Work Package 2

Downstream targets of Wnt signalling: molecules for cancer intervention

Introduction
The colorectal epithelium contains large numbers of invaginations termed the crypts of Lieberkühn. Pluripotent stem cells at the crypt bottom generate progenitors that occupy the lower third of the crypt. Cells in this amplification compartment divide approximately every 12 hours. In the mid-crypt region, the cells differentiate into one of the functional cell types of the colon. At the epithelial surface, cells undergo apoptosis and/or extrusion into the lumen. The entire process takes approximately 3-5 days. The Wnt signaling cascade is the major driving force of cell proliferation in the crypt. The so-called destruction complex consisting of the proteins APC, axin and GSK3ß resides at the heart of the Wnt cascade. In this complex, phosphorylation by GSK3ß targets b?catenin for ubiquitination and subsequent degradation by the proteasome. Wnt signaling inhibits GSK3ß activity. Then, b-catenin accumulates in the nucleus where it binds members of the TCF family and converts these WNT effectors from transcriptional repressors into activators. In colorectal cancer, truncating mutations in APC and axin/conductin, as well as mutations in the GSK3ß-target residues in b-catenin all lead to the formation of constitutive nuclear b-catenin/TCF4 complexes. Activating mutations of the WNT pathway are the only known genetic alterations in early premalignant lesions in the intestine, such as aberrant crypt foci and small polyps. Thus, these mutations may initiate the transformation of colorectal epithelial cells.

Objective
It is generally accepted that the aberrant expression of Tcf target genes constitutes the primary transforming event. It is the aim of this proposal to identify key Tcf target genes that play a central role in the physiological Wnt response, but also are the primary suspects responsible for the early transformation of epithelial cells.

Approach By DNA array technology we will capture the entire Tcf target gene program as activated in relevant biological models, i.e. in our engineered colorectal cancer cell lines (Clevers), in our Apc-mutant embryonic stem cell lines (Fodde), and in our mutant, cancer-prone mouse models (Apc: Fodde; Tcf1/4: Clevers). By state-of-the-art bioinformatics approaches in combination with “classical” literature searches, we will make educated guesses about putative candidate genes in our lists. These candidate genes will be analysed intitially in the cell lines using an inducible siRNA gene knockdown approach recently established in the Clevers lab. Ultimately, in vivo validation will be sought in transgenic or knockout mouse models.

Links with other parts of the program and other research: WP1) The cell lines that are developed in this project can serve as the basis for cell-based rapid screening methods for developmental signaling pathways; WP4) When available, the rapid transposon mediated insertional mutagenesis in ES cells will be applied for the generation of novel mutant mouse models; WP6) TCF target genes that encode surface receptors or secreted molecules will be used to isolate single chain monoclonal antibodies; WP7) The TCF target gene programs almost by definition identify stem cell programs of the various tissues under study (intestinal stem cells, embryonic stem cells).

Keywords: Colorectal cancer, Wnt cascade, stem cells, DNA array, cell-based screening.

Participants: Semaia Pharmaceuticals BV, Clevers, Fodde, Semaia Pharmaceuticals BV