Dysregulation of WNT5A/ROR2 Signaling Characterizes The

Published OnlineFirst April 15, 2016; DOI: 10.1158/1541-7786.MCR-15-0484

Signal Transduction Molecular Cancer Research Dysregulation of WNT5A/ROR2 Signaling Characterizes the Progression of Barrett- Associated Esophageal Adenocarcinoma Orestis Lyros1,2, Linghui Nie3, Tami Moore3, Rituparna Medda3, Mary Otterson3, Behnaz Behmaram4, Alexander Mackinnon4, Ines Gockel2, Reza Shaker1, and Parvaneh Raﬁee3

Abstract

The mechanism underlying the progression of normal survival, and migration in a dose-dependent fashion. esophageal mucosa to esophageal adenocarcinoma remains rhWNT5AwasfoundtoinhibitTOPflash activity in ROR2 elusive. WNT5A is a noncanonical WNT, which mainly func- wild-type cells, whereas increased TOPflash activity in ROR2- tionsviathereceptortyrosinekinase-likeorphanreceptor2 knockdown OE33 cells. In addition, ROR2 knockdown alone (ROR2), and has an unclear role in carcinogenesis. In this abolished cell proliferation andweakenedthemigrationprop- study, we aimed to determine the role of WNT5A/ROR2 erties of OE33 cells. These findings support an early dysregula- signaling in esophageal adenocarcinoma. Analysis of WNT5A tion of the noncanonical WNT5A/ROR2 pathway in the path- and ROR2 expression patterns in healthy controls, Barrett and ogenesis of esophageal adenocarcinoma, with the loss of esophageal adenocarcinoma patients' esophageal clinical spe- WNT5A expression together with the ROR2 overexpression to cimens as well as in various esophageal cell lines demonstrated be consistent with tumor promotion. a ROR2 overexpression in esophageal adenocarcinoma tissues compared with Barrett and healthy mucosa, whereas WNT5A Implications: The dysregulation of WNT5A/ROR2 noncanonical expression was found significantly downregulated toward WNT signaling in Barrett-associated esophageal adenocarcinoma esophageal adenocarcinoma formation. Treatment of esoph- introduces possible prognostic markers and novel targets for ageal adenocarcinoma OE33 cells with human recombinant tailored therapy of this malignancy. Mol Cancer Res; 14(7); 647–59. WNT5A (rhWNT5A) significantly suppressed proliferation, 2016 AACR.

Introduction (GERD; ref. 3). The course of Barrett esophagus is mainly benign; however, this condition is the main predisposing Esophageal adenocarcinoma is noted by having both the factor for esophageal adenocarcinoma and is associated with fastest growing incidence of all cancers in the western world an increased risk of progression to cancer (4). The molecular over the last several decades (1) and a high mortality rate that mechanisms underlying the development and the aggressive exceeds 80% (2). Despite improvements in chemotherapy phenotype of esophageal adenocarcinoma remain largely regimens and the use of multimodal strategies (e.g., surgery, undefined. neoadjuvant therapy, and adjuvant therapy), esophageal ade- The embryologic WNT signaling pathway is important for nocarcinoma remains a therapeutic challenge for oncologists development and tissue homeostasis, and its dysregulation has (2) due to high rates of local recurrence and early regional and been associated with tumorigenesis (5). It has been established systemic metastases. Esophageal adenocarcinoma is often that WNT proteins regulate at least three distinct intracellular studied in conjunction with Barrett esophagus, the condition signaling pathways: b-catenin–dependent (canonical) and -inde- in which the normal squamous epithelium in the distal pendent (noncanonical) signaling pathways, the planar cell esophagus is replaced by a metaplastic columnar mucosa in þ polarity/convergent extension pathway, and the Ca2 pathway response to chronic severe gastroesophageal reflux disease (summarized in ref. 6). The canonical WNT pathway regulates the cell cycle, growth, and proliferation (5), whereas the noncanonical WNT signaling is purported to have crucial functions in the 1 Division of Gastroenterology & Hepatology, Medical College of Wis- regulation of cell migration and polarity (6). Cross-talk between consin, Milwaukee, Wisconsin. 2Department of Visceral, Transplant, Thoracic and Vascular Surgery, University Hospital, Leipzig, Germany. canonical and noncanonical WNT pathways still remains elusive, 3Department of Surgery, Medical College of Wisconsin, Milwaukee, but inhibitory interaction of the canonical by the noncanonical 4 Wisconsin. Department of Pathology, Medical College of Wisconsin, has been shown (7, 8). Milwaukee, Wisconsin. Aberrant activation of the canonical WNT/b-catenin signal- Corresponding Author: Parvaneh Rafiee, Department of Surgery, Medical ing has been associated with the development of esophageal College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. adenocarcinoma (9). However, genetic alterations in compo- Phone: 414-955-4667; Fax: 414-955-6361; E-mail: prafi[email protected] nents of the canonical WNT pathway such as b-catenin, APC, doi: 10.1158/1541-7786.MCR-15-0484 and Axin, which are observed in other human cancers and result 2016 American Association for Cancer Research. in upregulated canonical signaling, are not frequently detected

