Abstract P6-02-01: Metabolic stress induces GD2 expression and cancer stem cell phenotype in triple negative breast cancer

Abstract

Breast cancer stem cells (BCSCs) have been characterized as a fraction of cells in primary tumors that are drug resistant and have metastatic potential. Ganglioside GD2 has been shown by us and others as a marker for BCSCs. Furthermore, nutrient deprivation associated metabolic stress seen during tumor progression is reportedly associated with the cancer stem cell phenotype. We hypothesized that metabolic stress could induce spontaneous generation of GD2+ BCSCs during tumor progression. To test our hypothesis, we cultured breast cancer cell lines MDA-MB-231 and SUM159 at low seeding density and measured percentage and absolute number of GD2+ cells daily. Flow cytometry analysis revealed that the percentage of GD2+ cells increased from 4.5 ± 2.5 on day 2 to 15 ± 3.8% on day 5 in MDA MB-231 cells and from 8.5 ± 2.8% on day2 to 28 ± 6.2% on day 5 in SUM159 cells (both designated as triple-negative breast cancer, TNBC). To investigate this phenomenon in-vivo, we injected GFP+ MDA-MB-231 cells in NSG mice mammary fat pads and examined GD2 expression in the implanted tumors weekly. Interestingly, we noticed that the percentage of GD2+ also increased from 12 ± 1.5% on week 1 to 30 ± 2.5% on week 6. Next, SUM159 cells were cultured in either nutrient rich (NR, i.e., 10% serum) or nutrient deprived (ND, 1% serum) for 4 days. We found that the percentage of GD2+ cells in NR medium at the end of 4 day culture was ~20% of the total cell population, whereas in ND medium was almost 50%. We then tested the effects of nutrient rich environment on GD2 expression by refreshing the media daily. Interestingly, cells that received fresh media had lower number of GD2+ cells (15 ± 1.5%) compared to cells cultured in the same medium for 4 days (33 ± 2.5%). Our data suggests that nutrient deprivation induces a stem cell phenotype in TNBC cells. Next, we performed global metabolic profiling (i.e., for a total of 300 biochemical metabolites) using a mass spectroscopy-based approach. We profiled SUM159 cells cultured with NR vs. ND medium (set-1); GD2+ vs GD2- SUM159 cells (set -2); GD2+ vs GD2- MDA-MB-231 cell (set-3). Metabolites associated with amino acid metabolism, in particular glutathione metabolism, including glutamyl-alanine, 5-oxy-proline, proline, glutamine, and glutathione itself were found to be most highly up-regulated in GD2+ compared to GD2- cells and also in cells cultured in serum starved compared to serum rich conditions. Further analysis of these metabolites and their association with GD2+ cell signature raveled that gamma-glutamyl transferase (GGT5), was one of the most highly up-regulated (>150-fold) gene across all the groups. GGT is expressed on cell surface and transfers glutamyl group to amino acids, which then get transported across the membrane. In cancer, cells expressing GGT has been shown to be resistant to chemotherapeutic agents including cisplatin. Targeting glutathione metabolism could be future therapeutic strategy to inhibit BCSC growth in TNBC. Citation Format: Battula VL, Piyaranthna B, Nguyen K, Sun JC, Jin F, Coarfa C, Nagireddy P, Andreeff M. Metabolic stress induces GD2 expression and cancer stem cell phenotype in triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-02-01.

Surface expression of the immunotherapeutic target GD2 in osteosarcoma depends on cell confluency

“… 36 , 37 More specifically, cellular hypoxia can induce expression of GD3S 38 and also Sialin, 39 a sialic acid transporter, thereby enhancing expression of sialogangliosides in tumor cells. Finally, oxidative stress caused by nutrient deprivation was found to induce expression of G D2 in breast cancer cells, 40 , 41 concomitant with a cancer stem cell‐like phenotype. 42 Overall, it is a common observation that ganglioside expression patterns in tumor cells vary with environmental conditions, as mimicked in vitro by cellular confluency.…”

“…Finally, oxidative stress caused by nutrient deprivation was found to induce expression of G D2 in breast cancer cells, 40,41 concomitant with a cancer stem cell-like phenotype. 42 Overall, it is a common observation that ganglioside expression patterns in tumor cells vary with environmental conditions, as mimicked in vitro by cellular confluency.…”

Targeting IκappaB kinases for cancer therapy

“…Interestingly, the agent was found to be safe in mice [58]. BMS-345541 can also suppress breast tumorigenesis and metastases by targeting GD2+ cancer stem cells [59]. In another study, BMS-345541 was found to reduce proliferation and induce apoptosis in PC-3 cells [60].…”

“…• BMS-345541: Selectively targets an allosteric site of IKKα (IC 50 : 4 μM) and of IKKβ (IC 50 : 0.3μM) [57]; suppressed breast tumorigenesis and metastases by targeting GD2+ cancer stem cells [59]; inhibited EMT, induced apoptosis and suppressed proliferation of prostate cancer cells in a dose-dependent manner [60]; inhibited constitutive IKK activity and melanoma cell survival both in vitro and in vivo [61]; induced mitochondria-mediated apoptosis in melanoma cells [61].…”