SEARCH

SEARCH BY CITATION

Keywords:

  • DKK-1;
  • wnt signal;
  • pancreatic cancer;
  • ductal adenocarcinoma

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

The protein products of the Dickkopf (DKK) genes are antagonists of Wnt glycoproteins, which participate in tumor development and progression by binding to frizzled receptors. In this study, the expression of DKK-1 was analyzed in a panel of 43 human cultured carcinoma cell lines. DKK-1 expression was consistently and significantly upregulated in pancreatic carcinoma cell lines. Low level of DKK-3 expression was also seen. In contrast, the expression of DKK-2 and -4 was not detectable in most pancreatic carcinoma cell lines. The overexpression of DKK-1 was confirmed in surgically resected human pancreatic cancer tissues, in which the mRNA level was evaluated in paired samples from cancerous and noncancerous pancreatic tissues. In ductal adenocarcinomas (23 cases), DKK-1 mRNA levels were significantly upregulated compared to corresponding noncancerous tissues in a statistically significant level. To test the biological role of DKK-1 in pancreatic carcinoma cells, we performed a knockdown of DKK-1 in SUIT-2 human pancreatic adenocarcinoma cell line and S2-CP8, its metastatic subline, using a retroviral short hairpin RNA expression vector. DKK-1 knockdown resulted in reduced migratory activity of SUIT-2 in vitro. The in vitro growth rate and Matrigel invasion were also suppressed by DKK-1 knockdown in S2-CP8 cells. Collectively, the evidence suggests that, despite of its presumed antagonistic role in Wnt signaling, DKK-1 may have a role in the aggressiveness of pancreatic carcinoma cells and could, therefore, serve as a novel biomarker of pancreatic cancer.

The Dickkopf (DKK) genes are Wnt antagonists that were originally identified as inducers of head formation in Xenopus.1 Wnt glycoproteins participate in tumor development and progression by binding to frizzled receptors and signaling through the canonical and noncanonical Wnt pathways.2 The DKK gene family consists of DKK-1, -2, -3, -4, and a unique DKK-3-related gene soggy.2 The expression of these genes is temporally and spatially regulated, and all DKK proteins show distinct and elevated expression patterns in tissues that mediate the epithelial-mesenchymal transformation.3 This finding suggests that they may participate in the epithelial to mesenchymal transition that is important not only in embryogenesis, but also in cancer progression.4 DKK-1 is a secreted protein that has been clearly defined as a direct inhibitor of Wnt binding to LRP5/6 coreceptors of frizzled.2 Evidence for the potential involvement of DKK-1 inactivation in human cancers is accumulating. The DKK-1 gene is frequently hypermethylated in colorectal cancers, whereas overexpression of DKK-1 reduced the growth of colorectal carcinoma cells.5, 6 The expression of DKK-1 is also reportedly downregulated in melanoma cells,7 lung cancer cells with neuroendocrine differentiation8 and in a metastatic subline of hepatocellular carcinoma cell line.9 In contrast, overexpression of DKK-1 has also been found in some tumors, such as Wilms' tumor, hepatoblastoma, hepatocellular carcinoma and ovarian endometrioid adenocarcinoma.10–13 Moreover, gene expression profiles have revealed that DKK-1 is overexpressed in nonsmall cell lung carcinoma and esophageal squamous cell carcinoma, serving as a serologic and prognostic biomarker.14 DKK-1 may also have roles in bone metastases of breast and prostatic carcinomas.15, 16 Therefore, the function and role of DKK-1 in cancer appears to depend on the histological types of the cancer cells and the tissue microenvironment.

Pancreatic cancer has the worst prognosis of any major malignancy and its incidence is increasing.17 Delayed diagnosis and an intrinsic biological aggressiveness contribute to the abysmal prognosis of this disease. The identification of sensitive biomarkers and further understanding of the cellular biology of pancreatic ductal adenocarcinoma (DAC) cells are required. In this study, expression analysis of a panel of human carcinoma cell lines showed that DKK-1 was consistently upregulated in pancreatic carcinoma cell lines. Using surgically resected tissues, we confirmed the upregulation of DKK-1 mRNA levels in DAC of the pancreas. We also utilized a short hairpin RNA (shRNA)-based knockdown system to examine the role of DKK-1 in cultured pancreatic carcinoma cells.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

