DICKKOPF-4 activates the noncanonical c-Jun-NH2 kinase signaling pathway while inhibiting the Wnt-canonical pathway in human renal cell carcinoma

Authors

  • Hiroshi Hirata MD, PhD,

    1. Department of Urology, San Francisco Veterans Affairs Medical Center and University of California at San Francisco, San Francisco, California
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  • Yuji Hinoda MD, PhD,

    1. Department of Oncology and Laboratory Medicine, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
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  • Shahana Majid PhD,

    1. Department of Urology, San Francisco Veterans Affairs Medical Center and University of California at San Francisco, San Francisco, California
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  • Yi Chen MD, PhD,

    1. Department of Urology, San Francisco Veterans Affairs Medical Center and University of California at San Francisco, San Francisco, California
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  • Mohd S. Zaman PhD,

    1. Department of Urology, San Francisco Veterans Affairs Medical Center and University of California at San Francisco, San Francisco, California
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  • Koji Ueno PhD,

    1. Department of Urology, San Francisco Veterans Affairs Medical Center and University of California at San Francisco, San Francisco, California
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  • Koichi Nakajima MD,

    1. Department of Urology, Toho University Faculty of Medicine, Tokyo, Japan
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  • Z. Laura Tabatabai MD,

    1. Department of Pathology, San Francisco Veterans Affairs Medical Center and University of California at San Francisco, San Francisco, California
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  • Nobuhisa Ishii MD, PhD,

    1. Department of Urology, Toho University Faculty of Medicine, Tokyo, Japan
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  • Rajvir Dahiya PhD

    Corresponding author
    1. Department of Urology, San Francisco Veterans Affairs Medical Center and University of California at San Francisco, San Francisco, California
    • Professor and Director, Urology Research Center (112F), Veterans Affairs Medical Center and University of California at San Francisco, 4150 Clement Street, San Francisco, CA 94121
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    • Fax: (415) 750-6639


Abstract

BACKGROUND:

To the authors' knowledge, the functional significance of the Wnt antagonist dickkopf homolog 4 (DKK4) has not been investigated previously in renal cancer.

METHODS:

The authors initially observed that the expression of DKK4 was significantly higher in renal cancer tissues compared with adjacent normal kidney tissues. To assess the function of DKK4, stable DKK4-transfected cells were established, and functional analyses were performed, including a T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter assay and tests for cell viability, colony formation, apoptosis, cell cycle, invasive capability, wound-healing capability, and in vivo tumor growth.

RESULTS:

The relative TCF/LEF activity was significantly lower in DKK4-transfected cells compared with empty vector, and nuclear β-catenin expression was decreased in DKK4 transfectants. In addition, expression levels of the β-catenin downstream effector proteins cyclin D1 and c-Myc were decreased in DKK4 transfectants. However, greater invasiveness and migration were observed in stably transfected DKK4 cells. Increased growth of DKK4-transfected tumors also was observed in nude mice. Members of the Wnt noncanonical/c-Jun-NH2 kinase (JNK) signaling pathway also were effected, such as c-Jun, which had significantly increased expression and phosphorylation in DKK4-stable transfectants, and matrix metalloproteinase-2, which had significantly increased expression in DKK4-stable transfectants.

CONCLUSIONS:

This is the first study to indicate that DKK4 expression is increased in renal cancer tissues and that DKK4 activates the noncanonical JNK signaling pathway while inhibiting the Wnt-canonical pathway. Cancer 2011. © 2010 American Cancer Society.

