbers. GLI1 expression values and secondary sphere number were determined for the same samples. (G–I) Quantification of the number (G), clonogenicity after MACS (H) and gene expression analyses (I) by RT–qPCR of Prominin1+ cerebellar stem cells (see Supplementary Figure 6B). Here and in all figures asterisks denote significative changes (P 160 KD) and a three-fold increase of a shorter form running ±130 KD (Figure 5F), quantified by densitometry. Similarly, analysis of postnatal wt SVZ NS for endogenous mouse Gli1 with an anti-GLI1 antibody to the C-terminus (α-GLI1803−818; GeneTex) showed a 40% increase in full-length mouse Gli1 (>160 KD) and a 2.3-fold increase in a shorter form running at ∼115 KD (Figure 5G). As control, and as α-GLI1∼420 does not recognise mouse Gli1 (Figure 6C), we used α-GLI1803−818 to probe extracts of P10 wt cerebellum, adult (∼4 mo) Cb and a Ptch1+/−;p53−/− medulloblastoma (MB) (Figure 5H). As expected from the transient perinatal expansion of cerebellar GLI1+ granule neuron progenitors and the strong expression of GLI1 in MB (Dahmane and Ruiz i Altaba, 1999; Dahmane et al, 2001), the full-length and the shorted 115 KD form were clearly detected at P10 and were nearly undetectable in adult cerebellum, whereas the full-length form was 14-fold and the 115 KD form 6.5-fold more abundant in MB than at P10 (Figure 5H). Figure 6.GLI1 protein isoforms and endogenous repression of p53 by GLI1. (A) Transfected myc-GLI1 isoforms detected as indicated in samples treated with cycloheximide (CHX) as shown. GLI1FL migrates >160 KD. (B) Deduced relationship of GLI1130 and GLI1100 to GLI1FL, and location of Ab epitopes and of the transactivating (TA) domain. (C) Analyses of human GLI1 protein in transgenic P0 mice as indicated. All three antibodies used recognise GLI1FL and GLI1130, but only those to the domain immediately C-terminal to the zinc fingers (ZF in B) recognise GLI1100. (D) Analysis of GLI1 isoforms in human cell lines and in a GBM stem cell culture (gliomasphere). (E) GLI1 kd with a specific siRNA for 8 h, but not a control siRNA, depletes all three isoforms in U87 cells, while enhancing total and phospho-serine15 p53 (p53-P) levels. Non-specific bands are denoted by asterisks. (F) Levels of Mdm2 in the blot shown in panel C middle for GLI1 and Hsp90. (G) Comparison of the levels of GLI1130, p53, phospho-serine15 p53 and HSP90 in two gliomaspheres that proliferate at different rates. Download figure Download PowerPoint GLI1 represses p53 The mutual compensatory effect of GLI1 and p53 (Figure 4D) and the dominance of GLI1 kd over p53 kd (Figure 4B) raised the possibility that in addition to the negative regulation of GLI1 by p53, p53 itself could be under GLI1 regulation. Analyses of p53 in GLI1 kd cells (see below) revealed that endogenous p53 is under the control of endogenous GLI1 as assessed by the levels of total p53 and of active phospho-serine15 p53 (p53-P) in cells lipofected with siGLI1 versus siC (Figure 6E). The kd of endogenous GLI1 function increased p53 ∼5-fold and phospho-serine15 p53 ∼2-fold. This indicates that in addition to the inhibition of GLI1 by p53 described earlier, endogenous GLI1 regulates the levels of endogenous p53, suggesting a negative regulatory loop. Enhanced GLI1 levels can inhibit p53 regulation in vitro through the activation of the p53 inhibitor Hdm2 (Abe et al, 2008). We find that Mdm2, the mouse homologue of Hdm2, is greatly enhanced in DT+dox versus DT−dox brains (Figure 6F), extending this possible mechanism of action to an in vivo context. The inhibitory loop between GLI1 and p53 is consistent with the inversely reciprocal levels of GLI1 and p53 observed in two primary glioblastoma stem cell cultures (Figure 6G; Clement et al, 2007). The level of GLI1 correlated with their proliferation rate (Figure 6G). GBM12 harbours a p53 gene with a frameshift mutation corresponding to aa 209 (M Vukicevic and ARA, unpublished) and predicted to encode an unstable protein as no p53 or phospho-serine15 p53 proteins were detected (Figure 6G). GBM13 expresses full-length p53 with an R->H substitution at aa 158 within the DNA-binding domain (M Vukicevic and ARA, unpublished) predicted