BRAF activation induces transformation and then senescence in human neural stem cells

A pilocytic astrocytoma model

Eric Raabe, Kah Suan Lim, Julia Minjung Kim, Alan Keith Meeker, Xing Gang Mao, Guido Nikkhah, Jarek Maciaczyk, Ulf Kahlert, Deepali Jain, Eli Bar, Kenneth J Cohen, Charles G Eberhart

Research output: Contribution to journalArticle

Abstract

Purpose: BRAF is frequently activated by gene fusion or point mutation in pilocytic astrocytoma, the most common pediatric brain tumor. We investigated the functional effect of constitutive BRAF activation in normal human neural stem and progenitor cells to determine its role in tumor induction in the brain. Experimental Design: The constitutively active BRAF V600E allele was introduced into human neurospheres, and its effects on MAPK (mitogen-activated protein kinase) signaling, proliferation, soft agarose colony formation, stem cell phenotype, and induction of cellular senescence were assayed. Immunohistochemistry was used to examine p16 INK4a levels in pilocytic astrocytoma. Results: BRAF V600E expression initially strongly promoted colony formation but did not lead to significantly increased proliferation. BRAF V600E-expressing cells subsequently stopped proliferating and induced markers of oncogene-induced senescence including acidic β-galactosidase, PAI-1, and p16 INK4a whereas controls did not. Onset of senescence was associated with decreased expression of neural stem cell markers including SOX2. Primary pilocytic astrocytoma cultures also showed induction of acidic β-galactosidase activity. Immunohistochemical examination of 66 pilocytic astrocytomas revealed p16 INK4a immunoreactivity in the majority of cases, but patients with tumors negative for p16 INK4a had significantly shorter overall survival. Conclusions: BRAF activation in human neural stem and progenitor cells initially promotes clonogenic growth in soft agarose, suggesting partial cellular transformation, but oncogene-induced senescence subsequently limits proliferation. Induction of senescence by BRAF may help explain the low-grade pathobiology of pilocytic astrocytoma, whereas worse clinical outcomes associated with tumors lacking p16 INK4a expression could reflect failure to induce senescence or an escape from oncogene-induced senescence.

Original languageEnglish (US)
Pages (from-to)3590-3599
Number of pages10
JournalClinical Cancer Research
Volume17
Issue number11
DOIs
StatePublished - Jun 1 2011

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Neural Stem Cells
Astrocytoma
Oncogenes
Galactosidases
Stem Cells
Sepharose
Neoplasms
Cell Aging
Gene Fusion
Plasminogen Activator Inhibitor 1
Mitogen-Activated Protein Kinases
Point Mutation
Brain Neoplasms
Research Design
Immunohistochemistry
Alleles
Pediatrics
Phenotype
Survival
Brain

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

BRAF activation induces transformation and then senescence in human neural stem cells : A pilocytic astrocytoma model. / Raabe, Eric; Lim, Kah Suan; Kim, Julia Minjung; Meeker, Alan Keith; Mao, Xing Gang; Nikkhah, Guido; Maciaczyk, Jarek; Kahlert, Ulf; Jain, Deepali; Bar, Eli; Cohen, Kenneth J; Eberhart, Charles G.

In: Clinical Cancer Research, Vol. 17, No. 11, 01.06.2011, p. 3590-3599.

Research output: Contribution to journalArticle

Raabe, Eric ; Lim, Kah Suan ; Kim, Julia Minjung ; Meeker, Alan Keith ; Mao, Xing Gang ; Nikkhah, Guido ; Maciaczyk, Jarek ; Kahlert, Ulf ; Jain, Deepali ; Bar, Eli ; Cohen, Kenneth J ; Eberhart, Charles G. / BRAF activation induces transformation and then senescence in human neural stem cells : A pilocytic astrocytoma model. In: Clinical Cancer Research. 2011 ; Vol. 17, No. 11. pp. 3590-3599.
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T1 - BRAF activation induces transformation and then senescence in human neural stem cells

