Author Manuscript Published OnlineFirst on September 4, 2018; DOI: 10.1158/0008-5472.CAN-18-1749 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. p63-dependent Dickkopf3 expression promotes esophageal cancer cell proliferation via CKAP4 Chihiro Kajiwara1#, Katsumi Fumoto1#, Hirokazu Kimura1, Satoshi Nojima2, Keita Asano1, Kazuki Odagiri3, Makoto Yamasaki3, Hayato Hikita4, Tetsuo Takehara4, Yuichiro Doki3, Eiichi Morii2, and Akira Kikuchi1* 1Departments of Molecular Biology and Biochemistry, 2Pathology, 3Gastroenterological Surgery, 4Gastroenterology and Hepatology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan. # These authors contributed equally to this work. Running title: DKK3-CKAP4 axis promotes esophageal cancer proliferation All authors have declared no conflict of interest. *Correspondence author. Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan. Phone, 81-6-6879-3410. Fax, 81-6-6879-3419. E-mail:[email protected] Word count, 5268/5000. Total number of figures, 7. 1 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 4, 2018; DOI: 10.1158/0008-5472.CAN-18-1749 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Dickkopf3 (DKK3) is a secretory protein that belongs to the DKK family but exhibits structural divergence from other family members; and its corresponding receptors remain to be identified. Although DKK3 has been shown to have oncogenic functions in certain cancer types, the underlying mechanism by which DKK3 promotes tumorigenesis remains to be clarified. We show here that DKK3 stimulates esophageal cancer cell proliferation via cytoskeleton-associated protein 4 (CKAP4), which acts as a receptor for DKK3. DKK3 was expressed in ~50% of tumor lesions of esophageal squamous cell carcinoma (ESCC) cases; simultaneous expression of DKK3 and CKAP4 was associated with poor prognosis. Anti-CKAP4 antibody inhibited both binding of DKK3 to CKAP4 and xenograft tumor formation induced by ESCC cells. p63, a p53-related transcriptional factor frequently amplified in ESCC, bound to the upstream region of the DKK3 gene. Knockdown of p63 decreased DKK3 expression in ESCC cells, and re-expression of DKK3 partially rescued cell proliferation in p63-depleted ESCC cells. Expression of ΔNp63α and DKK3 increased the size of tumor-like esophageal organoids, and anti-CKAP4 antibody inhibited growth of esophageal organoids. Taken together, these results suggest that the DKK3-CKAP4 axis might serve as a novel molecular target for ESCC. 2 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 4, 2018; DOI: 10.1158/0008-5472.CAN-18-1749 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction There are four Dickkopf (DKK) family members in vertebrates, including DKK1, DKK2, DKK3, and DKK4, all of which are secretory proteins and contain two cysteine-rich domains (CRD1 and CRD2) (1). DKK1 was originally identified as a head inducer in Xenopus embryos and the most extensively studied among DKK family proteins. DKK1 antagonizes -catenin-dependent Wnt signaling by binding to and internalizing low-density lipoprotein receptor-related protein (LRP) 5 or 6, which are Wnt co-receptors (1-4). The Dkk1 null mutation is embryonic lethal in mouse as DKK1 plays important roles in several developmental processes, including fetal anterior-proximal axial patterning and limb formation (5) as well as in postnatal stages, such as bone formation (6). DKK2 and DKK4 also bind to LRP6 via CRD2 and inhibit Wnt signaling, similar to DKK1 (2, 7). Dkk2 null mutant mice were viable but blind due to a complete transformation of the cornea epithelium into the stratified epithelium (8). No information regarding Dkk4 knockout mice are available at present. DKK3 exhibits structural divergence from the rest of the DKK family (1). Overall protein sequence homology between DKK1, DKK2, and DKK4 ranges around 50%, but that between DKK3 and other DKKs is less than 40% (9). Two CRDs are separated by a non-conserved linker region that spans 50-55 aa in DKK1, DKK2, and DKK4, but only 12 aa in DKK3. In addition, the soggy domain is found only in DKK3, but not in other DKK proteins. DKK3 neither interacts with LRP6 nor antagonizes Wnt signaling unlike DKK1, DKK2, and DKK4 (1, 2, 10). Therefore, DKK3 is a divergent member of the DKK family and possesses functions independent of Wnt signaling (11). Importantly, no corresponding cell surface receptor for DKK3 has been identified to date. 3 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 4, 2018; DOI: 10.1158/0008-5472.CAN-18-1749 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Dkk3 knockout mice exhibit no obvious phenotype during the