Differential responsiveness to BRAF inhibitors of melanoma cell lines BRAF V600E-mutated.
BRAF mutation
Next-generation sequencing
QPCR
Sanger sequencing
Journal
Journal of translational medicine
ISSN: 1479-5876
Titre abrégé: J Transl Med
Pays: England
ID NLM: 101190741
Informations de publication
Date de publication:
11 05 2020
11 05 2020
Historique:
received:
08
03
2020
accepted:
24
04
2020
entrez:
13
5
2020
pubmed:
13
5
2020
medline:
15
5
2021
Statut:
epublish
Résumé
Most mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation. DNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation. Three cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines. Cell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.
Sections du résumé
BACKGROUND
Most mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation.
METHODS
DNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation.
RESULTS
Three cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines.
CONCLUSION
Cell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.
Identifiants
pubmed: 32393282
doi: 10.1186/s12967-020-02350-8
pii: 10.1186/s12967-020-02350-8
pmc: PMC7216681
doi:
Substances chimiques
Protein Kinase Inhibitors
0
Vemurafenib
207SMY3FQT
BRAF protein, human
EC 2.7.11.1
Proto-Oncogene Proteins B-raf
EC 2.7.11.1
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
192Références
Acta Histochem. 2018 Apr;120(3):159-167
pubmed: 29496266
Clin Exp Metastasis. 2013 Oct;30(7):867-76
pubmed: 23673558
Nat Genet. 2003 Jan;33(1):19-20
pubmed: 12447372
J Transl Med. 2012 Jul 09;10:85
pubmed: 22554099
Br J Cancer. 2016 Mar 29;114(7):801-8
pubmed: 26924424
Nature. 2002 Jun 27;417(6892):949-54
pubmed: 12068308
J Transl Med. 2019 Apr 5;17(1):112
pubmed: 30953523
Nat Commun. 2015 Apr 09;6:6683
pubmed: 25865119
N Engl J Med. 2005 Nov 17;353(20):2135-47
pubmed: 16291983
BMC Genet. 2013 Feb 19;14:6
pubmed: 23418865
Br J Cancer. 2012 Jul 10;107(2):345-51
pubmed: 22713664
J Invest Dermatol. 2004 Feb;122(2):342-8
pubmed: 15009715
N Engl J Med. 2011 Jun 30;364(26):2507-16
pubmed: 21639808
Genes Chromosomes Cancer. 2013 May;52(5):503-11
pubmed: 23362162
Cell Death Differ. 2014 Dec;21(12):1936-49
pubmed: 25215949
Expert Opin Biol Ther. 2014 May;14(5):663-86
pubmed: 24625306
Lab Invest. 2017 Feb;97(2):146-157
pubmed: 28067895
Am J Pathol. 1996 Sep;149(3):883-93
pubmed: 8780392
Nat Rev Cancer. 2015 May;15(5):290-301
pubmed: 25877329
PLoS One. 2012;7(8):e43842
pubmed: 22952784
BMC Genomics. 2012 Apr 26;13:156
pubmed: 22537248
Br J Cancer. 2013 Mar 5;108(4):924-31
pubmed: 23403819
Lancet. 2012 Jul 28;380(9839):358-65
pubmed: 22735384
Mol Oncol. 2015 Jan;9(1):93-104
pubmed: 25174651