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Duplication of 7q34 is specific to juvenile pilocytic astrocytomas and a hallmark of cerebellar and optic pathway tumours.

Jacob K, Albrecht S, Sollier C, Faury D, Sader E, Montpetit A, Serre D, Hauser P, Garami M, Bognar L, Hanzely Z, Montes JL, Atkinson J, Farmer JP, Bouffet E, Hawkins C, Tabori U, Jabado N - Br. J. Cancer (2009)

Bottom Line: We demonstrate that 7q34 duplication is specific to sporadic JPA (35 of 53 - 66%) and does not occur in other LGA subtypes (0 of 27) or NF1-associated-JPA (0 of 4).Gain of function studies performed on hTERT-immortalised astrocytes show that overexpression of wild-type BRAF does not increase cell proliferation or baseline MAPK signalling even if it sensitises cells to EGFR stimulation.Our results suggest that variants of JPA might arise from a unique site-restricted progenitor cell where 7q34 duplication, a hallmark of this tumour-type in association to MAPK-kinase pathway activation, potentially plays a site-specific role in their pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Human Genetics, Montreal Children's Hospital, McGill University Health Center, Montreal H3Z 2Z3, Canada. nada.jabado@mcgill.ca

ABSTRACT

Background: Juvenile pilocytic astrocytomas (JPA), a subgroup of low-grade astrocytomas (LGA), are common, heterogeneous and poorly understood subset of brain tumours in children. Chromosomal 7q34 duplication leading to fusion genes formed between KIAA1549 and BRAF and subsequent constitutive activation of BRAF was recently identified in a proportion of LGA, and may be involved in their pathogenesis. Our aim was to investigate additional chromosomal unbalances in LGA and whether incidence of 7q34 duplication is associated with tumour type or location.

Methods and results: Using Illumina-Human-Hap300-Duo and 610-Quad high-resolution-SNP-based arrays and quantitative PCR on genes of interest, we investigated 84 paediatric LGA. We demonstrate that 7q34 duplication is specific to sporadic JPA (35 of 53 - 66%) and does not occur in other LGA subtypes (0 of 27) or NF1-associated-JPA (0 of 4). We also establish that it is site specific as it occurs in the majority of cerebellar JPA (24 of 30 - 80%) followed by brainstem, hypothalamic/optic pathway JPA (10 of 16 - 62.5%) and is rare in hemispheric JPA (1 of 7 - 14%). The MAP-kinase pathway, assessed through ERK phosphorylation, was active in all tumours regardless of 7q34 duplication. Gain of function studies performed on hTERT-immortalised astrocytes show that overexpression of wild-type BRAF does not increase cell proliferation or baseline MAPK signalling even if it sensitises cells to EGFR stimulation.

Conclusions and interpretation: Our results suggest that variants of JPA might arise from a unique site-restricted progenitor cell where 7q34 duplication, a hallmark of this tumour-type in association to MAPK-kinase pathway activation, potentially plays a site-specific role in their pathogenesis. Importantly, gain of function abnormalities in components of MAP-Kinase signalling are potentially present in all JPA making this tumour amenable to therapeutic targeting of this pathway.

