Published OnlineFirst June 15, 2010; DOI: 10.1158/0008-5472.CAN-09-3879 Published OnlineFirst on June 15, 2010 as 10.1158/0008-5472.CAN-09-3879 Molecular and Cellular Pathobiology Cancer Research Wnt Inhibitor Dickkopf-1 as a Target for Passive Cancer Immunotherapy Nagato Sato1,2, Takumi Yamabuki1, Atsushi Takano1,6, Junkichi Koinuma1, Masato Aragaki1, Ken Masuda1, Nobuhisa Ishikawa1, Nobuoki Kohno3, Hiroyuki Ito4, Masaki Miyamoto2, Haruhiko Nakayama4, Yohei Miyagi5, Eiju Tsuchiya5, Satoshi Kondo2, Yusuke Nakamura1, and Yataro Daigo1,6 Abstract Dickkopf-1 (DKK1) is an inhibitor of Wnt/β-catenin signaling that is overexpressed in most lung and esoph- ageal cancers. Here, we show its utility as a serum biomarker for a wide range of human cancers, and we offer evidence favoring the potential application of anti-DKK1 antibodies for cancer treatment. Using an original ELISA system, high levels of DKK1 protein were found in serologic samples from 906 patients with cancers of the pancreas, stomach, liver, bile duct, breast, and cervix, which also showed elevated expression levels of DKK1. Additionally, anti-DKK1 antibody inhibited the invasive activity and the growth of cancer cells in vitro and suppressed the growth of engrafted tumors in vivo. Tumor tissues treated with anti-DKK1 displayed sig- nificant fibrotic changes and a decrease in viable cancer cells without apparent toxicity in mice. Our findings suggest DKK1 as a serum biomarker for screening against a variety of cancers, and anti-DKK1 antibodies as potential theranostic tools for diagnosis and treatment of cancer. Cancer Res; 70(13); OF1–11. ©2010 AACR. Introduction growth factor (VEGF) used for the treatment of metastatic colorectal and lung cancers in combination with chemother- The concept of specific molecular targeting therapy has been apy (4). The results were promising when compared with applied to the development of innovative cancer-treatment responses of advanced cancers to conventional cytotoxic strategies. At present, two main approaches are available in agents, but the proportion of patients showing good response clinical practice: therapeutic monoclonal antibodies (mAb) is still limited, and in some cases, the medication caused se- and small-molecule agents (1). There is an increasing interest rious adverse effects including life-threatening ones. Hence, in the use of antibody-based immunotherapy for the treat- the development of new therapeutic antibodies targeting ment of malignant diseases, and some dramatic clinical transmembrane and/or secreted proteins, which show a responses have enhanced the activity in this field (1). For cancer-specific expression pattern and have an oncogenic example, rituximab (Rituxan) is the chimeric anti-CD20 anti- function, is eagerly awaited. body that revolutionized lymphoma treatment (2). Trastuzu- To develop new diagnostics and therapeutics improving mab (Herceptin) is the humanized Ab against the human cancer treatment, we have been screening the candidate tar- epidermal growth factor receptor (HER)/ERBB2 for the treat- get molecules by the following strategy: (a) identifying genes ment of patients with metastatic breast cancer in which HER/ upregulated in lung and esophageal cancers by genome-wide ERBB2 gene was highly overexpressed (3). Bevacizumab cDNA microarray system (5–11), (b) verifying the candidate (Avastin) is the humanized Ab against vascular endothelial genes for the absence of or very low level of expression in nor- mal tissues (5), (c) validating biological significance of their overexpression by means of tissue microarray containing Authors' Affiliations: 1Laboratory of Molecular Medicine, Human hundreds of archived lung cancer samples and functional as- Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; 2Department of Surgical Oncology, Hokkaido University says for examining their activity in cell growth and/or invasive Graduate School of Medicine, Sapporo, Japan; 3Department of activity (12–33), (d) evaluating them for usefulness as a serum Molecular and Internal Medicine, Graduate School of Biomedical diagnostic/prognostic biomarker for lung cancer by ELISA Sciences, Hiroshima University, Hiroshima, Japan; Divisions of – 4Thoracic Surgery and 5Molecular Pathology and Genetics, Kanagawa (29 33), if they are tumor-specific transmembrane or secretory Cancer Center, Yokohama, Japan; and 6Department of Medical proteins, and (e) screening the epitopes recognized by human Oncology, Shiga University of Medical Science, Otsu, Japan histocompatibility leukocyte HLA-*A0201/*A2402-restricted Note: Supplementary data for this article are available at Cancer Research CTL, if they are categorized into cancer testis antigens and Online (http://cancerres.aacrjournals.org/). are indispensable for cancer cell growth/survival (34–36). Corresponding Author: Yataro Daigo, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Using this approach, we had identified Dickkopf-1 (DKK1) Japan. Phone: 