Rewiring of energy metabolism and adaptation of mitochondria are considered to impact on prostate cancer development and progression. Here, we report on mitochondrial respiration, DNA mutations and gene expression in paired benign/malignant human prostate tissue samples. Results reveal reduced respiratory capacities with NADH-pathway substrates glutamate and malate in malignant tissue and a significant metabolic shift towards higher succinate oxidation, particularly in high-grade tumors. The load of potentially deleterious mitochondrial-DNA mutations is higher in tumors and associated with unfavorable risk factors. High levels of potentially deleterious mutations in mitochondrial Complex I-encoding genes are associated with a 70% reduction in NADH-pathway capacity and compensation by increased succinate-pathway capacity. Structural analyses of these mutations reveal amino acid alterations leading to potentially deleterious effects on Complex I, supporting a causal relationship. A metagene signature extracted from the transcriptome of tumor samples exhibiting a severe mitochondrial phenotype enables identification of tumors with shorter survival times.

Alterations of mitochondrial DNA (mtDNA) copy number have been associated with a wide variety of phenotypes and diseases. Unfortunately, the literature provides scarce methodical information about duplex targeting of nuclear and mtDNA that meets the quality criteria for qPCR. Therefore, we established a method for mtDNA copy number quantification using a quantitative PCR assay that allows for simultaneous targeting of a single copy nuclear gene (beta-2-microglobulin) and the t-RNALeu gene on the mtDNA. We include a plasmid containing both targets in order to normalize against differences in emission intensities of the fluorescent dyes Yakima Yellow and FAM. Applying the plasmid calibrator on an internal control reduced the intra-assay variability from 21% (uncorrected) to 7% (plasmid-corrected). Moreover, we noted that DNA samples isolated with different methods revealed different numbers of mtDNA copies, thus highlighting an important influence of the pre-analytical procedures. In summary, we developed a precise assay for mitochondrial copy number detection relative to nuclear DNA. Our method is applicable to comparative mitochondrial DNA copy number studies since the use of the dual insert plasmid allows correcting for the unequal emission intensities of the different fluorescent labels of the two targets.

Association of mitochondrial DNA copy number with metabolic syndrome and type 2 diabetes in 14 176 individuals (Original Article).