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, Ó2016 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 noncanon- ical 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 Ó2016 American Association for Cancer Research. in upregulated canonical signaling, are not frequently detected www.aacrjournals.org 647 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst April 15, 2016; DOI: 10.1158/1541-7786.MCR-15-0484 Lyros et al. 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 esophage- al 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