developmental stages, but do differ in hematological and immunological parameters as well as pulmonary ventilation (12). DKK3/REIC has also been shown to exhibit reduced expression gene in human immortalized cells (13) and its expression is frequently suppressed by promoter hypermethylation in human cancer cells, including the highly aggressive basal breast cancer (14), non-small cell lung cancer (15), hepatocellular carcinoma (16), gastric cancers, and colon cancers (17). DKK3 consistently suppressed cancer cell proliferation when ectopically overexpressed in various cancer cell types (14, 18). In this context, DKK3 seems to function as a tumor-suppressor. By contrast, it has also been reported that DKK3 may have tumor promoting functions. For instance, DKK3 was overexpressed in esophageal adenocarcinoma and oral squamous cell carcinoma (SCC) tissues, promoting cancer cell proliferation and migration (18, 19). DKK3 also induced stromal proliferation and differentiation in prostate cancer, influencing the angiogenesis program (20). Thus, the role of DKK3 in cancer development remains to be clarified. Esophageal cancer is the 6th leading cause of cancer-related death world-wide (21, 22). There are two histological types of esophageal cancer: SCC and adenocarcinoma (23). Esophageal SCC (ESCC) is believed to be affected by environmental factors, including alcohol and tobacco as well as genetic factors, such as a somatic mutation in p53 or overexpression of epidermal growth factor receptor (21, 23). Recent genomic analysis has revealed that p63, a p53-related transcriptional factor, is a major oncogenic protein in esophageal cancer; the gene locus is frequently amplified in ESCC and its expression in ESCC is significantly higher than non-tumor and adenocarcinoma tissues (24). Due to alternative splicing, there are at least six distinct p63 variants with two different N-termini (TA or N) and three different C-termini (or) (25). ΔNp63 and TAp63 show very different expression patterns, depending on the source of cell lines and tissues (26). Np63 is the main p63 4 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 4, 2018; DOI: 10.1158/0008-5472.CAN-18-1749 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. isoform expressed in ESCC and is required for ESCC cell proliferation (27), but the relationship between p63 and DKK3 remains to be clarified. We recently found that cytoskeleton-associated protein 4 (CKAP4) is a novel DKK1 receptor and that simultaneous expression of DKK1 and CKAP4 is negatively correlated with prognosis in pancreatic, lung, and esophageal cancers (28, 29). Here we show that the DKK3-CKAP4 and the DKK1-CKAP4 signaling axes are activated in distinct populations of ESCC tumors and that Np63 induces the expression of DKK3 in cells with mutations of Kras and p53. 5 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 4, 2018; DOI: 10.1158/0008-5472.CAN-18-1749 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Materials and Methods Materials and chemicals. MDCK cells were provided by Dr. S. Tsukita (Osaka University, Osaka, Japan). T24 bladder cancer cells and U-251 MG were purchased from the National Institutes of Biomedical Innovation, Health, and Nutrition (Osaka, Japan). TE-1, TE-4, TE-5, TE-6, TE-8, TE-9, TE-10, TE-11, and TE-14 ESCC cells were obtained from the Riken Bioresource Center Cell Bank (Tsukuba, Japan) in November 2008 (TE-5 and TE-11), May 2009 (TE-6, TE-9, and TE-14), or January 2015 (TE-1, TE-4, TE-8, and TE-10). KYSE-410 and KYSE-960 cells were obtained from the Japanese Cancer Research Resources Bank (Osaka, Japan) in May 2015. SW480 and DLD-1 colorectal cancer cells and TMK-1, KKLS, MKN1, and MKN45 gastric cancer cells were provided by Dr. W. Yasui (Hiroshima University, Hiroshima, Japan) in September 1997 (SW480 and DLD-1), September 2006 (TMK-1), August 2006 (KKLS), February 2006 (MKN1) or September 2005 (MKN45). HCT116 and Caco-2 colorectal cancer cells were provided by Dr. T. Kobayashi (Hiroshima University, Hiroshima, Japan) in November 2003 and were purchased from RIKEN Bioresource Center Cell bank in April 2013, respectively. AGS gastric cancer cells were provided by Dr. M. Hatakeyama (Tokyo University, Tokyo, Japan) in April 2014. HepG2, HLE, and HLF hepatic cancer cells were purchased from the ATCC in July 2017 (HepG2) and the Japanese Collection of Research Bioresources (Osaka, Japan) in June 2015 (HLE and HLF), respectively. A549 and Calu-6 lung adenocarcinoma cells were provided by Dr. Y. Shintani (Osaka University, Suita, Japan) in January 2014 and Shionogi Pharmaceutical Research (Osaka, Japan), respectively.