Abstract 1421: The spontaneous generation of GD2+ breast cancer stem-like cells is directly proportional to tumor progression and mediated by epithelial to mesenchymal transition and NFκB activation

Abstract

Abstract Breast cancer metastases are caused by extravasation of cancer cells from the primary tumor; once released, the cells travel through the circulation and initiate secondary tumors at distant sites. However, it is not clear how these metastasizing mesenchymal-like breast cancer stem cells (BCSCs) are generated in the primary tumor. We recently discovered that ganglioside GD2 is expressed on BCSCs and that induction of epithelial-to- mesenchymal transition (EMT) induced GD2 and GD3S expression. GD2+ cells expressed higher levels of mesenchymal markers (N-cadherin, vimentin, twist, snail) and lower levels of epithelial markers (E-cadherin) than GD2- cells. Interestingly, FACS-sorted GD2- cells spontaneously produced GD2+ cells within 4-6 days in culture. To investigate the factors associated with the spontaneous generation of GD2+ BCSCs, we cultured breast cancer cell lines MDA-MB-231and SUM159 cells in vitro and measured the percentages of GD2+ cells over time. Interestingly, we noticed that the percentage and absolute numbers of GD2+ cells increased in a time-dependent manner: from 4.5%±2.5% on day 1 to 18%±4.5% on day 6 suggesting spontaneous generation of GD2+ cells. Considering that GD2+ cells grow slower compared to GD2- cells, these data suggest that GD2- cells undergo cellular transformation into GD2+ cells. As induction of EMT generates stem cell-like cells, we determined the expression of mesenchymal-related markers, including vimentin, N-cadherin, and twist, in the breast cancer cells in a time-course experiment. Results suggest 3 to 6 - fold induction of mesenchymal markers in these breast cancer cells in a time-dependent manner. To investigate the spontaneous generation of GD2+ cells in vivo, 1×106 GFP+ MDA-MB-231 cells were transplanted into mammary fat pads of NOD/SCID mice (n = 18). Each week, a group of mice (n = 3) was sacrificed, tumors were extracted and the percentage of GFP+GD2+ cells was measured by flow cytometry. In line with our in vitro results, we observed significant increases in GD2+ BCSCs with increasing tumor volume. The frequency of GD2+ cells increased from 15.1%±4.6% to 37%±8.7% in 6 weeks, indicating that breast cancer cells spontaneously undergo EMT during tumor progression and generate GD2+ BCSCs. To identify possible targets to inhibit EMT in breast cancer cells, we performed Kinexus® proteomic analysis of GD2+ and GD2- cells and discovered activation of NFκB signaling in GD2+ cells. Inhibition of NFκB signaling by the small molecule inhibitor BMS345541 reduced the frequency of GD2+ cells by >95% in breast cancer cell lines in vitro and increased survival in tumor bearing mice. (Battula VL, et al., AACR, 2015). Inhibition of EMT using BMS345541 might reduce EMT-driven generation of GD2+ BCSCs and thus reduce their metastatic spread in breast cancer patients. Experiments testing this hypothesis are under way. Citation Format: Jeffrey Sun, Gabriel N. Hortobagyi, Michael Andreeff, Venkata Lokesh Battula. The spontaneous generation of GD2+ breast cancer stem-like cells is directly proportional to tumor progression and mediated by epithelial to mesenchymal transition and NFκB activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1421. doi:10.1158/1538-7445.AM2015-1421