Mutations in the Wnt/β-catenin pathway occur in most colorectal cancers (CRCs), and these mutations lead to increased nuclear accumulation of the β-catenin transcriptional co-activator. In the nucleus, β-catenin associates with TCF/LEF sequence specific transcription factors to activate target gene expression. The Hippo pathway restricts cellular growth by preventing nuclear accumulation of the Yes-associated protein (YAP) transcriptional co-activator. YAP expression is elevated in CRCs suggesting that, like Wnt/β-catenin signaling, the Hippo pathway may contribute to colorectal carcinogenesis. Regulation of YAP at the post-translational level has been well studied but the transcription factors that control YAP gene expression are unknown. Here we demonstrate that β-catenin/TCF4 complexes bind a DNA enhancer element within the first intron of the YAP gene to drive YAP expression in CRC cells. As such, reducing β-catenin expression in CRC cells using shRNAs leads to decreased YAP mRNA and protein levels. YAP is abundantly expressed in the cytoplasm and nuclei of several established human colon cancer cell lines and this localization pattern is insensitive to plating density. Finally, we show that YAP expression is elevated in the majority of a panel of primary human colorectal tumors compared with its expression in uninvolved colonic mucosa, and that YAP and β-catenin localize to the nuclear compartment of tumor cells. Together, these results implicate YAP as an oncogene whose expression is driven by aberrant Wnt/β-catenin signaling in human CRC cells.

Deregulation of the Wnt/β-catenin signaling pathway is a hallmark of colon cancer. Mutations in the adenomatous polyposis coli (APC) gene occur in the vast majority of colorectal cancers and are an initiating event in cellular transformation. Cells harboring mutant APC contain elevated levels of the β-catenin transcription coactivator in the nucleus which leads to abnormal expression of genes controlled by β-catenin/T-cell factor 4 (TCF4) complexes. Here, we use chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq) to identify β-catenin binding regions in HCT116 human colon cancer cells. We localized 2168 β-catenin enriched regions using a concordance approach for integrating the output from multiple peak alignment algorithms. Motif discovery algorithms found a core TCF4 motif (T/A–T/A–C–A–A–A–G), an extended TCF4 motif (A/T/G–C/G–T/A–T/A–C–A–A–A–G) and an AP-1 motif (T–G–A–C/T–T–C–A) to be significantly represented in β-catenin enriched regions. Furthermore, 417 regions contained both TCF4 and AP-1 motifs. Genes associated with TCF4 and AP-1 motifs bound β-catenin, TCF4 and c-Jun in vivo and were activated by Wnt signaling and serum growth factors. Our work provides evidence that Wnt/β-catenin and mitogen signaling pathways intersect directly to regulate a defined set of target genes.

Regulation of MYC gene expression by aberrant Wnt/β-catenin signaling in colorectal cancer

Aberrant MYC gene expression by the Wnt/β-catenin pathway is implicated in colorectal carcinogenesis. Wnt/β-catenin signaling stimulates association of the β-catenin coactivator complex with two Wnt responsive enhancers (WREs) located in close proximity to MYC gene boundaries. Each enhancer directly binds members of the TCF/Lef family of transcription factors that, in turn, recruit β-catenin. In a previous report, we showed that the downstream MYC enhancer ( MYC 3′ WRE) cooperated with the upstream enhancer ( MYC 5′ WRE) to activate expression of a heterologous reporter gene in response to Wnt/β-catenin and mitogen signaling. Here we use chromatin conformation capture (3C) to show that the MYC 5′ and 3′ WREs are juxtaposed at the genomic MYC locus during active transcription. This MYC 5′3′ chromatin loop is present in HCT116 human colorectal cancer cells that contain high levels of nuclear β-catenin and is absent in HEK293 cells that contain trace amounts of nuclear β-catenin. Depletion of functional β-catenin/TCF complexes blocks formation of the MYC 5′3 chromatin loop. Furthermore, we find that the chromatin loop is absent in quiescent cells, but is rapidly and transiently induced by serum mitogens in a β-catenin-dependent manner. Thus, we propose that a distinct chromatin architecture coordinated by β-catenin/TCF-bound WREs accompanies transcriptional activation of MYC gene expression.

Traditionally, signaling pathways have been perceived to act in an autonomous manner to regulate tissue morphology, size, differentiation, and development. Recent evidence suggests that these pathways often intersect and regulate one another to elicit an appropriate response to a complex set of stimuli. Two pathways known to be important for development, growth, and homeostasis are the Wnt/β-catenin and the Hippo/YAP pathways. Growing data indicate that these two pathways influence each other in a number of ways to properly regulate tissue growth and repair. Deregulation of these pathways often contributes to tumorigenesis. In this review, we will discuss the points of intersection between the Wnt/β-catenin and Hippo/YAP pathways and how these interactions contribute to homeostasis, organ repair, and tumorigenesis.

Deregulated Wnt/β-catenin signaling promotes colorectal cancer (CRC) by activating expression of the c-MYC proto-oncogene (MYC). In the nucleus, the β-catenin transcriptional co-activator binds T-cell factor (TCF) transcription factors, and together TCF/β-catenin complexes activate MYC expression through Wnt responsive DNA regulatory elements (WREs). The MYC 3' WRE maps 1.4-kb downstream from the MYC transcription stop site and binds TCF4/β-catenin transcription complexes to activate MYC. However, the underlying mechanisms for how this element operates are not fully understood. Here, we report that the TCF family member, TCF3, plays an important role in regulating MYC expression in CRCs. We demonstrate that TCF3 binds the MYC 3' WRE to repress MYC. When TCF3 is depleted using shRNAs, the MYC 3' WRE is more available to bind TCF4/β-catenin complexes. Stimulating downstream Wnt/β-catenin signaling by inhibiting GSK3β causes an exchange of TCF3 with TCF4/β-catenin complexes to activate MYC. Finally, this transcription factor switch at the MYC 3' WRE controls MYC expression as quiescent cells re-enter the cell cycle and progress to S phase. These results indicate that a dynamic interplay of TCF transcription factors governs MYC gene expression in CRCs.