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Functional analysis of BRAF and HIPK2 overexpression in hTERT-immortalised astrocytes. (A) Immunofluorescence analysis of hTERT-immortalised astrocytes transfected with CMYC/BRAF or the empty vector (EV) at 48 h using an antibody recognising the C-MYC tag (red), BRAF (green) and DAPI counterstaining (blue). (B) Immunofluorescence analysis of hTERT-immortalised astrocytes transfected with FLAG/HIPK2 or the EV at 48 h using an antibody recognising the FLAG tag (red) and DAPI counterstaining (blue). (C) Proliferation of hTERT-immortalised astrocytes was assessed at 48, 72 and 96 h following transfection with mock, BRAF, HIPK2 or both genes. No difference in the rate of cell growth was observed between the transfectant cells. Results represent the median of three separate experiments performed in triplicates. (C) Total cell extracts of hTERT-immortalised astrocytes transfected with the empty-vector or C-Myc-tagged BRAF. Cells were serum starved overnight before protein lysate extraction. (D) Left panel: hTERT-immortalised astrocytes were transiently transfected with CMYC/BRAF or the empty vector. Cells were serum starved overnight and total protein lysates extracted at 48 h posttransfection. Western blot analysis for C-Myc, BRAF, phopshoERK (pERK) and β-actin (loading control) was performed. Right panel: Empty-vector (EV) and C-Myc/BRAF transfectant hTERT-immortalised astrocytes were stimulated with 200 ng of epidermal growth factor (EGF) for 5 and 15 min. Total cell lysates extracted at baseline (0) and following activation (5, 15 min) with EGF were immunoblotted using antibodies against β-actin (loading control) and phosphorylated ERK (pERK), C-Myc and BRAF. Note the shift of BRAF immunoreactive band following EGF activation which indicates phosphorylation of the kinase and increase in its molecular weight. Importantly, increase in pERK levels relative to baseline following EGF stimulation was two fold higher in C-MYC/BRAF transfectant cells than in EV transfectants. Results represent the median of three separate experiments (P<0.001).
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fig4: Functional analysis of BRAF and HIPK2 overexpression in hTERT-immortalised astrocytes. (A) Immunofluorescence analysis of hTERT-immortalised astrocytes transfected with CMYC/BRAF or the empty vector (EV) at 48 h using an antibody recognising the C-MYC tag (red), BRAF (green) and DAPI counterstaining (blue). (B) Immunofluorescence analysis of hTERT-immortalised astrocytes transfected with FLAG/HIPK2 or the EV at 48 h using an antibody recognising the FLAG tag (red) and DAPI counterstaining (blue). (C) Proliferation of hTERT-immortalised astrocytes was assessed at 48, 72 and 96 h following transfection with mock, BRAF, HIPK2 or both genes. No difference in the rate of cell growth was observed between the transfectant cells. Results represent the median of three separate experiments performed in triplicates. (C) Total cell extracts of hTERT-immortalised astrocytes transfected with the empty-vector or C-Myc-tagged BRAF. Cells were serum starved overnight before protein lysate extraction. (D) Left panel: hTERT-immortalised astrocytes were transiently transfected with CMYC/BRAF or the empty vector. Cells were serum starved overnight and total protein lysates extracted at 48 h posttransfection. Western blot analysis for C-Myc, BRAF, phopshoERK (pERK) and β-actin (loading control) was performed. Right panel: Empty-vector (EV) and C-Myc/BRAF transfectant hTERT-immortalised astrocytes were stimulated with 200 ng of epidermal growth factor (EGF) for 5 and 15 min. Total cell lysates extracted at baseline (0) and following activation (5, 15 min) with EGF were immunoblotted using antibodies against β-actin (loading control) and phosphorylated ERK (pERK), C-Myc and BRAF. Note the shift of BRAF immunoreactive band following EGF activation which indicates phosphorylation of the kinase and increase in its molecular weight. Importantly, increase in pERK levels relative to baseline following EGF stimulation was two fold higher in C-MYC/BRAF transfectant cells than in EV transfectants. Results represent the median of three separate experiments (P<0.001).

Mentions: To assess the potential of increased levels of wild-type BRAF to drive increased activity of mitogenic pathways in cells of glial origin we used hTERT-immortalised astrocytes (Kamnasaran et al, 2007) as recipients of transient overexpression of genes from the 7q34 region, wild-type (WT) BRAF, HIPK2 or both. Mutant V600E BRAF was used as a positive control for transformation of astrocytes. Transfection efficiency was estimated by immunofluorescence analysis against the C-Myc and FLAG tags to be approximately ∼40–50% at 48, 72 and 96 h (Figure 4A and B). No foci of transformation or morphological changes were observed in cells transfected with WT constructs, whereas transformation foci were readily distinguishable in V600E transfectant cells. Proliferation assays revealed no change in growth of cells overexpressing of BRAF, HIPK2 or both, compared to their mock-transfected controls (Figure 4C). Similarly, overexpression of BRAF had no discernible effect on the baseline level of ERK expression or phosphorylation (Figure 4D). Indeed, in serum starved EV and C-Myc-BRAF transfectant cells pERK was similar in intensity in both settings, in keeping with previous findings in COS cells (Ciampi et al, 2005). Remarkably, exposure of BRAF overexpressing cells to ligands of the epidermal growth factor receptor (EGFR), such as transforming growth factor-α or EGF increased pERK levels by a mean of ∼5.9-fold relative to 2.3-fold in empty-vector transfectants (mean of three distinct experiments, Figure 4D). These results indicate that although BRAF overexpression may not be sufficient to drive proliferative responses of astrocytic cells it may sensitise them to exogenous growth factors.


