BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas

Abstract

The molecular pathogenesis of pediatric astrocytomas is still poorly understood. To further understand the genetic abnormalities associated with these tumors, we performed a genome-wide analysis of DNA copy number aberrations in pediatric low-grade astrocytomas by using array-based comparative genomic hybridization. Duplication of the BRAF protooncogene was the most frequent genomic aberration, and tumors with BRAF duplication showed significantly increased mRNA levels of BRAF and a downstream target, CCND1, as compared with tumors without duplication. Furthermore, denaturing HPLC showed that activating BRAF mutations were detected in some of the tumors without BRAF duplication. Similarly, a marked proportion of low-grade astrocytomas from adult patients also had BRAF duplication. Both the stable silencing of BRAF through shRNA lentiviral transduction and pharmacological inhibition of MEK1/2, the immediate downstream phosphorylation target of BRAF, blocked the proliferation and arrested the growth of cultured tumor cells derived from low-grade gliomas. Our findings implicate aberrant activation of the MAPK pathway due to gene duplication or mutation of BRAF as a molecular mechanism of pathogenesis in low-grade astrocytomas and suggest inhibition of the MAPK pathway as a potential treatment.

Figure 1. DNA copy-number aberrations in pediatric low-grade astrocytomas.

(A) Frequencies of DNA copy number gains and losses of tumors (n = 66) were plotted against their chromosomal position. (B) Complete array-CGH trace of a pilocytic astrocytoma, with a BRAF duplication at 7q34 representing the only detectable genomic aberration in this tumor. This constellation was found in 13 of 66 (20%) tumors (arrow indicates clones with copy number gain). (C) Array-CGH profile of chromosome 7 in another pilocytic astrocytoma with duplication of the BRAF locus at 7q34 (arrow indicates clones with copy number gain). (D) Duplication of the BRAF locus in a pilocytic astrocytoma as assessed by FISH. The probe covering the BRAF locus (3 copies) was labeled in green, and the centromeric control probe (2 copies) in red. Original magnification, ×100.

Figure 2. BRAF and CCND1 expression in primary pilocytic astrocytomas.

(A) BRAF mRNA expression levels in 5 pilocytic astrocytomas with 2 copies of BRAF (left) and 8 pilocytic astrocytomas with 3 copies of BRAF due to gene duplications (right) in relation to the BRAF transcript levels in nonneoplastic brain tissue. Note that the median BRAF mRNA expression level is significantly higher in tumors with BRAF duplications as compared with tumors without this aberration (P = 0.01). (B) CCND1 mRNA expression in the same tumors. Median CCND1 mRNA expression levels are significantly higher in tumors with BRAF duplications than in tumors with 2 gene copies (P = 0.008). (C) Western blot analysis demonstrating BRAF protein expression (upper panel) and ERK1/2 phosphorylation (pERK1/2, middle panel) in 6 tumors with BRAF duplication (lanes 2–7) and in nonneoplastic brain tissue (lane 1, pooled protein fractions from 5 brain tissue samples from 5 different individuals) showing that BRAF is exclusively expressed in the tumors and always associated with activation of MAPK signaling. Total ERK1/2 protein was used as a loading control (ERK1/2, lower panel). nb, normal brain; PA, pilocytic astrocytoma.

Figure 3. Pharmacological inhibition of MAPK signaling in cell lines derived from low-grade gliomas.

(A) Proliferation of glioma cells in vitro after treatment of 4 different cell lines derived from primary low-grade gliomas with the MEK1/2 inhibitor U0126 at a concentration of 20 μM as assessed by MTT assay over a time course of 48 hours. A °II, diffuse astrocytoma; OA, oligoastrocytoma. (B) Effective growth inhibition in all 4 cell lines can be achieved over a broad spectrum of concentrations ranging from 1 μM up to 20 μM. Medians and SDs of triplicate measurements at 48 hours are shown. (C) Dephosphorylation of ERK1/2 is readily detectable after 30 minutes and is maintained for 48 hours after a single dose of inhibitor at a concentration of 1 μM. (D) Maximal downregulation of CCND1 mRNA expression after treatment with MEK1/2 inhibitor U0126 at a concentration of μM was observed after 24 hours.

Figure 4. Functional analyses of low-grade glioma cell lines after pharmacological inhibition of MAPK signaling.

Cell-cycle analysis by measurement of DNA content in NCH492 cells prior to (A) and 48 hours after treatment with 10 μM U0126 (C). Cells were arrested in G2/M upon treatment with U0126 and lacked a subG1-peak, indicating no increase in apoptosis. Early apoptosis rate was additionally determined by annexin V staining and was only slightly increased 24 hours after drug treatment (B and D). Puromycin was used as a positive control for apoptosis induction (E and F).

Figure 5. Stable silencing of BRAF expression in pilocytic astrocytoma cells.

(A) Proliferation of NCH492 pilocytic astrocytoma cells after shRNA-mediated silencing of BRAF using 3 different shRNAs as assessed by MTT assay 24 hours after plating equal numbers of cells. Nontargeting shRNA was used as a reference. Severe growth inhibition was observed for all 3 shRNAs targeting BRAF. Error bars represent the SD between replicates. (B) Microscopic examination of the same samples at the time of MTT analysis showing growth arrest of cells upon BRAF knockdown. Original magnification, ×10. (C–F) Cell-cycle analysis 24 hours after plating equal numbers of cells either carrying nontargeting shRNA (C) or one of the shRNAs targeting BRAF (D–F). As observed for the treatment with MEK1/2 inhibitor, cells accumulated in the G2/M phase of the cell cycle.