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 approximately 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 reexpression 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.

Significance: In esophageal cancer, findings identify DKK3 as a poor prognostic indicator and demonstrate CKAP4 inhibition as an effective therapeutic strategy. Cancer Res; 78(21); 6107–20. ©2018 AACR.

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; ref. 1). DKK1 was originally identified as a head inducer in Xenopus embryos and the most extensively studied among DKK family proteins. DKK1 antagonizes β-catenin–dependent Wnt signaling by binding to and internalizing low-density lipoprotein receptor–related protein (LRP) 5 or 6, which are Wnt coreceptors (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 nonconserved 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.

Dkk3 knockout mice exhibit no obvious phenotype during the developmental stages, but do differ in hematologic and immunologic 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. In 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 sixth leading cause of cancer-related death worldwide (21, 22). There are two histologic 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 EGFR (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 nontumor and adenocarcinoma tissues (24). Because of alternative splicing, there are at least six distinct p63 variants with two different N-termini (TA or ΔN) and three different C-termini (α, β, or γ; ref. 25). ΔNp63 and TAp63 show very different expression patterns, depending on the source of cell lines and tissues (26). ΔNp63α is the main p63 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 ΔNp63α induces the expression of DKK3 in cells with mutations of Kras and p53.

Materials and chemicals

Madin-Darby canine kidney (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 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 KYSE960 cells were obtained from the Japanese Cancer Research Resources Bank 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 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, respectively.

S2-CP8, SUIT-2, and PANC-1 pancreatic cancer cells were purchased from the Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer of Tohoku University in April 2014 (S2-CP8 and SUIT-2) and the Riken Bioresource Center Cell Bank in October 2014 (PANC-1), respectively. Lenti-X 293T (X293T) cells were purchased from Takara Bio Inc. in October 2011. TE-5, TE-6, TE-9, TE-11, and TE-14 cells were authenticated in February 2017 using short tandem repeat analysis. Mycoplasma testing was not conducted.

Additional methods and antibodies, siRNAs, and shRNA used in this study are described in Supplementary Methods and Supplementary Table S1.

Polarized secretion of DKKs

Polarized secretion assays of DKK molecules were performed as described previously (30). MDCK cells expressing DKK-FLAG (2 × 105 cells) were seeded on Transwell polycarbonate filters (Corning Costar). DKKs secreted into the culture medium from the apical and basolateral chambers were collected and probed with anti-FLAG antibody. For DKK4, the same conditioned medium was precipitated with anti-FLAG antibody and protein G beads. After washing three times with NP40 buffer (20 mmol/L Tris-HCl pH 8.0, 10% glycerol, 137 mmol/L NaCl, and 1% NP40), the precipitates were probed with anti-FLAG antibody.

Polarized MDCK cells proliferation assay

Proliferation assays were performed as described previously (28, 31) with modification. MDCK cells (2 × 105 cells) were grown and polarized on Transwell polycarbonate filters (Corning Costar) in growth medium and subsequently starved by changing to DMEM containing 0.1% BSA. Then, DKK-FLAG conditioned medium was added to MDCK cells apically or basolaterally.

Generation of CKAP4 knockout cells

Single guide RNA oligo against canine CKAP4 (GTCGGGCGGCGCGGATGAC) was inserted into pX330 (Addgene plasmid #42230) that expresses hCas9. The cloned plasmids were introduced into MDCK cells using Viafect reagent (Promega) with blasticidin-resistant gene expressing plasmids. After transfection, MDCK cells were replated at low density in growth medium containing blasticidin (10 μg/mL); cells were allowed to grow until single-cell colonies became visible. Then, single colonies were picked mechanically, amplified, and analyzed.

Two-dimensional cell proliferation assay

MDCK cells (1 × 105 cells) were seeded into 3.5-cm dishes and cultured in DMEM supplemented with 1% FBS. TE-11 and KYSE960 cells (1 × 105 cells) were seeded in a 3.5-cm dish and cultured in growth medium. Cell numbers were counted on the indicated days.

Three-dimensional cell proliferation assay

Three-dimensional cell proliferation assays were performed as described previously (28, 31) with modification. Forty microliters of Matrigel (BD Biosciences) were solidified on a round coverslip by incubating for 30 minutes at 37°C. MDCK cells (5 × 104 cells) suspended in DMEM containing 2% Matrigel and 10% FBS were seeded on the solidified Matrigel and cultured for the indicated days.

Detection of cell surface CKAP4

Detection of cell surface CKAP4 was performed as described previously (28). MDCK cells treated with DKK-FLAG, HCT116, TE-11, and KYSE960 cells were incubated with 0.5 mg/mL sulfo-NHS-LC-biotin (Pierce Biotechnology) for 30 minutes at 4°C. Biotinylated cells were lysed in 500 μL of NP40 buffer with protease inhibitors. Lysates containing biotinylated proteins were precipitated using NeutrAvidin agarose beads (Pierce Biotechnology). Then, beads were washed twice with NP40 buffer and once with 10 mmol/L Tris-HCl pH 7.5; the bound complexes were probed with anti-CKAP4 antibody.

Patients and IHC studies of DKK1 and DKK3

Patients with ESCC (n = 72) who underwent surgery at Osaka University Hospital (Osaka, Japan) from January 2006 to May 2012 were examined in this study. All experimental protocols were approved by the Ethical Review Board of the Graduate School of Medicine, Osaka University (Osaka, Japan; no. 13455) under Declaration of Helsinki, and were performed in accordance with the Committee guidelines and regulations. The written informed consent was obtained from all patients. Among them, 48 patients overlapped with patients used in our previous study (29), and were reexamined in this study.

All tissue sections (5 μm thickness) were stained using a DakoRealTMEnVision Detection System (Dako) in accordance with the manufacturer's recommendations. After deparaffinization and heat-induced antigen retrieval, endogenous peroxidase activity was quenched with peroxidase-blocking solution (Dako) and the sections were blocked with 5% goat serum in buffer (5% BSA, 0.5% Triton X-100 in PBS) for 16 hours at 4°C. Then, the blocked specimens were stained with anti-DKK1 (1:100), anti-CKAP4 (1:100), or anti-DKK3 (1:50) antibody for 16 hours at 4°C. After washing, the specimens were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour and subsequently detected with diaminobenzidine (DAB; Dako). All tissue sections were also counterstained with 0.1% (w/v) hematoxylin. For DKK1 and CKAP4, tumors in which the positively stained area covered >5% were classified as positive cases. Because DKK3 was expressed specifically in the marginal regions of tumor nests near stroma, we regarded tumor areas in which marginal regions were positively stained (> 5%) as DKK3-positive tumor nests. Four researchers including a pathologist evaluated the staining.