Duplication of 7q34 is specific to juvenile pilocytic astrocytomas and a hallmark of cerebellar and optic pathway tumours.

Jacob K, Albrecht S, Sollier C, Faury D, Sader E, Montpetit A, Serre D, Hauser P, Garami M, Bognar L, Hanzely Z, Montes JL, Atkinson J, Farmer JP, Bouffet E, Hawkins C, Tabori U, Jabado N - Br. J. Cancer (2009)

Functional analysis of BRAF and HIPK2 overexpression in hTERT-immortalised astrocytes. (A) Immunofluorescence analysis of hTERT-immortalised astrocytes transfected with CMYC/BRAF or the empty vector (EV) at 48 h using an antibody recognising the C-MYC tag (red), BRAF (green) and DAPI counterstaining (blue). (B) Immunofluorescence analysis of hTERT-immortalised astrocytes transfected with FLAG/HIPK2 or the EV at 48 h using an antibody recognising the FLAG tag (red) and DAPI counterstaining (blue). (C) Proliferation of hTERT-immortalised astrocytes was assessed at 48, 72 and 96 h following transfection with mock, BRAF, HIPK2 or both genes. No difference in the rate of cell growth was observed between the transfectant cells. Results represent the median of three separate experiments performed in triplicates. (C) Total cell extracts of hTERT-immortalised astrocytes transfected with the empty-vector or C-Myc-tagged BRAF. Cells were serum starved overnight before protein lysate extraction. (D) Left panel: hTERT-immortalised astrocytes were transiently transfected with CMYC/BRAF or the empty vector. Cells were serum starved overnight and total protein lysates extracted at 48 h posttransfection. Western blot analysis for C-Myc, BRAF, phopshoERK (pERK) and β-actin (loading control) was performed. Right panel: Empty-vector (EV) and C-Myc/BRAF transfectant hTERT-immortalised astrocytes were stimulated with 200 ng of epidermal growth factor (EGF) for 5 and 15 min. Total cell lysates extracted at baseline (0) and following activation (5, 15 min) with EGF were immunoblotted using antibodies against β-actin (loading control) and phosphorylated ERK (pERK), C-Myc and BRAF. Note the shift of BRAF immunoreactive band following EGF activation which indicates phosphorylation of the kinase and increase in its molecular weight. Importantly, increase in pERK levels relative to baseline following EGF stimulation was two fold higher in C-MYC/BRAF transfectant cells than in EV transfectants. Results represent the median of three separate experiments (P<0.001).
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Related In: Results  -  Collection

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fig4: Functional analysis of BRAF and HIPK2 overexpression in hTERT-immortalised astrocytes. (A) Immunofluorescence analysis of hTERT-immortalised astrocytes transfected with CMYC/BRAF or the empty vector (EV) at 48 h using an antibody recognising the C-MYC tag (red), BRAF (green) and DAPI counterstaining (blue). (B) Immunofluorescence analysis of hTERT-immortalised astrocytes transfected with FLAG/HIPK2 or the EV at 48 h using an antibody recognising the FLAG tag (red) and DAPI counterstaining (blue). (C) Proliferation of hTERT-immortalised astrocytes was assessed at 48, 72 and 96 h following transfection with mock, BRAF, HIPK2 or both genes. No difference in the rate of cell growth was observed between the transfectant cells. Results represent the median of three separate experiments performed in triplicates. (C) Total cell extracts of hTERT-immortalised astrocytes transfected with the empty-vector or C-Myc-tagged BRAF. Cells were serum starved overnight before protein lysate extraction. (D) Left panel: hTERT-immortalised astrocytes were transiently transfected with CMYC/BRAF or the empty vector. Cells were serum starved overnight and total protein lysates extracted at 48 h posttransfection. Western blot analysis for C-Myc, BRAF, phopshoERK (pERK) and β-actin (loading control) was performed. Right panel: Empty-vector (EV) and C-Myc/BRAF transfectant hTERT-immortalised astrocytes were stimulated with 200 ng of epidermal growth factor (EGF) for 5 and 15 min. Total cell lysates extracted at baseline (0) and following activation (5, 15 min) with EGF were immunoblotted using antibodies against β-actin (loading control) and phosphorylated ERK (pERK), C-Myc and BRAF. Note the shift of BRAF immunoreactive band following EGF activation which indicates phosphorylation of the kinase and increase in its molecular weight. Importantly, increase in pERK levels relative to baseline following EGF stimulation was two fold higher in C-MYC/BRAF transfectant cells than in EV transfectants. Results represent the median of three separate experiments (P<0.001).
Mentions: To assess the potential of increased levels of wild-type BRAF to drive increased activity of mitogenic pathways in cells of glial origin we used hTERT-immortalised astrocytes (Kamnasaran et al, 2007) as recipients of transient overexpression of genes from the 7q34 region, wild-type (WT) BRAF, HIPK2 or both. Mutant V600E BRAF was used as a positive control for transformation of astrocytes. Transfection efficiency was estimated by immunofluorescence analysis against the C-Myc and FLAG tags to be approximately ∼40–50% at 48, 72 and 96 h (Figure 4A and B). No foci of transformation or morphological changes were observed in cells transfected with WT constructs, whereas transformation foci were readily distinguishable in V600E transfectant cells. Proliferation assays revealed no change in growth of cells overexpressing of BRAF, HIPK2 or both, compared to their mock-transfected controls (Figure 4C). Similarly, overexpression of BRAF had no discernible effect on the baseline level of ERK expression or phosphorylation (Figure 4D). Indeed, in serum starved EV and C-Myc-BRAF transfectant cells pERK was similar in intensity in both settings, in keeping with previous findings in COS cells (Ciampi et al, 2005). Remarkably, exposure of BRAF overexpressing cells to ligands of the epidermal growth factor receptor (EGFR), such as transforming growth factor-α or EGF increased pERK levels by a mean of ∼5.9-fold relative to 2.3-fold in empty-vector transfectants (mean of three distinct experiments, Figure 4D). These results indicate that although BRAF overexpression may not be sufficient to drive proliferative responses of astrocytic cells it may sensitise them to exogenous growth factors.