Renal cell carcinoma (RCC) is the third leading cause of death among urologic tumors and accounts for 2% of adult malignancies.1 Although the rate of detection of incidental RCC has increased with improved diagnostic techniques, metastatic lesions still are identified at diagnosis in approximately 30% of patients with RCC.2 Wnt/β-catenin signaling includes β-catenin-dependent (canonical) and β-catenin-independent (noncanonical) pathways.3-5 The canonical Wnt signaling pathway regulates cell fate and proliferation, and that signaling is initiated by the binding of Wnt ligands to frizzled (FZD) family receptors and the low-density lipoprotein receptor-related protein 5 and 6 (LRP5/LRP6) coreceptors. Subsequently, β-catenin interacts with members of the lymphoid enhancer factor 1/T-cell factor (LEF1/TCF) family, resulting in a functional transcription factor complex and the expression of downstream target genes.3, 4 The noncanonical Wnt ligands also bind to FZD family receptors and to the receptor tyrosine kinase-like orphan receptor 2 (ROR2) and receptor-like tyrosine kinase (RYK) coreceptors.4-7 The noncanonical signaling pathways include 3 pathways (the Wnt/Ca2+, Wnt/G protein, and Wnt/planar cell polarity [PCP] signaling pathways), and noncanonical signaling regulates cell polarity and movement.4-7 Among the 5 Wnt antagonist families (secreted frizzled-related protein [sFRP]; Wnt inhibitory factor 1 [Wif1]; and the Xenopus Cerberus, Wise, and Dickkopf [DKK] families), the DKK family consists of 4 main members (DKK1-DKK4) that contain 2 distinct cysteine-rich domains.3, 8 In our laboratory, we have studied several Wnt antagonist genes and their function in renal cancer.9-13 Previously, it was believed that DKK4 acted as an inhibitor of Wnt/β-catenin signaling in colorectal cancer.14, 15 Recently, 2 groups observed that DKK4 expression was increased in colon cancer tissues compared with matched normal colon tissues and that DKK4 was induced by activated β-catenin, although DKK4 itself significantly inhibited TCF/LEF reporter activity in colon cancer cell lines.16, 17 To our knowledge, there have been no reports regarding DKK4 and renal cancer. Therefore, first, we performed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analyses to clarify whether DKK4 is up-regulated in human renal cancer tissues compared with matched normal kidney tissues, and the results indicated that the expression level of DKK4 was significantly higher in renal cancer tissues compared with matched normal kidney tissues. Next, we used a TCF/LEF reporter assay to confirm the effect of DKK4 effect on the β-catenin-dependent pathway (canonical pathway) and observed that relative TCF/LEF activity was inhibited significantly in DKK4-transfected cells. In addition, β-catenin expression in the nucleus was decreased in DKK4-transfected renal cancer cells compared with empty vector cells, and protein expression of major TCF/β-catenin down-stream effectors (c-Myc and cyclinD1) was down-regulated in DKK4 transfectants. However, cell invasion and migratory ability were greater in DKK4-transfected renal cancer cells. On the basis of these results, we hypothesized that DKK4 may be functionally oncogenic in renal cancer despite its inhibitory effect on the β-catenin dependent pathway. To verify this hypothesis, we examined the effects of DKK4 expression on 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-)4o-sulfophenyl)-2H-tetrazolium (MTS) colony-formation, apoptosis, cell-cycle, invasion, and migration assays using DKK4-transfected renal cancer cells.

MATERIALS AND METHODS

Clinical Samples

In total, 30 patients (17 men and 13 women) with pathologically confirmed, conventional RCC were enrolled in this study (Toho University Hospital, Tokyo, Japan). The mean patient age was 60 years (range, 41-77 years) (Table 1). The patients were classified according to World Health Organization criteria and were staged according to the tumor-lymph node-metastasis (TNM) classification system, in which T refers to the size of the renal cancer and whether it has invaded nearby tissue, N refers to whether or not regional lymph nodes are involved, and M indicates whether or not there is distant metastasis. The pathology for all patients was clear cell renal carcinoma. Samples were obtained from the patients after written informed consent was obtained in Toho University Hospital.

Table 1. Characteristics of Patients With Renal Cancer (n=30)
CharacteristicNo. of Patients (%)
  1. SD indicates standard deviation.

Age: Mean±SD, y60.3±8.5
 Men17 (57)
 Women13 (43)
Tumor grade 
 16 (20)
 220 (67)
 34 (13)
Pathologic disease stage 
 I16 (53)
 II8 (27)
 III2 (7)
 IV4 (13)
Pathologic tumor classification 
 pT116 (53)
 pT29 (30)
 pT34 (13)
 pT41 (3)
 pT3+pT45 (17)
Lymph node invasion 
 Negative28 (93)
 Positive2 (7)
Distant metastasis 
 Negative27 (90)
 Positive3 (10)
Clear cell pathology30 (100)

Cell Culture

The renal cancer cell lines A-498 (American Type Culture Collection [ATCC] number HTB-44) and Caki-1 (ATCC number HTB-46) were purchased from the ATCC (Manassas, Va). The renal cancer cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum.

Plasmid Construction

Plasmids that contained the human full-length combinational DNA fragment of DKK4 (GenBank accession number NM_014420, catalog number RC221217) were purchased from Origene (Rockville, Md). This clone (pCMV6-DKK4) expresses the complete DKK4 open reading frame with a Tag (MYC/DDK) at the C terminal.

