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Molecular Biology, Pathobiology, and Genetics |
1 Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Departments of 2 Surgical Oncology and 3 Surgical Pathology, Hokkaido University Graduate School of Medicine; 4 Keiyukai Sapporo Hospital, Sapporo, Japan; Divisions of 5 Thoracic Surgery and 6 Molecular Pathology and Genetics, Kanagawa Cancer Center, Kanagawa, Japan; and 7 Department of Molecular and Internal Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan
Requests for reprints: Yataro Daigo, Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-Ward, Tokyo 108-8639, Japan. Phone: 81-3-5449-5457; Fax: 81-3-5449-5406; E-mail: ydaigo{at}ims.u-tokyo.ac.jp.
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Introduction |
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To isolate potential molecular targets for diagnosis, treatment, and/or prevention of lung and esophageal carcinomas, we did a genome-wide analysis of gene expression profiles of cancer cells from 101 lung cancer and 19 ESCC patients by means of a cDNA microarray consisting of 27,648 genes (9–14). To verify the biological and clinicopathologic significance of the respective gene products, we have established a screening system by a combination of the tumor tissue microarray analysis of clinical lung and esophageal cancer materials with RNA interference technique and cell growth/invasion assays (15–23). In this process, we identified Dikkopf-1 (DKK1) as a novel serologic and histochemical biomarker as well as a therapeutic target for lung and esophageal cancers.
DKK1 encodes a secreted protein, which plays a crucial role in head formation in vertebrate development, and is known as a negative regulator of the Wnt signaling pathway in colon cancer cells (24, 25). DKK1 binds to LRP5/6 and Kremen proteins and induces LRP endocytosis, which prevents the formation of Wnt-Frizzled-LRP5/6 receptor complexes (25). Other studies have shown overexpression of DKK1 in Wilms' tumor, hepatoblastoma, and hepatocellular carcinoma (HCC), indicating a potential oncogenic function of DKK1 (26, 27). In spite of these studies, there has been no report describing the significance of activation of DKK1 in human cancer progression and its potential as a diagnostic and therapeutic target.
We report here identification of DKK1 as a novel diagnostic and prognostic biomarker and a potential target for therapeutic agents/antibodies and also provide evidence for its possible role in human pulmonary and esophageal carcinogenesis.
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Materials and Methods |
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Serum samples. Serum samples were obtained with written informed consent from 207 healthy control individuals (168 males and 39 females; median age of 50.3 with a range of 31 to 61 years) and from 88 nonneoplastic lung disease patients with chronic obstructive pulmonary disease (COPD) enrolled as a part of the Japanese Project for Personalized Medicine (BioBank Japan) or admitted to Hiroshima University Hospital (78 males and 10 females; median age of 67.6 with a range of 54 to 84 years). All of these patients were current and/or former smokers [the mean (±1 SD) of pack-year index (PYI) was 70.0 ± 42.7; PYI was defined as the number of cigarette packs (20 cigarettes per pack) consumed a day multiplied by years]. Serum samples were also obtained with informed consent from 125 lung cancer patients (78 males and 47 females; median age of 68.0 with a range of 40 to 86 years) admitted to Hiroshima University Hospital as well as Kanagawa Cancer Center Hospital and from 140 patients with lung cancer who were registered in the BioBank Japan (100 males and 40 females; median age of 64.5 with a range of 41 to 89 years). These 265 lung cancer cases included 112 adenocarcinomas, 68 SCCs, and 85 SCLCs. Serum samples were also obtained with informed consent from 81 ESCC patients who were admitted to Keiyukai Sapporo Hospital or who were registered in the BioBank Japan (69 males and 12 females; median age of 62.0 with a range of 37 to 74 years). These serum samples from a total of 346 cancer patients were selected for the study based on the following criteria: (a) patients were newly diagnosed and previously untreated and (b) their tumors were pathologically diagnosed as lung or esophageal cancers (stages I–IV). Serum was obtained at the time of diagnosis and stored at –150°C.
Semiquantitative reverse transcription-PCR. A total of 3 µg aliquot of mRNA from each sample was reversely transcribed to single-stranded cDNAs using random primer (Roche Diagnostics, Basel, Switzerland) and SuperScript II (Invitrogen, Carlsbad, CA). Semiquantitative reverse transcription-PCR (RT-PCR) experiments were carried out with the following sets of synthesized primers specific to DKK1 or with ß-actin (ACTB)-specific primers as an internal control: DKK1, 5'-TAGAGTCTAGAACGCAAGGATCTC-3' and 5'-CAAAAACTATCACAGCCTAAAGGG-3'; ACTB, 5'-GAGGTGATAGCATTGCTTTCG-3' and 5'-CAAGTCAGTGTACAGGTAAGC-3'. PCRs were optimized for the number of cycles to ensure product intensity to be within the linear phase of amplification.
