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Disrupting Metabolism to Eliminate Cancer Stem Cells

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Original article from STEM CELLS

"Effective Elimination of Cancer Stem Cells By a Novel Drug Combination Strategy"

Effective means to target cancer stem cells (CSCs) using pharmaceutical agents have been hindered by their seemingly intrinsic resistance to chemotherapeutic agents (Gilbert and Ross).   CSCs in Glioblastoma multiforme (GBM) have been shown to be resistant to common chemotherapeutics and radiation (Kang and Kang and Beier et al), and glioblastoma cancer stem cells (GSCs) have been found to reside in hypoxic niches which further promotes drug resistance (Lei et al, Heddleston et al and Li et al).   This suggests that the glycolytic pathway may be utilised in these cells to generate energy, giving researchers a potential target for chemotherapeutic intervention.   Now researchers from the laboratories of Peng Huang from the State Key Laboratory of Oncology in South China, Guangdong, China and The University of Texas MD Anderson Cancer Center, Texas, USA, in a study published in Stem Cells, have found that 3-bromo-2-oxopropionate-1-propyl ester (3-BrOP), which affects key enzymes in the glycolytic pathway and reduces ATP generation (Ko et al, Geschwind et al and Xu et al), allows the efficient elimination of CD133+ GSCs when used in combination with other common chemotherapeutic agents (Yuan and Wang et al).

Two human GSC lines (GSC11 and GSC23) were used in this study which both exhibited typical neurosphere morphology and expressed Nestin at high levels while having low mitochondrial respiration (low oxygen consumption rate).   However, upon differentiation, cells adhered to culture plates, decreased expression of Nestin and glioma stem cell markers (CD133, SOX2, and Notch1) and mitochondrial respiration was substantially activated (75% increase).   The addition of drugs commonly used in the treatment of glioblastoma (temozolomide (TMZ) and bis-chloroethylnitrosourea (BCNU)) demonstrated the relative resistance of the cells to the drugs, although the addition of 3-BrOP under hypoxic conditions did lead to the inhibition of growth.   The addition of both 3-BrOP and BCNU led to a significant increase in the cytotoxic effect over the drugs alone, with these effects heightened under hypoxic conditions.   Further analysis demonstrated that BCNU and 3-BrOP exposure led to the abolishment of neurosphere formation of GSCs and subsequent in vivo analysis, using orthotopic inoculation of GSCs into the brains of immunodeficient mice, found that while control cells treated with PBS led to the death of all mice by 80 days, cells treated with BCNU or 3-BrOP exhibited a large delay in death, while in mice treated with both drugs 2 died at 12 months but 3 showed no signs of tumor development.   Importantly, the concentrations of drugs required to kill the GSCs caused only minimum cytotoxicity in non-malignant human astrocytes (NHAs), suggesting that the targeting of the GSCs through their differential metabolism is an effective strategy to explore.

Specific analysis of the metabolism of these cells found that incubation of GSCs with BCNU or 3-BrOP alone led only to a slight inhibition of lactate production, while the combination of these drugs led to a significant inhibition of lactate and a 50% decrease in ATP production prior to cell death.   However, TMZ exposure did not affect ATP production nor synergise with 3-BrOP to cause depletion of ATP in GSCs.   BCNU or 3-BrOP exposure to GSCs was also shown to lead to a 60% decrease in GAPDH activity, with the combination of these drugs leading to an 80% reduction.

Finally, the mechanism of this synergism was explored, concentrating on the fact that BCNU is known to cause DNA damage by formation of DNA adducts and double-strand breaks (DSBs) (Batista et al and Cui et al).   Formation of γH2AX foci is a rapid cellular response to the presence of DSBs (Rogakou et al), but requires ATP.   Comet assays, which measure DNA damage, found that BCNU alone or combination with 3-BrOP for 6 hours caused severe DNA damage in GSCs, with repair permitted only in cells treated with BCNU.   Addition of BCNU and 3-BrOP inhibited DNA damage repair, and led to the increase in DNA damage after 6 hours treatment.   BCNU treatment only led to a substantial increase in γH2AX, however cells treated with 3-BrOP or in combination with BCNU led to a substantial decrease in γH2AX formation, likely due to the lack of ATP.

Overall, this exciting study has found that GSCs exhibit a high glycolytic activity, which can be selectively targeted through drugs such as 3-BrOP which cause a decrease in ATP production, an increase in cell death and ultimately the impairment of tumourigenesis when combined with other chemotherapeutic drugs, leaving normal cells relatively unaffected.   Mechanistically, GAPDH was identified as a major target for 3-BrOP, leading to a lack of DNA damage repair after chemotherapeutic drug use due to the lack of γH2AX formation as a consequence of a lack of ATP formation.   Importantly, the drug combination effect was boosted under hypoxia, thought to mimic the conditions of the cancer stem cell niche, and so further enhancing the potential of this novel drug combination in overcoming resistance and selectively killing cancer stem cells; a confident stride towards moving such research to a clinical setting.

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Study originally appeared in Stem Cells.

Stem CellCorrespondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.