Effect of dabrafenib on melanoma cell lines harbouring the BRAFV600D/R mutations.

0.69 MB·English

by  Gentilcore, Giusy

Gentilcoreetal.BMCCancer2013,13:17
http://www.biomedcentral.com/1471-2407/13/17
RESEARCH Open Access
Effect of dabrafenib on melanoma cell lines
V600D/R
harbouring the BRAF mutations
Giusy Gentilcore1, Gabriele Madonna1, Nicola Mozzillo1, Antoni Ribas2, Antonio Cossu3, Giuseppe Palmieri4
and Paolo A Ascierto1,5*
Abstract
Background: Conventional therapeutic agents are largely unsatisfactory into the treatment ofmalignant
melanoma. Recently, an innovativeapproach based oninhibitors of themutated BRAF gene (which represents the
most prevalent alteration inmelanoma patients) appears very promising from theclinical point of view. On this
regard, a new compound, dabrafenib (GSK2118436), has been demonstrated to be effective inpatientscarrying the
BRAFV600E/K mutations.We here tested dabrafenib for its capability to inhibit cell growthon primary melanoma
cell lines, established from patients' tumour tissues and carrying the BRAFV600D/R mutations.
Methods: Three melanoma cell lines were tested: M257 wild-type BRAF, LCP BRAFV600R and WM266 BRAFV600D.
The MTT assays were performed using standardized approaches.To evaluate the inhibition of MAPKpathway and
theconsequent inhibition ofcellular proliferation, thephosphorylation of ERKwas examined by Western Blot
analysis performed ontotal protein extracts from cell lines after treatment with dabrafenib.
Results: Our experiments demonstrated an effective action ofDabrafenib (GSK2118436)and theinhibition ofMAPK
pathway inmelanoma cell lines carrying BRAFV600D/R mutations.
Conclusion: These results could be helpful to enlarge thenumber of melanoma patients who maybenefitof a
more effective targeted treatment.
Keywords: BRAF inhibitor, Dabrafenib, Growth inhibition, Melanoma therapy
Background ConstitutivelyactivatedERKthenstimulatescellprolifera-
The mitogen-activated protein kinase (MAPK) pathway tion and survival, sustaining tumour maintenance and
is a key regulator of cell cycle progression, commonly growth [2]. The remaining BRAF mutations are mostly
activated in human tumours through somatic oncogenic represented by other V600 subtypes (V600K,V600D, and
mutationsinRAS,RAF,andMEKgenes [1]. V600R), which account for about 8% of the pathogenetic
In melanoma, the most commonly mutated compo- gene sequence variants [3]. In our experience, mutations
nent of the MAPK pathway is the BRAF gene; among in BRAF gene occur in 43% of primary melanomas and
others, the most prevalent BRAF mutation (nearly, 90% 48%ofmetastaticmelanomas[4].
of cases) is represented by a substitution of valine with A significant benefit in melanoma treatment has been
glutamic acid at position 600 (V600E) [2]. This amino recently achieved with two selective inhibitors: vemura-
acid change leads to oncogenic activation of BRAF, with fenib (PLX4032), which seems to particularly act on
an increase of its kinase activity, and subsequent induc- BRAFV600E mutants (although it has been demonstrated
tion of phosphorylation of the downstream ERK protein. toalsoinhibitproliferationofmelanomacelllinesexpres-
sing other codon 600 BRAF mutations: V600D, V600K,
and V600R) [5-7], and dabrafenib (GSK2118436), which
*Correspondence:[email protected]
1DepartmentMelanoma,IstitutoNazionaleTumoriFondazionePascale, hasbeendemonstratedtomostlyinhibitthekinaseactivi-
Naples,Italy tyinBRAFV600E/Kmutants[8,9].
5UnitofMedicalOncologyandInnovativeTherapy,IstitutoNazionaleperlo
Toinvestigatewhetherthislattercompoundmayexert
StudioelaCuradeiTumori“FondazioneG.Pascale”,ViaMarianoSemmola,
80131,Naples,Italy inhibiting effects on a wider range of BRAF mutants
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Gentilcoreetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,
distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
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(similarly to those previously reported with vemurafe- measured in duplicates every 24 hours for a period of
nib/PLX4720 [5,7]), melanoma cell lines carrying the 7days,usingBurkercellcounts.
two remaining most prevalent BRAF mutations (V600D
andV600R)were here treatedwith dabrafeniband cellu- Results and discussion
larproliferationwasthen assessed. Using a panel of melanoma cell lines derived from the