Xenograft tumor formation assay

The xenograft tumor formation assay was performed as described previously (28, 31) with modification. KYSE960 cells (1 × 107 cells) were transplanted using dorsal subcutaneous transplantation of 6-week-old male BALB/cAnNCrj-nu immunodeficient mice (Charles River Laboratory Japan Inc.). Control cells (KYSE960 stably expressing control shRNA) were transplanted on the left dorsal region while the other cells (KYSE960 stably expressing DKK3 shRNA and CKAP4 shRNA) were on transplanted on the right dorsal region. The mice were sacrificed at 40 days after transplantation and mice with a control tumor size of 100 mm3 or less at 18 days after transplantation were excluded from the study.

To examine antitumor effects of the anti-CKAP4 antibody, mice were divided into anti-CKAP4 antibody or control IgG administration groups when the tumor size reached 100 mm3. Two hundred and fifty μg antibody was intraperitoneally administrated twice a week. The mice were sacrificed at 22 days after antibody injection. Tumor volumes were calculated using the following formula: (major axis) × (minor axis) × (minor axis) × 0.5.

Reporter gene assay

X293T cells were transfected with pGL4.27 (Promega)-containing wild-type (WT) or mutated (Mut) genomic regions of −8466 to −8118 from transcription start site (TSS) of DKK3 and the indicated constructs. The cells were lysed at 24 hours posttransfection, and the luciferase activity was measured with PicaGene reagent (Toyo Ink) as described previously (32). β-Galactosidase activities were measured to standardize the transfection efficiency.

Esophageal organoid culture

Esophageal organoid culture was performed as described previously (33) with modification. The esophagus mucosa from wild-type or KP (KrasG12DLSL from National Cancer Institute–Frederick, Bethesda, MD and p53lox/lox from The Jackson Laboratory) adult mouse was physically separated and minced into small pieces. Cells were dissociated in DMEM/F12 medium containing 0.25% Trypsin-EDTA and 100 U DNase I (Sigma, D4527) for 30 minutes at 37°C with agitation. After passing the cells through a 40-μm sterile filter (BD Biosciences, 352340), 5,000 esophageal epithelial cells were suspended in ice-cold 20 μL Matrigel solution (BD Biosciences). Matrigel-containing esophageal epithelial cells were placed as a droplet in a 24-well tissue culture plate and solidified by incubating for 20 minutes at 37°C. Complete growth medium was prepared at the time of use and consisted of 1:1 mixture of DMEM and F12, 1 × N-2 (Life Technologies, 17502048), 1 × B-27 Supplements (Life Technologies, 17504044), 1 × Glutamax (Life Technologies), 10 mmol/L HEPES/NaOH (pH 7.4), 50 ng/mL EGF (R&D Systems, 236-EG), 100 ng/mL Noggin (R&D Systems, 1967-NG), 100 ng/mL R-Spondin 1 (R&D Systems, 7150-RS), 100 ng/mL FGF10 (PeproTech, 100–26), and 10 μmol/L Y27632 (WAKO, 253-00513).

Statistical analyses

Experiments were performed repeatedly at least three times and results are presented as the mean ± SD. A paired or unpaired Student t test with a P < 0.05 and Mann–Whitney U test with P < 0.001 was used to determine statistical significance.

Study approval

The protocol for utilization of human specimens was approved by the ethical review board of the Graduate School of Medicine, Osaka University (Osaka, Japan; no. 13455). All protocols used for animal experiments in this study were approved by the Animal Research Committee of Osaka University (Osaka, Japan; no. 21-048-1).

All DKK family proteins bind to CKAP4 and promote cellular proliferation

To examine whether DKK family proteins other than DKK1 act on CKAP4, FLAG-tagged DKK family proteins were stably expressed in MDCK cells (Fig. 1A). When MDCK cells were cultured on a membrane filter to allow them to be polarized two dimensionally, DKK2-FLAG, DKK3-FLAG, and DKK4-FLAG were primarily secreted apically as efficiently as DKK1 (Fig. 1A; ref. 28). CKAP4 was expressed on the apical cell surface of MDCK cells (28). The addition of DKK proteins to the apical side (Ap), but not the basolateral side (Bl), promoted cell proliferation as assessed using Ki-67 positivity (Fig. 1B). Like DKK1, DKK2, DKK3, and DKK4 also bound to CKAP4 (Fig. 1C) and induced the internalization of CKAP4 from the cell surface membrane (Fig. 1D). DKK(s)-induced cellular proliferation was inhibited in CKAP4 knockout (KO) MDCK cells, which were generated by using CRISPR/Cas9-based genome editing (Fig. 1E and F). In three-dimensional culture conditions using Matrigel, MDCK cells formed cysts (34). The size of MDCK cysts increased and Ki-67–positive cells were simultaneously increased by the expression of DKKs (Fig. 1G). CRD1 was required for the binding of DKK1 to CKAP4 (28). CRD1 deletion mutants (ΔCRD1) of DKK2, DKK3, and DKK4 did not increase the size of the cyst or increase cellular proliferation as assessed using Ki-67 (Fig. 1G). These results suggest that all DKK family proteins promote MDCK cell proliferation through CKAP4, suggesting that CKAP4 is a common receptor of DKK family proteins.

Figure 1.

DKKs promote cell proliferation. A, Left, schematic diagrams of individual DKK proteins. aa, amino acids; SP, signal peptide; CRD1 or CRD2, cysteine rich domain 1 or 2. Middle, MDCK cells expressing FLAG-tagged DKK1 to DKK4 were generated, and their lysates were probed with anti-FLAG and HSP90 antibodies. Right, MDCK cells were subjected to the apical-basolateral sorting assay. Conditioned media were probed with anti-FLAG antibody. For DKK4, DKK4-FLAG (arrowhead) in conditioned media was immunoprecipitated with anti-FLAG antibody. *, Ig. B, MDCK cells were apically (Ap) or basolaterally (Bl) stimulated with 250 ng/mL DKK1 and 3 or DKK2 and 4 conditioned media, and then the cells were stained with anti-Ki-67 antibody (red) and DRAQ5 DNA Dye (blue). Ki-67–postitive cells are expressed as the percentage of positively stained cells as compared with total cells per field (n = 5 fields). NT, nontreatment. *, P < 0.01 (Student t test). C, Lysates of MDCK cells expressing DKKs-FLAG were immunoprecipitated with FLAG antibody and the immunoprecipitates were probed with the indicated antibodies. *, Ig and DKK3-FLAG overlapped with Ig. D, MDCK cells were stimulated with DKKs-FLAG conditioned media and biotinylated at the indicated time points. Cell surface proteins were precipitated with NeutrAvidin beads and probed with anti-CKAP4 antibody. E, Lysates of CKAP4 knockout (KO) MDCK cells were probed with the indicated antibodies. HSP90 was used as a loading control. F, Wild-type or CKAP4 KO MDCK cells were stimulated with DKKs-FLAG conditioned media for 2 days and the number of cells were quantified. *, P < 0.01 (Student t test). G, DKKs-FLAG–expressing MDCK cells were grown three dimensionally and stained with Ki-67 (red), F-actin (green), and DRAQ5 DNA Dye (blue; top). The cross-sectional areas of individual cysts were measured and are shown as cyst size (left bottom). Ki-67–positive cells were counted and are shown as the percentages of cells in a cyst (right bottom). *, P < 0.0001 (Mann–Whitney U test); **, P < 0.01 (Student t test). Scale bar, 20 μm.

