NANOG regulates glioma stem cells and is essential in vivo acting in a cross-functional network with GLI1 and p53

Abstract

A cohort of genes associated with embryonic stem (ES) cell behaviour, including NANOG, are expressed in a number of human cancers. They form an ES-like signature we first described in glioblastoma multiforme (GBM), a highly invasive and incurable brain tumour. We have also shown that HEDGEHOG-GLI (HH-GLI) signalling is required for GBM growth, stem cell expansion and the expression of this (ES)-like stemness signature. Here, we address the function of NANOG in human GBMs and its relationship with HH-GLI activity. We find that NANOG modulates gliomasphere clonogenicity, CD133(+) stem cell cell behavior and proliferation, and is regulated by HH-GLI signalling. However, GLI1 also requires NANOG activity forming a positive loop, which is negatively controlled by p53 and vice versa. NANOG is essential for GBM tumourigenicity in orthotopic xenografts and it is epistatic to HH-GLI activity. Our data establish NANOG as a novel HH-GLI mediator essential for GBMs. We propose that this function is conserved and that tumour growth and stem cell behaviour rely on the status of a functional GLI1-NANOG-p53 network.

Figure 1

NANOG expression in human gliomas. (A) Quantification of NANOG/P8 expression by qRT–PCR in normal brain and in different normal brain regions (black bars), and in human brain tumours (red bars): A, astrocytoma; GBM, glioblastoma multiforme; OG, oligodendroglioma; MB, medulloblastoma. Roman numerals refer to WHO tumour grade. Arabic numerals refer to tumour sample as in Clement et al (2007) except GBM-15, -17. Expression values were normalized by using the geometrical mean of TBP and β ACTIN. (B) Diagram of the structure of NANOG alleles a (NW_001838051) and b (NC_000012.11), and of NANOGP8 (NT_010194.17). NANOG allele b contains 22 extra bp in the 3′UTR (dark grey box). These differences suggest the identification of these conserved polymorphic variants as alleles. The diagram also highlights the bp differences (crosses) and the 2 or 3 aa variants. Of these, K82N is not conserved in different species. The right part shows the frequency of alleles as determined from sequencing a portion of the 3′UTR that is diagnostic for the different alleles in different primary GBMs and in U87 cells. (C) Indirect immunofluorescence localization of NANOG protein with YAb (top left) or KAb antibody in patient-derived GBM cells grown as gliomaspheres and attached U87 cells as indicated with confocal microscopy. Nuclei are counterstained with DAPI (blue). Labelling is seen in multiple nuclear puncta (arrows). Cells express NANOG at different levels (arrowheads). Asterisks mark small cells with no labelling that likely represent mouse stromal cells in xenografts. Control staining was performed on LV-cNANOG- and LV-shNANOG-1-transduced cells (top right) and on cells that lacked primary antibodies (see Supplementary Figure S1). (D) Scheme of the NANOG reporter construct used to drive RFP expression (left) and the resulting RFP fluorescence (red) (middle; a high-expressing cell is denoted by an arrowhead) in a small GBM-13 gliomasphere derived from a transduced cell. The sphere was allowed to attach to the dish for 30 min before processing. The same image is shown only with DAPI staining (blue; right) to highlight nuclei. Scale bar=3 μm (C, top and middle rows), 10 μm (C, bottom row), 15 μm (D).

Figure 2

NANOG regulates stem cell self-renewal and GBM cell proliferation. (A) Western blot of U87 cells expressing LV-GFPcontrol-1, showing endogenous levels of NANOG protein (middle), LV-cNANOG (left; with only 1/4th, 20 μg, of the amount of total protein as in the other lanes loaded; expressing NANOG cDNA) and LV-shNANOG-1 (right, 80 μg), with greatly reduced NANOG protein levels. (B, C) Quantification of clonogenic single-cell gliomasphere formation by transduced GBM samples after expression of shNANOG-1 (B), shNANOG-2 (C) or controls (LV-GFPcontrol-1 (B) or LV-control-2 (C)); >900 clonogenic events were counted per condition, plated in 96-well plates. (D, E) Representative photomicrographs of GFP⁺ (green) gliomaspheres in suspension (E) and measurement of their sizes (D) from GBM-8 cells transduced with LV-GFPcontrol-1 or LV-shNANOG-1 as indicated. (F) Representative examples of anti-BrdU labelling (red) in GBM-8 cells transduced with LV-GFPcontrol-1 or LV-shNANOG-1 as indicated. GBM-8 gliomaspheres were dissociated and allowed to attach to facilitate immunohistochemistry and counting. (G) Histograms showing the quantification of the rescue of the inhibition of clonogenicity by shNANOG-1 through the overexpression of NANOG cDNA (LV-cNANOG) in GBM-12 gliomaspheres. (H, I) Quantification of BrdU incorporation in multiple primary GBM and GBM cell lines after NANOG kd with shNANOG-1 (H) or shNANOG-2 (I), shown in comparison with controls (GFP-control-1 (H) or control-2 (I)). Asterisks denote significative changes (P<0.05). ns, not significative. Error bars represent s.e.m. For clonogenic (sphere) assays, 900 clonogenic events were counted per condition. Scale bar=120 μm (E), 45 (F).