Cell culture and cell growth assay

Human pancreatic adenocarcinoma cell line SUIT-218 and its metastatic sublines, S2-VP10 and S2-CP8,19 were kindly provided by Dr. Takeshi Iwamura (Junwakai Memorial Hospital). S2-CP8 and S2-VP10 were established by Cutis-Pulmonary metastasis-culture (8 times) and Vein-Pulmonary metastasis-culture (10 times), via injection of parental SUIT-2 cells subcutaneously (S2-CP8) or intravenously (S2-VP10) into nude mice.19 Other 6 human pancreas cancer cell lines (SUIT-4, AsPC-1, MIA PaCa-2, PANC-1, HPAF and BxPC-3), 13 colon cancer cell lines (RCM-1, RCM-2, RCM-3, CaCo-2, HCT116, SW837, DLD-1, LoVo, WiDr, Colo205, COCM-1, CaR-1 and Colo320DM), 11 lung cancer cell lines (LC-1/sq, RERF-LC-AI, LC2/ad, RERF-LC-KJ, HLC-1, A549, PC14, Lu135, Lu139, T3M-11 and MS-1), 3 gastric cancer cell lines (MKN28, MKN45 and KATO III), 1 duodenal cancer cell line (HUTU80), 2 hepatocellular carcinoma cell lines (HepG2 and HuH7), 3 breast cancer cell lines (MCF-7, BT-20 and SKBR3), 1 cervical cancer cell line (HeLa), 1 renal cell carcinoma cell line (MRT-1) and 1 prostate cancer cell line (PC-3) were used in this study. RCM-1, RCM-2, RCM-3, COCM-1, LC-1/sq, LC-2/ad and MRT-1 were established in our laboratory. SUIT-4, HPAF, BxPC-3, HUTU 80, MCF-7, BT-20 and SKBR-3 were also provided by Dr. T. Iwamura, Junwakai Memorial Hospital. HLC-1 was kindly provided by the Department of Pathology, Keio University. LoVo, Hela, RERF-LC-AI, RERF-LC-KJ, A549, PC-14, Lu139, MS-1, T3M11 and Lu139 were obtained from Riken Cell Bank (Tsukuba, Japan). WiDr, CaCO-2, SW837, Colo205, KATO III, HepG2, HuH7, AsPC-1, PANC-1, MIA PaCa-2 and PC-3 were obtained from Dainihon Seiyaku (Osaka, Japan). IBL (Fujioka, Japan) provided MKN-28 and -45. DLD-1, CaR-1 and Colo320DM were received from the Health Science Research Resources Bank (Osaka, Japan). HCT116 was purchased from the American Type Culture Collection (Manassas, VA). The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) or a mixture of RPMI 1640 and Ham's F-12 containing 10% fetal bovine serum (FBS), streptomycin (100 μg/ml), penicillin G (100 U/ml) and 20 mM N-2-hydroxyethyl piperazine-N′-2-ethane sulfonic acid, pH 7.25, at 37°C in a humidified atmosphere containing 5% CO2.

Preparation of tissue samples

All fresh tumor tissues were obtained from surgical specimens of patients with pancreatic tumors in the University of Miyazaki Hospital, Miyazaki, Japan. Informed consent was obtained from the patients and the protocol was approved by the ethical board of the Faculty of Medicine, University of Miyazaki. A total of 36 cases were evaluated in this study. Tissue samples were crashed and frozen in liquid nitrogen in the operating room as soon as the tumor was resected, and stored at −80°C freezer until analysis. Total cellular RNA was extracted with Trizol reagent (Gibco BRL, Gaithersburg, MD), followed by DNase I (Roche Applied Science, Indianapolis, IN) treatment and phenol-chloroform-isoamylalcohol extraction.

Immunohistochemistry and in situ hybridization

Formalin-fixed and paraffin-embedded sections were subjected to antigen retrieval by autoclaving for 5 min in 10 mM citrate buffer, pH 6.0. After peroxidase blocking, the sections were blocked in 3% bovine serum albumin (BSA) and 10% goat serum in phosphate-buffered saline (PBS) for 1 hr at room temperature. Subsequently, the sections were incubated with anti-DKK-1 rabbit polyclonal antibody (5 μg/ml in 1% BSA/PBS; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. Negative controls consisted of an omission of the primary antibodies. The sections were rinsed in PBS and incubated with Envision-labeled polymer (DAKO) for 30 min at 37°C. After washing, the sections were visualized with nickel, cobalt-3,39-diaminobenzidine (Pierce, Rockford, IL) and counter stained with haematoxylin. For in situ hybridization (ISH) study, formalin-fixed and paraffin-embedded sections (4-μm thick) were fixed in 4% paraformaldehyde/PBS, dehydrated and used for ISH reaction with a fully automated ISH apparatus (Ventana, Yokohama, Japan) as described previously. A 708-bp cDNA fragment corresponding to bases 182–889 of the human DKK-1 cDNA sequence20 was used as a template to generate digoxigenin-labeled RNA probes. The same amount of each antisense or sense probe (200 ng/slide) was used. The reaction was visualized with BlueMap Kit (Roche, GmbH, Penzberg, Germany) and counterstained with nuclear fast red.

Knockdown of DKK-1

The knockdown vector was constructed using an shRNA expression retroviral vector pSINsi-hU6 (TAKARA Bio, Shiga, Japan) as described previously.21 The target sequence of the DKK-1 gene was 5′-GGAATAAGTACCAGACCATTG-3′ and its scramble control was 5′-GAGGAATCATCAGACGCATTA-3′. For infection of retroviral vectors, Amphopack-293 packaging cells cultured in 6-well plates were incubated with 1 μg of recombinant retroviral vector and 3 μl of TransFectin (Bio-Rad, Hercules, CA) for 12 hr. After 48 hr of cultivation, the supernatant containing the retroviral particles was collected, filtered through a 0.45-μm filter, and used to infect target cells. Cultured SUIT-2 cells were trypsinized and resuspended in the viral supernatant in the presence of 5 μg/ml of polybrene (Aldrich, Milwaukee, WI) for 12 hr. The cells were then incubated with a 1:1 mixture of fresh medium and viral supernatant with magnetofection reagent (CombiMag™, OZ Biosciences, Marseille, France), and placed on a magnetic plate for an additional 12 hr. This process was repeated 3 times. The transfected cells were subcultured at an appropriate density in fresh DMEM containing 0.5 mg/ml G418 (Nacalai Tesque, Kyoto, Japan). G418-resistant cell pools were readily established within 2 weeks.

