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Decreased mitochondrial DNA mutagenesis in human colorectal cancer.

Ericson NG, Kulawiec M, Vermulst M, Sheahan K, O'Sullivan J, Salk JJ, Bielas JH - PLoS Genet. (2012)

Bottom Line: Remarkably, tumor tissue exhibited a decreased prevalence of these mutations relative to adjacent non-tumor tissue.The difference in mutation burden was attributable to a reduction in C:G to T:A transitions, which are associated with oxidative damage.We demonstrate that the lower random mutation frequency in tumor tissue was also coupled with a shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis, as compared to non-neoplastic colon.

View Article: PubMed Central - PubMed

Affiliation: Molecular Diagnostics Program, Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

ABSTRACT
Genome instability is regarded as a hallmark of cancer. Human tumors frequently carry clonally expanded mutations in their mitochondrial DNA (mtDNA), some of which may drive cancer progression and metastasis. The high prevalence of clonal mutations in tumor mtDNA has commonly led to the assumption that the mitochondrial genome in cancer is genetically unstable, yet this hypothesis has not been experimentally tested. In this study, we directly measured the frequency of non-clonal (random) de novo single base substitutions in the mtDNA of human colorectal cancers. Remarkably, tumor tissue exhibited a decreased prevalence of these mutations relative to adjacent non-tumor tissue. The difference in mutation burden was attributable to a reduction in C:G to T:A transitions, which are associated with oxidative damage. We demonstrate that the lower random mutation frequency in tumor tissue was also coupled with a shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis, as compared to non-neoplastic colon. Together these findings raise the intriguing possibility that fidelity of mitochondrial genome is, in fact, increased in cancer as a result of a decrease in reactive oxygen species-mediated mtDNA damage.

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Metabolic Shift in Human Colorectal Cancer.(A) Western blot analysis of the expression levels of markers of oxidative phosphorylation (ß-F1-ATPase), structural function of the mitochondria (Hsp60), and the glycolytic pathway [GAPDH and pyruvate kinase (PK)], fractionated by SDS-PAGE and blotted with the corresponding antibodies from eight patient-matched normal (N) and tumor (T) human colorectal tissues. (B) The comparative cellular content of each glycolytic protein marker relative to the expression of tubulin in normal (blue) and tumor (red) tissue. (C) The bioenergetic competence of the mitochondria (ß-F1-ATPase/Hsp60 ratio) from tissues, and (D) overall mitochondrial potential of the cell, defined as the BioEnergetic Cellular Index (BEC index). BEC index is assessed by the ß-F1-ATPase/Hsp60/GAPDH ratio, providing a normalized proteomic evaluation of the metabolic shift in colorectal tumors. (E) Gas chromatography/mass spectrometry (GC/MS) metabolite analysis revealing significantly higher levels of lactate and lower levels of citrate in cancer tissues when compared to patient-matched normal colorectal tissue controls. Decreased citrate in tumors indicates reduced flux through the tricarboxylic acid cycle. Coupled with increased lactate levels, these metabolic alterations are consistent with a shift in glucose metabolism from oxidative phosphorylation (OXPHOS) to glycolysis among tumors. Box and whisker plots depict the median, distribution, and data range. The median is indicated by the black line, the box shows the interquartile range, and the ends of the whiskers the maxima and minima. * P<0.05; ** P<0.01.
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pgen-1002689-g004: Metabolic Shift in Human Colorectal Cancer.(A) Western blot analysis of the expression levels of markers of oxidative phosphorylation (ß-F1-ATPase), structural function of the mitochondria (Hsp60), and the glycolytic pathway [GAPDH and pyruvate kinase (PK)], fractionated by SDS-PAGE and blotted with the corresponding antibodies from eight patient-matched normal (N) and tumor (T) human colorectal tissues. (B) The comparative cellular content of each glycolytic protein marker relative to the expression of tubulin in normal (blue) and tumor (red) tissue. (C) The bioenergetic competence of the mitochondria (ß-F1-ATPase/Hsp60 ratio) from tissues, and (D) overall mitochondrial potential of the cell, defined as the BioEnergetic Cellular Index (BEC index). BEC index is assessed by the ß-F1-ATPase/Hsp60/GAPDH ratio, providing a normalized proteomic evaluation of the metabolic shift in colorectal tumors. (E) Gas chromatography/mass spectrometry (GC/MS) metabolite analysis revealing significantly higher levels of lactate and lower levels of citrate in cancer tissues when compared to patient-matched normal colorectal tissue controls. Decreased citrate in tumors indicates reduced flux through the tricarboxylic acid cycle. Coupled with increased lactate levels, these metabolic alterations are consistent with a shift in glucose metabolism from oxidative phosphorylation (OXPHOS) to glycolysis among tumors. Box and whisker plots depict the median, distribution, and data range. The median is indicated by the black line, the box shows the interquartile range, and the ends of the whiskers the maxima and minima. * P<0.05; ** P<0.01.

