Abstract B111: Differential expression of c-Met/HGF axis and epithelial-mesenchymal transition (EMT)-related gene markers between primary colorectal carcinomas and liver metastases in primary tissue, isolated cell types and in vivo

Abstract

Abstract Introduction: EMT is frequently activated in a subset of tumor cells during disease progression. EMT is described as downregulation of E-cadherin with a corresponding upregulation of mesenchymal markers such as Vimentin. EMT is linked to carcinoma progression stemming from increased tumor cell motility, growth, survival, and invasion. The c-Met receptor and MACC1 (metastasis-associated in colon cancer-1) are often upregulated in CRC metastases, have been associated with increased metastasis, and represent potential drug targets. Here we assessed the expression of genes associated with EMT in human CRCs and liver metastases (LMs), in epithelial cells isolated from primary tissues and in mouse passaged primary tumor xenografts. Methods: Human primary CRC (n = 11) and LM (n = 21) samples, along with matched normal tissues, were obtained under full ethical approval from the Queen's Medical Centre, Nottingham, UK. Real-time quantitative PCR was used to determine expression levels of EMT-related markers (growth factors, transcriptional repressors, mesenchymal markers) relative to the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase, from (i) primary tissues, (ii) epithelial cells and fibroblasts isolated from disaggregated primary tissues using cell type-specific antibodies bound to magnetic beads, (iii) primary tumor tissues subcutaneously passaged in nude mice. A student's t-test was used for statistical analyses. Results: MACC1 and c-Met were significantly upregulated, whereas E-cadherin was significantly downregulated, in CRC and LM primary tissues compared to normal colonic mucosa, indicating that EMT had occurred. In LMs, hepatocyte growth factor (HGF), Snail, Slug, Twist, Vimentin, TGFβ1, and matrix metalloproteinase 9 were also significantly upregulated (P <0.05), whereas expression of Zeb1, s100a4, and MMP2 were not altered. Gene expression profiles from isolated epithelial cells and fibroblasts showed that hgf, vimentin, and TGFβ1 were predominantly expressed by mesenchymal cells, whereas c-met, e-cadherin, and snail expression were predominantly epithelial. In xenograft tumor tissues, hgf and vimentin expression was lost within the first passage, whereas e-cadherin and c-met expression was maintained, indicating that human stroma was rapidly replaced. Conclusions: Phenotypic markers consistent with EMT were significantly increased in LMs compared to CRCs, and Snail, Twist, Vimentin, TGFβ1, and MMP9 provide the best markers for LM. In CRCs and LMs, paracrine signalling of HGF (mesenchymal) to the c-Met receptor (epithelial) is likely to occur, and this signalling pathway appears to be lost during growth of the primary tumor in nude mice. This has important implications in therapy studies targeting the HGF-MACC1-cMet axis. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B111.