Abstract 5507: Activation of a subset of growth factor-associated genes in cetuximab-sensitive colorectal cancer GEO cells with KRAS mutation
Authors: Dai Yao , Siemann Dietmar , Shi Wenyin 
Published: May 2015

Abstract
KRAS mutation has been widely used in the clinic as a predictive biomarker in metastatic colorectal cancer (mCRC) for insensitivity to the anti-EGFR antibody cetuximab (Erbitux). However recent clinical studies indicate that a subset of mCRC patients with mutated KRAS may still retain sensitive to the drug. Thus it is important to identify alternative signaling pathways that bypass the activated KRAS pathway and mediate cetuximab-induced tumor killing effects. The mCRC cell line GEO harbors mutated KRAS (G12A) but is one of the most sensitive cell lines to cetuximab. The underlying mechanisms by which KRAS-mutated cells remain responsive to EGFR inhibitors have not been explored. In the current study, we compared the expression of KRAS and EGFR in four CRC cells (GEO, LOVO, HT-29, Caco2) with varying KRAS status and evaluated the treatment effects of cetuximab in these models. Cell proliferation was detected by MTT assay. Drug effects in xenografts were assessed by monitoring tumor growth delay. At the molecular level, active K-Ras was detected by pull-down assay and EGFR-associated signaling pathways were determined by Western blotting. KRAS gene expression was tested by qPCR, and EGFR expression in tumor tissues was tested by immunohistochemical staining. Finally, GEO xenografted tumors with or without cetuximab treatment were processed for PCR array analysis to detect the gene expression of molecular components associated with growth factor (EGF, PDGF) signaling networks. In accordance with the sequencing information, we confirmed the constitutively active K-Ras in GEO cells compared to KRAS-wildtype counterparts. However, consistent with other studies, we found that of GEO cell proliferation and xenograft tumor growth were significantly inhibited by cetuximab, a phenotype similar to that in KRAS normal HT-29 but not observed in the KRAS mutated LOVO model. Further, cetuximab inhibited expression of p-Akt but not p-ERK, p-STAT3, p-Src and total EGFR. Interestingly, PCR arrays showed that a subset of genes was highly activated in cetuximab treated GEO tumors, including LTA, MAP2K7, MAPK10 and STAT1. These gene products have been previously implicated in growth arrest and apoptosis however their association with EGFR/K-Ras signaling remains unknown. Studies on examining the functional role of these candidate genes in mCRC cells in response to cetuximab are ongoing. These preliminary findings support further investigations to identify alternative molecular signatures as potential biomarkers that may predict cetuximab-mediated tumor response in a subset of mCRC cells that harbors KRAS mutation.
Citation Format: Yao Dai, Dietmar Siemann, Wenyin Shi. Activation of a subset of growth factor-associated genes in cetuximab-sensitive colorectal cancer GEO cells with KRAS mutation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5507. doi:10.1158/1538-7445.AM2014-5507