Author Manuscript Published OnlineFirst on February 24, 2020; DOI: 10.1158/1078-0432.CCR-19-2912 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. A Novel Splice Variant of HYAL-4 Drives Malignant Transformation and Predicts Outcome in Bladder Cancer Patients. Vinata B. Lokeshwar1,♠,, Daley S. Morera1,♠, Sarrah L. Hasanali1, Travis J. Yates2,+, Marie C. Hupe3, Judith S. Knapp3, Soum D. Lokeshwar4, Jiaojiao Wang1, Martin Hennig3, Rohitha Baskar1, Diogo O. Escudero5,+, Ronny R. Racine6,+, Neetika Dhir6,+, Andre R. Jordan1,2, Kelly Hoye2,+, Ijeoma Azih7, Murugesan Manoharan8, Zachary Klaassen9, Sravankumar Kavuri10, Luis E. Lopez1, Santu Ghosh11, Bal L. Lokeshwar12 Departments of Biochemistry and Molecular Biology (1); Clinical Trials Office (7), Surgery, Division of Urology (9); Pathology (10); Department of Population Health Sciences (11), Georgia Cancer Center (12), Medical College of Georgia, Augusta University, Augusta, GA, 1410 Laney Walker Blvd., 30912, USA Sheila and David Fuente Graduate Program in Cancer Biology (2), Honors Program in Medical Education (4), Molecular Cell and Developmental Biology Graduate Program (5), Department of Urology (6) University of Miami-Miller School of Medicine, Miami, 1600 NW 10th Avenue, Florida, 33136, USA Department of Urology, University-Hospital Schleswig-Holstein, Campus Luebeck, Luebeck, Germany (3) Division of Urologic Oncology Surgery, Miami Cancer Institute, Baptist Health South Florida, Miami, Florida (8) ♠: Contributed equally and are joint first authors Running title: HYAL4-V1 Chase in Bladder Cancer *Address for Correspondence: Vinata B. Lokeshwar, Ph.D. Professor and Chair; Department of Biochemistry and Molecular Biology Medical College of Georgia, Augusta University 1410 Laney Walker Blvd., Room CN 1177A, Augusta, GA 30912-2100 Office: (706) 721-7652; Fax: (706) 721-6608; E-mail: [email protected] +Present address: Travis Yates: QualTek Molecular Laboratories, King of Prussia, PA; Diogo Escudero: George Washington University Kelly Hoye: University of North Carolina Lineberger Comprehensive Cancer Center; Ronny Racine: Astellas Institute for Regenerative Medicine (AIRM); Neetika Dhir: deceased. The authors declare no competing interests. 1 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 24, 2020; DOI: 10.1158/1078-0432.CCR-19-2912 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abbreviations used: aa: amino acid(s); ALDH: aldehyde dehydrogenase 1; BC: bladder cancer; Chase: chondroitinase; CM: conditioned medium; CS: chondroitin sulfate; CSC: cancer stem cell CSPG: chondroitin sulfate proteoglycan; EV: empty vector; HG: high-grade; LG: low-grade; MIBC: muscle invasive bladder cancer; NBL: normal bladder; sCD44: soluble CD44; stub: Neoepitope generated after chondroitin-6-sulfate chains are removed from a CSPG; V1: splice variant of HYAL- 4; V1-FL: V1-floating; Wt: wild type 2 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 24, 2020; DOI: 10.1158/1078-0432.CCR-19-2912 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT Purpose: Poor prognosis of patients with muscle-invasive bladder cancer (BC) that often metastasizes drives the need for discovery of molecular determinants of BC progression. Chondroitin sulfate proteoglycans, including CD44, regulate cancer progression; however, the identity of a chondroitinase (Chase) that cleaves chondroitin sulfate from proteoglycans is unknown. HYAL-4 is an understudied gene suspected to encode a Chase, with no known biological function. We evaluated HYAL-4 expression and its role in BC. Experimental design: In clinical specimens HYAL-4 wild-type (Wt) and V1 expression was evaluated by RT-qPCR, immunohistochemistry and/or immunoblotting; a novel assay measured Chase activity. Wt and V1 were stably expressed or silenced in normal urothelial and three BC cell lines. Transfectants were analyzed for stem cell phenotype, invasive signature and tumorigenesis, and metastasis in four xenograft models, including orthotopic bladder. Results: HYAL-4 expression, specifically a novel splice variant (V1), was elevated in bladder tumors; Wt expression was barely detectable. V1 encoded a truncated 349 amino acid protein that was secreted. In BC tissues, V1 levels associated with metastasis and cancer-specific- survival with high efficacy and encoded Chase activity. V1 cleaved chondroitin-6-sulfate from CD44, increasing CD44 secretion. V1 induced stem cell phenotype, motility/invasion, and an invasive signature. CD44 knockdown abrogated these phenotypes. V1-expressing urothelial cells developed angiogenic, muscle-invasive tumors. V1-expressing BC cells formed tumors at low-density and formed metastatic bladder tumors when implanted orthotopically. Conclusion: Our study discovered the first naturally-occurring eukaryotic/human Chase and connected it to disease pathology, specifically cancer. V1-Chase is a driver of malignant BC and potential predictor of outcome in BC patients. 