Inducible Hyaluronan Production Reveals Differential Effects on Prostate Tumor Cell Growth and Tumor Angiogenesis

Abstract

Prostate cancer progression can be predicted in human tumor biopsies by abundant hyaluronan (HA) and its processing enzyme, the hyaluronidase HYAL1. Accumulation of HA is dictated by the balance between expression levels of HA synthases, the enzymes that produce HA polymers, and hyaluronidases, which process polymers to oligosaccharides. Aggressive prostate tumor cells express 20-fold higher levels of the hyaluronan synthase HAS3, but the mechanistic relevance of this correlation has not been determined. We stably overexpressed HAS3 in prostate tumor cells. Adhesion to extracellular matrix and cellular growth kinetics in vitro were significantly reduced. Slow growth in culture was restored either by exogenous addition of hyaluronidase or by stable HYAL1 coexpression. Coexpression did not improve comparably slow growth in mice, however, suggesting that excess hyaluronan production by HAS3 may alter the balance required for induced tumor growth. To address this, we used a tetracycline-inducible HAS3 expression system in which hyaluronan production could be experimentally controlled. Adjusting temporal parameters of hyaluronan production directly affected growth rate of the cells. Relief from growth suppression in vitro but not in vivo by enzymatic removal of HA effectively uncoupled the respective roles of hyaluronan in growth and angiogenesis, suggesting that growth mediation is less critical to establishment of the tumor than early vascular development. Collectively results also imply that HA processing by elevated HYAL1 expression in invasive prostate cancer is a requirement for progression.Despite improved detection and diagnosis of prostate cancer, this disease remains the second leading cause of malignant mortality in United States males (1, 2). Metastasis may occur with no prior indication of an invasive tumor (3), so prostate cancer progression is difficult to predict. Changes in levels of extracellular matrix molecules such as hyaluronan (HA), 2 a high molecular weight polysaccharide, and its processing enzyme, the hyaluronidase HYAL1, within the prostate extracellular matrix have been correlated to invasive prostate cancer progression (4 -11). The molecular mechanisms underlying this correlation could provide important insights for therapeutic development or improved diagnosis of prostate cancer.HA production is a tightly regulated process that impacts cellular transformation and motility during development (12)(13)(14). Dynamic HA turnover within tissues controls many acute processes such as wound healing or immune function. HA accumulation is the outcome of a balance between the activity of HA synthases (HAS), enzymes that synthesize the linear polymers (15), and hyaluronidases, which process the polymers to biologically potent oligosaccharides (16). Excess quantities of large HA polymers have been reported to suppress cellular growth (17, 18) and angiogenesis (19,20), whereas processed oligosaccharides dramatically stimulate angiogenesis (21-23), and fully degraded oligosaccharides induce apoptosis (1...

Hyaluronic Acid Synthase-1 Expression Regulates Bladder Cancer Growth, Invasion, and Angiogenesis through CD44

“…Overexpression of HAS3 slows the growth of 22Rv1 prostate cancer cells. Furthermore, HAS3-overexpressing tumors are less angiogenic and the growth-inhibitory effects of HAS3 overexpression can be reversed by stable expression of HYAL1 (49). Taken together, these findings show that the tumor-associated HA-HYAL1 system is important in tumor growth and progression.…”

Hyaluronidase Activity of Human Hyal1 Requires Active Site Acidic and Tyrosine Residues

“…Initial quantitative comparisons of wild-type and mutant Hyal1 activity were done in an HA-coated microplate assay (13,(32)(33)(34). Briefly, serial dilutions of concentrated conditioned media adjusted for equal Hyal1 content were applied in triplicate to 96-well plates that had been precoated overnight at 4°C in a solution of 50 g/ml human umbilical cord HA (200 mM carbonate buffer, pH 9.6), and blocked with Pierce Superblock reagent.…”

Spontaneous Metastasis of Prostate Cancer Is Promoted by Excess Hyaluronan Synthesis and Processing

“…Vascularization of the tumors was assessed in acetone-fixed frozen sections (8 m thickness) by antibody staining for CD31 as described previously. 14,16,19,21 Angiogenesis Quantification CD31-phycoerythrin-conjugated antibody staining of frozen sections was visualized by fluorescence microscopy. Ten random sections from each of three tumors per cell line were digitally photographed with 5 seconds of exposure time, saved as TIF files, and processed.…”