Figure 5

NANOGP8 is expressed and required in GBMs. (A) RT–qPCR quantification of the expression levels of NANOG, NANOGP8, GLI1 and NESTIN after NANOGP8 kd with a specific shRNA to NANOGP8 in a lentivector in U87 cells. Control U87 cells were transduced with a GFP-control1 lentivector. Values are shown after normalization. (B) Western blot analysis of NANOG protein with KAb in U87 GBM cells transfected with LV-cNANOG, overexpressing NANOG protein, control GFP-expressing LV-GFPcontrol-1 and two lentivectors-expressing shRNAs against NANOG plus NANOGP8 (LV-shNANOG-1) or against NANOGP8 (LV-shNANOGP8) separately. shNANOG-1 or shNANOGP8 abolishes detectable NANOG protein expression. HSP90 is shown as loading control; 80 μg of total protein were loaded per lane for SDS–PAGE. (C) Quantification of BrdU incorporation showing similar efficiencies of shNANOG-1 and shNANOGP8 in inhibiting cell proliferation in U87 cells. The graph shows the percentage of BrdU⁺ cells over the total number of DAPI-labelled cells. (D) FACS plots of in vivo red/green assays in orthotopic xenografts of GBM-8 with a GFP⁺ population also expressing shNANOGP8. Parental cells at passage 0 (P0) show about equal subpopulations of RFP⁺ and GFP⁺ cells. After the first in vivo passage (P1), the GFP⁺ cells with NANOGP8 kd have disappeared. (E) Dorsal views of dissected brain harbouring grafts of GFP⁺ cells co-expressing shNANOGP8 and RFP⁺ cells after tumour growth (at first passage). The tumours are composed exclusively of RFP⁺-control cells. Scale bar=3.5 mm (E). Asterisks denote significative changes (P<0.05).

Figure 3

Expression and function of NANOG in CD133⁺ GBM cells. (A) Gene expression levels determined by RT–qPCR shown as the ratios of the expression values of CD133⁺ cells over those of CD133⁻ cells, of GBM-8 and GBM-12 cells, after normalization with housekeeping genes. Enhanced levels ⩾1.5-fold are in red and repressed levels ⩽50% are in blue. CD133⁺ cells express higher levels of NANOG/P8 as compared with CD133⁻ cells. (B) Quantification of the levels of mRNA expression of NANOG/P8 (NANOG + NANOGP8) and of NANOGP8 alone in freshly MACS-sorted CD133⁺ versus CD133⁻ GBM-8 cells. Normalization was set so that CD133⁻ levels equal 1. NANOGP8 shows a similar enrichment to that of the combined genes in CD133⁺ cells. (C) Normalized enrichment of NANOG → RFP⁺ cells in CD133⁺ versus CD133⁻ GBM-8 cells. Normalization was performed by equating the level in the unsorted parental population to 1. (D) Acute (16 h) BrdU incorporation assay on freshly sorted CD133⁺ and CD133⁻ cells of GBM-8 showing a selective effect of NANOG kd in CD133⁺ cells at this early time point. (E) Red/green competition assay in vitro testing for the changes in GFP⁺ experimental cells co-expressing shNANOG-1 or controlGFP-1 versus RFP⁺-control cells. Cells were transduced, FACS sorted to obtain 100% transduced cell populations, mixed and 5 days later sorted for CD133 by MACS. MACS-sorted cells were then FACS analysed to determine the relative GFP/RFP ratios per culture condition shown in the graph. Asterisks denote significative changes (P<0.05).

Figure 4

NANOG function is essential for GBM growth in vivo. (A) Scheme of the red/green orthotopic assay with GBMs. For illustration purposes, gliomaspheres are shown as starting material, but adherent cells have also been used (see Supplementary Figure S2). In addition, also for illustration purposes, an aggregate of red and green cells is shown before intracerebral transplantation into immunocompromised mice, although FACS-sorted cells are routinely mixed and injected before aggregation. (B) Representative images of dissected whole brains with developed ‘red/green' brain tumours after orthotopic xenotransplantation of GBM gliomaspheres transduced with control red (RFP⁺) plus either control green (GFP⁺) or GFP⁺/shNANOG-expressing lentivectors as indicated. The same samples are shown in each row under visible and fluorescent light, the latter with filters to selectively detect green or red fluorescence. Far right panels show the FACS profiles and green/red ratios. (C) Quantification of FACS ratios in red/green competition assays in vivo after normalization with controls, which are equated to 1. The number of mice analysed at each passage (P) is also given (n) for each case. Scale bar=3.5 mm (B).

Figure 6

Functional NANOG, GLI1 and p53 interactions. (A) Gene expression changes at 4 days post-transduction in primary GBMs and U87 cells after NANOG kd with LV-shNANOG-1 shown as ratios over the levels obtained in cells transduced with control lentivectors and after normalization. Bottom panel shows the decrease of GLI1 and PTCH1 at 3 days post-transduction, whereas at 4 days PTCH1 levels recover, suggesting different dynamics for different GLI1 targets. (B) Inhibition of GLI1¹³⁰ and GLI1¹⁰⁰ protein isoforms detected with a specific affinity-purified polyclonal antibody (see Stecca and Ruiz i Altaba, 2009) in U87 cells after kd of NANOG. This confirms the relevance of 70% inhibition at the mRNA level (A). GLI1^FL was not detectable. (C) GLI-binding site → luciferase reporter assays testing for the activity of endogenous GLI1 in U87 cells. Reporter activity is strongly reduced by NANOG kd. Note that luciferase levels were normalized so that normal endogenous level is set to 100%. Luciferase levels were firefly over control renilla ratios. (D) Enhancement of NANOG/P8 and the HH-GLI pathway after p53 kd with LV-shp53 in U87 cells. Values are ratios over control lentivectors after normalization. (E) Rescue of the proliferative (BrdU incorporation) defect induced by NANOG kd by simultaneous kd of p53, and rescue of the proliferative enhancement of p53 kd by simultaneous kd of NANOG in U87 cells. (F) Western blot analyses of p53 protein in U87 GBM cells after p53 kd or NANOG kd with appropriate lentivectors. HSP90 is shown as quantification control. Ctrl, control LV-GFPcontrol-1-transduced cells. (G) Antagonistic effects of NANOG kd and p53 kd on GLI1 mRNA levels and rescue by double kd in U87 cells. Gene expression levels (shown as fold changes) were quantified and normalized and are shown as ratios of the experimental condition over control (parental lentivector-transfected cells). The arrow points to the rescue of GLI1 levels by concomitant kd of p53 and NANOG. (H) Quantification of U87 cell numbers in control and NANOG kd populations after treatments with 0, 10 and 30 μM temozolomide for 5 days. NANOG kd dampens temozolomide effects, passing from a two-fold (2 × ) reduction in cell numbers in controls to 1.6-fold in shNANOG-expressing cells at 10 μM, and from 6.2- to 4.8-fold at 30 μM. Asterisks denote significative changes (P<0.05). NS, not significative. Error bars represent s.e.m.

Figure 7

NANOG is regulated by HH-GLI signalling and NANOG function is epistatic to an active HH-GLI pathway. (A) Gene expression changes after enhancement (through PTCH1 kd or GLI1 expression) or repression (through SMOH kd) of HH-GLI signalling in GBM-8, -12 and U87 cells. Values are RT–qPCR ratios over controls after normalization at 4 days after transduction. (B) Western blot analysis of NANOG protein using KAb in U87 cells transduced with LV-GFPcontrol-1, LV-GLI1, LV-shSMOH or LV-GLI3R. HSP90 is shown as loading control. NANOG protein is greatly diminished in cells with compromised HH-GLI signalling. (C) Representative images of dissected whole brains with developed ‘red/green' brain tumours after orthotopic xenotransplantation of GBM-8 gliomaspheres transduced with control red (RFP⁺) plus green (GFP⁺) lentivectors expressing different shRNAs as indicated. Each row shows the images of dorsal brains with anterior to the left under visible, green or red fluorescence. Green/red FACS ratios are given in the right column. (D) Quantification and evolution of FACS ratios in red/green competition assays in vivo after normalization with controls, which are equated to 1. The number of mice analysed at each passage (P) is also given (n) for each case. Scale bar, 3.5 mm (C).

Figure 8

Model of the GLI1-NANOG module and cross-functional interactions with p53. Scheme of NANOG action downstream of HH-GLI signalling, establishing a positive feedback loop with GLI1 and being repressed by p53, which acts in a negative-regulatory loop with GLI1 in brain tumours (Stecca and Ruiz i Altaba, 2009). The outcome determines tumour growth and stem cell behaviour.