Immunohistochemical, pharmacovigilance, and omics analyses reveal the involvement of ATP-sensitive K+ channel subunits in cancers: role in drug-disease interactions.

Background: ATP-sensitive-K+ channels (KATP) are involved in diseases, but their role in cancer is poorly described. Pituitary macroadenoma has been observed in Cantu' syndrome (C.S.), which is associated with the gain-of-function mutations of the ABCC9 and KCNJ8 genes. We tested the role of the ABCC8/Sur1, ABCC9/Sur2A/B, KCNJ11/Kir6.2, and KCNJ8/Kir6.1 genes experimentally in a minoxidil-induced renal tumor in male rats and in the female canine breast cancer, a spontaneous animal model of disease, and in the pharmacovigilance and omics databases. Methods: We performed biopsies from renal tissues of male rats (N = 5) following a sub-chronic high dosing topical administration of minoxidil (0.777-77.7 mg/kg/day) and from breast tissues of female dogs for diagnosis (N = 23) that were analyzed by immunohistochemistry. Pharmacovigilance and omics data were extracted from EudraVigilance and omics databases, respectively. Results: An elevated immunohistochemical reactivity to Sur2A-mAb was detected in the cytosol of the Ki67+/G3 cells other than in the surface membrane in the minoxidil-induced renal tumor and the breast tumor samples. KCNJ11, KCNJ8, and ABCC9 genes are upregulated in cancers but ABCC8 is downregulated. The Kir6.2-Sur2A/B-channel opener minoxidil showed 23 case reports of breast cancer and one case of ovarian cancer in line with omics data reporting, respectively, and the negative and positive prognostic roles of the ABCC9 gene in these cancers. Sulfonylureas and glinides blocking the pancreatic Kir6.2-Sur1 subunits showed a higher risk for pancreatic cancer in line with the positive prognostic role of the ABCC8 gene but low risks for common cancers. Glibenclamide, repaglinide, and glimepiride show a lower cancer risk within the KATP channel blockers. The Kir6.2-Sur1 opener diazoxide shows no cancer reactions. Conclusion: An elevated expression of the Sur2A subunit was found in proliferating cells in two animal models of cancer. Immunohistochemistry/omics/pharmacovigilance data reveal the role of the Kir6.1/2-Sur2A/B subunits as a drug target in breast/renal cancers and in C.S.

Decreased Mitochondrial DNA Content Drives OXPHOS Dysregulation in Chromophobe Renal Cell Carcinoma.

Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are closely related, rare kidney tumors. Mutations in complex I (CI)-encoding genes play an important role in dysfunction of the oxidative phosphorylation (OXPHOS) system in renal oncocytoma, but are less frequently observed in chRCC. As such, the relevance of OXPHOS status and role of CI mutations in chRCC remain unknown. To address this issue, we performed proteome and metabolome profiling as well as mitochondrial whole-exome sequencing to detect mitochondrial alterations in chRCC tissue specimens. Multiomic analysis revealed downregulation of electron transport chain (ETC) components in chRCC that differed from the expression profile in renal oncocytoma. A decrease in mitochondrial (mt)DNA content, rather than CI mutations, was the main cause for reduced OXPHOS in chRCC. There was a negative correlation between protein and transcript levels of nuclear DNA- but not mtDNA-encoded ETC complex subunits in chRCC. In addition, the reactive oxygen species scavenger glutathione (GSH) was upregulated in chRCC due to decreased expression of proteins involved in GSH degradation. These results demonstrate that distinct mechanisms of OXPHOS exist in chRCC and renal oncocytoma and that expression levels of ETC complex subunits can serve as a diagnostic marker for this rare malignancy. SIGNIFICANCE: These findings establish potential diagnostic markers to distinguish malignant chRCC from its highly similar but benign counterpart, renal oncocytoma.

Investigating Human Mitochondrial Genomes in Single Cells.

Mitochondria host multiple copies of their own small circular genome that has been extensively studied to trace the evolution of the modern eukaryotic cell and discover important mutations linked to inherited diseases. Whole genome and exome sequencing have enabled the study of mtDNA in a large number of samples and experimental conditions at single nucleotide resolution, allowing the deciphering of the relationship between inherited mutations and phenotypes and the identification of acquired mtDNA mutations in classical mitochondrial diseases as well as in chronic disorders, ageing and cancer. By applying an ad hoc computational pipeline based on our MToolBox software, we reconstructed mtDNA genomes in single cells using whole genome and exome sequencing data obtained by different amplification methodologies (eWGA, DOP-PCR, MALBAC, MDA) as well as data from single cell Assay for Transposase Accessible Chromatin with high-throughput sequencing (scATAC-seq) in which mtDNA sequences are expected as a byproduct of the technology. We show that assembled mtDNAs, with the exception of those reconstructed by MALBAC and DOP-PCR methods, are quite uniform and suitable for genomic investigations, enabling the study of various biological processes related to cellular heterogeneity such as tumor evolution, neural somatic mosaicism and embryonic development.

