Abstract B130: BRAFV600E negatively regulates the PI3K/AKT pathway in melanoma cell lines.
Authors: Chen Brenden , Tardell Christine , Higgins Brian , Packman Kathryn , Boylan John F. , Niu Huifeng 
Published: June 2012

The BRAFV600E mutation is frequently found in human cancers (∼ 8%), with the highest incidence in melanoma (60–70%). Small molecules targeting BRAFV600E or MEK have shown significant clinical activity in melanoma patients carrying the BRAFV600E mutation. However, rapid acquisition of drug resistance is an emerging problem that limits patient survival benefits from these agents. MEK inhibition abrogates the intrinsic negative feedback on both RAS/RAF/MEK and PI3K/AKT pathways leading to induction of MEK and AKT phosphorylation. Induction of MEK and AKT phosphorylation may confer resistance and limit the clinical activity of MEK inhibitors. In this study, we investigated the molecular mechanisms of crosstalk between the two pathways. In NIH3T3 cells, engineered expression of BRAFV600E activates the MEK/ERK pathway leading to a significant growth advantage compared to control cells both in vitro and in vivo. MEK inhibition in NIH3T3 control cells strongly induces AKT phosphorylation (Thr308 and Ser473) and downstream signalling, whereas in NIH3T3 cells expressing BRAFV600E the AKT phosphorylation is significantly reduced. Knockdown of BRAFV600E restores MEK inhibition-induced elevation of AKT phosphorylation (pAKT). In the melanoma cell line with wild-type BRAF, MEK inhibition also induces pAKT (Thr308 and Ser473), whereas in the melanoma cell lines harbouring BRAFV600E, MEK inhibition-induced pAKT is not seen. Knockdown of BRAFV600E in those melanoma cell lines significantly elevates basal pAKT levels. These results suggest that BRAFV600E negatively regulates AKT pathway activation. In BRAFV600E melanoma cell lines, knockdown of MEK1/2 or ERK1/2 and inhibition of BRAF by RAF inhibitor have minimal effects on pAKT, suggesting that BRAFV600E suppresses AKT activation independent of the MEK/ERK downstream signals and its kinase activity. Furthermore, the elevation of pAKT(Thr308 and Ser473) caused by BRAFV600E knockdown can be abolished by Rictor, but not Raptor, knockdown, suggesting that the pAKT induction is Rictor dependent. Lack of pAKT induction in cells carrying BRAFV600E is associated with co-purification of Rictor-mTOR and Raptor-mTOR complexes, which is rarely seen in cells with wild type BRAF. Taken together, our data demonstrate that in BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT phosphorylation in a Rictor-dependent, MEK/ERK and kinase-independent manner, possibly via altering Rictor-mTOR complex formation. This study reveals a novel molecular mechanism of crosstalk between the RAF/MEK and the PI3K/AKT pathways, suggesting an underlying mechanism whereby a subset of BRAFV600E melanoma cells are exquisitely sensitive to MEK inhibition.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B130.