Genetic Aberrations Leading to MAPK Pathway Activation ... · Karine Jacob 1, Dongh-Anh...

2011;17:4650-4660. Published OnlineFirst May 24, 2011.Clin Cancer Res Karine Jacob, Dongh-Anh Quang-Khuong, David T.W. Jones, et al. Pilocytic AstrocytomasMediate Oncogene-Induced Senescence in Sporadic Genetic Aberrations Leading to MAPK Pathway Activation

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Human Cancer Biology

Genetic Aberrations Leading to MAPK Pathway Activation MediateOncogene-Induced Senescence in Sporadic Pilocytic Astrocytomas

Karine Jacob1, Dongh-Anh Quang-Khuong1, David T.W. Jones5,8, Hendrik Witt5, Sally Lambert8, Steffen Albrecht2,Olaf Witt6, Catherine Vezina3, Margret Shirinian3, Damien Faury1,3, Miklos Garami9, Peter Hauser9, Almos Klekner10,Laszlo Bognar10, Jean-Pierre Farmer4, Jose-Luis Montes4, Jeffrey Atkinson4, Cynthia Hawkins11,Andrey Korshunov7, V. Peter Collins8, Stefan M. Pfister5,6, Uri Tabori12, and Nada Jabado1,3

AbstractPurpose: Oncogenic BRAF/Ras or NF1 loss can potentially trigger oncogene-induced senescence (OIS)

through activation of the mitogen-activated protein kinase (MAPK) pathway. Somatic genetic abnorm-

alities affecting this pathway occur in the majority of pilocytic astrocytomas (PA), the most prevalent brain

neoplasm in children. We investigated whether OIS is induced in PA.

Experimental Design: We tested expression of established senescence markers in three independent

cohorts of sporadic PA. We also assessed for OIS in vitro, using forced expression of wild-type and V600E-

mutant BRAF in two astrocytic cell lines: human telomerase reverse transcriptase (hTERT)-immortalized

astrocytes and fetal astrocytes.Results:Our results indicate that PAs are senescent as evidenced bymarked senescence-associated acidic

b-galactosidase activity, low KI-67 index, and induction of p16INK4a but not p53 in the majority of 52 PA

samples (46 of 52; 88.5%). Overexpression of a number of senescence-associated genes [CDKN2A (p16),

CDKN1A (p21), CEBPB, GADD45A, and IGFBP7] was shown at the mRNA level in two independent PA

tumor series. In vitro, sustained activation of wild-type or mutant BRAF induced OIS in both astrocytic cell

lines. Loss of p16INK4a in immortalized astrocytes abrogated OIS, indicative of the role of this pathway in

mediating this phenomenon in astrocytes. OIS is a mechanism of tumor suppression that restricts the

progression of benign tumors. We show that it is triggered in PAs through p16INK4a pathway inductionfollowing aberrant MAPK activation.

Conclusions: OISmay account for the slow growth pattern in PA, the lack of progression to higher-grade

astrocytomas,andthehighoverallsurvivalofaffectedpatients.ClinCancerRes;17(14);4650–60.’2011AACR.

Introduction

Primary brain tumors are the second most common typeof cancer in children (after leukemia) and the leading causeof cancer-relatedmortality andmorbidity in young patients(1, 2). Astrocytomas account for 50% of all central nervoussystem (CNS) tumors and are commonly regrouped intolow-grade (WHO grade I and II, LGA) or high-grade (WHOgrade III and IV, HGA) tumors (3). Pilocytic astrocytomas(PA) are WHO grade I tumors. They are the predominanthistologic subtype in LGA and the most prevalent CNSneoplasm in childhood, accounting for 23% of all pediatricbrain tumors (2). They occur sporadically throughout child-hood and in 15% to 40% of children affected with neurofi-bromatosis type 1 (NF1; ref. 4). These tumors arisethroughout the CNS but have a predilection for the cere-bellum and the optic pathways (5). PAs are slow growingtumors, which often harbor a cystic component. Theyexhibit distinct features, readily distinguishable from otherLGAs, including an improved clinical course and prognosisin children, limited proliferation index, and no TP53muta-tions or platelet-derived growth factor (PDGF) A/PDGFreceptor a (PDGFRa) amplification (6–10). Maximal