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in esophageal adenocarcinoma (10, 11). Instead, esophageal Materials and Methods adenocarcinoma is marked by changes, such as loss of the Reagents and antibodies negative regulators of the pathway, Wnt inhibitory factor 1, Recombinant human WNT5A (rhWNT5A) was obtained from and secreted frizzled receptor proteins), as well as induction of R&D Systems Inc. Antibodies against ROR2 and WNT5A were Wnt-2 expression, which are expected to increase signaling obtained from Abcam; active b-catenin antibody (ABC; unphospho- along the WNT axis (9, 12, 13). Thus, the mechanism of rylated in Ser37 and Thr41 sites) was from Millipore, total b-catenin canonical WNT activation in esophageal adenocarcinoma is and phospo-GSK3b (Ser9) antibodies were obtained from Cell still unknown, whereas the involvement of the noncanonical Signaling Technology. GSK3b and horseradish peroxidase second- WNT pathway has not been addressed yet. ary antibodies were obtained from Santa Cruz Biotechno- WNT5A is classified as a nontransforming WNT family member logy. b-Actin antibody, DNA demethylation drug; 5-azacytidine that activates noncanonical WNT signaling by interacting with (5-Aza-CR), protease inhibitor cocktail, and all other reagents and receptor tyrosine kinase–like orphan receptors (ROR; ref. 14). chemicals were obtained from Sigma unless otherwise specified. ROR2 is a member of the ROR family of receptor tyrosine kinases, acts as a receptor or coreceptor for WNT5A, and has shown to have essential functions in developmental morphogenesis (15). Human esophageal specimens fi WNT5A/ROR2 signaling has been shown to inhibit the canonical Archival paraf nized human esophageal specimens of esoph- n ¼ WNT signaling, which is often activated in tumor cells, indicating ageal adenocarcinoma patients ( 11) who underwent esopha- that WNT5A might act as a tumor suppressor (16). Indeed, loss or gectomy in the Department of Thoracic Surgery at Froedtert low expression of WNT5A is associated with increased invasion Hospital and Medical College of Wisconsin from 2010 to 2012 and aggressiveness of many carcinomas (17, 18). Nevertheless, were retrospectively analyzed (Table 1). Laser microdissection fi WNT5A has also demonstrated oncogenic properties based on was undertaken to isolate RNA from tumor cells from paraf n fi – findings depicting upregulation of WNT5A levels in melanoma, blocks (puri cation up to 70% 80% was achieved). In addition, lung, and breast cancers (19–21). Although, the exact function human esophageal biopsy specimens were collected from of WNT5A is highly dependent on the receptor context, the ROR2 patients 18 years or older undergoing endoscopy for Barrett n ¼ n ¼ receptor on the other hand, appears to demonstrate oncogenic esophagus ( 15) and healthy individuals ( 10) undergoing properties regardless the presence of ligand (22). Thus, the role endoscopy for nonesophageal indications (Table 2). Studies were of WNT5A/ROR2 signaling in carcinogenesis remains still approved by the Human Research Review Committee of the ambiguous. Medical College of Wisconsin, and study participants gave written The current study was undertaken to explore the role of the informed consent prior to their studies. noncanonical WNT5A/ROR2 signaling pathway in esophageal adenocarcinoma with respect to the canonical WNT/b-cate- Cell lines nin signaling activation. We hypothesized that alteration in Human squamous esophageal telomerase-immortalized cells, the WNT5A/ROR2 signaling pathway may underlie the aber- EPC1 (EPC1-hTERT) and EPC2 (EPC2-hTERT) with intact p53 rant activation of b-catenin transcriptional activity in esoph- and p16 genes (23) were the generous gifts of Dr. Hiroshi ageal adenocarcinoma cells. By analyzing human esophageal Nakagawa (Gastroenterology Division, University of Pennsylva- specimens and esophageal cell lines, we present evidence nia, Philadelphia, PA). CP-A cells are a nondysplastic columnar suggesting that the progression of esophageal adenocarcino- cell line derived from Barrett esophagus that was obtained from ma is characterized by deregulation of the noncanonical the ATCC. OE33 cells are poorly differentiated esophageal ade- WNT5A/ROR2 signaling pathway. The loss of WNT5A expres- nocarcinoma cancer cells (Sigma). Cells have been recently tested sion along with upregulation of ROR2 receptor appears to and authenticated by immunofluorescence staining for cytoker- contribute to an aggressive phenotype of the esophageal atin expression and were grown according to a standard protocol adenocarcinoma cells. Our findings underscore the signifi- as described previously (24). cance of the WNT5A/ROR2 signaling pathway in esophageal adenocarcinoma and provide rationale for the development Cellular assays of future target therapies along with new insights into the Cell proliferation was assessed either by [3H] thymidine mechanisms of noncanonical WNT signaling in the context of uptake, as described previously (25), or by 3-(4,5-dimethylthia- tumor progression. zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as

Table 1. Demographic and histologic ﬁndings in esophageal adenocarcinoma patients Presence of Patient Age Sex TNM classiﬁcation Barrett mucosa Neoadjuvant #1 77 M pT1a, N0, Mx, (G1) Yes No #2 56 M pT1b, N0, Mx (G2) Yes No #3 62 F pT1b, N0, Mx, (G1) Yes No #4 46 F pT3, pN1a, pMX (G3) No No #5 76 M pT1b, pN0, pMX (G2) Yes No #6 74 M pT1b, pN0, pMX (G3) Yes No #7 60 M pT1b, pN0, pM0 (G2) No No #8 61 M pT1b, pN0, pMX (G3) No No #9 55 M pT3, pN1, pMX (G3) No No #10 68 M pTcis, N0, Mx (G1) Yes No #11 68 M pT3, pN1, Mx (G2) Yes No

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Table 2. Demographic, endoscopic, and histologic findings in Barrett patients and healthy controls Patient Age Sex Indication for upper GI-endoscopy Endoscopy report Pathology report Healthy Controls #1 61 M Abdominal pain Normal looking squamous mucosa No abnormalities #2 46 F Weight loss, diarrhea Normal looking squamous mucosa No abnormalities #3 23 M Abdominal pain Normal looking squamous mucosa No abnormalities #4 37 M Vitamin B deficiency Normal looking squamous mucosa No abnormalities #5 60 F Abdominal pain Normal looking squamous mucosa No abnormalities #6 24 F Abdominal pain Normal looking squamous mucosa No abnormalities #7 25 M Crohn disease Normal looking squamous mucosa No abnormalities #8 46 F Family history of esophageal cancer Normal looking squamous mucosa No abnormalities #9 39 F Severe iron deficiency anemia Normal looking squamous mucosa No abnormalities #10 54 M Not reported Normal looking squamous mucosa No abnormalities

