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Keywords:

  • colon cancer;
  • protein kinase C-α;
  • β-catenin;
  • phosphorylation;
  • degradation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We reported previously that protein kinase C-α (PKC-α) negatively regulates Wnt/β-catenin signalling pathway. The current study explores the role of PKC-α in the regulation of proliferation of colon cancer cells, which contain aberrant up-regulation of intracellular β-catenin. In colon tissue and cells, an inverse correlation was observed between the expression levels of PKC-α and intracellular β-catenin. Activation of PKC-α inhibited β-catenin response transcription by down-regulation of intracellular β-catenin and induced phosphorylation of the N-terminal serine and threonine residues (Ser33/Ser37/Thr41) of β-catenin, marking it for proteasomal degradation, in colon cancer cells. Pharmacological inhibition or depletion of PKC-α-abrogated PKC-α-mediated β-catenin down-regulation and phosphorylation in colon cancer cells. Notably, the Ser45 residue of β-catenin was essential for PKC-α-induced β-catenin down-regulation in colon cancer cells. Moreover, PKC-α activation repressed the expression of cyclin D1 and c-myc, which are known β-catenin target genes, and thus inhibited the growth of colon cancer cells. These findings suggest that PKC-α negatively regulates colon cancer cell proliferation viaβ-catenin phosphorylation/down-regulation and may facilitate the development of new strategies to treatment of colon cancer.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Colon cancer is the most common type of cancer and the third leading cause of cancer-related deaths in Western countries [1]. The progression from normal colonic epithelium to colonic neoplasm is associated with the accumulation of a series of genetic and epigenetic alterations [2, 3]. Several lines of evidence from human and animal studies suggest the involvement of protein kinase C (PKC) isozymes and the PKC-mediated signalling pathway in colonic tumorigenesis. Modified expression and activity of specific PKC isozymes have been detected in human and rodent colonic tumours. PKC activity is frequently lower in human colon cancers than in surrounding normal epithelium [4–8], and expression of several PKC isozymes was found to be markedly reduced in adenomas of ApcMIN mice compared to adjacent normal colonic mucosa [9]. These findings indicate that alterations in the PKC signalling pathway may contribute to uncontrolled colonic cell growth and transformation.

Considerable evidence also exists for important roles of PKC in growth arrest and differentiation during the renewal of normal intestinal epithelium. The intestinal epithelium consists of proliferating, differentiating and functional cells located in distinct compartments in the basal, middle and upper parts of crypts, respectively. In the proliferating cells of the basal crypt, relatively low levels of PKC isozymes are expressed diffusely throughout the cytosol. The levels of membrane-associated PKC isozymes start to increase in the mid-crypt, where cell proliferation ceases and cell differentiation begins, and its expression is sustained in the villi [10–12].

Several evidence indicated the PKC-mediated signalling pathway is involved in regulation of the Wnt/β-catenin pathway [13, 14]. The Wnt/β-catenin pathway is activated by the association of Wnts (Wnt1, Wnt3a, Wnt8) with Frizzled (Fz) receptors and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) co-receptors [15]. This pathway is primarily controlled by the level of cytoplasmic β-catenin, which stimulates its target genes [16]. In the absence of a Wnt signal, cytoplasmic β-catenin is sequentially phosphorylated by casein kinase 1 (CK1) and glycogen synthase kinase-3β (GSK-3β), which form a complex with adenomatous polyposis coli (APC) and axin, resulting in the degradation of β-catenin via a ubiquitin-dependent mechanism [17]. Activation of the receptor by its Wnt ligands negatively regulates GSK-3β, leading to the stabilization of β-catenin in the cytoplasm [18].

Abnormal activation of the Wnt/β-catenin pathway is a frequent early event in intestinal epithelial cells during the development of colon cancer [19, 20]. Mutations of the APC gene occur in the majority of sporadic colorectal cancers, as well as in familial adenomatous polyposis [21]. In addition, mutations in the N-terminal phosphorylation motif of the β-catenin gene have been observed in colorectal cancer [22]. These mutations lead to the excessive accumulation of β-catenin in the nucleus, where β-catenin forms a complex with members of the T-cell factor (TCF)/lymphocyte enhancer factor transcription factor family, activating the expression of Wnt/β-catenin responsive genes, including cyclin D1, myc, matrix metalloproteinase-7 and PPAR-δ, which play important roles in colorectal tumorigenesis [23–26].