Northern blot analysis. Human multiple tissue blots covering 23 tissues (BD Biosciences, Palo Alto, CA) were hybridized with an [
-32P]dCTP-labeled, 776-bp PCR product of DKK1 that was prepared as a probe using primers 5'-CATCAGACTGTGCCTCAGGA-3' and 5'-CAAAAACTATCACAGCCTAAAGGG-3'. Prehybridization, hybridization, and washing were done following the manufacturer's specifications. The blots were autoradiographed with intensifying screens at –80°C for 7 days.
Western blotting. Tumor tissues or cells were lysed in lysis buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.5% NP40, 0.5% sodium deoxycholate, Protease Inhibitor Cocktail Set III (EMD Biosciences, Inc., San Diego, CA)]. The protein content of each lysate was determined by a Bio-Rad protein assay (Bio-Rad, Hercules, CA) with bovine serum albumin (BSA) as a standard. Each lysate (10 µg) was resolved on 10% to 12% denaturing polyacrylamide gels (with 3% polyacrylamide stacking gel) and transferred electrophoretically to a nitrocellulose membrane (GE Healthcare Biosciences, Piscataway, NJ). After blocking with 5% nonfat dry milk in TBS-Tween 20 (TBST), the membrane was incubated with primary antibodies for 1 h at room temperature. Immunoreactive proteins were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare Biosciences) for 1 h at room temperature. After washing with TBST, the reactants were developed using the enhanced chemiluminescence kit (GE Healthcare Biosciences). A commercially available rabbit polyclonal antibody to human DKK1 (hDKK1; Santa Cruz Biotechnology, Santa Cruz, CA) was hybridized by Western blot analysis using lysates of lung cancer and ESCC tissues and cell lines as well as normal tissues.
Immunocytochemical analysis. Cells were plated on glass coverslips (Becton Dickinson Labware, Franklin Lakes, NJ), fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in PBS for 3 min at room temperature. Nonspecific binding was blocked by Casblock (ZYMED, San Francisco, CA) for 10 min at room temperature. Cells were then incubated for 60 min at room temperature with primary antibodies diluted in PBS containing 3% BSA. After being washed with PBS, the cells were stained by FITC-conjugated secondary antibody (Santa Cruz Biotechnology) for 60 min at room temperature. After another wash with PBS, each specimen was mounted with Vectashield (Vector Laboratories, Inc., Burlingame, CA) containing 4',6-diamidino-2-phenylindole and visualized with Spectral Confocal Scanning Systems (TSC SP2 AOBS; Leica Microsystems, Wetzlar, Germany).
Immunohistochemistry and tissue microarray. To investigate the DKK1 protein in clinical samples that had been embedded in paraffin blocks, we stained the sections in the following manner. Briefly, 3.3 µg/mL of a rabbit polyclonal anti-hDKK1 antibody (Santa Cruz Biotechnology) were added to each slide after blocking of endogenous peroxidase and proteins, and the sections were incubated with HRP-labeled anti-rabbit IgG [Histofine Simple Stain MAX PO (G), Nichirei, Tokyo, Japan] as the secondary antibody. Substrate-chromogen was added, and the specimens were counterstained with hematoxylin.
Tumor tissue microarrays were constructed with formalin-fixed 279 primary lung cancers and 280 primary esophageal cancers as described elsewhere (30–32). The tissue area for sampling was selected based on visual alignment with the corresponding H&E-stained section on a slide. Three, four, or five tissue cores (diameter, 0.6 mm; depth, 3–4 mm) taken from a donor tumor block were placed into a recipient paraffin block with a tissue microarrayer (Beecher Instruments, Sun Prairie, WI). A core of normal tissue was punched from each case, and 5-µm sections of the resulting microarray block were used for immunohistochemical analysis. Three independent investigators semiquantitatively assessed DKK1 positivity without prior knowledge of clinicopathologic data as reported previously (16–21). The intensity of DKK1 staining was evaluated using the following criteria: strong positive (scored as 2+), dark brown staining in >50% of tumor cells completely obscuring cytoplasm; weak positive (1+), any lesser degree of brown staining appreciable in tumor cell cytoplasm; and absent (scored as 0), no appreciable staining in tumor cells. Cases were accepted as strongly positive only if reviewers independently defined them as such.
