Abstract
BackgroundThyroid 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.MethodsSuppression 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.ResultsBoth BRAF RNAi and sorafenib inhibited proliferation in all the cell lines independently of the genetic background, mostly in cells with BRAFV600E mutation. In BRAFV600E mutated cells inhibition of BRAF pathway lead to a decrease in ERK1/2 phosphorylation and cyclin D1 levels and an increase in p27Kip1. Specific inhibition of BRAF by RNAi in cells with BRAFV600E mutation had no effect on apoptosis. In the case of sorafenib treatment, cells harbouring BRAFV600E mutation showed increase levels of apoptosis due to a balance of the anti-apoptotic proteins Mcl-1 and Bcl-2.ConclusionOur results in thyroid cancer cells, namely those harbouring BRAFV600Emutation 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 BRAFV600E 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.
Highlights
Thyroid carcinomas show a high prevalence of mutations in the oncogene BRAF which are inversely associated with RAS or RET/PTC oncogenic activation
We tested the effect of BRAF inhibition by RNA interference (RNAi) and sorafenib in cell lines representing the various genetic profiles found in thyroid tumours in vivo: 8505C harbours a homozygous BRAFV600E mutation, TPC1 harbours a RET/ PTC1 rearrangement and wild-type for BRAF, and C643 harbours a RASG13R mutation and wild-type BRAF [21], recently demonstrated to be unique thyroid cancer cell lines origin [22]
In this study we described for the first time, to the best of our knowledge, the effect of both sorafenib and specific Small interference RNAs (siRNAs) for BRAF in thyroid cancer cells and associated molecules
Summary
Thyroid carcinomas show a high prevalence of mutations in the oncogene BRAF which are inversely associated with RAS or RET/PTC oncogenic activation. 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. In melanomas, which harbour BRAFV600E mutations, it has been demonstrated that BRAFV600E activates the MAPK pathway and controls proliferation of melanoma cells through the regulation of cyclin D1 and of the cyclindependent kinase inhibitor p27Kip1 [4,5,6,7]. In colon cancer suppression of BRAF in cell lines with BRAFV600E showed significant decreased proliferation through cyclin D1 and p27Kip and induces apoptosis by a significant decrease in the levels of anti-apoptotic protein Bcl-2 [14] The suppression of BRAFV600E in melanoma cells was demonstrated to inhibit proliferation, transformation, invasion and promote apoptosis [8,9,10,11,12,13].
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