Abstract
Hyaluronic acid (HA) promotes tumor metastasis and is an accurate diagnostic marker for bladder cancer. HA is synthesized by HA synthases HAS1, HAS2, or HAS3. We have previously shown that HAS1 expression in tumor tissues is a predictor of bladder cancer recurrence and treatment failure. In this study, we stably transfected HT1376 bladder cancer cells with HAS1-sense (HAS1-S), HAS1-antisense (HAS1-AS), or vector cDNA constructs. Whereas HAS1-S transfectants produced approximately 1.7-fold more HA than vector transfectants, HA production was reduced by approximately 70% in HAS1-AS transfectants. HAS1-AS transfectants grew 5-fold slower and were approximately 60% less invasive than vector and HAS1-S transfectants. HAS1-AS transfectants were blocked in G(2)-M phase of the cell cycle due to down-regulation of cyclin B1, cdc25c, and cyclin-dependent kinase 1 levels. These transfectants were also 5- to 10-fold more apoptotic due to the activation of the Fas-Fas ligand-mediated extrinsic pathway. HAS1-AS transfectants showed a approximately 4-fold decrease in ErbB2 phosphorylation and down-regulation of CD44 variant isoforms (CD44-v3, CD44-v6, and CD44-E) both at the protein and mRNA levels. However, no decrease in RHAMM levels was observed. The decrease in CD44-v mRNA levels was not due to increased mRNA degradation. Whereas CD44 small interfering RNA (siRNA) transfection decreased cell growth and induced apoptosis in HT1376 cells, HA addition modestly increased CD44 expression and cell growth in HAS1-AS transfectants, which could be blocked by CD44 siRNA. In xenograft studies, HAS1-AS tumors grew 3- to 5-fold slower and had approximately 4-fold lower microvessel density. These results show that HAS1 regulates bladder cancer growth and progression by modulating HA synthesis and HA receptor levels.
Highlights
Hyaluronic acid (HA) and its degrading enzyme, hyaluronidase (HAase), are intricately involved in tumor growth and metastasis
HAS1 immunoblotting reveals that HAS1 expression in HAS1-S and HAS1-AS transfectants mirrors HA production by these cells (Fig. 1A-b)
Unlike HAS2, where increased HAS1 and HYAL1 expression compensates for the loss of HAS2 expression [28], HAS1 expression did not result in a compensatory increase in other HA synthase (HAS) or HYAL1 genes
Summary
Hyaluronic acid (HA) and its degrading enzyme, hyaluronidase (HAase), are intricately involved in tumor growth and metastasis. HA is a nonsulfated glycosaminoglycan made up of repeating disaccharide units D-glucuronic acid and N-acetyl-D-glucosamine [1]. HAase is an endoglycosidase that breaks HA into fragments, some of which are angiogenic [2]. HA levels are elevated in many tumors [3,4,5,6,7,8,9,10]. We have shown that HA levels are elevated in the urine of bladder cancer patients and, together with urinary HAase levels, serve as an accurate diagnostic marker [11,12,13].
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