In this report, we show that PKC-α phosphorylated the N-terminal Ser/Thr residues of β-catenin and subsequently induced its degradation, thereby suppressing the growth of colon cancer cells. Thus, our results indicate that PKC-α regulated the proliferation/growth cessation of colon cancer cells by modulating intracellular β-catenin levels.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Cell culture, chemicals and plasmids

CCD-18co, CCD-33co, SW480, HCT15, DLD-1, SW620, HCT116 and LS174T cells were obtained from the American type culture collection. These cells, except for DLD-1, were maintained in DMEM and DLD-1 cells were in Roswell Park Memorial Institute Medium (RPMI)1640 supplemented with 10% foetal bovine serum, 120 μg/ml penicillin and 200 μg/ml streptomycin (Hyclone Laboratories, Logen, UT, USA) at 37°C in 5% CO2. A23187, phobol 12-myristat 13-acetate (PMA), MG132, Gö6976, KN-93, CsA and GF-109203X (bisindolymaleimide I) were purchased from Calbiochem (San Diego, CA, USA). The pTOPFlash and pFOPFlash reporter plasmids were obtained from Upstate Biotechnology (Lake Placid, NY, USA). pCMV-RL plasmid was purchased from Promega (Madison, WI, USA). The dominant negative β-TrCP expression plasmid was kindly provided by Dr. M. Davis (Hebrew University-Hadassah Medical School, Israel). In addition, pGFP3 PKC-α wild-type and pHACE PKC-α wild-type expression plasmids were kindly provided by Dr. J.W. Soh (Inha University, Incheon, Korea).

Small interfering RNA (siRNA), transfection and luciferase assay

PKC-α siRNA was designed as previously described [27]. Specific for PKC-α siRNA (5′-AAGCACAAGTTCAAAATCCAC-3′) were synthesized by Invitrogen (Valenica, CA, USA). Negative control siRNA (Silencer™) was purchased from Ambion (Austin, TX, USA). Transfection was carried out with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Luciferase assays were performed with the Dual Luciferase Assay kit (Promega).

Western blot and antibodies

The cytosolic fraction was prepared as previously described [28]. Proteins were separated using 4–12% gradient SDS-PAGE (Invitrogen) and transferred to Polyvinyliclene Difluoride (PVDF) membranes (Amersham Bioscience, Buckinghamshire, UK) by wet blotting. The membranes were blocked with 5% non-fat milk in Tris-buffered saline (TBS) (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% tween 20) and probed with primary antibodies (1:1000). The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Santa Cruz Biotechnology, California, CA, USA, 1:2500) and visualized using the ECL chemiluminescence (Santa Cruz Biotechnology). The antibodies against β-catenin and PKC-α were purchased from BD Transduction Laboratories (San Diego, CA, USA). β-actin and phospho-β-catenin antibodies (Ser33/Ser37/Thr41) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Preparing the membrane fraction

SW480 cells grown in 100-mm culture dishes were washed with ice-cold phosphate buffered saline (PBS). The cells were then suspended in 1 ml of ice-cold extraction buffer [20 mM Tris [pH 7.5], 0.5 mM ethylenediaminetetraacetic acid and 0.5 mM Ethylene glycol-bis(aminoethyl ether)N, N,N′,N′-tetraacetic acid (EGTA)]; homogenized using a syringe (26G); and incubated on ice for 30 min. The homogenate was centrifuged at 13,400 ×g for 2 min. at 4°C. The supernatant was centrifuged at 100,000 ×g for 30 min. at 4°C in a 100Ti rotor (Beckman, Fullerton, CA, USA). The pellet was suspended in extraction buffer containing 0.5% (w/v) Triton X-100.

Cell viability assay

Cells were inoculated into 96-well plates at 5000 cells per well and then treated with A23187 for 2 days. Cell viability was determined using a CellTiter-Glo assay kit (Promega).

