Colorectal cancer (CRC) is still one of the most common causes of cancer-related death worldwide (World Health Organization, Fact sheet N°297, October 2011). Though, therapeutic options improved over the past years, the prognosis of metastatic colorectal cancer (mCRC) remains poor with a median survival of 18-21 months. Monoclonal antibodies targeting the epidermal growth factor receptor (EGFR) are used for the palliative treatment of metastatic colorectal cancer (mCRC) carrying a KRAS-wildtype. Among the tumors with a KRAS-wildtype, only 35% respond, and alterations of other signaling molecules and pathways modulate therapeutic response. In this study we analyzed the effect of KRAS-mutations on intracellular signaling cascades to find new therapeutic approaches for patients with mCRC. Reverse-phase protein array and gene array technology was applied to sixteen adenocarcinomas of the sigmoid colon carrying either wildtype (n=8) or mutant (n=8) KRAS. Differential expression was validated on 49 sigmoid cancers by RT-PCR, Western blot and immunohistochemistry. In a screening for members of signaling pathways known to have a high impact on tumor development in colon cancer, we found 14 proteins downregulated in tumors with mutated KRAS. Subsequently, we compared the data of the protein arrays with the results of gene expression arrays performed with the same samples. We confirmed the differential expression of eight genes as a function of the KRAS genotype of the tumor. To further validate the differential expression of these candidates, we performed quantitative expression analysis and immunohistochemical staining of an independent validation series comprising 49 CRC patient samples. It was clearly demonstrated that the expression of EGFR is decreased in colorectal carcinomas with a mutant KRAS genotype.
Overall design
mRNA was obtained from 16 patients with colorectal cancer (8 with and 8 without KRAS-mutation). mRNA was extracted from neoplastic and non-neoplastic colon mucosa and analyzed separately by Affymetrix Human Gene 1.1 ST GeneChips finally generating 32 separate data sets.
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