Evaluation of hyaluronan metabolism as a therapeutic target in the treatment of breast cancer

Hyaluronan (HA) is a ubiquitous component of the extracellular matrix where it has important roles in the maintenance of tissue hydration and structural integrity. Malignant transformation is typically accompanied by changes in the cellular microenvironment, including altered composition of the extracellular matrix. Increased deposition of HA in the extracellular environment is frequently reported, where increased concentrations of cell-associated and stromal hyaluronan are considered poor prognostic indicators in several cancers. Upregulation of the expressions of the transmembrane HA synthetic proteins, the hyaluronan synthases (HAS), are implicated in the generation of heightened HA concentrations observed in cancer, where the interaction of the synthetic product with its cellular receptors generates signalling cascades which promote malignant progression. This thesis has explored the contribution of hyaluronan synthase 2 to breast cancer progression by overexpression of HAS2 in the ZR-75-1 human breast epithelial ductal carcinoma cell line, which has been previously reported to exhibit low HA synthetic capacity and CD44 expression. Overexpression of HAS2 induced up to a ~10 fold increase in the concentration of liberated HA, which stimulated expression of both CD44s and CD44v9. The increased HA-CD44 interactions afforded by HAS2 overexpression mediated activation of the PI3K/Akt pathway and increased the propensity for glycolytic metabolism, thereby implicating HAS2 in the dynamics of cellular energy metabolism. Furthermore, HAS2 modulated the expression of key breast cancer glycolytic markers including TKTL1 and PKM2. Downregulation of the glucose transporter GLUT1 contrasted the classic glycolytic phenotype, however may be indicative of the capacity of HA to act as an alternate energy source. The acquisition of an invasive, proliferative phenotype which is associated with both the induction of aerobic glycolysis (Warburg effect) and the increased production of HA was apparent in >6 fold increases in endpoint tumor volume and decreased overall survival in a model of metastases in the HAS2 transfectants relative to the control cell lines. The multifaceted roles of HA production in breast cancer that have been highlighted by findings of this thesis implicate HAS in the malignant process, and indicate that the isoforms warrant targeting by novel therapies. The inhibition of HA translocation across the cell membrane represents an approach to inhibition of HA synthesis. The recently described role of ABC transporter, MRP5, in HA translocation prompted investigation of the effect of pharmacological inhibition of ABC transporters on the export of HA and properties of breast cancer. Findings generated in this thesis however refuted a role for ABC transporters in breast cancer, demonstrating no reduction in liberated HA nor alteration of cellular HA localisation in response to inhibition of a wide range of ABC transporters. In accordance with this result, low endogenous expression of transporters was identified in a panel of breast cancer cell lines, where there was no significant correlation identified between HAS expression, HA production and ABC transporter expression. Furthermore, the HA glycocalyx was well retained in the presence of ABC transporter inhibitors, disproving the role of ABC transporters in HA translocation in breast cancer and possibly suggesting the means of HA export is cell-type dependent. Given the lack of inhibitory activity of ABC transporter inhibitors on HA synthesis, novel HAS antibody inhibitors were designed, synthesised and characterised in breast cancer and non-malignant cell lines in vitro. Antibodies were targeted to distinct epitopes within HAS1, where western blotting and FACS analysis confirmed the specific targeting of the HA synthase. The designated INT-1 and EX-1 antibodies were localised to their hypothesised intracellular and extracellular localisations, whilst the INT-2 antibody binding epitope was localised to the intracellular environment in non-malignant cell lines, and the extracellular environment in a panel of breast cancer cell lines. The capacity of the INT-2 antibody to discriminate between the hyaluronan synthase in malignant and non-malignant tissue and have specific inhibitory activity in malignant tissue only, has unique implications for both the targeted cancer-specific inhibition of HAS, and the structure and topology of the protein within the plasma membrane. Functionally, the inhibitory effects of the antibodies translated to reductions in liberation of HA and cell proliferation, which were consistent with the characterised HA synthesis inhibitor, 4-methylumbelliferone. Analysis of cell morphology by scanning electron microscopy suggested that HAS antibodies may be inducing necrosis. The collective work in this thesis highlights the importance of HAS and HA production in breast cancer, and the potential therapeutic value in their inhibition. Whilst the ABC transporters are not legitimate targets for inhibition of HA export in breast cancer, the design and generation of novel antibody inhibitors represents a highly significant finding in HA research. The seemingly altered organisation of the HA synthase in breast cancer which results in the accessibility of the INT-2 binding domain and the resultant inhibitory activity that it mediates, could be exploited in the development of novel breast cancer therapeutics, and in turn abrogate the many pro-malignant effects of the once seemingly innocent structural molecule of hyaluronan.