Clonal mutations represent identical mutant mtDNA molecules that are present in the majority of genomes within a cell population. Such mutations occur via propagation of the genotype of a single mutant genome in a single founder cell to all cellular descendants during clonal proliferation. In a tumor, clonal mutations reflect the genotype of the founding cell of the terminal clonal outgrowth. Random mutations, in contrast, are mutations that arise in cell divisions after the founding of a clonal population and are present in only a subset of cells. The frequency of random mutations in a population is proportional to the rate of mutation and dependent on the number of cell divisions having led to the generation of the sampled population.
First, to stratify our colorectal tissue samples with respect to the abundance of clonally expanded mutations, we sequenced the entire mitochondrial genome of each of our samples. We found that 55% (11 of 20) of the carcinomas carried at least one clonally expanded mutation in their mtDNA (). Furthermore, when located inside a protein-coding gene, the mutations identified in our tumors uniformly resulted in frameshift mutations (2/13) or non-synonymous changes (11/13). Although the observed frequency of non-synonymous point mutations (11/11) exceeded that expected by chance, the difference did not reach significance in our sample set (P
0.06, 2-tailed chi-square test, n
11). To assess whether the expansion of mtDNA mutations is an early or a late event during carcinogenesis, we also sequenced the entire mitochondrial genome of 19 patient-matched adenomas. We found that 32% (6 of 19) of the adenomas carried clonally expanded mutations (), revealing that clonal expansion of mtDNA mutations can occur prior to overt malignancy.
Clonal Mitochondrial DNA Mutations Identified in Colorectal Tissue.
Next, we quantified the frequency of random mutations in colorectal tissue using the RMC assay. We found that in normal colorectal tissue, mutations occurred at an average absolute frequency of 3.2±0.5×10−5
per base pair at a site in the 12S rRNA subunit (), and 1.2±0.2×10−4
at a second site in the COXI gene (). At both sites, mutation frequency was independent of patient age (). Unexpectedly, when we examined patient-matched colorectal carcinomas we found that, on average, tumor cells displayed an approximately three-fold lower random mutation frequency compared to normal colonic tissue (1.4±0.4×10−5
0.004 for 12S rRNA, 4.1±1.2×10−5
, P<0.001 for COXI, 2 tailed paired t-test) (). No appreciable difference in mtDNA copy number was observed between the tissues (). These mtDNA findings are in stark contrast to previous observations in which we demonstrated a>100-fold increase in the frequency of random mutations in the nuclear DNA of human tumors 
Decreased Random Mitochondrial DNA Mutations in Colorectal Cancer.
Random Mitochondrial DNA Mutations and Genome Copy Number in Colorectal Tissue.
of random mutations within a tissue is proportional to both the rate
of de novo
mutations per generation and the total number
of cell divisions having occurred during development and aging. The observed decrease in mutation frequency within tumors could, thus, either be the result of a reduced mutation rate during tumor growth, or simply a consequence of the fact that the number of cell divisions having occurred since founding of the final clonal tumor outgrowth may be fewer than the number having occurred between development and sampling of normal adult colon tissue 
. Genetic bottlenecking during the clonal formation of a carcinoma from a single founder cell effectively purges mutational diversity that has accumulated in normal colonic mucosa over a lifetime by repopulating it with closely related progeny 
To assess whether clonal expansion alone might account for the decreased mutation load associated with tumor mtDNA, we quantified the frequency of random mutations in adenoma tissues. Advanced adenomas are generally believed to be clonally derived 
, and the presence of clonal mtDNA mutations empirically demonstrates this to be true in at least 32% of our samples (). Given this, if clonal expansion alone were to underlie the decrease in random mutation burden, we would expect the frequency of random mutations in adenoma mtDNA (4.9±2.3×10−5
per base pair for 12S RNA, and 1.7±0.6×10−4
for the COXI gene) to be lower than that of patient-matched normal colon mtDNA (3.2±0.5×10−5
per base for 12S rRNA and 1.2±0.2×10−4
at the COXI gene), but this was not the case () (P
0.73 for 12S RNA and P
0.15 for the COXI gene, 2-tailed paired t-test). Moreover, the decrease in the level of genetic heterogeneity in tumors was independent of whether or not the cancers bore a separate clonal mutation (). Thus, this leaves the possibility of a reduction in mtDNA mutation rate during late tumorigenesis.
To gain further insight into possible mechanisms responsible for reducing mtDNA mutagenesis in tumor cells, we examined the mutation spectrum of 796 random mutation events (297 normal, 275 adenoma, 224 carcinoma). We found that in normal and adenoma tissues, C
G to T
A transitions predominated (). Similar spectra have been previously reported in mice and are consistent with deamination of cytosine bases as the primary source of mutagenesis 
, a process that is driven by oxidative damage 
. While all tissue types exhibited similar levels of T
A to C
G mutations, there was >3-fold decrease in C
G to T
A transitions among the tumor samples relative to normal colon, suggesting the source of these latter oxidatively-mediated mutations to be specifically reduced during carcinoma outgrowth. Interestingly, the majority of the clonally expanded mutations observed in carcinomas are C
G to T
A transitions (), which is similar to that previously reported in human colorectal tumors 
and to the spectrum of random mutations in normal and adenoma tissue (), but is markedly different from the spectrum of mutations generated by polymerase γ on undamaged template 
. This suggests that the biological change responsible for reducing the frequency of C
G to T
A mutations occurs after the initiation of neoplastic clonal expansion.
Figure 3 Decreased mtDNA Mutagenesis in Colorectal Carcinoma Is Attributable to a Reduction in CG to TA Transitions.
One of the biochemical hallmarks of tumor cells involves the reprogramming of energy metabolism from primarily oxidative phosphorylation (OXPHOS) to anaerobic glycolysis 
, a phenomenon termed the Warburg effect 
. 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 
. 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 (). Consistent with an upregulation of glycolysis, we observed a significant increase in the glycolytic markers pyruvate kinase (PK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 
in tumors relative to normal colon (). Moreover, as assessed by the abundance of the catalytic subunit OXPHOS marker ATP synthase (ß-F1-ATPase) 
normalized to the structural mitochondrial protein Hsp60, mitochondrial bioenergetic competence (i.e. OXPHOS) 
is significantly decreased in colon carcinoma compared to normal controls () (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 
, is reduced and consistent with a shift to Warburg metabolism in tumor tissue () (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 
, was significantly reduced in carcinomas relative to normal colon () (P<0.01, 2-tailed paired t-test), whereas the level of lactate, the end product of anaerobic glycolysis 
, was increased () (P<0.05, 2-tailed paired t-test).
Metabolic Shift in Human Colorectal Cancer.
Finally, to examine the relationship between energy metabolism and mtDNA mutation frequency, we plotted the random mutation frequency of all samples against the ratio of citrate to lactate (). We found that in both normal and tumor tissue, mutation frequency decreases concomitantly with reduced mitochondrial respiration (linear regression: slope −10.17±0.9894, significance of non-zero slope P<0.0001, R2
0.71). This is consistent with the hypothesis that a large fraction of mtDNA mutagenesis is a consequence of oxidative damage generated as a byproduct during OXPHOS and provides a plausible rationale for the decreased frequency of random mutations in tumor cells–specifically, a decrease in C
G to T
A transitions (), the most commonly observed mutations resulting from oxidative damage 
Decreased mtDNA Mutagenesis Is Coupled to a Shift in Glucose Metabolism.