Inhibition of β-catenin signaling by nongenomic action of orphan nuclear receptor Nur77.

Dysregulation of β-catenin turnover due to mutations of its regulatory proteins including adenomatous polyposis coli (APC) and p53 is implicated in the pathogenesis of cancer. Thus, intensive effort is being made to search for alternative approaches to reduce abnormally activated β-catenin in cancer cells. Nur77, an orphan member of the nuclear receptor superfamily, has a role in the growth and apoptosis of cancer cells. Here, we reported that Nur77 could inhibit transcriptional activity of β-catenin by inducing β-catenin degradation via proteasomal degradation pathway that is glycogen synthase kinase 3β and Siah-1 independent. Nur77 induction of β-catenin degradation required both the N-terminal region of Nur77, which was involved in Nur77 ubiquitination, and the C-terminal region, which was responsible for β-catenin binding. Nur77/ΔDBD, a Nur77 mutant lacking its DNA-binding domain, resided in the cytoplasm, interacted with β-catenin, and induced β-catenin degradation, demonstrating that Nur77-mediated β-catenin degradation was independent of its DNA binding and transactivation, and might occur in the cytoplasm. In addition, we reported our identification of two digitalis-like compounds (DLCs), H-9 and ATE-i2-b4, which potently induced Nur77 expression and β-catenin degradation in SW620 colon cancer cells expressing mutant APC protein in vitro and in animals. DLC-induced Nur77 protein was mainly found in the cytoplasm, and inhibition of Nur77 nuclear export by the CRM1-dependent nuclear export inhibitor leptomycin B or Jun N-terminal kinase inhibitor prevented the effect of DLC on inducing β-catenin degradation. Together, our results demonstrate that β-catenin can be degraded by cytoplasmic Nur77 through their interaction and identify H-9 and ATE-i2-b4 as potent activators of the Nur77-mediated pathway for β-catenin degradation.

Introduction

Wnt/β-catenin signal pathway plays a critical role in embryonic development, tissue homeostasis and carcinogenesis (Logan and Nusse 2004, Moon et al 2002, Peifer 1997, Peifer and Polakis 2000, Polakis 2000, Polakis 2007). Abnormal subcellular localization and aberrant accumulation of β-catenin is often observed in human cancers, contributing to tumorigenesis. The mechanism responsible for β-catenin-associated tumorigenesis involves the stabilization of β-catenin and its interaction with T-cell factor/lymphoid enhancer factor (TCF/LEF) that bind to the promoters of downstream target genes, such as c-myc and cyclin D1, involved in cell proliferation, survival, and migration. The cellular levels of β-catenin protein are tightly regulated by two distinct adenomatous polyposis coli (APC)-dependent proteasomal degradation pathways, namely, a glycogen synthase kinase 3β (GSK3β)-regulated pathway involving the APC-axin complex (Logan and Nusse 2004, Moon et al 2002, Peifer 1997, Peifer and Polakis 2000, Polakis 2000, Polakis 2007) and a p53-inducible pathway involving Siah-1 (Liu et al 2001, Matsuzawa and Reed 2001). Since the canonical GSK3β/APC and p53/Siah-1/APC pathways for down-regulation of β-catenin are often mutated in human cancers, identifying new mechanisms that induce β-catenin turnover will have great therapeutic significance for β-catenin-related cancers.

Nur77 (also called TR3 or NGFI-B), an orphan member of the nuclear receptor superfamily and an early immediate-response gene, plays a critical role in regulating the growth and survival, differentiation, and apoptosis of cancer cells in response to a pleiotropy of stimuli, including growth factors, inflammatory stimuli, cytokines, peptide hormones, and cellular stress (Chao et al 2008, Maxwell and Muscat 2006, Safe et al 2008, Zhang 2007, Zhao and Bruemmer 2010). Numerous studies have shown that Nur77 is overexpressed in precancerous or cancer cells to maintain their growth and survival (Ke et al 2004, Kolluri et al 2003, Uemura and Chang 1998, Wu et al 2010, Wu et al 1997a, Wu et al 1997b). In contrast, many others have found that underexpression of Nur77 in cancer cells is involved in tumor development and metastasis as well as drug resistance (Ke et al 2004, Lee et al 2010, Li et al 2000, Ramaswamy et al 2003, Shipp et al 2002). Overexpression of Nur77 in transgenic mice results in massive apoptosis in thymocytes (Liu et al 1994, Woronicz et al 1994), and transgenic mice lacking both Nur77 and the related Nor-1 genes developed lethal acute myeloid leukemia (Mullican et al 2007). Thus, Nur77 can exert both tumor promoting and tumor suppressive effects, depending on cell types and cellular environment (Zhang 2007). Recent studies have provided important insight into the mechanisms responsible for the diverse and sometimes opposing biological activities of Nur77 in cancer cells. Like other nuclear receptors, Nur77 can act in the nucleus as a transcription factor by binding to its DNA response element as monomers (Wilson et al 1991), homodimers (Philips et al 1997), or heterodimer (Forman et al 1995, Wu et al 1997a). Such a genomic action of Nur77 can mediate the mitogenic effect of growth factors and appears to play a role in the promotion of cancer cell growth and survival (Kolluri et al 2003, Lee et al 2010, Wu et al 1997a, Wu et al 1997b). In addition to its transcriptional regulation in the nucleus, Nur77 has extranuclear effects in response to a variety of apoptotic agents, including tetradecanoylphorbol-13-acetate (Li et al 2000), the retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437/AHPN) (Dawson et al 2001, Kolluri et al 2003), 1,1-Bis(3'-indoly)-1-(p-substituted phenyl)methanes (C-DIM) (Cho et al 2007), and cytosporone B (Zhan et al 2008). In response to these apoptosis-inducing agents, Nur77 expression is usually induced and the induced Nur77 protein subsequently translocates from the nucleus to the cytoplasm where it targets mitochondria through interaction with Bcl-2, leading to cytochrome c release and apoptosis (Cao et al 2004a, Kolluri et al 2003, Kolluri et al 2008, Li et al 2000, Lin 2004). Thus, subcellular localization of Nur77 also plays a critical role in the survival and death of cancer cells, which has been extensively targeted for developing new cancer therapies. Several small molecule and Nur77-derived short peptide modulators of Nur77 have been identified, which induce apoptosis of cancer cells by either directly or indirectly acting on the nongenomic pathways of Nur77 (Kolluri et al 2008, Safe et al 2008, Zhan et al 2008, Zhang 2007). However, Bcl-2 interaction and mitochondrial targeting is unlikely the sole nongenomic action of Nur77. Nur77 was found to target endoplasmic reticulum during stress-induced apoptosis of cancers (Liang et al 2007). In colon cancer cells, induction of apoptosis was associated with Nur77 nuclear export but not its mitochondrial targeting (Wilson et al 2003). Thus, the cytoplasmic effects of Nur77 remain to be explored.

In this study, we showed that Nur77, through its cytoplasmic action, potently induced β-catenin degradation through a mechanism that is independent of GSK3β and Siah-1. Our data demonstrated that DNA-binding and transcriptional function of Nur77 were dispensable while Nur77 cytoplasmic localization was essential for its induction of β-catenin degradation. Mutational analysis revealed that Nur77 induction of β-catenin degradation required both the N-terminal A/B region of Nur77, which was involved in Nur77 ubiquitination, and the C-terminal region, which was responsible for β-catenin binding. In addition, we identified two natural products belonging to the family of digitalis-like compounds (DLC), which potently induced β-catenin turnover through their induction of Nur77 expression and its nuclear export. Together, our results reveal a novel mechanism by which Nur77 acts nongenomically to suppress the β-catenin signaling pathway and identify two Nur77 inducers as potent inhibitors of the growth of cancer cells with abnormally activated β-catenin due to APC and/or p53 mutations.

Results

Nur77 reduces β-catenin protein levels and inhibits its transcriptional activity

We recently reported that β-catenin was directly involved in the regulation of Nur77 transcription by binding to the Nur77 promoter (Wu et al 2010). Since there is extensive crosstalk between Nur77 and Wnt signaling pathways (Camacho et al 2009, Chtarbova et al 2002, Kitagawa et al 2007, Wu et al), we studied whether there was a regulatory loop between Nur77 and β-catenin by determining the effect of Nur77 on β-catenin turnover. We transfected HEK-293T cells with Myc-tagged Nur77 (Myc-Nur77) and HA-tagged β-catenin (HA-β-catenin) expression vectors to examine whether Nur77 cotransfection affected the expression of HA-β-catenin protein. Immunoblotting analysis showed that transfection of Myc-Nur77 led to decrease in the level of HA-β-catenin protein in a Nur77 concentration dependent manner (Figure 1a). In contrast to its effect on β-catenin, transfection of Myc-Nur77 had no effect on protein levels of GSK3β and p53. The effect of Myc-Nur77 was not due to the Myc epitope as cotransfection of the wild-type Nur77 expression vector also led to β-catenin degradation (Supplemental Figure 1). To determine whether Nur77 could also affect the signaling activity of β-catenin, Myc-Nur77 and β-catenin were cotransfected into HeLa cells together with the TCF/LEF-1 reporter plasmid TOPFLASH. Transfection of β-catenin strongly enhanced the transcriptional activity of the TOPFLASH reporter. When Myc-Nur77 was cotransfected, β-catenin-induced TOPFLASH reporter activity was suppressed in a Myc-Nur77 concentration dependent manner (Figure 1b). Thus, Nur77 could inhibit β-catenin expression and its transcriptional activity.

Figure 1

Regulation of β-catenin protein levels and transcriptional activity by Nur77

(a) Reduction of protein levels of β-catenin but not GSK3β and p53 by Nur77. (b). Inhibition of transcriptional activity of β-catenin by Nur77. (c) Reduction of β-catenin/S33Y protein levels by Nur77. (d) Inhibition of transcriptional activity of β-catenin/S33Y by Nur77. (e) Reduction of β-catenin/ΔN protein levels by Nur77. (f) Inhibition of transcriptional activity of β-catenin/ΔN by Nur77. To determine the effect of Nur77 on the protein levels of β-catenin and mutants, HEK293T cells were transfected with the indicated β-catenin expression vector (2 μg) and the indicated concentration of Myc-Nur77 expression vector. Lysates were prepared and analyzed by immunoblotting. Anti-Myc antibody was used to detect Myc-Nur77 expression. One of three similar experiments is shown. To determine the effect of Nur77 on the transcriptional activity of β-catenin and mutants, HEK293T cells were transiently transfected with 100 ng of TOPFLASH reporter gene with or without β-catenin (100 ng) and the indicated Nur77 expression vectors. All reporter activity is expressed as mean ± S.E. of 6 samples from two independent experiments. The total amount of DNA in all transfections was kept constant using appropriate parental empty expression vectors.

APC independent effect of Nur77

Phosphorylation of β-catenin at the Ser33 site by GSK3β is essential for its degradation by proteasome system in an APC dependent manner and mutant β-catenin/S33Y is refractory to proteasomal degradation (Hart et al 1999, Liu et al 1999). We therefore examined whether Nur77 could inhibit β-catenin expression through GSK3β/APC dependent pathway by determining its effect on the level of β-catenin/S33Y. To this end, HA-β-catenin/S33Y and Myc-Nur77 were cotransfected, and immunoblotting analysis revealed that levels of HA-β-catenin/S33Y were reduced by Myc-Nur77 cotransfection (Figure 1c), similar to its effect on β-catenin. Myc-Nur77 cotransfection also inhibited β-catenin/S33Y-mediated TOPFLASH reporter transcription (Figure 1d). These data suggested that Nur77-induced β-catenin degradation was GSK3β/APC independent. The N-terminal 50-amino acid-spanning domain of β-catenin is targeted not only by the GSK-3β-regulated β-catenin degradation pathway but also by the Siah-1-dependent proteasomal pathway (Liu et al 2001, Matsuzawa and Reed 2001). We next determined the effect of Nur77 on levels of GFP-fused β-catenin/ΔN, a β-catenin mutant lacking its N-terminal 50 amino acid residues. Similar to its effect on β-catenin/S33Y, Myc-Nur77 cotransfection resulted in decrease of levels of GFP-β-catenin/ΔN (Figure 1e) and GFP-β-catenin/ΔN-activated the transcription of the TOPFLASH reporter (Figure 1f). The expression of both endogenous full-length β-catenin and transfected β-catenin/ΔN showed similar sensitivity to inhibition by Nur77 transfection (Supplemental Figure 2). Thus, the N terminus of β-catenin was dispensable for the inhibitory effect of Nur77 on β-catenin expression, suggesting that the effect of Nur77 on β-catenin expression occurs via a distinct pathway that is also p53/Siah-1/APC independent.

