Microsatellite instability (MSI), caused by defective DNA mismatch repair, occurs frequently in colorectal carcinomas (8, 9). MSI is categorized as high (MSI-H), low, (MSI-L), or negative (MSS, or microsatellite-stable), according to the frequency of microsatellite alterations at anonymous (non-coding) loci (10). There is strong evidence that tumors with high-frequency MSI (MSI-H tumors) have significant differences in their clinical behavior that distinguish them from MSS and MSI-L tumors (11, 12). In addition, evidence is accumulating that tumors with low-frequency MSI (MSI-L) tumors have unique features (13). Nevertheless, our understanding of both MSI-H and MSI-L tumors remains incomplete, and the existence of MSI-L tumors as a distinct subgroup has been questioned. Hypothesis: MSI-H, MSIL, and MSS gastrointestinal tumors are phenotypically unique. These distinct biologies can be better defined and understood through comprehensive genomic approaches, including instabilotyping and microarray-based bioinformatics. Moreover, valuable insights into molecular pathways underlying these entities can be gained by identifying and further studying candidate genes. This hypothesis will be explored with the following Specific Aims: 1) To broaden and extend unbiased instabilotyping of MSI-H colorectal cancers and cell lines, in order to identify additional genes targeted by frequent frameshift mutation; 2) To examine functional consequences of mutations in selected coding region targets of microsatellite instability; 2.a To demonstrate biallelic inactivation of genes showing frequent frameshift mutation by analyzing for loss of heterozygosity, point mutation, and altered expression; 2.b To determine functional differences between WT and mutant candidate proteins. To ascertain the effect(s) of mutant proteins on cell biology and behavior by transfecting WT candidate genes into biallelically mutated cells and ascertaining effect(s) on cell biology; 2.b.i To assess cell proliferation, anchorage-independent growth, invasion (Matrigel assay), mobility (wound assay), apoptosis (TUNEL, DAPI staining and caspase assays), and differentiation (morphology); 2.b.ii To evaluate the effects of WT transfected ACTR2 and other candidate genes on protein expression and signal transduction, including phosphorylated and total SMAD2, total SMAD4, caspase 1, and TTK; 2.b.ii) Using cDNA microarrays, to compare colon cancer cells before and after transfection with WT ACTR2, TTK, HDCMA18, CASP 1, and other as-yet unidentified genes containing frequently mutated microsatellites; 3 To increase our understanding of the biologies of MSI-H, MSI-L, and MSS colorectal carcinomas by comparing the transcriptomes of these cancers, using cDNA microarrays and bioinformatics strategies; 3.a To generate global gene expression data from MSI-H, MSI-L, and MSS status colorectal tumors; 3.a.i To produce and probe cDNA microarrays with RNAs extracted from MSI-H and MSS cells. 3.a.ii To hybridize microarrays to MSI-H, MSI-L, and MSS primary colorectal tumors; 3.b To determine whether MSI-L tumors comprise a biologically distinct subgroup; 3.b.i To apply bioinformatics strategies, including hierarchical clustering, significance analysis of microarrays (SAM) and principal components analysis (PCA) in order to confirm the existence and provide clues to the biology of a distinct MSI-L tumor subgroup; 3.c To identify genes defining molecular genetic pathways underlying MSI-H, MSS, and MSI-L tumors; and 3.c.i To utilize PCA components to find genes segregating with MSI status, and SAM to identify genes significantly differentially expressed among these three groups.
