Scientific Abstract

Proposal No. IBD-0065R
Principal Investigator: Randall F. Holcombe, M.D.
Applicant Organization: University of California, Irvine (U.S.A.)
Project Title: Wnt signaling in inflammatory bowel disease-related colon cancer
Period of Award: November 1, 2003 - October 31, 2005

Wnt ligands induce a signaling cascade by binding to cell surface frizzled receptors. This leads to increases in cellular β-catenin and, ultimately, the induction of transcription of growth promoting genes. This process, mimicked by mutations in the APC gene, is integrally associated with the process of sporadic colon carcinogenesis. Mutations in APC and β-catenin appear to be rare in inflammatory bowel disease (IBD)-related tumors. However, abnormal expression of other components of the Wnt pathway, including frizzled receptors, SARP1 and disheveled (DVL) have been described.

In this proposal, the expression of several elements of the Wnt signal transduction pathway will be defined in normal, dysplastic and malignant colonic tissues from patients with IBD. First, we will examine the expression of FZ receptors and DVL proteins by immunohistochemistry in IBD-associated colon cancer tissues and compare this expression to adjacent mucosal tissues and sporadic colon cancers. In situ hybridization with anti-sense RNA probes specific for each of the ten FZ receptor subtypes as well as for specific frizzled-inhibitory proteins (FIPs) and selected Wnt ligands will then be utilized to define their expression in these tissues.

We will also test the hypothesis that genetic mutations in axin and casein kinase Iε (CK1ε) occur in IBD-associated colon cancer. This analysis will involve dHPLC, evaluation of heteroduplex formation and direct sequencing. Next, colon biopsies of uninflamed, inflamed and dysplastic mucosa will be obtained prospectively from patients with IBD undergoing surveillance colonoscopy. These tissues will be evaluated for expression of frizzled receptor subtypes, DVL, Wnts and FIPs and also for activation of Wnt signaling, indicated by nuclearization of β-catenin and increased expression and activity of a downstream pathway component, LEF1.

Finally, the role of specific FZ receptor subtypes in the initiation and propagation of Wnt pathway signals will be defined utilizing a novel, in vitro, co-culture expression system. Those Fz receptor subtypes which exhibit differential expression in IBD tissues will be evaluated in this system. Expression constructs for individual FZ receptor subtypes will be utilized for more in depth analysis in colon cancer cell lines to define signal pathway specificity of specific Fz:Wnt combinations. The expression of genes regulated by these specific Fz:Wnt interactions will be evaluated by microarray techniques.