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. 2012 Apr 6;287(15):11730-9.
doi: 10.1074/jbc.M111.327767. Epub 2012 Feb 15.

Wnt/β-catenin signaling regulates Yes-associated protein (YAP) gene expression in colorectal carcinoma cells

Wesley M Konsavage Jr  1 , Sydney L Kyler, Sherri A Rennoll, Ge Jin, Gregory S Yochum
Affiliations

Wnt/β-catenin signaling regulates Yes-associated protein (YAP) gene expression in colorectal carcinoma cells

Wesley M Konsavage Jr et al. J Biol Chem. .

Abstract

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.

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Figures

FIGURE 1.
FIGURE 1.
β-Catenin, TCF4, and RNAP bind the first intron of the YAP gene in vivo. A, diagram of the YAP gene locus and the β-catenin-enriched regions identified in a ChIP-Seq screen. Top, gray vertical lines represent peak density plot from the β-catenin ChIP-Seq screen. The arrow indicates the accumulated ChIP-Seq reads that mapped near the YAP transcription start site. Bottom, enlargement of the 5′-end of the YAP1 gene with Y1, Y2, Y3, and Y4 indicating the position of the PCR primer sets used in B, C, and D. B, quantitative real-time PCR analysis of DNA fragments isolated in ChIP assays conducted in HCT116 cells using β-catenin specific antibodies or an irrelevant IgG as a control. The primer sets Y1-Y4 were used to detect β-catenin binding and primers designed against regions in GAPDH (Ga.) and Tubulin (Tu.) were used as negative controls. C and D, as in B except anti-TCF4 antibodies were used in C and anti-RNAP antibodies were used in D to detect TCF4 and RNAP binding in ChIP assays, respectively. In B, C, and D, error is S.E.
FIGURE 2.
FIGURE 2.
The β-catenin/TCF4 binding region of YAP enhances SV40-driven luciferase gene expression. A, diagram of the pGL3-promoter plasmids used in luciferase assays with the SV40 minimal promoter and firefly luciferase gene shown as white rectangles and the 624 bp YAP enhancer in gray. A black vertical line represents the position of the consensus TCF motif within the YAP enhancer. The TCF sequence was changed by site-directed mutagenesis to generate the pGL3-YAP mutant plasmid (Mut.). B, HCT116 cells were transfected with vector control, pGL3-YAP wild-type (WT) or pGL3-YAP mutant (Mut.) firefly luciferase plasmids and a plasmid expressing Renilla luciferase. Twenty-four hours later, luciferase levels were measured and firefly luciferase levels were normalized to Renilla luciferase levels to control for differences in transfection efficiency between samples. C, cells transfected with the indicated reporters were untreated (black bars) or treated (gray bars) with the GSK3β inhibitor BIO for 12 h prior to measuring luciferase levels. D, cells were transfected with vector control or the pGL3-YAP (WT) luciferase reporter and, where indicated, the cells were co-transfected with a dominant negative TCF4 plasmid. Luciferase levels were measured as in B. E, nuclear proteins prepared from HCT116 cells were incubated with a 28 bp biotinylated and double-stranded DNA probe that contained either the wild-type or mutant TCF consensus sequence. Where indicated, unlabeled YAP duplexes were included in the binding reactions. Western blot analysis was used to detect bound β-catenin or TCF4. In B, C, and D, error is standard deviation.
FIGURE 3.
FIGURE 3.
β-Catenin controls YAP expression in HCT116 colon cancer cells. A, Western blot analysis of β-catenin protein levels in HCT116 cells expressing a control shRNA or an shRNA targeting β-catenin. HCT116 cells were infected with lentiviruses and Western blots were conducted 5 days after infection. B, quantitative real-time PCR analysis of DNA precipitated using β-catenin, TCF4, or RNAP specific antibodies in ChIP assays conducted in HCT116 cells expressing the indicated shRNAs. Primer set Y3 listed in Fig. 1A was used to detect β-catenin, TCF4, and RNAP binding to the YAP gene. C, quantitative real-time PCR analysis of cDNAs synthesized from RNA isolated from HCT116 cells expressing control (black bars) or β-catenin shRNA (gray bars). Specific primers were used to detect expression of YAP, CTGF, and GAPDH as a control. D, Western blot analysis of YAP protein levels in cells expressing the indicated shRNAs. Whole cell lysates were prepared and analyzed 5 days following lentiviral infection. In B and C error is S.E.
FIGURE 4.
FIGURE 4.
YAP enhances CRC cell metabolism, anchorage-independent growth in soft agar and tumorigenesis. A, quantitative real time PCR analysis of cDNAs prepared from mRNAs isolated from HCT116 cells 5 days after they were infected with a lentivirus expressing a control (black bars) or YAP specific (gray bars) shRNAs. Specific primers were used to measure relative expression of YAP, CTGF, and GAPDH. B, as in A, except that SW620 CRC cells were used and a second YAP specific shRNA (YAP shRNA2) was included as an additional control. C, Western blot analysis of YAP protein levels in HCT116 and SW620 cells expressing the indicated shRNAs. Blots were reprobed with tubulin antibodies to demonstrate that equivalent amounts of proteins were loaded. D and E, MTT assays were conducted in HCT116 and SW620 cells expressing the indicated shRNAs. Assays were performed 1 and 5 days after lentiviral infection of the cells. F and G, anchorage independent growth assays were conducted in HCT116 and SW620 cells expressing the indicated shRNAs. The day following infection with the indicated lentiviruses, cells were plated in soft agar and colony formation was assessed and quantified after 21 days. H, growth curves of tumors that formed after injection of control or YAP shRNA2 expressing SW620 cells into the flanks of athymic mice. I, explanted tumors on day 21 following injection. J, tumor weights at day 21. Error denotes S.E. and *, p < 0.005 and **, p < 0.0005.
FIGURE 5.
FIGURE 5.
Nuclear YAP expression is retained in densely grown CRC cells. A, Western blot analysis of YAP and β-catenin expression in six CRC cell lines. Tubulin served as a loading control. B, immunocytochemical analysis of YAP and β-catenin expression in HCT116 and SW480 cells. C, Western blot analysis of YAP protein levels in cytoplasmic and nuclear fractions from HCT116 and SW480 cells. Blots were reprobed with anti-histone H3 antibodies and anti-tubulin antibodies to mark the nuclear and cytoplasmic fractions, respectively. D, immunocytochemical analysis of YAP and β-catenin expression in HCT116 and SW480 cells that were densely cultured on glass coverslips. E, Western blot analysis of YAP protein levels in cytoplasmic and nuclear proteins prepared from densely cultured HCT116 and SW480.
FIGURE 6.
FIGURE 6.
β-catenin and YAP are expressed in the nuclear compartment of cells comprising primary and metastatic colorectal tumors. A, immunohistochemical analysis of tissue arrays prepared from primary colorectal tumors, normal tissue, and colorectal tumors that metastasized to the lymph node and liver. Shown are representative images of the 9 uninvolved colonic mucosa samples, the 36 primary tumors, and the 10 metastatic tumors examined. Hematoxylin staining was used to evaluate tissue architecture and β-catenin, and YAP localization was detected using specific antibodies. White arrows identify representative cells with nuclear β-catenin or nuclear YAP staining. The scale bar is 20 μm. B, tumor sections were scored for cells with nuclear β-catenin only (red), nuclear YAP only (blue), nuclear YAP and nuclear β-catenin (white), or no detectable staining (gray).
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