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
Authors: Sun Jeffrey , Hortobagyi Gabriel N. , Andreeff Michael , Battula Venkata Lokesh 
Published: August 2015

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 &gt;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