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Blocking anaplerotic entry of glutamine into the TCA cycle sensitizes K-Ras mutant cancer cells to cytotoxic drugs.

Saqcena M, Mukhopadhyay S, Hosny C, Alhamed A, Chatterjee A, Foster DA - Oncogene (2014)

Bottom Line: One of the key shunts is the exit of citrate from the mitochondria and the TCA cycle for the generation of cytosolic acetyl-coenzyme A that can be used for fatty acid and cholesterol biosynthesis.Inhibition of K-Ras effector pathways was able to revert cells to G1 arrest upon Q deprivation.Significantly, Q deprivation or suppression of anaplerotic Q utilization created synthetic lethality to the cell cycle phase-specific cytotoxic drugs, capecitabine and paclitaxel.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Hunter College of the City University of New York, New York, NY, USA.

ABSTRACT
Cancer cells undergo a metabolic transformation that allows for increased anabolic demands, wherein glycolytic and tricarboxylic acid (TCA) cycle intermediates are shunted away for the synthesis of biological molecules required for cell growth and division. One of the key shunts is the exit of citrate from the mitochondria and the TCA cycle for the generation of cytosolic acetyl-coenzyme A that can be used for fatty acid and cholesterol biosynthesis. With the loss of mitochondrial citrate, cancer cells rely on the 'conditionally essential' amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates. Although Q deprivation causes G1 cell cycle arrest in non-transformed cells, its impact on the cancer cell cycle is not well characterized. We report here a correlation between bypass of the Q-dependent G1 checkpoint and cancer cells harboring K-Ras mutations. Instead of arresting in G1 in response to Q-deprivation, K-Ras-driven cancer cells arrest in either S- or G2/M-phase. Inhibition of K-Ras effector pathways was able to revert cells to G1 arrest upon Q deprivation. Blocking anaplerotic utilization of Q mimicked Q deprivation--causing S- and G2/M-phase arrest in K-Ras mutant cancer cells. Significantly, Q deprivation or suppression of anaplerotic Q utilization created synthetic lethality to the cell cycle phase-specific cytotoxic drugs, capecitabine and paclitaxel. These data suggest that disabling of the G1 Q checkpoint could represent a novel vulnerability of cancer cells harboring K-Ras and possibly other mutations that disable the Q-dependent checkpoint.