Reverse-transcriptase/polymerase-chain reaction (RT-PCR) and quantitative real-time RT-PCR

Three micrograms of total RNA was reverse-transcribed with a mixture of oligo dT and random primer using 200 units of SuperScript™ Reverse Transcriptase (Gibco BRL), and 1/90 of the resultant cDNA was processed for each PCR reaction with 0.1 μM of both forward and reverse primers and 2.5 units of HotStar™ Taq DNA Polymerase (Qiagen, Tokyo, Japan). The PCR products were analyzed by 1.5% agarose gel electrophoresis. The following primers were used for conventional RT-PCR: DKK-1 (247-bp product), 5′-AGGAAG CGCCGAAAACGCTGCATG-3′ (forward) and 5′-AGGCACAGTCTGATGACCGGAGAC-3′ (reverse); DKK-2 (334 bp), 5′-GCAGTGATAAGGAGTGTGAAGTT-3′ (forward) and 5′-AATGCAGTCTGATGATCGTAGGC-3′ (reverse); DKK-3 (349 bp), 5′-AGGCAGAAGAA GCTGCTGCTAA-3′ (forward) and 5′-AGCTGGTCTCCACAGCACTCACT-3′ (reverse); DKK-4 (315 bp), 5′-ACGGACT GCAATACCAGA AAGTT-3′ (forward) and 5′-CAAAGTCCAG GGCCACAGTCAA-3′ (reverse); c-Myc (338 bp) 5′-TCCAGC TTG TACCTGCAGGATCTGA-3′ (forward) and 5′-CCT CCAGCAGAAGGTGATCCAGACT-3′ (reverse); cyclin D1 (319 bp) 5′-GGTCCTGCCGTCCATGCGGA-3′ (forward) and 5′-CGGGGT CATTGCGGCCAGGT-3′ (reverse); glyceraldehydes-3-phosphate dehydrogenase (GAPDH) (300 bp), 5′-GTGAAGGTCGGAGTCAACG-3′ (forward) and 5′-GGTGAAGACGCCAGTGGACTC-3′ (reverse).

Quantitative real-time RT-PCR using SYBR green was performed in a LightCycler™ (Roche Applied Science) according to the manufacturer's instructions. Levels of internal control, β-actin mRNA, were measured as described previously.21

In vitro motility and invasion assays

A monolayer wounding (scratch) assay was performed to evaluate the in vitro motility of cultured cells. Cells were allowed to form a monolayer on a culture dish, and a wound was made by scratching the monolayer with a pipette tip. After the scratched cells were removed, the cells were cultivated for the indicated time periods. In vitro invasive capability was evaluated using the Matrigel invasion assay performed with Chemotaxicells (polycarbonate filter, pore size 8 μm) (Kurabo, Osaka, Japan) coated with 25 μg/filter of Matrigel (Gibco BRL). Cells (1 × 105) in 100 μl of DMEM/0.1% BSA were placed in the upper compartment and incubated for 48 hr. As a chemoattractant, 1% FBS was added to the lower compartment. After incubation, the cells on the upper surface of the filter were wiped off with a cotton swab, and the cells on the lower surface were stained with hematoxylin. Migration activity on Type IV collagen was quantified by counting the cells in 10 randomly selected fields (200× original magnification).

Immunoblot analysis

To detect DKK-1 protein, subconfluent cultured cells were washed 3 times with PBS and cultured with serum-free DMEM for 72 hr. The serum-free conditioned media were harvested and dialyzed against 2.5 mM Tris-HCl (pH 7.6). The dialyzed samples were lyophilized and stored at −80°C until analyzed. Equal amounts of proteins were electrophoresed by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, transferred to Immobilon membrane (Millipore, Bedford, MA), and detected by immunoblotting as described previously.21 Anti-human DKK-1 rabbit polyclonal antibody (Santa Cruz Biotechnology) was used as the primary antibody. To evaluate β-catenin and glycogen synthase kinase-3β (GSK-3β), cellular proteins were extracted at 80% confluency with cell lysis buffer (CelLytic™-M; Sigma-Aldrich, St. Louis, MO) supplemented with protease inhibitor and phosphatase inhibitor mixtures (Sigma-Aldrich) on ice. To examine the cellular localization of β-catenin, cellular proteins were extracted and fractionated in cytosolic and nuclear fractions using CelLytic NuCLEAR Extraction Kit (Sigma-Aldrich). After centrifugation (16,000g for 10 min), equal amounts of proteins were separated by SDS-PAGE and subjected to immunoblot analysis as described.21 After blocking with 5% phosphatase-free BSA in Tris-buffered saline (TBS) with 0.05% Tween 20 (TBS-T), the membrane was incubated with primary antibody diluted in Can Get Signal™ (TOYOBO, Osaka, Japan) solution1 for 2 hr at room temperature. The following primary antibody were used: anti-β-catenin (Santa Cruz Biotechnology) and anti-GSK-3β (BD Transduction Laboratories, San Jose, CA) mouse monoclonal antibodies; anti- phospho-β-Catenin, anti-phospho-GSK-3β, anti-c-Jun N-terminal kinase (JNK), anti-phospho-JNK, anti-lamin A/C, anti-heat shock protein 70 (HSP70) rabbit polyclonal antibodies (Cell Signaling Technology, Danvers, MA). Then, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies diluted in Can Get Signal solution for 1 hr at room temperature. The labeled proteins were visualized as described.21

Subcutaneous injection of tumor cells in nude mice

All animal work was carried out under protocols approved by the University of Miyazaki Animal Research Committee, in accordance with international guiding principles for biomedical research involving animals. Injections of 5 × 106 cells/0.2 ml PBS were subcutaneously administered at the abdominal flank of 6-week-old male nude mice (Balb/cAJc1-nu). Tumor volume was estimated by the formula V = L × W × W/2 (V: volume [mm3]; L: length [mm]; W: width [mm]). All mice were observed everyday and sacrificed at 8 weeks postimplantation. Their lungs were excised and fixed in 4% paraformaldehyde for 24 hr. The number of metastatic lesions was counted in the largest cross-sectional specimens from both lungs of each mouse.

