S1684 Sprouty-2 Enhances Cell Proliferation and Induces Resistance to Apoptosis Through IGF-1R Pathway in Colon Cancer Cells

Abstract

Background: The Caudal-related homeobox (Cdx) transcription factor Cdx2 is a homeodomain protein related to the Drosophila Caudal gene. It is an important regulator of intestinespecific gene expression and columnar morphogenesis. It plays a key role in intestinal cell differentiation, cell to cell adhesion, columnar morphogenesis, and proliferation. Cdx2 has been implicated in a number of human disease processes including colon cancer and intestinal metaplasia. The purpose of this study is to develop a Cre-regulated Cdx2 expression model to facilitate the study of these complex and multi-staged processes. Other inducible systems generally yield a leaky expression that can blunt the Cdx2 response. A Cre-inducible system would not share this limitation. Methods: We have subcloned a Flag-Cdx2 cDNA into a Cre-regulated expression vector. In this construct, Cdx2 is linked in frame to a histone 2BRFP(Cherry) reporter. In between is a picornavirus 2A peptide directing cotranslational cleavage of the linked proteins, thereby releasing the Flag-Cdx2. Upstream to this sequence is a _eta_actin promoter sequence (with CMV enhancer sequence) to drive expression of the bicistronic mRNA. In between these is a loxP flanked cassette that includes a PGK-neo selectable marker and a SV40 virus tpA transcriptional stop sequence. Once integrated, this locus will remain silent until expression of Cre recombinase. This expression vector is cotransfected along with an expression vector for CreER(T2). Subsequent use of Tamoxifen enables drug-dependent control of Cre-mediated DNA recombination. Results: The Creresponsive Cdx2 clone was created and confirmed by DNA sequencing. 293T cells were transiently transfected with this construct, with and without a Cre expression vector. In the absence of Cre recombinase, there was immunofluorescent H2B detected. Conversely, in the presence of Cre co-transfection, reporter immunofluorescent product was strongly observed within cells. In addition, a whole cell lysate was prepared from these cells and analyzed by Western blot using anti-Cdx2 and anti-Flag-Cdx2 probes. Results showed that Flag-Cdx2 was identified exclusively in the Cdx2 clone + Cre co-transfection sample only. Stably transfected HCT116, DLD1, and Colo 205 cells are currently being raised for studies of the effects of Cdx2 induction on cell proliferation and cell to cell adhesion. Conclusions: We have developed a novel Cre-recombinase regulated Cdx2 expression system allowing for the spatiotemporal study of Cdx2 in both In Vitro and In Vivo models. Further studies with stable transfection of our Cdx2-inducible clone into colon cancer cell lines are underway.