Papillary Renal Cell Carcinomas Rewire Glutathione Metabolism and Are Deficient in Both Anabolic Glucose Synthesis and Oxidative Phosphorylation.

Papillary renal cell carcinoma (pRCC) is a malignant kidney cancer with a prevalence of 7-20% of all renal tumors. Proteome and metabolome profiles of 19 pRCC and patient-matched healthy kidney controls were used to elucidate the regulation of metabolic pathways and the underlying molecular mechanisms. Glutathione (GSH), a main reactive oxygen species (ROS) scavenger, was highly increased and can be regarded as a new hallmark in this malignancy. Isotope tracing of pRCC derived cell lines revealed an increased de novo synthesis rate of GSH, based on glutamine consumption. Furthermore, profound downregulation of gluconeogenesis and oxidative phosphorylation was observed at the protein level. In contrast, analysis of the The Cancer Genome Atlas (TCGA) papillary RCC cohort revealed no significant change in transcripts encoding oxidative phosphorylation compared to normal kidney tissue, highlighting the importance of proteomic profiling. The molecular characteristics of pRCC are increased GSH synthesis to cope with ROS stress, deficient anabolic glucose synthesis, and compromised oxidative phosphorylation, which could potentially be exploited in innovative anti-cancer strategies.

Renal oncocytoma characterized by the defective complex I of the respiratory chain boosts the synthesis of the ROS scavenger glutathione.

Renal oncocytomas are rare benign tumors of the kidney and characterized by a deficient complex I (CI) enzyme activity of the oxidative phosphorylation (OXPHOS) system caused by mitochondrial DNA (mtDNA) mutations. Yet, little is known about the underlying molecular mechanisms and alterations of metabolic pathways in this tumor. We compared renal oncocytomas with adjacent matched normal kidney tissues on a global scale by multi-omics approaches, including whole exome sequencing (WES), proteomics, metabolomics, and metabolic pathway simulation. The abundance of proteins localized to mitochondria increased more than 2-fold, the only exception was a strong decrease in the abundance for CI subunits that revealed several pathogenic heteroplasmic mtDNA mutations by WES. We also observed renal oncocytomas to dysregulate main metabolic pathways, shunting away from gluconeogenesis and lipid metabolism. Nevertheless, the abundance of energy carrier molecules such as NAD+, NADH, NADP, ATP, and ADP were significantly higher in renal oncocytomas. Finally, a substantial 5000-fold increase of the reactive oxygen species scavenger glutathione can be regarded as a new hallmark of renal oncocytoma. Our findings demonstrate that renal oncocytomas undergo a metabolic switch to eliminate ATP consuming processes to ensure a sufficient energy supply for the tumor.

A multi-parametric workflow for the prioritization of mitochondrial DNA variants of clinical interest.

Assigning a pathogenic role to mitochondrial DNA (mtDNA) variants and unveiling the potential involvement of the mitochondrial genome in diseases are challenging tasks in human medicine. Assuming that rare variants are more likely to be damaging, we designed a phylogeny-based prioritization workflow to obtain a reliable pool of candidate variants for further investigations. The prioritization workflow relies on an exhaustive functional annotation through the mtDNA extraction pipeline MToolBox and includes Macro Haplogroup Consensus Sequences to filter out fixed evolutionary variants and report rare or private variants, the nucleotide variability as reported in HmtDB and the disease score based on several predictors of pathogenicity for non-synonymous variants. Cutoffs for both the disease score as well as for the nucleotide variability index were established with the aim to discriminate sequence variants contributing to defective phenotypes. The workflow was validated on mitochondrial sequences from Leber's Hereditary Optic Neuropathy affected individuals, successfully identifying 23 variants including the majority of the known causative ones. The application of the prioritization workflow to cancer datasets allowed to trim down the number of candidate for subsequent functional analyses, unveiling among these a high percentage of somatic variants. Prioritization criteria were implemented in both standalone ( http://sourceforge.net/projects/mtoolbox/ ) and web version ( https://mseqdr.org/mtoolbox.php ) of MToolBox.

