Abstract This study is to determine if triplex DNA-binding proteins in human cells play an important role in processes that lead to cancer such as genetic instability or growth deregulation. Cytoplasmic and nuclear extracts from 60 colorectal cancer patients’ tumor (T) and normal (N) tissues were isolated and examined by gel shifts (EMSA) for triplex DNA-binding proteins. Total tumor extracts showed significantly higher triplex DNA-binding activities of the major H3 complex than normal tissue extracts (Wilcoxon test). Neither T-staging nor M-staging at diagnosis was associated with any of the factors, but N-Stage was significantly associated with the ratio of tumor/normal (T/N) for the total of cytoplasmic and nuclear extracts. This means all patients without lymph node affectation had decreased binding ratios (T/N) in both cytoplasmic and nuclear extracts. Kaplan-Meier survival analysis using a cut-off of 1.5 (rounded-up median) for the nuclear binding activity ratio (T/N) showed significantly poorer survival for those patients whose nuclear binding was greater than 1.5. This suggests that although the major triplex DNA-binding protein is present in normal tissue extracts, it is more abundant in tumor extracts, and that a specific abundance of the major triplex-binding protein (H3) in the tumor nucleus is associated with poorer patient survival. Biotinylated triplex DNA-protein complexes were isolated from the RKO colorectal cancer cell line with streptavidin-agarose and analyzed by nano-HPLC ESI-MS-MS. We identified 100 and 60 kDa proteins from nuclear extracts as PSF (polypyrimidine tract binding-associated splicing factor) and p54nrb (NonO), known to function as RNA polymerase II-associated splicing factors, bind as heterodimers, and implicated in the regulation of expression of the myc family of oncoproteins and COX2. We also identified a 75 kDa protein from cytoplasmic extracts as U2AF65 (U2 small nuclear RNA auxiliary factor 2 isoform b), also a splicing factor that can directly interact with p54 nrb and shuttle continuously between the nucleus and cytoplasm. To examine differences between tumor and normal tissues in multiple signaling proteins and proteins known to be relevant in colorectal cancer, reverse phase protein array (RPPA), Pearson correlation and cluster analysis of tumor nuclear lysates ranked from lowest to highest H3 EMSA values shows a highly significant correlation between decreased β-catenin expression in tissues that have the lowest H3 EMSA values and increased β-catenin expression in tissues that have the highest H3 EMSA values. We found this pattern in all tissue lysate types: normal-cytoplasm, normal-nucleus, tumor-cytoplasm, and tumor-nucleus. We believe these splicing factors are overexpressed in tumor tissues and could cause deregulated growth of cells by influencing other proteins associated with tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4566.