Authors' Affiliations: 1Department of Human Genetics, McGill UniversityHealth Center Research Institute; 2Department of Pathology, 3Departmentof Pediatrics, Division of Hematology-Oncology, and 4Department of Sur-gery, Division of Neurosurgery, Montreal Children's Hospital, McGill Uni-versity Health Center, Montreal, Canada; 5Division of Molecular Genetics,German Cancer Research Center; 6Department of Pediatric Oncology,Hematology and Immunology, University Hospital Heidelberg; 7Depart-ment of Neuropathology, Institute of Pathology, University of Heidelberg,Heidelberg, Germany, and Clinical Cooperation Unit Neuropathology, Ger-man Cancer Research Centre; 8Division of Molecular Histopathology,Department of Pathology, University of Cambridge, Cambridge, UnitedKingdom; 92ndDepartment of Paediatrics andDepartment of Neuropathol-ogy, Faculty ofMedicine, SemmelweisUniversity, Budapest; 10Departmentof Neurosurgery, Medical and Health Science Center, University of Deb-recen, Debrecen, Hungary; 11Division of Pathology and The Labatt BrainTumour Research Centre, The Hospital for Sick Children; and 12Division ofPediatric Hematology-Oncology and The Labatt Brain Tumour ResearchCentre, The Hospital for Sick Children, Toronto, Ontario, Canada

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Nada Jabado, Montreal Children's HospitalResearch Institute, 4060 Ste Catherine West, PT-239, Montreal, Quebec,Canada H3Z 2Z3. Phone: (514) 412-4400 ext 23270; Fax: (514) 412-4331;E-mail: [email protected] or Stefan M. Pfister, German CancerResearch Center, Molecular Genetics of Pediatric Brain Tumors (B062),Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Phone: 49-6221-424618; Fax: 49-6221-424639; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-0127

’2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 17(14) July 15, 20114650





surgical resection is the mainstay of therapy, and failure toachieve it remains the main therapeutic concern (3).Although cerebellar PAs are usually readily amenable tocomplete surgical resection, residual tumor in other lessanatomically accessible locations can require adjuvanttherapies for tumor control (9, 11). Thus, despite an excel-lent 5-year overall survival rate of more than 90% andprogression-free survival rate of more than 65% for PA(1, 11, 12), affected children may experience significantmorbidity due to the tumor location and/or the side effectsof adjuvant therapies in case of residual disease.Several consecutive articles, including from our groups,

recently showed aberrant activation of the mitogen-acti-vated protein kinase (MAPK) pathway to be the mainmolecular abnormality in PA (13–18). Indeed, genefusions and activating mutations involving key regulatorsof the MAPK pathway have been found in the majority ofPAs tested. Duplication of 7q34 leading to in-frameKIAA1549-BRAF fusions and constitutive BRAF activationhave been identified in up to 65% of sporadic PAs tested(13–18). KRAS activating mutations affecting codons 12,13, and 61 have been identified in approximately 4% to 7%of PAs (13, 19–21) and similarly low incidence rates havebeen found for the V600E BRAF activating mutation in PA(13, 14, 16, 17). RAF1 fusions leading to constitutiveactivation of RAF1 have been detected in 4 PAs to date(13, 15) and a novel BRAF activating mutation was alsorecently identified at low frequency in PA (15, 22, 23).Moreover, in NF1 patients, NF1 gene inactivation leads toincreased Ras activity and subsequent MAPK pathwayactivation. All of these observations converge to indicatea major role of MAPK pathway activation in the clinicalcourse and biology of PA.

Senescence is a physiologic phenomenon characterizedby a permanent cell-cycle arrest. Oncogene-induced senes-cence (OIS) was first reported when the expression ofoncogenic Ras in primary human or rodent cells resultedin a permanent G1 arrest and was accompanied by accu-mulation of p53 and p16 (24). Collectively, investigatorshave subsequently observed that mutations in KRAS, BRAF,PTEN, and NF1 can trigger cellular senescence in vivo andhave implied that OIS is a mechanism of tumor suppres-sion that restricts the progression of benign tumors in theabsence of additional cooperating mutations (25–28).Human melanocytic nevi, which are common benignlesions of cutaneous melanocytes that rarely progress tomelanoma, constitute an intriguing example. Somaticmutations leading to constitutive activation of BRAF(V600E) are present in up to 80% of melanocytes fromthis benign tumor and lead to growth arrest of cells throughOIS (27, 29). Human neurofibromas developing in thecontext of neurofibromatosis type I also show senescencein vivo due to loss of NF1 activity (25).