Figure 1.

DKKs promote cell proliferation. A, Left, schematic diagrams of individual DKK proteins. aa, amino acids; SP, signal peptide; CRD1 or CRD2, cysteine rich domain 1 or 2. Middle, MDCK cells expressing FLAG-tagged DKK1 to DKK4 were generated, and their lysates were probed with anti-FLAG and HSP90 antibodies. Right, MDCK cells were subjected to the apical-basolateral sorting assay. Conditioned media were probed with anti-FLAG antibody. For DKK4, DKK4-FLAG (arrowhead) in conditioned media was immunoprecipitated with anti-FLAG antibody. *, Ig. B, MDCK cells were apically (Ap) or basolaterally (Bl) stimulated with 250 ng/mL DKK1 and 3 or DKK2 and 4 conditioned media, and then the cells were stained with anti-Ki-67 antibody (red) and DRAQ5 DNA Dye (blue). Ki-67–postitive cells are expressed as the percentage of positively stained cells as compared with total cells per field (n = 5 fields). NT, nontreatment. *, P < 0.01 (Student t test). C, Lysates of MDCK cells expressing DKKs-FLAG were immunoprecipitated with FLAG antibody and the immunoprecipitates were probed with the indicated antibodies. *, Ig and DKK3-FLAG overlapped with Ig. D, MDCK cells were stimulated with DKKs-FLAG conditioned media and biotinylated at the indicated time points. Cell surface proteins were precipitated with NeutrAvidin beads and probed with anti-CKAP4 antibody. E, Lysates of CKAP4 knockout (KO) MDCK cells were probed with the indicated antibodies. HSP90 was used as a loading control. F, Wild-type or CKAP4 KO MDCK cells were stimulated with DKKs-FLAG conditioned media for 2 days and the number of cells were quantified. *, P < 0.01 (Student t test). G, DKKs-FLAG–expressing MDCK cells were grown three dimensionally and stained with Ki-67 (red), F-actin (green), and DRAQ5 DNA Dye (blue; top). The cross-sectional areas of individual cysts were measured and are shown as cyst size (left bottom). Ki-67–positive cells were counted and are shown as the percentages of cells in a cyst (right bottom). *, P < 0.0001 (Mann–Whitney U test); **, P < 0.01 (Student t test). Scale bar, 20 μm.

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The expression of DKK3 and CKAP4 is associated with poor ESCC prognosis

The protein expression levels of DKK2, DKK3, and DKK4 in cancer cells was examined. DKK3 was highly expressed in KYSE960 and TE-11 esophageal, T24 bladder, U-251 glioblastoma, PANC-1 pancreatic, and Caco-2 colon cancer cells; moderately detected KKLS gastric, HLE liver, S2-CP8 and SUIT-2 pancreatic, SW480 colon, and Calu-6 and A549 lung cancer cells (Fig. 2A); and hardly detected endogenously in other cell lines, including TMK1, AGS, MKN1, and MKN45 gastric, HLF and HepG2 liver, and DLD-1 and HCT116 colon cancer cells in addition to X293T and MDCK nontumor cells (Fig. 2A). Transcriptome data from matched tumor and nontumor tissues (12 paired cases) in The Cancer Genome Atlas (TCGA) showed that DKK3 is expressed in ESCC tumor lesions more significantly than in nontumor tissue (Fig. 2B). The TCGA dataset also revealed that DKK3 is expressed highly in ESCC (86 cases) rather than esophageal adenocarcinoma (85 cases) and nontumor esophageal tissues (12 cases; Fig. 2B).

Figure 2.

Expression of DKK3 in human ESCC is associated with poor prognosis. A, Lysates of embryonic kidney cells (X293T), parental, or DKK3-FLAG–expressing MDCK cells, ESCC cells (KYSE960 and TE-11), bladder cancer cells (T24), glioblastoma cells (U-251), gastric cancer cells (TMK1, AGS, KKLS, MKN1, and MKN45), liver cancer cells (HLE, HLF, and HepG2), pancreatic cancer cells (S2-CP8, SUIT-2, and PANC1), colon cancer cells (SW480, Caco-2, DLD-1, and HCT116), and lung adenocarcinoma cells (Calu-6 and A549) were probed with anti-DKK3 or anti-HSP90 antibodies. HSP90 was used as a loading control. B, The levels of DKK3 expression in 12 paired cancerous and noncancerous tissue samples of ESCC cases (left) or in nontumor (n = 12), ESCC (n = 86), and esophageal adenocarcinoma (n = 85; right) from TCGA datasets are expressed as RPKM (reads per kilobase of exon per million mapped). *, P < 0.01 (paired samples t test); **, P < 0.001 (Mann–Whitney U test). C, Lysates of ESCC cell lines were probed with anti-DKK3 or anti-HSP90 antibodies. D, ESCC tissues (n = 72) were stained with anti-DKK3 or anti-CKAP4 antibody and hematoxylin. Scale bars, 100 μm. Percentages of DKK3- and/or CKAP4-positive cases are shown on the right. E and F, The relationship between overall (E) or relapse-free survival (F) and the expression of DKK3 and CKAP4 in patients with ESCC was analyzed. The log-rank test was used for statistical analysis.

Figure 2.

Expression of DKK3 in human ESCC is associated with poor prognosis. A, Lysates of embryonic kidney cells (X293T), parental, or DKK3-FLAG–expressing MDCK cells, ESCC cells (KYSE960 and TE-11), bladder cancer cells (T24), glioblastoma cells (U-251), gastric cancer cells (TMK1, AGS, KKLS, MKN1, and MKN45), liver cancer cells (HLE, HLF, and HepG2), pancreatic cancer cells (S2-CP8, SUIT-2, and PANC1), colon cancer cells (SW480, Caco-2, DLD-1, and HCT116), and lung adenocarcinoma cells (Calu-6 and A549) were probed with anti-DKK3 or anti-HSP90 antibodies. HSP90 was used as a loading control. B, The levels of DKK3 expression in 12 paired cancerous and noncancerous tissue samples of ESCC cases (left) or in nontumor (n = 12), ESCC (n = 86), and esophageal adenocarcinoma (n = 85; right) from TCGA datasets are expressed as RPKM (reads per kilobase of exon per million mapped). *, P < 0.01 (paired samples t test); **, P < 0.001 (Mann–Whitney U test). C, Lysates of ESCC cell lines were probed with anti-DKK3 or anti-HSP90 antibodies. D, ESCC tissues (n = 72) were stained with anti-DKK3 or anti-CKAP4 antibody and hematoxylin. Scale bars, 100 μm. Percentages of DKK3- and/or CKAP4-positive cases are shown on the right. E and F, The relationship between overall (E) or relapse-free survival (F) and the expression of DKK3 and CKAP4 in patients with ESCC was analyzed. The log-rank test was used for statistical analysis.

