Abstract 5174: HYAL4: a new player in bladder cancer progression and diagnosis

Abstract

Abstract BACKGROUND: Hyaluronidase (HAase) family of enzymes degrades hyaluronic acid (HA). In the human genome there are 6 HAase genes clustered on chromosomes 3p21.3 (HYAL-1, -2, -3) and 7q31.3 (HYAL-4, PH20, HYALP1). While PH20 and HYAL-2 are testicular and lysosomal HAases, HYAL1 is a tumor-derived HAase. HYAL1 promotes tumor growth, invasion, and angiogenesis and is a diagnostic and prognostic marker for bladder cancer (BCa). The expression of other HAases has not been investigated in either normal cells or tumor cells and tissues. In this study we examined the expression of all 6 HAases in bladder tissues, cells and urine. METHODS: Using time RT-PCR, we examined the mRNA levels of 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). The 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 0.05). 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, and 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, like HYAL1, HYAL4 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 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5174. doi:10.1158/1538-7445.AM2011-5174