Cooperativity within and among Pten, p53, and Rb pathways induces high-grade astrocytoma in adult brain

Abstract

Mutations in the PTEN, TP53, and RB1 pathways are obligate events in the pathogenesis of human glioblastomas. We induced various combinations of deletions in these tumor suppressors in astrocytes and neural precursors in mature mice, resulting in astrocytomas ranging from grade III to grade IV (glioblastoma). There was selection for mutation of multiple genes within a pathway, shown by somatic amplifications of genes in the PI3K or Rb pathway in tumors in which Pten or Rb deletion was an initiating event. Despite multiple mutations within PI3K and Rb pathways, elevated Mapk activation was not consistent. Gene expression profiling revealed striking similarities to subclasses of human diffuse astrocytoma. Astrocytomas were found within and outside of proliferative niches in the adult brain.

Figure 1

Pten deletion in adult astrocytes does not disrupt brain architecture or activate Akt. (AC) Sagittal sections were immunostained for Gfap (brown) and counterstained with hematoxylin. Magnified views of boxed regions for Gfap IHC (D–F) and pAkt S473 IHC (G–I, brown) are shown. (A, D, and G) Pten^loxP/loxP mouse injected with tamoxifen. (B, E, and H) CreER; Pten^loxP/loxP mouse injected with vehicle. (C, F and I) CreER; Pten^loxP/loxP mouse injected with tamoxifen. Scale bar in (A) is 1 mm and applies to (A–C). Scale bar in (D) is 50 μm and applies to (D–I). See also Figure S1.

Figure 2

High-grade astrocytomas arising from Pten; p53 cKO mice. (A) Kaplan-Meier survival analysis on tamoxifen-induced cohorts consisting of Pten cKO (N=34; blue), Pten^loxP/+; p53 cKO (N=6; green) and Pten; p53 cKO mice (N=148; red). (B–G) H&E stain of Pten; p53 cKO brains showing recurrent histological features: glioblastoma with multinucleated giant cell phenotype (B, arrow), necrotic foci (C, nec) and sometimes microvascular proliferation (D, arrow), and anaplastic astrocytoma with myxoid degeneration (E) and diffusely infiltrating tumor cells (F) sometimes consistent with gliomatosis (G). (H–J) IHC for Pten (brown) counterstained with hematoxylin highlighting areas of perivascular invasion (I) and leptomeningeal spread (J). Arrows in (I) indicate a blood vessel while the arrow in J points to the pial membrane overlying tumor cells. Scale bar in (B) is 20 μm and applies to (B–F, H). Scale bar in (G) is 200 μm. Scale bar in (J) is 50 μm and applies to (I, J). See also Figure S2.

Figure 3

Immunophenotype of Pten; p53 cKO HGAs. (A–C) Sections from a brain containing anaplastic astrocytoma were immunostained for pAkt S473 (A, brown), Ki67 (B, purple) and active Caspase-3 (C, purple), counterstained with hematoxylin (A) or methyl green (B, C). The dashed line indicates the approximate interface between normal cerebral cortex above and tumor below. (D–F) IHC (brown) of anaplastic astrocytoma for Gfap (D), the light chain of Neurofilament (E) and Nestin (F), counterstained with hematoxylin. The arrow in (E) indicates the soma of a neuron. (G–I) H&E stain showing tumors in the cerebellum (Cbl), spinal cord (SpC) and growing as an exophytic mass from the pons. Scale bar in (D) is 50 μm and applies to (A–F). Scale bars in (G, H) are 200 μm. Scale bar in (I) is 1 mm. See also Figure S3.

Figure 4

Focal amplification, signaling pathway activation and overepression of RTKs in Pten; p53 cKO HGA. (A) Recurrent focal amplification of Met in Pten; p53 cKO HGA. Left panel: aCGH plot of Chr6 location on the x-axis and log₂ ratio on the y-axis is shown for a representative tumor. Amplified region containing Met is indicated. Middle panel: heatmap of log₂ ratio showing focal copy number gains in 13 tumors (horizontal bars) in the amplified region surrounding Met. The boxed region contains Met. Color scale with corresponding log₂ ratio value is below. Right panel: FISH on same tumor as left panel showing amplification of Met (green) compared to control Chr6 probe (red). Scale bar is 2 μm. (B) Survey of pathway activation and RTK expression in Pten; p53 cKO HGAs. Lysates from Pten;p53 cKO mice were prepared from cortex that was grossly free of tumor (lanes 1, 2) or from HGAs demonstrating a variety of histological features (lanes 3–16) and analyzed by western blots with the indicated antibodies. The Met, Egfr and Pdgfra gene CNs below the corresponding blots are calculated from log₂ ratio values obtained from aCGH on the same tumor sample. See also Figure S4 and Tables S1 and S2.