Mentions: One of the biochemical hallmarks of tumor cells involves the reprogramming of energy metabolism from primarily oxidative phosphorylation (OXPHOS) to anaerobic glycolysis [20], a phenomenon termed the Warburg effect [21]. This transition effectively decreases OXPHOS, and by extension, the production of reactive oxygen species (ROS) in the mitochondrial matrix, which have the potential to damage mtDNA [22]. To investigate whether the switch between oxidative phosphorylation and glycolysis is associated with a change in mtDNA mutation frequency, we analyzed the relative expression of protein markers for glycolysis and oxidative phosphorylation (Figure 4A). Consistent with an upregulation of glycolysis, we observed a significant increase in the glycolytic markers pyruvate kinase (PK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [23] in tumors relative to normal colon (Figure 4B). Moreover, as assessed by the abundance of the catalytic subunit OXPHOS marker ATP synthase (ß-F1-ATPase) [23], [24] normalized to the structural mitochondrial protein Hsp60, mitochondrial bioenergetic competence (i.e. OXPHOS) [25], [26], [27] is significantly decreased in colon carcinoma compared to normal controls (Figure 4C) (P<0.05, 2-tailed paired t-test). As such, the BioEnergetic Cellular (BEC) index, which gives the ratio of OXPHOS protein content to glycolytic protein content [26], [27], is reduced and consistent with a shift to Warburg metabolism in tumor tissue (Figure 4D) (P<0.05, 2-tailed paired t-test). In addition, to generate an instantaneous snapshot of the competing forms of glucose metabolism, we directly measured the relative amount of citrate and lactate in colorectal tissues by gas chromatography/mass spectrometry (GC/MS). We found that citrate, a tricarboxylic acid (TCA) cycle intermediate that correlates tightly with the level of mitochondrial respiration [28], [29], was significantly reduced in carcinomas relative to normal colon (Figure 4E) (P<0.01, 2-tailed paired t-test), whereas the level of lactate, the end product of anaerobic glycolysis [30], was increased (Figure 4E) (P<0.05, 2-tailed paired t-test).


Decreased mitochondrial DNA mutagenesis in human colorectal cancer.

Ericson NG, Kulawiec M, Vermulst M, Sheahan K, O'Sullivan J, Salk JJ, Bielas JH - PLoS Genet. (2012)

Metabolic Shift in Human Colorectal Cancer.(A) Western blot analysis of the expression levels of markers of oxidative phosphorylation (ß-F1-ATPase), structural function of the mitochondria (Hsp60), and the glycolytic pathway [GAPDH and pyruvate kinase (PK)], fractionated by SDS-PAGE and blotted with the corresponding antibodies from eight patient-matched normal (N) and tumor (T) human colorectal tissues. (B) The comparative cellular content of each glycolytic protein marker relative to the expression of tubulin in normal (blue) and tumor (red) tissue. (C) The bioenergetic competence of the mitochondria (ß-F1-ATPase/Hsp60 ratio) from tissues, and (D) overall mitochondrial potential of the cell, defined as the BioEnergetic Cellular Index (BEC index). BEC index is assessed by the ß-F1-ATPase/Hsp60/GAPDH ratio, providing a normalized proteomic evaluation of the metabolic shift in colorectal tumors. (E) Gas chromatography/mass spectrometry (GC/MS) metabolite analysis revealing significantly higher levels of lactate and lower levels of citrate in cancer tissues when compared to patient-matched normal colorectal tissue controls. Decreased citrate in tumors indicates reduced flux through the tricarboxylic acid cycle. Coupled with increased lactate levels, these metabolic alterations are consistent with a shift in glucose metabolism from oxidative phosphorylation (OXPHOS) to glycolysis among tumors. Box and whisker plots depict the median, distribution, and data range. The median is indicated by the black line, the box shows the interquartile range, and the ends of the whiskers the maxima and minima. * P<0.05; ** P<0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3369930&req=5

