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Oxidative stress inhibits distant metastasis by human melanoma cells.

Piskounova E, Agathocleous M, Murphy MM, Hu Z, Huddlestun SE, Zhao Z, Leitch AM, Johnson TM, DeBerardinis RJ, Morrison SJ - Nature (2015)

Bottom Line: We show that melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficiently metastasizing melanomas.Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours.Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence on NADPH-generating enzymes in the folate pathway.

View Article: PubMed Central - PubMed

Affiliation: Children's Research Institute and the Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

ABSTRACT
Solid cancer cells commonly enter the blood and disseminate systemically, but are highly inefficient at forming distant metastases for poorly understood reasons. Here we studied human melanomas that differed in their metastasis histories in patients and in their capacity to metastasize in NOD-SCID-Il2rg(-/-) (NSG) mice. We show that melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficiently metastasizing melanomas. Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours. Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence on NADPH-generating enzymes in the folate pathway. Antioxidants promoted distant metastasis in NSG mice. Folate pathway inhibition using low-dose methotrexate, ALDH1L2 knockdown, or MTHFD1 knockdown inhibited distant metastasis without significantly affecting the growth of subcutaneous tumours in the same mice. Oxidative stress thus limits distant metastasis by melanoma cells in vivo.

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Metastasizing melanoma cells experience high levels of oxidative stressa) GSH/GSSG ratio in subcutaneous tumours as compared to metastatic nodules (n=15 mice from 2 independent experiment with 3 melanomas, M481, M405, UT10; note that extractions were performed with 0.1% formic acid to prevent spontaneous oxidation47). Total amounts of GSH and GSSG are shown in Extended data Figure 5h and 5i. b) GSH/GSSG ratio in subcutaneous tumours as compared to circulating melanoma cells (n=7 mice from 3 independent experiments with 2 melanomas, M405 and UT10; these were different experiments than those in panel a, performed under different technical conditions). c, d) cytoplasmic (c) and mitochondrial (d) ROS levels in dissociated melanoma cells from subcutaneous tumours, the blood, and metastatic nodules obtained from the same mice (n=9 mice from 3 independent experiments using 3 different melanomas). e, f) Mitochondrial mass (e) and mitochondrial membrane potential (f) in dissociated melanoma cells from subcutaneous tumours, the blood, and metastatic nodules obtained from the same mice (n=6 mice from 2 independent experiments using 3 different melanomas). g) Melanoma cells underwent reversible changes in mitochondrial mass during metastasis: mitochondrial mass in dissociated melanoma cells from subcutaneous tumours versus metastatic nodules obtained from the same mice transplanted with subcutaneous, circulating, or metastatic melanoma cells. All data represent mean±sd. Statistical significance was assessed using two-tailed Student’s t-tests (a and b) and one-way analyses of variance (ANOVAs) followed by Dunnett’s tests for multiple comparisons (c–g; *, p<0.05; ***, p< 0.0005; ****, p<0.00005).
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Figure 1: Metastasizing melanoma cells experience high levels of oxidative stressa) GSH/GSSG ratio in subcutaneous tumours as compared to metastatic nodules (n=15 mice from 2 independent experiment with 3 melanomas, M481, M405, UT10; note that extractions were performed with 0.1% formic acid to prevent spontaneous oxidation47). Total amounts of GSH and GSSG are shown in Extended data Figure 5h and 5i. b) GSH/GSSG ratio in subcutaneous tumours as compared to circulating melanoma cells (n=7 mice from 3 independent experiments with 2 melanomas, M405 and UT10; these were different experiments than those in panel a, performed under different technical conditions). c, d) cytoplasmic (c) and mitochondrial (d) ROS levels in dissociated melanoma cells from subcutaneous tumours, the blood, and metastatic nodules obtained from the same mice (n=9 mice from 3 independent experiments using 3 different melanomas). e, f) Mitochondrial mass (e) and mitochondrial membrane potential (f) in dissociated melanoma cells from subcutaneous tumours, the blood, and metastatic nodules obtained from the same mice (n=6 mice from 2 independent experiments using 3 different melanomas). g) Melanoma cells underwent reversible changes in mitochondrial mass during metastasis: mitochondrial mass in dissociated melanoma cells from subcutaneous tumours versus metastatic nodules obtained from the same mice transplanted with subcutaneous, circulating, or metastatic melanoma cells. All data represent mean±sd. Statistical significance was assessed using two-tailed Student’s t-tests (a and b) and one-way analyses of variance (ANOVAs) followed by Dunnett’s tests for multiple comparisons (c–g; *, p<0.05; ***, p< 0.0005; ****, p<0.00005).

Mentions: In a total of six independent experiments that involved four different technical approaches, the glutathione (GSH) to oxidized glutathione (GSSG) ratio was always significantly higher in subcutaneous tumours as compared to metastatic nodules or circulating melanoma cells (Figure 1a and 1b and Extended data figure 4f). This was true irrespective of whether melanoma cells were isolated by dissection or by flow cytometry (to eliminate stromal cells). The lower GSH/GSSG ratio in circulating melanoma cells and metastatic nodules suggested that metastasizing cells experienced oxidative stress not observed in established subcutaneous tumours, and they consumed GSH in an effort to maintain redox homeostasis.