Stable Clone Establishment

To prepare stable cell lines that overexpressed DKK4, we transfected A-498 cells with the pCMV6-DKK4 expression vector that encodes DKK4 combinational DNA using FuGENE HD (Roche Diagnosis, Basel, Switzerland) according to the manufacture's instructions. Transfected cells were selected by culturing with G418 (150 μg/mL) for 2 months. Empty vector transfectants were used as controls. Single colonies of stable transfectants were isolated and expanded for further analysis based on the level of DKK4 expression. We selected the top 2 stable clones that had the highest DKK4 messenger (mRNA) expression levels compared with empty vector transfectants. We named these DKK4 Clone 1 and Clone 2 and used them for further experiments (MTS, colony formation, invasion, apoptosis, cell cycle analysis, and in vivo studies). When the cells (stable empty vector cells and stable DKK4 Clone 1 and Clone 2 cells) were confluent, they were trypsinized and resuspended in media according to the protocol. At the same time, we confirmed DKK4 expression levels in the cells before carrying out the experiments. All experiments were done in triplicate.

Total RNA and Protein Extraction

Total RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) human renal cancer tissues and matched, adjacent, noncancerous, normal tissues using an miRNeasy FFPE kit (Qiagen, Valencia, Calif) after microdissection. To digest DNA, the Qiagen RNase-Free DNase kit was used. Total RNA also was extracted from cell lines using an RNeasy mini kit (Qiagen). Cells were lysed with radioimmunoprecipitation assay buffer (Pierce, Brebieres, France) that contained protease inhibitors (Sigma Chemical Company, St. Louis, Mo). We also extracted nuclear protein and cytoplasmic protein separately using a CelLytic NuCLEAR extraction kit (Sigma-Aldrich, St. Louis, Mich) to confirm β-catenin expression. Protein quantification was done using a bicinchoninic acid protein assay kit (Pierce, Brebieres, France).

T-Cell Factor/Lymphoid Enhancer Factor Reporter Assay

To monitor the activity of Wnt/β-catenin signal transduction, we used the TCF/LEF reporter assay with TCF-reporter plasmids (TOPFLASH, which contains the wild-type TCF binding site; FOPFLASH, which contains a mutant-type TCF binding site; Millipore, Billerica, Mass). The pRL-TK renilla luciferase (Promega, Madison, Wis) was cotransfected to normalize transfection efficiency. A-498 renal cancer cells were stimulated with recombinant mouse Wnt3a protein (100 ng/mL; rmWnt3a, catalog number 1324-WN; R & D Systems, Minneapolis, Minn). FuGENE HD (Roche Diagnosis) was used for transfection according to the manufacture's instructions. All experiments were performed in triplicate. Luciferase activity was assayed at 48 hours after transfection using a dual-Luciferase reporter assay system (Promega).

Cell Viability Assay

Stably transfected DKK4 Clone 1 and Clone 2 cells or empty vector A-498 cells were maintained in medium supplemented with 150 μg/mL G418. Cell viability was measured after 4 days with MTS (CellTiter 96 Aqueous 1 Solution cell proliferation assay; Promega). To verify the effect of DKK4 on renal cancer cells, we also performed similar MTS assays using another renal cancer cell line (Caki-1). Data are reported as the mean ± standard deviation of 6 independent experiments.

Soft Agar Colony-Formation Assay

Soft agar colony formation was assayed with empty vector A-498 cells and A-498 DKK4 stably transfected cells (Clones 1 and 2) using a Cell BioLabs CytoSelect Cell Transformation Assay kit (Cell BioLabs Inc., San Diego, Calif). The cells were incubated for 7 days in a semisolid agar media before they were solubilized and detected by using the provided (3-[4,5-dimethylthiazol-2-yl]-2-5-diphenyltetrazolium bromide) solution in a microplate reader (optical density [OD], 570 nm). The absorbance was compared between empty vector cells and stably transfected DKK4 cells (Clones 1 and 2). Data are reported as the mean ± standard deviation of 8 independent experiments.

Cell Invasion Assay

A cell-invasion assay was performed with the CytoSelect 24-well Cell Invasion Assay Kit as described previously (Cell BioLabs Inc.). Empty vector A-498 cells and stable DKK4 Clone 1 and 2 cells were resuspended to the upper chamber in triplicate. To verify the effect of DKK4 on renal cancer cells, we performed invasion assays using another renal cancer cell line (Caki-1). Cells migrating through the membrane were stained and counted with a microscope. Five random fields were chosen for each membrane, and the results were expressed as migrated cells quantified at OD 560 nm after extraction.