3 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 24, 2020; DOI: 10.1158/1078-0432.CCR-19-2912 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. TRANSLATIONAL RELEVANCE Notwithstanding cystectomy, 50% or more of patients with muscle invasive bladder cancer (MIBC) develop metastasis. With a dismal 14-month median survival despite chemotherapy, early stratification of BC patients who have a higher risk of developing metastasis is pivotal for improving clinical outcome. Currently, no predictive biomarkers are incorporated into the clinical management of BC patients. The discovery of HYAL4-V1, the first human chondroitinase (Chase), establishes V1-Chase as a potential independent predictor of metastasis and cancer- specific mortality following cystectomy. The high-throughput Chase and q-PCR assays demonstrate the potential for clinical translation of V1-Chase as a prognostic biomarker. Identification of V1-Chase as a molecular driver of malignant transformation of bladder uro- epithelium and metastasis reveals the “why” and “how” V1-Chase is a potential prognostic biomarker. The results demonstrated here also reveal that V1-Chase, a previously untapped class of molecules, could pave the way for developing strategies to target V1-Chase for the treatment of metastatic BC. 4 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 24, 2020; DOI: 10.1158/1078-0432.CCR-19-2912 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. INTRODUCTION Bladder cancer is a common cancer of the urinary tract with two distinct characteristics, frequent recurrence and heterogeneity in tumor progression. Ninety percent of bladder tumors arise from malignant transformation of the urothelial lining. For bladder tumors, grade indicates invasive potential, while stage indicates depth of invasion. Low-grade tumors are confined to the mucosa (stage Ta) and rarely invade the lamina propria (stage T1). However, high-grade tumors, if not detected early, will present as muscle invasive bladder cancer (MIBC). This results in about 2/3rd of high-grade BC patients presenting with MIBC at initial diagnosis. MIBC is associated with poor prognosis; 50% of these patients develop metastasis within two years. Although the standard systemic cisplatin-based chemotherapy for metastatic BC yields a reasonable initial response, the majority of these responses are only partial, resulting in a median survival of 14 months. Since the efficacy of the standard systemic chemotherapy has plateaued and the response rate for newer immunotherapy and checkpoint inhibitors is 30% or less, molecular profiling holds the promise of individualizing treatment for MIBC patients (1,2). Discovery of molecular drivers of BC progression could improve the clinical management of patients with MIBC through better prognostic predictions and potential targeted treatments. Molecular subtypes of MIBC were one such discovery; however, we recently reported that routine pathology parameters (e.g. grade, lymph node invasion) are more accurate prognostic predictors than the subtypes (3). Therefore, there is still a major unmet clinical need for newly discovered molecular drivers of BC. Proteoglycans regulate normal physiology and disease processes, including cancer growth and metastasis (4). Chondroitin sulfate proteoglycans (CSPGs), such as Versican, Biglycan, and Decorin, regulate tumor growth, disease progression, and chemoresistance (5). CD44, a stem cell marker and hyaluronic acid receptor, is post-translationally modified by 5 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 24, 2020; DOI: 10.1158/1078-0432.CCR-19-2912 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. addition of chondroitin sulfate (6-12). Furthermore, our published studies