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Pharmacological inhibition of Q anaplerosis sensitizes K-Ras mutant cancer cells to cytotoxic drugs. (a) Schematic overview of anaplerotic Q utilization. Q is deaminated to glutamate by glutaminase (GLS). Glutamate is then converted to α-ketoglutarate via transamination catalyzed by GOT, which uses oxaloacetate as the amino group acceptor to generate aspartate. AOA inhibits GOT and therefore suppresses generation of α‐ketoglutarate from Q-derived glutamate. Aspartate is critical for redox balance and NAPDH production and the generation of citrate for fatty acid synthesis. (b) Cells were plated at 20% confluence in 10-cm plates in complete medium (CM). After 24 hr, cells were shifted to CM, or medium lacking Q, or CM containing 0.5 mM AOA (C13408, Sigma-Aldrich) for 48 hr – at which time the cells were observed using phase-contrast microscopy. (c) MCF7 and MDA-MB-231 cells were plated and treated as in (b) for 48 hr, at which time cells were analyzed for cell cycle distribution as in Figure 1. In addition to AOA, the MDA-MB-231 cells were treated with cell permeable analogues of α-ketoglutarate (DMKG; 4 mM) (349631, Sigma-Aldrich) and aspartate (β-MD; 4 mM) (A8921, Sigma-Aldrich). Error bars represent standard error of mean for experiments repeated three times. (d) Cells were plated and treated as in (b), harvested at indicated time points, and scored after staining with crystal violet using light microscopy. Error bars represent the standard error of the mean for experiments repeated three times. (e) MCF7 and MDA-MB-231 cells were plated as in (b) and shifted to CM or treated with 0.5 mM AOA for 48 hr. The cells were additionally treated with 50 nM Pac or 1 μg/ml Cap for 24 hr, at which time the percentage non-viable cells were determined using trypan blue exclusion assay. Error bars represent the standard error of mean for experiments repeated three times. Cell lysates were also collected, and the levels of cleaved PARP (antibody from Cell Signaling) were determined by Western blot analysis. Data shown are representative of experiments repeated two times. (f) Model depicting that AOA treatment mimics Q deprivation causing G1 cell cycle arrest in K-Ras wild type cells and S- and G2/M-phase arrest in K-Ras mutant human cancer cell lines, which creates synthetic lethality to cell cycle phase-specific cytotoxic drugs causing apoptotic cell death.
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Figure 4: Pharmacological inhibition of Q anaplerosis sensitizes K-Ras mutant cancer cells to cytotoxic drugs. (a) Schematic overview of anaplerotic Q utilization. Q is deaminated to glutamate by glutaminase (GLS). Glutamate is then converted to α-ketoglutarate via transamination catalyzed by GOT, which uses oxaloacetate as the amino group acceptor to generate aspartate. AOA inhibits GOT and therefore suppresses generation of α‐ketoglutarate from Q-derived glutamate. Aspartate is critical for redox balance and NAPDH production and the generation of citrate for fatty acid synthesis. (b) Cells were plated at 20% confluence in 10-cm plates in complete medium (CM). After 24 hr, cells were shifted to CM, or medium lacking Q, or CM containing 0.5 mM AOA (C13408, Sigma-Aldrich) for 48 hr – at which time the cells were observed using phase-contrast microscopy. (c) MCF7 and MDA-MB-231 cells were plated and treated as in (b) for 48 hr, at which time cells were analyzed for cell cycle distribution as in Figure 1. In addition to AOA, the MDA-MB-231 cells were treated with cell permeable analogues of α-ketoglutarate (DMKG; 4 mM) (349631, Sigma-Aldrich) and aspartate (β-MD; 4 mM) (A8921, Sigma-Aldrich). Error bars represent standard error of mean for experiments repeated three times. (d) Cells were plated and treated as in (b), harvested at indicated time points, and scored after staining with crystal violet using light microscopy. Error bars represent the standard error of the mean for experiments repeated three times. (e) MCF7 and MDA-MB-231 cells were plated as in (b) and shifted to CM or treated with 0.5 mM AOA for 48 hr. The cells were additionally treated with 50 nM Pac or 1 μg/ml Cap for 24 hr, at which time the percentage non-viable cells were determined using trypan blue exclusion assay. Error bars represent the standard error of mean for experiments repeated three times. Cell lysates were also collected, and the levels of cleaved PARP (antibody from Cell Signaling) were determined by Western blot analysis. Data shown are representative of experiments repeated two times. (f) Model depicting that AOA treatment mimics Q deprivation causing G1 cell cycle arrest in K-Ras wild type cells and S- and G2/M-phase arrest in K-Ras mutant human cancer cell lines, which creates synthetic lethality to cell cycle phase-specific cytotoxic drugs causing apoptotic cell death.