81-3-5449-5457; Fax: 81-3-5449-5406; E-mail: ydaigo@ as a novel serologic and histochemical biomarker as well as ims.u-tokyo.ac.jp. a possible therapeutic target for lung and esophageal cancers doi: 10.1158/0008-5472.CAN-09-3879 (31). Our data indicated that DKK1 expression was associated ©2010 American Association for Cancer Research. with poor prognosis for patients with non–small cell lung www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst June 15, 2010; DOI: 10.1158/0008-5472.CAN-09-3879 Sato et al. cancer (NSCLC) or esophageal squamous cell carcinoma tained at 37°C in humidified air with 5% CO2. Surgically (ESCC), and that serum DKK1 levels were significantly higher resected primary cancers from six pancreatic cancer pa- in lung and esophageal cancer patients than in healthy con- tients, five gastric cancer patients, seven HCC patients, trols; the proportions of the serum DKK1–positive cases was six bile duct cancers, six breast cancer patients, and five 74.1% for lung adenocarcinoma (83 of 112), 63.2% for lung cervical cancer patients were obtained with informed con- squamous cell carcinoma (43 of 68), 69.4% for small cell lung sent at Kanagawa Cancer Center (Yokohama, Japan). Pri- cancer (59 of 85), and 63.0% for ESCC (51 of 81; ref. 31). In mary bile duct cancers were obtained with informed addition, we identified that the exogenous expression of consent from six patients who had undergone surgery at DKK1 increased the invasive activity of mammalian cells. Hokkaido University Hospital (Sapporo, Japan). Primary The evidence further prompted us to focus on DKK1 as a lung cancers in the same set of 18 lung cancer cases whose potential target for therapeutic antibodies as well as a serum serum had been collected before surgery (nine patients biomarker for a wide range of human cancers. with DKK1-positive tumors and nine with DKK1-negative DKK1 encodes a secreted protein that plays a crucial role tumors) were obtained at Hiroshima University Hospital in head formation in vertebrate development (37), and is (Hiroshima, Japan) and Kanagawa Cancer Center. This known as a negative regulator of the Wnt-signaling path- study and the use of all clinical materials used were way in colon cancer cell lines (38). During specific period approved by individual institutional Ethical Committees. of embryogenesis, DKK1 is secreted and binds to the LRP5/6 coreceptor (39), resulting in the blocking of the Serum samples interaction with secreted Wnt protein, inducing degrada- Serum samples were obtained with informed consent from tion of β-catenin, and then reducing the expression of 207 healthy control individuals (168 males and 39 females; genes regulated by T-cell factor. This mechanism of DKK1 median age of 50.3 y with a range of 31–61 y) and from action is important in limb and head development (37). 179 pancreatic cancer patients (114 males and 65 females; Inhibition of the Wnt pathway by secreted DKK1 is also median age of 66 y with a range of 30–83 y), 101 gastric can- known to initiate cardiogenesis in vertebrate embryos (40). cer patients (71 males and 30 females; median age of 62 y On the other hand, the overexpression of DKK1 was de- with a range of 29–82 y), 168 HCC patients (146 males and scribed in multiple myeloma, hepatoblastoma, Wilms' 22 females; median age of 70 y with a range of 32–84 y), 107 tumor, prostate cancer, kidney cancer, and breast cancer bile duct cancer patients (75 males and 32 females; median as well as lung and esophageal cancers (31, 41–44). Serum age of 67 y with a range of 45–75 y), 169 breast cancer concentrations of DKK1 protein were reported to be in- patients (169 females; median age of 51 y with a range of creased in patients with multiple myeloma or osteosarcoma 27–72 y), and 182 cervical cancer patients (182 females; me- (41, 45). In spite of recent accumulating reports that indi- dian age of 48 y with a range of 31–77 y) who were registered cate the oncogenic potential of DKK1 activation and its in the Japanese Project for Personalized Medicine (BioBank usefulness as a cancer biomarker, there was no report indi- Japan). These serum samples from a total of 906 cancer pa- cating the direct antitumor effect of anti-DKK1 antibodies. tients were selected for the study on the basis of the follow- We here show evidence suggesting DKK1 as a diagnostic ing criteria: (a) patients were newly diagnosed and (b) their biomarker for a wide variety of cancers and results implicat- tumors were pathologically diagnosed as cancers (stages I–IV). ing the therapeutic potential of anti-DKK1 antibody to neu- Serum was obtained at the time of diagnosis and stored at tralize the activity of DKK1 function for cancer cell invasion −150°C. The sample size of each cancer group (pancreas, and growth. stomach, liver, bile duct, breast, and cervix) and a healthy control group provided 90% statistical power (α =0.05; Materials and Methods β = 0.1) to detect group differences in mean levels of serum DKK1.