Close modal

Because the antibodies for DKK2 and DKK4 were not available for Western blotting, a public database analysis was performed to test their expression levels. When compared with DKK1 and DKK3, the mRNA levels of DKK2 and DKK4 were quite low in cultured cell lines from various tissues (Supplementary Fig. S1; http://medical-genome.kribb.re.kr/GENT/). In addition, DKK2 and DKK4 were barely detectable in various tissues and cancer cell lines (RefExA, http://157.82.78.238/refexa/main_search.jsp). Therefore, the role of DKK3 in ESCC was further investigated.

Among ESCC cell lines, DKK3 was highly expressed in TE-6, TE-11, and KYSE960 cells; moderately expressed in TE-8, TE-9, and TE-10 cells; and little expressed in TE-1, TE-4, TE-5, TE-14, and KYSE410 cells (Fig. 2C). In ESCC cases, DKK3 and CKAP4 were detected in 37 of 72 (51.4%) and 47 of 72 (65.2%) cases, respectively, whereas positive expression was minimally detected in nontumor regions under our staining conditions (Fig. 2D).

Clinicopathologic examination revealed that the protein expression levels of DKK3 or CKAP4 are not associated with various clinicopathologic parameters including tumor invasion, lymph node metastasis, or venous invasion (Supplementary Table S2). Overall survival was decreased in patients positive for DKK3 compared with patients that were negative for DKK3 (P = 0.0402; Fig. 2E). In addition, ESCC cases positive for both DKK3 and CKAP4 significantly shortened the duration of overall survival and relapse-free survival as compared with ESCC cases positive for either DKK3 or CKAP4 (P = 0.0139 and 0.0333, respectively; Fig. 2E and F). Univariate analysis demonstrated that patients with pN1-3, lymphatic vessel invasion, or DKK3 and/or CKAP4 expression were associated with inferior overall survival (Supplementary Table S3). Multivariate analysis identified that, along with lymph node metastasis (pN1-3), being both DKK3 and CKAP4 positive was indicative of poor prognosis (Supplementary Table S4). Taken together, these results suggest that the expression of DKK3 and CKAP4 negatively correlates with prognosis in ESCC.

Because DKK1 was also shown to be expressed in ESCC (29, 35), the expression levels of DKK1 and DKK3 were compared in ESCC. The results revealed that the expression of DKK1 and DKK3 is mutually exclusive in ESCC cell lines that we examined (Fig. 3A). Both DKK1 and DKK3 were expressed in 27% of the ESCC cases used in Fig. 2 (Fig. 3B); furthermore, they were present in different tumor lesions in the same patient (Fig. 3C). Whereas DKK1 was expressed throughout the tumor lesion, DKK3 showed a tendency to be localized to peripheral regions of the tumors (Fig. 3C). ESCC cases were classified according to the expression of ligands (DKK1 and/or DKK3) and a receptor (CKAP4; Fig. 3D). Interestingly, patients expressing both CKAP4 and ligands (either DKK1 and/or DKK3) showed poorer overall survival and relapse-free survival compared with other patients (Fig. 3E). Thus, DKK1 and DKK3 can be expressed in ESCC independently, while the simultaneous expression of both ligands and CKAP4 is associated with increased aggressiveness of ESCC.

Figure 3.

DKK3 and DKK1 are exclusively expressed in ESCC. A, The protein levels of DKK1 (Fig. 2A in ref. 34) and DKK3 (Fig. 2C in this study) normalized with HSP90 were expressed as arbitrary unit. B, ESCC tissues (n = 72) were stained with anti-DKK1 or anti-DKK3 antibodies and hematoxylin. The percentages of DKK1- and/or DKK3-positive cases are shown. C, Serial sections of an ESCC tissue were stained with anti-DKK1 or anti-DKK3 antibodies and hematoxylin. Regions 1 and 2 were positive for DKK3 and DKK1, respectively. Scale bar, 1 mm. D, ESCC tissues (n = 72) were stained with anti-DKK1, anti-DKK3, or anti-CKAP4 antibodies. The number of DKK1-, DKK3-, and/or CKAP4-positive cases are shown. E, The relationship between overall (left) or relapse-free survival (right) and the expression of ligand [DKK3(+) and/or DKK1(+)] and/or CKAP4 in patients with ESCC was analyzed. The log-rank test was used for statistical analysis.

Figure 3.

DKK3 and DKK1 are exclusively expressed in ESCC. A, The protein levels of DKK1 (Fig. 2A in ref. 34) and DKK3 (Fig. 2C in this study) normalized with HSP90 were expressed as arbitrary unit. B, ESCC tissues (n = 72) were stained with anti-DKK1 or anti-DKK3 antibodies and hematoxylin. The percentages of DKK1- and/or DKK3-positive cases are shown. C, Serial sections of an ESCC tissue were stained with anti-DKK1 or anti-DKK3 antibodies and hematoxylin. Regions 1 and 2 were positive for DKK3 and DKK1, respectively. Scale bar, 1 mm. D, ESCC tissues (n = 72) were stained with anti-DKK1, anti-DKK3, or anti-CKAP4 antibodies. The number of DKK1-, DKK3-, and/or CKAP4-positive cases are shown. E, The relationship between overall (left) or relapse-free survival (right) and the expression of ligand [DKK3(+) and/or DKK1(+)] and/or CKAP4 in patients with ESCC was analyzed. The log-rank test was used for statistical analysis.

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DKK3 and CKAP4 are required for ESCC cell proliferation in vitro

DKK3 and CKAP4 formed an endogenous complex in TE-11 cells (Fig. 4A). When compared with HCT116 colon cancer cells, CKAP4 was significantly localized to the cell surface membrane of TE-11 and KYSE960 cells (Fig. 4B). Knockdown of DKK3 or CKAP4 using siRNA in TE-11 and KYSE960 cells resulted in the inhibition of cell proliferation under two-dimensional culture conditions (Fig. 4C–F). Impaired cell proliferation caused by DKK3 or CKAP4 knockdown was rescued by the expression of DKK3-FLAG or CKAP4-HA, respectively (Fig. 4G–J). A DKK3 mutant lacking the CRD1 domain (DKK3-ΔCRD1) did not rescue the phenotype (Fig. 4K and L). Depletion of DKK3 or CKAP4 in TE-11 and KYSE960 cells inhibited AKT activity, but not Src and ERK activities (Fig. 4G–I; Supplementary Fig. S2A and S2B). AKT activity was also rescued by the expression of DKK3 and CKAP4 in siRNA-transfected cells (Fig. 4G–I). These results suggest that the binding of DKK3 to CKAP4 activates AKT and is required for DKK3-mediated ESCC cell proliferation.