Statistics

Comparisons between 2 paired groups or 2 unpaired groups were performed with the Wilcoxon signed rank test or Mann–Whitney U-test, respectively, using Statview 5.0 (Brainpower, Calabasas, CA). Significance was set at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

Consistent upregulation of DKK-1 expression in cultured human pancreatic carcinoma cell lines

DKK-1 expression was characterized in a panel of cultured human carcinoma cell lines; lung (11 lines), uterine (1), colorectal (13), gastroduodenal (4), pancreatic (7), 2 hepatocellular (2), breast (3), prostate (1) and renal (1) (Fig. 1). Among the 43 cell lines examined, DKK-1 mRNA was detectable in 27, and its expression level was distinct in 20. Remarkably, all pancreatic carcinoma cell lines (7/7) showed distinct levels of DKK-1 mRNA, suggesting that DKK-1 may be consistently upregulated in pancreatic cancer (Fig. 1). Pulmonary nonsmall cell lung carcinoma cell lines also showed relatively consistent DKK-1 expression. In contrast, none of the small cell lung carcinoma or gastric carcinoma cell lines expressed DKK-1 mRNA (0/4 and 0/3, respectively). Detection was also low in colorectal carcinoma cell lines (5/13).

thumbnail image

Figure 1. DKK-1 expression in human tumor cell lines (RT-PCR: 30 cycles of amplification). GAPDH mRNA was used as the internal control.

Download figure to PowerPoint

The expression of the other DKK family genes such as DKK-2, DKK-3 and DKK-4 was tested in a panel of human pancreatic carcinoma cell lines (Fig. 2). In this panel, in addition to 7 pancreatic cell lines, 6 subclones of SUIT-2 with different histopathological differentiation and metastatic capabilities were also included.19, 22 Notably, all pancreatic carcinoma cell lines examined abundantly expressed DKK-1 mRNA. In contrast, DKK-2 and DKK-4 expression was hardly detectable in most cell lines. A low level of DKK-3 mRNA was observed in 68% (8/13) of pancreatic cancer cell lines. These data suggested that DKK-1 may have an important role in the biology of pancreatic cancer and may also serve as a novel biomarker of this disease.

thumbnail image

Figure 2. Expression of DKK-1, -2, -3 and -4 in human pancreas carcinoma cell lines. Representative results of 2 RT-PCR experiments (30 cycles and 34 cycles of amplification) are shown.

Download figure to PowerPoint

Upregulation of DKK-1 in pancreatic cancer tissue in vivo

Next, we asked whether DKK-1 is also upregulated in pancreatic cancer tissue in vivo by examining DKK-1 mRNA levels in surgically resected pancreatic cancer tissues and corresponding normal tissues. Among 23 cases of invasive DAC (6 cases of well-differentiated tubular adenocarcinoma, 14 cases of moderately differentiated tubular adenocarcinoma, 2 cases of poorly differentiated tubular adenocarcinoma and 1 case of metastatic liver tumor from pancreatic moderately differentiated tubular adenocarcinoma), 17 showed significantly upregulated expression of DKK-1 in cancer tissue compared with corresponding normal tissue (Fig. 3a). Quantitative real-time RT-PCR analyses also confirmed this trend, and the difference was statistically significant (Fig. 3c). Intraductal papillary mucinous carcinoma (IPMC) (4 cases) also showed increased DKK-1 expression (Figs. 3b and 3c). We also examined DKK-1 mRNA levels in 2 cases of mucinous cystadenocarcinoma (MCAC) and 1 case showed increased DKK-1 expression (Fig. 3b). In contrast, solid pseudopapillary neoplasm (SPN) (1 case), serous cystadenoma (SCA) (2 cases) and endocrine carcinoma (EC) (4 cases) did not show enhanced expression of DKK-1 (Figs. 3b and 3c). Although there may be a trend that advanced DAC (Stage III/IV) showed higher DKK-1 mRNA levels than Stage I/II, the difference was not statistically significant (Fig. 3d).

thumbnail image

Figure 3. DKK-1 expression in pancreatic tumor tissues and corresponding normal pancreatic tissues. (a) RT-PCR (30 cycles) of pancreatic ductal adenocarcinoma (DAC, 23 cases). N, normal tissue; T, tumor tissue. (b) RT-PCR (30 cycles) of intraductal papillary mucinous carcinoma (IPMC, 4 cases), mucinous cystadenocarcinoma (MCAC, 2 cases), endocrine carcinomas (EC, 4 cases) and solid and pseudopapillary neoplasm (SPN, 1 case). (c) Quantitative real-time RT-PCR analysis of the tumor tissues. The data were normalized by β-actin mRNA level and the ratio of DKK-1 in tumor tissue to that in corresponding normal tissue is indicated as a logarithmic scale. SCA, serous cystadenoma. Values are mean of log10T/N ± standard error. *, DKK-1 level in the tumor tissue is significantly higher than that in the corresponding normal tissue, p < 0.01 (Wilcoxon signed rank test). (d) Relationship between DKK-1 expression (log10T/N) and disease stage.