Barrett Patients #1 66 M History of Barrett, repaired hiatus hernia Barrett esophagus 3 cm BE-Metaplasia, no dysplasia #2 72 F Hx of Barrett, GERD Two tongues of Barrett esophagus <1 cm BE-Metaplasia, no dysplasia #3 60 M Hx of Barrett, GERD Barrett mucosa with scattered patchy BE-Metaplasia, no dysplasia distribution #4 45 M Hx of Barrett, GERD Irregular Z-line with possible 1 cm segment of BE-Metaplasia, no dysplasia Barrett esophagus #5 51 M Hx of Barrett Irregular Z-line with islands of salmon colored BE-Metaplasia, no dysplasia Barrett type mucosa in the vicinity of the Z-line. #6 68 F GERD Salmon-colored tongue-shaped extensions of up BE-Metaplasia, no dysplasia to 2 cm into distal esophagus #7 64 M Hx of Barrett Area of short-segment Barrett esophagus, visible BE-Metaplasia, no dysplasia on retroﬂexion within hernia sac #8 60 F Hx of erosive esophagitis Short segment of Barrett extending for 2cm BE-Metaplasia, no dysplasia segment in two tongues #9 45 F Hx of BE, GERD symptoms Barrett mucosa, with a few islands, extends about BE-Metaplasia, no dysplasia 1 cm above anatomic GE junction (Prague C0M1). #10 59 M Hx of Barrett Multiple islands Barrett mucosa—short segment BE-Metaplasia, no dysplasia #11 64 F Hx of Barrett Not available BE-Metaplasia, no dysplasia #12 59 M Hx of Barrett Circumferential salmon colored mucosa 34–36 Barrett esophagus with focal cm without nodule low-grade dysplasia #13 88 M Hx of Barrett 1 cm Barrett esophagus (C0M1) in hiatal hernia BE-Metaplasia, no dysplasia #14 46 F Hx of GERD Barrett appearing mucosa C1, M3 (upper limit BE-Metaplasia, no dysplasia of long segment 33 cm, upper limit of circumferential segment 35 cm GE junction 36 cm and diaphragmatic indentation 38 cm) #15 74 M Hx of Barrett with LGD Barrett esophagus, Prague classiﬁcation C8M10 Barrett esophagus with focal low-grade dysplasia Abbreviations: BE, Barrett esophagus; GE, gastroesophageal; Hx, history; GI, gastrointestinal. suggested by the manufacturer (Roche Diagnostics). Migration of Green Supermix (Bio-Rad), with 250 nmol/L primer and 1 mLof OE33 cancer cells was assayed with Wound Scratch Assay and cell cDNA per 20-mL reaction as described previously (24). 5-Aza-CR transmigration by using 24-well HTS FluoroBlok inserts in triplets was administrated to the OE33 cells at the concentration of 0.5 (8-mm pore size; Becton Dickinson). Later, cells were serum mmol/L and RNA was collected after 6, 24, 48, and 72 hours and starved for 24 hours before initiation of the assay. Cells (4 analyzed as described above. Gene expression was analyzed with 104) were resuspended in serum-free RPMI and added to the top iQ5 software (Bio-Rad). The primers used are; ROR2: (F: 50-GGC chamber. Consecutively, RPMI with or without rhWNT5A was AGA ACC CAT CCT CGT G-30,R:50-CGA CTG CGA ATC CAG GAC added to the bottom chamber. Chambers were incubated either C-30), WNT5A: (F: 50-GTG CAA TGT CTT CCA AGT TCT T-30,R:50- 0 0 for 24 hours or 48 hours at 37 Cin5%CO2. After incubation, the ATG AGC CGG ACA CCC CAT GGC A-3 ), GAPDH: (F: 5 - TGC number of migrated cells in the bottom chamber was determined ACC ACC AAC TGC TTA GC-30,R:50-GGC ATG GAC TGT GGT by luminescence assay according to the recommendations of the CAT GAG-30), b-actin: (F: 50-CAC TCT TCC AGC CTT CCT TC-30,R: manufacturer (Celltiter-Glo Cell Viability assay, Promega Corpo- 50-GGT GTA ACG CAA CTA AGT CAT), Axin2: (F: 50-TAC CGG ration) following incubation with Calcein AM Fluorescent Dye AGG ATG CTG AAG GC-30,R:50-CCA CTG GCC GAT TCT TCC TT- (BD Biosciences). 30), Ki67:(F: 50-ATT ATC GTT CCT TCA GGT ATG-30,R:50-TCA TCA GGG TCA GAA GAG AA-3). RNA isolation and qRT-PCR RNA was isolated from biopsies and OE33 cell using Arcturus Western blot analysis PicoPure RNA (Life Technologies) and the RNeasy Kit (Qiagen), Cell extracts were prepared and subjected to SDS-PAGE and respectively. cDNA was synthesized from 1 mg of total RNA using Western blotting using antibodies against ROR2, WNT5A, b-cate- the iScript cDNA synthesis kit according to the manufacturer's nin, active b-catenin, phospho GSK3, total GSK3, and b-actin as protocol (Bio-Rad). qRT-PCR was performed with SsoFast Eva- described previously (26).

