Gary R. Brownlee

Antisense-Mediated Suppression of Hyaluronan Synthase 2 Inhibits the Tumorigenesis and Progression of Breast Cancer

The progression of several cancers is correlated with the increased synthesis of the glycosaminoglycan, hyaluronan. Hyaluronan is synthesized at the plasma membrane by various isoforms of hyaluronan synthases (HAS). The importance of HAS2 expression in highly invasive breast cancer was characterized by the antisense inhibition of HAS2 (ASHAS2). The effect of HAS2 inhibition on cell proliferation, migration, hyaluronan metabolism, and receptor status was characterized in vitro, whereas the effect on tumorigenicity and metastasis was established in vivo. HAS2 inhibition resulted in a 24-hour lag in proliferation that was concomitant to transient arrest of 79% of the cell population in G0-G1. Inhibition of HAS2 did not alter the expression of the other HAS isoforms, whereas hyaluronidase (HYAL2) and the hyaluronan receptor, CD44, were significantly down-regulated. ASHAS2 cells accumulated greater amounts of high molecular weight hyaluronan (>10,000 kDa) in the culture medium, whereas mock and parental cells liberated less hyaluronan of three distinct molecular weights (100, 400, and 3,000 kDa). The inhibition of HAS2 in the highly invasive MDA-MB-231 breast cancer cell line inhibited the initiation and progression of primary and secondary tumor formation following s.c. and intracardiac inoculation into nude mice, whereas controls readily established both primary and secondary tumors. The lack of primary and secondary tumor formation was manifested by increased survival times where ASHAS2 animals survived 172% longer than the control animals. Collectively, these unique results strongly implicate the central role of HAS2 in the initiation and progression of breast cancer, potentially highlighting the co-dependency between HAS2, CD44, and HYAL2 expression.

Inhibition of hyaluronan degradation by dextran sulphate facilitates characterisation of hyaluronan synthesis: An in vitro and in vivo study

The concentration and molecular weight of hyaluronan often dictates its physiological function. Consequently full characterisation of the anabolic products and turnover rates of HA could facilitate understanding of the role that HA metabolism plays in disease processes. In order to achieve this it is necessary to interrupt the dynamic balance between concurrent HA synthesis and degradation, achievable through the inhibition of the hyaluronidases, a group of enzymes which degrade HA. The sulphated polysaccharide, dextran sulphate has been demonstrated to competitively inhibit testicular hyaluronidase in a non-biological system, but its application to in vitro biological systems had yet to be developed and evaluated. This study determined the inhibitory concentrations of dextran sulphate against both testicular and Streptomyces hyaluronidase in a cell-free and breast cancer model followed by characterisation of the effect that hyaluronidase inhibition exerted on HA synthesis and degradation. The IC100 of dextran sulphate for both hyaluronidases in a cell-free and biological system was determined to be ≥400 μg/ml. At concentrations up to 10 mg/ml the dextran sulphate did not effect breast cancer cell proliferation or morphology, while at 400 μg/ml HA degradation was totally inhibited, enabling an accurate quantitation of HA production as well as characterisation of the cell-associated and liberated HA. FACS quantitation of the HA receptor CD44, HA synthase and the hyaluronidases HYAL 1 and HYAL 2 demonstrated that dextran sulphate down-regulated CD44 and HA synthase while upregulating the hyaluronidases. These results suggest dynamic feedback signalling and complex mechanisms occur in the net deposition of HA in vivo. Published in 2004.

HYALURONAN SYNTHASE EXPRESSION IN HUMAN ENDOMETRIUM DURING THE MENSTRUAL CYCLE

ABSTRACT The human endometrium undergoes extensive remodelling during the menstrual cycle in preparation of a favorable environment for implantation. Biphasic fluctuations of hyaluronan (HA) in the cycling endometrium have been demonstrated with peaks occurring during the early proliferative (days 6–8) and mid secretory (days 19–23) stages of the cycle. The second, mid secretory, HA peak encompasses the time of embryo implantation suggesting a possible role for HA in this process. The aim of the present study was to investigate expression patterns of the 3-hyaluronan synthase (HAS) isoforms: HAS-1, HAS-2 and HAS-3. In situ hybridization was carried out on formalin fixed endometrial tissue across the cycle to determine whether these enzymes were expressed in human endometrium and if so, their cellular source. Riboprobes were constructed for each HAS: HAS-1 (64bp), HAS-2 (60bp) and HAS-3 (550bp) and labeled with digoxygenin (DIG). The HAS protein was also localized immunohistochemically using a polyclonal antibody against extracellular HAS peptides. All three forms of HAS mRNA were expressed in the human endometrium. Expression was observed in both stromal and glandular epithelial cells with greatest intensity in epithelial cells and considerable cyclical changes. In all cases expression of mRNA was low during menstruation (days 1–5). Levels increased throughout the proliferative stage (days 6–14) of the cycle and for HAS-2 and HAS-3 remained elevated until the mid secretory (days 19–23) / implantation phase. HAS-1 expression temporarily decreased during the late proliferative stage (days 12–14). Following implantation expression of all HAS types decreased dramatically. HAS protein localization was demonstrated on the plasma membrane of both glandular epithelial and stromal cells. The partial correlation between HA and HAS supports a role for these enzymes in endometrial HA regulation during the menstrual cycle and also suggests tight control of enzyme expression.