Mentions: Glutamine, via anaplerotic entry to the TCA cycle, replenishes the intermediates lost by the exit of citrate from the mitochondria for fatty acid and cholesterol biosynthesis.27 Glutaminase catalyzes the deamination of Q to generate glutamate. Glutamate can then be converted to α-ketoglutarate by either glutamate dehydrogenase or transaminase. Kimmelman and colleagues recently reported that K-Ras-driven pancreatic cancer cells preferentially utilize the transaminase pathway for anaplerotic glutamine utilization.28 In the transaminase pathway, glutamate acts as an amino donor to oxaloacetate – a reaction catalyzed by glutamate oxaloacetate transaminase (GOT), which generates aspartate and α‐ketoglutarate (schematic shown in Figure 4a). Anaplerotic entry of Q into the TCA cycle can be inhibited by aminooxyacetate (AOA) – a pan-transaminase inhibitor, which inhibits GOT and consequently the entry of glutamine into the TCA cycle.29, 30 Treatment of both MCF-7 and MDA-MB-231 cells with AOA for 48 hr led to morphological changes similar to that observed with Q deprivation (Figure 4b). As seen in Figure 4c, AOA treatment caused G1 arrest in the MCF-7 cells and S- and G2/M-phase arrest in the MDA-MB-231 cells and also blocked proliferation (Figure 4d) as was observed with Q deprivation in Figure 1a and b – indicating that AOA mimics Q deprivation in both cell types. We also investigated whether the effect of AOA on cell cycle progression could be reversed by providing cell permeable analogues of α-ketoglutarate and aspartate – the products of the transamination reaction between glutamate and oxaloacetate. Dimethyl-α-ketoglutarate (DMKG) and β-methyl-aspartate (β-MD) were included along with AOA for the MDA-MB-231 cells. As shown in Figure 4e, neither compound by itself was able to completely reverse S- and G2/M arrest seen in the MDA-MB-231 cells, however the combination of both DMKG and β-MD did reverse the S- and G2/M arrest in these cells – indicating that generating both α-ketoglutarate and aspartate in the transaminase reaction is critical for passing through S- and G2/M phases. This finding is similar to that observed by Kimmelman and colleagues who showed a requirement for both α-ketoglutarate and non-essential amino acids (which included aspartate) for colony formation by pancreatic cancer cells. The need for aspartate as well as α-ketoglutarate indicates that the aspartate generated by the transaminase reaction is important. Kimmelman and colleagues28 demonstrated a critical need for conversion of aspartate → oxaloacetate, followed by conversion of oxaloacetate → malate, and then oxidative decarboxylation to pyruvate by malic enzyme in order to generate NADPH and maintain redox balance. However, a substantial amount of anaplerotic Q is converted to fatty acids.4 Thus, the aspartate generated by the transaminase reaction between glutamate and oxaloacetate is likely destined to re-enter the TCA cycle via conversion to oxaloacetate followed by condensation with acetyl-CoA to form citrate (see Figure 4a).


Blocking anaplerotic entry of glutamine into the TCA cycle sensitizes K-Ras mutant cancer cells to cytotoxic drugs.

Saqcena M, Mukhopadhyay S, Hosny C, Alhamed A, Chatterjee A, Foster DA - Oncogene (2014)

Pharmacological inhibition of Q anaplerosis sensitizes K-Ras mutant cancer cells to cytotoxic drugs. (a) Schematic overview of anaplerotic Q utilization. Q is deaminated to glutamate by glutaminase (GLS). Glutamate is then converted to α-ketoglutarate via transamination catalyzed by GOT, which uses oxaloacetate as the amino group acceptor to generate aspartate. AOA inhibits GOT and therefore suppresses generation of α‐ketoglutarate from Q-derived glutamate. Aspartate is critical for redox balance and NAPDH production and the generation of citrate for fatty acid synthesis. (b) Cells were plated at 20% confluence in 10-cm plates in complete medium (CM). After 24 hr, cells were shifted to CM, or medium lacking Q, or CM containing 0.5 mM AOA (C13408, Sigma-Aldrich) for 48 hr – at which time the cells were observed using phase-contrast microscopy. (c) MCF7 and MDA-MB-231 cells were plated and treated as in (b) for 48 hr, at which time cells were analyzed for cell cycle distribution as in Figure 1. In addition to AOA, the MDA-MB-231 cells were treated with cell permeable analogues of α-ketoglutarate (DMKG; 4 mM) (349631, Sigma-Aldrich) and aspartate (β-MD; 4 mM) (A8921, Sigma-Aldrich). Error bars represent standard error of mean for experiments repeated three times. (d) Cells were plated and treated as in (b), harvested at indicated time points, and scored after staining with crystal violet using light microscopy. Error bars represent the standard error of the mean for experiments repeated three times. (e) MCF7 and MDA-MB-231 cells were plated as in (b) and shifted to CM or treated with 0.5 mM AOA for 48 hr. The cells were additionally treated with 50 nM Pac or 1 μg/ml Cap for 24 hr, at which time the percentage non-viable cells were determined using trypan blue exclusion assay. Error bars represent the standard error of mean for experiments repeated three times. Cell lysates were also collected, and the levels of cleaved PARP (antibody from Cell Signaling) were determined by Western blot analysis. Data shown are representative of experiments repeated two times. (f) Model depicting that AOA treatment mimics Q deprivation causing G1 cell cycle arrest in K-Ras wild type cells and S- and G2/M-phase arrest in K-Ras mutant human cancer cell lines, which creates synthetic lethality to cell cycle phase-specific cytotoxic drugs causing apoptotic cell death.
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Related In: Results  -  Collection