pgen-1002689-g004: Metabolic Shift in Human Colorectal Cancer.(A) Western blot analysis of the expression levels of markers of oxidative phosphorylation (ß-F1-ATPase), structural function of the mitochondria (Hsp60), and the glycolytic pathway [GAPDH and pyruvate kinase (PK)], fractionated by SDS-PAGE and blotted with the corresponding antibodies from eight patient-matched normal (N) and tumor (T) human colorectal tissues. (B) The comparative cellular content of each glycolytic protein marker relative to the expression of tubulin in normal (blue) and tumor (red) tissue. (C) The bioenergetic competence of the mitochondria (ß-F1-ATPase/Hsp60 ratio) from tissues, and (D) overall mitochondrial potential of the cell, defined as the BioEnergetic Cellular Index (BEC index). BEC index is assessed by the ß-F1-ATPase/Hsp60/GAPDH ratio, providing a normalized proteomic evaluation of the metabolic shift in colorectal tumors. (E) Gas chromatography/mass spectrometry (GC/MS) metabolite analysis revealing significantly higher levels of lactate and lower levels of citrate in cancer tissues when compared to patient-matched normal colorectal tissue controls. Decreased citrate in tumors indicates reduced flux through the tricarboxylic acid cycle. Coupled with increased lactate levels, these metabolic alterations are consistent with a shift in glucose metabolism from oxidative phosphorylation (OXPHOS) to glycolysis among tumors. Box and whisker plots depict the median, distribution, and data range. The median is indicated by the black line, the box shows the interquartile range, and the ends of the whiskers the maxima and minima. * P<0.05; ** P<0.01.
Mentions: One of the biochemical hallmarks of tumor cells involves the reprogramming of energy metabolism from primarily oxidative phosphorylation (OXPHOS) to anaerobic glycolysis [20], a phenomenon termed the Warburg effect [21]. This transition effectively decreases OXPHOS, and by extension, the production of reactive oxygen species (ROS) in the mitochondrial matrix, which have the potential to damage mtDNA [22]. To investigate whether the switch between oxidative phosphorylation and glycolysis is associated with a change in mtDNA mutation frequency, we analyzed the relative expression of protein markers for glycolysis and oxidative phosphorylation (Figure 4A). Consistent with an upregulation of glycolysis, we observed a significant increase in the glycolytic markers pyruvate kinase (PK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [23] in tumors relative to normal colon (Figure 4B). Moreover, as assessed by the abundance of the catalytic subunit OXPHOS marker ATP synthase (ß-F1-ATPase) [23], [24] normalized to the structural mitochondrial protein Hsp60, mitochondrial bioenergetic competence (i.e. OXPHOS) [25], [26], [27] is significantly decreased in colon carcinoma compared to normal controls (Figure 4C) (P<0.05, 2-tailed paired t-test). As such, the BioEnergetic Cellular (BEC) index, which gives the ratio of OXPHOS protein content to glycolytic protein content [26], [27], is reduced and consistent with a shift to Warburg metabolism in tumor tissue (Figure 4D) (P<0.05, 2-tailed paired t-test). In addition, to generate an instantaneous snapshot of the competing forms of glucose metabolism, we directly measured the relative amount of citrate and lactate in colorectal tissues by gas chromatography/mass spectrometry (GC/MS). We found that citrate, a tricarboxylic acid (TCA) cycle intermediate that correlates tightly with the level of mitochondrial respiration [28], [29], was significantly reduced in carcinomas relative to normal colon (Figure 4E) (P<0.01, 2-tailed paired t-test), whereas the level of lactate, the end product of anaerobic glycolysis [30], was increased (Figure 4E) (P<0.05, 2-tailed paired t-test).

Bottom Line: Remarkably, tumor tissue exhibited a decreased prevalence of these mutations relative to adjacent non-tumor tissue.The difference in mutation burden was attributable to a reduction in C:G to T:A transitions, which are associated with oxidative damage.We demonstrate that the lower random mutation frequency in tumor tissue was also coupled with a shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis, as compared to non-neoplastic colon.

View Article: PubMed Central - PubMed

Affiliation: Molecular Diagnostics Program, Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

ABSTRACT
Genome instability is regarded as a hallmark of cancer. Human tumors frequently carry clonally expanded mutations in their mitochondrial DNA (mtDNA), some of which may drive cancer progression and metastasis. The high prevalence of clonal mutations in tumor mtDNA has commonly led to the assumption that the mitochondrial genome in cancer is genetically unstable, yet this hypothesis has not been experimentally tested. In this study, we directly measured the frequency of non-clonal (random) de novo single base substitutions in the mtDNA of human colorectal cancers. Remarkably, tumor tissue exhibited a decreased prevalence of these mutations relative to adjacent non-tumor tissue. The difference in mutation burden was attributable to a reduction in C:G to T:A transitions, which are associated with oxidative damage. We demonstrate that the lower random mutation frequency in tumor tissue was also coupled with a shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis, as compared to non-neoplastic colon. Together these findings raise the intriguing possibility that fidelity of mitochondrial genome is, in fact, increased in cancer as a result of a decrease in reactive oxygen species-mediated mtDNA damage.

Show MeSH
Related in: MedlinePlus