RNA extraction and semiquantitiative RT-PCR

Total RNA was isolated with TRIzol reagent (Invitrogen) in accordance with the manufacturer’s instructions and analysed with RT-PCR using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen). To amplify cyclin D1, β-catenin and GAPDH mRNA, PCR was performed for 23 cycles (94°C for 30 sec.; 59°C for 30 sec. and 72°C for 30 sec.), which were in exponential phase (Fig. S1), using the following primers: (a) cyclin D1 fwd, 5′-GGA TGC TGG AGG TCT GCG AGG AAC-3′ and cyclin D1 rev, 5′-GAG AGG AAG CGT GTG AGG CGG TAG-3′ (b) β-catenin fwd, 5′-GGG ATG TTC ACA ACC GAA TTG TTA TC-3′ and β-catenin rev, 5′-ACC AGA GTG AAA AGA ACG ATA GCT AGG A-3′ and (c) GAPDH fwd, 5′-GCT CAC TGG CAT GGC CTT CCG-3′ and GAPDH rev, 5′-GTG GGC CAT GAG GTC CAC CAC-3′. The final reaction volume was 20 μl, containing approximately 50–100 ng template DNA, 1 unit of rTaq polymerase (Takara Bio, Inc., Kyoto, Japan), 0.2 μM of each primer, 200 μM of each dNTP (Takara Bio, Inc.) and 1×PCR buffer. The PCR amplification products were separated on a 1.5% (w/v) agarose gel and visualized by ethidium bromide staining.

Immunoprecipitation

For immunoprecipitation experiments, SW480 cells were transiently transfected with β-catenin wild-type flag or Ser45Ala β-catenin-flag and then treated with A23187 and MG132. The final reaction volume was 500 μl, containing approximately 20 μl of protein A-agarose (Roche Diagnostics, Meylan, France), 200 μg of cell lysate, 1 μg of antibody and buffer A [10 mM HEPES [pH 7.4 at 4°C], 1.5 mM MgCl2, 10 mM KCl and 0.5 mM Dithiothreitol (DTT)] at 4°C for overnight. Immunocomplexes were washed five times with PBS and boiled in the SDS-PAGE loading buffer, and the proteins were detected by Western blotting.

Immunohistochemical staining

Tissue samples (SuperBioChip Lab., Seoul, Korea) were deparaffinized and then hydrated. To achieve optimal antigen retrieval, the sections were incubated in a microwave oven for 5 min. with 0.01 M citrate buffer at pH 6.0. The slides were incubated in 3% (v/v) H2O2 without solvent for 6 min. to quench endogenous peroxidase activity. The sections were immunostained for 2 hrs at room temperature with the indicated antibodies (β-catenin, BD Transduction Laboratories, 1:200, PKC-α, BD Transduction Laboratories, 1:100), and antibodies were detected with the VECTASTAIN Universal ABC kit (Vector Laboratories, Burlingame, CA, USA). Staining pattern (membrane, cytoplasm, or nuclear) and intensity were observed.

[3H]Thymidine incorporation assay

SW480 and DLD-1 cells were seeded in 96-well plates at 5000 cells per well and then treated with A23187 for 2 days. During the last 6 hrs of incubation, 1 μCi/well of 3H were added and media was removed at the end of incubation and cells were rinsed with ice-cold PBS. The cells were then treated with 5% trichloroacetic acid for 30 min. at 4°C, rinsed with PBS, and lysed with 0.5 M NaOH. The content of each well was transferred to scintillation vials, scintillation cocktail added and counts per minute (cpm) determined. Radioactivity was counted in a Beckman scintillation counter (LS 5801, Beckman Instruments, Mississauga, Canada). [3H] thymidine incorporation is expressed as total counts per minute per well.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

PKC-α is inversely correlated with β-catenin expression in colon cancer cells

To investigate the potential relationship between PKC-α and intracellular β-catenin in colon cancer, we determined the level of membrane PKC-α and cytosolic β-catenin in various colon cell lines. PKC-α was readily detected in the membrane fraction of CCD-18co and CCD-33co cells, which are normal colon epithelial cells (Fig. 1A). In contrast, we could not observe membrane PKC-α in colon cancer cells (Fig. 1A). Cytosolic β-catenin was strongly expressed in colon cancer cells, but not in normal colon epithelial cells (Fig. 1A). We also performed immunohistochemical staining for PKC-α and β-catenin in colon adenocarcinoma and paired adjacent normal epithelium. In normal colonic epithelium, PKC-α was strongly expressed in whole epithelium and β-catenin was stained in membrane (Fig. 1B, C, F and G). In contrast, in carcinoma areas, the expression of PKC-α was lower than in normal epithelium and β-catenin was expressed in nucleus and cytoplasm rather than in membrane (Fig. 1D, E, H and I). The two images show adjacent sections of the same tissue stained immunohistochemically with antibodies against PKC-α and β-catenin.

image

Figure 1. PKC-α inversely correlates with β-catenin expression in colon cells and tissue. (A) Extracts of normal cell lines (CCD-18co, CCD-33co) and colon cancer cell lines (DLD-1, HCT15, SW620, SW480) were examined for PKC-α (membranes fraction) and β-catenin (cytosolic fraction) expression by Western blot analysis. The blots were re-probed with anti-actin as a loading control. (BI) Serial tissue sections from colon adenocarcinoma (D, E, H, I) and paired normal adjacent epithelium (B, C, F, G) were stained with anti-PKC-α (white arrow) and anti-β-catenin (black arrow) antibody magnification was at 200×.