The natural history of PA is unique, as these tumors areself-containing, almost never progress to higher-gradeastrocytomas, and their initial growth is usually followedby a decrease in tumor activity, after which they enter adormant phase and either remain quiescent or restart cycle(s) of growth followed by dormancy, similar to what isobserved in OIS in vitro. On the basis of these observations,we hypothesized that activation of the MAPK pathwayleads to OIS in PA, potentially accounting for the benignnature of these tumors. Thus, we investigated a number ofestablished senescence markers in 52 primary sporadicpediatric PAs, including expression levels of p16INK4a,p53, and KI-67, a marker of proliferation index, in archivaltissues and senescence-associated acidic b-galactosidase(SA-b-Gal) activity in fresh tissue samples where available(30). Gene expression profiling in 2 independent PAcohorts also strongly supported the presence of an activatedsenescence program in PA.

In addition, we overexpressed wild-type or V600Emutant BRAF in human immortalized astrocytes and infetal astrocytes and assessed for OIS induction in vitro inthese surrogate astrocytic models. Our results indicate thatMAPK activation triggers OIS in PA. As MAPK activation islikely the major abnormality driving proliferation in PA,OIS may be responsible for the tendency of PA for sponta-neous growth arrest. Furthermore, the absence of addi-tional genetic alterations and the presence of functionalcell-cycle control mechanisms could explain the lack oftransformation of PA into higher-grade tumors, accountingfor the relatively benign course of this grade I tumor whenconsidering overall survival.

Materials and Methods

Sample characteristics and pathologic reviewAll samples were obtained with informed consent from

patients after approval of the Institutional Review Board ofthe respective hospitals they were treated in and were

Translational Relevance

Pilocytic astrocytomas are the most common braintumor in children. Despite an overall good prognosis,children may experience significant morbidity due totumor location and/or the side effects of adjuvanttherapies in case of residual disease. Our study providesa comprehensive molecular profiling of the effect of therecently uncovered, genetically driven, mitogen-acti-vated protein kinase (MAPK) pathway activation inPA. It shows prominent hallmarks of oncogene-inducedsenescence (OIS) that is potentially at the origin ofclinical behavior and overall good prognosis of PAs.Moreover, artificial acceleration of this OIS programcould present an interesting therapeutic possibility inpatients with progressive disease. In this area ofresearch, the strength of associations defines the trans-lational implications of the findings. In this regard, thecombined results from 3 independent cohorts and ourin vitro data that provide inherent validation for themechanisms we propose are limiting the potential fortransformation in this tumor.

BRAF Mediated-Senescence in Pilocytic Astrocytoma

www.aacrjournals.org Clin Cancer Res; 17(14) July 15, 2011 4651





independently reviewed by senior pediatric neuropathol-ogists according to the WHO guidelines (3, 31). Patients’characteristics in the Montreal, Cambridge, and Heidelbergseries are detailed in Table 1 and Supplementary Tables S1and S2.

Cell lines, antibodies, and transfectionsHuman telomerase reverse transcriptase (hTERT)-

immortalized human astrocytes (kind gift of Dr A Guha,Labatt Brain Tumour Research Centre, Toronto, Ontario,Canada) were grown as previously described (32). Humanfetal astrocytes were obtained from 18- to 22-week-old fetalbrain specimens provided by the Human Fetal TissueRepository (Albert Einstein College of Medicine, Bronx,NY; collaboration with Dr Jacques Antel, Montreal Neu-rologic Institute) following approved guidelines from theCanadian Institutes of Health Research and processed asdescribed in Supplementary Table S1. Primary antibodieswere from Cell Signaling Technology (GFAP/Myc-tag/b-actin) and from Santa Cruz Biotechnology (pERK/BRAF/p53/p16INK4a). Cells were transfected with wild-typeMyc-tagged BRAF or V600E BRAF (a kind gift of Dr RichardMarais, Cancer Research, London, UK) as previouslydescribed (33). Transfection efficiency was assessed usingimmunofluorescence and Western blot analysis as pre-viously described (33).

BRAF tandem duplication screeningReverse transcriptase-PCR to detect the BRAF fusion

transcripts was carried out as previously described (16).

Gene expression profilingCambridge series. Samples were analyzed on the Illu-

mina HT12 v3 expression platform. Selected genes wereassessed for differential expression between tumor andcontrol samples, using a 2-tailed Mann–Whitney U test.

Heidelberg series. Samples were analyzed on the Illu-mina WG6 v3 expression platform. The mean of the nor-mal brain expression was subtracted from the expressionvalue for each sample and for each gene of interest, to give alog2 fold-change value. Detailed overview is provided inSupplementary Table S1.

Analysis of SA-b-Gal activityCells were plated in a 6-well plate at 50% confluence

overnight prior to the assay. A detailed overview of theexperimental procedure is provided in SupplementaryTable S1. The appearance of a blue color was determinedunder the microscope as previously described (27, 30).

