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. 2013 Oct;15(10):1317-29.
doi: 10.1093/neuonc/not084. Epub 2013 Jun 27.

Cooperativity between MAPK and PI3K signaling activation is required for glioblastoma pathogenesis

Mark Vitucci  1 , Natalie O Karpinich, Ryan E Bash, Andrea M Werneke, Ralf S Schmid, Kristen K White, Robert S McNeill, Byron Huff, Sophie Wang, Terry Van Dyke, C Ryan Miller
Affiliations

Cooperativity between MAPK and PI3K signaling activation is required for glioblastoma pathogenesis

Mark Vitucci et al. Neuro Oncol. 2013 Oct.

Abstract

Background: Glioblastoma (GBM) genomes feature recurrent genetic alterations that dysregulate core intracellular signaling pathways, including the G1/S cell cycle checkpoint and the MAPK and PI3K effector arms of receptor tyrosine kinase (RTK) signaling. Elucidation of the phenotypic consequences of activated RTK effectors is required for the design of effective therapeutic and diagnostic strategies.

Methods: Genetically defined, G1/S checkpoint-defective cortical murine astrocytes with constitutively active Kras and/or Pten deletion mutations were used to systematically investigate the individual and combined roles of these 2 RTK signaling effectors in phenotypic hallmarks of glioblastoma pathogenesis, including growth, migration, and invasion in vitro. A novel syngeneic orthotopic allograft model system was used to examine in vivo tumorigenesis.

Results: Constitutively active Kras and/or Pten deletion mutations activated both MAPK and PI3K signaling. Their combination led to maximal growth, migration, and invasion of G1/S-defective astrocytes in vitro and produced progenitor-like transcriptomal profiles that mimic human proneural GBM. Activation of both RTK effector arms was required for in vivo tumorigenesis and produced highly invasive, proneural-like GBM.

Conclusions: These results suggest that cortical astrocytes can be transformed into GBM and that combined dysregulation of MAPK and PI3K signaling revert G1/S-defective astrocytes to a primitive gene expression state. This genetically-defined, immunocompetent model of proneural GBM will be useful for preclinical development of MAPK/PI3K-targeted, subtype-specific therapies.

Keywords: Pten; astrocytes; genetically engineered mouse; glioblastoma; invasion.

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Figures

Fig. 1.
Fig. 1.
MAPK and PI3K signaling and growth of G1/S-defective astrocytes with activated Kras and/or Pten deletion. Representative immunoblots showing MAPK and PI3K pathway signaling in G1/S-defective astrocytes with activated Kras, Pten deletion, or both (A). Growth of G1/S defective astrocytes in vitro. Cell number was assessed by counting cells at days 1–7 (B). Mean doubling times ± 95% confidence intervals were calculated from the exponential growth curves in B (C). Growth rates were significantly different across genotypes (P < .0001). Apoptosis in G1/S defective astrocytes in vitro (D). Colors compare genotypes with and without activated Kras. Error bars represent standard error (SEM).
Fig. 2.
Fig. 2.
Kras activation and Pten loss increase G1/S-defective astrocyte migration. Representative photomicrographs of wound closure in T, TR, and TRP−/− astrocytes at 0 and 24 h (A). Mean percent wound closure ± SEM at 24 h (B). Colors compare genotypes with and without activated Kras. Mean velocity ± SEM of individual astrocytes measured using time-lapse microscopy for 1 h (C). Colors compare genotypes with and without activated Kras. Wound closure of TRP−/− astrocytes treated with 10 nM rapamycin (Rapa), 50 µM LY294002 (LY), 10 µM U0126, or both LY294002 and U0126 (D). Mean percent wound closure ± SEM is shown relative to untreated (No Drug) TRP−/− astrocytes.
Fig. 3.
Fig. 3.
Pten deletion is necessary for maximum G1/S-defective astrocyte invasion. Representative photomicrographs of collagen invasion of T, TR, and TRP−/− astrocytes at 4 days (A). Mean percent invasion ± SEM into collagen after 4 days (B). Colors compare genotypes with and without activated Kras. Collagen invasion of TRP−/− astrocytes treated with 10 nM rapamycin (Rapa), 50 µM LY294002 (LY), 10 µM U0126, or both LY294002 and U0126 (C). Mean percent invasion ± SEM is shown relative to untreated (No Drug) TRP−/− astrocytes.
Fig. 4.
Fig. 4.
Restoration of Pten expression limits growth, migration, and invasion in TRP−/− astrocytes. Representative immunoblot of MAPK and PI3K pathway signaling in TRP−/− astrocytes after infection with retrovirus containing Pten or GFP cDNA (A). Growth (B), doubling time (C), mean percent wound closure at 24 h (D), and mean percent invasion into collagen at 1, 3, and 5 days (E) of Pten rescued versus nonrescued (GFP) TRP−/− astrocytes. Mean doubling times ± 95% confidence intervals in C were calculated from the exponential growth curves in B. All experiments are the mean of at least three independent experiments using different astrocyte isolations. Error bars are SEM.
Fig. 5.
Fig. 5.
Gene expression profiling of G1/S-defective astrocytes with activated Kras and/or Pten deletion. Consensus clustering of 22 independently isolated astrocyte cultures identifies 3 clusters (A). Individual isolates are repeated on the X and Y axes. Darker shades of blue signify isolates that cluster together most often. Single sample GSEA (ssGSEA) of the 15 most significantly enriched gene signatures from MsigDB in Class 3 (green) astrocytes (B). ssGSEA of human GBM signatures (C). ssGSEA of murine neural lineage signatures (D). Red signifies higher enrichment scores of signature genes.
Fig. 6.
Fig. 6.
A PI3K signature defined in TRP−/− astrocytes upon release from PI-103-mediated inhibition of PI3K signaling is enriched in human proneural GBM. Heatmap of 518 genes with significantly increased expression in TRP−/− astrocytes after release from PI-103 (A). A box and whiskers plot of the distribution of mean expression of PI3K signature genes (centroid) (B) and ssGSEA (C) shows that the PI3K signature is significantly enriched in human proneural (PN), but not neural (N), classical (Cl), and mesenchymal (Mes) GBM from TCGA.
Fig. 7.
Fig. 7.
G1/S-defective astrocytes form astrocytomas after orthotopic injection into syngeneic, immunocompetent mouse brains. Astrocytoma incidence in terminally aged mice after orthotopic injection of 105 astrocytes (A). The number of mice injected per genotype is indicated. The fraction of astrocytomas in panel A with histological features of high-grade astrocytomas (HGA) (B). The number of astrocytomas detected per genotype is indicated. Kaplan–Meier survival analysis of astrocytoma-bearing mice (C). Median survivals were 36, 57, and 207 days for TRP−/−, TRP+/−, and TR astrocytes, respectively (P < .0001). The incidence of astrocytomas in mice sacrificed between 7 and 28 days after injection with astrocytes of the indicated genotypes (D).
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