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PKC-α activation down-regulates intracellular β-catenin in colon cancer cells

We first examined whether PKC-α was activated by treatment with PMA and A23187, well-known PKC activators [29, 30], in colon cancer cells. Since activated PKC-α translocates from the cytoplasm to the plasma membrane, we isolated the membrane fraction from SW480 cells treated or not treated with PMA and A23187 and then measured the amount of PKC-α using an anti-PKC-α antibody. Consistent with the results in other cells [30, 31], both compounds led to the translocation of PKC-α to the membrane (Fig. 2A), indicating that PKC-α was activated by PMA and A23187 in colon cancer cells.

image

Figure 2. Activation of PKC induces down-regulation of β-catenin in colon cancer cells. (A) Activation of PKC-α in colon cancer cells. Cytosolic and membrane fractions were prepared from SW480 cells treated with A23187 (0.625 μM) for 12 hrs or PMA (100 nM) for 6 hrs and then analysed by Western blotting with anti-PKC-α antibody. (B) SW480 and DLD-1 cells were co-transfected with TOPFlash or FOPFlash and pCMV-RL plasmids and incubated with A23187 (0.625, 1.25 μM) for 12 hrs or PMA (50, 100 nM) for 6 hrs. Luciferase activities were measured 36 hrs after transfection. Results are the average of three experiments, and the bars indicate standard deviations. *, P < 0.05 and **, P < 0.01, compared with the vehicle control group. (C) Cytosolic proteins were prepared from SW480 and DLD-1 cells treated with A23187 (0.625, 1.25 μM) for 12 hrs or PMA (50, 100 nM) for 6 hrs and then analysed by Western blotting with anti-β-catenin antibody. (D) SW480 cells were co-transfected with β-catenin wild-type flag and pGFP3 PKC-α wild-type or β-catenin wild-type-flag and pHACE PKC-α wild-type. Cytosolic fractions were prepared and then subjected to Western blot analysis with anti-PKC-α and anti-β-catenin antibody. (E) Semiquantitative RT-PCR for β-catenin and GAPDH was performed with total RNA prepared from SW480 and DLD-1 treated with A23187 (0.625, 1.25 and 2.5 μM) for 12 hrs or PMA (50, 100 and 200 nM) for 6 hrs. In (A), (C) and (D), the blots were reprobed with anti-actin antibody as a loading control.

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We next determined the effects of PKC signalling on β-catenin response transcription (CRT) in colon cancer cells. SW480 and DLD-1 colon cancer cells harboring wild-type β-catenin and a truncated form of APC were transfected with TOPFlash, a synthetic β-catenin/TCF-dependent luciferase reporter, followed by incubation with PMA and A23187. As shown in Fig. 2B, activation of PKC efficiently reduced CRT in both cells in a concentration-dependent manner. In contrast, the activity of FOPFlash, a negative control reporter with a mutated β-catenin/TCF binding site, was not changed by incubation with PMA and A23187 (Fig. 2B). CRT is primarily regulated by the level of intracellular β-catenin, which stimulates the expression of downstream target genes. Thus, we tested whether PKC activation reduced the level of intracellular β-catenin by Western blotting. Treatment of colon cancer cells with PMA and A23187 resulted in down-regulation of intracellular β-catenin, consistent with their effects on CRT (Fig. 2C). In addition, ectopic expression of PKC-α dramatically decreased the level of intracellular β-catenin (Fig. 2D). In contrast to the protein level of β-catenin, mRNA expression of β-catenin was not altered by activation of PKC-α (Fig. 2D). Under these conditions, PMA and A23187 exhibited no cytotoxic effects on colon cancer cells (data not shown). Together, these results indicate that PKC-α activation inhibited CRT via down-regulation of intracellular β-catenin in colon cancer cells.