Bottom Line: We demonstrate that 7q34 duplication is specific to sporadic JPA (35 of 53 - 66%) and does not occur in other LGA subtypes (0 of 27) or NF1-associated-JPA (0 of 4).Gain of function studies performed on hTERT-immortalised astrocytes show that overexpression of wild-type BRAF does not increase cell proliferation or baseline MAPK signalling even if it sensitises cells to EGFR stimulation.Our results suggest that variants of JPA might arise from a unique site-restricted progenitor cell where 7q34 duplication, a hallmark of this tumour-type in association to MAPK-kinase pathway activation, potentially plays a site-specific role in their pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Human Genetics, Montreal Children's Hospital, McGill University Health Center, Montreal H3Z 2Z3, Canada. nada.jabado@mcgill.ca

ABSTRACT

Background: Juvenile pilocytic astrocytomas (JPA), a subgroup of low-grade astrocytomas (LGA), are common, heterogeneous and poorly understood subset of brain tumours in children. Chromosomal 7q34 duplication leading to fusion genes formed between KIAA1549 and BRAF and subsequent constitutive activation of BRAF was recently identified in a proportion of LGA, and may be involved in their pathogenesis. Our aim was to investigate additional chromosomal unbalances in LGA and whether incidence of 7q34 duplication is associated with tumour type or location.

Methods and results: Using Illumina-Human-Hap300-Duo and 610-Quad high-resolution-SNP-based arrays and quantitative PCR on genes of interest, we investigated 84 paediatric LGA. We demonstrate that 7q34 duplication is specific to sporadic JPA (35 of 53 - 66%) and does not occur in other LGA subtypes (0 of 27) or NF1-associated-JPA (0 of 4). We also establish that it is site specific as it occurs in the majority of cerebellar JPA (24 of 30 - 80%) followed by brainstem, hypothalamic/optic pathway JPA (10 of 16 - 62.5%) and is rare in hemispheric JPA (1 of 7 - 14%). The MAP-kinase pathway, assessed through ERK phosphorylation, was active in all tumours regardless of 7q34 duplication. Gain of function studies performed on hTERT-immortalised astrocytes show that overexpression of wild-type BRAF does not increase cell proliferation or baseline MAPK signalling even if it sensitises cells to EGFR stimulation.

Conclusions and interpretation: Our results suggest that variants of JPA might arise from a unique site-restricted progenitor cell where 7q34 duplication, a hallmark of this tumour-type in association to MAPK-kinase pathway activation, potentially plays a site-specific role in their pathogenesis. Importantly, gain of function abnormalities in components of MAP-Kinase signalling are potentially present in all JPA making this tumour amenable to therapeutic targeting of this pathway.

Show MeSH
Related in: MedlinePlus