Download figure to PowerPoint

To verify the overexpression of DKK-1 in pancreatic DAC cells in vivo, we performed an immunohistochemical analysis using surgically resected DAC tissues. Immunoreactive DKK-1 proteins were, in fact, overexpressed in DAC cells compared with non-neoplastic ductal epithelium (Figs. 4a and 4b). This finding was further confirmed by ISH for DKK-1 mRNA in the serial sections (Figs. 4c and 4d). The specific mRNA signal was observed in DAC cells but not in normal ductal epithelial cells, showing similar staining pattern to that in the immunohistochemistry. The DAC cells at the invasion front tended to show increased levels of DKK-1 mRNA, and no signal could be identified in the endocrine cells of Langerhans islands (Figs. 4e and 4f).

thumbnail image

Figure 4. Expression of DKK-1 in pancreatic DAC cells in vivo. Serial tissue sections of 2 pancreatic DAC cases (ad and ef ) were stained with hematoxylin and eosin (a, e), immunostained for DKK-1 (b), and used for in situ hybridization with DKK-1 sense probe (negative control) (c) and antisense probe (d, f ). Significantly increased DKK-1 protein and mRNA levels were observed in carcinoma cells compared with non-neoplastic epithelium of the pancreatic duct (duct) (ad ). DAC cells at the invasion front also showed enhanced DKK-1 mRNA levels (e, f ). islet, Langerhans islet. Bar, 50 μm.

Download figure to PowerPoint

Reduced cellular invasiveness by knockdown of DKK-1 in human pancreatic carcinoma cell lines

To test the biological role of DKK-1 in pancreatic carcinoma cells, we attempted to knockdown of DKK-1 in the human pancreas carcinoma cell line SUIT-2 using a retroviral vector expressing DKK-1 shRNA (Fig. 5a). A significant reduction in the levels of both DKK-1 protein and mRNA was observed in DKK-1 knockdown cell pools (SUIT2-KD) compared with controls treated with scrambled shRNA (SUIT2-scr). Subsequent real-time RT-PCR revealed that the mRNA level of DKK-1 in SUIT2-KD was suppressed by 17% compared with levels in SUIT2-scr (not shown). The knockdown of DKK-1 did not alter cellular proliferation (Fig. 5b). However, migratory activity was suppressed, as judged by a monolayer wounding assay (Fig. 5c). Matrigel invasion assay was also performed using SUIT-scr and SUIT2-KD, but invaded cells through Matrigel were hardly visible in both cases (data not shown).

thumbnail image

Figure 5. Effect of DKK-1 knockdown on SUIT-2 cells. (a) Immunoblot (upper panel) and RT-PCR (lower panel) analyses. Both DKK-1 protein and mRNA levels were significantly decreased in a SUIT-2 cell pool infected with a DKK-1 shRNA retroviral vector (SUIT2-KD) compared with parental (SUIT-2) and scrambled control (SUIT2-scr) cells. As a control, U251 human glioblastoma cells with engineered overexpression of DKK-1 in a plasmid vector (U251 DKK-1) were also used in the analyses. (b) Knockdown of DKK-1 did not alter the in vitro cellular growth of SUIT-2 cells. (c) Cell scratch assay. The confluent monolayer was wounded by scratching with a pipette tip. Photographs were taken of the wound 0, 4 and 6 hr after scratching. Knockdown of DKK-1 suppressed cellular restitution of the wounded area. The ratio (%) of nonclosed area at 4 hr to that at 0 hr was calculated and shown as a bar graph (n = 4). Values are mean ± standard deviation. *p < 0.05 (Mann–Whitney U-test).

Download figure to PowerPoint

To test the effect on cellular invasiveness, DKK-1 gene silencing was also performed using S2-CP8, a metastatic subline of SUIT-2 established by Cutis-Pulmonary metastasis-culture (8-cycle selection).19 S2-CP8 cells show loss of E-cadherin expression and are more invasive than SUIT-2.23 DKK-1 was more highly expressed in S2-CP8 than in SUIT-2 cells (Fig. 2). Both mRNA and protein levels of DKK-1 were significantly downregulated by shRNA (Fig. 6a), and real time RT-PCR revealed a 72.3% reduction in mRNA levels (not shown). Interestingly, in vitro growth rate in serum (10% FBS)-containing medium was significantly suppressed by DKK-1 silencing in S2-CP8 (Fig. 6b). As S2-CP8 cells easily lost their cohesiveness and were not suitable for the monolayer wounding assay, we used the Matrigel invasion assay to test the effect of DKK-1 knockdown on cellular invasion. In this assay, the cells were cultured in serum-free medium containing 0.1% BSA in order to minimize the growth advantage of the control (S2CP8-scr) cells and 1% FBS was used as a chemoattractant in the lower well. As shown in Figure 6c, knockdown of DKK-1 expression resulted in significantly reduced cellular invasiveness. In an attempt to analyze a possible molecular mechanism underlying DKK-1 konckdown-induced phenotype, we examined the phosphorylation of β-catenin and GSK-3β, both of which are involved in canonical Wnt signaling, and JNK. However, the phosphorylation status of these proteins was not altered by the DKK-1 silencing (Fig. 6d). Moreover, nuclear β-catenin level and mRNA levels of possible transcriptional targets of canonical Wnt/β-catenin signaling, such as c-Myc and cyclin D1, were not altered either.

thumbnail image

Figure 6. Effect of DKK-1 knockdown on S2-CP8 cells. (a) Immunoblot (upper panel) and RT-PCR (lower panel) analyses indicated that both DKK-1 protein and mRNA levels were significantly decreased in S2-CP8 cell pools infected with a DKK-1 shRNA retroviral vector (S2CP8-KD) compared with scrambled control (S2CP8-scr) cells. (b) Knockdown of DKK-1 in S2-CP8 cells resulted in reduced growth rate and saturation density. (c) Effects of DKK-1 knockdown on invasiveness of S2-CP8 cells through Matrigel. Values are means ± standard deviation of triplicate experiments. *p < 0.001, Mann–Whitney U test. (d) Immunoblot analysis for the effects of DKK-1 knockdown on phosphorylation of β-catenin, GSK-3β and JNK. The β-actin signal is also shown as an internal blot control of each sample. The levels of cytosolic and nuclear β-catenin were also analyzed, and HSP70 and lamin A/C were used as internal blot controls. The effects of DKK-1 knockdown on c-Myc and cyclin D1 mRNA levels were also analyzed by RT-PCR (30 cycles).

