Abstract
mTOR pathway regulates proliferation in several carcinoma models. Rapamycin is a specific inhibitor of Raptor-mTOR complex Predictors of tumour sensitivity to rapamycin are not well established and there is evidence that oncogenic activation of other pathways may hamper rapamycin effects. To study whether mTOR inactivation has any impact on cell proliferation of thyroid carcinoma cell lines. Furthermore we aim to test oncogene dependency of rapamycin effects. We studied the effect of 40nM of rapamycin on the proliferation of three thyroid carcinoma cell lines positive for RET/PTC-1 (TPC-1), V600E BRAF (B-CPAP) and G13C HRAS (C643). BrdU incorporation was analysed after 24h of culture in the presence of rapamycin. DNA synthesis was inhibited two fold in B-CPAP and three fold in C643. In TPC-1, rapamycin had no effect on DNA synthesis. To understand the different behaviour of TPC-1 we prepared cell extracts from cultures treated for one and 24h with or without 40nM rapamycin. we analysed the phosphorylation pattern of several members of the mTOR pathway. As expected, in B-CPAP and C643 the phosphorylation of mTOR, thr 308 AKT, S6K1, S6 and 4EBP1 were strongly inhibited by rapamycin treatment both at one and 24 hours. In TPC-1, despite inhibition of phosphorylations of mTOR, S6K1 and S6, we detected increased phosphorylations of both AKT and ERK1/2. In this cell line, mTOR inhibition induces MAPK and PI-3K activation. This likely explains the resistance of TPC-1 cells to growth inhibition by rapamycin, since inhibitors of PI-3K and MAPK pathways restored the inhibition by rapamycin of DNA synthesis. The RET/PTC-1 positive cell line was resistant to the growth inhibitory effects of mTOR inhibition through a positive feedback loop involving the activation of PI-3K and MAPK pathways. Although for RET/PTC tumours our findings may not be clinically relevant they might raise important questions in the therapy of RET mutated medullary thyroid carcinomas.