Immunofluorescence, immunohistochemical analysis,Western blotting, and cell-cycle analysis

Immunofluorescence and immunohistochemical stain-ing and Western blot analysis were carried out and scoredas previously described (24, 28, 34). Detailed overview ofthe experimental procedures and data analysis using theFlowJow software are provided in Supplementary Table S1.All experiments were carried out at least 3 independenttimes in triplicate and at least 40,000 cells were acquiredfrom each sample for fluorescence-activated cell-sortinganalysis.

Results

Genetic alterations in the MAPK pathway in PAincluded in the Montreal cohort

We first confirmed the nature of the genetic abnormalityaffecting the MAPK pathway in the cohort of 52 sporadicPAs from the Montreal series investigated in this study. Tothis end, we used specific exonic primer pairs for PCRamplification of KIAA1549-BRAF fusion products. Fusiontranscripts were detected in 37 of 52 (71.1%) PA samples(24 of 28 posterior fossa, 4 of 7 brainstem, 5 of 8 opticpathway, 1 of 4 cerebrum, and 3 of 5 spinal PAs). We alsosequenced BRAF exons 11 and 15 in 50 of 52 PAs andidentified the hotspot V600E mutation in 2 of the 15tumors negative for KIAA1549-BRAF fusion and 1 in aPA with KIAA1549-BRAF fusion. We then screened formutations of exons 2 to 7 of RAF1, exons 2 to 3 of KRASandNRAS, and exon 13 of PTPN11, and for SRGAP3-RAF1fusion, and identified 2 samples with mutated KRAS (2posterior fossa) in the 13 samples that were negative forKIAA1549-BRAF fusion or V600E BRAF mutation (Table 1,Supplementary Table S2). These data, as well as overallsurvival and event-free survival in our patient cohort (Sup-plementary Fig. S1), are in concordance with current lit-erature, showing the preponderance of KIAA1549-BRAFfusions as MAPK activating events in PA (1, 11, 12, 35).

PAs express senescence markersActivation of the INK4a/ARF locus by oncogenes is well

documented as a tumor-suppressive mechanism, notablythrough induction of OIS. Senescent cells have been shownto display increased expression of p16INK4a, p21CIP, or p53

Table 1. Patient characteristics and geneticalterations affecting the MAPK pathway (Mon-treal cohort)

Gender 24 M; 28 FAge [average and (range), years] 6.22 (0.33–18)Tumor location

Posterior fossa 28 (53.8%)Optic pathway 8 (15.4%)Brainstem 7 (13.5%)Cerebrum 4 (7.7%)Spinal cord 5 (9.6%)

MAPK pathway aberrationKIAA1549-BRAF fusion 37 (71.2%)V600E BRAF 2 (3.8%)SRGAP3-RAF1 0KRAS mutation 2 (3.8%)Unknown 11 (21.2%)

Jacob et al.

Clin Cancer Res; 17(14) July 15, 2011 Clinical Cancer Research4652





while having a low to null mitotic index (36–38). Thep16INK4a protein is a major tumor suppressor, often highlyexpressed in senescent cells in vitro and inactivated in avariety of human cancers, including 30% to 70% of HGAs(39, 40). OIS is characterized by cycle arrest, which isaccompanied by the induction of both p16INK4a and SA-b-Gal activity, a commonly used senescence marker (27,

28, 37). We investigated whether senescence was triggeredin PA in vivo using SA-b-Gal activity and levels of p16INK4a,p53, and KI-67 (a marker of cellular proliferation). FreshPA tumor samples from 6 consecutive patients includinglesions from the cerebellum (N ! 4), brainstem (N ! 1),and spinal cord (N ! 1) were markedly positive for SA-b-Gal (Fig. 1; Montreal cohort). Five of these samples

Figure 1. SA-b-Gal activity isincreased in primary PAs. Six freshtissue samples from patients withPA (Montreal cohort, Table 1;Supplementary Table S2) wereprocessed as described (27) andshowed marked SA-b-Gal activity.Control immortalized astrocytes(NHA), cells from a primary cell lineestablished from a pediatricglioblastoma patient (SF188), or afresh tissue sample obtained froma pediatric patient withglioblastoma (GBM) wereprocessed similarly and did notshow SA-b-Gal activity (negativecontrols).

