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Tong-Chuan He, Andrew B. Sparks, Carlo Rago, Heiko Hermeking, Leigh Zawel, Luis T. da Costa, Patrice J. Morin, Bert Vogelstein, Kenneth W. Kinzler*
The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. Here, the c-MYC oncogene is identified as a target gene in this signaling pathway. Expression of c-MYC was shown to be repressed by wild-type APC and activated by beta-catenin, and these effects were mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers.
Most human colorectal tumors are initiated by inactivation of the APC tumor suppressor gene, located on chromosome 5q21 (1). APC is a cytoplasmic protein that can bind to and promote the degradation of beta-catenin (2). Among beta-catenin functions is the ability to bind members of the Tcf family of transcription factors and activate gene transcription (3). Accordingly, human colorectal tumors with APC or beta-catenin mutations exhibit increased beta-catenin/Tcf-mediated transcription (4, 5). However, the downstream targets of this beta-catenin/Tcf-4-regulated transcription are unknown. This study was undertaken to define those targets and thereby gain clues to the mechanisms through which APC affects cellular growth.
To evaluate the transcriptional effects of APC, we studied a human colorectal cancer cell line (HT29-APC) containing a zinc-- inducible APC gene and a control cell line (HT29-beta-Gal) containing an analogous inducible lacZ gene (6). Both endogenous APC alleles in HT29 cells contain truncating mutations, and restoration of wild-type (WT) APC expression results in growth inhibition and apoptosis. Upon induction, APC protein is synthesized rapidly and reaches maximal levels by 9 hours (7). By 12 hours, a significant fraction of the cells display morphological signs of apoptosis. Because we were interested in identifying changes in gene expression that directly relate to restoration of APC function and not apoptosis, we analyzed the HT29-APC cells 9 hours after APC induction.
To evaluate changes in gene expression, we used serial analysis of gene expression (SAGE), a technique that allows the quantitative evaluation of cellular mRNA in an unbiased manner (8). In brief,...