Wound-Healing Assay

The wound-healing process begins with tissue matrix remodeling, migration, and eventual closing of the wound area. Therefore, this assay frequently is used to assess cancer cell migration. A wound-healing assay was performed with the CytoSelect 24-well Wound Healing Assay Kit as described previously (Cell BioLabs Inc.). To generate a wound field, the cells were cultured until they formed a monolayer around the insert. After removing insert, a 0.9 mm open wound field was left. Cells migrated from either side of the gap. The wound closure was monitored and the percentage closure was measured at 10 hours between empty vector cells and DKK4 Clone 1 and Clone 2 cells. To verify the effect of DKK4 on renal cancer cell, we also performed migration assays using another renal cancer cell line (Caki-1) according to the following formula: percentage closure rate (%) = migrated cell surface area/total surface area × 100.

Apoptosis and Cell Cycle Analysis

Empty vector A-498 cells and stably transfected DKK4 Clone 1 and 2 cells were washed twice in phosphate-buffered saline and trypsinized. After inactivating trypsin in complete medium, the cells were resuspended in ice-cold 1-μL binding buffer (70 μL). Annexin V-fluorescein isothiocyanate (V-FITC) solution (10 μL) and 7-aminoactinomycin D viability dye (20 μL) were added to 70 μL of the cell suspensions. After incubation for 15 minutes in the dark, 400 μL of ice-cold 1-μL binding buffer were added. The apoptotic distribution of the cells in each sample was then determined using fluorescence-activated cell sorting (FACS) (Cell Lab QUANTA SC; Beckman Coulter, Fullerton, Calif). The various cell phases were determined using a DNA stain (4,6-diamidino-2-phenylindole). Cell populations in G0/G1 phase, S phase, and G2/M phase were measured using fluorescence and were contrasted against cell volumes. Data are reported as the mean ± standard deviation of 4 independent experiments. To verify the effect of DKK4 on renal cancer cells, we performed apoptosis and cell cycle analyses using another renal cancer cell line (Caki-1).

Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction

Quantitative real-time RT-PCR was performed in triplicate with an Applied Biosystems Prism 7500 Fast Sequence Detection System using TaqMan universal PCR master mix according to the manufacture's protocol (Applied Biosystems Inc., Foster City, Calif). TaqMan probes and primers were purchased from Applied Biosystems Inc. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. Levels of RNA expression were determined using the 7500 Fast System SDS software package (version 1.3.1; Applied Biosystems Inc.).

Western Blot Analysis

Total cell protein (20 μg) was used for Western blot analysis. Samples were resolved in 4% to 20% Precise Protein Gels (Pierce, Brebieres, France) and transferred to polyvinylidene fluoride membranes (Amersham Biosciences, Fairfield, Conn). The membranes were immersed in 0.3% skim milk in Tris-buffered saline (TBS) that contained 0.1% Tween 20 for 1 hour and probed with primary polyclonal and monoclonal antibodies against cyclin D1 (catalog number 556470; BD Biosciences, San Jose, Calif), c-Myc (catalog number 551102; BD Biosciences), caspase 3 (catalog number 610322; BD Biosciences), cleaved caspase 3 (catalog number 9664; Cell Signaling Technology, Danvers, Mass), β-catenin (catalog number 9562; Cell Signaling Technology), Bax (catalog number 2772; Cell Signaling Technology), matrix metalloproteinase 2 (MMP-2) (catalog number 4022; Cell Signaling Technology), GAPDH (catalog number 2118; Cell Signaling Technology), c-Jun-NH2 kinase (JNK) (catalog number 9258; Cell Signaling Technology), phosphorylated JNK (catalog number 4668; Cell Signaling Technology), c-Jun (MAB3732; Millipore), and phosphorylated c-Jun (catalog number 9164; Cell Signaling Technology) overnight at 4°C. To confirm the expression of DKK4 in stably transfected cells, we used anti-DKK4 antibody (catalog number TA302466; Origene). Blots were washed in TBS that contained 0.1% Tween 20 and were labeled with horseradish peroxidase-conjugated secondary antimouse or antirabbit antibody (Cell Signaling Technology). Proteins were enhanced by chemiluminescence (ECL plus Western Blotting Detection System; Amersham Biosciences, Piscataway, NJ) for visualization. The reported protein expression levels are expressed relative to GAPDH levels.