establishment of excised primaryand metastatic tumours,
Methods wehaveinvestigatedtheabilityofdabrafenibtobothexert
To determine effects on proliferation, melanoma cell an antiproliferative activity on cultured melanoma cells
linesweretreatedintriplicatewithincreasingconcentra- and block the ERK signalling induced by the mutated
tions (3to100nM)ofdabrafenib for 72hrs. To evaluate BRAF. In our assay, the LCP melanoma cell line carried
the occurrence of inhibitory effects on the ERK activa- theBRAFV600Rmutation,whereastheWM266melanoma
tion, a Western Blot analysis was performed on total celllinepresentedtheBRAFV600D variant(Figure1A);the
proteins extracted from cell lines after the 72-hrs treat- M257 melanoma cell line, with a wild-type BRAF, was
ment with the BRAF inhibitor. In particular, the rate of usedascontrol.
phosphorylated ERK was estimated on equal amounts Inourseries,dabrafenibshowedaremarkableinhibition
of total protein for cell lysates; GAPDH was used as ofcellproliferationinbothmelanomacelllinescarryinga
an internal control for total protein expression levels. mutated BRAF (regardless of the type of mutation,
Cell doubling time was determined from cell numbers BRAFV600D or BRAFV600R), with lack of significant
Figure1Effectsofdabrafenibinmelanomacelllines.A.BRAFmutationalstatusinourseriesofmelanomacelllines.B.Antiproliferative
activityofincreasingconcentrationsofdabrafenibineachcellline,ascomparedwiththeuntreated(0)control.C.Inhibitoryeffectsofdabrafenib
onERK phosphorylation,underthesameexperimentalconditions(drugconcentrationsandtimeoftreatment)asabove.
1-2
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8. TrefzerUD,MinorD,RibasA,LebbeC,SiegfriedA,AryaN,GuckertM,
Althoughcarriedoutonalimitedseriesofmelanomacell
SchadendorfD,KeffordR,GrobJJ,HamidO,AmaravadiR,SimeoneE,
lines, our study provided evidence that dabrafenib may TWilhelmT,KimK,GoodmanV,AsciertoPA:BREAK-2:aphaseIIAtrialof
exert a wider inhibitory activity on oncogenic variants theselectiveBRAFkinaseinhibitorGSK2118436inpatientswithBRAF
ofBRAF. mutation-positive(V600E/K)metastaticmelanoma.PigmentCellRes2011,
24:1020.abstractLBA1-1.
9. FalchookGS,LongGV,KurzrockR,KimKB,ArkenauTH,BrownMP,
Abbreviation HamidO,InfanteJR,MillwardM,PavlickAC,O’DaySJ,BlackmanSC,
MAPK:Mitogen-activatedproteinkinase. CurtisCM,LebowitzP,MaB,OuelletD,KeffordRF:Dabrafenibinpatients
withmelanoma,untreatedbrainmetastases,andothersolidtumours:
Competinginterests aphase1dose-escalationtrial.Lancet2012,379:1893–1901.
DrPaoloA.AsciertoparticipatedtoAdvisoryBoardfromBristolMyers
Squibb,MSD,RocheGenentech,GSK,andreceivedhonorariafromBrystol doi:10.1186/1471-2407-13-17
MyersSquibb,MSDandRoche-Genentech.Allremainingauthorsdeclarethe Citethisarticleas:Gentilcoreetal.:Effectofdabrafenibonmelanoma
absenceofanyCompetingInterest. celllinesharbouringtheBRAFV600D/Rmutations.BMCCancer201313:17.
Authors’contributions
GGperformeddataacquisition,dataanalysis,preparationoftheillustration,
anddraftedthemanuscript;GMcontributedindataanalysisanddraftofthe
manuscript;NMcontributedininterpretationofthedata;ARcriticallyrevised
themanuscript;ACcontributedindataanalysis;GPcollaboratedinthedraft
ofthemanuscript;PAAconceivedandsupervisedthestudy,andrevisedthe
manuscript.Allauthorsreadandapprovedthefinalversionofthe
manuscript.
Acknowledgments
AuthorsthankGlaxoSmithandKlineforprovidingthedabrafenibforthis
study.AspecialthankstotheFondazioneMelanomaOnlusforpartiallygrant
thepresentwork.
Authordetails
1DepartmentMelanoma,IstitutoNazionaleTumoriFondazionePascale,
Naples,Italy.2DepartmentofMedicine,JonssonComprehensiveCancer Submit your next manuscript to BioMed Central
Center,UCLA,LosAngeles,CA,USA.3DepartmentofPathology, and take full advantage of:
Hospital-UniversityHealthUnit(AOU),Sassari,Italy.4InstituteofBiomolecular
Chemistry,NationalResearchCouncil(ICB-CNR),Sassari,Italy.5UnitofMedical
OncologyandInnovativeTherapy,IstitutoNazionaleperloStudioelaCura • Convenient online submission
deiTumori“FondazioneG.Pascale”,ViaMarianoSemmola,80131,Naples, • Thorough peer review
Italy.
• No space constraints or color figure charges
Received:25March2012Accepted:13November2012 • Immediate publication on acceptance
Published:14January2013 • Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
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