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Immunohistochemical analysis in esophageal tissue sections obtained from esophageal adeno- Mucosal esophageal biopsy specimens were embedded in carcinoma patients (n ¼ 11) undergoing esophagectomy, with- paraffin blocks and cut into 5-mm sections. The sections were out previous treatment. High levels of ROR2 were detected in routinely stained with hematoxylin and eosin for histologic esophageal adenocarcinoma tissues, whereas ROR2 expression diagnosis, and additional sequential sections were subjected to was weak in the corresponding Barrett esophagus and even IHC using antibodies against WNT5A and ROR2, as described weaker and isolated in the proliferative basal layer of the previously (24). Expression of ROR2 immunohistochemical corresponding squamous esophageal mucosa (Fig. 1A). A sim- staining was quantified by both, determination of the intensity ple grading system (0–4; Fig. 1B) as well as an image analysis score of ROR2 expression in all biopsy samples using a 0–4 software were used to quantify the intensity and region score simple scoring system by a specialized pathologist as well as (%; Fig. 1C) of ROR2 staining in the different tissues as using an Automated Cellular Imaging System III (ACIS III, described in Materials and Methods. Statistical analysis, using DAKO) in a single case as described previously (27). This case Friedman–Kendall W test to analyze the overall results of was representative of the entire cohort and contained areas of intensity score between the three related conditions yielded a normal squamous mucosa, Barrett mucosa, and adenocarcino- Kendall coefficient of concordance of 0.363, which approached ma, and each of these histologic areas were analyzed indepen- statistical significance (P ¼ 0.079). Finally, significance between dently and graphed. The percentage of ROR2-positive tissue two-group comparisons was determined by Wilcoxon signed- and the intensity of ROR2 were measured independently and rank tests, followed by correction for multiple comparisons. used to create a region score. Obvious artifacts including tissue Overall, ROR2 expression was significantly higher in esophageal folding, edge effect, nonspecific chemical precipitation, and adenocarcinoma compared with squamous mucosa and Barrett dust or debris artefacts were excluded. esophagus tissues.

WNT5A and ROR2 gene silencing WNT5A expression is downregulated in esophageal OE33 and EPC2-hTERT cells were transfected with predesigned adenocarcinoma short interfering RNA (siRNA) targeting human WNT5A and/or Next, we examined the level of WNT5A mRNA expression in ROR2 gene and a control negative siRNA (Dharmacon) according esophageal biopsy specimens from three independent patients' to the manufacturer's protocol and described previously (24). The groups, healthy controls (n ¼ 10), Barrett (n ¼ 15), and esoph- ratio of siRNA to DharmaFECT reagent was 25 nmol/L siRNA to ageal adenocarcinoma patients (n ¼ 5). To ensure tissue homo- 1.5 mL of transfection reagent. geneity in esophageal adenocarcinoma tissues, laser microdissection was utilized to isolate tumor lesions. qRT-PCR analysis fi WNT5A Luciferase reporter assay demonstrated signi cantly lower level of mRNA in The assay was performed as described previously (24). In esophageal adenocarcinoma samples compared with squamous summary, OE33 cells in 60% confluence in 24-well plates were mucosa and Barrett esophagus biopsy samples (Fig. 2A). We WNT5A transfected with DNA plasmids of b-catenin-LEF/TCF-sensitive compared mRNA expression of gene between groups by – (TOP-flash) or b-catenin-LEF/TCF-insensitive (FOP-flash) report- using a global nonparametric Kruskal Wallis test, which fi P ¼ er vectors (Addgene) using Lipofectamine 2000 (Invitrogen) for showed a signi cant overall difference ( 0.0069). Subse- 48 hours. One microgram of TOPflash as well as 1 mg of FOPflash quent comparisons between groups (squamous mucosa vs. were used per well of a 24-well plate. phRL-TK plasmid (Promega) Barrett esophagus, squamous mucosa vs. esophageal adenocar- was cotransfected as control for transfection efficiency. Reporter cinoma, and Barrett esophagus vs. esophageal adenocarcino- – assay was performed using dual luciferase reporter system (Pro- ma) was done by the nonparametric Mann Whitney test. mega). Luciferase activity was measured via GLOMAX 20/20 Corresponding with the gene expression, Western blot analysis Luminometer (Promega). Values for each reporter were normal- of biopsy samples from esophageal adenocarcinoma patients ized to phRL-TK values. showed a lower level of WNT5A protein in tumor site compared with the corresponding healthy esophageal mucosa (Fig. 2B). Statistical analysis Conversely, the level of ROR2 protein expression was higher in IBM SPSS Statistics 19 software was used for statistical analysis. All tumor biopsy compared with healthy mucosa. Immunohisto- experiments were performed in triplicate, and values are means SE. chemical analysis of WNT5A expression in healthy esophageal Expression patterns between squamous mucosa, Barrett metaplasia, mucosa revealed a nuclear staining throughout all mucosal and esophageal adenocarcinoma tissue were compared within layers, whereas ROR2 expression was detected mainly in cell esophageal adenocarcinoma patients and across different patients' cytoplasm and membrane of the basal mucosal layer (Fig. 2C). fi groups (healthy controls, Barrett patients, and esophageal adeno- Further immunohistochemical analysis con rmed the negative carcinoma patients) by using nonparametric methods followed by correlation of ROR2 and WNT5A expression in esophageal Wilcoxon signed-rank test and Mann–Whitney test, respectively. adenocarcinoma tissue (Fig. 2D). Significant differences in cellular assays were evaluated by Student t test. P 0.05 was considered statistically significant. ROR2/WNT5A expression in esophageal cell lines The baseline level of WNT5A and ROR2 gene and protein expression in esophageal cell lines derived from normal esoph- Results agus (EPC1, EPC2), nondysplastic Barrett esophagus (CP-A), ROR2 is overexpressed in human esophageal adenocarcinoma and esophageal adenocarcinoma (OE33) were evaluated by To demonstrate whether ROR2 expression alters in the course RT-PCR and Western blotting. The level of WNT5A mRNA of progression from normal squamous epithelium to esoph- expression in EPC1 and EPC2 cells was higher than Barrett ageal adenocarcinoma, ROR2 expression was evaluated by IHC esophagus–derived cells (CP-A), and WNT5A expression was