Download figure to PowerPoint

Finally, we tested the effect of DKK-1 knockdown on the in vivo metastatic capability of S2-CP8 in a nude mouse model. After subcutaneous implantation of S2CP8-KD or S2CP8-scr cells, the cells were examined for pulmonary metastasis. There was no significant difference in either tumor growth rate or incidence of pulmonary metastasis between S2CP8-KD and S2CP8-scr cells, though there may be a tendency toward decreases in tumor size and number of metastases per each lung following DKK-1 knockdown (Table 1).

Table 1. Effects of DKK-1 knockdown on growth and pulmonary metastasis of S2-CP8 in nude mice
inline image

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

The family of human DKK proteins is composed of DKK-1, DKK-2, DKK-3, DKK-4 and soggy.2 DKK-1 and DKK-4 suppress the Wnt-induced signaling that is frequently involved in tumor progression.24 Consequently, reduced expression in tumor cells and tumor-suppressive activity have been reported for DKK-1. For example, DKK-1 expression is significantly reduced in gastrointestinal cancers and malignant melanoma,5–7, 25 and mesenchymal stem cells inhibit cancer cell proliferation by secreting DKK-1.26, 27 However, DKK-1 is upregulated in some tumor types including esophageal and nonsmall cell lung carcinomas, despite its Wnt inhibitory activity.10–14 In this study, we show that DKK-1 is also consistently overexpressed in pancreatic carcinomas, particularly DAC, and may promote invasive growth of the cancerous cells.

To date, little is known regarding the expression and function of DKK-1 in the pancreas. Many attempts of expression profiling have been made in order to identify molecules involved in the progression of pancreatic carcinomas.28–34 Enhanced expression of DKK-1 has not been found in most studies. However, in one report of global gene expression, the DKK-1 gene is included on a list of 217 known genes that were highly expressed in pancreatic DAC, without further validation of the expression.30 We confirmed in this study that DKK-1 mRNA is in fact upregulated in cultured human pancreatic carcinoma cell lines in vitro and also in pancreatic DAC cells in vivo. Furthermore, using retroviral expression of DKK-1 shRNA, we showed that knockdown of DKK-1 suppresses the migration and invasion of human pancreatic DAC cell line SUIT-2 and its metastatic subline S2-CP8 in vitro. Notably, DKK-1 silencing also suppressed the cell growth in S2-CP8. In accordance with our finding, Yamabuki et al. showed that overexpression of DKK-1 in NIH3T3 and COS-7 cells results in significantly enhanced Matrigel invasion in vitro.14 However, it has also been reported that overexpression of DKK-1 in human hepatocellular carcinoma cell line M-H7402 suppresses cell growth and migration.9 Therefore, DKK-1 might possess diverse functional roles in tumor cells depending on the cell types involved.

Our study suggests that DKK-1 may have a positive role in the development or progression of pancreatic DAC, though DKK-1 expression has been implicated in a negative feedback mechanism for activated Wnt signaling.2, 5 Wnt/β-catenin signaling is an important factor in the development of normal pancreatic tissue.35 However, only a limited number of reports have been published regarding the role of Wnt signaling in pancreatic cancer,36–38 and the function of DKK-1 in the pancreatic cancer has not been clarified. Interestingly, a recent study of a mutant mouse model indicates that activation of β-catenin, a critical member of canonical Wnt signaling, results in SPN of the pancreas, while it blocks the formation of pancreatic intraepithelial neoplasia (PanIN) in the presence of an activating mutation in K-ras,38 suggesting that canonical Wnt signaling may suppress the development of PanIN. As PanIN is a precursor lesion of DAC, the upregulation of DKK-1 may have a role in the development of PanIN and, thereby, DAC of the pancreas via its inhibitory effect on the canonical pathway. Alternatively, DKK-1 may have Wnt/β-catenin-independent functions. For example, ectopic expression of DKK-1 in HeLa cells did not alter cellular β-catenin localization or expression of Wnt target genes.39 In lung and esophageal cancer cells that overexpress the DKK-1 gene, there was no relationship between the mRNA expression patterns of DKK-1 and LRP5/6, the binding target of DKK-1 in Wnt signaling, suggesting that there may be unknown binding partners and/or receptors of DKK-1.14 In accordance with this hypothesis, phosphorylation of β-catenin or GSK-3β and expression of Wnt target genes were not altered by DKK-1 knockdown in S2-CP8 in our study. JNK may also be a candidate for the target of DKK-1 activity.40 However, JNK phosphorylation level was not altered by DKK-1 knockdown in S2-CP8 cells. Another possibility regarding the function of cancer cell-derived DKK-1 is its effect on the stromal cells, as Wnt signaling may also be activated in the stroma of pancreatic cancer.37 Whatever the underlying biological function may be, further clinicopathological study will be required to test the prognostic impact of DKK-1 expression using a large number of pancreatic DAC cases.