In Vivo Study

Groups of 6 female nude mice (strain BALB/c nude; Charles River Laboratories, Inc., Wilmington, Mass), ages 4 to 5 weeks, received subcutaneous injections of 1 × 107 A-498 empty vector (n = 6) or A-498-DKK4 stably transfected cells (Clone 1 [n = 5] or Clone 2 [n = 5]) in the right flank area at a volume of 200 μL. Tumor size was determined using calipers once weekly for 35 days, and tumor volume was calculated on the basis of width (x) and length (y) as follows: x2y/2, where x<y. After the mice were killed, the tumors were resected and weighted. In addition, tumor tissues were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin and with DKK4 (catalog number AP1524a; ABGENT, San Diego, Calif) for histologic examination. Animal experiments were approved by the Animal Studies Subcommittee of the Veterans Affairs Medical Center (Protocol 08-003-01).

Statistical Analysis

All statistical analyses were performed using StatView software (version 5; SAS Institute Inc., Cary, NC). P values <.05 were regarded as statistically significant.

RESULTS

DKK4 Expression Levels in Renal Cancer Tissues and Adjacent Normal Kidney Tissues

We compared DKK4 mRNA expression levels between renal cancer tissues and matched, adjacent, normal kidney tissues (n = 30) using the expression level in each patient's normal tissue sample as a reference (expression = 1). DKK4 mRNA expression was high in renal cancer tissues compared with that in matched normal kidney tissues in 19 of 30 paired tissue specimens (63.3%) of normal kidney and renal cancer (Fig. 1).

Figure 1.

Dickkopf homolog 4 (DKK4) messenger RNA (mRNA) expression levels in human renal cancer tissues (T) and in adjacent normal kidney tissues (N) are illustrated in samples from 30 patients with clear cell carcinoma. When the relative expression of DKK4 mRNA (DKK4/glyceraldehyde 3-phosphate dehydrogenase) was higher in renal cancer tissues compared with matched normal renal tissues, the patient was included in the “high DKK4 expression in cancer tissues” category.

Relation Between DKK4 Expression Level and Clinical Characteristics

All patients had clear cell renal carcinoma pathology. DKK4 mRNA expression was classified into 2 categories based on real-time RT-PCR results. When the relative DKK4 mRNA expression (DKK4/GAPDH) was higher in renal cancer tissues compared with matched normal renal tissues, the patient was included in the “high DKK4 expression in cancer tissues” category (Table 2). We investigated the relation between DKK4 mRNA expression levels and clinical factors, including sex, grade, pathologic tumor classification (pT), pathologic lymph node status (pN), pathologic metastasis status (pM), and outcomes (survival and recurrence). There was no significant association between DKK4 expression and clinical parameters except for sex (Table 2).

Table 2. Association of Dickkofp-4 Expression and Renal Cancer Clinical Parameters (n=30)
 High DKK4 Expression: No. of Patients (%) 
ParameterIn Normal Tissues, N=11In Cancer Tissues, N=19P
  1. DKK4 indicates the Wnt antagonist Dickkopf homolog 4; SD, standard deviation.

Age: Mean±SD, y58.0±7.764.4±8.5.045
Sex
 Men, n=178 (47)9 (53)Reference
 Women, n=133 (23)10 (77).17
Tumor grade
 1, n=61 (17)5 (83)Reference
 2, n=209 (45)11 (55).45
 3, n=41 (25)3 (75).74
Pathologic stage
 I, n=166 (37.5)10 (62.5) 
 II, n=83 (37.5)5 (62.5) 
 I+II9 (37.5)15 (62.5)Reference
 III, n=20 (0)2 (100) 
 IV, n=42 (50)2 (50) 
 III+IV2 (33.3)4 (66.7).84
Pathologic tumor classification
 pT1, n=166 (37.5)10 (62.5) 
 pT2, n=94 (44.4)5 (55.6) 
 pT1+pT210 (40)15 (60)Reference
 pT3, n=41 (25)3 (75) 
 pT4, n=10 (0)1 (100) 
 pT3+pT41 (20)4 (80).39
Lymph node metastasis
 Negative, n=2810 (35.7)18 (64.3)Reference
 Positive, n=21 (50)1 (50).68
Distant metastasis
 Negative, n=2710 (37)17 (63)Reference
 Positive, n=31 (33.3)2 (66.7).89
Overall survival
 Alive, n=237 (30.4)16 (69.6)Reference
 Dead, n=74 (57.1)3 (42.9).19
Recurrence
 No, n=227 (31.8)15 (68.2)Reference
 Yes, n=84 (50)4 (50).36

T-Cell Factor/Lymphoid Enhancer Factor Reporter Assay

The relative TCF/LEF activity was significantly lower in DKK4 transfected cells compared with empty vector cells (Fig. 2A).

