Genomically amplified Akt3 activates DNA repair pathway and promotes glioma progression

Abstract

Akt is a robust oncogene that plays key roles in the development and progression of many cancers, including glioma. We evaluated the differential propensities of the Akt isoforms toward progression in the well-characterized RCAS/Ntv-a mouse model of PDGFB-driven low grade glioma. A constitutively active myristoylated form of Akt1 did not induce high-grade glioma (HGG). In stark contrast, Akt2 and Akt3 showed strong progression potential with 78% and 97% of tumors diagnosed as HGG, respectively. We further revealed that significant variations in polarity and hydropathy values among the Akt isoforms in both the pleckstrin homology domain (P domain) and regulatory domain (R domain) were critical in mediating glioma progression. Gene expression profiles from representative Akt-derived tumors indicated dominant and distinct roles for Akt3, consisting primarily of DNA repair pathways. TCGA data from human GBM closely reflected the DNA repair function, as Akt3 was significantly correlated with a 76-gene signature DNA repair panel. Consistently, compared with Akt1 and Akt2 overexpression models, Akt3-expressing human GBM cells had enhanced activation of DNA repair proteins, leading to increased DNA repair and subsequent resistance to radiation and temozolomide. Given the wide range of Akt3-amplified cancers, Akt3 may represent a key resistance factor.

Keywords: Akt; DNA repair; RCAS/tv-a mouse model; glioma.

Fig. 1.

Akt3 frequently amplified in cancer and tops hierarchy in glioma. (A) Copy number variation of Akt isoforms in TCGA cancers, shown in order of increasing Akt3 amplification percentage. (B) Percentage of HGG and representative tumors shown from Akt isoforms injected in conjunction with PDGFB. P values represent Fisher’s exact test. *P < 0.05, ***P < 0.001. Scale bar for images and insets, 100 and 50 µm, respectively.

Fig. 2.

Variable regions in pleckstrin homology and regulatory domains contribute to Akt isoform function. (A) Grantham polarity values for each amino acid plotted. Enlarged portions depict variable regions. (B) Akt2/Akt1 and Akt3/Akt1 chimeras were generated by domain swapping and injected in combination with PDGFB. Representative H&E HGG images are shown. (Scale bar, 100 µm.)

Fig. 3.

Akt3 regulates DNA repair pathways. (A) Venn diagram representing number of shared and unique differentially expressed genes among mouse tumors generated from PDGFB with Akt1, Akt2, or Akt3. (B) GO enrichment analysis based on Akt2 differentially expressed genes. (C) Heatmap of the Akt2-related pathway genes from B. (D) GO enrichment analysis based on Akt3 differentially expressed genes. (E) Heatmap of Akt3-related pathway genes from D. (F) Partial correlation among Akt isoforms and Akt3 signature genes across 166 TCGA GBM samples.

Fig. 4.

Specific role for Akt3 in activating DNA damage response pathways. (A, Left) Micronuclei were detected in U87 cells with Akt isoforms. (A, Right) Quantification of MN relative to control cells was assessed by counting more than 100 DAPI-stained nuclei in U87 cells with Akt isoforms exposed to 2 Gy irradiation. ***P < 0.0001 ANOVA with Dunnett’s post hoc test. (B, Left) γH2AX foci in U87 nuclei with 2 Gy irradiation, fixed and stained 30 min postirradiation. (Scale bar, 10 µm.) (B, Right) Quantification of γH2AX foci. Data represent foci in irradiated nuclei normalized to untreated nuclei. *P = 0.01, t test, corrected by multiple testing. (C) Semiquantitative PCR measuring end joining in U87 nuclear extracts. Extent of end joining assayed with primers spanning the breakpoint. (D) Homologous recombination of U87 cells stably expressing both an Akt isoform and a nonfunctional GFP cassette. Cells were transfected with I-SceI. Percent recombination represents GFP-positive cells as measured via flow cytometry. **P < 0.01, ANOVA with Dunnett’s post hoc test. (E and F) MTT measurement of U87 cell viabilty 5 d postirradiation or TMZ treatment. *P < 0.05, ***P < 0.001, two-way ANOVA with Tukey’s Post hoc test. (G) Number of mutations in TCGA GBM samples according to Akt3 copy number. P < 0.05, one tailed t test.