KRAS p.G13D mutations are associated with sensitivity to anti-EGFR antibody treatment in colorectal cancer cell lines.

PURPOSE: Targeted therapies using the anti-EGFR antibodies panitumumab (Pmab) or cetuximab (Cmab) are currently restricted to patients with metastatic colorectal adenocarcinoma whose tumours do not show a mutation in KRAS. However, recent retrospective studies indicated that patients with tumours mutated in codon 13 of KRAS may benefit from treatment with Cmab in contrast to patients with tumours mutated in KRAS codon 12. METHODS: To study the functional impact of the subtype of KRAS mutations on the efficiency of EGFR-targeted therapies, we correlated the KRAS mutation status of 15 colorectal carcinoma cell lines with the in vitro sensitivity of these cells to Cmab/Pmab. Mutations in the potential predictive biomarkers BRAF and PIK3CA as well as protein expression of EGFR and PTEN were also determined. RESULTS: Four out of seven KRAS-mutated cell lines were characterised by the p.G13D mutation. Treatment of these cells using Cmab/Pmab induced a significant growth inhibition in contrast to cell lines showing a KRAS mutation at codon 12 or 61. Out of the eight KRAS wild-type cell lines, five were insensitive to Cmab/Pmab. These cell lines were characterised either by BRAF mutation or by absence of EGFR or PTEN protein expression. CONCLUSIONS: Since KRAS p.G13D-mutated tumour cells may respond to EGFR-targeted therapy, we suggest including subtype analysis of KRAS mutations in prospective clinical trials. In KRAS wild-type tumour cells, BRAF mutations and loss of EGFR or PTEN expression may lead to resistance to EGFR-targeted therapy and should be considered as additional negative predictive biomarkers.

Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines.

Three-dimensional (3D) tumor cell cultures grown in laminin-rich-extracellular matrix (lrECM) are considered to reflect human tumors more realistic as compared to cells grown as monolayer on plastic. Here, we systematically investigated the impact of ECM on phenotype, gene expression, EGFR signaling pathway, and on EGFR inhibition in commonly used colorectal cancer (CRC) cell lines. LrECM on-top (3D) culture assays were performed with the CRC cell lines SW-480, HT-29, DLD-1, LOVO, CACO-2, COLO-205 and COLO-206F. Morphology of lrECM cultivated CRC cell lines was determined by phase contrast and confocal laser scanning fluorescence microscopy. Proliferation of cells was examined by MTT assay, invasive capacity of the cell lines was assayed using Matrigel-coated Boyden chambers, and migratory activity was determined employing the Fence assay. Differential gene expression was analyzed at the transcriptional level by the Agilent array platform. EGFR was inhibited by using the specific small molecule inhibitor AG1478. A specific spheroid growth pattern was observed for all investigated CRC cell lines. DLD-1, HT-29 and SW-480 and CACO-2 exhibited a clear solid tumor cell formation, while LOVO, COLO-205 and COLO-206F were characterized by forming grape-like structures. Although the occurrence of a spheroid morphology did not correlate with an altered migratory, invasive, or proliferative capacity of CRC cell lines, gene expression was clearly altered in cells grown on lrECM as compared to 2D cultures. Interestingly, in KRAS wild-type cell lines, inhibition of EGFR was less effective in lrECM (3D) cultures as compared to 2D cell cultures. Thus, comparing both 2D and 3D cell culture models, our data support the influence of the ECM on cancer growth. Compared to conventional 2D cell culture, the lrECM (3D) cell culture model offers the opportunity to investigate permanent CRC cell lines under more physiological conditions, i.e. in the context of molecular therapeutic targets and their pharmacological inhibition.