Figure 2.

Illustrated are a T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter assay, the establishment of the Wnt antagonist dickkopf homolog 4 (DKK4) transfectants (transient, stable), a cell-viability assay, and colony-formation assays in DKK4 transfectants. (A) A TCF/LEF reporter assay is illustrated (72 hours of transient transfection). TOP flash indicates a TCF-reporter plasmid that contained the wild-type T-cell factor (TCF) binding site; FOP flash, TCF-reporter plasmid that contained a mutant-type TCF binding site. (B) The expression of DKK4 was analyzed in stable transfectants (A-498 cells) and in transient transfectants (Caki-1 cells) by using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses. GAPDH indicates glyceraldehyde 3-phosphate dehydrogenase. (C) Results are shown from a cell-viability assay in empty vector and DKK4-transfected cells (A-498 cells, Clones 1 and 2; Caki-1 cells). MTS indicates 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; h, hour. (D) A colony-formation assay is illustrated in empty vector and in stably DKK4-transfected cells (Clones 1 and 2). OD indicates outer diameter.

Establishment of DKK4 Stable Clones and Effects of DKK4 on Cell Viability, Colony Formation, and Cell Invasion

After transfection of A-498 or Caki-1 cells with a pCMV6-DKK4 expression plasmid, the DKK4 expression level was confirmed by real-time RT-PCR and Western blot analyses (Fig. 2B). DKK4 protein expression was observed in both DKK4 stably transfected A-498 cells and in DKK4 transiently transfected Caki-1 cells. Then, cell viability analyses (MTS assay), colony-forming assays, and cell-invasion assays were performed using stable DKK4 transfectants of A-498 cells or transient DKK4 transfectants of Caki-1 cells. We observed enhanced growth of A-498 and Caki-1 cells after DKK4 transfection by MTS and colony-formation assays (Fig. 2C,D). DKK4 also promoted the in vitro invasion and migration ability of A-498 and Caki-1 cells (Fig. 3A,B).

Figure 3.

Illustrated are the results from (A) an invasion assay; (B) a wound-healing assay (for A-498 cells at 0 hours [h] and at 10 hours; for Caki-1 cells at 0 hours and at 8 hours); and (C) in vivo studies with empty vector and with dickkopf homolog 4 (DKK4)-transfected renal cancer cells, including (C-1) an assessment of tumor growth promotion in nude mice with empty vector and DKK4 Clones 1 and 2 and (C-2) representative nude mice with DKK4-transfected and empty vector cells: DKK4 protein expression was confirmed by immunohistochemistry (IHC). OD indicates outer diameter.

In Vivo Study

A-498 cells that were stably transfected with either empty vector or the DKK4 gene (stable Clones 1 and 2) were injected subcutaneously into the right flank of nude mice. Tumor growth was promoted significantly in the mice that had DKK4-transfected cells compared with those that had empty vector-transfected cells (Fig. 3C). We performed immunohistochemistry for DKK4 in grown tumors and confirmed that DKK4 protein was highly expressed in DKK4-transfectant tumors (Fig. 3C).

Apoptosis, Cell Cycle Analyses, and Cleaved Caspase 3 Expression

Apoptosis and cell cycle analyses were performed to investigate whether DKK4 overexpression affected these parameters in renal cancer cells. However, no significant difference was observed in the number of apoptotic cells between DKK4-transfected Clones 1 and 2 and control cells (Fig. 4A,B). There was no significant difference observed in any of the cell cycle phases (G0/G1 phase, S phase, or G2/M phase) between DKK4 transfectants and controls (data not shown). To verify the effect of DKK4 on renal cancer cells (A-498 cells), we performed apoptosis assays using another renal cancer cell line (Caki-1). Similar what we observed in A-498 cells, no significant difference in apoptosis was observed in DKK4 transiently transfected Caki-1 cells. We examined the expression of procaspase 3 and cleaved caspase 3 in DKK4 transfectants (stable Clone 2) and in controls using Western blot analysis, but no difference was observed in procaspase expression between the transfectants, and no cleaved caspase 3 expression was observed in either transfectant (Fig. 4C). This result was also verified in another renal cancer cell line (Caki-1 cells).

Figure 4.

