Activated BRAF induces gliomas in mice when combined with Ink4a/Arf loss or Akt activation

Abstract

Mutations in receptor tyrosine kinase (RTK) growth factor receptors (epidermal growth factor receptor, platelet-derived growth factor receptor, MET and ERBB2), which result in downstream activation of the RAS/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway and PI(3)K/Akt pathway, are found in almost all high-grade gliomas and MAPK signaling is necessary for continued glioma maintenance. In addition, BRAF is mutated in the majority of low-grade gliomas and its expression and activity is significantly increased in the majority of high-grade gliomas. Although the importance of RTKs and RAS signaling in glioma development has been shown, the role of BRAF has yet to be characterized. We evaluated the effect of activated BRAF in glioma formation using the retroviral replication-competent avian leukosis virus long terminal repeat, splice acceptor (RCAS)/TVA system to transfer genes encoding activated forms of BRAF, KRas, Akt and Cre to nestin-expressing neural progenitor cells in Ink4a/Arf(lox/lox) mice in vivo. Although expression of activated BRAF alone is not sufficient for tumorigenesis, the combination of activated BRAF and Akt or BRAF with Ink4a/Arf loss is transforming. Interestingly, activated BRAF generates gliomas with characteristics similar to activated KRas in the context of Akt but not Ink4a/Arf loss. Our studies show a role for BRAF activation and signaling in glioma development and as potential target for glioma therapy.

Figure 1. KRas^G12D and BRAF^V600E cooperate with activated Akt or Ink4a/Arf loss to induce gliomas in N-TVA mice

(a) In vitro expression of GFP in N-TVA astrocytes. RCASBP(A)GFP infection of N-TVA positive and negative (control) cells in culture confirmed stable and targeted gene delivery of GFP into N-TVA expressing cells. (b) Western blot analysis of gene expression. N-TVA-positive immortalized astrocytes were infected in culture with RCASBP(A)GFP (lane 1), RCASBP(A)Akt (lane 2), RCASBP(A)KRas^G12D (lane 3), RCASBP(A)BRAF^V600E (lane 4), RCASBP(A)BRAF^V600E and RCASBP(A)Akt (lane 5), or RCASBP(A)Akt and RCASBP(A)KRas^G12D (lane 6). Lysates were separated on a 4-20% Tris-glycine polyacrylamide gel, transferred to nitrocellulose, and probed with antibodies directed against V5 (BRAF), HA (Akt), pan-RAS (KRas), phoshporylated Erk (P-Erk), or total Erk (Erk) as indicated. The membrane was re-probed with an antibody against α-tubulin (bottom) to ensure equal loading. The Ras blot shows endogenous Ras (lower band) and the virally delivered KRas (upper band). The virally delivered KRas is a higher molecular weight as a result of the FLAG epitope tag. (c) Kaplan-Meier percent survival curve. N-TVA;Ink4a/Arf^lox/lox mice were injected with the indicated viruses at birth. Censored survival data was analyzed using a log-rank test of the Kaplan-Meier estimate of survival. No tumors were observed in mice injected with BRAF^V600E or Cre alone n = 36 (solid black line) and it has previously been shown that no tumors form in mice injected with either KRas^G12D or Akt alone (Holland et al., 2000). There was no significant difference in survival between KRas^G12D + Akt n = 50 (red line) BRAF^V600E + Cre n = 26 (blue line) or BRAF^V600E + Akt n = 28 (green line). A significant difference in survival was observed between KRas^G12D + Cre n = 17 (dashed black line) and all of the other conditions, P < 3 × 10⁻⁷.

Figure 2. Representative tumors induced by KRas^G12D or BRAF^V600E in cooperation with activated Akt or Cre (Ink4a/Arf loss)

Histological comparison of gliomas induced in N-TVA;Ink4a/Arf^lox/lox mice after injection with (a-b) BRAF^V600E + Akt, (c-d) BRAF^V600E + Cre, (e-f) KRas^G12D + Akt and (g-h) KRas^G12D + Cre. Although the CNS border was well defined (g-h) and the tumors were not infiltrative, invasion was seen to progress through the subarachnoid space and Virchow-Robin perivascular spaces (arrow, h). Scale bars represent 500 μm.

Figure 3. Immunohistochemical confirmation of gene expression in gliomas arising in N-TVA;Ink4a/Arf^lox/lox mice

(a) IHC for the V5 epitope tag on BRAF^V600E in gliomas induced by BRAF^V600E + Cre. (b) IHC for the V5 epitope tag on BRAF^V600E in gliomas induced by BRAF^V600E + Akt. (c) IHC for the FLAG epitope tag on KRas in gliomas induced by KRas^G12D +Akt. (d) IHC for the FLAG epitope tag on KRas^G12D in gliomas induced by KRas^G12D + Cre. (e) IHC for the HA epitope tag on Akt in gliomas induced by KRas^G12D + Akt. (f) IHC for the HA epitope tag on Akt in gliomas induced by BRAF^V600E + AKT. IHC for p19 indicates loss of p19 expression in (g) KRas^G12D, (f) BRAF^V600E + Cre induced gliomas. Scale bars represent 200 μm.

Figure 4. Expression of astrocyte and oligodendrocyte markers in gliomas induced with KRas^G12D

(a-b) Corresponding H&E sections for histological comparison with IHC analysis. (c-d) Expression of the astrocyte marker GFAP. (e-f) Expression of the oligodendrocyte marker Olig2. (g-h) Expression of the neural progenitor cell marker Nestin. Scale bars represent 200 μm.

Figure 5. Expression of astrocyte and oligodendrocyte markers in gliomas induced with BRAF^V600E

(I-II) BRAF^V600E gliomas induced with activated Akt were highly pleomorphic in expression and appearance so two examples are highlighted for comparison with (III-IV) the highly monomorphic nature of BRAF^V600E gliomas in the context of Ink4a/Arf loss. (a-b and i-j) Corresponding H&E sections for histological comparison with IHC analysis. (c-f and k-l) Expression of the astrocyte marker GFAP. (e-f and m-n) Expression of the oligodendrocyte marker Olig2. (g-h and o-p) Expression of the neural progenitor cell marker Nestin. Scale bars represent 200 μm.

Figure 6. All gliomas demonstrate MAPK pathway activation and high levels of proliferation

(A-D) Confirmation of MAPK activation. IHC for P-Erk confirmed MAPK activation in all gliomas induced with either KRas^G12D or BRAF^V600E. (e-h) Analysis of tumor vasculature. IHC for the endothelial cell marker endomucin highlights the highly abnormal vasculature of Akt/KRas^G12D gliomas compared with the other tumor samples. (i-l) Assessment of mitotic activity. IHC for the cell division marker Ki67 demonstrates that all of the tumors are highly proliferative. Scale bars represent 200 μm.