Figure 4.

DKK3-CKAP4 signaling is required for ESCC proliferation. A, Lysates (input) of TE-11 cells were immunoprecipitated with anti-DKK3 antibody or nonimmune IgG. The immunoprecipitates (IP) were probed with anti-CKAP4 and anti-DKK3 antibodies. B, Cell surface proteins were biotinylated and precipitated with NeutrAvidin Agarose beads. Total protein and the precipitates (cell surface) were probed with anti-CKAP4 antibody. C and D, Lysates of TE-11 and KYSE960 cells transfected with DKK3 siRNA (C) or CKAP4 siRNA (D) were probed with the indicated antibodies. HSP90 was used as a loading control. E and F, Numbers of TE-11 and KYSE960 cells transfected with DKK3 siRNA (E) or CKAP4 siRNA (F) were counted. *, P < 0.005 (Student t test). G and H, Wild-type or DKK3-FLAG–expressing TE-11 and KYSE960 cells were transfected with DKK3 siRNA and the lysates were probed with indicated antibodies (G). Cell numbers of the same cells were counted (H). *, P < 0.005 (Student t test). I and J, Wild-type or CKAP4-HA–expressing TE-11 and KYSE960 cells were transfected with CKAP4 siRNA and the lysates were probed with indicated antibodies (I). Cell numbers of the same cells were counted (J). *, P < 0.005 (Student t test). K and L, Wild-type, DKK3-FLAG, or DKK3-ΔCRD1-FLAG–expressing TE-11 and KYSE960 cells were transfected with DKK3 siRNA and the lysates were probed with the indicated antibodies (K). Cell numbers of the same cells were counted (L). *, P < 0.005 (Student t test).

Figure 4.

DKK3-CKAP4 signaling is required for ESCC proliferation. A, Lysates (input) of TE-11 cells were immunoprecipitated with anti-DKK3 antibody or nonimmune IgG. The immunoprecipitates (IP) were probed with anti-CKAP4 and anti-DKK3 antibodies. B, Cell surface proteins were biotinylated and precipitated with NeutrAvidin Agarose beads. Total protein and the precipitates (cell surface) were probed with anti-CKAP4 antibody. C and D, Lysates of TE-11 and KYSE960 cells transfected with DKK3 siRNA (C) or CKAP4 siRNA (D) were probed with the indicated antibodies. HSP90 was used as a loading control. E and F, Numbers of TE-11 and KYSE960 cells transfected with DKK3 siRNA (E) or CKAP4 siRNA (F) were counted. *, P < 0.005 (Student t test). G and H, Wild-type or DKK3-FLAG–expressing TE-11 and KYSE960 cells were transfected with DKK3 siRNA and the lysates were probed with indicated antibodies (G). Cell numbers of the same cells were counted (H). *, P < 0.005 (Student t test). I and J, Wild-type or CKAP4-HA–expressing TE-11 and KYSE960 cells were transfected with CKAP4 siRNA and the lysates were probed with indicated antibodies (I). Cell numbers of the same cells were counted (J). *, P < 0.005 (Student t test). K and L, Wild-type, DKK3-FLAG, or DKK3-ΔCRD1-FLAG–expressing TE-11 and KYSE960 cells were transfected with DKK3 siRNA and the lysates were probed with the indicated antibodies (K). Cell numbers of the same cells were counted (L). *, P < 0.005 (Student t test).

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DKK3 and CKAP4 are required for ESCC proliferation in vivo

KYSE960 cells exhibited xenograft tumor formation when they were inoculated into immunodeficient mice; KYSE960 cells stably expressing shRNAs for DKK3 or CKAP4 showed decreased cell proliferation ability in vitro and tumor-forming ability in vivo (Fig. 5A and B; Supplementary Fig. S3A and S3B). Rabbit anti-CKAP4 polyclonal antibody (anti-CKAP4 pAb) inhibited the binding of DKK3 and CKAP4 (Fig. 5C; Supplementary Fig. S3C). In DKK3-depleted KYSE960 cells, anti-CKAP4 pAb suppressed DKK3-dependent internalization of cell surface CKAP4 (Fig. 5D) and activation of AKT (Fig. 5E). After xenograft tumors were allowed to grow to a size of approximately 100 mm3, anti-CKAP4 pAb or control IgG was administrated intraperitoneally and tumor development was observed for an additional 22 days. The mice receiving anti-CKAP4 pAb reduced tumor volumes (Fig. 5F). These results suggest that the DKK3-CKAP4 axis is required for tumor growth in vivo and that anti-CKAP4 antibody is effective as an anticancer agent against ESCCs with DKK3 and CKAP4 expression.

Figure 5.

DKK3-CKAP4 signaling is required for xenograft tumor formation. A and B, KYSE960 stably expressing control (scramble) shRNA or DKK3 shRNA (n = 6; A), control shRNA or CKAP4 shRNA (n = 6; B) were subcutaneously implanted into immunodeficient mice. Representative appearance of one mouse (left top) and extirpated xenograft tumors (left bottom) are shown. Dashed lines show the outline of xenograft tumors. The volumes (middle) and weights (right) of the xenograft tumors were measured. Results are plotted as box and whiskers, where the median is represented with a line, the box represents the 25th–75th percentile, and error bars show the 5th–95th percentile. Scale bars, 10 mm. *, P < 0.005 (Student t test). C, Purified DKK3-FLAG was incubated with GST-CKAP4 ECD in the presence of control IgG or anti-CKAP4 antibody (CKAP4 pAb) and the mixtures were precipitated with glutathione sepharose beads. The precipitates were probed with DKK3 antibody. ECD, extracellular domain. D, KYSE960 cells expressing DKK3 shRNA were treated with or without DKK3-FLAG conditioned media and biotinylated for 60 minutes. Cell surface proteins were precipitated from total cell lysates by using NeutrAvidin beads and probed with anti-CKAP4 antibody. E, KYSE960 cells expressing DKK3 shRNA were pretreated with control IgG or anti-CKAP4 antibody and then stimulated with DKK3-FLAG CM. The lysates were probed with the indicated antibodies. An arrow indicates the position of pAKT (activated AKT). F, KYSE960 cells were subcutaneously implanted into immunodeficient mice. Anti-CKAP4 antibody or control IgG (250 μg/body; n = 7) was injected into the intraperitoneal cavity twice per week. Left, representative appearance of one mouse (left) and extirpated xenograft tumors (right) is shown. Dashed lines show the outline of xenograft tumors. The volumes (middle) and weights (right) of the xenograft tumors were measured. Scale bars, 10 mm. *, P < 0.005 (Student t test).

Figure 5.