Apoptosis assays in stable (A-498)-transfected or transient (Caki-1)-transfected empty vector and dickkopf homolog 4 (DKK4) renal cancer cells are illustrated along with protein expression of apoptosis markers. (A) Flow cytometry analyses of apoptosis are shown in empty vector, in stable DKK4 Clones 1 and 2 cells, and in transient DKK4 transfectants of Caki-1 cells. Annexin V-fluorescein isothiocyanate and 7-aminoactinomycin D levels were measured by flow cytometry. N.S. indicates nonsignificant. (B) Representative results also are shown (data are the mean ± standard deviation of 4 independent experiments). (C) Protein expression levels of procaspase 3 and cleaved caspase 3 are illustrated in empty vector and stable DKK4 transfectants (A-498 cells) and in DKK4 transient transfectants (Caki-1 cells). GAPDH indicates glyceraldehyde 3-phosphate dehydrogenase.

Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction and Western Blot Analyses in DKK4-Overexpressing Cells

We observed decreased TCF/LEF reporter activity in DKK4 transfectants, suggesting that DKK4 affects the Wnt canonical pathway. Therefore, we examined the expression of TCF/LEF down-stream effectors, such as cyclin D1 and c-Myc, in Western blot analyses (Fig. 5A). We observed decreased expression of these proteins in DKK4 stable transfectants. In addition, we compared the protein expression of β-catenin in cytoplasmic and nuclear fractions between empty vector and DKK4 transfectants. Consistent with the TCF/LEF reporter assay results and the decreased TCF/LEF down-stream effector expression, β-catenin expression in the nucleus was decreased significantly in DKK4 transfectants (Fig. 5B). In contrast to the β-catenin-dependent pathway protein expression, we observed tumor growth promotion (in vitro and in vivo) and increased invasion and migration ability in DKK4-transfected cells, suggesting that DKK4 influenced other cancer pathways. Therefore, we investigated the expression of genes in the β-catenin-independent pathway Wnt-JNK, and observed that c-Jun expression and phosphorylation were increased significantly in DKK4 stable transfectants, as indicated in Figure 5C. In addition, 1 of the major down-stream effectors, MMP-2, also had increased expression in DKK4 transfectants.

Figure 5.

Protein expression of signaling pathway genes is illustrated in empty vector and in stable dickkopf homolog 4 (DKK4)-transfected A-498 cells. (A) Wnt-canonical pathway-related gene expression is shown (Western blot assay). Bax indicates BCL2-associated X protein; c-Myc; v-myc myelocytomatosis viral oncogene homolog; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (B) β-Catenin expression is shown in cytoplasm (C) and nuclei (N). (C) Noncanonical pathway gene expression is shown (Western blot assay). JNK indicates c-Jun-NH2 kinase; p-JNK, phosphorylated JNK; p-c-Jun; phosphorylated c-Jun; MMP-2, matrix metalloproteinase-2.

DISCUSSION

In this study, we observed that the expression of DKK4 was up-regulated in renal cancer tissues compared with matched, normal kidney tissues. These results suggested that DKK4 may have oncogenic functions in renal cancer. Recently, several reports have indicated that DKK4 expression was high in cancer tissues compared with normal tissues in patients with colon cancer.16, 17 Our current results are consistent with those previous reports.16, 17

DKK4 is 1 of the Wnt antagonist genes and generally has been regarded as an inhibitor of the Wnt canonical pathway.14 Initially, to investigate the relation between DKK4 and the canonical pathway, we performed TCF/LEF reporter assays and observed that DKK4 inhibited TCF/LEF reporter activity and that β-catenin expression in the nuclear protein fraction was down-regulated in DKK4-transfected renal cancer cells. Expression of c-Myc and cyclin D1, which are major TCF/LEF down-stream effector proteins, also was down-regulated in DKK4-transfected cells. This finding means that DKK4 may be involved in the β-catenin-dependent pathway and that DKK4 is a β-catenin-dependent pathway inhibitor, as described in previous reports in patients with colon cancer.16, 17