PA2

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PA5

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SF188

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PA37

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GBM

20X

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(PA 2-3-7-12) had KIAA1549-BRAF fusions and 1 (PA 37)had no identified genetic abnormality of the MAPK path-way (Table 1, Supplementary Table S2). We then investi-gated fixed material from the 52 PA samples included inour cohort, including these 6 samples for which weobtained fresh material at initial surgery, for the expressionof p16INK4a, p53, and KI-67 by using specific antibodiestargeting these proteins. Staining for p16INK4a showedmarkedly increased expression in 46 of 52 (89.5%) PAscompared with normal brain (Fig. 2, Table 2). PAs werepositive for p16INK4a regardless of their anatomic locationand regardless of the presence or type of the geneticalteration affecting the MAPK pathway. Immunopositivityfor p16INK4a was both cytoplasmic and nuclear. Its extentvaried widely, in terms of both the fraction of positive cellsand its intensity (Table 2), and was found predominantlyin the nonpilocytic component, particularly in those PAsthat had the classical biphasic "loose-dense" pattern(Fig. 2). Six PAs had p16INK4a staining in 5% of cells orless counted across any field (Fig. 2, Table 2). These PAs hadan active MAPK pathway as shown by the positive pERKstaining on the respective slides (Supplementary Fig. S2B).No PAs showed increased nuclear p53 expression consis-tent with previous publications indicating absence of TP53mutations in PA (reviewed in ref. 9). As also expected in PA,Ki-67 index was low in all cases (1%–2%; rarely up to 5%).The MAPK pathway was triggered in all PA cases (14), asshown by the positivity of the antibody against the phos-phorylated (activated) forms of ERK1/2 (pERK; Supple-mentary Fig. S2B; ref. 14). Slides from normal control brainwere negative for p16INK4a, p53, and Ki-67 expression(Supplementary Fig. S2A, Table 2).

Taken together, these data show that at the protein level,PAs display features of OIS. Several key publications showcentral roles for both CDKN2A (p16) and CDKN1A (p21)in OIS, and these are now widely accepted markers of thisphenomenon [e.g. refs. (24, 41)]. More recently, IGFBP7

has been proposed as being a central mediator of BRAF-induced OIS in melanocytes and also as having growthregulatory properties in thyroid carcinogenesis (a tumorthat is also closely associated with BRAFmutation; refs. 42,43). CEBPB, encoding CCAAT/enhancer binding proteinbeta, has also recently been shown to have a role in MAPKactivation–mediated OIS in fibroblasts (44, 45), andGADD45A has been linked with apoptosis and growtharrest in Ras-driven breast cancer and UV-induced skincancer (46, 47). To investigate the transcriptional changesunderlying this senescent behavior, we examined transcrip-tome data from 2 independent tumor series (Cambridgeand Heidelberg) to look at these specific candidate geneswith well defined or putative roles in senescence. Strikingly,both datasets provided strong evidence for overexpressionof these genes in the tumors compared with normal brain(Fig. 3, Supplementary Fig. S3). In the Cambridge series, allgenes showed significantly increased expression in primarytumors compared with normal brain controls (P < 0.0001for all comparisons, Mann–Whitney U test; Fig. 3). In theHeidelberg series, the median log2 fold change of tumorversus control ranged from 0.69 to 5.01, with all differencesalso significant at P < 0.05 (2-tailed Mann–Whitney U test;Supplementary Fig. S3). Thus, the alterations in behaviorand in tumor protein expression seen in the Montrealcohort are closely mirrored by a program of OIS geneexpression changes in 2 additional PA cohorts assayedon 2 different platforms.

Short-term overexpression of BRAFWT or BRAFV600E

induces features typical of OIS in two independentastrocytic cell line models

A wealth of data support a model in which somatic cellsin culture possess at least 2 independent mechanismslimiting their life span and leading to senescence: First, atelomere-dependent pathway registers the cumulativenumber of cell divisions and triggers senescence following

Table 2. Levels of Ki-67, nuclear p53, and p16INK4a staining in all 52 primary PAs and in control brainsamples

PA Control brain(N ! 6)

All PA samples(N ! 52)

KIAA1549-BRAFfusion (N ! 37)

V600E BRAF(N ! 2)

KRAS(N ! 2)

Unknown(N ! 11)

Ki-67 <1% 9 (18.8%) 7 (13.4%) 0 0 2 (18%) 6 (100%)1%–5% 39 (86.5%) 29 (78.4%) 2 (100%) 2 (100%) 7 (64%) 0>5% 2 (3.8%) 0 0 0 2 (18%) 0NA 1 1 0 0

p53 0% 42 (80.7%) 32 (86.5%) 0 2 (100%) 8 (72.7%) 6 (100%)1%–20% 7 (13.5%) 4 (11%) 2 (100%) 0 1 (9%) 0>20% 0 0 0 0 0 0NA 3 1 2 0

p16 <10% 6 (11.5%) 3 (8%) 0 0 3 (27%) 6 (100%)10%–50% 30 (57.7%) 20 (54%) 2 (100%) 2 (100%) 6 (54.5%) 0"50% 16 (30.8%) 14 (37.8%) 0 0 2 (18%) 0

Jacob et al.