DKK3-CKAP4 signaling is required for xenograft tumor formation. A and B, KYSE960 stably expressing control (scramble) shRNA or DKK3 shRNA (n = 6; A), control shRNA or CKAP4 shRNA (n = 6; B) were subcutaneously implanted into immunodeficient mice. Representative appearance of one mouse (left top) and extirpated xenograft tumors (left bottom) are shown. Dashed lines show the outline of xenograft tumors. The volumes (middle) and weights (right) of the xenograft tumors were measured. Results are plotted as box and whiskers, where the median is represented with a line, the box represents the 25th–75th percentile, and error bars show the 5th–95th percentile. Scale bars, 10 mm. *, P < 0.005 (Student t test). C, Purified DKK3-FLAG was incubated with GST-CKAP4 ECD in the presence of control IgG or anti-CKAP4 antibody (CKAP4 pAb) and the mixtures were precipitated with glutathione sepharose beads. The precipitates were probed with DKK3 antibody. ECD, extracellular domain. D, KYSE960 cells expressing DKK3 shRNA were treated with or without DKK3-FLAG conditioned media and biotinylated for 60 minutes. Cell surface proteins were precipitated from total cell lysates by using NeutrAvidin beads and probed with anti-CKAP4 antibody. E, KYSE960 cells expressing DKK3 shRNA were pretreated with control IgG or anti-CKAP4 antibody and then stimulated with DKK3-FLAG CM. The lysates were probed with the indicated antibodies. An arrow indicates the position of pAKT (activated AKT). F, KYSE960 cells were subcutaneously implanted into immunodeficient mice. Anti-CKAP4 antibody or control IgG (250 μg/body; n = 7) was injected into the intraperitoneal cavity twice per week. Left, representative appearance of one mouse (left) and extirpated xenograft tumors (right) is shown. Dashed lines show the outline of xenograft tumors. The volumes (middle) and weights (right) of the xenograft tumors were measured. Scale bars, 10 mm. *, P < 0.005 (Student t test).

Close modal

p63 regulates DKK3 expression in ESCC

The underlying mechanism that induces high expression of DKK3 in ESCC is not known. p63, a p53-related transcription factor, is frequently amplified in ESCC and is required for cell proliferation (24, 27). p63 was expressed in the basal layer of esophageal epithelium in nontumor regions as well as throughout entire tumor lesions (Fig. 6A). DKK3-positive areas seemed to partially overlap with p63-positive areas in the peripheral legions of tumors (Fig. 6A). ΔNp63α was primarily expressed in TE-11 and KYSE960 cells (Fig. 6B; Supplementary Fig. S4). ΔNp63β and ΔNp63γ were also weakly expressed in TE-11 cells. Knockdown of p63, using a common siRNA for ΔNp63 isoforms, in TE-11 and KYSE960 cells reduced the expression of DKK3; DKK3 expression was rescued by exogenously expressed ΔNp63α in KYSE960 cells (Fig. 6B). In addition, although overexpression of DKK3 did not increase proliferation, knockdown of p63 inhibited the proliferative ability of TE-11 and KYSE960 cells; the inhibition was partially rescued by ectopic expression of DKK3 (Fig. 6C).

Figure 6.

p63 upregulates DKK3 in ESCC. A, ESCC tissues were stained with anti-p63 (left) or anti-DKK3 (right) antibody and hematoxylin. These sections obtained from the same patient with ESCC. Scale bar, 100 μm. Top, nontumor region; bottom, tumor lesion. B, TE-11 and KYSE960 cells (left) or KYSE960 cells expressing ΔNp63α (right) were transfected with p63 siRNA, and the lysates were probed with the indicated antibodies. Black, gray, and white arrowheads indicate ΔNp63α, ΔNp63β, and ΔNp63γ, respectively. Clathrin was used as a loading control. C, Wild-type or DKK3-FLAG–expressing TE-11 and KYSE960 cells were transfected with p63 siRNA and the lysates were probed with the indicated antibodies (left). Numbers of the same cells were counted (right). Black and white arrowheads indicate exogenous and endogenous DKK3, respectively. *, P < 0.005; **, P < 0.05 (Student t test). D, A consensus binding sequence for p63 was found in the DKK3 genome locus, which is located in the approximately 8-kb upstream region of the DKK3 transcription starting site. Wild-type (WT) and mutated (Mut) sequences of p63-binding site are shown. The consensus sequence of p63 is represented graphically as a sequence logo obtained from JASPAR web site. E, A chromatin immunoprecipitation (ChIP) assay was performed using KYSE960 cells. The chromatin precipitated with control IgG, anti-p63, or anti-acetyl histone H4 antibodies was analyzed by PCR with region-specific primers. Band intensities of PCR products were quantified and are expressed as the percentage of the input. p21 is a target gene of p63. F, Left, after transfection of reporter constructs shown in D with the indicated constructs in X293T cells, luciferase activities were measured. The results are shown as fold increases compared with empty vector-expressing cells. Right, the DKK3 mRNA levels in ΔNp63α and/or p53 shRNA expressing X293T cells were measured using quantitative PCR. Relative mRNA levels were normalized to GAPDH and are shown as fold changes compared with mock-transfected cells (*, P < 0.05; Student t test).

Figure 6.

p63 upregulates DKK3 in ESCC. A, ESCC tissues were stained with anti-p63 (left) or anti-DKK3 (right) antibody and hematoxylin. These sections obtained from the same patient with ESCC. Scale bar, 100 μm. Top, nontumor region; bottom, tumor lesion. B, TE-11 and KYSE960 cells (left) or KYSE960 cells expressing ΔNp63α (right) were transfected with p63 siRNA, and the lysates were probed with the indicated antibodies. Black, gray, and white arrowheads indicate ΔNp63α, ΔNp63β, and ΔNp63γ, respectively. Clathrin was used as a loading control. C, Wild-type or DKK3-FLAG–expressing TE-11 and KYSE960 cells were transfected with p63 siRNA and the lysates were probed with the indicated antibodies (left). Numbers of the same cells were counted (right). Black and white arrowheads indicate exogenous and endogenous DKK3, respectively. *, P < 0.005; **, P < 0.05 (Student t test). D, A consensus binding sequence for p63 was found in the DKK3 genome locus, which is located in the approximately 8-kb upstream region of the DKK3 transcription starting site. Wild-type (WT) and mutated (Mut) sequences of p63-binding site are shown. The consensus sequence of p63 is represented graphically as a sequence logo obtained from JASPAR web site. E, A chromatin immunoprecipitation (ChIP) assay was performed using KYSE960 cells. The chromatin precipitated with control IgG, anti-p63, or anti-acetyl histone H4 antibodies was analyzed by PCR with region-specific primers. Band intensities of PCR products were quantified and are expressed as the percentage of the input. p21 is a target gene of p63. F, Left, after transfection of reporter constructs shown in D with the indicated constructs in X293T cells, luciferase activities were measured. The results are shown as fold increases compared with empty vector-expressing cells. Right, the DKK3 mRNA levels in ΔNp63α and/or p53 shRNA expressing X293T cells were measured using quantitative PCR. Relative mRNA levels were normalized to GAPDH and are shown as fold changes compared with mock-transfected cells (*, P < 0.05; Student t test).

