P63-Dependent Dickkopf3 Expression Promotes Esophageal Cancer Cell

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.

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.

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.

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

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.

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.

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 (Tsukuba, Japan) in 6

October 2014 (PANC-1), respectively. Lenti-X™ 293T (X293T) cells were purchased from Takara

Bio Inc. (Shiga, Japan) 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 Table S1.

Polarized secretion of DKKs. Polarized secretion assays of DKK molecules was performed as

described (30). Madin-Darby canine kidney (MDCK) cells expressing DKK-FLAG (2 x 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 mM Tris-HCl pH 8.0, 10%

glycerol, 137 mM NaCl, and 1% NP40), the precipitates were probed with anti-FLAG antibody.

Polarized MDCK cells proliferation assay. Proliferation assays were performed as described (28,

31) with modification. MDCK cells (2 x 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 7

expresses hCas9. The cloned plasmids were introduced into MDCK cells using Viafect reagent

(Promega) with blasticidin resistance gene expressing plasmids. After transfection, MDCK cells

were re-plated 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 (2D) cell proliferation assay. MDCK cells (1 x 105 cells) were seeded into 3.5-cm

dishes and cultured in DMEM supplemented with 1% FBS. TE-11 and KYSE960 cells (1 x 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 (3D) cell proliferation assay. Three-dimensional cell proliferation assays were

performed as previously described (28, 31) with modification. Forty µl of Matrigel (BD Biosciences)

were solidified on a round coverslip by incubating for 30 min at 37°C. MDCK cells (5 x 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 (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 min 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 mM Tris-HCl pH 7.5; the bound complexes were 8

probed with anti-CKAP4 antibody.

Patients and immunohistochemical studies of DKK1 and DKK3. ESCC patients (n = 72) who underwent surgery at Osaka University Hospital 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 (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 re-examined in this study.

All tissue sections (5 μm thickness) were stained using a DakoRealTMEnVisionTM 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 h 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 h at 4 °C. After washing, the specimens were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h 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. Since DKK3 was expressed specifically in the marginal regions of tumor nests near stroma, we regarded tumor areas of 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

previously described (28, 31) with modification. KYSE960 cells (1 x 107 cells) were transplanted using dorsal subcutaneous transplantation of six-week-old male BALB/cAnNCrj-nu

immunodeficient mice (Charles River Laboratory Japan Inc., Osaka, Japan). 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 anti-tumor 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 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) x (minor axis) x (minor axis) x 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) and the indicated constructs. The cells were lysed at 24 h post-transfection, and the luciferase activity was measured with PicaGene reagent (Toyo Ink, Tokyo, Japan) as described previously (32).

-Galactosidase activities were measured to standardize the transfection efficiency.

Esophageal organoid culture. Esophageal organoid culture was performed as described (33) with modification. The esophagus mucosa from wild-type or KP (KrasG12DLSL from National Cancer

Institute–Frederick, Bethesda, Maryland and p53lox/lox from The Jackson laboratory) adult mouse was 10

physically separated and minced into small pieces. Cells were dissociated in DMEM/F12 medium

containing 0.25% Trypsin-EDTA and 100 unit DNase I (Sigma, D4527) for 30 min at 37°C with

agitation. After passing the cells through a 40 m sterile filter (BD Biosciences, 352340), 5000 esophageal epithelial cells were suspended in ice-cold 20 l Matrigel solution (BD Biosciences).

Matrigel containing esophageal epithelial cells was placed as a droplet in a 24-well tissue culture plate and solidified by incubating for 20 min at 37°C. Complete growth medium was prepared at the time of use and consisted of 1:1 mixture of DMEM and F12, 1 x N-2 (Life Technologies, 17502048),

1 x B-27 Supplements (Life Technologies, 17504044), 1 x Glutamax (Life Technologies), 10 mM

HEPES/NaOH [pH 7.4], 50 ng/ml EGF (R&D, 236-EG), 100 ng/ml Noggin (R&D, 1967-NG), 100

ng/ml R-Spondin 1 (R&D, 7150-RS), 100 ng/ml FGF10 (Peprotech, 100-26), and 10 M Y27632

(WAKO, 253-00513).

Statistical analyses. Experiments were performed repeatedly at least three times and results are

presented as the mean ± standard deviation (s.d.). A paired or unpaired Student’s t test with a P value

of < 0.05 and Mann-Whitney's U test with P value of < 0.0001 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, Japan (No. 13455). All protocols used

for animal experiments in this study were approved by the Animal Research Committee of Osaka

University, Japan (No.21-048-1).

Results

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 (28) (Fig. 1A). 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 (3D) 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 (CRD1) 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.

