Abstract 3801: Oxidative stress induces glutamine-dependent GD2+ triple negative breast cancer stem cells
Authors: Jaggupilli Appalaraju , Ly Stanely J. , Borkar Roshan , Nguyen Khoa , Yuan Bin , Putluri Nagireddy , Andreeff Michael , Battula V. Lokesh 
Published: September 2020

Breast cancer stem cells (BCSCs) have been shown to be associated with tumor recurrence, metastasis and drug resistance. We reported that the ganglioside GD2 selectively identifies BCSCs and its over-expression in Triple-Negative Breast Cancer (TNBC). However, the factors regulating GD2+ BCSCs in a growing tumor are not known. Here we hypothesize that nutrient deprivation-induced oxidative stress upregulates GD2+ BCSCs in TNBC. TNBC cell lines including SUM159 and MDA-MB-231 were cultured under different nutrient-deprived conditions and analyzed for GD2 expression by flow cytometry. We have identified a steady increase of GD2+ cells from 5%±2% on day 3 to 20%±5% on day 6 in cells cultured in nutrient deprived condition. We further performed the in vivo by injecting GFP+ MDA-MB-231 cells into NSG mice and measured GD2+ expression in the growing tumor once a week. Interestingly, we also noticed a positive correlation between the percentage of GD2+ cells and tumor volume in vivo with r=0.9829 and p&lt;0.0004 suggesting nutrient deprived conditions in the growing tumors results in higher GD2 expression. Next, the cells were cultured in medium with or without glucose or media containing 2-deoxy glucose (2DG, glycolysis inhibitor). We found that the percentage of GD2+ cells increased 2-fold in the absence of glucose or in the presence of 2DG (10mM) compared to cells cultured with glucose (6g/L) or without 2DG, suggesting that glucose deprivation enhances GD2 biosynthesis in TNBC cells. Global metabolomics profiling using liquid chromatography-mass spectrometry followed by KEGG pathway analysis identified signatures such as glutathione mediated detoxification and glutathione biosynthesis to be most highly upregulated in GD2+ compared to GD2- cells. As glutamine is a key precursor for glutathione biosynthesis, we cultured SUM159 and MDA-MB-231 cells in media with or without glutamine and found that the percentage of GD2+ cells positively correlates with glutamine concentration in the medium. Next, to target glutamine metabolism, we treated TNBC cells with glutaminase inhibitor, CB-839 or glutamine transporter inhibitor, V9302. This resulted in a 70-80% reduction of GD2 expression in a dose dependent manner. CB-839 and V9302 also inhibited mammosphere formation by 40-50% and increased reactive oxygen species (ROS) in a dose-dependent manner in TNBC cells. In conclusion, oxidative stress results in GD2+ BCSC phenotype in TNBC cells. Inhibition of glutamine uptake or its utilization by V9302 or CB-839 inhibits GD2+ BCSC phenotype and alters redox homeostasis in BCSCs by inducing ROS levels. Targeting glutamine transporter or glutaminase could complement conventional chemotherapy by targeting BCSCs in TNBC.

Citation Format: Appalaraju Jaggupilli, Stanely J. Ly, Roshan Borkar, Khoa Nguyen, Bin Yuan, Nagireddy Putluri, Michael Andreeff, V. Lokesh Battula. Oxidative stress induces glutamine-dependent GD2+ triple negative breast cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3801.