Statistical analysis. Statistical analyses were done using the StatView statistical program (SAS, Cary, NC). Tumor-specific survival curves were calculated from the date of surgery to the time of death related to non-SCLC (NSCLC) or ESCC or to the last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and for DKK1 expression; differences in survival times among patient subgroups were analyzed using the log-rank test. Univariate and multivariate analyses were done with the Cox proportional hazard regression model to determine associations between clinicopathologic variables and cancer-related mortality. First, we analyzed associations between death and possible prognostic factors, including age, gender, pathologic tumor classification, and pathologic node classification, taking into consideration one factor at a time. Second, multivariate Cox analysis was applied on backward (stepwise) procedures that always forced strong DKK1 expression into the model, along with any and all variables that satisfied an entry level of a P value of <0.05. As the model continued to add factors, independent factors did not exceed an exit level of P < 0.05.
ELISA. Serum levels of DKK1 were measured by ELISA system, which had been originally constructed. First, a rabbit polyclonal antibody specific to DKK1 was added to a 96-well microplate (Nunc, Roskilde, Denmark) as a capture antibody and incubated for 2 h at room temperature. After washing away any unbound antibody, 5% BSA was added to the wells and incubated for 16 h at 4°C for blocking. After a wash, 3-fold diluted sera were added to the wells and incubated for 2 h at room temperature. After washing away any unbound substances, a biotinylated polyclonal antibody specific for DKK1 using Biotin Labeling Kit-NH2 (DOJINDO, Kumamoto, Japan) was added to the wells as a detection antibody and incubated for 2 h at room temperature. After a wash to remove any unbound antibody-enzyme reagent, HRP-streptavidin was added to the wells and incubated for 20 min. After a wash, a substrate solution (R&D Systems, Inc., Minneapolis, MN) was added to the wells and allowed to react for 30 min. The reaction was stopped by adding 100 µL of 2 N sulfuric acid. Color intensity was determined by a photometer at a wavelength of 450 nm, with a reference wavelength of 570 nm. Levels of CEA in serum were measured by ELISA with a commercially available enzyme test kit (Hope Laboratories, Belmont, CA) according to the supplier's recommendations. Levels of ProGRP in serum were measured by ELISA with a commercially available enzyme test kit (TFB, Tokyo, Japan) according to the manufacturer's protocol. Differences in the levels of DKK1, CEA, and ProGRP between tumor groups and a healthy control group were analyzed by Mann-Whitney U tests. The levels of DKK1, CEA, and ProGRP were evaluated by receiver operating characteristic (ROC) curve analysis to determine cutoff levels with optimal diagnostic accuracy and likelihood ratios. The correlation coefficients between DKK1 and CEA/ProGRP were calculated with Spearman rank correlation. Significance was defined as P < 0.05.
Matrigel invasion assay. NIH3T3 and COS-7 cells transfected either with p3XFLAG-tagged (COOH terminus) plasmids expressing DKK1 or with mock plasmids were grown to near confluence in DMEM containing 10% FCS. The cells were harvested by trypsinization, washed in DMEM without addition of serum or proteinase inhibitor, and suspended in DMEM at concentration of 1 x 105/mL. Before preparing the cell suspension, the dried layer of Matrigel matrix (Becton Dickinson Labware) was rehydrated with DMEM for 2 h at room temperature. DMEM (0.75 mL) containing 10% FCS was added to each lower chamber in 24-well Matrigel invasion chambers, and 0.5 mL (5 x 104 cells) of cell suspension was added to each insert of the upper chamber. The plates of inserts were incubated for 24 h at 37°C. After incubation, the chambers were processed; cells invading through the Matrigel were fixed and stained by Giemsa as directed by the supplier (Becton Dickinson Labware).
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Results |
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Northern blot analysis using a DKK1 cDNA fragment as a probe identified a transcript of 
1.8 kb that was highly expressed in placenta and at a very low level in prostate, but its transcript was hardly detectable in any other normal tissues (Fig. 2A
). We did immunofluorescence analysis to examine the subcellular localization of endogenous DKK1 in ESCC cell line TE8 and NSCLC cell line LC319 and found that DKK1 was located at cytoplasm of tumor cells with granular appearance (representative data of TE8 cells are shown in Fig. 2B).