Effect of digitalis-like compounds on Nur77 expression and β-catenin degradation

We recently reported that several cardenolides isolated from Antiaris toxicaria LESCH (Moraceae) could potently induce Nur77 expression in cancer cells at very low concentrations (low nM) (Jiang et al 2008). To determine whether induction of endogenous Nur77 expression regulated the expression of β-catenin, we treated HCT116 cells with ATE-i2-b4 (Figure 2a), one of the cardenolides, to examine its effect on the expression of Nur77 and β-catenin. Lithium chloride (LiCl), which inhibits GSK3β and consequently stabilizes free cytosolic β-catenin (Klein and Melton 1996, Yochum et al 2007)(Supplemental Figure 3), was added to the cells for 6 hr prior to ATE-i2-b4 treatment. As shown in Figure 2b, ATE-i2-b4 potently induced levels of Nur77 protein, which occurred when cells were treated with as low as 10 nM of the compound. Induction of Nur77 by ATE-i2-b4 was accompanied with decrease in the level of β-catenin protein. Time-course study demonstrated that treatment of HCT116 cells with 50 nM ATE-i2-b4 for as short as 90 min induced Nur77 expression, and a significant amount of Nur77 protein was induced when cells were treated for 8 hr (Figure 2c). Such induction of Nur77 was again closely correlated with reduction of β-catenin.

Figure 2

Induction of Nur77 expression by ATE-i2-b4 and H-9 is accompanied with reduction of β-catenin and cyclin D1 and phosphorylation of c-Jun

(a) Chemical structures of ATE-i2-b4 and H-9 (hellebritoxin). (b, d) Dose dependent effect of ATE-i2-b4 (b) and H-9 (d). HCT116 cells were pretreated with LiCl (10 mM) for 6 hr and then treated with the indicated concentration of ATE-i2-b4 (ATE) or H-9 for another 6 hr. Lysates were prepared and analyzed by immunoblotting with appropriate antibodies. (c, e) Time-course analysis of the effect of ATE-i2-b4 (c) and H-9 (e). HCT116 cells were pretreated with LiCl (10 mM) for 6 hr and then treated with 50 nM ATE-i2-b4 or 75 nM H-9 for the indicated time. Lysates were prepared and analyzed by immunoblotting with appropriate antibodies. One of three to five similar experiments is shown.

ATE-i2-b4 is structurally related to a class of DLC that also include bufotoxins present in the body of toads (Lopez-Lazaro 2007, Nesher et al 2007, Steyn and van Heerden 1998). We thus examined several bufotoxins purified from the skin of toad and found that H-9 (Figure 2a), known as hellebritoxin (Hutchinson et al 2007), could potently induce Nur77 expression in various cancer cell lines (Figure 2d and data not shown), similar to the effect of ATE-i2-b4. Induction of Nur77 by H-9 was also accompanied with reduction of β-catenin in dose (Figure 2d) and time (Figure 2e) dependent manners. Because H-9 could be isolated and purified in large quantity, it was then used for most of our studies.

We next examined the effect of H-9 on β-catenin degradation by immunofluorescence analysis in SW620 colon cancer cells that express mutant APC protein (Ilyas et al 1997). Immunostaining of SW620 cells with anti-β-catenin antibody showed that β-catenin was distributed in the membrane as well as in the cytoplasm and nucleus of the cells. Incubation of these cells with H-9 resulted in a dramatic reduction in the levels of cytoplasmic and nuclear β-catenin, while it had little effect on the levels of the membrane-bound β-catenin (Figure 3a). These results further demonstrated the APC-independent effect of H-9 on β-catenin stability. To determine whether Nur77 expression mediated H-9-induced reduction of β-catenin level, we analyzed the effect of Nur77 siRNA transfection in SW620 cells. Transfection of Nur77 siRNA efficiently reduced the level of Nur77 induced by H-9 (Figure 3b). Concomitantly, the reduction of β-catenin level by H-9 was inhibited. Similar results were obtained in HCT116 cells (data not shown). Thus, induction of Nur77 expression by H-9 is essential for its inhibitory effect on β-catenin expression.

Figure 3

Effects of H-9 on Nur77-mediated β-catenin degradation and tumor growth in vitro and in animals

(a) Membrane-bound β-catenin is resistant to H-9. SW620 cells were treated with or without 300 nM H-9 6 hr and cells were subjected to immunofluorescence analysis with anti-β-catenin antibody. Nuclei were visualized by DAPI staining. One of three similar experiments is shown. (b) Nur77 expression is required for β-catenin degradation by H-9. HCT116 cells transfected with Nur77 siRNA or scrambled control siRNA were treated with LiCl (10 mM) for 6 hr, and then with 200 nM H-9 for another 6 hr. Lysates were prepared and analyzed by immunoblotting. One of four similar experiments is shown. (c) Inhibition of cell cycle progression by H-9. HCT116 cells transfected with scrambled control siRNA or Nur77 siRNA were treated with LiCl (10 mM) for 6 hr, and then with 200 nM H-9 for another 6 hr. Cell cycle progression was examined by flow cytometry. The percentages of cells in G1/G0, G2, and S are indicated. (d) Nude mice (n=6) with SW620 xenografts were administered intragastrically daily with H-9 (0.5 mg/kg) for 20 days. Tumors were removed and measured. (e) Time-dependent inhibition of SW620 xenografts (n=6) by H-9 (0.5 mg/kg). (f) Effect of H-9 on gene expression in tumor samples. Lysates prepared from three tumors treated with or without H-9 (0.5 mg/kg) for 3 weeks were analyzed for gene expression by immunoblotting with appropriate antibodies. Error bars represent SEM.

Effect of DLCs on cyclin D1 expression and cell cycle progression

The proliferative effect of β-catenin has been linked to its upregulation of target genes such as cyclin D1, which is often overexpressed in cancer cells (Fu et al 2004). To determine whether Nur77 induction by ATE-i2-b4 and H-9 modulated the expression of β-catenin target genes, we examined their effect on cyclin D1 expression in HCT116 cells. Induction of Nur77 expression and reduction of β-catenin levels by ATE-i2-b4 (Figures 2b and c) and H-9 (Figures 2d and e) was associated with their inhibition of cyclin D1 expression in both time and dose dependent manners. DNA content analysis using flow cytometry showed that treatment of HCT116 cells with H-9 induced G1 arrest, which was prevented when cells were transfected with Nur77 siRNA (Figure 3c). Thus, reduction of levels of β-catenin by H-9-induced Nur77 expression could lead to inhibition of cyclin D1 expression and cell cycle progression.

H-9 inhibits β-catenin expression and tumor growth in animals

To study the effect of DLCs on β-catenin expression in vivo, mice with SW620 tumor xenografts were treated with H-9 (0.5 mg/kg). Administration of H-9 inhibited the growth of SW620 tumor in a time dependent manner, resulting in a 43.3% reduction of tumor volume after a three-week treatment (Figures 3d–e). Consistent with our in vitro observation, the inhibition of tumor growth by H-9 was associated with induction of Nur77 and reduction of β-catenin and cyclin D1 (Figure 3f). Thus, H-9 inhibits tumor growth is likely mediated though its induction of Nur77, which in turn down-regulates the expression of β-catenin and cyclin D1.

H-9-induced β-catenin degradation is proteasomal dependent

To gain insight into the mechanism by which Nur77 decreased the protein levels of β-catenin, real-time RT-PCR was performed to determine if H-9 decreased β-catenin gene transcription. Figure 4a showed no significant change in β-catenin mRNA levels following exposure of SW620 cells to different concentrations of H-9, which under the same conditions down-regulated β-catenin protein expression (Figure 2d). Thus, down-regulation of β-catenin gene transcription by H-9 was not involved in its inhibition of β-catenin protein expression. We then studied whether the inhibitory effect of H-9 was mediated through proteasome-dependent pathway. To this end, we first determined whether H-9 inhibition of β-catenin expression could be prevented by the proteasomal inhibitor MG132. SW620 (Figure 4b) and HCT116 (Figure 4c) cells were treated with 100 nM H-9 in the absence or presence of 20 μM MG132. Immunoblotting showed that the inhibitory effect of H-9 on the expression of β-catenin protein was completely abolished when cells were cotreated with MG132. We next determined whether β-catenin was ubiquitinated in cells treated with H-9. Cell lysates were prepared from HCT116 cells treated with H-9 in the presence of MG132 and immunoprecipitated by anti-β-catenin antibody. Analysis of β-catenin immunoprecipitates by immunoblotting using anti-ubiquitin antibody showed that β-catenin was ubiquitinated in a H-9 concentration dependent manner (Figure 4d). Thus, Nur77-mediated β-catenin degradation is mediated by the proteasomal pathway.

Figure 4

H-9-induced β-catenin degradation is mediated by proteasomal pathway

(a) H-9 does not affect β-catenin gene transcription. RNAs prepared from SW620 cells treated with the indicated concentration of H-9 for 8 hr were analyzed by RT-PCR. (b, c) Inhibition of H-9-induced β-catenin degradation in SW620 (b) and HCT116 cells (c) by proteasome inhibitor MG132. Cells were pretreated with 20 μM MG132 for 2 hr, and then 100 nM H-9 for 6 hr. Lysates were prepared and analyzed by immunoblotting. (d) HCT116 cells were treated with 20 μM MG132 and the indicated concentration of H-9 for 6 hr. Lysates were prepared and subjected to immunoprecipitation by anti-β-catenin antibody. Immunoprecipitates were analyzed by immunoblotting using anti-ubiquitin antibody. One of two to five similar experiments is shown.

The A/B region of Nur77 is ubiquitinated and required for β-catenin degradation

To determine the functional domains of Nur77 required for its induction of β-catenin degradation, several Nur77 mutants (Figure 5a) were analyzed for their effect on the stability of β-catenin. While transfection of Nur77 resulted in reduction of β-catenin/S33Y, transfection of several Nur77 mutants lacking the A/B domain, including Nur77/ΔAB, Nur77/ΔAB/ΔAF2 and the Nur77 ligand-binding domain (Nur77/LBD), failed to show any effect (Figure 5b), suggesting that the N-terminal A/B domain is essential for Nur77 to induce β-catenin degradation. However, the levels of β-catenin were not changed when cells were transfected with the A/B region of Nur77 (Nur77/AB) or Nur77/LBD (Figure 5c). Thus, the A/B region of Nur77 was essential but not sufficient for inducing β-catenin degradation. Interestingly, a mutant lacking the entire DNA-binding domain of Nur77, Nur77/ΔDBD, effectively induced β-catenin degradation (Figure 5d). Consistent with their effect on β-catenin stability, Nur77/ΔDBD but not the other mutants inhibited β-catenin-mediated TOPFLASH reporter activity (Figure 5e). These data demonstrated that Nur77-induced β-catenin degradation requires its N-terminal region but not its DNA binding and transcriptional function.

Figure 5

Nur77-inducced β-catenin degradation involves the N-terminal region of Nur77

(a) Schematic representation of Nur77 mutants. (b, c) The N-terminal A/B region of Nur77 is essential for its induction of β-catenin degradation. HA-β-catenin/S33Y (b) or GFP-β-catenin/ΔN (c) was cotransfected with the indicated Nur77 and mutants into HEK293T cells. 24 hr later lysates were prepared and analyzed by immunoblotting. Anti-HA or anti-GFP antibody was used to detect HA-β-catenin/S33Y or GFP-β-catenin/ΔN, respectively. Anti-GFP or anti-Myc antibody was used to detect Nur77 and mutants. (d) DNA-binding domain in Nur77 is dispensable for its effect on β-catenin stability. HA-β-catenin/S33Y was cotransfected with the indicated Nur77 and mutants into HEK293T cells. 2 4 hr later lysates were prepared and analyzed by immunoblotting. Anti-HA antibody was used to detect HA-β-catenin/S33Y, while anti-GFP antibody was used to detect Nur77 and mutants. (e) Effect of Nur77 and its mutants on the transcriptional activity of β-catenin. HEK293T cells were transiently transfected with 100 ng of TOPFLASH reporter gene with or without Nur77 or mutant expression vector as indicated. All reporter activity is expressed as mean ± S.E. of 6 samples from 2 independent experiments. (f) The A/B region of Nur77 is involved in Nur77 ubiquitination. HEK293T cells transfected with Myc-Nur77 or Myc-Nur77/ΔAB for 24 hr were treated with 10 μM MG132 for 2 hr. Lysates were then immunoprecipitated using anti-Myc antibody, and probed with anti-ubiquitin antibody. (g) HEK293T cells were transfected with Myc-Nur77 or mutants together with or without HA-ubiquitin plasmid for 40 hr, and then treated with 10 μM MG132 for 2 hr. Lysates prepared were then immunoprecipitated with anti-Myc antibody, and immunoprecipitates were immunoblotted with anti-HA antibody. One of three to five similar experiments is shown.

Nuclear receptors can be degraded through the ubiquitin-proteasome pathway (Ward and Weigel 2009, Wu and Mo 2007). To study the possible mechanism by which the A/B region of Nur77 mediated β-catenin degradation, we determined whether the region was involved in Nur77 ubiquitination. Our results showed an increase of ubiquitination of Myc-Nur77 transfected into HEK293T cells by MG132 (Figure 5f). However, the effect of MG132 was largely reduced when the ubiquitination of Nur77/ΔAB was analyzed, demonstrating that the A/B region of Nur77 plays a critical role in Nur77 ubiquitination. This was consistent with our analysis of several Nur77 point mutants, in which several serine residues in the A/B region were replaced with alanine. Mutations of Ser39 and Ser140 inhibited Nur77 ubiquitination, whereas mutation of Ser95 and Ser152 showed a clear increase (Figure 5g).

Interaction of Nur77 with β-catenin was required for β-catenin degradation

The finding that the N-terminal region of Nur77 was essential but not sufficient for β-catenin degradation prompted us to determine whether Nur77-induced β-catenin degradation was mediated through their physical interaction. HEK293T cells were transfected with Myc-Nur77 and HA-β-catenin expression vectors and their interaction was examined by coimmunoprecipitation (Co-IP) assays. As shown in Figure 6a, HA-β-catenin was coimmunoprecipitated by anti-Myc antibody but not by control IgG. Immunoprecipitation of HA-β-catenin by anti-HA antibody also resulted in co-immunoprecipitation of Myc-Nur77 (Figure 6b). We also examined whether endogenous Nur77 and β-catenin interacted. Treatment of HCT116 cells with H-9 resulted in not only induction of Nur77 but also its phosphorylation (Figure 6c). Immunoprecipitation of β-catenin by anti-β-catenin antibody resulted in coimmunoprecipitation of Nur77 only in cells treated with H-9, demonstrating that H-9 induced interaction of endogenous Nur77 and β-catenin. These data clearly demonstrated that Nur77 interacted with β-catenin. To further characterize the interaction, HA-β-catenin/S33Y interaction with GFP-Nur77, GFP-Nur77/ΔDBD, or GFP-Nur77/ΔAB/ΔAF2 was examined by Co-IP using anti-GFP antibody. Immunoprecipitation of GFP-Nur77 or GFP-Nur77/ΔDBD but not GFP-Nur77/ΔAB/ΔAF2 resulted in co-immunoprecipitation of HA-β-catenin/S33Y (Figure 6d). The fact that GFP-Nur77 and GFP-Nur77/ΔDBD but not GFP-Nur77/ΔAB/ΔAF2 could induce the degradation of β-catenin (Figure 5) suggested that interaction of Nur77 with β-catenin was critical for its effect on β-catenin stability. Interestingly, Nur77/ΔAB could interact strongly with β-catenin (Figure 6e), despite its inability to induce β-catenin degradation (Figures 5b–c). These results demonstrated that the C-terminal region of Nur77 was responsible for β-catenin binding. In addition, they showed that induction of β-catenin degradation by Nur77 required not only the N-terminal A/B region but also the C-terminal region of Nur77.

Figure 6

Interaction of μ-catenin with Nur77

(a, b) Myc-Nur77 and HA-β-catenin expression vectors were transfected into HEK293T cells. Lysates prepared were subjected to immunoprecipitation by anti-Myc antibody (a) or anti-HA antibody (b) and the presence of Myc-Nur77 and HA-β-catenin were examined by immunoblotting using anti-Myc and anti-HA antibody, respectively. (c) H-9 induces interaction of endogenous Nur77 and β-catenin. HCT116 cells pretreated with LiCl for 6 hr were treated with H-9. Nur77-β-catenin interaction was analyzed by co-immunoprecipitation assays using anti-β-catenin antibody. Nur77 was detected by anti-Nur77 antibody. (d) Interaction of β-catenin/S33Y with Nur77 mutants. HEK293T cells were transfected with HA-β-catenin/S33Y and the indicated GFP-fused Nur77 or mutant expression vector. Lysates were immunoprecipitated with anti-GFP antibody and the presence of HA-β-catenin/S33Y in the GFP immunoprecipitates was examined by immunoblotting using anti-HA antibody. (e) Deletion of the A/B region of Nur77 does not affect Nur77 interaction with β-catenin. HEK293T cells were transfected with Myc-Nur77/ΔAB and HA-β-catenin. Lysates were immunoprecipitated with anti-HA antibody and the presence of Myc-Nur77/ΔAB in the HA immunoprecipitates was examined by immunoblotting using anti-Myc antibody.

Nuclear export of Nur77 is required for its induction of β-catenin degradation

We previously reported that Nur77/ΔDBD was predominantly resided in the cytoplasm (Li et al 2000, Lin 2004). Our observation that Nur77/ΔDBD could interact with β-catenin and induce its degradation suggested that Nur77-mediated β-catenin degradation might occur in the cytoplasm. Indeed, GFP-Nur77/ΔDBD expressed in HEK293T cells was found mainly in the cytoplasm, colocalizing with β-catenin (Figure 7a). We also studied whether inhibition of Nur77 nuclear export by leptomycin B (LMB), a specific inhibitor of CRM1-dependent protein nuclear export (Kudo et al 1999), could affect its degradation of β-catenin (Figure 7b). Nuclear (N) and cytoplasmic (C) fractions were prepared from HEK293T cells transfected with Myc-Nur77 to determine its subcellular localization (Figure 7c). The purity of the nuclear and cytoplasmic fractions was confirmed by the presence of nuclear protein PARP and cytoplasmic protein α-tubulin, respectively. Examination of the expression of Myc-Nur77 revealed its presence in both the cytoplasm and nuclear fractions (Figure 7c). When cells transfected with Myc-Nur77 were treated with LMB, Myc-Nur77-induced degradation of HA-β-catenin/S33Y was inhibited (Figure 7b). Treatment of HCT116 cells with LMB also inhibited the ability of H-9 to induce β-catenin degradation (Figure 7d). Thus, Nur77 might interact with β-catenin in the cytoplasm, leading to its degradation.

Figure 7

Cytoplasmic localization of Nur77 is required for its induction of β-catenin degradation

(a) Colocalization of Nur77/ΔDBD with β-catenin in the cytoplasm. HEK-293T cells were transfected with GFP-Nur77/ΔDBD expression vector in the presence of MG132 to prevent β-catenin degradation. Cells were immunostained with anti-β-catenin antibody and the subcellular localization of β-catenin and GFP-Nur77/ΔDBD was visualized by confocal microscopy. Nuclei were visualized by DAPI staining. (b) Inhibition of Nur77-induced β-catenin/S33Y degradation by nuclear export inhibitor LMB. HEK293T cells were transfected with Myc-Nur77 and HA-β-ctenin/S33Y in the absence or presence of LMB (2.5 ng/ml). 24 hr later lysates were prepared and analyzed by immunoblotting using anti-HA and anti-Myc antibody. (c) Subcellular localization of transfected Myc-Nur77. HEK293T cells were transfected with Myc-Nur77, and nuclear (N) and cytoplasmic (C) cellular fractions were prepared and analyzed for the presence of Myc-Nur77 by immunoblotting using anti-Myc antibody. The purity of fractions was examined for the presence of PARP, a nuclear protein, and α-tubulin, a cytoplasmic protein. (d) Inhibition of H-9-induced β-catenin degradation by LMB. HCT116 grown in the presence of LiCl (10 mM) for 6 hr were treated with 200 nM H-9 together with or without 2.5 ng/ml LMB for another 6 hr. Cell lysates were prepared and analyzed by immunoblotting using anti-β-catenin or anti-Nur77 antibody. One of three similar experiments is shown.

JNK inhibitor prevents Nur77 nuclear export and induction of β-catenin degradation

Migration of Nur77 from the nucleus to the cytoplasm requires its phosphorylation by JNK (Han et al 2006). Interestingly, both ATE-i2-b4 and H-9 strongly activated JNK (Figure 2). Thus, we determined whether JNK activation contributed to their effect on β-catenin stability through induction of Nur77 nuclear export. Treatment of HCT116 cells with SP600125, a JNK inhibitor, reduced the effect of H-9 on inducing β-catenin degradation. Phosphorylation of c-Jun was completely blocked by SP600125, demonstrating that the inhibitor was active. Thus, JNK activation by H-9 was involved in its degradation of β-catenin. JNK activation by anisomycin however had no effect on β-catenin stability (Figure 8b), indicating that JNK activation alone was not sufficient to induce β-catenin degradation. Interestingly, a strong synergistic effect on inducing β-catenin turnover was observed when anisomycin was used together with H-9 (Figure 8b). Thus, both JNK activation and Nur77 induction were required for induction of β-catenin degradation by H-9.

Figure 8

Role of JNK inhibitor on cytoplasmic localization of Nur77 and its induction of β-catenin degradation

(a) Inhibition of H-9-induced β-catenin degradation by JNK inhibitor SP600125. SW620 cells grown in serum-free medium for 2 hr were treated with 200 nM H-9 with or without 10 μM SP600125 for 6 hr. Cell lysates were analyzed by immunoblotting using anti-β-catenin, anti-nur77, and anti-p-c-Jun antibody. (b) JNK activation alone is insufficient to induce Nur77 expression and β-catenin degradation. SW620 cells grown in serum-free medium were treated with 25 nM H-9 and/or 10 ng/ml anisomycin for 6 hr. Cell lysates were analyzed by immunoblotting. (c, d) Inhibition of H-9-induced cytoplasmic localization of GFP-Nur77 by JNK inhibitor SP600125. HCT116 cells transfected with GFP-Nur77 were treated with 200 nM H-9 in the presence of 10 μM SP600125 for 6 hr and the subcellular localization of β-catenin and GFP-Nur77 was examined by immunofluorescence microscopy (c) or by subcellular fractionation (d). For immunofluorescence study, cells were immunostained with anti-β-catenin. For cellular fractionation, the purity of the cytoplasmic (C) and nuclear (N) fractions was studied for the presence of PARP and α-tubulin. One of three similar experiments is shown.

To determine the effect of H-9 on the subcellular localization of Nur77, HCT116 cells transfected with GFP-Nur77 were treated with H-9 together with or without SP600125. Immunofluorescence assays showed that GFP-Nur77 was mainly found in the nucleus of control cells, while it was found predominantly in the cytoplasm of cells when cells were treated with H-9 (Figure 8c). Treatment of cells with SP600125 abolished the effect of H-9 on inducing Nur77 nuclear export. Similar results were found when treated with other cardenolides (not shown). To further confirm the effect of H-9, cellular fractionation was carried out. In the absence of H-9 treatment, Nur77 was mainly accumulated in the nuclear fraction. However, upon H-9 treatment Nur77 was phosphorylated and the heavily phosphorylated Nur77 was found in the cytoplasmic fraction. Treatment of cells with H-9 in the presence of SP600125 inhibited not only the phosphorylation of Nur77 but also its accumulation in the cytoplasmic fraction (Figure 8d). Together, these results demonstrated that H-9 induced Nur77 nuclear export through its activation of JNK that phosphorylated Nur77.

Discussion

Previous studies have shown that Nur77 functionally interacts with Wnt/β-catenin signaling at multiple levels (Camacho et al 2009, Chtarbova et al 2002, Kitagawa et al 2007, Wu et al). Nur77 expression was induced by LiCl that inhibits GSK3β activity in follicular thyroid carcinoma cells (Camacho et al 2009) and colon cancer cells (Wu et al 2010), by activation of the Wnt-1 signaling in mammary epithelial cells (Chtarbova et al 2002), or by expression of stabilized β-catenin mutant β-catenin/S33Y (Wu et al 2010). The present study demonstrated that Nur77 through its cytoplasmic localization and interaction with β-catenin induced β-catenin degradation, resulting in inhibition of its transcriptional activity and target gene expression. We also identified two DLCs, which could potently induce Nur77 expression and β-catenin degradation in vitro and in animals. Thus, inhibition of the β-catenin signaling pathway likely represents one of the mechanisms by which Nur77 exerts its tumor suppressive effect.

Abnormal activation of β-catenin due to dysregulation of its turnover is implicated in the pathologenesis of cancer (Logan and Nusse 2004, Moon et al 2002, Peifer 1997, Peifer and Polakis 2000, Polakis 2000, Polakis 2007). Major pathways that direct β-catenin to the proteasome for degradation include p53/Siah-1/APC and GSK3β/APC pathways, which are abnormally inactivated due to mutations in APC and/or p53 in cancer cells. Several pieces of evidence presented here showed that Nur77-induced β-catenin degradation is independent of the activation of either p53/Siah-1/APC or GSK3β/APC pathway. First, we found that Nur77 was capable of inducing degradation of β-catenin/S33Y (Figure 1c), which is insensitive to GSK3β phosphorylation. In addition, we showed that the N-terminal domain, which is targeted by p53/Siah-1/APC-dependent proteasomal pathway, was also dispensable for the destabilizing effect of Nur77 (Figure 1e). Furthermore, we found that β-catenin was effectively degraded in SW620 cells that express mutant APC protein (Ilyas et al 1997) (Figures 3a and b). Because APC and p53 are often mutated in cancer cells, our results suggest that targeting Nur77 for inducing β-catenin degradation may hold promise for treating cancers with abnormally activated β-catenin due to APC and/or p53 mutations.

Nur77-dependent pathway for β-catenin degradation involved ubiquitination of β-catenin and was mediated through proteasome-dependent pathways. Preincubation of cells with the proteasomal inhibitor MG132 abolished the effect of Nur77 (Figures 4b–c). In addition, treatment of cells with H-9 that induced β-catenin degradation caused ubiquitination of β-catenin (Figure 4d). Nur77 could be ubiquitinated (Figure 5f) and degraded through the proteasome-dependent pathway (data not shown). Interestingly, deletion of the A/B region of Nur77, which was essential for inducing β-catenin degradation (Figure 5), inhibited Nur77 ubiquitination (Figure 5f). Thus, β-catenin and Nur77 were likely co-degraded via the same mechanism. In support of the notion, we found that Nur77 could interact with β-catenin (Figure 6). Moreover, both Nur77 and Nur77/ΔDBD, which induced β-catenin degradation, interacted with β-catenin, while mutant that failed to induce β-catenin degradation did not (Figures 6d–e), suggesting that the interaction was critical for β-catenin degradation. The Nur77-dependent pathway required both the A/B region of Nur77, which was critical for Nur77 ubiquitination, and the C-terminal region of Nur77, which mediated Nur77 interaction with β-catenin, were essential. It can be envisioned that β-catenin becomes a target of the ubiquitin-proteasome machinery upon binding to Nur77 and that Nur77 and β-catenin are present in the same degradation complex.

An important finding reported here is the nongenomic action of Nur77 in inducing β-catenin degradation. Previous studies demonstrated that Nur77 could act in the cytoplasm to induce differentiation (Katagiri et al 2000) and apoptosis (Li et al 2000). In response to a number of apoptosis-inducing agents, Nur77 migrates from the nucleus to the cytoplasm where it triggers apoptosis by converting Bcl-2 from an anti-apoptotic to a pro-apoptotic molecule (Cao et al 2004b, Li et al 2000, Lin 2004). Our current studies showed that cytoplasmic Nur77 could also act to inhibit the β-catenin signaling by interacting with β-catenin and inducing its degradation. The mutant Nur77/ΔDBD, which resided predominantly in the cytoplasm (Li et al 2000), colocalized with β-catenin in the cytoplasm (Figure 7a), induced β-catenin degradation (Figure 5d), and inhibited its transcriptional activity (Figure 5e). These results demonstrated that Nur77-mediated β-catenin degradation was independent of its DNA binding and transcriptional function, and moreover they showed that the cytoplasmic localization of Nur77 was essential for its effect on β-catenin instability. In support of this notion, we showed that LMB that inhibits CRM-1 dependent nuclear export prevented the effect of Nur77 and H-9 on inducing β-catenin degradation (Figure 7). Thus, Nur77 could serve as a targeting molecule that carries its interacting proteins to certain cytoplasmic compartments for modification and degradation. Previous studies showed that RXRα, the Nur77 heterodimerization partner, could also target β-catenin for degradation through GSK3β and p53/Siah-independent mechanisms (Dillard and Lane 2007, Dillard and Lane 2008, Xiao et al 2003). Whether RXRα is involved in Nur77-mediated pathway remains to be determined.

In searching for compounds that target Nur77 for inducing β-catenin degradation, we showed here our characterization of ATE-i2-b4 and H-9 as potent inducers of Nur77-mediated pathway for β-catenin degradation. Both compounds belong to the family of DLC (also called cardenolides), which have been widely used in the therapy of congestive heart failure and other cardiac disorders as well as cancer (Lopez-Lazaro 2007, Nesher et al 2007, Steyn and van Heerden 1998). These compounds are known to bind to the α-subunit of the sodium/potassium ATPase, a plasma membrane ion transporter responsible for translocating sodium and potassium ions across cell membranes. In addition to pumping ions, the sodium/potassium pump interacts with neighboring membrane proteins upon binding to DLCs, resulting in modulation of various signaling pathways. Consistently, the cancer therapeutic effect of certain DLCs has been attributed to their inhibition of cancer cell growth, and induction of cancer cells differentiation and/or apoptosis (Lopez-Lazaro 2007, Nesher et al 2007, Steyn and van Heerden 1998). We showed here that both ATE-i2-b4 and H-9 could act at nM concentrations to induce Nur77 expression (Figure 2), inhibit cell cycle progression (Figure 3c), and promote apoptosis (not shown), suggesting that induction of Nur77 may be an important mechanism by which this class of compounds exert their anti-cancer activity. Induction of Nur77 expression and β-catenin degradation by H-9 could be also observed in animals, which likely contributed to its potent effect on inhibiting tumor growth (Figure 3). It is noteworthy that H-9-induced β-catenin degradation depended on not only its ability to induce Nur77 expression but also its activation of JNK, which is known to promote Nur77 nuclear export (Han et al 2006). Treatment of cells with H-9 or ATE-i2-b4 resulted in Nur77 induction and JNK activation (Figure 2). Interestingly, the hyperphosphorylated Nur77 was predominantly in the cytoplasm (Figure 8d) and interacted strongly with β-catenin (Figure 6c). Furthermore, JNK inhibitor SP600125 inhibited the cytoplasmic localization of Nur77 (Figure 8) and its effect on inducing β-catenin degradation. Thus, digitalis-like compounds such as ATE-i2-b4 and H-9 are important activators of the Nur77-mediated pathway for inducing β-catenin degradation and they may serve as important leads for developing new therapeutics for treating cancers with abnormally activated β-catenin due to APC and/or p53 mutations.

Materials and methods

Reagents

Lipofectamin 2000 (Invitrogen); enhanced chemiluminescence reagents (Thermo Fisher Scientific, Inc); goat anti-rabbit anti-mouse secondary antibody conjugated to horseradish peroxidase (Thermo Fisher Scientific, Inc); anti-mouse IgG conjugated with Cy3 (Chemicon Intenational); polyclonal antibodies against Nur77(P15) (Cell Signaling Technology), and c-Jun-P (phospho S73)(E107) (Abcam); monoclonal antibodies against β-catenin(E-5), β-actin(C-2), myc(9E10) (Santa Cruz), α-tubulin (Millipore), PARP (BD), HA, and GFP (Roche); and cocktail of proteinase inhibitors (Roche) were used in this studies.

Cell culture

HCT-116 and SW620 human colon cancer, HeLa cervical cancer, and HEK293T human embroyonic kidney cells were cultured in DMEM containing 10% fetal bovine serum (FBS). Subconfluent cells with exponential growth were used throughout the experiments. Cell transfections were carried out by using Lipofectamine 2000.

Plasmid constructions

Nur77 expression vectors were described previously (Cao et al 2004b, Li et al 2000, Lin 2004). The expression vectors for HA-β-catenin, HA-β-catenin/S33Y, HA-ΔNβ-catenin/ΔN, a β-catenin mutant with an N-terminal deletion (amino acid residues 1–50), TOPFLASH, which contains TCF/LEF binding sites placed in front of the TK-Luc reporter gene, were kindly provided by Dr. Zhuohua Zhang (Sanford-Burnham Medical Research Institute).

Purification of ATE-i2-b4 and H-9

ATE-i2-b4, 3-beta-((6-Deoxy-beta-D-allopyranosyl)oxy)-5-beta,14-dihydroxy-19-oxocard-20(22)-enolide, is a cardenolide isolated from the chromatographic fractionation of the 60% ethanol extract of the stem of Anitaris toxicaria Lesch based on its induction of Nur77 expression (Jiang et al 2008). H-9 (Hellebritoxin), which was isolated from the skin of toad, was described previously (Zhao et al 2010). The purity of both compounds was about 95%.

Reporter Gene Assays

For measuring the TOPFLASH activity, cells were seeded at 30,000 cells per well in 24-well plates coated with poly-D-lysine (BD Biosciences). Twenty-four hours later, TOPflash and expression vectors were co-transfected into cells in DMEM containing 10% FBS. To monitor transfection efficiency, 10 ng of β-galactosidase (β-gal) were co-transfected and β-gal activity was measured as described (Kolluri et al 2008, Li et al 2000, Lin 2004, Wu et al 1997a, Wu et al 1997b, Zhou et al 2010).

Nur77 siRNA and transfections

Small interfering RNAs (siRNAs) against Nur77 used were from Dharmacon Research Inc (Kolluri et al 2008). The following four mixed siRNA sequences were used: Nur77 siRNA, 5'-UCG AGG ACU UCC AGG UGU A dTdT-3'; 5'-GGA CAG AGC AGC UGC CCA A dTdT-3'; 5'-GAA GGC CGC UGU GCU GUG U dTdT-3'; 5'-CGG CUA CAC AGG AGA GUU U dTdT-3'; A 5-μl aliquot of 20 μM siRNA/well was transfected into cells in six-well plates using Oligofectamine reagent (Invitrogen) as per the manufacturer's recommendations.

Western blotting

Western blotting was conducted as described (Kolluri et al 2008, Li et al 2000, Lin 2004, Wu et al 1997a, Wu et al 1997b, Zhou et al 2010). The dilutions of the primary antibodies were anti-Nur77 in 1:1,000, anti-β-catenin in 1:1,000, anti-p-c-Jun in 1:1,000, anti-PARP in 1:1,000, anti-α-tubulin in 1:10,000, anti-Myc in 1:1,000, anti-HA in 1:3,000, anti-β-actin in 1:10,000, and anti-GFP in 1:5,000.

Ubiquitination assays

For β-catenin ubiquitination assays, HCT116 cells cultured in serum-free medium for 2 hr were treated with 20 μM MG132 and H-9 for 6 hr. Lysates prepared were subjected to immunoprecipitation by anti-β-catenin antibody and immunoprecipitates were analyzed by immunoblotting using anti-ubiquitin antibody. For Nur77 ubiquitination, HEK293T cells were transfected with Myc-Nur77 or Myc-tagged mutants together with or without HA-ubiquitin plasmid, and treated with 10 μM MG132 for 2 hr. Lysates prepared were then immunoprecipitated using anti-Myc antibody, and probed with anti-ubiquitin antibody or anti-HA antibody.

Reverse transcription-PCR analysis

Total RNAs were isolated by Trizol LS. The first-strand synthesis was performed with RevertAid First-Strand cDNA synthesis kits (Fermentas) using primers for β-catenin (forward primer, 5'-TCA GAG GGT CCG AGC TGC CA-3'; reverse primer, 5'-TGT CAG CTC AGG AAT TGC AC-3') and GAPDH (forward primer, 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3'; reverse primer, 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3'). PCR reactions were performed in Eppendorf AG 22331 Hamburg (Eppendorf), and PCR products were electrophoresed on 2% agarose gels and gel images were captured with a Gel logic 200 system (Kodak).

Co-immunoprecipitation assays

For co-immunoprecipitation assay (Kolluri et al 2008, Li et al 2000, Lin 2004, Wu et al 1997a, Wu et al 1997b, Zhou et al 2010), Cells grown in 10 cm dishes were transfected with various plasmids and lysates were prepared 36 hr after transfection. Cell lysates were incubated with the appropriate antibody for 1hr, and subsequently incubated with protein A-Sepharose beads for 1 hr. The protein–antibody complexes recovered on beads were subjected to western blotting using appropriate antibodies after separation by SDS–PAGE.

Immunofluorescence microscopy

Cells were seeded (40,000/cm2) on glass coverslips in six-well plates. Twenty-four hours after treatment, cells were permeabilized with 0.1% Triton X-100 and 0.1 mol/L of glycine for 15 min on ice, and stained with anti-β-catenin and detected by Cy3-labeled anti-mouse IgG. Cells were costained with 4'6'-diamidino-2-phenylindole (DAPI) to visualize nuclei. The images were taken under a LSM-510 confocal laser scanning microscope system (Carl Zeiss) (Kolluri et al 2008, Li et al 2000, Lin 2004, Wu et al 1997a, Wu et al 1997b, Zhou et al 2010).

Cellular fractionation

For cellular fractionation, cells were lysed in cold buffer A [10 mmol/L HEPES-KOH (pH 7.9), 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT, 10mmol/L of NaF and 1mmol/L Na3VO4] with a cocktail of proteinase inhibitors on ice for 10 min as described (Kolluri et al 2008, Li et al 2000, Lin 2004, Wu et al 1997a, Wu et al 1997b, Zhou et al 2010). Cytoplasmic fraction was collected by centrifuging at 6,000 rpm for 30 sec. Pellets containing nuclei were resuspended in cold high-salt buffer C [20 mmol/L HEPES-KOH (pH 7.9), 25% glycerol, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 10mmol/L of NaF and 1mmol/L Na3VO4] with a cocktail of proteinase inhibitors on ice for 30 min. Cellular debris was removed by centrifugation at 12,000 rpm at 4°C for 15 min.

SW620 Xenografts

Nude mice (BALB/c, 4–5 weeks old) were injected subcutaneously with 100 μl SW620 cells (2×106). For drug treatment, mice were administered intragastrically after 7 days of transplantation with H-9 (0.5 mg/kg) or vehicle (tween-80) once a day. Body weight and tumor sizes were measured every 4 days. Mice were scarified after drug treatment and tumors were removed for various assessments. All manipulations involving live mice were approved by the Animal Care and Use Committee of Xiamen University.