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Figure 1. Immunohistochemical analysis of ROR2 expression in human esophageal tissue. ROR2 protein expression was evaluated by IHC in full-thickness esophageal tissue sections obtained from esophageal adenocarcinoma (EAC) patients (n ¼ 11). A, level of ROR2 protein expression was higher in esophageal adenocarcinoma tissues compared with the corresponding Barrett esophagus (BE) and squamous mucosa (SQ; original magnification, 20; scale bar, 300 mm). B, the intensity score of ROR2 expression in biopsy samples using 0–4 simple grading system, Wilcoxon signed-rank test, P ¼ 0.026 for SQ versus esophageal adenocarcinoma, P ¼ 0.096 for SQ versus BE and P ¼ 0.343 for BE versus esophageal adenocarcinoma. C, the region score (%) of ROR2 expression was created by combining the percentage of ROR2-positive tissue and the intensity of ROR2 expression by using the automated Computerized Imaging System III (ACIS III), Wilcoxon signed-rank test, P ¼ 0.015 for SQ versus BE, P ¼ 0.008 for BE versus esophageal adenocarcinoma. further reduced in OE33 cells (Fig. 3A). Conversely, the ROR2 (rhWNT5A). Application of rhWNT5A drastically decreased mRNA expression was significantly higher in CP-A and OE33 OE33 cell proliferation (Fig. 4A), cell survival (Fig. 4B), invasion cells compared with EPC1 and EPC2 cells (Fig. 3B). In agree- (Fig. 4C), and cell migration (Fig. 4D) in a dose- and time- ment with the gene expression, ROR2 protein expression in dependent manner, as shown by 3[H] thymidine uptake, MTT CP-A and OE33 cells was higher than in normal epithelial assay, transmigration, and wound scratch assays, respectively. cells, whereas WNT5A protein expression analysis demonstrat- These findings suggest that WNT5A functions as tumor suppres- ed a reverse pattern of expression (Fig. 3C). To investigate sor in esophageal adenocarcinoma. whether WNT5A loss of expression could be caused by promoter methylation based on previous reports (28), OE33 cells ROR2 overexpression induces tumor growth in OE33 cells were treated with 5-Aza-CR, a drug capable of demethylating Given the overexpression of ROR2, despite the loss of WNT5A, WNT5A's promoter (29). WNT5a mRNA expression was we aimed to assess the functional role of ROR2 receptor with increased following 0.5 mmol/L 5-Aza-CR treatment (Fig. respect to the tumor growth by siRNA-mediated gene silencing. 3D), suggesting that promoter hypermethylation may contrib- After 48 hours of siRNA transfection, ROR2 gene expression in ute to the downregulation of WNT5A. OE33 cells was effectively decreased as estimated 24 and 48 hours In all, the data derived from the in vitro model verify a high posttransfection by qRT-PCR (Fig. 5A). The knockdown of ROR2 expression of ROR2 receptor together with a simultaneous loss gene expression was further confirmed by Western blotting of WNT5A ligand, underlying the deregulation of the WNT5A/ (Fig. 5B). Remarkably, treatment with rhWNT5a for 24 hours ROR2 axis toward esophageal adenocarcinoma progression. resulted in similar downregulation of ROR2 protein expression, suggesting that the overexpression of ROR2 receptor may be Recombinant WNT5A suppresses OE33 cells' tumor growth compensatory to the loss of WNT5A ligand (Fig. 5C). Similarly, Next, we examined the functional consequences of the down- WNT5A gene expression was found upregulated following ROR2 regulated WNT5A expression in the esophageal adenocarcino- silencing, supporting a compensatory feedback loop between ma tumor growth. We evaluated the role of WNT5A by treating ligand and receptor (Fig. 5D). Functional assays revealed that OE33 cells with exogenous recombinant WNT5A protein silencing of ROR2 decreased OE33 cell proliferation (Fig. 5E) and

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Figure 2. WNT5A/ROR2 expressions in esophageal tissues. WNT5A gene expression in esophageal biopsy samples was determined by qRT-PCR. A, low level of WNT5A mRNA expression was found in esophageal adenocarcinoma (EAC) tissues compared with independent squamous mucosa (SQ) (n ¼ 10) and Barrett esophagus (BE; n ¼ 15). Mann–Whitney test, P ¼ 0.002; esophageal adenocarcinoma versus SQ, P ¼ 0.086; BE versus SQ and P ¼ 0.05; esophageal adenocarcinoma versus BE. B, representative Western blot analysis of esophageal human tissues from one esophageal adenocarcinoma patient using antibodies against ROR2 and WNT5A, demonstrates that esophageal adenocarcinoma samples express low level of WNT5A protein and high level of ROR2 protein. Graph is the summarized data of relative density. b-Actin served as an internal loading control. C, immunohistochemical analysis of the cellular and mucosal localization of ROR2 and WNT5A protein expression in healthy esophageal squamous mucosa (original magniﬁcation, 40; scale bar, 100 mm). D, IHC revealed strong ROR2 expression in absence of WNT5a expression in esophageal adenocarcinoma tissue (original magniﬁcation, 10; scale bar, 500 mm).

transmigration (Fig. 5F), signifying that ROR2 overexpression OE33 cells with rhWNT5a for 24 hours after sufficient siRNA- induces tumor growth. mediated ROR2 gene silencing. TOPflash activity was inhibited by rhWNT5A treatment in wild-type OE33 cells; however, it was WNT5A suppresses canonical Wnt/b-catenin signaling markedly increased in ROR2-knockdown cells (Fig. 6D). We in OE33 cells further confirmed this finding by increased level of ABC by To determine the nature of tumor suppressor properties of rhWNT5A stimulation in the ROR2-knockdown cells (Fig. 6E). WNT5A, we investigated whether WNT5a inhibits the canonical These findings signify that the presence of ROR2 receptor dictates Wnt/b-catenin signaling in OE33 cells. Treatment with rhWNT5a the function of WNT5A regarding inhibition or induction of the for 24 hours significantly decreased TOPFlash activity in a dose- canonical WNT/b-catenin signaling. dependent manner (Fig. 6A). In agreement with a reduced b-catenin transcriptional activity, the gene expression of the b-catenin target WNT5A knockdown induces proliferation in normal EPC2 gene, AXIN2, was suppressed by rhWNT5A (Fig. 6B). At the molec- epithelial cells ular level, rhWNT5A was found to reduce the level of phosphor- Finally, to reversely address, whether WNT5A silencing in normal ylated GSK3b (Ser 9; Fig. 6C), indicating activation of GSK3b and epithelial cells could induce a tumorigenic phenotype, we investi- subsequent inhibition of b-catenin–mediated signaling (30). Of gated the proliferative properties of EPC2 normal epithelial cells note, the WNT5A-mediated dephosphorylation of GSK3b was not following by knocking down the WNT5A gene expression using rescued by the canonical rhWNT3A, suggesting independent down- siRNA tranfection. Sufficient WNT5A gene silencing in EPC2 cells stream signaling transduction between the two WNTs. was demonstrated after 48 hours of siRNA transfection and was To further demonstrate the role of the ROR2 receptor in the found to result in significant increase of Ki67 expression (Fig. 7A) WNT5A-induced inhibition of the canonical WNT/b-catenin sig- as well as in marked induction of cell proliferation, mainly 72 hours naling, the level of TOPFlash activity was evaluated by treating posttransfection (Fig. 7B) as shown by qRT-PCR and 3[H]

