Abstract 3339: A role for CXCR4 signalling in the regulation of normal and malignant breast stem cell activity

Abstract

Abstract CXCR4 is a chemokine receptor specific for stromal derived factor-1 (SDF-1) and is highly expressed in breast cancer cell lines and primary breast cancer samples. Elevated expression of CXCR4 in breast cancer is linked to poor prognosis. CXCR4 signalling has been reported to be important in breast cancer migration and metastasis formation but its role is yet to be fully elucidated. Our previously published data have shown that a stem cell-enriched population of cells can be obtained by harvesting anoikis resistant cells which survive in early mammosphere culture (Harrison et al., Cancer Res; 2010, 70 (2), 709-18). We collected anoikis resistant cells 12 hours after plating out into the mammosphere culture to obtain stem cell-enriched populations from 2 immortalised (MCF10A, 226L) and 3 malignant breast cell lines (MCF7, T47D, SKBR3). Anoikis resistant cells harvested at 12 hours had a significantly higher mammosphere colony-forming efficiency than their unsorted monolayer cells (2-4 fold increase, p < 0.05) suggesting stem cell enrichment. In limiting dilution transplantation assays into athymic Nude mice, MCF7 and T47D anoikis resistant cells had a 12 and 16-fold enrichment for tumor formation respectively, compared to the unsorted monolayer cells. Using an Agilent custom microarray, we found that CXCR4 expression was consistently upregulated in these stem cell-enriched populations compared with monolayer cells (2.0 and 6.8 fold; normal and malignant cell line averages respectively). We further investigated the function of CXCR4 in vitro using the mammosphere assay to assess stem cell activity. AMD3100 (Sigma), a CXCR4 antagonist and lentiviral CXCR4 shRNA were used to inhibit and recombinant soluble SDF-1 to stimulate the pathway. In the normal breast cell line MCF10a, SDF-1 addition significantly reduced mammosphere formation in a dose dependent manner (200ng/ml; 25% decrease, t = 4.320, p < 0.001) and this could be rescued by the addition of AMD3100. Inhibiting the CXCR4 pathway stimulated primary mammosphere formation in the breast cancer cell lines T47D and MCF7 using either 20uM AMD3100 (T47D; 34% increase, t = 3.017, p < 0.01) or lentiviral CXCR4 shRNA (T47D; 97% increase, t = 6.288 p < 0.005, MCF7; 48% increase, t = 3.026, p < 0.05). In vivo data has already shown the importance of the CXCR4/SDF-1 pathway in metastasis formation in breast cancer. Our data takes suggests that CXCR4 signalling, as well as its role in migration and homing, is important in maintaining stem cell activity. Thus CXCR4 targeted therapy, alongside current standards of care, may improve breast cancer outcomes. Funding: Biotechnology and Biological Sciences Research Council (BBSRC), Vertex Pharmaceuticals (Europe) Limited, Breast Cancer Campaign and Cancer Research UK (CRUK). 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 3339. doi:10.1158/1538-7445.AM2011-3339