T2 - A pilocytic astrocytoma model

AU - Raabe, Eric

AU - Lim, Kah Suan

AU - Kim, Julia Minjung

AU - Meeker, Alan Keith

AU - Mao, Xing Gang

AU - Nikkhah, Guido

AU - Maciaczyk, Jarek

AU - Kahlert, Ulf

AU - Jain, Deepali

AU - Bar, Eli

AU - Cohen, Kenneth J

AU - Eberhart, Charles G

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N2 - Purpose: BRAF is frequently activated by gene fusion or point mutation in pilocytic astrocytoma, the most common pediatric brain tumor. We investigated the functional effect of constitutive BRAF activation in normal human neural stem and progenitor cells to determine its role in tumor induction in the brain. Experimental Design: The constitutively active BRAF V600E allele was introduced into human neurospheres, and its effects on MAPK (mitogen-activated protein kinase) signaling, proliferation, soft agarose colony formation, stem cell phenotype, and induction of cellular senescence were assayed. Immunohistochemistry was used to examine p16 INK4a levels in pilocytic astrocytoma. Results: BRAF V600E expression initially strongly promoted colony formation but did not lead to significantly increased proliferation. BRAF V600E-expressing cells subsequently stopped proliferating and induced markers of oncogene-induced senescence including acidic β-galactosidase, PAI-1, and p16 INK4a whereas controls did not. Onset of senescence was associated with decreased expression of neural stem cell markers including SOX2. Primary pilocytic astrocytoma cultures also showed induction of acidic β-galactosidase activity. Immunohistochemical examination of 66 pilocytic astrocytomas revealed p16 INK4a immunoreactivity in the majority of cases, but patients with tumors negative for p16 INK4a had significantly shorter overall survival. Conclusions: BRAF activation in human neural stem and progenitor cells initially promotes clonogenic growth in soft agarose, suggesting partial cellular transformation, but oncogene-induced senescence subsequently limits proliferation. Induction of senescence by BRAF may help explain the low-grade pathobiology of pilocytic astrocytoma, whereas worse clinical outcomes associated with tumors lacking p16 INK4a expression could reflect failure to induce senescence or an escape from oncogene-induced senescence.

AB - Purpose: BRAF is frequently activated by gene fusion or point mutation in pilocytic astrocytoma, the most common pediatric brain tumor. We investigated the functional effect of constitutive BRAF activation in normal human neural stem and progenitor cells to determine its role in tumor induction in the brain. Experimental Design: The constitutively active BRAF V600E allele was introduced into human neurospheres, and its effects on MAPK (mitogen-activated protein kinase) signaling, proliferation, soft agarose colony formation, stem cell phenotype, and induction of cellular senescence were assayed. Immunohistochemistry was used to examine p16 INK4a levels in pilocytic astrocytoma. Results: BRAF V600E expression initially strongly promoted colony formation but did not lead to significantly increased proliferation. BRAF V600E-expressing cells subsequently stopped proliferating and induced markers of oncogene-induced senescence including acidic β-galactosidase, PAI-1, and p16 INK4a whereas controls did not. Onset of senescence was associated with decreased expression of neural stem cell markers including SOX2. Primary pilocytic astrocytoma cultures also showed induction of acidic β-galactosidase activity. Immunohistochemical examination of 66 pilocytic astrocytomas revealed p16 INK4a immunoreactivity in the majority of cases, but patients with tumors negative for p16 INK4a had significantly shorter overall survival. Conclusions: BRAF activation in human neural stem and progenitor cells initially promotes clonogenic growth in soft agarose, suggesting partial cellular transformation, but oncogene-induced senescence subsequently limits proliferation. Induction of senescence by BRAF may help explain the low-grade pathobiology of pilocytic astrocytoma, whereas worse clinical outcomes associated with tumors lacking p16 INK4a expression could reflect failure to induce senescence or an escape from oncogene-induced senescence.

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