COLON CELL DIFFERENTIATION--DCC ROLE IN MUC2 EXPRESSION

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The DCC gene is a potential tumor suppressor gene whose deletion or inactivation plays a role in colonic tumor progression. Recent evidence suggests that it plays a role in colonic goblet cell differentiation and mucin production, consistent with its less can also be effected by inducers which interact with metabolic processes and signal transduction pathways. Some of these inducers may be related to dietary components. We are in a unique position to dissect this link among DCC, mucin synthesis and cell differentiation since we have initiated detailed studies on the mechanisms of regulation of the MUC2 gene, the gene which encodes the principal colonic mucin peptide backbone. We have now cloned, mapped and partially sequenced approximately 90 kilobases of the locus on chromosome II encompassing the MUC2 gene and its promotor and enhancer, and have found this contig contains another mucin backbone gene, probably MUC5b, which is also expressed in colonic cells both in vivo and in vitro, that could be coordinately regulated with MUC2. We will investigate the interaction between DCC and MUC2 expression at the RNA and protein level, as well as the elaboration and secretion of mucus and growth parameters. Colonic carcinoma cell lines which have deleted at least one copy of DCC will be used as recipients of expression vectors which contain the entire wild-type DCC cDNA, or which encode specific extracellular and intracellular domains of the DCC protein, and the influence on constitutive and inducible MUC2 expression determined. The experiments will be extended to mucin synthesis and secretion, since we have demonstrated that expression of the gene does not necessarily lead to production and secretion of mature mucins. Further, different promotor constructs will be used to define the relationship between specific levels of DCC expression and cell and molecular phenotype. The promoter and enhancer for MUC2 will be dissected to delineate sequence elements and their boundaries necessary to establish responsiveness of MUC2 to DCC. Finally, we will investigate this relationship of mutations in the promotor of the MUC2 gene to the sub-class of colonic carcinomas having a mucinous phenotype. These experiments will provide detailed understanding of the interaction of a gene important in the differentiation and transformation of colonic epithelial cells with the principal hallmark of differentiation along one lineage. In addition, since mucinous colonic tumors in which MUC2 is deregulated exhibit a poorer prognosis than most common colorectal tumors, understanding this interaction may define prognostic factors which are not reflected in tumor histology or pathology.