The Beta-Catenin Target Gene Bfgf is Expressed in A Small Fraction of Tumor Cells with Characteristics of Cancer Stem Cells in Human Colorectal Cancer

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 5002 Many human colorectal cancers (CRC) are characterized by the deregulation of the Wnt signalling pathway leading to the stabilization and accumulation of β-catenin in tumor cells. Depending on the subcellular localization, β-catenin exerts two functions: at the cell membrane in a complex with E-cadherin, it is an integral part of the zonula adherens, thus mediating epithelial organization. This phenotype is found in central areas of CRCs. At the invasive front β-catenin is expressed in the nucleus which is associated with a gain of mesenchymal characteristics and strong transcriptional activity of the Wnt/β-catenin pathway. Thus, in these cells Wnt/β-catenin target genes conferring migration, invasion and dissemination -hallmarks of malignant progression- are transcriptionally up-regulated. Therefore, nuclear β-catenin induces in the context of the invasive front an epithelio-mesenchymal transition (EMT). As the cancer stem cell marker CD133 is also a Wnt/β-catenin target gene, nuclear β-catenin expressing tumor cells might thus be migrating cancer stem cells (MCSC). Finally, in cell culture, the maintenance of dedifferentiated, as spheroids growing colorectal tumor cells with the capability of initiating tumor growth in mice strictly depends on bFGF (basic fibroblast growth factor), indicating bFGF as an essential stemness supporting growth factor. Therefore, we hypothesized that bFGF might be another Wnt/β-catenin target gene which is produced in an autocrine manner as part of an inherent stemness maintenance program of mesenchymally transformed MCSCs at the invasive front of human colorectal carcinomas. In a first set of experiments we show evidence that bFGF is indeed a Wnt/β-catenin target gene employing electromobillity shift assays (EMSA) and chromatin immunoprecipitations (ChIP), luciferase-gene reporter assays, expression analysis in cultivated colorectal tumor cell lines using RT-PCR and Western blotting as well as β-catenin specific RNA interference (RNAi). In a second line of experiments we demonstrate that the cultivated human colorectal cancer cell line CaCo2 contains at least two different types of cells with respect to Hoechst 33342 dye exclusion. A tiny (0.1 %) Hoechst 33342 low or negative side population (SP) of cells initiated tumor growth in a xenograft mouse model at low cell numbers in contrast when cells were taken from the bulk culture or Hoechst 33342 positive population. Interestingly, the SP cells expressed high amounts of bFGF as well as CD133 compared to non-SP CaCo2 cells. Taken together we demonstrate that β-catenin regulates bFGF expression and that tumor initiating cells are characterized by high expression of bFGF and CD133. This is in support with the model that tumor cells at the invasive front resemble MCSC which is strengthened by the immunohistochemically detectable expression of bFGF in these cells.