In recent years, our group and others have generated much new knowledge regarding the pathogenesis of ulcerative colitis-associated neoplasias and preneoplasias (UCANs), but much more remains to be discovered. We now know, for example, that tumor suppressor gene is a very early event in UCANs. We have also learned that microsatellite instability (MI) is relatively common and occurs early in UC-associated neoplastic progression, and that in doing so, it targets at least very important growth-suppressive genes, TGF-beta1RII and IGFIIR. The central unifying hypothesis of this proposal is that the p53 inactivation and MI pathways are separate but equally important routes to cancer occurring in ulcerative colitis, and that both are involved very early in this process. We will test this hypothesis by addressing the following Specific Aims: 1. To study MI at anonymous noncoding loci in a large cohort of UCAN patients. 1.a. To determine the occurrence of MI both with neoplastic or preneoplastic lesions and remote from them, as well as in patients without known or previously diagnosed colorectal dysplasia or cancer. 1.b. To apply a novel microassay based on the cloning of single microsatellite alleles, with the aim of detecting MI in histologically normal tissues as well as sera from UC patients. 2. To assess MI at coding region targets in a large cohort of UCAn patients. 2.a. To assess inactivation of IGFIR and TGF-beta1RII by coding region MI, loss of heterozygosity, or diminished IGFIIR or TGF-betaRII expression at the mRNA and protein levels. 2.b. To demonstrate deficient activation of TGF-beta1 by examining expression of the active and latent precursor forms of TGF-beta1, using antibodies specific for each protein, and excessive expression of IGFII ligand, in lesions with or without IGFIR mutations. 2.c. To discover additional, novel coding region targets of MI in UCANs. 3. To develop a dynamic in vitro model of IGFIR function and dysfunction. 3.a. To discover colon cancer cell lines containing IGFIIR inactivating mutations. 3.b. To utilize SW48 colon cells, which possess an IGFIRR mutation, in cell complementation and transfection studies to test the growth suppressive activity of IGFIIR. 4. To investigate p53 tumor suppressor gene alterations in a large cohort of UCAN patients. 4.a. To determine whether the p53 inactivation and MI pathways are nonoverlapping. 4.b. To investigate the specific p53 base substitutions in all UC tissues studied, in order to prove the existence of a unique p53 mutational spectrum in UC. 5. To determine the prognostic significance and/or early detection value of p53 inactivation, along with that of MI occurring in noncoding regions and at specific targets, including IGFIR and TGF-beta1RII, using initial and followup clinical correlative parameters in a large cohort of UC patients.
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