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Figure 4: Pharmacological inhibition of Q anaplerosis sensitizes K-Ras mutant cancer cells to cytotoxic drugs. (a) Schematic overview of anaplerotic Q utilization. Q is deaminated to glutamate by glutaminase (GLS). Glutamate is then converted to α-ketoglutarate via transamination catalyzed by GOT, which uses oxaloacetate as the amino group acceptor to generate aspartate. AOA inhibits GOT and therefore suppresses generation of α‐ketoglutarate from Q-derived glutamate. Aspartate is critical for redox balance and NAPDH production and the generation of citrate for fatty acid synthesis. (b) Cells were plated at 20% confluence in 10-cm plates in complete medium (CM). After 24 hr, cells were shifted to CM, or medium lacking Q, or CM containing 0.5 mM AOA (C13408, Sigma-Aldrich) for 48 hr – at which time the cells were observed using phase-contrast microscopy. (c) MCF7 and MDA-MB-231 cells were plated and treated as in (b) for 48 hr, at which time cells were analyzed for cell cycle distribution as in Figure 1. In addition to AOA, the MDA-MB-231 cells were treated with cell permeable analogues of α-ketoglutarate (DMKG; 4 mM) (349631, Sigma-Aldrich) and aspartate (β-MD; 4 mM) (A8921, Sigma-Aldrich). Error bars represent standard error of mean for experiments repeated three times. (d) Cells were plated and treated as in (b), harvested at indicated time points, and scored after staining with crystal violet using light microscopy. Error bars represent the standard error of the mean for experiments repeated three times. (e) MCF7 and MDA-MB-231 cells were plated as in (b) and shifted to CM or treated with 0.5 mM AOA for 48 hr. The cells were additionally treated with 50 nM Pac or 1 μg/ml Cap for 24 hr, at which time the percentage non-viable cells were determined using trypan blue exclusion assay. Error bars represent the standard error of mean for experiments repeated three times. Cell lysates were also collected, and the levels of cleaved PARP (antibody from Cell Signaling) were determined by Western blot analysis. Data shown are representative of experiments repeated two times. (f) Model depicting that AOA treatment mimics Q deprivation causing G1 cell cycle arrest in K-Ras wild type cells and S- and G2/M-phase arrest in K-Ras mutant human cancer cell lines, which creates synthetic lethality to cell cycle phase-specific cytotoxic drugs causing apoptotic cell death.
Mentions: Glutamine, via anaplerotic entry to the TCA cycle, replenishes the intermediates lost by the exit of citrate from the mitochondria for fatty acid and cholesterol biosynthesis.27 Glutaminase catalyzes the deamination of Q to generate glutamate. Glutamate can then be converted to α-ketoglutarate by either glutamate dehydrogenase or transaminase. Kimmelman and colleagues recently reported that K-Ras-driven pancreatic cancer cells preferentially utilize the transaminase pathway for anaplerotic glutamine utilization.28 In the transaminase pathway, glutamate acts as an amino donor to oxaloacetate – a reaction catalyzed by glutamate oxaloacetate transaminase (GOT), which generates aspartate and α‐ketoglutarate (schematic shown in Figure 4a). Anaplerotic entry of Q into the TCA cycle can be inhibited by aminooxyacetate (AOA) – a pan-transaminase inhibitor, which inhibits GOT and consequently the entry of glutamine into the TCA cycle.29, 30 Treatment of both MCF-7 and MDA-MB-231 cells with AOA for 48 hr led to morphological changes similar to that observed with Q deprivation (Figure 4b). As seen in Figure 4c, AOA treatment caused G1 arrest in the MCF-7 cells and S- and G2/M-phase arrest in the MDA-MB-231 cells and also blocked proliferation (Figure 4d) as was observed with Q deprivation in Figure 1a and b – indicating that AOA mimics Q deprivation in both cell types. We also investigated whether the effect of AOA on cell cycle progression could be reversed by providing cell permeable analogues of α-ketoglutarate and aspartate – the products of the transamination reaction between glutamate and oxaloacetate. Dimethyl-α-ketoglutarate (DMKG) and β-methyl-aspartate (β-MD) were included along with AOA for the MDA-MB-231 cells. As shown in Figure 4e, neither compound by itself was able to completely reverse S- and G2/M arrest seen in the MDA-MB-231 cells, however the combination of both DMKG and β-MD did reverse the S- and G2/M arrest in these cells – indicating that generating both α-ketoglutarate and aspartate in the transaminase reaction is critical for passing through S- and G2/M phases. This finding is similar to that observed by Kimmelman and colleagues who showed a requirement for both α-ketoglutarate and non-essential amino acids (which included aspartate) for colony formation by pancreatic cancer cells. The need for aspartate as well as α-ketoglutarate indicates that the aspartate generated by the transaminase reaction is important. Kimmelman and colleagues28 demonstrated a critical need for conversion of aspartate → oxaloacetate, followed by conversion of oxaloacetate → malate, and then oxidative decarboxylation to pyruvate by malic enzyme in order to generate NADPH and maintain redox balance. However, a substantial amount of anaplerotic Q is converted to fatty acids.4 Thus, the aspartate generated by the transaminase reaction between glutamate and oxaloacetate is likely destined to re-enter the TCA cycle via conversion to oxaloacetate followed by condensation with acetyl-CoA to form citrate (see Figure 4a).