Close modal

A consensus binding motif for p63 was found in the approximately 8-kb upstream promoter region of the DKK3 transcription starting site (Fig. 6D). A chromatin immunoprecipitation (ChIP) assay revealed that p63 binds to the predicted binding region in KYSE960 cells (Fig. 6E). Loss-of-function mutations in p53 have been frequently found in ESCC and ESCC cell lines as well (24, 36, 37). It was examined whether the p63-binding genomic region that we identified is involved in the transcription of DKK3 using the reporter gene assay. ΔNp63α expression stimulated luciferase activity under the control of the genomic region including −8466 to −8118 of DKK3 in X293T cells depleting p53 (Fig. 6F). When the possible p63-binding sites were mutated, ΔNp63α-dependent luciferase activity was diminished (Fig. 6D–F). Similarly, although the expression of ΔNp63α or depletion of p53 alone did not induce the endogenous DKK3 expression, their combination stimulated the DKK3 expression (Fig. 6F). These results suggest that p63 could function as a transcription factor to stimulate DKK3 expression in ESCC, likely in the setting of a p53 mutational background; thus, one reason why DKK3 is overexpressed in ESCC may be due to the expression of ΔNp63α.

ΔNp63α and DKK3 promote the growth of esophageal organoids

As shown in some murine and human tissues, including gastrointestinal organs, liver, and pancreas (38), organoid cultures were generated from adult mouse esophagus (33). The organoids consisted of an outer layer of basal cells positive for cytokeratin (CK) 14 and inner differentiated cells expressing CK13 (Fig. 7A). Growth factors, including EGF, noggin, R-spondin, and FGF10, were required for esophageal organoid proliferation (Fig. 7B).

Figure 7.

p63 and DKK promote the growth of esophageal organoids. A, Organoids from wild-type adult mouse esophagus were stained with the indicated antibodies. B, Approximately 5,000 epithelial cells derived from esophageal organoids were embedded in Matrigel and cultured in complete media or EGF-, noggin-, R-spondin 1-, or FGF10-depleted media for 5 days (left). C, Left, the KP or KPC organoids were cultured in complete growth media (control) or growth media without EGF and FGF10 for 5 days. Right, the cross-sectional areas of individual organoid were measured and are shown as the organoid size. *, P < 0.0001 (Mann–Whitney U test). D, The KP or KPC organoids were trypsinized and dissociated cells were biotinylated. The lysates were precipitated with Neutravidin beads and probed with anti-CKAP4 antibody. KPC, KP-organoids expressing Cre recombinase. E, The KPC organoids expressing DKK1-FLAG, DKK3-FLAG, or ΔNp63α were cultured in growth media without EGF and FGF10 for 4 days (top). The cross-sectional areas of individual organoid were measured and are shown as the organoid size (left bottom; *, P < 0.05; **, P < 0.001; Mann–Whitney U test). The mRNA expression levels of DKK1 or DKK3 were measured (right bottom; *, P < 0.05; Student t test). F, The KPC organoids shown in E were stained with the indicated antibodies. G, Left, the KPC organoids expressing ΔNp63α treated with 20 μg/mL control IgG or anti-CKAP4 antibody (CKAP4 pAb) were cultured in growth media without EGF and FGF10 for 5 days. Right, the cross-sectional areas of individual organoids were measured and are shown as the organoid size. Scale bars, 100 μm (A, B, C, E, F, and G; *, P < 0.0001; Mann–Whitney U test).

Figure 7.

p63 and DKK promote the growth of esophageal organoids. A, Organoids from wild-type adult mouse esophagus were stained with the indicated antibodies. B, Approximately 5,000 epithelial cells derived from esophageal organoids were embedded in Matrigel and cultured in complete media or EGF-, noggin-, R-spondin 1-, or FGF10-depleted media for 5 days (left). C, Left, the KP or KPC organoids were cultured in complete growth media (control) or growth media without EGF and FGF10 for 5 days. Right, the cross-sectional areas of individual organoid were measured and are shown as the organoid size. *, P < 0.0001 (Mann–Whitney U test). D, The KP or KPC organoids were trypsinized and dissociated cells were biotinylated. The lysates were precipitated with Neutravidin beads and probed with anti-CKAP4 antibody. KPC, KP-organoids expressing Cre recombinase. E, The KPC organoids expressing DKK1-FLAG, DKK3-FLAG, or ΔNp63α were cultured in growth media without EGF and FGF10 for 4 days (top). The cross-sectional areas of individual organoid were measured and are shown as the organoid size (left bottom; *, P < 0.05; **, P < 0.001; Mann–Whitney U test). The mRNA expression levels of DKK1 or DKK3 were measured (right bottom; *, P < 0.05; Student t test). F, The KPC organoids shown in E were stained with the indicated antibodies. G, Left, the KPC organoids expressing ΔNp63α treated with 20 μg/mL control IgG or anti-CKAP4 antibody (CKAP4 pAb) were cultured in growth media without EGF and FGF10 for 5 days. Right, the cross-sectional areas of individual organoids were measured and are shown as the organoid size. Scale bars, 100 μm (A, B, C, E, F, and G; *, P < 0.0001; Mann–Whitney U test).

Close modal

It has been shown that the activation of RAS/MAPK pathway was found in human esophageal cancer in addition to genomic mutations in p53 locus (24). Furthermore, expression of KrasG12D in the foregut is able to generate esophageal cancer in mice treated with genotoxic agents (39). Therefore, it was examined to generate tumor-like organoids by introducing KrasG12D and depleting p53. Esophageal organoids were generated from KrasG12DLSL/p53lox/lox (KP) mice–derived esophageal epithelial cells and Cre recombinase was transduced into KP organoids to generate another type of organoid (KPC). KP organoids were morphologically and histologically similar to wild-type esophageal organoids and required complete growth media, including EGF and FGF10 (Fig. 7C). In contrast, KPC organoids grew rapidly and exhibited increased organoid size in the absence of EGF and FGF10 (Fig. 7C).

CKAP4 was confirmed to be expressed on the cell surface membrane of the organoid cells when single cells were prepared from the organoids (Fig. 7D). Additional expression of ΔNp63α, DKK3, or DKK1 into KPC organoids further increased the size of organoids, and expression of ΔNp63α increased levels of DKK3 mRNA but not that of DKK1 mRNA (Fig. 7E). These organoids were able to differentiate as they expressed CK13, but CK13 expression was distributed irregularly in places other than the inner region (Fig. 7F), suggesting that these organoids may lose the epithelial polarity and exhibit tumor-like characteristics. Notably, anti-CKAP4 pAb inhibited the growth of the KPC organoids expressing ΔNp63α (Fig. 7G). These results suggest that DKK3 promotes organoid growth via CKAP4 under conditions in which driver genes, such as Kras and p53, are genetically manipulated.