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

non-tumor cells (Fig. 2A). Transcriptome data from matched tumor and non-tumor tissues (12 paired

cases) in the cancer genome atlas (TCGA) showed that DKK3 is expressed in ESCC tumor lesions

more significantly than in non-tumor tissue (Fig. 2B). The TCGA data set also revealed that DKK3 is

expressed highly in ESCC (86 cases) rather than esophageal adenocarcinoma (85 cases) and

non-tumor esophageal tissues (12 cases) (Fig. 2B).

Since 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 37/72

(51.4%) and 47/72 (65.2%) cases, respectively, whereas positive expression was minimally detected in non-tumor regions under our staining conditions (Fig. 2D). 13

Clinicopathological examination revealed that the protein expression levels of DKK3 or CKAP4

are not associated with various clinicopathological 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.

Since 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 14

ESCC.

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 2D 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-CRD1) 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 and I;

Supplementary Fig. S2A and B). AKT activity was also rescued by the expression of DKK3 and

CKAP4 in siRNA-transfected cells (Fig. 4G and I). These results suggest that the binding of DKK3

to CKAP4 activates AKT and is required for DKK3-mediated ESCC cell proliferation.

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 formation ability in vivo (Fig. 5A, B, and Supplementary Fig. S3A and B). Rabbit

anti-CKAP4 polyclonal antibody (anti-CKAP4 pAb) inhibited the binding of DKK3 and CKAP4

(Fig. 5C, and 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, 15

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 anti-cancer agent against ESCCs with DKK3 and

CKAP4 expression.

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 non-tumor 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). Np63 was primarily expressed in TE-11 and KYSE960 cells (Fig. 6B, and

Supplementary Fig. S4). Np63andNp63 were also weakly expressed in TE-11 cells.

Knockdown of p63, using a common siRNA for Np63 isoforms in TE-11 and KYSE960 cells

reduced the expression of DKK3; DKK3 expression was rescued by exogenously expressed Np63

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).

A consensus binding motif for p63 was found in the ~8kb 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 16

using the reporter gene assay. Np63 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, Np63-dependent luciferase activity was diminished

(Fig. 6D and F). Similarly, although the expression of Np63 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 Np63.

Np63 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).

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). Futhermore, 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 17

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 Np63, DKK3, or DKK1 into KPC organoids further increased the size of organoids, and expression of Np63 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 Np63 (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.

Discussion

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-Δ

CRD1 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 pathological conditions have been reported. For instance,

DKK3 regulated MMP2/9 levels to control normal acinar morphogenesis in prostate epithelial cells and inhibited TGF-β-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 pathological

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 present study

demonstrates that DKK3 activates AKT via CKAP4 like DKK1 and that the DKK3-CKAP4 and 19

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 monoclonal antibody 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 ESCC patient, 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 which 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 re-expression of Np63. 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 Np63, but not DKK3, is expressed in normal basal layer

tissue, suggesting that Np63-dependent DKK3 expression occurs in a tumor-dependent manner. It

is possible that in the environment of a tumor, there may be unknown co-activators of Np63 that can promote DKK3 expression, since overexpression of Np63 alone did not affect DKK3 20

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

Np63.

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 non-quiescent 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

Np63, 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 anti-cancer drugs, such as anti-CKAP4 antibody. It is also possible to establish organoids from surgically-resected human intestinal tissues and endoscopic biopsies of cancer patients (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.

Acknowledgements

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.A. is supported by the Osaka University Medical Doctor Scientist Training Program. This work was supported by Grants-in-Aid for Scientific Research (2016-2021) (No. 16H06374) to A. Kikuchi, and Grants-in-Aid for Young Scientists (Start-up) (2016-2017) (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) (No. DNW-16002) to K. Fumoto and the Project for Cancer Research And Therapeutic Evolution (P-CREATE) (2016-2017) (No. 16cm0106119h0001) and (2018-2019) (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.

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Figure legend

Figure 1. DKKs promote cell proliferation.

(A) Left panel, schematic diagrams of individual DKK proteins. aa, amino acids; SP, signal peptide;

CRD1 or CRD2, cysteine rich domain 1 or 2. Middle panel, MDCK cells expressing FLAG-tagged

DKK1 to DKK4 were generated, and their lysates were probed with anti-FLAG and HSP90 antibodies. Right panels, MDCK cells were subjected to the apical-basolateral sorting assay.