Figure 2. p16INK4A is induced inthe majority of PAs.Immunohistochemical analyses ofp16INK4A (p16) were done on 52pediatric PA samples (Montrealcohort) and control brains (CB)from age-matched children. Arepresentative staining of 3 CBsand 18 PAs from different regionswithin the brain is shown. PA1–PA15 had marked p16 induction,whereas PA16–PA18 and the CBhad limited p16 staining.

CB1 CB2 CB3

PA1 PA2 PA3

PA4 PA5 PA6

PA7 PA8 PA9

PA10 PA11 PA12

PA13 PA14 PA15

PA16 PA17 PA18







telomere attrition. Second, an INK4a/ARF-dependentmechanism reacts to the exposure of cells to mitogenicstimulation and may in some cases trigger a permanentcell-cycle arrest (36). Activation of this INK4a/ARF pathwaycan be accelerated by introducing high levels of promito-genic oncogenes (36). We elected to use normal humanhTERT-immortalized astrocytes (NHA), where telomereshortening–induced senescence is inhibited by constitutiveoverexpression of human hTERT, thereby resulting inimmortalization. We used these cells and fetal astrocytesas models to determine in vitrowhether increased wild-typeBRAF (BRAFWT) or V600E mutant BRAF (BRAFV600E)expression levels induce OIS and increased levels ofp16INK4a in cells from the astrocytic lineage, similar towhat has been observed in fibroblasts and melanocytesand as we observed in primary PA.

NHA and fetal astrocytes were transiently transfectedwith BRAFWT and its mutant form BRAFV600E. Transfectionefficiency was estimated by immunofluorescence analysisagainst the Myc-tag to be approximately 60% at 48, 72, and96 hours (Fig. 4A). No foci of transformation wereobserved in cells transfected with either construct, whereasmorphologic changes including an enlarged cytoplasm androunded flattened shape consistent with a senescent phe-notype were noted in cells transfected with the BRAFconstructs (Fig. 4B). As we had previously described(14), proliferation assays revealed limited changes in thegrowth of cells overexpressing BRAFWT or BRAFV600E com-pared with empty vector transfectants. Increased SA-b-Galactivity in BRAFWT and BRAFV600E transfectant cells com-pared with empty vector controls was also observed(Fig. 4B). Western blotting analysis showed induction ofp16INK4a expression in BRAFWT and BRAFV600E transfectedNHA and fetal astrocytes, whereas p53 levels remainedunchanged (Fig. 4C) compared with the empty vector.Moreover, forced BRAFWT and BRAFV600E expression inNHA and fetal astrocytes induced a growth arrest at theG1–M phase as shown by increased G1 and decreased S-phase in these cells compared with empty vector transfec-tant cells following cell-cycle analysis (Fig. 4D).

These results show that forced overexpression of WT andmutant BRAF induces p16INK4a expression, SA-b-Galactivity and cell-cycle arrest in 2 astrocytic cell lines. Themaintenance of telomere length through hTERT immorta-lization of NHA argues for an active oncogene-drivensenescence process, rather than a loss of replicative poten-tial in these cells, which can normally be serially passagedsuccessfully.

Loss of p16 abrogates the senescent features of NHAcells stably overexpressing BRAFWT or BRAFV600E

To better understand the cellular responses to expressionof BRAF, we sought to generate stable NHA clones over-expressing BRAFWT or BRAFV600E. In several independentexperiments, we observed unusually low transfection effi-ciency and were able to select only 4 clones overexpressingBRAFWT from 75 that were screened and 2 clones over-expressing BRAFV600E from 60 screened. Interestingly,when we investigated p16INK4a and p53 levels in clones,we observed that p16INK4a expression was lost in all BRAF-overexpressing clones whereas p53 expression remainedunchanged (Fig. 5A). This suggests that inactivation ofp16INK4a in NHA may allow the cells to bypass BRAF-induced senescence and to divide enough to constitutivelyincorporate the DNA plasmid and become transformed. Inkeeping with this hypothesis, sustained expression ofBRAFWT and BRAFV600E did not induce senescent-asso-ciated features in NHA, as was seen following transientoverexpression. Indeed, cells had no morphologic changesindicative of senescence, no significant induction of SA-b-Gal activity (Fig. 5B), showed foci of transformation(Fig. 5C), and were able to grow and form colonies inanchorage-independent conditions (soft agar) comparedwith EV transfectants (Fig. 5D). These data indicate thatp16INK4a may protect against BRAF-driven proliferationand that its loss may diminish the physiologic protectionprovided by this cell-cycle regulator through induction ofsenescence.