A comprehensive characterization of mitochondrial DNA mutations in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant brain cancer in adults, with a poor prognosis, whose molecular stratification still represents a challenge in pathology and clinics. On the other hand, mitochondrial DNA (mtDNA) mutations have been found in most tumors as modifiers of the bioenergetics state, albeit in GBM a characterization of the mtDNA status is lacking to date. Here, a characterization of the burden of mtDNA mutations in GBM samples was performed. First, investigation of tumor-specific vs. non tumor-specific mutations was carried out with the MToolBox bioinformatics pipeline by analyzing 45 matched tumor/blood samples, from whole genome or whole exome sequencing datasets obtained from The Cancer Genome Atlas (TCGA) consortium. Additionally, the entire mtDNA sequence was obtained in a dataset of 104 fresh-frozen GBM samples. Mitochondrial mutations with potential pathogenic interest were prioritized based on heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity. A preliminary biochemical analysis of the activity of mitochondrial respiratory complexes was also performed on fresh-frozen GBM samples. Although a high number of mutations was detected, we report that the large majority of them does not pass the prioritization filters. Therefore, a relatively limited burden of pathogenic mutations is indeed carried by GBM, which did not appear to determine a general impairment of the respiratory chain. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

A platform independent RNA-Seq protocol for the detection of transcriptome complexity.

BACKGROUND: Recent studies have demonstrated an unexpected complexity of transcription in eukaryotes. The majority of the genome is transcribed and only a little fraction of these transcripts is annotated as protein coding genes and their splice variants. Indeed, most transcripts are the result of antisense, overlapping and non-coding RNA expression. In this frame, one of the key aims of high throughput transcriptome sequencing is the detection of all RNA species present in the cell and the first crucial step for RNA-seq users is represented by the choice of the strategy for cDNA library construction. The protocols developed so far provide the utilization of the entire library for a single sequencing run with a specific platform. RESULTS: We set up a unique protocol to generate and amplify a strand-specific cDNA library representative of all RNA species that may be implemented with all major platforms currently available on the market (Roche 454, Illumina, ABI/SOLiD). Our method is reproducible, fast, easy-to-perform and even allows to start from low input total RNA. Furthermore, we provide a suitable bioinformatics tool for the analysis of the sequences produced following this protocol. CONCLUSION: We tested the efficiency of our strategy, showing that our method is platform-independent, thus allowing the simultaneous analysis of the same sample with different NGS technologies, and providing an accurate quantitative and qualitative portrait of complex whole transcriptomes.

Respiratory complex I is essential to induce a Warburg profile in mitochondria-defective tumor cells.

BACKGROUND: Aerobic glycolysis, namely the Warburg effect, is the main hallmark of cancer cells. Mitochondrial respiratory dysfunction has been proposed to be one of the major causes for such glycolytic shift. This hypothesis has been revisited as tumors appear to undergo waves of gene regulation during progression, some of which rely on functional mitochondria. In this framework, the role of mitochondrial complex I is still debated, in particular with respect to the effect of mitochondrial DNA mutations in cancer metabolism. The aim of this work is to provide the proof of concept that functional complex I is necessary to sustain tumor progression. METHODS: Complex I-null osteosarcoma cells were complemented with allotopically expressed complex I subunit 1 (MT-ND1). Complex I re-assembly and function recovery, also in terms of NADH consumption, were assessed. Clones were tested for their ability to grow in soft agar and to generate tumor masses in nude mice. Hypoxia levels were evaluated via pimonidazole staining and hypoxia-inducible factor-1α (HIF-1α) immunoblotting and histochemical staining. 454-pyrosequencing was implemented to obtain global transcriptomic profiling of allotopic and non-allotopic xenografts. RESULTS: Complementation of a truncative mutation in the gene encoding MT-ND1, showed that a functional enzyme was required to perform the glycolytic shift during the hypoxia response and to induce a Warburg profile in vitro and in vivo, fostering cancer progression. Such trigger was mediated by HIF-1α, whose stabilization was regulated after recovery of the balance between α-ketoglutarate and succinate due to a recuperation of NADH consumption that followed complex I rescue. CONCLUSION: Respiratory complex I is essential for the induction of Warburg effect and adaptation to hypoxia of cancer cells, allowing them to sustain tumor growth. Differently from other mitochondrial tumor suppressor genes, therefore, a complex I severe mutation such as the one here reported may confer anti-tumorigenic properties, highlighting the prognostic values of such genetic markers in cancer.