Conditioned media (CM) were probed with anti-FLAG antibody. For DKK4, DKK4-FLAG

(arrowhead) in CM 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 CM, 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 to total cells per field (n = 5 fields). NT, non-treatment. *, P < 0.01 (Student’s 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 CM 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 CM for 2 days and

the number of cells were quantified. *, P < 0.01 (Student’s 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 panels). The cross-sectional areas of

individual cysts were measured and are shown as cyst size (left bottom panel). Ki-67-postitive cells

were counted and are shown as the percentages of cells in a cyst (right bottom panel). *, P < 0.0001

(Mann-Whitney's U test); **, P < 0.01 (Student’s t test). Scale bar, 20 µm.

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 panel) or in non-tumor (n = 12), ESCC (n = 86), and esophageal adenocarcinoma (n

= 85) (right panel) from TCGA datasets were expressed as RPKM (reads per kilobase of exon per million mapped). *, P < 0.01 (paired samples t test). **, P < 0.001 (Mann-Whitney's U test)

(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 in the right

panel.

(E and F) The relationship between overall (E) or relapse-free survival (F) and the expression of

DKK3 and CKAP4 in ESCC patients 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 (Figure 2A in Ref. 34) and DKK3 (Figure 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.

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 panel) or relapse-free survival (right panel) and the expression of ligand (DKK3(+) and/or DKK1(+)) and/or CKAP4 in ESCC patients was analyzed.

The log-rank test was used for statistical analysis.

Figure 4. DKK3-CKAP4 signaling is required for ESCC proliferation.

(A) Lysates (input) of TE-11 cells were immunoprecipitated with anti-DKK3 antibody or non-immune 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. 29

(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’s 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’s 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’s t test).

(K and L) Wild-type, DKK3-FLAG, or DKK3-CRD1-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’s 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 (A) (n = 6),

control shRNA or CKAP4 shRNA (B) (n = 6) were subcutaneously implanted into immunodeficient

mice. Representative appearance of one mouse (left top panel) and extirpated xenograft tumors (left

bottom panel) are shown. Dashed lines show the outline of xenograft tumors. The volumes (middle

panel) and weights (right panel) 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’s t test).

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 CM and biotinylated for 60 min. 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 pre-treated 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 panels, representative appearance of one mouse (left pictures) and extirpated xenograft tumors (right pictures) are shown. Dashed lines show the outline of xenograft tumors. The volumes

(middle panel) and weights (right panel) of the xenograft tumors were measured. Scale bars, 10 mm.

*, P < 0.005 (Student’s t test).

Figure 6. p63 upregulates DKK3 in ESCC.

(A) ESCC tissues were stained with anti-p63 (left panels) or anti-DKK3 (right panels) antibody and

hematoxylin. These sections obtained from the same ESCC patient. Scale bar, 100 m. Top panels,

non-tumor region; bottom panels, tumor lesion.

(B) TE-11 and KYSE960 cells (left panels) or KYSE960 cells expressing Np63 (right panels) were transfected with p63 siRNA, and the lysates were probed with the indicated antibodies. Black,

gray and white arrowheads indicate Np63,Np63and Np63, respectively. Clathrin was used as a loading control.

siRNA and the lysates were probed with the indicated antibodies (left panels). Numbers of the same

cells were counted (right panels). Black and white arrowheads indicate exogenous and endogenous

DKK3, respectively. *, P < 0.005; **, P < 0.05 (Student’s t test).

(D) A consensus binding sequence for p63 was found in the DKK3 genome locus which is located to the ~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 panel, 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 panel, the DKK3 mRNA levels in Np63 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’s t 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. 32

(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 panels).

without EGF and FGF10 for 5 days (left panels). The cross-sectional areas of individual organoid

were measured and are shown as the organoid size (right panel). *, P < 0.0001 (Mann-Whitney's 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 Np63 were cultured in

growth media without EGF and FGF10 for 4 days (top panels). The cross-sectional areas of

individual organoid were measured and are shown as the organoid size (left bottom panel, *, P <

0.05; **, P < 0.001 (Mann-Whitney's U test)). The mRNA expression levels of DKK1 or DKK3 were measured (right bottm panel, *, P < 0.05 (Student’s t test)).

(F) The KPC organoids shown in (E) were stained with the indicated antibodies.

(G) The KPC organoids expressing Np63 treated with 20 g/ml control IgG or anti-CKAP4

antibody (CKAP4 pAb) were cultured in growth media without EGF and FGF10 for 5 days (left

panels). The cross-sectional areas of individual organoids were measured and are shown as the

organoid size (right panel). Scale bars, 100 m (A, B, C, E, F, and G). *, P < 0.0001

(Mann-Whitney's U test).

p63-dependent Dickkopf3 expression promotes esophageal cancer cell proliferation via CKAP4

Chihiro Kajiwara, Katsumi Fumoto, Hirokazu Kimura, et al.

Cancer Res Published OnlineFirst September 4, 2018.

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