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Figure 3. WNT5A/ROR2 expression in esophageal cell lines. WNT5A and ROR2 gene and protein expression were determined in various esophageal cell lines at rest. A and B, qRT-PCR shows higher levels of WNT5A mRNA expression in normal esophageal cell lines (EPC1 and EPC2) cells compared with Barrett (CP-A) and esophageal adenocarcinoma (OE33) cells. In contrast, ROR2 mRNA levels in CP-A and OE33 cells were significantly higher compared with EPC1 and EPC2 cells. C, Western blot analysis of OE33 whole cell lysates demonstrates that WNT5A protein expression in CP-A and OE33 cells was lower than EPC1 and EPC2 cells, whereas ROR2 protein was highly expressed in CP-A and OE33 cells compared with EPC1 and EPC2 cells. b-Actin served as an internal loading control. D, qRT-PCR analysis of WNT5A mRNA levels in OE33 cells following treatment with 0.5 mmol/L of 5-Aza-CR showed for different time points as indicated. thymidine uptake, respectively. Further analysis with qRT-PCR ROR2 overexpression was found to characterize esophageal ade- demonstrated an increase in AXIN2 gene expression following nocarcinoma compared with Barrett and healthy esophageal WNT5A silencing in EPC2 cells, indicating an enhanced canonical tissues. Similarly, in vitro analysis demonstrated low level of WNT signaling, whereas ROR2 gene expression remained WNT5A expression and ROR2 overexpression in OE33 cells unchanged (data not shown). Those findings are consistent with compared with the normal squamous (EPC1, EPC2) and Barrett a tumorigenic phenotype induced by WNT5a loss of expression in metaplastic (CP-A) cell lines. Treatment of OE33 cells with drug 5- the context of normal epithelial esophageal cells. Aza-CR an epigenetic modifier which results in DNA demethylation (29) restored the WNT5A expression, suggesting the epigenetic nature of WNT5a loss in esophageal adenocarcinoma. Discussion Moreover, treatment of OE33 cells with recombinant WNT5A In the current study, we examined the role of noncanonical inhibited cell proliferation, migration, and invasion, whereas, at WNT5A/ROR2 signaling pathway in the Barrett-associated esoph- the molecular level, significantly reduced TOPflash luciferase ageal adenocarcinoma. Loss of WNT5A expression along with activity, AXIN2 gene expression, and GSK3b phosphorylation at

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Figure 4. Effect of rhWNT5A on OE33 cell's proliferation, survival, and migration. Following treatment with rhWNT5A functional assays were performed in OE33 cells. A and B, rhWNT5A dose- and time-dependently decreased OE33 cell proliferation measured by [H]3 thymidine uptake and cell viability (MTT assay). C and D, rhWNT5a decreased cell transmigration and approximation in wound-scratch assay (, P < 0.05) dose dependently. Cells were serum starved (2% FCS) for 24 hours before initiation of the assay. Medium with 30% FCS in the bottom chamber for the transmigration assay served as positive control.

serine 9, indicating antagonism of the canonical WNT signaling. found to induce TOPﬂash activity and to increase the transcrip- This antagonism required the presence of ROR2 receptor, as tional level of active b-catenin in ROR2-knockdown OE33 cells. exogenous WNT5A, in contrast to wild-type OE33 cells, and was Notably, ROR2 knockdown itself resulted in decreased cell

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Noncanonical WNT5A-ROR2 Axis in Esophageal Adenocarcinoma

Figure 5. ROR2 silencing in OE33 cancer cells. Transfection of OE33 cells with ROR2 siRNA. A, ROR2 gene was effectively silenced at 24 and 48 hours posttransfection analyzed by RT-PCR. B, Western blot analysis demonstrated that expression of ROR2 protein was impaired following ROR2 gene silencing. C, stimulation of OE33 cell with rhWNT5A (500 ng/mL) for 24 hours resulted in decreased ROR2 protein expression. D, ROR2 gene silencing resulted in increased expression of WNT5A gene as measured after 48 hours posttransfection by qRT-PCR. E and F, ROR2 knockdown resulted in decreased cell proliferation ([H]3 thymidine uptake) and cell transmigration. Control at 48 hours posttransfection was considered as 100% proliferation, P < 0.05 (NT siRNA vs. ROR2 siRNA). proliferation and migration, pointing to a WNT5a ligand–inde- Altered expression patterns of the noncanonical WNT5A and its pendent role in tumor progression. Together, our ﬁndings high- receptor ROR2 have already been reported in various malignan- light a deregulation of WNT5A/ROR2 signaling pathway in the cies, indicating the critical roles of WNT5A-ROR2 axis in tumor pathogenesis of esophageal adenocarcinoma, with its exact role, progression (31, 32). In this study, we report a gradual loss of however, to be dictated by the ligand/receptor context. WNT5A expression along the esophageal adenocarcinoma

