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

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-specificsurvival 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.

HYAL4-V1/Chondroitinase (Chase) Drives Gemcitabine Resistance and Predicts Chemotherapy Failure in Patients with Bladder Cancer

“…In this study we utilized empty vector (EV), HYAL-4 Wt (Wt) and HYAL4-V1 (V1) stable transfectants of BC cells (T24, HT1376 and 253J) and an immortalized urothelial cell line (Urotsa). These transfectants, as well as, HYAL-4 shRNA and CD44 siRNA transfectants have been previously characterized (16). All transfectants were cultured in RPMI 1640 plus FBS and gentamicin.…”

“…We recently discovered the first known example of a eukaryotic chondroitinase (Chase) and demonstrated that it is a driver of malignant BC and a potential prognostic marker for metastasis and BC-specific survival (16). This Chase is encoded by a novel splice variant of HYAL-4, a member of the hyaluronidase family of genes.…”

“…This Chase is encoded by a novel splice variant of HYAL-4, a member of the hyaluronidase family of genes. We demonstrated that while the wild-type protein (Wt; 481 amino acids) is not expressed in the bladder, the variant (V1; 349 amino acids) is expressed in BC and has Chase activity (16). V1 is generated by the insertion of a novel exon (which was designated as a part of an intron); consequently, V1 protein is secreted.…”

“…Publically available databases would not include V1, because we only recently deposited V1 sequence in GenBank (accession nos. BankIt2227219 Seq1MN902225 and BankIt227219 Seq2 MN902226; (16)). Nevertheless, in a clinical cohort we found that both V1 transcript and protein levels are highly elevated in BC, especially in high-grade tumors, and predict clinical outcome.…”

“…V1-expressing urothelial cells formed angiogenic muscle-invasive tumors and V1-expressing BC cells formed metastatic bladder tumors. CD44 was found to be the major substrate of V1 and V1's Chase activity generated soluble CD44 (sCD44) (16). Further, the malignant effects of V1 were driven through CD44.…”