Abstract 3023: The characterisation of hyaluronan-related enzymes in breast cancer cell subpopulations
Authors: Evtimov Vera , Brown Tracey 
Published: May 2015

Abstract
Background:Hyaluronan (HA) has been associated with various facets of tumor biology where it provides structural integrity, maintains hydration homeostasis, and in conjunction with its metabolic receptor, CD44, has been implicated in tumor cell proliferation and migration. There is a direct correlation between treatment-resistant breast carcinomas and the increased synthesis of HA via the over-expression of the HA anabolic enzyme, HA synthase (HAS). It is becoming generally accepted that the failure of cancer treatments can be due to a small subpopulation of slow growing, invasive and drug resistant cells known as cancer stem cells (CSCs) which are often characterized by the presence of CD44. As CD44 is the sole HA metabolic receptor within the tumor microenvironment, this study aimed to characterize the role of HA metabolism in the breast CSC subpopulation and its potential contribution to the persistence of treatment-resistant cancer.
Methodology:HA metabolic enzymes [HAS 1-3 and hyaluronidase 1-2 (Hyal)] in breast cancer cell subpopulations were characterised in five cancer cell lines after FACS separation into putative CSC (CD44+/CD24-) and non-stem cell (CD44-/CD24+) subpopulations using quantitative real time PCR. Cell subpopulations were further characterised for cell-growth kinetics and resistance to chemotherapy using an Excelligence proliferation assay. The most highly expressed HAS isoform, HAS2, was knocked down or inserted in different cell lines using stable transfection and the resultant changes in proliferation, invasive and migratory potential and the direct effect on the CSC subpopulation was investigated both in vitro and in vivo.
Results:HAS2 and Hyal2 were determined to be preferentially expressed in invasive breast cancer cell lines, where the chemotherapy-resistant CSC subpopulations (CD44+/CD24-) expressed the highest level of these HAS and Hyal isoforms. Transfection-induced attenuation of HAS2 mediated a reduction in the CSC subpopulation while over-expression of HAS2 increased the size of the CD44+/CD24- subpopulation. Both in vitro and in vivo HAS 2 over-expression increased HA production with a concomitant increase in tumorigenesis and metastasis while the counter-effect was observed in HAS2 knock-down breast cancer cells.
Conclusions:These preliminary data suggest that HA metabolism is a tightly regulated process and a key component within breast cancer subpopulations and the CSC niche. The direct relationship between increased HAS expression, HA production and enlargement of the CD44+/CD24- subpopulation highlights HA metabolic enzymes as potential therapeutic targets in treatment-resistant breast cancer.
Citation Format: Vera Evtimov, Tracey Brown. The characterisation of hyaluronan-related enzymes in breast cancer cell subpopulations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3023. doi:10.1158/1538-7445.AM2014-3023