CKAP4 is a receptor for DKK3

In our preceding study, we found that CKAP4 is a DKK1 receptor (28, 29, 40); our current study suggests that CKAP4 also functions as a receptor for other DKK family proteins including DKK2, DKK3, and DKK4. This finding is especially important for DKK3, because its receptor has been unknown to date. We showed that DKK3 binds to CKAP4, activates AKT, and induces the internalization of CKAP4. Furthermore, we also demonstrated that DKK3-ΔCRD1 does not rescue the phenotypes induced by DKK3 knockdown. These results strongly support the conclusion that CKAP4 on the cell surface membrane is a receptor for DKK3.

Possible functions of DKK3 in other pathologic conditions have been reported. For instance, DKK3 regulated MMP2/9 levels to control normal acinar morphogenesis in prostate epithelial cells and inhibited TGFβ-induced prostate cancer cell migration and invasion (41). DKK3 was also expressed in macrophages found in atherosclerotic plaques, while Dkk3 and ApoE simultaneous knockout mice exhibited decreased atherosclerotic pathogenesis (42). Therefore, it is intriguing to speculate that DKK3 has pleiotropic functions and that CKAP4 mediates several pathologic functions of DKK3.

DKK1-CKAP4 and DKK3-CKAP4 signaling are independently activated in ESCC

Despite recent advances in endoscopic detection of early esophageal tumor lesions and in therapeutic advances such as endoscopic resection, surgery, radiation, and chemotherapy, the survival of patients with ESCC is still poor. Thus, a novel molecularly targeted therapy is required. Taken together with our previous report that the DKK1-CKAP4 signaling is a therapeutic target for ESCC (29), our current study demonstrates that DKK3 activates AKT via CKAP4 like DKK1 and that the DKK3-CKAP4 and DKK1-CKAP4 axes are activated in distinct populations of ESCC cells, because DKK1 and DKK3 are separately expressed in the different cultured ESCC cells and ESCC cases and in the different tumor lesions of the same cases.

The anti-DKK1 antibody DKN-01, a humanized therapeutic mAb against DKK1, underwent phase I evaluation for advanced esophageal cancer in a combination with paclitaxel (43). Given that DKK1- or DKK3-expressing tumors compose a different subset of cancer cells even in the same patient with ESCC, anti-CKAP4 antibody effectively suppresses both types of DKK signaling in ESCC. Therefore, therapeutic agents directed against CKAP4 could be more useful than antibodies against each DKK family member. The AKT activity and proliferation of TE-14 cells, where CKAP4 is expressed on the cell surface membrane but DKK1 and DKK3 are hardly expressed, were not decreased by the anti-CKAP4 antibody (29). These results support the idea that anti-CKAP4 antibody specifically affects cancer cells that express both DKKs and CKAP4.

p63 is a transcriptional factor that can increase DKK3 expression in ESCC

The underlying mechanism by which DKK3 expression is increased in cancer tissue has not yet been elucidated. We found that p63 knockdown reduces the expression of DKK3 in ESCC cells and the reduced expression of DKK3 was rescued by the reexpression of ΔNp63α. Furthermore, the predicted enhancer region was associated with p63, suggesting that p63 is a possible transcription factor that promotes DKK3 expression. On the other hand, ΔNp63 is physiologically required for epidermal development and homeostasis (44) and ΔNp63, but not DKK3, is expressed in normal basal layer tissue, suggesting that ΔNp63-dependent DKK3 expression occurs in a tumor-dependent manner. It is possible that in the environment of a tumor, there may be unknown coactivators of ΔNp63 that can promote DKK3 expression, because overexpression of ΔNp63α alone did not affect DKK3 expression in X293T cells but increased DKK3 expression in p53-depleted cells. In addition, the expression of DKK3 partially rescued the inhibition of cell proliferation in p63-depleted cells, suggesting that not only DKK signaling but other pathways are activated in ESCC expressing ΔNp63.

Recent progress in organoid technology has allowed the establishment of epithelial organoid culture from many murine and human tissues including gastrointestinal organs, liver, and pancreas (33, 38). Esophageal organoids retain a similar cell-surface phenotype as compared with primary tissues, including a nonquiescent stem cell population residing in the basal epithelium. By expressing KrasG12D and depleting p53, the organoids were able to grow in the absence of EGF and FGF10 in vitro. Organoid growth was promoted by the expression of either DKK3, DKK1, or ΔNp63α, while treatment with anti-CKAP4 pAb suppressed organoid growth. Thus, these tumor-like organoids could be useful for the evaluation of the effects of new anticancer drugs, such as anti-CKAP4 antibody. It is also possible to establish organoids from surgically resected human intestinal tissues and endoscopic biopsies of patients with cancer (45). It would be interesting to evaluate the efficacy of anti-CKAP4 antibody in esophageal cancer organoids from patients that are positive for both DKK3 and CKAP4.

No potential conflicts of interest were disclosed.

Conception and design: C. Kajiwara, K. Fumoto, M. Yamasaki, A. Kikuchi

Development of methodology: C. Kajiwara, K. Fumoto, K. Asano, A. Kikuchi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Kajiwara, K. Fumoto, K. Asano, K. Odagiri, M. Yamasaki, E. Morii

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Kajiwara, K. Fumoto, H. Kimura, M. Yamasaki, E. Morii

Writing, review, and/or revision of the manuscript: K. Fumoto, M. Yamasaki, E. Morii, A. Kikuchi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Fumoto, S. Nojima, M. Yamasaki, H. Hikita, T. Takehara, Y. Doki

Study supervision: K. Fumoto, A. Kikuchi

Other (analyzed the expression of DKK1, DKK3, and CKAP4 in cancer specimens): H. Kimura

We would like to thank Drs. S. Tsukita, M. Hatakeyama, Y. Matsuura, K. Matsumoto, A. Shintani, Y. Watanabe, and K. Shinjo for donating cells and helping data analysis. K. Asano is supported by the Osaka University Medical Doctor Scientist Training Program. This work was supported by Grants-in-Aid for Scientific Research (2016-2021; grant no. 16H06374 to A. Kikuchi), and Grants-in-Aid for Young Scientists (Start-up; 2016-2017; grant no. 16H06944 to H. Kimura) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and also supported by the Project Promoting Support for Drug Discovery (2016-2018; grant no. DNW-16002 to K. Fumoto) and the Project for Cancer Research and Therapeutic Evolution (P-CREATE; 2016–2017; grant no. 16cm0106119h0001 and 2018–2019; grant no. 18cm0106132h0001; to A. Kikuchi) from the Japan Agency for Medical Research and Development, AMED, and by grants to A. Kikuchi from the Yasuda Memorial Foundation.

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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