We confirmed that PKC-α activity was required for the down-regulation of β-catenin in colon cancer cells by testing the effects of a pharmacological inhibitor of PKC on the restoration of A23187-mediated CRT repression. As shown in Fig. 3A, inhibition of PKC activity using the PKC inhibitor bisindoylmaleimide I (BIM I) abrogated the suppression of CRT by A23187 (Fig. 3A). Moreover, the down-regulation of β-catenin induced by A23187 was inhibited when SW480 cells were incubated with PKC inhibitors, BIM I (Fig. 3B) and Gö6976 (Fig. S2A). Under the same conditions, other kinase inhibitors, such as KN-93 and H-89, did not rescue A23187-mediatd β-catenin degradation (Fig. S2B). Notably, selective depletion of endogenous PKC-α protein without altering the levels of other PKC family proteins in SW480 cells using siRNA also nullified A23187-mediated β-catenin down-regulation (Figs 3C and S2C). These results suggest that PKC-α is responsible for the down-regulation of β-catenin in colon cancer cells.

image

Figure 3. PKC-α is essential for down-regulation of β-catenin in colon cancer cells. (A) SW480 cells were transfected with pTOPFlash and then transfected cells were incubated with A23187 (0.625 μM) for 12 hrs in the presence or absence of BIM I (5 μM), and luciferase activity was determined. The results are shown as the average of three experiments the bars indicate standard deviations. *, P < 0.05 and **, P < 0.01, compared with the vehicle control group. (B) Cytosolic proteins were prepared from SW480 cells treated with A23187 (0.625 μM) for 12 hrs in the presence or absence of BIM I (5 μM) and then analysed by Western blotting with anti-β-catenin antibody. (C) SW480 cells were transfected with negative control siRNA (NC, 40 nM) and PKC-α siRNA (40 nM) for 36 hrs and then incubated with A23187 for 12 hrs. Cell lysates were subjected to Western blot analysis with anti-PKC-α, anti-phospho-Ser33/37/Thr41-β-catenin and anti-β-catenin. (D) SW480 cells were incubated with vehicle [Dimethyl sulfoxide (DMSO)] or A23187 (0.625 μM) and MG132 (10 μM) for 12 hrs. Cytosolic fractions were prepared and subjected to Western blot analysis with anti-phospho-Ser33/37/Thr41-β-catenin and anti-β-catenin antibody. In (B), (C) and (D), the blots were reprobed with anti-actin antibody as a loading control.

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PKC-α mediates the N-terminal phosphorylation of β-catenin in colon cancer cells

Several studies have demonstrated that phosphorylation at the N-terminal Ser residues of β-catenin plays an essential role in its ubiquitin-dependent degradation [17, 32]. Thus, we next determined whether PKC-α activity was necessary for phosphorylation of β-catenin Ser residues in SW480 cells. Western blot analysis using a phospho-specific β-catenin antibody showed that phosphorylation of β-catenin at Ser33/Ser37/Thr41 residues was induced by treatment with A23187 (Fig. 3D), but A23187-mediated β-catenin phosphorylation was abolished by the addition of BIM I (Fig. 3D). We also confirmed PKC-α-mediated β-catenin phosphorylation at Ser33/Ser37/Thr41 residues in SW480 cells using PKC-α siRNA. As expected, A23187 consistently induced phosphorylation of β-catenin, but the effect of A23187 on the induction of β-catenin phosphorylation was abolished by a PKC-α siRNA (Fig. 3C). These results indicate that PKC-α phosphorylated β-catenin Ser33/Ser37/Thr41 residues, leading to β-catenin degradation, in colon cancer cells.

The Ser 45 residue is essential for PKC-α-mediated β-catenin degradation

We next evaluated the effects of PKC-α signalling on the degradation of β-catenin in HCT116 (heterozygous deletion with a Ser45) and LS174T (homozygous missense mutation Ser to Phe with a Ser45) colon cancer cells. HCT116 and LS174T cells were transfected with TOPFlash and then incubated with A23187. In contrast to results in SW480 and DLD-1 cells, incubation with A23187 did not lead to an inhibition of TOPFlash activity in HCT116 or LS174T cells (Fig. 4A). Consistently, the intracellular β-catenin level was not altered in A23187-treated HC116 and LS174T cells (Fig. 4B), indicating that the Ser45 residue may play an important role in the PKC-α-mediated down-regulation of β-catenin.

image

Figure 4. The effects of PKC signalling on β-catenin level in Ser45-mutated colon cancer cells. (A) HCT116 and LS174T cells were co-transfected with TOPFlash and pCMV-RL plasmids and incubated with A23187 (0.625, 1.25 μM) for 12 hrs. Luciferase activities were measured 36 hrs after transfection. Results are the average of three experiments, and the bars indicate standard deviations. (B) Cytosolic proteins were prepared from HCT116 and LS174T cells treated with vehicle (DMSO) or A23187 (0.625, 1.25 μM) for 12 hrs and then analysed by Western blotting with anti-β-catenin and anti-actin antibody.

