GENE INTERACTIONS IN COLON CANCER

The c-myc gene is overexpressed in human colon tumors and rigorous quantitative and statistical analysis has demonstrated that the gene is frequently amplified from 2 to 5 fold in these tumors. Moreover, in a phase III study of 5FU based adjuvant therapy for colon cancer, amplification of the c-myc gene identified the subset of patients who show improved disease free and overall survival in response to treatment. c-myc may influence outcome in at least 3 ways: both c-myc and thymidylate synthase (TS) bind TS protein and are translationally regulated by formation of these ribonucleoprotein structures, establishing a potentially complex interaction between c-myc and TS mRNA levels in regulating TS activity; stimulation of proliferation may stimulate TS expression, a gene regulated in the cell cycle; and c-myc may influence proliferation, differentiation and apoptosis of colonic epithelial cells independent of affects on TS. The goal of this application is two fold: 1st, to understand how c-myc amplification and expression modulate TS mRNA, protein, RNP complex formation, and final TS activity and sensitivity to 5FU. This will be done by investigating these interactions in a panel of colon carcinoma cell lines which we have characterized as to level of c-myc amplification. We will then utilize expression vectors containing wild- type c-myc, the c-myc: TS binding site, c-myc with deleted or mutated TS binding site, or a chimeric c-myc:estrogen receptor construct to dissect the influence of expression of c-myc mRNA, protein, and/or c-myc activity on TS mRNA, protein and activity and sensitivity to 5FU. Preliminary data already indicate that c-myc and TS mRNA levels appear to be tightly linked. 2nd, we will utilize novel automated cytometry, fluorescent probe methodology and imaging technology, recently reported in Science by our coinivestigator, which is capable of analysis of DNA and RNA levels in situ with single molecule sensitivity and resolution, to address the heterogeneity of c-myc and TS gene copy number and mRNA content. Following initial analysis of cell lines for calibration and base line measurements, the DiscoveryTM system will be applied to tumor and normal tissue sections (already in our possession) from over 700 patients entered into two phase III studies of adjuvant therapy for colon cancer conducted by ECOG. A continuation of our successful collaboration with ECOG biostatistics will test whether the interaction of c-myc and TS, and the cellular distribution of amplification and/or expression of these genes, is prognostic for either outcome or response to adjuvant therapy.