Abstract P3-02-05: Targeting glutamine metabolism inhibits GD2+ breast cancer stem cell function in triple negative breast cancer
Authors: Ly Stanley , Nguyen Khoa , Andreeff Michael , Battula Venkata Lokesh 
Published: March 2020

Breast cancer stem cells (BSCs) constitute a fraction of primary tumor cells that exhibit drug resistance and have metastatic potential. Ganglioside GD2 has been shown by us and others as a marker for BCSCs. Nutrient deprivation-associated metabolic stress observed during tumor progression is reportedly associated with the cancer stem cell phenotype, and we have shown that oxidative stress caused by serum deprivation induces GD2 expression in vitro and in vivo. To identify metabolic signatures associated with GD2+ cells and their potential adaptive mechanisms to oxidative stress, global metabolic profiling was performed using a mass spectroscopy-based approach. We found that metabolites associated with amino acid metabolism, particularly glutathione metabolism, to be most highly upregulated in GD2+ compared to GD2− cells. In addition, glutathione biosynthesis and oxidation to glutathione disulfide was also increased in cells undergoing oxidative stress. These data suggest that the glutathione-mediated detoxification pathways play a key role in GD2+ or basal type breast cancer cells.

As glutamine is a major precursor molecule for glutathione biosynthesis, we hypothesized that glutamine, at least in part, regulates cellular redox tone and the GD2+ phenotype in breast cancer cells. To test our hypothesis, we cultured breast cancer cell lines MDA-MB-231 and SUM159 in media containing 0%, 0.5%, 1%, 2%, or 4% of L-glutamine for 3 days. Flow cytometry analysis revealed that the percentage of GD2+ increased from 6% to 12% and from 12% to 22% in MDA-MB-231 and SUM159 cells, respectively when the cells were cultured at 0 or 4% glutamine. Glutaminase, an enzyme that converts glutamine to glutamate, which further leads to glutathione biosynthesis, has been shown to be overexpressed in basal-type breast cancer cells. To investigate the role of glutaminase on stem cell redox homeostasis and phenotype, we treated TNBC cell lines MDA-MB-231 and SUM159 cells with glutaminase inhibitors CB-839 or 968 compound for 3 days. Flow analysis revealed 70-80% reduction in GD2 expression after treatment in a dose dependent manner. In addition, treatment with CB-839 inhibited cell proliferation by over 80% in a dose-dependent manner in MDA-MB-231 cells and SUM159 cells (p&lt;0.001). Next, to determine the effect of glutaminase inhibition on BCSC function, we cultured MDA-MB-231 and SUM-159 cells in low adherent cell culture dishes with or without glutaminase inhibitors 968 or CB-839 for 7 days. Interestingly, we observed a 40-50% reduction in mammosphere formation in both treated cell lines compared to the untreated controls, suggesting that inhibition of glutaminase is critical for BCSC function. In conclusion, the anti-oxidant glutathione is highly upregulated in GD2+ BCSCs, and this appears to be associated with the concentration of glutamine, and appears to regulate, perhaps via the regulation of redox homeostaisis, GD2 expression in BCSCs. Targeting glutaminase and redox homeostasis using CB-839 or CB968 inhibits BCSC as measured by GD2 positivity and mammosphere formation. Glutaminase inhibition in combination with chemotherapy could be a valuable future therapeutic strategy for targeting BCSCs in TNBC.

Citation Format: Stanley Ly, Khoa Nguyen, Michael Andreeff, Venkata Lokesh Battula. Targeting glutamine metabolism inhibits GD2+ breast cancer stem cell function in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-05.