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GLYCOSAMINOGLYCANS, CHONDROITINASE, AND MOLECULAR SUBTYPES IN BLADDER CANCERThere is a need for novel prognostic biomarkers and targeted treatments in bladder cancer (BC), even more so for muscle-invasive disease (MIBC). Discovery of molecular markers to predict outcome in BC patients may lead to identification of impactful therapeutic targets. The hyaluronic acid (HA) family of molecules and distinct molecular subtypes have both been investigated as potential prognostic markers. HA family and chondroitin sulfate proteoglycans, such as CD44, have been implicated in driving aggressiveness of disease; however, a chondroitinase enzyme that cleaves chondroitin sulfate proteoglycans has not been identified in any eukaryotic system. We evaluated molecular markers of BC and the first known eukaryotic/human chondroitinase, that we identified, for their ability to predict clinical outcome in patients, and for their roles as drivers of disease. We also investigated the anti-tumor effects of HA synthesis inhibitor 4-methylumbelliferone (4MU), a non-toxic orally bioavailable supplement. This study demonstrates that transcript levels of HA family members can predict metastasis and poor survival in BC patients. HA- family expression also correlated with epithelial mesenchymal transition (EMT) markers β -Catenin, Twist, Snail, and E-Cadherin. HA signaled through its receptors CD44/RHAMM and the PI3-K/AKT axis. 4MU targeted HA signaling, inhibiting proliferation and motility/invasion, inducing apoptosis in vitro, and preventing tumor growth in vivo. We discovered that a previously unidentified splice variant of HYAL-4 was elevated in bladder tumors. We named this variant "V1". Our studies showed that V1 had chondroitinase activity, cleaved chondroitin-6-sulfate from CD44, and consequently increased CD44 secretion. In vivo, V1-expressing urothelial cells formed muscle-invasive tumors and V1-expressing cancer cells developed metastatic tumors. Evaluation of the prognostic significance of the molecular subtypes of MIBC that were recently identified by other groups, showed the subtypes to have little to no predictive ability for clinical outcome in multivariate analyses that included standard clinical parameters. Consistently, clinical parameters such as histopathologic tumor grade, T-stage, and lymph node status, outperformed the molecular subtypes. Contrarily, V1 levels could independently predict metastasis and survival with high efficacy, suggesting that focusing on V1 as a functional biomarker may be a better strategy to improve clinical outcome of BC patients.