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To confirm these results, we ectopically expressed flag-tagged wild-type β-catenin and mutant β-catenin (Ser45Ala) in SW480 cells and analysed the levels of β-catenin protein using an anti-flag antibody in response to A23187. The level of wild-type β-catenin was significantly reduced by A23187 treatment, whereas the mutant β-catenin (Ser45Ala) was largely unaffected in response to A23187 (Fig. 5A). Because phosphorylation of β-catenin and its association with β-TrCP leads to β-catenin ubiquitination [33], we measured the interaction between β-catenins (wild-type and Ser45Ala) and β-TrCP with A23187 treatment using immunoprecipitation experiments with an anti-flag antibody. As expected, β-TrCP was associated with wild-type β-catenin in the presence of A23187; however, this interaction was largely decreased with the mutant β-catenin (Ser45Ala; Fig. 5B). Finally, A23187 treatment resulted in an increase in wild-type β-catenin ubiquitination (Fig. 5C). In contrast to wild-type β-catenin, ubiquitination of mutant β-catenin (Ser45Ala) was not detected after A23187 treatment (Fig. 5C).

image

Figure 5. A Ser 45 residue is essential for PKC-α-mediated β-catenin degradation. (A) SW480 cells were transfected with β-catenin wild-type flag or Ser45Ala β-catenin-flag and then incubated with either the vehicle (DMSO) or A23187 (0.625, 1.25 μM) for 12 hrs. Cytosolic proteins were subjected to Western blotting with anti-flag antibody. The blots were reprobed with anti-actin antibody as a loading control. (B) SW480 cells were co-transfected with β-catenin wild-type flag and β-TrCP or Ser45Ala β-catenin-flag and β-TrCP and then incubated with MG132 (10 μM) and A23187 (0.625 μM) for 12 hrs. Cytosolic proteins were subjected to immunoprecipitated with anti-flag M2 agarose beads (normal IgG as a negative control). Proteins in the β-catenin complex were analysed by Western blotting with anti-flag and anti-HA antibodies. (C) SW480 cells were transfected with HA-ubiquitin, β-catenin wild-type flag or Ser45Ala β-catenin-flag and β-TrCP or F-box-deleted β-TrCP as indicated, and cells were then treated with A23187 (0.625 μM) and MG132 (10 μM). Flag was immunoprecipitated and then analysed by Western blot with anti-flag and anti-HA antibody.

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PKC-α activation represses the expression of β-catenin-dependent genes and inhibits the proliferation of colon cancer cell

We examined the effects of PKC-α signalling on the expression of β-catenin downstream genes in colon cancer cells. To this end, SW480 and DLD-1 cells were transfected with a reporter construct containing the cyclin D1 promoter, which contains a β-catenin/TCF-4 responsive region, and subsequently incubated with increasing amounts of A23187. Cyclin D1 promoter activity was repressed in SW480 and DLD-1 cells (Fig. 6A), whereas A23187 did not affect cyclin D1 promoter activity in HCT116 or LS174T cells (Fig. S3A). In conjunction with this experiment, we determined the mRNA and protein levels of cyclin D1 in A23187-treated SW480 and DLD-1 cells. Consistent with the results of the reporter assay, dose-dependent decreases in cyclin D1 mRNA and protein levels were observed in response to A23187 (Fig. 6B and C). Notably, BIM I nullified A23187-mediated down-regulation of cyclin D1 expression in SW480 cells (Fig. S3B). Additionally, expression of c-myc, an established downstream target of β-catenin [24], was also significantly reduced in SW480 and DLD-1 cells by incubation with A23187 (Fig. 6C). Recently, inactivation of β-catenin function by antisense or siRNA strategies has been shown to specifically suppress the proliferation of human colon cancer cells [34]. Therefore, we evaluated whether PKC-α stimulation inhibited the growth of colon cancer cells. SW480 and DLD-1 cells were incubated with varying concentrations of A23187, and cell growth was monitored. A23187 efficiently suppressed the growth of these colon cancer cells in a concentration-dependent manner (Fig. 6D and E).