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Figure 6. WNT5A inhibits canonical WNT/b-catenin signaling in OE33 cancer cells. Recombinant WNT5A inhibits the canonical WNT/b-catenin signaling in OE33 cancer cells. A, luciferase assay demonstrated that treatment of OE33 cells with rhWNT5A for 24 hours significantly decreased TOPFlash activity in a dose-dependent manner. Treatment with rhWNT5A (500 ng/mL) for 24-hour suppressed AXIN2 gene expression as measured by qRT-PCR (B) and reduced the phosphorylated levels of GSK3b (Ser 9), despite the costimulation with rhWNT3A (C). D, rhWNT5A decreased TOPflash activity in wild-type OE33 cells, but increased the TOPflash activity in ROR2-knockdown OE33 cells. E, rhWNT5a increased the expression of active b-catenin (ABC) in ROR2-knockdown OE33 cells.

progression and attribute it to WNT5A promoter methylation, increased invasion and aggressiveness of many solid tumors, such proposing that deactivation of WNT5a-induced downstream sig- as thyroid cancer (16), breast cancer (18), neuroblastoma, (17) naling may induce tumorigenesis in esophageal adenocarcinoma. and colon cancer (19, 28). Furthermore, loss of WNT5A expres- This notion was further supported in vitro, as rhWNT5A signiﬁ- sion has been shown to correlate with cell transformation (33), cantly inhibited OE33 cell proliferation, migration, and invasion. whereas ectopic expression of human WNT5A reversed the phe- Indeed, loss or low expression of WNT5A is associated with notype of a mouse epithelial cell line from tumorigenic to

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Noncanonical WNT5A-ROR2 Axis in Esophageal Adenocarcinoma

Figure 7. WNT5A silencing in EPC2 normal epithelial cells. Transfection of EPC2 cells with WNT5A siRNA. A, qRT-PCR analysis of WNT5A (top) and Ki67 (bottom) gene expression following transfection of WNT5A siRNA for 48 hours. B, [H]3 thymidine uptake assay in EPC2 cells as assessed 24, 48, and 72 hours posttransfection. , P < 0.05 (NT siRNA vs. WNT5A siRNA). nontumorigenic (34). Consistent with these observations, we both, cell proliferation and migration, in OE33 cancer cells. Intrigu- demonstrated that WNT5A silencing induces cell proliferation ingly, these effects were observed in wild-type OE33 cells, which are in normal epithelial EPC2 cells, indicating a protective role for characterized by WNT5A loss, signifying that the ROR2 receptor WNT5A against tumorigenesis. However, WNT5A has also been activates a downstream signaling independently of WNT5A. In all, shown to possess oncogenic properties, based on findings depict- these observations imply the tumorigenic role of ROR2 regardless ing upregulation of WNT5A levels in melanoma, lung, and gastric the presence of WNT5A ligand. cancers (20, 21, 35). These diverse functions of WNT5a toward Cross-talk between WNT5A/ROR2 signaling and the canonical tumor progression could reflect differences in receptor context or WNT signaling has been proposed (36). Particularly, in vivo studies cell context of cancer cells (14, 31). in mice have shown that when ROR2 or WNT5A expression is On the other hand, the receptor of WNT5A, ROR2 was found knocked down, WNT/b-catenin signaling is enhanced (39). Given overexpressed, suggesting a ROR2-mediated, however WNT5A- that b-catenin signaling induces tumor progression, these observa- independent, downstream signaling activation in esophageal ade- tions justify the WNT5A-mediated tumor suppression, in which nocarcinoma progression. On the basis of its high levels of expres- ROR2 appears to have an essential role as receptor (37, 39, 40). sion, ROR2 has already been reported to promote tumorigenesis in Indeed, in ovarian cancer, WNT5A-mediated inhibition of b-catenin various malignancies, such as osteosarcoma, melanoma, renal cell transcriptional activity has been associated with inhibition of tumor carcinoma (RCC), as well as gastrointestinal stromal tumor and progress via cellular senescence (41). Taking into consideration that oral cancers (36). Especially, strong correlation between absence of the canonical WNT/b-catenin signaling is activated in esophageal ROR2 and decreased cell migration has been shown in siRNA adenocarcinoma cancer cells (24), we could attribute the suppres- studies of osteosarcoma cells (37), whereas ROR2 knockdown in sion of OE33 cell proliferation by WNT5A to the inhibition of RCC cells decreased cell migration and inhibited tumor growth in b-catenin transcriptional activity, as shown by WNT5A-mediated an orthotopic xenograft mouse model (22). Furthermore, ROR2 decrease in TOPflash activity and AXIN2 gene expression. Interest- silencing inhibited invasion of melanoma cells in a murine in vivo ingly, following ROR2 knockdown, rhWNT5A activated b-catenin– tumor model (38). In this study, we confirmed similar tumor- mediated gene transcription instead of inhibiting it as shown in promoting profile for ROR2 in esophageal adenocarcinoma by ROR2 wild-type cells, pointing to the essential role of ROR2 showing that ROR2 knockdown results in significant decrease of receptor in dictating WNT5A's function; whether it will suppress

