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Novel drug targets can be identified by differential analysis of RNA transcripts isolated from cancer cell lines and tissues. We have extended this approach by analyzing differences in gene expression resulting from the drug treatment of transformed and nontransformed cells. A mouse mammary epithelial cell line (C57MG), which conditionally expresses the Wnt-1 proto-oncogene, was left untreated or treated with retinoic acid in the presence or absence of Wnt-1 expression. The experiment was performed in triplicate, and RNA extracted from the four samples was analyzed by hybridization to over 12,000 unique oligonucleotide probe sets. Reproducible alterations in gene expression that occurred in response to retinoic acid, Wnt-1, or retinoic acid plus Wnt-1 relative to untreated cells were identified. Greater attention was given to genes encoding cell surface antigens that were selectively up-regulated by the combination of Wnt-1 and retinoic acid. These genes included the tumor necrosis factor family 4-1BB ligand, ephrin B1,stra6, autotaxin, and ISLR. Administration of retinoic acid to mice bearing tumors driven by activation of the Wnt-1/β-catenin pathway resulted in increased expression of stra6in the tumors but not in normal tissue. In principal, the therapeutic index of antibodies directed against these antigens should be enhanced by co-administration of retinoic acid.

Mutational activation of the Wnt signaling pathway is a common early event in colorectal tumorigenesis, and the identification of target genes regulated by this pathway will provide a better understanding of tumor progression. Gene expression profiling on oligonucleotide microarrays revealed reduced expression of the immediate early genes fosand fosBfollowing stimulation of cells by Wnt-1. Further analysis demonstrated that serum or 12-O-tetradecanoylphorbol-13-acetate activation of several immediate early genes including fos,fosB, junB, and egr1was inhibited by Wnt signaling. Wnt signaling inhibited transcriptional activation driven by the serum response element without altering the activation of the extracellular signal-regulated kinase cascade or ternary complex formation at the fosserum response element promoter. The Wnt-mediated repression of c-Fos, FosB, and JunB expression was consistent with a decrease in their binding to an AP-1 promoter element and decreased target gene transcription. The expression offos, fosB, junB, andegr1was also repressed in human colon tumors relative to patient matched normal tissue. By contrast, the fosfamily member fra-1was up-regulated in the human colon tumors, suggesting a compensatory mechanism for the reduction infosand fosBexpression. The results indicate that Wnt signaling can repress the expression of certain immediate early genes, and that this effect is consistent with changes in gene expression observed in human colorectal tumors.