Abstract 1163: HYAL4 is a novel potential prognostic and diagnostic biomarker for bladder cancer

Abstract Introduction and Objectives: The Hyaluronidase (HAase) family of enzymes is responsible for the degradation of hyaluronic acid (HA). There are 6 HAase genes in the human genome, clustered on chromosomes 3p21.3 (HYAL-1, -2, -3) and 7q31.3 (HYAL-4, PH20, HYALP1). Studies have shown that, while PH20 and HYAL-2 are basically testicular and lysosomal HAases, HYAL1 is the tumor-derived HAase, promoting tumor growth, invasion and angiogenesis. Additionally, HYAL1 is a diagnostic and prognostic marker for bladder cancer (BCa). The expression of other HAases has not been investigated in either normal and/or tumor cells and tissues. In this study we examined the expression of all 6 HAases in bladder tissues, cells and urine. Methods: Real time RT-PCR was used to examine the mRNA levels of the 6 HAase genes in BCa cell lines, 59 bladder tissues (normal (NBL) = 22; tumor (TBL) = 37) and 160 urine specimens (BCa = 52; normal = 18; history of BCa = 30; benign conditions = 59). Transcript levels were normalized to β-actin. HYAL1 and HYAL4 were localized in bladder tissues by immunohistochemistry using anti-HYAL1 and anti-HYAL4 antibodies, respectively. The staining was graded for intensity (0 - 3) and area of staining. HYAL4 function was analyzed by siRNA transfection of HT1376 and HT5637 cell lines. Results: HYAL1, HYAL4 and HYALP1 mRNA levels were significantly (6-13-fold) elevated in TBL tissues (12.5 ± 2.7; 29.7 ± 21.9; 26.7 ± 21.9) when compared to NBL tissues (1.2 ± 0.6; 2.2 ± 2.4; 4.2 ± 6.6; P < 0.001). HYAL3 and PH20 were expressed at 100-fold less level. HYAL2 expression was comparable to HYAL1 and HYAL4, but the difference in the expression between NBL and TBL was not significant (P > 0.05). cDNA cloning revealed that HYALP1 is a pseudo gene that encodes a non-functional truncated protein. HYAL1 and HYAL4 staining was significantly elevated in TBL tissues (188 ± 57; 232 ± 91) when compared to NBL tissues (68.5 ± 21.3; 93 ± 46; P < 0.001). The staining for both HAases was exclusively localized in tumor cells. When compared to HYAL3, PH20, HYAL2 mRNA levels, HYAL1, HYAL4 and HYALP1 mRNA levels were significantly elevated in BCa patients’ urine (P < 001). Urinary HYAL4 mRNA levels had higher sensitivity (78.9%) and specificity (86.2%) than HYAL1 mRNA levels (72.3%; 76.2%) to detect BCa. Both HAases detected high-grade BCa with 83% - 86% sensitivity. Down regulation of HYAL4 expression by siRNA decreased proliferation of HT1376 and HT5637 cells (2.3-fold) and induced apoptosis (2.6-fold). Conclusion: This is the first study showing that HYAL4, like HYAL1, is a tumor derived HAase that is exclusively expressed in BCa cells. HYAL4 may be a potentially accurate marker for BCa diagnosis and may promote BCa growth and progression. Support: Grant NCI/NIH R01 CA-72821-09; Bankhead Coley Cancer Research Program Sylvester Comprehensive Cancer Center and CURED Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1163. doi:1538-7445.AM2012-1163