Introduction: The MEK/ERK pathway is mutated in approximately 7% of human cancers. Extracellular ligands signal via RAS to recruit RAF kinase and activate MEK and its downstream effectors. This pathway is involved in a range of biological pathways including cell proliferation, differentiation and senescence. The BRAF isoform is the most potent activator of MEK and is frequently mutated in cancer. A single thymine to adenine base pair change at position 1799 accounts for greater than 95% of somatic mutation in BRAF. BRAFV600E can gain 500-fold increased activation, stimulating the constitutive activation of the MEK/ERK pathway in the absence of extracellular signalling. The strategic position of MEK within the pathway makes it a promising target for drug development. Although MEK is downstream of both RAF and RAS, RAF but not RAS mutation has been demonstrated to predict sensitivity to MEK inhibition.

Materials and Methods: Human thyroid cell lines were SNP typed for the T1799A mutation using a custom TaqMan® SNP genotyping assay. The optimal IC50 and length of treatment for the MEK inhibitor PD0325901 for each cell line were determined via a series of MTT assays. Each cell line was subjected to PD0325901 treatment and surviving cells were harvested for mRNA and protein extraction. A comparative study for relative expression of MAP2K1 (MEK1), MAP2K2 (MEK2), MAPK3 (ERK1), MAPK1 (ERK2) and the downstream target ELK1 was performed using TaqMan assays with GAPDH as the endogenous control.

Results: Human thyroid cell line 8505c carries the T1799A mutation and N-Thy-Ori cell lines carry wild type BRAF. BRAFV600E cells had visually evident patches of inhibition following PD0325901 treatment which were not evident in media-only treated controls or in the BRAFWT cell line. In BRAFV600E, all 5 genes were down-regulated in PD0325901 treated cells compared to their media-only treated counterparts. This pattern was not seen with the BRAFWT cells. BRAFV600E cells exhibited this pattern following both a single treatment and two successive treatments with PD0325901.

Discussion: This pattern of gene expression is consistent with the hypothesis that resistance to small molecule MEK inhibition occurs via upregulation of an alternative pathway or activation of a redundant signalling pathway rather than through reactivation off the BRAF/MEK/ERK pathway. It also reinforces the theory that MEK inhibition induces differential effects in target cells dependent upon BRAF status.

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 B135.