Identification and functional analyses of beta-catenin/TCF target genes.

Abstract

The canonical Wnt signaling pathway plays an essential role in development, and control of beta-catenin stability is central to Wnt signaling. Mutational defects in the Wnt pathway are believed to result in an abnormal accumulation of beta-catenin in the cytoplasm and nucleus and constitutive activation of specific target genes that may be involved in cancer development. To identify novel beta-catenin/TCF-regulated target genes, two different strategies were pursued. A representational difference analysis comparing parental rat RK3E cells to those transformed by mutant beta-catenin proteins led to the identification of a candidate beta-catenin/TCF regulated gene termed beta-<italic> CUP3</italic> (beta-catenin upregulated). Database analysis showed beta-<italic> CUP3</italic> belongs to the Kelch repeat superfamily of proteins characterized by a single BTB/POZ domain and a variable number of Kelch motifs. Functional analyses of beta-CUP3 in a chick chorioallantoic membrane assay suggested beta-CUP3 may play a role in cell invasion. Oligonucleotide arrays to identify transcripts activated in RK3E cells after neoplastic transformation by mutant beta-catenin proteins or after ligand-induced activation of a beta-catenin-estrogen receptor fusion protein, led to the identification of the AXIN2 gene as a direct transcriptional target of beta-catenin signaling. Interestingly, AXIN2 has previously been shown to function as a scaffold to negatively regulate Writ signaling, thus suggesting a negative feedback pathway. Attempts to further define the function of AXIN2 led to the identification of a novel AXIN2 interacting protein containing SMC (structural of maintenance chromosome) and DIX (disheveled-AXIN) domains recently termed CCD1 (coiled-coiled domain). Further analyses showed CCD1 could subtly increase TCF activity induced by wild type beta-catenin. Finally, prior work in the literature has suggested an important role for NF-kappaB activation in neoplastic transformation by beta-catenin. Inhibition of NF-kappaB activity, with a dominant negative form of IkappaBalpha did not affect the ability of mutant beta-catenin-transformed RK3E cells to grow in soft agar. My observations collectively provide additional insights to the mechanism of beta-catenin regulation and its role in the pathogenesis of cancer.