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Serum levels of DKK1 in patients with lung cancer or ESCC. Because DKK1 encodes a secreted protein, we investigated whether the DKK1 protein is secreted into sera of patients with lung or esophageal cancer. ELISA experiments detected DKK1 protein in serologic samples from the great majority of the 346 patients with lung or esophageal cancer. The mean (±1 SD) of serum levels of DKK1 in 265 lung cancer patients were 30.7 ± 22.0 units/mL and those in 81 ESCC patients were 38.8 ± 34.6 units/mL. In contrast, the mean (±1 SD) serum levels of DKK1 in 207 healthy individuals were 6.1 ± 5.0 units/mL and those in 88 patients with COPD, who were current and/or former smokers, were 7.2 ± 6.2 units/mL. The levels of serum DKK1 protein were significantly higher in lung or esophageal cancer patients than in healthy donors or in COPD patients (P < 0.001, Mann-Whitney U test); the difference between healthy individuals and COPD patients was not significant (P = 0.427). According to histologic types of lung cancer, the serum levels of DKK1 were 32.2 ± 21.6 units/mL in 112 adenocarcinoma patients, 25.6 ± 17.6 units/mL in 68 SCC patients, and 33.0 ± 24.8 units/mL in 85 SCLC patients (Fig. 4A ); the differences among the three histologic types were not significant. High levels of serum DKK1 were detected even in patients with earlier-stage tumors (Fig. 4B). Using ROC curves drawn with the data of these 346 cancer patients and 207 healthy controls (Fig. 4C), the cutoff level in this assay was set to provide optimal diagnostic accuracy and likelihood ratios (minimal false-negative and false-positive results) for DKK1 [i.e., 14.7 units/mL with a sensitivity of 68.2% (236 of 346) and a specificity of 95.2% (197 of 207)]. According to tumor histology, the proportions of the serum DKK1-positive cases was 74.1% for adenocarcinoma (83 of 112), 63.2% for SCC (43 of 68), 69.4% for SCLC (59 of 85), and 63.0% for ESCC (51 of 81). The proportions of the serum DKK1-positive cases were 11.4% (10 of 88) for COPD. We then did ELISA experiments using paired preoperative and postoperative (2 months after the surgery) serum samples from lung cancer and ESCC patients to monitor the levels of serum DKK1 in the same patients. The concentration of serum DKK1 was dramatically reduced after surgical resection of primary tumors (Fig. 4D, top and bottom). The results independently support the high specificity and the great potentiality of serum DKK1 as a biomarker for detection of cancer at an early stage and for monitoring of the relapse of the disease.
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= –0.026; P = 0.728), indicating that measuring both markers in serum can improve overall sensitivity for detection of NSCLC to 82.2% (148 of 180); for diagnosing NSCLC, the sensitivity of CEA alone is 32.2% (58 of 180) and that of DKK1 is 70.0% (126 of 180). False-positive rates for either of the two tumor markers among normal volunteers (control group) were 7.7% (16 of 207), although the false-positive rates for each of CEA and DKK1 in the same control group were 2.9% (6 of 207) and 4.8% (10 of 207), respectively.
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= –0.012; P = 0.910), also indicating that measurement of serum levels of both markers can improve overall sensitivity for detection of SCLC to 89.4% (76 of 85); for diagnosing SCLC, the sensitivity of ProGRP alone was 63.5% (54 of 85) and that of DKK1 was 69.4% (59 of 85). False-positive cases for either of the two tumor markers among normal volunteers (control group) were 6.3% (13 of 207), although the false-positive rates for ProGRP and DKK1 in the same control group were 1.5% (3 of 207) and 4.8% (10 of 207), respectively. Activation of cellular migration by DKK1. As the immunohistochemical analysis on tissue microarray had indicated that lung and esophageal cancer patients with DKK1-positive tumors showed shorter cancer-specific survival period than those with DKK1-negative tumors, we examined a possible role of DKK1 in cellular migration and invasion using Matrigel assays using NIH3T3 and COS-7 cells. As shown in Fig. 6 , transfection of DKK1 cDNA into either cell line significantly enhanced its invasive activity through Matrigel compared with cells transfected with mock vector.
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Discussion |
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DKK1 is a 35-kDa protein that contains a signal peptide sequence and two cysteine-rich domains (33) and is a secreted protein that functions as a negative regulator of the Wnt signaling and plays a crucial role in head formation in vertebrate development (33–37). In addition, DKK1 was reported to be a downstream target of ß-catenin/T-cell factor and participates in a negative feedback loop in the Wnt signaling in colon cancer cells (24, 25).
