) Carcinogenesis is a complex genome-wide process of mutation and altered genetic programing that is incompletely understood at the molecular level. Global molecular biologic approaches are needed if progress is to be made in our understanding of detection and treatment of this deadly group of diseases. Microsatellite instability (MI) occurs early and often in human carcinoma development, and it affects the entire genome, often known as the replication error-positive (RER+) phenotype. How can MI contribute to carcinogenesis? One prevailing notion is that by occurring within the coding regions of genes, MI causes gene innovation or dysfunction leading to cancer. The first of such targets to be identified was the transforming growth factor between beta 1 type II receptor gene (TGF-1RII), which mutates frequently at an intragenic microsatellicate in sporadic RER+ colorectal, gastric other neoplasms. We demonstrated that an identical mechanism exits in ulcerative colitis-associated premalignant lesions in that the insulin-like growth factor II receptor (IGFIIR), E2F-4, and phosphatase and tension homolog located on chromosome ten (PTEN 1) genes also mutate during RER+ human tumorigenesis. These data, along with evidence of MI within other cancer-related genes such as the anti-apoptotic gene BAX and some DNA mismatch repair genes themselves, appear to represent the """"""""tip of the iceberg"""""""" for this category of molecular alteration. Using these data as a launching point, we propose to embark on the task of identifying all MI occurring within coding portions of genes in one group of MI-prone cancers, colorectal carcinomas. We call this global profile the colorectal tumor instabilitome.
Our aim i s to deepen our understanding of RER+ colorectal carcinogenesis by identifying all genes within which microsatellite instability occurs in these tumors. We will utilize powerful computer-based algorithms to discover microsatellites within open reading frames and will test these microsatellite sites for instability in RER+ colorectal tumor specimens. Multiplex calorimetric-labeled semi-automated PCR coupled with simultaneous electrophoresis will allow concurrent evaluation of 10 or more coding region microsate loci per single gel lane, greatly lowering the amount of genomic DNA and time need per locus assayed. The detailed objectives of this proposal are to 1) map the entire colorectal cancer coding region """"""""instabilitome,"""""""" i.e., to discover all the coding region targets of MI in colon cancers; 2) develop robust automatable methods of """"""""instabilotyping"""""""" that will be generalizable to other tumor types, stages, and small tumor specimens; and 3) make available to the general scientific public a comprehensive database of coding region microsatellite loci and mutation for use in other laboratories.
