Sox17 and Sox4 Differentially Regulate β-Catenin/T-Cell Factor Activity and Proliferation of Colon Carcinoma Cells

The canonical Wnt signaling pathway is involved in many biological processes, ranging from embryonic development to stem cell maintenance in adult tissues, while the dysregulation of Wnt signaling is implicated in human tumorigenesis. The key effector of the canonical Wnt pathway is β-catenin, which forms complexes with T-cell factor (TCF)/lymphoid enhancer factor (LEF) high-mobility-group (HMG) box transcription factors to stimulate the transcription of Wnt-responsive genes (7). While numerous studies have shown that β-catenin is regulated at many levels, less is known about the regulation of TCF/LEF transcription factors. In the absence of a Wnt signal, levels of cytosolic β-catenin are kept low via the interaction of β-catenin with a protein complex including glycogen synthase kinase 3β (GSK3β), adenomatous polyposis coli (APC), and Axin. The phosphorylation of β-catenin by the kinase GSK3β allows β-catenin to be ubiquitinated and targeted for degradation by the proteasome (1). The binding of a canonical Wnt ligand to the frizzled-lipoprotein receptor-related protein 5/6 receptor complex results in the repression of GSK3β and the stabilization of β-catenin. Stabilized β-catenin accumulates in the nucleus, where it acts as a cofactor with the HMG box family of TCF/LEF transcription factors to regulate the expression of Wnt target genes, such as cyclin D1 and Cdx-1 (17, 22). Although the formation of a TCF-β-catenin complex is required for the activation of all Wnt target genes (36), Wnt signaling is involved in a wide array of biological processes, including cell proliferation, cellular transformation (14), and embryonic development (24), demonstrating that the output of this pathway is highly influenced by the cellular context. Given that aberrant activation of the canonical Wnt pathway can lead to unrestricted cell division and tumor formation (12, 26, 28, 31, 40), it is not surprising that this pathway is antagonized by several different mechanisms. For example, several extracellular antagonists that inhibit ligand-receptor interactions have been described previously, including Dickkopf (Dkk), Cerberus, and the secreted frizzled-related proteins (10, 21, 34, 35). In many instances, Wnt signaling is kept in check by a negative-feedback loop in which β-catenin/TCF activity induces the transcription of its own negative regulators, Axin and Dkk1 (4, 20, 39). Finally, in the absence of activated β-catenin, TCF/LEF transcription factors keep Wnt target genes off via their interaction with members of the Grouch family of transcriptional repressors (4, 20, 39). Structurally related to TCF/LEFs, several members of the Sox family of HMG box transcription factors, including Sox17, Sox3, Sox7, and Sox9, have also been implicated in repressing β-catenin activity by a mechanism that is not well understood (2, 48, 54, 55). In addition to acting as an antagonist, Sox17 cooperates with β-catenin to activate the transcription of its endoderm target genes in Xenopus laevis (44). These findings suggest that, dependent on the context, Sox proteins can utilize β-catenin as a cofactor or can antagonize β-catenin/TCF function. While the mechanism by which Sox proteins antagonize Wnt signaling is unknown, one possibility is that they compete with TCFs for binding to β-catenin (55). Here, we report that Sox proteins expressed in normal and neoplastic gut epithelia can modulate canonical Wnt signaling and the proliferation of gastrointestinal tumor cells. While several Sox factors, including Sox17, Sox2, and Sox9, are antagonists of canonical Wnt signaling, others, such as Sox4 and Sox5, promote Wnt signaling activity. Gain- and loss-of-function analyses demonstrate that the Wnt antagonist Sox17 represses colon carcinoma cell proliferation while the agonist Sox4 promotes proliferation. In contrast to a proposed model in which Sox17 protein antagonizes Wnt signaling by competing with TCFs for β-catenin binding, we found that Sox17 interacts with both TCF/LEF and β-catenin and that Sox17 and TCF/LEF proteins interact via their respective HMG domains. Binding experiments suggest that Sox17, TCF, and β-catenin cooperatively interact to form a complex. In contrast, Sox4 can bind to either TCF/LEF or β-catenin alone but does not appear to cooperatively bind both proteins. Structure-function analyses indicate that Sox17 must bind directly to both β-catenin and TCF in order to antagonize Wnt signaling and that Sox17 DNA binding activity is not required. Lastly, functional studies show that Sox17 promotes the degradation of TCF/LEF and β-catenin proteins via a GSK3β-independent mechanism that can be blocked by proteasome inhibitors. In contrast, Sox4 may function to stabilize β-catenin protein. Together, these findings suggest that Sox transcription factors act in a novel pathway to modulate the stability of β-catenin/TCF proteins and regulate the proliferation of colon carcinoma cells. These results have important implications for how Sox proteins regulate the transcriptional output of Wnt signaling in many developmental and pathological processes.