A Novel Splice Variant of HYAL-4 Drives Malignant Transformation and Predicts Outcome in Patients with Bladder Cancer.

PURPOSE: Poor prognosis of patients with muscle-invasive bladder cancer that often metastasizes drives the need for discovery of molecular determinants of bladder cancer progression. Chondroitin sulfate proteoglycans, including CD44, regulate cancer progression; however, the identity of a chondroitinase (Chase) that cleaves chondroitin sulfate from proteoglycans is unknown. HYAL-4 is an understudied gene suspected to encode a Chase, with no known biological function. We evaluated HYAL-4 expression and its role in bladder cancer. EXPERIMENTAL DESIGN: In clinical specimens, HYAL-4 wild-type (Wt) and V1 expression was evaluated by RT-qPCR, IHC, and/or immunoblotting; a novel assay measured Chase activity. Wt and V1 were stably expressed or silenced in normal urothelial and three bladder cancer cell lines. Transfectants were analyzed for stem cell phenotype, invasive signature and tumorigenesis, and metastasis in four xenograft models, including orthotopic bladder. RESULTS: HYAL-4 expression, specifically a novel splice variant (V1), was elevated in bladder tumors; Wt expression was barely detectable. V1 encoded a truncated 349 amino acid protein that was secreted. In bladder cancer tissues, V1 levels associated with metastasis and cancer-specific survival with high efficacy and encoded Chase activity. V1 cleaved chondroitin-6-sulfate from CD44, increasing CD44 secretion. V1 induced stem cell phenotype, motility/invasion, and an invasive signature. CD44 knockdown abrogated these phenotypes. V1-expressing urothelial cells developed angiogenic, muscle-invasive tumors. V1-expressing bladder cancer cells formed tumors at low density and formed metastatic bladder tumors when implanted orthotopically. CONCLUSIONS: Our study discovered the first naturally-occurring eukaryotic/human Chase and connected it to disease pathology, specifically cancer. V1-Chase is a driver of malignant bladder cancer and potential predictor of outcome in patients with bladder cancer.

Antitumor activity of hyaluronic acid synthesis inhibitor 4-methylumbelliferone in prostate cancer cells.

4-Methylumbelliferone (4-MU) is a hyaluronic acid (HA) synthesis inhibitor with anticancer properties; the mechanism of its anticancer effects is unknown. We evaluated the effects of 4-MU on prostate cancer cells. 4-MU inhibited proliferation, motility, and invasion of DU145, PC3-ML, LNCaP, C4-2B, and/or LAPC-4 cells. At IC(50) for HA synthesis (0.4 mmol/L), 4-MU induced >3-fold apoptosis in prostate cancer cells, which could be prevented by the addition of HA. 4-MU induced caspase-8, caspase-9, and caspase-3 activation, PARP cleavage, upregulation of Fas-L, Fas, FADD and DR4, and downregulation of bcl-2, phosphorylated bad, bcl-XL, phosphorylated Akt, phosphorylated IKB, phosphorylated ErbB2, and phosphorylated epidermal growth factor receptor. At IC(50), 4-MU also caused >90% inhibition of NF-kappaB reporter activity, which was prevented partially by the addition of HA. With the exception of caveolin-1, HA reversed the 4-MU-induced downregulation of HA receptors (CD44 and RHAMM), matrix-degrading enzymes (MMP-2 and MMP-9), interleukin-8, and chemokine receptors (CXCR1, CXCR4, and CXCR7) at the protein and mRNA levels. Expression of myristoylated-Akt rescued 4-MU-induced apoptosis and inhibition of cell growth and interleukin-8, RHAMM, HAS2, CD44, and MMP-9 expression. Oral administration of 4-MU significantly decreased PC3-ML tumor growth (>3-fold) when treatment was started either on the day of tumor cell injection or after the tumors became palpable, without organ toxicity, changes in serum chemistry, or body weight. Tumors from 4-MU-treated animals showed reduced microvessel density ( approximately 3-fold) and HA expression but increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells and expression of apoptosis-related molecules. Therefore, the anticancer effects of 4-MU, an orally bioavailable and relatively nontoxic agent, are primarily mediated by inhibition of HA signaling.