In summary, although the detailed function of DKK-1 in pancreatic carcinogenesis and progression of DAC is unknown, our results suggest a role for DKK-1 in the promotion of invasive growth of pancreatic cancer cells, and that it could serve as a novel tumor marker for pancreatic carcinoma.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  • 1
    Glinka A, Wu W, Delius H, Monaghan AP, Blumenstock C, Niehrs C. Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 1998; 391: 35762.
  • 2
    Niehrs C. Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene 2006; 25: 746981.
  • 3
    Monaghan AP, Kioschis P, Wu W, Zuniga A, Bock D, Poustka A, Delius H, Niehrs C. Dickkopf genes are co-ordinately expressed in mesodermal lineages. Mech Dev 1999; 87: 4556.
  • 4
    Moustakas A, Heldin CH. Signaling networks guiding epithelial-mesenchymal transitions during embryogenesis and cancer progression. Cancer Sci 2007; 98: 151220.
  • 5
    Gonzalez-Sancho JM, Aguilera O, Garcia JM, Pendas-Franco N, Pena C, Cal S, Garcia de Herreros A, Bonilla F, Munoz A. The Wnt antagonist DICKKOPF-1 gene is a downstream target of β-catenin/TCF and is downregulated in human colon cancer. Oncogene 2005; 24: 1098103.
  • 6
    Aguilera O, Fraga MF, Ballestar E, Paz MF, Herranz M, Espada J, Garcia JM, Munoz A, Esteller M, Gonzalez-Sancho JM. Epigenetic inactivation of the Wnt antagonist DICKKOPF-1 (DKK-1) gene in human colorectal cancer. Oncogene 2006; 25: 411621.
  • 7
    Kuphal S, Lodermeyer S, Bataille F, Schuierer M, Hoang BH, Bosserhoff AK. Expression of Dickkopf genes is strongly reduced in malignant melanoma. Oncogene 2006; 25: 502736.
  • 8
    Osada H, Tomida S, Yatabe Y, Tatematsu Y, Takeuchi T, Murakami H, Kondo Y, Sekido Y, Takahashi T. Roles of achaete-scute homologue 1 in DKK1 and E-cadherin repression and neuroendocrine differentiation in lung cancer. Cancer Res 2008; 68: 164755.
  • 9
    Qin X, Zhang H, Zhou X, Wang C, Zhang H, Zhang X, Ye L. Proliferation and migration mediated by Dkk-1/Wnt/ β-catenin cascade in a model of hepatocellular carcinoma cells. Transl Res 2007; 150: 28194.
  • 10
    Wirths O, Waha A, Weggen S, Schirmacher P, Kuhne T, Goodyer CG, Albrecht S, Von Schweinitz D, Pietsch T. Overexpression of human Dickkopf-1, an antagonist of wingless/WNT signaling, in human hepatoblastomas and Wilms' tumors. Lab Invest 2003; 83: 42934.
  • 11
    Koch A, Waha A, Hartmann W, Hrychyk A, Schuller U, Waha A, Wharton KA,Jr, Fuchs SY, von Schweinitz D, Pietsch T. Elevated expression of Wnt antagonists is a common event in hepatoblastomas. Clin Cancer Res 2005; 11: 4295304.
  • 12
    Patil MA, Chua MS, Pan KH, Lin R, Lih CJ, Cheung ST, Ho C, Li R, Fan ST, Cohen SN, Chen X, So S. An integrated data analysis approach to characterize genes highly expressed in hepatocellular carcinoma. Oncogene 2005; 24: 373747.
  • 13
    Chamorro MN, Schwartz DR, Vonica A, Brivanlou AH, Cho KR, Varmus HE. FGF-20 and DKK1 are transcriptional targets of beta-catenin and FGF-20 is implicated in cancer and development. EMBO J 2005; 24: 7384.
  • 14
    Yamabuki T, Takano A, Hayama S, Ishikawa N, Kato T, Miyamoto M, Ito T, Ito H, Miyagi Y, Nakayama H, Fujita M, Hosokawa M, et al. Dikkopf-1 as a novel serologic and prognostic biomarker for lung and esophageal carcinomas. Cancer Res 2007; 67: 251725.
  • 15
    Voorzanger-Rousselot N, Goehrig D, Journe F, Doriath V, Body JJ, Clezardin P, Garnero P. Increased Dickkopf-1 expression in breast cancer bone metastases. Br J Cancer 2007; 97: 96470.
  • 16
    Hall CL, Kang S, MacDougald OA, Keller ET. Role of Wnts in prostate cancer bone metastases. J Cell Biochem 2006; 97: 66172.
  • 17
    Eloubeidi MA, Desmond RA, Wilcox CM, Wilson RJ, Manchikalapati P, Fouad MM, Eltoum I, Vickers SM. Prognostic factors for survival in pancreatic cancer: a population-based study. Am J Surg 2006; 192: 32229.
  • 18
    Iwamura T, Katsuki T, Ide K. Establishment and characterization of a human pancreatic cancer cell line (SUIT-2) producing carcinoembryonic antigen and carbohydrate antigen 19-9. Jpn J Cancer Res 1987; 78: 5462.
  • 19
    Kitamura N, Iwamura T, Taniguchi S, Yamanari H, Kawano MA, Hollingsworth K, Setoguchi T. High collagenolytic activity in spontaneously highly metastatic variants derived from a human pancreatic cancer cell line (SUIT-2) in nude mice. Clin Exp Metastasis 2000; 18: 56171.
  • 20
    Fedi P, Bafico A, Nieto Soria A, Burgess WH, Miki T, Bottaro DP, Kraus MH, Aaronson SA. Isolation and biochemical characterization of the human Dkk-1 homologue, a novel inhibitor of mammalian Wnt signaling. J Biol Chem 1999; 274: 1946572.