image

Figure 6. Activation of PKC-α inhibits the expression of the TCF/β-catenin target gene. (A) SW480 and DLD-1 cells were transfected with cyclin D1 promoter-pRL plasmids and incubated with A23187 (0.625, 1.25, 2.5 and 5 μM) for 12 hrs. Luciferase activities were measured 36 hrs after transfection. Results are the average of three experiments, and the bars indicate standard deviations. *, P < 0.05 and **, P < 0.01, compared with the vehicle control group. (B) Semiquantitative RT-PCR for cyclin D1 and GAPDH was performed with total RNA prepared from SW480 and DLD-1 treated with the vehicle (DMSO) or A23187 (0.625, 1.25 and 2.5 μM) for 12 hrs. (C) SW480 and DLD-1 cells were incubated with the vehicle (DMSO) or A23187 (0.625, 1.25 and 2.5 μM) for 12 hrs. Whole-cell extracts was prepared for Western blotting with anti-cyclin D1 and anti-myc antibodies. To confirm equal loading, the blots were reprobed with anti-actin antibody. (D, E) The effect of A23187 on cell growth. Cells were incubated, in the indicated concentrations of A23187, for 48 hrs in 96-well plates. Cell viability was examined using the CellTiter-Glo assay (Promega) (D) and cell proliferation was determined by the incorporation of [3H] thymidine (E). In (D) and (E), the results are shown as the average of three experiments, *, P < 0.05 and **, P < 0.01, compared with the vehicle control group, and the bars indicate standard deviations and the value at vehicle (DMSO) treated cells was set as 100%.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Abnormal accumulation of β-catenin in nuclei is a well-recognized characteristic of colon cancer [35]. Moreover, a previous study has indicated that expression of PKC-α is markedly reduced in human colonic tumour tissue relative to adjacent normal colonic mucosa [36]. In the present study, we found that accumulation of β-catenin in human colon adenocarcinoma and paired adjacent normal epithelium as well as in colon cancer cell lines inversely correlated with PKC-α expression, and importantly, we demonstrated that PKC-α-mediated β-catenin phosphorylation at Ser33/Ser37 residues, followed by degradation, was a mechanistic basis for the regulation of colon cancer cell proliferation. PKC agonists (PMA and A23187)-induced PKC-α activation was found to inhibit β-CRT by down-regulation of intracellular β-catenin in colon cancer cells. Moreover, PKC-α activation repressed its downstream target genes, such as cyclin D1 and myc, which play important roles in cell cycle progression and tumorigenesis [37, 38], resulting in suppressed proliferation in colon cancer cells.

The level of intracellular β-catenin is predominantly controlled by two APC-dependent proteasomal degradation pathways, a GSK3β-dependent pathway and a Siah-dependent pathway. In the GSK3β-dependent pathway, GSK3β, which forms a complex with APC and axin, phosphorylates the N-terminal Ser/Thr residues of β-catenin, leading to the degradation of β-catenin through a ubiquitin-dependent mechanism [17]. In the Siah-dependent pathway, Siah-1 interacts with the carboxyl terminus of APC, which recruits the ubiquitination complex and promotes the degradation of β-catenin [39]. In this study, PKC-α activation could induce the degradation of intracellular β-catenin in SW480 and DLD-1 cells, which have mutations in APC, suggesting that PKC-α promotes the degradation of β-catenin in colon cancer cells through a mechanism other than those described above.

In our study, PKC-α catalysed the phosphorylation of β-catenin at Ser33/Ser37/Thr41 residues, thereby promoting β-catenin degradation in colon cancer cells. Additionally, a pharmacological inhibitor or siRNA of PKC-α abrogated PKC-α-mediated β-catenin phosphorylation/down-regulation, indicating that PKC-α is a relevant kinase for β-catenin phosphorylation/degradation in colon cancer cells. Several studies, together with our previous data, have suggested a potential role for PKC-α in the Wnt/β-catenin pathway. Wnt5a is involved in the mobilization of intracellular Ca2+, followed by the activation of PKC-α, and antagonizes the Wnt/β-catenin pathway [13, 14]. Orford and colleagues reported that PKC inhibitors caused β-catenin accumulation in human breast cancer cells [40]. Recently, we demonstrated that PKC-α negatively regulated the Wnt/β-catenin pathway in HEK293 cells, which contain a normal Wnt/β-catenin pathway [31]. Our present study extends those previous findings by showing that PKC-α regulates intracellular β-catenin levels in colon cancer cells, which have mutation in components of the Wnt/β-catenin pathway, such as APC.

