Intrinsic Resistance To the Antiproliferative Effects of Azd6244 a Selective Mek1/2 Inhibitor Is Associated With Concomitant K-ras and Pi3kca Gene Mutations in Colorectal Cancer Cells

[Background:] The EGFR-dependent growth pathway plays a key role in colorectal cancer. Gene mutations in EGFR downstream intracellular effectors correlate with resistance to EGFR targeting agents. Approximately 40% of colorectal cancers carry an activating K-RAS gene mutation mostly in codons 12 or 13; whereas in about 5 to 8% cases an activating V600E B-RAF gene mutation is observed. These mutations are mutually exclusive, whereas PI3KCA mutations (5-15%) can occur independently. We aimed to evaluate the role of AZD6244, a MEK1/2 inhibitor, in colon cancer cell lines with different mutation profiles in these three genes. [Methods:] Western blots were performed to evaluate the expression of total and activated EGFR, RAS, MAPK and AKT proteins in a panel of six (GEO, HCT15, HCT116, SW620, SW480 and LS174T) colon cancer cell lines. The antiproliferative effects of AZD6244 and gefitinib, a selective EGFR inhibitor, were determined using MTT assays. Induction of apoptosis was quantified by flow cytometry. [Results:] Different levels of total and phosphorylated EGFR were detected in five out of six colon cancer cell lines (except in SW620 cells). Four colon cancer cell lines were sensitive to AZD6244-induced growth inhibition, whereas two (HCT15 and LS174T) cell lines were resistant. In AZD6244-sensitive colon cancer cell lines, AZD6244 treatment induced early apoptosis (within 24 hours), which was enhanced between 48 and 72 hours. No correlation between sensitivity to AZD6244 and the expression of total and activated EGFR, RAS, MAPK and AKT proteins was observed. Of note, AZD6244 activity correlated with gene mutation status. K-RAS gene was mutated in GEO, HCT15, HCT116, SW480 and LS174T cells. PI3KCA was mutated in HCT15 and LS174T cells. All cancer cell lines harboured a wild type B-RAF gene. Cancer cells sensitive to AZD6244-induced growth inhibition carried a normal, wild type PI3KCA gene with either a wild type or a mutated K-RAS gene. On the contrary, the two AZD6244-resistant colon cancer cell lines harboured a double mutation in both the K-RAS and PI3KA genes. Finally, all six colon cancer cell lines resulted resistant to gefitinib, a selective EGFR inhibitor, independently from the K-RAS and PI3KA gene mutational status. [Conclusions:] A concomitant double mutation in bothK-RAS and PI3KCA genes status is a predictive factor for intrinsic resistance of colorectal cancer cells to AZD6244 treatment.