  • 21
    Fukushima T, Tezuka T, Shimomura T, Nakano S, Kataoka H. Silencing of insulin-like growth factor-binding protein-2 in human glioblastoma cells reduces both invasiveness and expression of progression-associated gene CD24. J Biol Chem 2007; 282: 1863444.
  • 22
    Iwamura T, Katsuki T. Kinetics of carcinoembryonic antigen and carbohydrate antigen 19-9 production in a human pancreatic cancer cell line (SUIT-2). Gastroenterol Jpn 1987; 22: 6406.
  • 23
    Cheng H, Fukushima T, Takahashi N, Tanaka H, Kataoka H. Hepatocyte growth factor activator inhibitor type 1 regulates epithelial to mesenchymal transition through membrane-bound serine proteinases. Cancer Res 2009; 69: 182835.
  • 24
    Klaus A, Birchmeier W. Wnt signaling and its impact on development and cancer. Nat Rev Cancer 2008; 7: 38798.
  • 25
    Sato H, Suzuki H, Toyota M, Nojima M, Maruyama R, Sasaki S, Takagi H, Sogabe Y, Sasaki Y, Idogawa M, Sonoda T, Mori M, et al. Frequent epigenetic inactivation of DICKKOPF family genes in human gastrointestinal tumors. Carcinogenesis 2007; 28: 245966.
  • 26
    Qiao L, Xu ZL, Zhao TJ, Ye LH, Zhang XD. Dkk-1 secreted by mesenchymal stem cells inhibits growth of breast cancer cells via depression of Wnt signalling. Cancer Lett 2008; 269: 6777.
  • 27
    Zhu Y, Sun Z, Han Q, Liao L, Wang J, Bian C, Li J, Yan X, Liu Y, Shao C, Zhao RC. Human mesenchymal stem cells inhibit cancer cell proliferation by secreting DKK-1. Leukemia 2009; 23: 92533.
  • 28
    Crnogorac-Jurcevic T, Efthimiou E, Nielsen T, Loader J, Terris B, Stamp G, Baron A, Scarpa A, Lemoine NR. Expression profiling of microdissected pancreatic adenocarcinomas. Oncogene 2002; 21: 458794.
  • 29
    Terris B, Blaveri E, Crnogorac-Jurcevic T, Jones M, Missiaglia E, Ruszniewski P, Sauvanet A, Lemoine NR. Characterization of gene expression profiles in intraductal papillary-mucinous tumors of the pancreas. Am J Pathol 2002; 160: 174554.
  • 30
    Logsdon CD, Simeone DM, Binkley C, Arumugam T, Greenson JK, Giordano TJ, Misek DE, Kuick R, Hanash S. Molecular profiling of pancreatic adenocarcinoma and chronic pancreatitis identifies multiple genes differentially regulated in pancreatic cancer. Cancer Res 2003; 63: 264957.
  • 31
    Grutzmann R, Foerder M, Alldinger I, Staub E, Brummendorf T, Ropcke S, Li X, Kristiansen G, Jesnowski R, Sipos B, Lohr M, Luttges J, et al. Gene expression profiles of microdissected pancreatic ductal adenocarcinoma. Virchows Arch 2003; 443: 50817.
  • 32
    Missiaglia E, Blaveri E, Terris B, Wang YH, Costello E, Neoptolemos JP, Crnogorac-Jurcevic T, Lemoine NR. Analysis of gene expression in cancer cell lines identifies candidate markers for pancreatic tumorigenesis and metastasis. Int J Cancer 2004; 112: 10012.
  • 33
    Grutzmann R, Boriss H, Ammerpohl O, Luttges J, Kalthoff H, Schackert HK, Kloppel G, Saeger HD, Pilarsky C. Meta-analysis of microarray data on pancreatic cancer defines a set of commonly dysregulated genes. Oncogene 2005; 24: 507988.
  • 34
    Harada T, Chelala C, Crnogorac-Jurcevic T, Lemoine NR. Genome-wide analysis of pancreatic cancer using microarray-based techniques. Pancreatology 2008; 9: 1324.
  • 35
    Murtaugh LC. The what, where, when and how of Wnt/β-catenin signaling in pancreas development. Organogenesis 2008; 4: 816.
  • 36
    Pasca di Magliano M, Biankin AV, Heiser PW, Cano DA, Gutierrez PJ, Deramaudt T, Segara D, Dawson AC, Kench JG, Henshall SM, Sutherland RL, Dlugosz A, et al. Common activation of canonical Wnt signaling in pancreatic adenocarcinoma. PLoS One 2007; 2: e1155.
  • 37
    Pilarsky C, Ammerpohl O, Sipos B, Dahl E, Hartmann A, Wellmann A, Braunschweig T, Lohr M, Jesnowski R, Friess H, Wente MN, Kristiansen G, et al. Activation of Wnt signalling in stroma from pancreatic cancer identified by gene expression profiling. J Cell Mol Med 2008; 12: 282335.
  • 38
    Heiser PW, Cano DA, Landsman L, Kim GE, Kench JG, Klimstra DS, Taketo MM, Biankin AV, Hebrok M. Stabilization of β-catenin induces pancreas tumor formation. Gastroenterology 2008; 135: 1288300.
  • 39
    Mikheev AM, Mikheeva SA, Liu B, Cohen P, Zarbl H. A functional genomics approach for the identification of putative tumor suppressor genes: Dickkopf-1 as suppressor of HeLa cells transformation. Carcinogenesis 2004; 25: 4759.
  • 40
    Endo Y, Beauchamp E, Woods D, Taylor WG, Toretsky JA, Uren A, Rubin JS. Wnt-3a and Dickkopf-1 stimulate neurite outgrowth in Ewing tumor cells via a Frizzled3- and c-Jun N-terminal kinase-dependent mechanism. Mol Cell Biol 2008; 28: 236879.