In the dual kinase mechanism, Ser45 phosphorylation of β-catenin by CK1 is indispensable for subsequent GSK3β-mediated Ser33/Ser37 phosphorylation and degradation of β-catenin [17]. In this study, we observed that activation of PKC-α could not induce β-catenin degradation in HCT116 and LS174T cells, which contain β-catenin with a mutation at the Ser45 residue. Additionally, ectopically expressed mutant β-catenin (Ser45Ala) was not degraded by PKC-α activation in SW480 cells, in contrast to wild-type β-catenin. We also found that β-TrCP and ubiquitin did not interact with Ser45Ala β-catenin in response to activation of PKC-α. These data suggest that the Ser45 residue of β-catenin is necessary for PKC-α-induced β-catenin degradation in colon cancer cells. We plan to investigate the role of the Ser45 residue in PKC-α-mediated β-catenin phosphorylation/degradation in colon cancer cells in future experiments.

Based on these data, we propose that in normal colorectal epithelial cells, activated PKC-α phosphorylates β-catenin and promotes its degradation (Fig. 7). In colorectal tumours, the reduced expression of PKC-α leads to the accumulation of β-catenin, which then promotes neoplastic growth (Fig. 7). This model also explains the inverse expression of PKC-α and β-catenin during intestinal epithelial renewal. In lower crypt cells, PKC-α is distributed diffusely throughout the cytosol. The expression of PKC-α starts to increase in the mid-crypt, where cell proliferation ceases and cell differentiation begins, and its expression is sustained in the villi [10]. In contrast, nuclear β-catenin is only detected in lower crypts [10]. Thus, activated PKC-α may decrease the β-catenin level from the mid-crypt to the villi.

image

Figure 7. Schematic model of regulation of colonic cell proliferation. A proposed model illustrated that the PKC-α pathway induces growth arrest viaβ-catenin phosphorylation/degradation in normal colon epithelium and its reduced activity causes accumulation of β-catenin, which promotes cell proliferation, in colon cancer cells.

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In conclusion, we propose a possible mechanism for the PKC-α-mediated regulation of colon cell proliferation. PKC-α induces growth inhibition of colon cancer cells via the promotion of β-catenin phosphorylation/degradation. In a support of our findings, it has been previously shown that a PKC-α activator, minerval exhibits antitumour effects both in cells and animal models [41]. Thus, a molecule that activates or induces expression of PKC-α would be a potential reagent to prevent colonic neoplasms.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank M. Davis for dominant-negative β-TrCP expression plasmid, J.H. Soh for pGFP-3 PKC-α wild-type and pHACE PKC-α wild-type expression plasmids and D.S. Min for PKC antibodies. This work was supported by the Korea Research Foundation Grant funded by the Korean government (MOEHRD, Basic Research Promotion Fund) (KRF-2007–313-C00440) (S Oh) and the Korea Institute of Science & Technology Evaluation and Planning (KISTEP) and Ministry of Science & Technology (MOST), Korean government, through its National Nuclear Technology Program (S Oh).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1 Semiquantitative RT-PCR for cyclin D1 and GAPDH was performed with total RNA prepared from SW480 and DLD-1 treated with A23187 for 15 h and then PCR amplification products were separated agarose gel using equal amount.

Fig. S2 PKC-α is required for degradation of β-catenin in colon cancer cells. (A), Cytosolic proteins were prepared from SW480 treated with A23187 for 12 h in the presence or absence of PKC inhibitors (BIM I, Gö6976) and then analyzed by Western blotting with anti-β-catenin antibody. (B), Cytosolic proteins were prepared from SW480 cells treated with A23 187 (0.625 μM) for 12 h in the presence or absence of KN-93 (10 μM), H-89 (10 μM) and BIM I (2.5, 5 μM) and then analyzed by Western blotting with anti-β-catenin antibody. (C), SW480 cells transfected with negative control siRNA (NC, 40 nM) and PKCα siRNA (40 nM) for 36 h and then analyzed by Western blotting with anti-PKCα, anti-PKCγI, anti-PKCγl and anti-PKCβII antibodies. In (A), (B) and (C), the blots were reprobed with anti-actin antibody as a loading control.

Fig. S3 The effects of TCF/β-catenin target gene on β-catenin level in Ser45-mutated colon cancer cells. (A), HCT116 and LS 174T cells were transfected with cyclin D1 promoter-pRL plasmids and incubated with A23187 (0.625, 1.25, 2.5, and 5 μM) for 12 h. Luciferase activities were measured 36 h after transfection. Results are the average of three experiments, and the bars indicate standard deviations. (B), Semiquantitative RT-PCR for cyclin D1 and GAPDH was performed with total RNA prepared from SW480 treated with A23187 for 12 h in the presence or absence of BIM I.

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