A Novel Splice Variant of HYAL-4 Drives Malignant Transformation and Predicts Outcome in Patients with Bladder Cancer

Purpose: Poor prognosis of patients with muscle-invasive bladder cancer that often metastasizes drives the need for discovery of molecular determinants of bladder cancer 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 bladder cancer.

Experimental Design: In clinical specimens, HYAL-4 wild-type (Wt) and V1 expression was evaluated by RT-qPCR, IHC, and/or immunoblotting; a novel assay measured Chase activity. Wt and V1 were stably expressed or silenced in normal urothelial and three bladder cancer 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 bladder cancer 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 bladder cancer cells formed tumors at low density and formed metastatic bladder tumors when implanted orthotopically.

Conclusions: 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 bladder cancer and potential predictor of outcome in patients with bladder cancer.