Oxidative stress inhibits distant metastasis by human melanoma cells.

Piskounova E, Agathocleous M, Murphy MM, Hu Z, Huddlestun SE, Zhao Z, Leitch AM, Johnson TM, DeBerardinis RJ, Morrison SJ - Nature (2015)

Metastasizing melanoma cells experience high levels of oxidative stressa) GSH/GSSG ratio in subcutaneous tumours as compared to metastatic nodules (n=15 mice from 2 independent experiment with 3 melanomas, M481, M405, UT10; note that extractions were performed with 0.1% formic acid to prevent spontaneous oxidation47). Total amounts of GSH and GSSG are shown in Extended data Figure 5h and 5i. b) GSH/GSSG ratio in subcutaneous tumours as compared to circulating melanoma cells (n=7 mice from 3 independent experiments with 2 melanomas, M405 and UT10; these were different experiments than those in panel a, performed under different technical conditions). c, d) cytoplasmic (c) and mitochondrial (d) ROS levels in dissociated melanoma cells from subcutaneous tumours, the blood, and metastatic nodules obtained from the same mice (n=9 mice from 3 independent experiments using 3 different melanomas). e, f) Mitochondrial mass (e) and mitochondrial membrane potential (f) in dissociated melanoma cells from subcutaneous tumours, the blood, and metastatic nodules obtained from the same mice (n=6 mice from 2 independent experiments using 3 different melanomas). g) Melanoma cells underwent reversible changes in mitochondrial mass during metastasis: mitochondrial mass in dissociated melanoma cells from subcutaneous tumours versus metastatic nodules obtained from the same mice transplanted with subcutaneous, circulating, or metastatic melanoma cells. All data represent mean±sd. Statistical significance was assessed using two-tailed Student’s t-tests (a and b) and one-way analyses of variance (ANOVAs) followed by Dunnett’s tests for multiple comparisons (c–g; *, p<0.05; ***, p< 0.0005; ****, p<0.00005).
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Figure 1: Metastasizing melanoma cells experience high levels of oxidative stressa) GSH/GSSG ratio in subcutaneous tumours as compared to metastatic nodules (n=15 mice from 2 independent experiment with 3 melanomas, M481, M405, UT10; note that extractions were performed with 0.1% formic acid to prevent spontaneous oxidation47). Total amounts of GSH and GSSG are shown in Extended data Figure 5h and 5i. b) GSH/GSSG ratio in subcutaneous tumours as compared to circulating melanoma cells (n=7 mice from 3 independent experiments with 2 melanomas, M405 and UT10; these were different experiments than those in panel a, performed under different technical conditions). c, d) cytoplasmic (c) and mitochondrial (d) ROS levels in dissociated melanoma cells from subcutaneous tumours, the blood, and metastatic nodules obtained from the same mice (n=9 mice from 3 independent experiments using 3 different melanomas). e, f) Mitochondrial mass (e) and mitochondrial membrane potential (f) in dissociated melanoma cells from subcutaneous tumours, the blood, and metastatic nodules obtained from the same mice (n=6 mice from 2 independent experiments using 3 different melanomas). g) Melanoma cells underwent reversible changes in mitochondrial mass during metastasis: mitochondrial mass in dissociated melanoma cells from subcutaneous tumours versus metastatic nodules obtained from the same mice transplanted with subcutaneous, circulating, or metastatic melanoma cells. All data represent mean±sd. Statistical significance was assessed using two-tailed Student’s t-tests (a and b) and one-way analyses of variance (ANOVAs) followed by Dunnett’s tests for multiple comparisons (c–g; *, p<0.05; ***, p< 0.0005; ****, p<0.00005).
Mentions: In a total of six independent experiments that involved four different technical approaches, the glutathione (GSH) to oxidized glutathione (GSSG) ratio was always significantly higher in subcutaneous tumours as compared to metastatic nodules or circulating melanoma cells (Figure 1a and 1b and Extended data figure 4f). This was true irrespective of whether melanoma cells were isolated by dissection or by flow cytometry (to eliminate stromal cells). The lower GSH/GSSG ratio in circulating melanoma cells and metastatic nodules suggested that metastasizing cells experienced oxidative stress not observed in established subcutaneous tumours, and they consumed GSH in an effort to maintain redox homeostasis.

Bottom Line: We show that melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficiently metastasizing melanomas.Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours.Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence on NADPH-generating enzymes in the folate pathway.

View Article: PubMed Central - PubMed

Affiliation: Children's Research Institute and the Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

ABSTRACT
Solid cancer cells commonly enter the blood and disseminate systemically, but are highly inefficient at forming distant metastases for poorly understood reasons. Here we studied human melanomas that differed in their metastasis histories in patients and in their capacity to metastasize in NOD-SCID-Il2rg(-/-) (NSG) mice. We show that melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficiently metastasizing melanomas. Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours. Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence on NADPH-generating enzymes in the folate pathway. Antioxidants promoted distant metastasis in NSG mice. Folate pathway inhibition using low-dose methotrexate, ALDH1L2 knockdown, or MTHFD1 knockdown inhibited distant metastasis without significantly affecting the growth of subcutaneous tumours in the same mice. Oxidative stress thus limits distant metastasis by melanoma cells in vivo.

Show MeSH
Related in: MedlinePlus