Proliferation and survival molecules implicated in the inhibition of BRAF pathway in thyroid cancer cells harbouring different genetic mutations.
BACKGROUND
Thyroid carcinomas show a high prevalence of mutations in the oncogene BRAF which are inversely associated with RAS or RET/PTC oncogenic activation. The possibility of using inhibitors on the BRAF pathway as became an interesting therapeutic approach. In thyroid cancer cells the target molecules, implicated on the cellular effects, mediated by inhibition of BRAF are not well established. In order to fill this lack of knowledge we studied the proliferation and survival pathways and associated molecules induced by BRAF inhibition in thyroid carcinoma cell lines harbouring distinct genetic backgrounds.


METHODS
Suppression of BRAF pathway in thyroid cancer cell lines (8505C, TPC1 and C643) was achieved using RNA interference (RNAi) for BRAF and the kinase inhibitor, sorafenib. Proliferation analysis was performed by BrdU incorporation and apoptosis was accessed by TUNEL assay. Levels of protein expression were analysed by western-blot.


RESULTS
Both BRAF RNAi and sorafenib inhibited proliferation in all the cell lines independently of the genetic background, mostly in cells with BRAF(V600E) mutation. In BRAF(V600E) mutated cells inhibition of BRAF pathway lead to a decrease in ERK1/2 phosphorylation and cyclin D1 levels and an increase in p27(Kip1). Specific inhibition of BRAF by RNAi in cells with BRAF(V600E) mutation had no effect on apoptosis. In the case of sorafenib treatment, cells harbouring BRAF(V600E) mutation showed increase levels of apoptosis due to a balance of the anti-apoptotic proteins Mcl-1 and Bcl-2.


CONCLUSIONS
Our results in thyroid cancer cells, namely those harbouring BRAF(V600E) mutation showed that BRAF signalling pathway provides important proliferation signals. We have shown that in thyroid cancer cells sorafenib induces apoptosis by affecting Mcl-1 and Bcl-2 in BRAF(V600E) mutated cells which was independent of BRAF. These results suggest that sorafenib may prove useful in the treatment of thyroid carcinomas, particularly those refractory to conventional treatment and harbouring BRAF mutations.
Preto
Ana
A
Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-465, Porto, Portugal. apreto@ipatimup.pt
Gonçalves
Joana
J
Rebocho
Ana P
AP
Figueiredo
Joana
J
Meireles
Ana M
AM
Rocha
Ana S
AS
Vasconcelos
Helena M
HM
Seca
Hugo
H
Seruca
Raquel
R
Soares
Paula
P
Sobrinho-Simões
Manuel
M
D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001557:Benzenesulfonates / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D006801:Humans; D009154:Mutation; D009536:Niacinamide / Q000031:analogs & derivatives; D010671:Phenylurea Compounds; D010766:Phosphorylation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics* / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D000077157:Sorafenib; D013964:Thyroid Neoplasms / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology*
D051017:Apoptosis Regulatory Proteins; D001557:Benzenesulfonates; D010671:Phenylurea Compounds; D011725:Pyridines; D009536:Niacinamide; D000077157:Sorafenib; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf