Small Molecule-Based Promotion of PKCα-Mediated β-Catenin Degradation Suppresses the Proliferation of CRT-Positive Cancer Cells

Aberrant accumulation of intracellular β-catenin is a well recognized characteristic of several cancers, including prostate, colon, and liver cancers, and is a potential target for development of anticancer therapeutics. Here, we used cell-based small molecule screening to identify CGK062 as an inhibitor of Wnt/β-catenin signaling. CGK062 promoted protein kinase Cα (PKCα)-mediated phosphorylation of β-catenin at Ser33/Ser37, marking it for proteasomal degradation. This reduced intracellular β-catenin levels and consequently antagonized β-catenin response transcription (CRT). Pharmacological inhibition or depletion of PKCα abrogated CGK062-mediated phosphorylation and degradation of β-catenin. In addition, CGK062 repressed the expression of the genes encoding cyclin D1, c-myc, and axin-2, β-catenin target genes, and thus inhibited the growth of CRT-positive cancer cells. Furthermore, treatment of nude mice bearing PC3 xenograft tumors with CGK062 at doses of 50 mg/kg and 100 mg/kg (i.p.) significantly suppressed tumor growth. Our findings suggest that CGK062 exerts its anticancer activity by promoting PKCα-mediated β-catenin phosphorylation/degradation. Therefore, CGK062 has significant therapeutic potential for the treatment of CRT-positive cancers.


Introduction
The Wnt/b-catenin pathway, which is activated by the interaction of Wnt1, Wnt3a, and Wnt8 with Frizzled (Fz) receptors and low-density lipoprotein receptor-related protein5/6 (LRP5/6) co-receptors, plays important roles in cell proliferation, differentiation, and oncogenesis [1]. Central to this pathway is the level of cytosolic b-catenin, which regulates its target genes. In the absence of a Wnt signal, b-catenin is phosphorylated by both casein kinase 1 (CK1) and glycogen synthase kinase-3b (GSK-3b), which form a complex with adenomatous polyposis coli (APC)/Axin (destruction complex). This is then recognized by F-box b-transducin repeatcontaining protein (b-TrCP), a component of the ubiquitin ligase complex, which results in the degradation of b-catenin [2][3][4]. Activation of the receptor by its Wnt ligands negatively regulates the destruction complex and leads to cytoplasmic b-catenin stabilization [5].
Abnormal activation of the Wnt/b-catenin pathway and subsequent up-regulation of b-catenin response transcription (CRT) is thought to contribute to the development and progression of certain cancers [6]. Oncogenic mutation in bcatenin or other components of the destruction complex (APC or Axin) are observed in colon cancer, hepatocelluar carcinoma, and prostate cancer [6][7][8]. These mutations lead to the excessive accumulation of b-catenin in cytoplasm and then b-catenin is translocated into the nucleus, where it complexes with T cell factor/lymphocyte enhancer factor (TCF/LEF) family transcription factors to activate the expression of Wnt/b-catenin responsive genes, such as c-myc, cyclin D1 and metalloproteinase-7 (MMP-7), which play important roles in tumorigenesis and metastasis [9][10][11]. The accumulation of b-catenin is also observed in other types of cancer, such as ovarian cancer, melanoma, endometrial cancer, medulloblastoma, and pilomatricoma [6][7][8]. Thus, aberrant activation of the Wnt/b-catenin pathway is a potential therapeutic target for chemoprevention and treatment of various cancers.
In the present study, we identified CGK062, which markedly inhibits the Wnt/b-catenin pathway and cell proliferation of CRT-positive cancer cells. CGK062 promoted the degradation of intracellular b-catenin through PKCa-mediated b-catenin phosphorylation.

Identification of CGK062 as an inhibitor of the Wnt/bcatenin pathway
To identify small molecule antagonists of the Wnt/b-catenin pathway, we used HEK293 reporter cells, which stably harbored TOPFlash reporter and human Frizzled-1 (hFz-1) plasmids. After the incubation of these reporter cells with Wnt3a-CM and each compound, we measured firefly luciferase activity using a microplate reader and then identified that CGK062 (3-(3,4dihydroxy-phenyl)-acrylic acid 2,2-dimethyl-8-oxo-3,4-dihydro-2H,8H-pyrano [3,2-g]chromen-3-yl ester) as an inhibitor of the Wnt/b-catenin pathway ( Figure 1A, B and S1). As shown in Figure 1C, treatment of HEK293 reporter cells with different concentrations of CGK062 resulted in a dose-dependent decrease in b-catenin response transcription (CRT) that had been induced by Wnt3a-CM (IC 50 = 12.3 mM). CGK062 did not affect FOP-Flash activity in HEK293 control cells and cell viability in HEK293 reporter cells ( Figure 1C and S2A). NF-kB and p53 reporter activities were not affected by CGK062 ( Figure S2B and S2C). These results suggest that CGK062 potently and specifically inhibits the Wnt/b-catenin pathway.
In the Wnt/b-catenin pathway, CRT is largely dependent on the level of intracellular b-catenin, which is controlled by ubiquitin-dependent proteolysis [12]. To investigate the effect of CGK062 on the intracellular level of b-catenin, we analyzed the amount of cytoplasmic b-catenin by Western blotting with anti-bcatenin antibody in CGK062-treated HEK293 reporter cells. The level of cytoplasmic b-catenin, which had been accumulated by Wnt3a-CM, was dramatically decreased by treatment of CGK062 ( Figure 1D), which is consistent with the CRT result. To examine whether the reduction of cytoplasmic b-catenin protein by this compound was due to the decrease of b-catenin mRNA level in HEK293 reporter cells, we performed semi-quantitative RT-PCR to determine the amount of b-catenin mRNA. As shown in Figure 1E, the b-catenin mRNA level did not change in response to different concentrations of CGK062 in HEK293 reporter cells.
Next, to explore whether down-regulation of b-catenin by CGK062 is mediated by the proteasome, we used MG-132 to inhibit proteasome-mediated protein degradation in HEK293 reporter cells. As shown in Figure 1F, CGK062 consistently led to a decrease in the b-catenin protein level; however, the addition of MG-132 abrogated the effect of CGK062 on the reduction in bcatenin. Ammonium chloride, a lysosome inhibitor, did not affect CGK062-mediated b-catenin degradation ( Figure S3). Taken together, these results indicate that CGK062 suppresses the Wnt/ b-catenin pathway via a mechanism involving the degradation of intracellular b-catenin.
CGK062-mediated b-catenin degradation requires b-TrCP but not GSK-3bactivity Given that the phosphorylation of b-catenin by GSK-3b and its subsequent association with b-TrCP leads to b-catenin degradation [13], we examined whether GSK-3b activity is necessary for the degradation of b-catenin induced by CGK062. When HEK293 reporter cells were incubated with LiCl or 6-bromoindirubin-39-oxim (BIO), inhibitors of GSK-3b, CRT was stimulated ( Figure 2A and 2B), consistent with previous reports [14,15]. As shown in Figure 2A and 2B, treatment with CGK062 resulted in the suppression of CRT in a dose-dependent manner. In addition, Western blot analysis using anti-b-catenin antibody consistently showed that CGK062 led to a down-regulation of intracellular bcatenin levels, which accumulated with LiCl ( Figure 2C), suggesting that CGK062-mediated b-catenin degradation is independent of GSK-3b.
We then determined whether b-TrCP is necessary for CGK062-induced degradation ofb-catenin. As shown in Figure 2D, ectopic expression of a dominant-negative form of b-TrCP (Db-TrCP), which interacts with phosphorylated bcatenin but is unable to form a SCF b-TrCP ubiquitin ligase complex [16], abrogated the CGK062-induced degradation of bcatenin. In addition, CGK062 did not affect CRT that had been activated by overexpression of b-catenin mutants, S45A and S37A ( Figure S4). These results indicate that b-TrCP and N-terminal residues of b-catenin are required for CGK062-mediated bcatenin degradation.

CGK062 induces PKCa-mediated b-catenin phosphorylation/degradation
Previous reports have demonstrated that activated PKCa catalyzes the phosphorylation of b-catenin at Ser33/37 and its subsequent association with b-TrCP leads to b-catenin degradation [17,18]. To further gain insight into the mechanism, we first examined whether PKCa is activated by treatment with CGK062. Since activated PKCa moves from the cytoplasm to the plasma membrane [19], we isolated membrane fractions from CGK062treated and -untreated cells, and measured the amount of PKCa by Western blot analysis. CGK062 treatment led to the accumulation of PKCa at the plasma membrane in HEK293 cells ( Figure 3A). We also observed CGK062-induced membrane translocation of PKCa in HEK293 cells by immunofluorescence analysis ( Figure S5). Consistently, CGK062 enhanced the kinase activity of PKCa in vitro, when we used synthetic peptide containing PKCa phosphorylation site as a substrate ( Figure 3B). Moreover, BIM I, a specific inhibitor of PKC abolished the effect of CGK062 ( Figure 3B), suggesting that CGK062 is a bona fide activator of PKCa.
We then examined whether PKCa activity is essential for CGK062-mediated b-catenin degradation. The inhibition of PKCa activity using BIM I abolished the down-regulation of bcatenin by CGK062 ( Figure 3C). Notably, the selective depletion of endogenous PKCa using small-interfering RNA (siRNA) also nullified the CGK062-induced degradation ofb-catenin ( Figure 3D), indicating that PKCa is responsible for the degradation of b-catenin by CGK062.
Next, to test whether CGK062 directly promotes PKCamediated b-catenin phosphorylation at Ser33/37, we performed an in vitro kinase assay using bacterially expressed b-catenin and purified PKCa. PKCa readily phosphorylated b-catenin in the presence of CGK062 and BIM I inhibited this phosphorylation ( Figure 3E). We also examined whether CGK062 promotes PKCa-mediated b-catenin phosphorylation at Ser33/37 and Ser45 in HEK293 reporter cells. Western blot analysis showed that Wnt3a-CM inhibited the phosphorylation of b-catenin at Ser33/37 and Ser45 ( Figure 3F, S6 and S7). In addition, CGK062 induced the phosphorylation of b-catenin at Ser33/37 and Ser45 ( Figure 3F, S6 and S7), and Ser33/37 phosphorylation was abrogated by adding BIM I ( Figure 3F). Consistently, CGK062 treatment rescued the phosphorylation of b-catenin at Ser33/37, which was inhibited by Wnt3a-CM, and the knockdown of PKCa markedly suppressed CGK062-induced Ser33/37 phosphorylation in HEK293 reporter cells ( Figure 3G).

CGK062 also promotes b-catenin degradation in CRTpositive cancer cells
We next tested whether CGK062 activates PKCa in CRTpositive cancer cells, such as PC3 (prostate cancer), SNU475 (hepatoma), and SW480 (colon cancer). Consistent with results from HEK293 cells, CGK062 promoted the translocation of PKCa to the plasma membrane in these cancer cells ( Figure 4A). To determine whether CGK062 also inhibits b-catenin function in CRT-positive cancer cells, TOPFlash plasmid was transfected into CRT-positive cancer cells followed by treatment with increasing concentrations of CGK062. As shown in Figure 4B, CGK062 consistently repressed CRT in PC3, SNU475, and SW480 cells. In parallel with this experiment, we determined the effect of CGK062 on the level of cytosolic b-catenin in these CRT-positive cancer cells by Western blot analysis. Consistently, treatment of CGK062 resulted in the down-regulation of intracellular b-catenin level in a concentration-dependent manner in PC3, SNU475, and SW480 cells ( Figure 4C). We also found that CGK062 promoted the phosphorylation of b-catenin at Ser33/37, and this phosphorylation was abolished by BIM I in SW480 cells ( Figure S8). These results indicate that CGK062 also induces b-catenin degradation in CRT-positive cancer cells.

CGK062 represses the expression of b-catenindependent genes
To determine whether CGK062 affects the expression of bcatenin-dependent genes, the promoter activity of cyclin D1, which is a known b-catenin-dependent gene, was evaluated. A reporter construct containing the cyclin D1 promoter, which contains a bcatenin/TCF-4 responsive region, was transfected into PC3, SNU475, and SW480 cells followed by treatment with different concentrations of CGK062. As shown in Figure 5A, cyclin D1 promoter activity was repressed by CGK062 in these CRTpositive cancer cells. We also evaluated the protein level of cyclin D1 in CGK062-treated CRT-positive cancer cells. Consistent with our result for the cyclin D1 promoter, a dose-dependent decrease in cyclin D1 protein expression was observed in response to CGK062 ( Figure 5B) in PC3, SNU475, and SW480 cells. In addition, the expression of c-myc and axin-2, established downstream targets of b-catenin [9], were also significantly reduced in these CRTpositive cancer cells following incubation with CGK062 ( Figure 5B). Under these conditions, the PKCa level did not change in response to different concentrations of CGK062 ( Figure  S9).

CGK062 inhibits the proliferation of CRT-positive cancer cells in vitro and in vivo
Recent studies have demonstrated that the disruption of bcatenin function by antisense, siRNA, or secreted Wnt antagonist strategies has been shown to specifically inhibit the growth of CRT-positive cancer cells [20][21][22]. Given that CGK062 promoted the degradation of intracellular b-catenin, we hypothesized that CGK062 also suppresses the proliferation of CRT-positive cancer cells. To explore this hypothesis, we evaluated the effect of CGK062 on the growth of various CRT-positive cancer cells. As shown in Table 1 and Figure   To further evaluate the antitumor activity of CGK062 in vivo, athymic nude mice bearing established s.c. PC3 xenograft tumors were treated daily with i.p. administration of CGK062 for 5 weeks at 50 and 100 mg/kg. In comparison to the vehicle (control), treatment of mice with CGK062 significantly inhibited PC3 tumor growth ( Figure 6A). A dose of 100 mg/kg CGK062 induced near complete inhibition of tumor growth. Even at the dose of 50 mg/ kg, CGK062 inhibited tumor growth by ,70% relative to the control group. All mice tolerated the treatment without significant loss in body weight ( Figure 6B).

Discussion
Aberrant up-regulation of intracellular b-catenin is involved in the development of several cancers, including prostate cancer, colorectal cancer, and hepatocellular carcinoma [6][7][8]. In this report, we used a cell-based screening to identify CGK062 as a potent inhibitor of the Wnt/b-catenin pathway. CGK062 provided considerable therapeutic advantage with respect to antitumor potency in various b-catenin response transcription (CRT)-positive cancer cells, such as colon cancer cells (SW480, DLD-1, and HCT15), hepatocellular carcinoma (SNU475), hormone-refractory prostate cancer cells (PC3).
CGK062-activated PKCa catalyzed the phosphorylation of bcatenin at Ser33/Ser37, and thereby reduced the intracellular bcatenin level by b-TrCP-dependent proteasomal degradation. In addition, pharmacological inhibition and PKCa siRNA abrogated CGK062-induced b-catenin phosphorylation/degradation, indicating that PKCa is responsible for CGK062-mediated inhibition of the Wnt/b-catenin pathway. The results of several studies, including some conducted previously in our laboratory, suggest a key role for PKCa in the regulation of Wnt/b-catenin signaling. Wnt5a is involved in the mobilization of intracellular Ca 2+ , the subsequent activation of PKCa, and inhibition of the Wnt/bcatenin pathway [23,24]. Orford et al. [25] reported that PKC Recently, retinoic acid-related orphan nuclear receptor a (ROR a) was shown to attenuate Wnt/b-catenin signaling in colon cancer by stimulating PKCa-dependent phosphorylation [26]. Previously, we demonstrated that PKCa negatively regulates Wnt/b-catenin signaling in HEK293 cells, which have normal Wnt/b-catenin pathway function [17] and that PKCa regulates the intracellular b-catenin level in colon cancer cells [18]. The present study extends those previous findings by showing that the activation of PKCa by a novel agonist, CGK062, promotes bcatenin degradation in three cancer cell lines -PC3 (prostate cancer), SNU475 (hepatoma), and SW480 (colon cancer) -which display aberrant up-regulation of the intracellular b-catenin level.
Small molecule inhibitors that antagonize CRT have been discovered by high-throughput screening. Small molecules that block the association between Tcf4 and b-catenin impair b- catenin-dependent activities, such as cell proliferation and duplication of the embryonic dorsal axis in Xenopus laevis [27]. Another small molecule, ICG-001, which blocks the interaction between b-catenin and cyclic AMP response element-binding protein (CBP), specifically induces apoptosis in colon cancer cells [28]. Two other small molecules, IWR-3 and XAV939, are recently shown to stimulate the degradation of b-catenin by stabilizing axin and thereby inhibiting the proliferation of DLD-1 colon cancer cells, which carry a mutation in APC [29,30]. In contrast to previously studied small molecules, CGK062 reduced the intracellular level of b-catenin through PKCa-mediated phosphorylation/degradation. Notably, it was able to promote b-catenin degradation in SNU475 hepatoma cells, which carry an axin mutation, as well as in SW480 colon cancer cells with APC mutation, and suppressed the growth of CRT-positive cancer cells. CGK062 markedly suppressed the growth of PC3 prostate tumors in a mouse xenograft model, consistent with the results of our in vitro analyses.
In conclusion, we identified CGK062 that promotes PKCamediated b-catenin phosphorylation and its degradation. CGK062 repressed the expression of its target genes, including those encoding cyclin D1, c-myc and axin-2, thereby suppressing tumor growth in vitro and in vivo. Taken together, CGK062 represents a promising candidate treatment of CRT-positive cancers.

Screening for a small-molecule inhibitor of Wnt/b-catenin signaling
The HEK293 reporter cells were inoculated into 96-well plates at 15,000 cells per well in duplicate and grown for 24 h. Wnt3a-CM was added, and then compounds (800 compounds) including coumarins, flavonoids, naphthoquinones, and terpenoids were added to the wells at a final concentration of 30 mM. After 15 h, the plates were assayed for firefly luciferase activity and cell viability.

Preparation of the membrane fraction
Cells grown in 100-mm culture dishes were washed with icecold PBS. The cells were then suspended in 1 ml of ice-cold extraction buffer (20 mM Tris (pH 7.5), 0.5 mM EDTA, and 0.5 mM EGTA); homogenized using a syringe (26G); and incubated on ice for 30 min. The homogenate was centrifuged at 13,4006g for 2 min at 4uC. The supernatant was centrifuged at 100,0006 g for 30 min at 4uC in a 100Ti rotor (Beckman, USA). The pellet was suspended in extraction buffer containing 0.5% (w/ v) Triton X-100.

RNA extraction and semi-quantitative RT-PCR
Total RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. cDNA synthesis, reverse transcription, and PCR (Polymerase chain reaction) were performed as previously described [32]. The amplified DNA was separated on 2% agarose gels and stained with ethidium bromide.

Immunofluorescence analysis
HEK293cells were cultured on glass chamber slides and then treated with DMSO or CGK062 for 15 h. After treatment, the cells were washed with PBS, fixed with 4% formaldehyde, permeabilized in 0.3% Triton X-100, and blocked in 4% bovine serum albumin for 1 h. The cells were stained with anti-PKCa or anti-E-cadherin (BD Transduction Laboratories) antibodies and then analyzed by confocal microscopy using a Zeiss LSM510 Meta microscope.

In vitro kinase assay
Kinase assays were performed with purified PKCa (Sigma) using GST-b-catenin (100 ng) as a substrate as previously described [17]. The proteins were subjected to SDS-PAGE and transferred onto nitrocellulose membranes. The transferred proteins were analyzed using Western blotting with antiphospho-b-catenin antibody (Cell Signaling Technology), and visualized using the ECL system (Santa Cruz Biotechnology). Cell viability assay Cells were inoculated into 96-well plates and treated with CGK062 for 48 h. The cell viability from each treated sample was measured in triplicate using CellTiter-Glo assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions.

In vivo xenograft experiment
Animal experiment was performed with approval of the Animal Study Committee of Inje University (Permit Number: 2008-051). PC3 prostate cancer cells (1610 7 ) cells were injected s.c. into 6week-old female athymic nude mice. One week after cell implantation, mice were randomly assigned to three experimental groups (n = 5 each) and then treated with CGK062 (50 mg/kg/ day or 100 mg/kg/day) by i.p. for 4 weeks. Tumors were measured with a caliper and their volumes were calculated using formula, width 2 6length60.52. Figure S1 Schematic diagram of CGK062 synthesis. CGK062 was synthesized by the following procedure. (+)-Decursinol was prepared form the roots of A. Gigas and was added to a solution of caffeic acid (5 g, 1eq) in pyridine (11.2 ml, 5eq) was added acetic anhydride (26.2 ml, 10eq) at room temperature. The reaction mixture was stirred for 1 day and extracted with ethylacetate/H 2 O (1:1) solution. The organic layer was dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude mixture was solidified with ethylacetate/nhexane (1:1) solution to give 3-(3,4-diacetoxy-phenyl)-acrylic acid in 74.8% yield (5.48 g). Thionyl chloride (2.37 ml, 5eq) was added to a solution of 3-(3,4-diacetoxy-phenyl)-acrylic acid (1.6 g, 1eq) in anhydrous benzene (18 ml) and catalytic amount of DMF (1 drop). The reaction mixture was refluxed for 6 h and then allowed to cooling to room temperature. (C) HEK293 cells were co-transfected with NF-kB-FL and pCMV-RL plasmids and incubated with CGK062 in the presence or absence of aspirin, an activator of NF-kB pathway, for 15 h. Luciferase activities were measured 39 h after transfection and reported as relative light unit (RLU) normalized to Renilla luciferase activities. (TIF) Figure S3 CGK062 induces b-catenin degradation through a mechanism independent of the lysosomal degradation pathway. Cytosolic proteins prepared from HEK293 reporter cells, which were incubated with vehicle (DMSO) or CGK062 (25 mM) in the presence or absence of Wnt3a CM, exposed to NH 4 C1 (10 mM), were subjected to Western blotting with anti-b-catenin antibody. (TIF) Figure S4 The N-terminus of b-catenin is required for CGK062-mediated b-catenin degradation.