Abstract 3409: Hyaluronan synthase 2 promotes tumor progression by stimulating interaction of cancer stem-like cells with tumor associated macrophage and stromal cells in the bone
Authors: Okuda Hiroshi , Kobayashi Aya , Watabe Misako , Pai Sudha K. , Liu Wen , Xing Fei , Pandey Puspa R. , Fukuda Koji , Iiizumi Megumi , Watabe Kounosuke 
Published: June 2012

Abstract
According to the recent cancer stem theory, metastatic cells must have stem cell-like capability for their self-renewal and invasive ability. Understanding how the cancer stem cells adapt themselves to the microenvironment of the distant organs and how the “niche” at the metastatic site promotes the proliferation of these cells is of paramount importance to develop more effective therapeutic strategy for metastatic disease. In this study, we have isolated cancer stem cell-like cells (CSC) from human breast cancer cell lines, MDA-MB231, MDA231BoM and MDA231BrM. The latter two cell lines are derivatives of MDA-MB231 and they preferentially metastasize to bone (MDA231BoM) or brain (MDA231BrM) when they are transplanted into mice. CSC were isolated from each line using surface markers (CD24-, CD44+, ESA+) and their tumor initiating abilities were tested in animal. We then performed an analysis of global expression profile for these CSCs using the Affymetrix expression array and found that among all up-regulated genes, two genes (HAS2 and MMP1) were significantly correlated with overall- and metastasis-free survival when they were cross-examined in GEO database. To further examine the role of HAS2 in tumor metastasis in vivo, we prepared CSC from MDA231BoM which carries shRNA expression vector for HAS2. The CSC were then implanted into nude mice by intra-cardiac injection followed by oral administration of 4MU, a specific inhibitor of HAS genes. We found that the injection of CSC of MDA231BoM which expresses shRNA significantly improved the survival rate of animals. In addition, administration of 4MU significantly suppressed tumor metastasis of CSC, suggesting that inhibition of HAS2 can indeed reduce the incidence of metastasis in vivo. We also examined the effect of tumor associated macrophage (TAM) on growth of CSC in animals by injecting CSC of MDA231-BoM with or without TAM into the tibial bone. We found that co-injection of TAM with CSC significantly augmented the growth of tumor in their legs. These results strongly support the idea that TAM plays a critical role in CSC growth in breast cancer. To understand how TAM promotes CSC proliferation, we attempted to identify secretory factor(s) from TAM by using a cytokine/growth factor antibody array system. We found that the secretion of PDGF from TAM was significantly augmented in conditioned medium of co-culture of CSC and TAM. Furthermore, we found that treatment of bone marrow stem cell (BMSC) with PDGF followed by co-culture of CSC significantly promoted proliferation of CSC. Collectively, our results suggest that TAM and BMSC promote self-renewal of CSC in bone microenvironment via direct interaction between hyaluronan and CD44. Our results also provide strong rationale to target HAS2 by using 4MU in order to block the metastatic progression of CSC in breast cancer.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3409. doi:10.1158/1538-7445.AM2011-3409