Abstract LB-193: Ganglioside GD2 identifies cancer stem cells and inhibition of GD2 biosynthesis by targeting GD3 synthase exerts antitumor effects
Authors: Battula Venkata Lokesh , Shi Yuexi , Evans Kurt , Wang Rui-Yu , Speath Erika L. , Jacamo Rodrigo , Guerra Rudy , Sahin Aysegul , Marini Frank , Mani Sendurai , Andreeff Micheal 
Published: August 2012

Abstract
Currently, only very few markers either as single or in combination are available to identify cancer stem cells (CSCs). In this report, we identified that the ganglioside GD2, a marker known to express on mesenchymal stromal cells (MSCs) is expressed on a small fraction (5.5% ± 3.4%) of transformed human mammary epithelial cells (HMLER) and breast cancer patient tumors. FACS sorted GD2+ cells appear spindle shaped and proliferate 5 fold slower compared to GD2- cells in-vitro. Analysis of breast cancer cell lines (n=12) indicated that GD2 expression varies and that the basal breast cancer cell lines have a higher percentage of GD2+ cells (median 9%, range 1.2-17%, n=6), as compared to their luminal counterparts (median 0.2%, range 0-3%, n=6, p&lt;0.001). Functional analysis revealed that GD2+ HMLER and MDA-MB-231 cells produced 2-5 fold more mammospheres in-vitro (p&lt;0.003) and tumors in-vivo than GD2- cells (p&lt;0.04). Recent reports suggest that breast tumor cells with CD44highCD24low phenotype exhibit CSCs characteristics. Interestingly, the majority (94% ± 3.5%) of GD2+ cells were characterized by the CD44highCD24low phenotype in HMLER cells. Analysis of primary breast cancer samples (n=10) revealed that GD2 is variably expressed in these samples (median 4.35%, range 0.5%-35.8%) and 95.5% ± 2.7% of GD2+ cells co-segregated with CD44highCD24lowCD45- phenotype. In contrast, only 2.4%± 0.4% of GD2- cells displayed this phenotype. Gene-chip and quantitative RT-PCR analysis revealed that GD3 synthase (GD3S), the enzyme responsible for synthesis of GD3, the precursor of GD2, was expressed 10-fold higher in GD2+ cells compared to GD2- HMLER and MDA-MB-231 cells. Analysis of GD2 expression in HMLER cells induced to undergo EMT revealed that the percentage of GD2+ cells increased 6-7 fold and the expression of GD3S &gt; 10 fold. Moreover, we observed spontaneous generation of GD2+ from GD2- cells and vice versa in both in-vitro and in-vivo, suggesting a role of EMT in this process. Stable knock-down of GD3S in MDA-MB-231 cells using shRNA impaired in-vitro matrigel invasion by more than 10-fold and completely abolished tumor growth in-vivo. Importantly, Triptolide, an anti-inflammatory and anti-cancer drug, which was recently shown to inhibit GD3S expression in melanoma cells, also inhibited GD3S expression in MDA-MB-231 and SUM159 cells by &gt;95% in a dose dependent manner and thereby inhibited growth of GD2+ cells in a time dependent manner. Intra-peritoneal administration of Triptolide (0.15mg/Kg/day) in NOD/SCID mice bearing MDA-MB-231 breast tumors completely eliminated tumors in 50% and reduced the tumor volume 7- to 8-fold in 25% of the mice. In conclusion, we identified GD2 as a new CSC specific cell surface marker and GD3 synthase as a potential therapeutic target for CSCs, with the potential of improving survival and cure rates of patients with breast cancer.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-193. doi:1538-7445.AM2012-LB-193