A family of hDKK-related genes was composed of DKK1, DKK2, DKK3, and DKK4 together with a unique DKK3-related protein, termed Soggy (Sgy). hDKK1, hDKK2, hDKK3, and hDKK4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved among family members. hDKK1 and hDKK4, but not hDKK2, hDKK3, or Sgy, were shown to suppress the Wnt-induced secondary axis induction in Xenopus embryos (38). DKK4 was found to show high specificity for gastric cancer by serial analysis of gene expression and quantitative RT-PCR (39). Other studies have shown overexpression of DKK1 in Wilms' tumor, hepatoblastoma, and HCC (26, 27), but clinical significance of DKK1 protein as a serologic/histochemical marker in human cancer or as a therapeutic target was not investigated previously. Like the DKK1 protein, Wnt inhibitory factor-1 and Frizzled-related protein (FRP) were known to be secreted molecules, which have been indicated to bind to Wnt proteins and inhibit their activity (40–42). These two proteins were associated with human cancers, including colorectal carcinoma (43). FRP-4 protein showed markedly increased expression levels in colorectal cancers compared with normal mucosa but no significant association with pathologic features or with patient outcome (44). Because various DKK family proteins had been described as being overexpressed in human cancers (39, 44), DKK1 seemed likely to have a potential role in development or progression of certain types of human tumor, although it was indicated as a negative feedback factor for the activated Wnt signaling pathway in colon cancer.
In this study, we showed that induction of exogenous expression of DKK1 enhanced the cellular migration/invasive activity of mammalian cells. Concordantly, tissue microarray analyses revealed correlation of the strong DKK1 staining in primary NSCLC and ESCC tissues with poorer prognosis of the patients. Although the detailed function of DKK1 in lung and esophageal carcinogenesis is unknown, our results implied that DKK1 expression could promote dissemination of tumors by stimulating cell invasion. DKK1 was described as a secreted protein, which could bind to LRP5/6 and Kremen proteins, induce LRP endocytosis, and prevent the formation of the Wnt-Frizzled-LRP5/6 receptor complexes (25). However, when we analyzed mRNA expression of DKK1 and LRP5/6 in lung and esophageal cancer cell lines and cancer tissues by semiquantitative RT-PCR, the expression pattern of LRP5/6 was not concordant with that of DKK1 (data not shown). Additional studies to identify unknown binding partners and receptors of DKK1 in human lung and esophageal cancers should contribute to a better understanding of the signaling pathway mediated by DKK1 overexpression.
We also found high levels of DKK1 protein in serologic samples from lung cancer and ESCC patients. As a half of the serum samples used for this study were derived from patients with early-stage cancers, DKK1 should be useful for diagnosis of even early-stage cancers. To examine the feasibility for applying DKK1 as the diagnostic tool, we compared serum levels of DKK1 with those of CEA or ProGRP, two conventional diagnostic markers for NSCLCs and SCLCs, about its sensitivity and specificity for diagnosis. An assay combining both markers (DKK1 + CEA or DKK1 + ProGRP) increased the sensitivity to about 80% to 90% for lung cancer (NSCLC as well as SCLC), significantly higher than that of CEA or ProGRP alone, whereas 6% to 8% of healthy volunteers were falsely diagnosed as positive. Although further validation using a larger set of serum samples covering various clinical stages will be required, our data presented here sufficiently show a potential clinical usefulness of DKK1 itself as a serologic/histochemical marker for lung and esophageal cancers.
In conclusion, we have shown DKK1 as a potential biomarker for diagnosis of lung and esophageal cancers as well as for prediction of the poor prognosis of the patients with these diseases. DKK1 was overexpressed in the great majority of lung and esophageal cancer tissues we examined, and its serum levels were elevated in the sera of a large proportion of the patients. DKK1, combined with other tumor markers, could significantly improve the sensitivity of cancer diagnosis, although it could be used at initial diagnosis to identify patients who might benefit from early systemic treatment. Moreover, this molecule is also a likely candidate for development of therapeutic approaches, such as antibody therapy, to inhibit the cancer progression.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Footnotes |
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Received 9/12/06. Revised 12/12/06. Accepted 1/11/07.
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in serum as predictors of poor response to gefitinib among patients with advanced non-small cell lung cancers. Cancer Res 2005;65:9176–84.This article has been cited by other articles:
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