Abstract #2856: mTORC1 and mTORC2 involvement in activation of MAPK pathway via a PI3K/AKT-dependent feedback loop influences glioblastoma multiforme dissemination

Numerous studies have demonstrated an association between aberrant AKT/mTOR (mammalian target of Rapamycin) activation and tumorigenesis in glioblastoma multiforme (GBM), thereby suggesting that inhibition of mTOR may have therapeutic potential. We investigated two distinct complexes of mTOR, mTORC1 and mTORC2, regulating cell survival, growth, and motility. Here we show that a substantial number of GBM tumors expressed activated AKT and mTOR as assessed by immunohistochemistry. We observed that inhibition of mTOR by Rapamycin (RAPA) resulted in time-dependent suppression of S6 kinase (pS6K^Ser235/236; mTORC1 substrate) and caused transient suppression of pAKT^Ser473 (mTORC2 substrate) at 1 to 3hrs followed by a consistent increase from 6 to 12hrs. Expression of pAKT was suppressed by siRNA silencing of mTORC2 co-protein Rictor, but not by mTORC1 co-protein Raptor. Treatment with RAPA or LY294002(LY; phosphoinositide 3-kinase [PI3K] inhibitor) suppressed platelet derived growth factor (PDGF)- or fibronectin (FN)-induced activation of p70S6K^Thr389. Furthermore, a combined treatmentwith RAPA and LY abrogated the activation of S6K^Ser235/236. In addition to these observations, RAPA activated the Ras/MAPK pathway in GBM assessed by an increase in pERK1/2^{Thr202/Tyr204}. This feedback loop is dependent on an S6K/PI3K/Ras pathway. Pre-treatment with MEK1/2 inhibitor U0126 blunted RAPA-induced ERK1/2 activation. Also, RAPA potentiated PDGF and FN activation of ERK1/2. In addition, a timed treatment with RAPA displayed a bimodal pattern of pERK1/2 expression that suggested mTORC1 regulates ERK1/2 activation transiently while a stable expression of pERK1/2 may require mTORC2. GBM cell proliferation and motility paralleled the activation of mTORC2. Both directional and non-directional GBM cell migration showed an increase following prolonged RAPA treatment (12 and 24hrs), while no change in cell motility was observed at earlier time points (1, 3 and 6hrs). RAPA induced migration involved RhoA, since RhoA activation coincided with enhanced cell motility also observed after prolonged RAPA treatment. Combined inhibition of mTOR and PI3K had an additive effect in suppressing cell growth and motility. Moreover, PDGF-induced nuclear localization of mTOR was blocked by pre-treatment with RAPA. These results demonstrated that an activation of mTORC2 occurs when mTORC1 is inhibited by RAPA treatments. Taken together, the results of these findings suggest a novel mTORC/MAPK feedback activation, which underscores the potential of a combined treatment strategy in GBM.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2856.