Figure 5

HGAs arising from triple cKO mice. (A) Kaplan-Meier survival analysis was performed on tamoxifen-induced cohorts consisting of Pten; p53 (N=148; red) and triple cKO mice (N=35; blue). (B) H&E stain of a triple cKO brain containing a giant cell glioblastoma. (C) IHC for Pten (brown) on a section corresponding to the same tumor counterstained with hematoxylin. (D) IHC for Ki67 (purple) on an adjacent section counterstained with methyl green. Scale bar in (C) is 50 μm and applies to (B–D). See also Figure S5.

Figure 6

Signaling pathway activation in triple cKO mice. Lysates from triple cKO mice were prepared from cortex that was grossly free of tumor (lanes 1–5) or from HGAs demonstrating a variety of histological features (lanes 6–19) and analyzed by western blots with the indicated antibodies. The Met, Egfr and Pdgfra gene CNs below the corresponding blots are calculated from log₂ ratio values obtained from aCGH on the same tumor sample. One tumor sample (lane 13) did not have a corresponding aCGH profile (ND).

Figure 7

Genome-wide CNAs in murine HGAs. Heatmap of genomic copy number imbalances showing segmented log₂ ratio from aCGH data to identify copy number gains (red) and losses (blue) in 50 Pten; p53 cKO (white bar) and 21 triple cKO (black bar) tumors. Amplifications and gains of genes encoding the RTKs Met (green bars), Egfr (orange bars) and Pdgfr-α (violet bars) are shown. Tumors with amplifications of the cell cycle-related genes shown in Table S2 are also indicated (brown bars). Chromosome numbers are indicated on the right and frequent losses of Chr12 and Chr14 are boxed. Color scale with corresponding log₂ ratio value is to the right. See also Figure S6.

Figure 8

Gene expression signatures of mouse HGAs are similar to human gene expression subgroups. (A) Gene expression profiles generated from Pten; p53 cKO (Model: white bars) and triple cKO (Model: black bars) HGAs were analyzed by unsupervised hierarchical clustering using the top 1,000 probe sets showing the greatest differential expression levels as selected by median absolute deviation scores. Three primary clusters were identified, HC1 – HC3, and the dendrogram is shown on top. The heatmap shows the most up-regulated probe sets for each cluster derived using linear models algorithm. The primary histological features (Histology) are glioblastoma (yellow bars), HGA with myxoid degeneration (light blue bars), anaplastic astrocytoma (purple bars), anaplastic oligoastrocytoma (dark green bar) and one tumor (grey bar) which was not determined. Tumors with RTK and cell cycle-related gene mutations are indicated as in Figure 6. Tumors located at the base of the brain are marked with arrowheads. Color scale with corresponding log₂ ratio value is to the right. (B) GSEA enrichment plots. Each primary cluster was compared to the other two clusters using gene sets defining expression subgroups derived from human glioblastoma studies. The three gene sets from Phillips et al. (2006): Phillips-PN (proneural), Phillips-Prolif (proliferative) and Phillips-Mes (mesenchymal) are the ones that are most significant for each cluster. The nominal p-value and false discovery rate-corrected (FDR) q-value are indicated below. (C) Heatmap of normalized enrichment scores calculated using single sample GSEA comparing individual mouse tumors arranged by cluster as in (A) to gene sets defining expression subgroups previously identified in human glioblastoma. TCGA-PN, TCGA-Neural, TCGA-Classical and TCGA-Mes are the four gene sets derived from Verhaak et al. (2010). Tumors located at the base of the brain are marked with arrowheads. Color scale with corresponding normalized enrichment score is to the right. See also Figure S7 and Table S3.