Bottom Line: One of the key shunts is the exit of citrate from the mitochondria and the TCA cycle for the generation of cytosolic acetyl-coenzyme A that can be used for fatty acid and cholesterol biosynthesis.Inhibition of K-Ras effector pathways was able to revert cells to G1 arrest upon Q deprivation.Significantly, Q deprivation or suppression of anaplerotic Q utilization created synthetic lethality to the cell cycle phase-specific cytotoxic drugs, capecitabine and paclitaxel.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Hunter College of the City University of New York, New York, NY, USA.

ABSTRACT
Cancer cells undergo a metabolic transformation that allows for increased anabolic demands, wherein glycolytic and tricarboxylic acid (TCA) cycle intermediates are shunted away for the synthesis of biological molecules required for cell growth and division. One of the key shunts is the exit of citrate from the mitochondria and the TCA cycle for the generation of cytosolic acetyl-coenzyme A that can be used for fatty acid and cholesterol biosynthesis. With the loss of mitochondrial citrate, cancer cells rely on the 'conditionally essential' amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates. Although Q deprivation causes G1 cell cycle arrest in non-transformed cells, its impact on the cancer cell cycle is not well characterized. We report here a correlation between bypass of the Q-dependent G1 checkpoint and cancer cells harboring K-Ras mutations. Instead of arresting in G1 in response to Q-deprivation, K-Ras-driven cancer cells arrest in either S- or G2/M-phase. Inhibition of K-Ras effector pathways was able to revert cells to G1 arrest upon Q deprivation. Blocking anaplerotic utilization of Q mimicked Q deprivation--causing S- and G2/M-phase arrest in K-Ras mutant cancer cells. Significantly, Q deprivation or suppression of anaplerotic Q utilization created synthetic lethality to the cell cycle phase-specific cytotoxic drugs, capecitabine and paclitaxel. These data suggest that disabling of the G1 Q checkpoint could represent a novel vulnerability of cancer cells harboring K-Ras and possibly other mutations that disable the Q-dependent checkpoint.

Show MeSH
Related in: MedlinePlus