Abstract 511: The role of hyaluronan-CD44 interaction in breast cancer.
Authors: Kultti Anne , Zimmerman Susan , Huang Lei , Chen Yanling , Cowell Jessica , Symons Rebecca C. , Jadin Laurence , Jiang Ping , Frost Gregory I. , Shepard Michael , Huang John 
Published: August 2014

Abstract
The tumor microenvironment is crucial for cancer cell survival and spreading. The glycosaminoglycan hyaluronan (HA) is accumulated in 50% of malignant breast cancer tumors and its accumulation correlates with poor survival of breast cancer patients. HA is synthesized at the cell surface by HA synthase enzymes (HAS1-3) and is extruded to the extracellular space where HA molecules can be attached to the cell surface via interactions with its receptors or HAS proteins. HA can also interact with its binding proteins and be incorporated into surrounding ECM. However, the origin and exact functions of HA in breast cancer are still unclear. The aim of this study was to explore the role of HA in the tumor microenvironment of breast cancer, especially in the interaction of tumor and stromal cells in vitro and in vivo. First, interaction of breast cancer cells and stromal cells were studied in mono- and co-cultures. Human bone marrow-derived mesenchymal stem cells (MSCs) and breast cancer-associated fibroblasts (CAFs) synthesized high amounts of HA, while this was the case for &lt;5% MDA-MB-468 breast cancer cells. In co-culture with MSCs or CAFSs, MDA-MB-468 and MDA-MB-231 cells formed distinct pericellular HA coats. Similar HA coats were observed after addition of exogenous FITC-labeled high molecular weight HA (1,2 MDa) to MDA-MB-468 and MDA-MB-231 cell cultures. Interestingly, binding of FITC-labeled HA was not efficiently blocked by unlabeled HA below 500 kDa. In co-cultures, the high molecular weight HA coats around MDA-MB-468 and MDA-MB-231 cells were prevented by antibody blockade of the HA receptor CD44, indicating that formation of HA coats is CD44-mediated. Knockdown of CD44 by shRNA also inhibited the formation of HA coats when FITC-HA was added to the cultures or when breast cancer cells were co-cultured with MSCs. MSCs also increased proliferation and migration of MDA-MB-468 (parental/Luc) cells, analyzed by luciferin and Transwell migration assays, respectively. MDA-MB-468 cell proliferation was slightly inhibited by removal of HA with pegylated human recombinant hyaluronidase PH20 (PEGPH20), and migration towards exogenous HA could be inhibited by CD44 knockdown. Importance of HA coats around breast cancer cells was also studied in vivo using MDA-MB-468 cells over-expressing HAS3 which forms 4.7-fold larger HA coats than parental MDA-MB-468 cells. MDA-MB-468 HAS3 cells exhibited much enhanced in vivo growth compared to MDA-MB-468 cells, and tumor growth of MDA-MB-468 HAS3 xenografts was inhibited up to 85% by PEGPH20. The results suggest that HA in tumor microenvironment, produced by tumor or stromal cells, provides growth benefit for breast cancer cells via promoting their proliferation and migration. Both phenomena seem to be mediated by CD44, which highlights the importance of HA-CD44 interaction in the growth of breast cancer.
Citation Format: Anne Kultti, Susan Zimmerman, Lei Huang, Yanling Chen, Jessica Cowell, Rebecca C. Symons, Laurence Jadin, Ping Jiang, Gregory I. Frost, Michael Shepard, John Huang. The role of hyaluronan-CD44 interaction in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 511. doi:10.1158/1538-7445.AM2013-511