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. 2012;8(6):e1002689.
doi: 10.1371/journal.pgen.1002689. Epub 2012 Jun 7.

Decreased mitochondrial DNA mutagenesis in human colorectal cancer

Nolan G Ericson  1 , Mariola Kulawiec, Marc Vermulst, Kieran Sheahan, Jacintha O'Sullivan, Jesse J Salk, Jason H Bielas
Affiliations

Decreased mitochondrial DNA mutagenesis in human colorectal cancer

Nolan G Ericson et al. PLoS Genet. 2012.

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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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Decreased Random Mitochondrial DNA Mutations in Colorectal Cancer.
(A) Mutation frequency (± s.e.m.) was determined at TaqI restriction sites 1215–1218 within the 12S rRNA gene and (B) 7335–7338 within the COXI gene in mitochondrial DNA isolated from patient-matched normal (blue) and carcinoma (red) colorectal tissues. The mean (n = 20) mutation burden (± s.e.m.) of mtDNA isolated from carcinoma versus normal tissue is reduced ∼3-fold at both mutational target sites. * P<0.01; ** P<0.001; two-tailed paired t-test.
Figure 2
Figure 2. Random Mitochondrial DNA Mutations and Genome Copy Number in Colorectal Tissue.
(A) Frequency of mitochondrial mutation as a function of age. Mutation frequency was determined at the 12S rRNA gene (blue) and COXI gene (red) in mtDNA isolated from normal human colorectal tissue. Each data point represents one patient. (B) Mitochondrial genome content (± s.e.m.) in normal and tumor tissue. (C) Average frequency of random mitochondrial mutations in colorectal tissue. Mean (± s.e.m.) mutation frequency within the 12S rRNA gene and COXI gene in mtDNA isolated from patient-matched normal (n = 20), adenoma (n = 8), and carcinoma (n = 20) colorectal tissues. (D) Patient-matched comparison of the mean (± s.e.m.) mtDNA random mutation frequency as stratified by carcinomas that harbored one or more clonal mutation.
Figure 3
Figure 3. Decreased mtDNA Mutagenesis in Colorectal Carcinoma Is Attributable to a Reduction in C∶G to T∶A Transitions.
Mitochondrial DNA mutation spectrum (± s.e.m.) per Mb in human colorectal tissue. Mutant mtDNA amplicons (n = 796) spanning two restriction sites (1215–1218 and 7335–7338) were recovered from the RMC assay and subjected to DNA sequencing to generate the mutational signature of mtDNA isolated from normal (n = 297), adenoma (n = 275), and carcinoma (n = 224) colorectal tissues. * P<0.0001; two-tailed paired t-test.
Figure 4
Figure 4. 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.
Figure 5
Figure 5. Decreased mtDNA Mutagenesis Is Coupled to a Shift in Glucose Metabolism.
Normal (blue) and tumor (red) patient-matched colorectal tissue comparison of the mean mtDNA mutation burden (± s.e.m.) as a function of the tissue metabolic signature. This plot illustrates the inverse correlation between the level of mitochondrial respiration and mutagenesis (linear regression with 95% confidence intervals, slope −10.17±0.9894, significance of non-zero slope P<0.0001, R2 = 0.71).
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