Resveratrol-mediated chemoprevention of diethylnitrosamine-initiated hepatocarcinogenesis: inhibition of cell proliferation and induction of apoptosis.

Hepatocellular carcinoma (HCC) is one of the most common cancers and lethal diseases. In view of the limited treatment and a grave prognosis of liver cancer, preventive control has been emphasized. Resveratrol, a polyphenol found in grape skins, peanuts, berries and red wine, has been shown to possess potent growth inhibitory effects against various human cancer cells. Although resveratrol has been found to exhibit chemopreventive actions in experimentally induced skin, breast, colon and esophagus rodent tumors, chemopreventive potential of this dietary constituent has not been explored well against experimental liver cancer. We evaluated the inhibitory effect of resveratrol using a two-stage model of rat hepatocarcinogenesis in Sprague-Dawley rats. Initiation was performed by a single intraperitoneal injection of diethylnitrosamine (DENA, 200 mg/kg), followed by promotion with phenobarbital (0.05%) in drinking water. The rats had free access to food supplemented with resveratrol equivalent to 50, 100 or 300 mg/kg body weight/day. Resveratrol treatment was started 4 weeks prior to the initiation and continued for 20 weeks. Resveratrol dose-dependently reduced the incidence, total number and multiplicity of visible hepatocyte nodules. Mean nodular volume and nodular volume as percentage of liver volume were also inhibited upon resveratrol treatment. Histopathological examination of liver tissue confirmed the protective effect of resveratrol. Immunohistochemical detection of cell proliferation and assay of apoptosis indicated a decrease in cell proliferation and increase of apoptotic cells in the livers of resveratrol-supplemented rats. Resveratrol also induced the expression of pro-apoptotic protein Bax, reduced anti-apoptotic Bcl-2 expression, with a concurrent increase in Bax/Bcl-2 ratio with respect to DENA control. The present study provides evidence, for the first time, that resveratrol exerts a significant chemopreventive effect on DENA-initiated hepatocarcinogenesis through inhibition of cell proliferation and induction of apoptosis. Resveratrol-induced apoptogenic signal during rat liver carcinogenesis may be mediated through the downregulation of Bcl-2 and upregulation of Bax expression. Due to a favorable toxicity profile, resveratrol can potentially be developed as a chemopreventive drug against human HCC.

Epigenetic regulation of HYAL-1 hyaluronidase expression. identification of HYAL-1 promoter.

HYAL-1 (hyaluronoglucosaminidase-1) belongs to the hyaluronidase family of enzymes that degrade hyaluronic acid. HYAL-1 is a marker for cancer diagnosis and a molecular determinant of tumor growth, invasion, and angiogenesis. The regulation of HYAL-1 expression is unknown. Real time reverse transcription-PCR using 11 bladder and prostate cancer cells and 69 bladder tissues showed that HYAL-1 mRNA levels are elevated 10-30-fold in cells/tissues that express high hyaluronidase activity. Although multiple transcription start sites (TSS) for HYAL-1 mRNA were detected in various tissues, the major TSS in many tissues, including bladder and prostate, was at nucleotide 27274 in the cosmid clone LUCA13 (AC002455). By analyzing the 1532 base sequence 5' to this TSS, using cloning and luciferase reporter assays, we identified a TACAAA sequence at position -31 and the minimal promoter region between nucleotides -93 and -38. Mutational analysis identified that nucleotides -73 to -50 (which include overlapping binding consensus sites for SP1, Egr-1, and AP-2), bases C(-71) and C(-59), and an NFkappaB-binding site (at position -15) are necessary for promoter activity. The chromatin immunoprecipitation assay identified that Egr-1, AP-2, and NFkappaB bind to the promoter in HYAL-1-expressing cells, whereas SP1 binds to the promoter in non-HYAL-1-expressing cells. 5-Aza-2'-deoxycytidine treatment, bisulfite DNA sequencing, and methylation-specific PCR revealed that HYAL-1 expression is regulated by methylation at C(-71) and C(-59); both Cs are part of the SP1/Egr-1-binding sites. Thus, HYAL-1 expression is epigenetically regulated by the binding of different transcription factors to the methylated and unmethylated HYAL-1 promoter.