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the b-catenin axis or enhance it. The most possible explanation for this notion is that in absence of ROR2, WNT5A activates b-catenin via Frizzled receptors binding (14). Someone would expect WNT5A to induce the canonical WNT signaling in normal epithelial cells, due to their low ROR2 expression. However, WNT5A silencing in EPC2 cells resulted in increased canonical Figure 8. WNT signaling as dictated by induction of AXIN2 gene expression, Dysregulation of WNT5A/ROR2 signaling in esophageal adenocarcinoma. indicating that in normal epithelial cells, WNT5A might antagonize Schematics illustrate the speculated deregulation of WNT5A/ROR2 signaling other WNTs. Thus, the function of WNT5A regarding the activation in esophageal adenocarcinoma. Loss of WNT5A with concurrent ROR2 of canonical WNT signaling is not only determined by the receptor overexpression promotes tumor growth in esophageal adenocarcinoma, high likely via activation of b-catenin signaling and WNT5A-independent context but also the presence of other WNTs. mechanisms downstream of ROR2 receptor. It remains questionable, how rhWNT5A impaired the invasive properties of OE33 wild-type cells, when WNT5A is known to In conclusion, we have examined for the first time that the induce cell migration and cancer metastasis by promoting epi- WNT5A/ROR2 axis in the pathogenesis of esophageal adenocar- thelial–mesenchymal transition and activating transcription of cinoma and we have shown that its deregulation is significantly matrix metalloproteases (MMP; ref. 42). Notably, exogenous involved to esophageal adenocarcinoma tumor progression. Loss WNT5A decreased ROR2 expression in OE33 cells indicating a of WNT5A with concurrent activation of WNT5A-independent possible reduction of receptor levels in the presence of ligand, ROR2 signaling appears to mark the progression of esophageal highly likely via receptor-mediated endocytosis and degradation. adenocarcinoma (Fig. 8). Although already speculated in other Given that cell motility regulation via WNT5A requires ROR2 malignancies, we confirmed that WNT5A ligand could function in receptor (43), we can speculate at this point that the subsequent two discreet pathways in esophageal adenocarcinoma based on ROR2 downregulation in OE33 cells following rhWNT5A simu- ROR2 receptor availability. It becomes evident that whether lation may underlay the impaired invasiveness. In favor of this WNT5A contributes to promote or suppress tumor progression notion, ROR2 knockdown induced WNT5A gene expression and is partly dependent on the cellular context, the receptor's context, was also found to inhibit invasion and migration. However, the and the balance of the canonical WNT signaling and noncanon- complex role of ROR2 receptor in esophageal adenocarcinoma ical WNT signaling. Thus, it is of great importance to understand invasiveness, in absence of WNT5A ligand, remains unclear and the respective molecular mechanisms governing WNT5A-medi- merits further study. ated tumor-progressive and tumor-suppressive functions, to Our current findings highlight that deregulation of WNT5A/ develop novel and proper diagnostic and therapeutic strategies ROR2 signaling may contribute to the high invasive properties and targeting WNT5A signaling for esophageal adenocarcinoma. subsequently to the poor prognosis of esophageal adenocarcinoma. Because of the limited number of patients in this study, we were unable to provide a powerful correlation between expression Disclosure of Potential Conflicts of Interest patterns and tumor stage or prognosis in this study, which is an No potential conflicts of interest were disclosed. acknowledged limitation. However, we observed a stronger ROR2 staining together with a further decreased WNT5A gene expression Authors' Contributions in the esophageal adenocarcinoma tissues classified in pT3/N1 Conception and design: O. Lyros, I. Gockel, R. Shaker, P. Rafiee stage compared with those in pT1/N0 stage (data not shown), Development of methodology: O. Lyros, L. Nie, T. Moore, A. Mackinnon, which allows us, at this point, to speculatively support the prog- P. Rafiee nostic potential of ROR2 and WNT5A expressions in esophageal Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): O. Lyros, T. Moore, A. Mackinnon, I. Gockel, P. Rafiee adenocarcinoma. Similar correlations, which allow conclusions on Analysis and interpretation of data (e.g., statistical analysis, biostatistics, the prognostic value of WNT5A and ROR2, have been performed in computational analysis): O. Lyros, T. Moore, A. Mackinnon, I. Gockel, P. Rafiee other malignancies (31). Intriguingly, in these studies, a poor Writing, review, and/or revision of the manuscript: O. Lyros, M. Otterson, outcome was associated with the overexpression of both WNT5A A. Mackinnon, I. Gockel, R. Shaker, P. Rafiee and ROR2 (38). Our report, however, indicates that is WNT5A loss- Administrative, technical, or material support (i.e., reporting or organizing of-expression in combination with ROR2 gain-of-expression that data, constructing databases): O. Lyros, T. Moore, R. Medda, A. Mackinnon, P. Rafiee contributes to the tumor growth and metastatic potential of esoph- fi in vitro Study supervision: O. Lyros, T. Moore, R. Shaker, P. Ra ee ageal adenocarcinoma cells. On the basis of our data, we can Other (the pathologist that contributed to the diagnosis and assessments of speculate that high WNT5a levels may be associated with a better the tissue.): B. Behmaram prognosis, whereas ROR2 overexpression with a poor one. Undoubtedly, our data make apparent that in the case of esoph- Acknowledgments ageal adenocarcinoma, the prognostic value of WNT5A cannot be This work was supported by Digestive Disease Center, Medical College of evaluated independently of ROR2 and vice versa. With respect to Wisconsin. The National Center for Advancing Translational Sciences, NIH, WNT5A's receptors, the expression ROR1 is yet not addressed in this through grant number 8UL1TR000055, in part by NIH grant R01DK025731, study. ROR1 has been shown to be highly expressed in several types and Laura Gralton on behalf of Daniel and Laura Gruber Charitable Lead of cancer, including mainly hematopoietic malignancies (44) and Trust #3. The costs of publication of this article were defrayed in part by the payment of might play a role in the esophageal adenocarcinoma tumor pro- page charges. This article must therefore be hereby marked advertisement in gression. However, in contrast to the well-established function of accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ROR2, it remains unclear whether ROR1 acts as a coreceptor for WNT5A-mediated signal in tumor biology (31) and such an Received December 15, 2015; revised March 16, 2016; accepted March 30, investigation was beyond the scopes of this study. 2016; published OnlineFirst April 15, 2016.

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Dysregulation of WNT5A/ROR2 Signaling Characterizes the Progression of Barrett-Associated Esophageal Adenocarcinoma

Orestis Lyros, Linghui Nie, Tami Moore, et al.

Mol Cancer Res 2016;14:647-659. Published OnlineFirst April 15, 2016.

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