Discussion

We show in vivo and in vitro that PAs display classicalhallmarks of senescence, suggesting that MAPK pathwayactivation, whatever the genetic defect at its origin, drivesOIS partly through the induction of p16INK4a pathwayactivation in astrocytes. Fresh samples from pediatric PAswere invariably positive for SA-b-Gal, and we observed aclear induction of p16INK4a protein in 46 of 52 (89.5%)primary pediatric PA samples. Tumor samples also had alow mitotic index indicative of a limited growth potentialand no increased p53 nuclear expression, suggesting that itis primarily the INK4a/RB cell-cycle control pathway that isbeing triggered in these tumors through induction ofp16INK4a (48). Similar to what we observed in vivo, weshow that transient BRAFWT or mutant BRAFV600E over-expression in human astrocytes (NHA and fetal astrocytes)induced morphologic changes evocative of senescent cells,which was accompanied by the induction of p16INK4a,

14 *** *** *** *** ***

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exp

ress

ion

valu

e

6

4

2

0CDKN1A (p21)CDKN2A (p16) CEBPB GADD45A IGFBP7

TumorControl

Figure 3. Expression of OIS-related candidate genes. Normalized log2expression values in PA tumors (dark gray, N ! 29) and control brainsamples (light gray, N ! 10) showing clear upregulation of thesegenes in the tumor samples. ***, P < 0.0001, 2-tailed Mann–WhitneyU test.

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Figure 4. Transient overexpression of wild-type (WT)-BRAF and V600E BRAF induces OIS in immortalized astrocytes (NHA) and fetal astrocytes (FA). A,transfection efficiency at 48 hours of NHA and FA following transient overexpression of Myc-tagged (red staining) WT-BRAF (top, green staining for BRAF) andV600E BRAF (bottom). 40, 6-Diamidino-2-phenylindole (DAPI) staining (blue) is provided to show the total number of cells per field. B, morphologic changesand induction of SA-b-Gal activity were seen in FA (top) and in NHA (bottom) cells transiently overexpressing WT-BRAF or mutant V600E BRAF comparedwith empty vector controls (EV). The percentage of cells showing positive SA-b-Gal activity following transient BRAF (WT and V600E) induction wasstatistically significantly increased compared with EV controls (P < 0.001, Fisher's exact test). C, a mean of 4-fold induction in p16INK4a expression wasseen in NHA (left) and FA (right) following overexpression of either WT-BRAF or mutant BRAF. D. Cell-cycle analysis of NHA and fetal astrocyte cell linestransiently overexpressing WT or mutant V600E BRAF shows an increase in G1 and a decrease in the S-phase in these cells compared withcells transfectedwith EV. This is suggestive of a G1–Mcell-cycle arrest induced by BRAF overexpression in both astrocytic cell lines. Results are representativeof 3 independent experiments carried out in triplicates for each cell line.







SA-b-Gal activity, and cell-cycle arrest. Conversely, we alsoshow that loss of p16INK4a expression in NHA abrogatedOIS, promoted cellular transformation, and increased pro-liferation following sustained BRAFWT and BRAFV600E

expression in vitro. This is of particular interest given arecent study reporting deletion of p16 in a subset ofclinically aggressive PAs (49). Moreover, in mice, loss ofp16INK4a allowed cultured astrocytes to grow withoutsenescing (37). These data are concordant with our resultson the role for p16INK4a pathway activation in the induc-tion of OIS in PAs.

Our data may provide a rationale for the lack of progres-sion to higher-grade tumors of PAs, in contrast to WHOgrade II and III astrocytomas. Senescence is not triggered bya single, linear series of events but, instead, is regulated by acomplex signaling network including the p53 and retino-blastoma (Rb) tumor suppressor pathways, which serve ascritical cell-cycle checkpoints that mediate both replicativeand oncogene-induced senescence. In contrast to PAs,nearly all grade II to IV astrocytomas, exhibit alterationsin genetic loci related to the p53 and Rb pathways govern-ing G1 arrest, including loss of the INK4a/ARF or Rb loci orgain of CDK4 (37, 38).

