Hyaluronic Acid Family of Molecules: Biomarkers and Functional Studies in Bladder Cancer

Profound changes in the extracellular matrix (ECM) in the vicinity of tumor cells occur as the tumor establishes its own microenvironment. Among these changes, elevated deposition of hyaluronic acid (HA), as well as abnormal expression and activity of the HA-family of molecules HAS1, HAS2, HAS3, HYAL-1, CD44, RHAMM are observed in a variety of tumors (Simpson and Lokeshwar 2008). In this study we evaluated whether the members of the HA-family of molecules, either alone or in combination with one another, are accurate diagnostic and prognostic markers for patients with bladder cancer (BCa). Additionally, we investigated whether HYAL-4 expression, a novel mammalian chondroitinase, is predictive of tumor outcome. Moreover, this study analyzed potential underlying molecular mechanisms through which HYAL-4 expression may be a modulator of tumor growth and progression. Using RT-qPCR, we found that the mRNA transcript levels of all 3 HA-synthases, HYAL-1, CD44v, and RHAMM were elevated by 4-16 fold in bladder cancer tissues, in comparison with normal bladder tissue (p\u3c0.001). Furthermore, the expression of HAS1 and HYAL-1 were predictive of metastasis, while HYAL-1 expression was also predictive of disease-specific mortality. Subsequently, in exfoliated urothelial cells, the transcript levels of all three HA synthases and HYAL-1 were significantly elevated in specimens from BCa patients, while the combination HAS2-HYAL-1 was a sensitive biomarker, detecting BCa with 85.4% sensitivity, 79.5% specificity, and 81.7% accuracy. Using commercially available qPCR tissue array plates, we found that HYAL-4 transcript levels are elevated in a number of tumors including bladder, kidney, stomach and breast. Furthermore, we successfully cloned and isolated HYAL-4, identifying in the process 2 novel and unpublished alternative splicing variants for this gene, namely HYAL-4 V1, and HYAL-4 V2. Interestingly, when analyzing the gene expression levels of these HYAL-4 isoforms in BCa tissue and exfoliated urothelial cells, we found that the levels of HYAL-4 V1 were elevated by 26-fold in bladder tissue and 2 to 6-fold in exfoliated cells from BCa patients. As a biomarker, HYAL-4 V1 showed 90% sensitivity and 83.3% specificity to detect BCa in bladder tissue specimens. However, in exfoliated urothelial cells, HYAL-4 V1 displayed excellent sensitivity (81.8%), but inadequate specificity (60%). Additionally, we successfully generated stable over-expression transfectants using 2 BCa cell lines (253J-Lung and T24) and a normal urothelial cell line (UROsta). Using antibodies specific for chondroitin sulfate 4 and chondroitin sulfate 6, we showed by immunoblotting that both HYAL-4 wt and V1 appear to have chondroitinase activity in 253J-Lung cells; HYAL-4 V1 showed chondroitinase activity in all cell lines tested. Analysis of versican expression, a chondroitin sulfate proteoglycan overexpressed in a number of solid tumors, suggested that HYAL-4 V1 may be a regulator of versican degradation and activity, possibly through hydrolysis of its chondroitin sulfate chains. Our cell proliferation studies indicate that HYAL-4 wt appears to have inhibitory effect, while HYAL-4 V1 promoted a slight increase in the proliferation of 253J-Lung and UROsta cells. Lastly, HYAL-4 V1 expression conferred increased chemoresistance to gemcitabine in both T24 and 253J-Lung cell lines, suggesting that it may be a potential novel therapeutic target for tumors refractory to certain chemotherapy agents