In contrast to the inhibitory effect of DKK4 on the β-catenin-dependent Wnt pathway, DKK4 promoted cell proliferation, invasion, and migration in renal cancer cells (the A-498 and Caki-1 cell lines). We also observed that DKK4 promoted the in vivo tumor growth of A-498 cells. The Wnt signaling pathway involves β-catenin-dependent (canonical) and β-catenin-independent (noncanonical) pathways.4, 18 The noncanonical pathway includes 3 pathways (the Wnt/Ca2+, Wnt/G protein, and Wnt/PCP signaling pathways).5 These pathways primarily regulate cell movement.5 The role of noncanonical pathways in renal cancer is less understood. Among these, it is believed that the JNK pathway is involved in the noncanonical pathway (the Wnt/PCP signaling pathway).5 Noncanonical Wnt signals are transduced through FZD receptors, and small G-proteins (RhoA, the ras-related protein Rac, cell division cycle 42 [CDC42]) and JNK are involved with dishevelled (Dvl)-dependent effector molecules.4 Generally, Rac1 mediates JNK activation. However, the exact role of JNK in Wnt-mediated signaling remains unknown.19-21 Recently Fukukawa et al reported that FZD homologue 10 was highly expressed in synovial sarcoma, caused the activation of the noncanonical Dvl-Rac1-JNK pathway, and also caused destruction of the actin cytoskeleton structure through the down-regulation of RhoA activation.22 The activation of JNK is associated with apoptosis in response to various cellular stresses and antiapoptotic and growth-promoting effects.23 Regarding cancer, several reports on JNK-related apoptosis in response to cellular stress have been published.24-28 In renal cancer, JNK-related apoptosis also has been reported.29, 30 Among DKK family members, DKK3 induces apoptosis in several cancers (prostate, testicular, and breast cancers) through the activation of JNK.24-26 DKK1 also plays an important role in the induction of apoptosis through the activation of JNK in placental choriocarcinoma and mesothelioma.27, 28 Regarding DKK4, to our knowledge, there have been no reports demonstrating its relation to the JNK pathway in cancer. Previous studies in colon cancer indicated that ectopic DKK4 expression increased the migration and invasion properties of colon cancer cells, and our results are consistent with those reports.16, 17 Khatlani et al reported that JNK was activated in nonsmall cell lung cancer biopsy samples and promoted oncogenesis.23 Wang et al also observed that the overexpression of active JNK in human breast cancer cells did not cause apoptosis but enhanced cell migration and invasion.31 In the current study, we examined apoptosis by using FACS and investigated active caspase 3 protein expression by using Western blot analysis in A-498 cells that were stably transfected with either DKK4 or empty vector. We did not observe a significant change in apoptotic cell numbers between DKK4 and control transfectants, and there was no cleaved caspase 3 expression in these cells. These results suggest that DKK4 is not associated with the induction of apoptosis. However, we did observe the significant promotion of cell proliferation and invasion in vitro. We also observed that DKK4 transfectants displayed enhanced tumor growth in nude mice compared with control transfectants. In addition to the phosphorylation of JNK, the expression and phosphorylation of c-Jun were increased in DKK4-transfected A-498 cells. This result is similar to that reported by Wang et al.31

The JNK pathway includes several down-stream proteins. The MMP family plays an important role in cell invasion and metastasis in renal cancer and is mediated by several intracellular pathways.32-35 Fromigue et al reported that atorvastatin (a member of the drug class known as statins) reduced MMP expression or activity in invading osteosarcoma cells and that the inhibition of JNK reduced MMP-2 activity.34 Inamoto et al reported that gamma-aminobutyric acid (GABA) stimulation significantly increased the expression of MMP-2 and MMP-9 and also increased the invasive activity of renal cancer cells.35 GABA stimulation promoted the phosphorylation of microtubule-associated protein kinases, including extracellular signal-regulated kinase 1/2, JNK, and p38.35 In the current study, we observed increased MMP-2 expression in DKK4-transfected A-498 cells. Thus, our results suggest that DKK4 promotes the noncanonical pathway by activating JNK, resulting in up-regulation of c-Jun expression and phosphorylation and increasing MMP-2 expression. Therefore, the up-regulation of MMP-2 may be involved in the increased growth of DKK4-transfectant tumors in nude mice. Although the exact molecular mechanism with which DKK4 regulates MMP-2 expression is unknown, our findings indicate that further studies will be warranted on the roles of DKK4 in renal cancer invasion and metastasis.

In conclusion, to our knowledge, this is the first report documenting higher DKK4 expression in renal cancer tissues compared with matched, normal kidney tissues. We examined the role of DKK4 in the canonical and noncanonical Wnt pathways and observed that DKK4 inhibited the canonical pathway. Although DKK4 did not induce apoptosis, it promoted renal cancer cell proliferation, invasion, and migration probably through the noncanonical JNK pathway, which also increased MMP-2 expression. Thus, the current findings contribute important information about the role of DKK4 in renal cancer cells.

Acknowledgements

We thank Dr. Roger Erickson for his support and assistance with preparing this article.

CONFLICT OF INTEREST DISCLOSURES

This study was supported by grants RO1CA130860, RO1CA111470, T32-DK07790 from the National Institutes of Health; by the Veterans Affairs Research Enhancement Award Program (REAP); by Merit Review grants, and by the Yamada Science Foundation.

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