Abstract 336: 4-methylumbeliferone, an HA synthesis inhibitor, inhibits EMT determinants in bladder cancer cell lines

Abstract

Abstract INTRODUCTION AND OBJECTIVE: Elevated Hyaluronic Acid (HA) levels are a molecular determinant of bladder cancer (BCa) metastasis. Furthermore, HA, HYAL-1 hyaluronidase and HA-synthesizing enzymes are potential markers for BCa diagnosis and predicting prognosis. 4-Methylumbelliferone (4-MU) is an orally bioavailable dietary supplement that inhibits HA synthesis. Epithelial mesenchymal transition (EMT) is the hallmark of invasion and metastasis, in which cancer cells acquire an invasive phenotype. Since HA promotes tumor metastasis, we evaluated the antitumor effects of 4-MU in vitro and in vivo and examined whether 4-MU can reverse the EMT by inhibiting HA synthesis. METHODS: Quantitative PCR was used to measure mRNA expression of EMT genes (β-catenin, Twist, and Snail) in 66 bladder tissue specimens (27 normal; 39 tumor); follow-up: 26±4.3 months; median 20 months. The effect of 4-MU (0.2 - 0.6 mM) on cell proliferation, apoptosis, intracellular signaling, and the expression of HA receptors and EMT genes were examined in BCa cell lines, 253J-Lung and HT1376. Effect of 4-MU on tumor growth was analyzed in subcutaneous xenografts. RESULTS: Among the EMT genes, both Snail and Twist were differentially expressed in BCa tissues when compared to normal bladder (P<0.001). Furthermore, Twist expression significantly correlated with metastasis (+: 0.25+0.15, -: 0.12+0.14; P=0.028). β-catenin expression negatively correlated with survival (chi square=4.3, p=0.038). 4-MU inhibited proliferation and induced apoptosis in the BCa cells. Following a 48 h exposure at the IC50 for HA synthesis (0.4 mM), 4-MU inhibited proliferation by 61% and increased apoptosis by 73%. 4-MU induced caspase-8, -9 and -3 activation and up-regulation of Fas and FADD. 4-MU downregulated the expression of HA-receptors - CD44, RHAMM, EMT promoters - β-catenin, Snail and Twist by 4-10-fold, but increased the expression of E-cadherin by 2-fold, suggesting EMT-reversal. In xenograft studies, 4-MU significantly decreased tumor growth (> 3-fold) when treatment was started either on the day of tumor cell injection or after tumors became palpable. No weight loss or serum or organ toxicity was observed in treated mice. Tumors showed reduced microvessel density (∼3-fold) and HA expression but increased TURNEL positive cells. CONCLUSION: This is the first study that shows inhibition of HA synthesis by an orally bioavailable, non-toxic supplement potentially reverses EMT and has potent anti-tumor activity. Support: R01 CA 123063-04 (VBL); R01 CA 72821-11 (VBL). 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 336. doi:1538-7445.AM2012-336