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.

Massive parallel sequencing technologies are promising a highly sensitive detection of low-level mutations, especially in mitochondrial DNA (mtDNA) studies. However, processes from DNA extraction and library construction to bioinformatic analysis include several varying tasks. Further, there is no validated recommendation for the comprehensive procedure. In this study, we examined potential pitfalls on the sequencing results based on two-person mtDNA mixtures. Therefore, we compared three DNA polymerases, six different variant callers in five mixtures between 50% and 0.5% variant allele frequencies generated with two different amplification protocols. In total, 48 samples were sequenced on Illumina MiSeq. Low-level variant calling at the 1% variant level and below was performed by comparing trimming and PCR duplicate removal as well as six different variant callers. The results indicate that sensitivity, specificity, and precision highly depend on the investigated polymerase but also vary based on the analysis tools. Our data highlight the advantage of prior standardization and validation of the individual laboratory setup with a DNA mixture model. Finally, we provide an artificial heteroplasmy benchmark dataset that can help improve somatic variant callers or pipelines, which may be of great interest for research related to cancer and aging.

Under aerobic conditions, some cancers switch to glycolysis to cover their energy requirements. Taking advantage of this process, functional imaging techniques such as PET-CT can be used to detect and assess tumorous tissues. The aim of this study was to investigate standardized uptake values and mitochondrial DNA mutations in oral squamous cell carcinoma. A cohort of 57 patients underwent ¹⁸[F]FDG-PET-CT and standardized uptake values were collected. In 15 patients, data on mitochondrial DNA mutations of the tumor were available. Kaplan–Meier curves were calculated, and correlation analyses as well as univariate Cox proportional hazard models were performed. Using ROC analysis to determine a statistical threshold for SUVmax in PET investigations, a cut-off value was determined at 9.765 MB/mL. Survival analysis for SUVmax in these groups showed a Hazard Ratio of 4 (95% CI 1.7–9) in the high SUVmax group with 5-year survival rates of 23.5% (p = 0.00042). For SUVmax and clinicopathological tumor features, significant correlations were found. A tendency towards higher mtDNA heteroplasmy levels in high SUVmax groups could be observed. We were able to confirm the prognostic value of SUVmax in OSCC, showing higher survival rates at lower SUVmax levels. Correlations between SUVmax and distinct tumor characteristics were highly significant, providing evidence that SUVmax may act as a reliable diagnostic parameter. Correlation analysis of mtDNA mutations suggests an influence on metabolic activity in OSCC.

Mitochondria play a vital role in cellular bioenergetics, providing energy via oxidative phosphorylation (OXPHOS) and metabolic intermediates. Aerobic ATP production is orchestrated by a multi-enzyme complex, which uses the electrons gained during oxidation of energy substrates to generate an electrochemical potential across the mitochondrial (mt) membrane that drives ATP synthase. Although there is evidence that mitochondrial function as well as mtDNA mutations might play a role in tumor formation and progression, combined analysis of mt-physiology and DNA alterations are lacking. We analyzed mitochondrial respiration using high-resolution respirometry (OROBOROS Oxygraph-2k). By applying various mitochondrial substrate-uncoupler-inhibitor combinations we dissected the contribution of mitochondrial electron transfer pathways to oxidative phosphorylation. Relative mtDNA copy numbers were determined by duplex qPCR, mtDNA mutations by next-generation sequencing (Ion Torrent Proton). Sequencing data were analyzed using our newly developed mtDNA-Server analysis pipeline (http://mtdna-server.uibk.ac.at). Significant differences in BE and CA tissue were found with respect to the respiratory capacities of Complex I- and Complex II-linked pathways (CI and CII). CA tissue exhibited lower CI-linked respiration which was compensated by higher CII-linked capacities. No correlation was found between mt-function and mtDNA copy number. A comparison of mtDNA sequence variations in BE and CA tissue revealed a significantly higher overall mutation rate in malignant tissue. Most interestingly, deleterious mutations in regions coding for Complex I components strongly correlated with a relative loss of CI-linked respiration in both CA and BE tissue. No statistically significant correlation of mt-mutation burden was found to tumor Gleason scores or patients’ serum PSA levels. Funding: COMET K1 Center Oncotyrol – Center for Personalized Medicine, K-Regio project MitoFit. Presenting Author: Bernd Schöpf, Division of Genetic Epidemiology, Department of Medical Genetics, Medical University of Innsbruck, Schöpfstraße 4, 6020 Innsbruck, Austria. Email: Bernd.schoepf@gmail.com