In this study, 6 PA samples from the Montreal cohort of52 samples showed limited p16INK4a expression. Thesesamples had marked pERK positivity, limited proliferationindex, no p53 nuclear staining, and did not belong to agiven age group. Three harbored KIAA1549-BRAF fusionsand were typical cerebellar PAs. In nevi, additional factorsto p16INK4a contribute to the protection of melanocytesagainst BRAFV600E-driven proliferation (27). Senescence in

these 6 PAs as well as in all of the other PA samplesinvestigated in this study could be additionally triggeredthrough other mechanisms. Indeed, at the transcriptionallevel, a number of genes associated with OIS showedsignificant upregulation in PA compared with normalbrain. These included CDKN2A (p16) and CDKN1A(p21), which are widely accepted markers of OIS (24,41), as well as IGFBP7, CEBPB, and GADD45A, which havealso been associated with MAPK-induced OIS (42–46, 50).Furthermore, telomerase activity has been shown to be lowin LGA and telomere attrition has been shown to increasewith recurrence in LGA including PA (51). Together, theseobservations indicate that there may be a more widespreadsenescence program that is activated in PAmore than solelyp16INK4a induction and that there may be a degree offunctional redundancy in these tumors.

In addition to benign melanocytic nevi, the V600EBRAF mutation has been identified in the majority ofmelanomas. It is unclear to date as to what leads tocellular transformation in this tumor type, as senescencecan still be partly triggered in tumor cells (29). Limitingsignals affecting induction and levels of OIS may bedictated not only by the initiating genetic alterationbut also by the threshold (intensity and duration) ofthe signaling and/or the tissue type (cell of origin, micro-environment including the stroma and immuneresponse). The lack of progression to higher-grade tumorsin senescent PAs may be due to the nature of the onco-genic aberration, as KIAA1549-BRAF fusions predominateand the V600E mutation accounts for only 4% to 7%of all genetic abnormalities in PAs, or to any of these

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Figure 5. Loss of p16INK4A decreases BRAF-mediated OIS in NHA and promotes cellular transformation. A, stable clones overexpressing wild-type(WT) or mutant V600E BRAF were obtained only from cells that lost p16 expression. B, loss of p16 decreased SA-b-Gal activity in NHA clones despitesustained WT-BRAF or V600E expression. It promoted the formation of foci of transformation (C) and allowed for anchorage independent growth(D), as shown by increased colony formation in soft agar of clones stably overexpressing WT-BRAF or V600E BRAF compared with empty vector transfectantNHA cells (*, P < 0.05, Fisher's exact test). Numbers are representative of 3 independent experiments.

Jacob et al.






other factors. Additional studies using larger cohorts ofpatients or engineered mouse models generated to thateffect (26) are needed to tease out the impact of thesevariables in PA.In summary, our study suggests that aberrant MAPK

signaling leads to OIS in PAs. This can explain the dichot-omy between activation of an oncogenic pathway, whichwas recently found to be constitutively activated in 75% to100% of PAs, and their tendency toward growth arrest. Thisunique phenomenon is at least partly due to the inductionof p16INK4a in cells and may account for the lack of progres-sion to higher-grade astrocytomas,which is a unique featureof PAs compared with other LGA, and for the better survivalof patients with this tumor. Further studies are warranted todetermine whether the nature of the genetic alteration, thecell of origin, or the microenvironment play an additionalrole in the maintenance of OIS and in the striking lack ofpotential for transformation seen in PAs. Finally, artificialacceleration of this OIS program could also present aninteresting therapeutic possibility in this tumor type.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was supported by the Canadian Institute of Health Research(CIHR) and the Brain Tumour Foundation (to N. Jabado, U. Tabori), theCole Foundation (to N. Jabado), the Hungarian Scientific Research Fund(OTKA) contract no. T-04639, the National Research and DevelopmentFund (NKFP) contract no. 1A/002/2004 (to P. Hauser, M. Garami, L.Bognar), the Addenbrooke’s Charitable Trust (to D.T.W. Jones), and theSamantha Dickson Brain Tumour Trust (to S. Lambert, V.P. Collins). K.Jacob is the recipient of a studentship award from CIHR, S.M. Pfister is therecipient of the Sybille Assmus award for 2009, and N. Jabado is therecipient of a Chercheur Boursier award from Fonds de Recherche en Santedu Quebec.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 19, 2011; revised April 19, 2011; accepted May 11,2011; published OnlineFirst May 24, 2011.

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