Somatic complex I disruptive mitochondrial DNA mutations are modifiers of tumorigenesis that correlate with low genomic instability in pituitary adenomas.

Mitochondrial DNA (mtDNA) mutations leading to the disruption of respiratory complex I (CI) have been shown to exhibit anti-tumorigenic effects, at variance with those impairing only the function but not the assembly of the complex, which appear to contribute positively to cancer development. Owing to the challenges in the analysis of the multi-copy mitochondrial genome, it is yet to be determined whether tumour-associated mtDNA lesions occur as somatic modifying factors or as germ-line predisposing elements. Here we investigated the whole mitochondrial genome sequence of 20 pituitary adenomas with oncocytic phenotype and identified pathogenic and/or novel mtDNA mutations in 60% of the cases. Using highly sensitive techniques, namely fluorescent PCR and allele-specific locked nucleic acid quantitative PCR, we identified the most likely somatic nature of these mutations in our sample set, since none of the mutations was detected in the corresponding blood tissue of the patients analysed. Furthermore, we have subjected a series of 48 pituitary adenomas to a high-resolution array comparative genomic hybridization analysis, which revealed that CI disruptive mutations, and the oncocytic phenotype, significantly correlate with low number of chromosomal aberrations in the nuclear genome. We conclude that CI disruptive mutations in pituitary adenomas are somatic modifiers of tumorigenesis most likely contributing not only to the development of oncocytic change, but also to a less aggressive tumour phenotype, as indicated by a stable karyotype.

Deep sequencing unearths nuclear mitochondrial sequences under Leber's hereditary optic neuropathy-associated false heteroplasmic mitochondrial DNA variants.

Leber's hereditary optic neuropathy (LHON) is associated with mitochondrial DNA (mtDNA) ND mutations that are mostly homoplasmic. However, these mutations are not sufficient to explain the peculiar features of penetrance and the tissue-specific expression of the disease and are believed to be causative in association with unknown environmental or other genetic factors. Discerning between clear-cut pathogenetic variants, such as those that appear to be heteroplasmic, and less penetrant variants, such as the homoplasmic, remains a challenging issue that we have addressed here using next-generation sequencing approach. We set up a protocol to quantify MTND5 heteroplasmy levels in a family in which the proband manifests a LHON phenotype. Furthermore, to study this mtDNA haplotype, we applied the cybridization protocol. The results demonstrate that the mutations are mostly homoplasmic, whereas the suspected heteroplasmic feature of the observed mutations is due to the co-amplification of Nuclear mitochondrial Sequences.

Searching for a needle in the haystack: comparing six methods to evaluate heteroplasmy in difficult sequence context.

Mitochondrial DNA (mtDNA) mutations have been involved in disease, aging and cancer and furthermore exploited for evolutionary and forensic investigation. When investigating mtDNA mutations the peculiar aspects of mitochondrial genetics, such as heteroplasmy and threshold effect, require suitable approaches which must be sensitive enough to detect low-level heteroplasmy and, precise enough to quantify the exact mutational load. In order to establish the optimal approach for the evaluation of heteroplasmy, six methods were experimentally compared for their capacity to reveal and quantify mtDNA variants. Drawbacks and advantages of cloning, Fluorescent PCR (F-PCR), denaturing High Performance Liquid Chromatography (dHPLC), quantitative Real-Time PCR (qRTPCR), High Resolution Melting (HRM) and 454 pyrosequencing were determined. In particular, detection and quantification of a mutation in a difficult sequence context were investigated, through analysis of an insertion in a homopolymeric stretch (m.3571insC).

Placing mitochondrial DNA mutations within the progression model of type I endometrial carcinoma.

Mitochondrial DNA (mtDNA) mutations have been described in almost all types of cancer. However, their exact role and timing of occurrence during tumor development and progression are still a matter of debate. A Vogelstein-like model of progression is well established for endometrial carcinoma (EC), however, mtDNA has been scarcely investigated in these tumors despite the fact that mitochondrial biogenesis increase has been shown to be a hallmark of type I EC. Here, we screened a panel of 23 type I EC tissues and matched typical hyperplasia for mutations in mtDNA and in four oncosupressors/oncogenes, namely PTEN, KRAS, CTNNB1 and TP53. Overall, mtDNA mutations were identified in 69% of cases, while mutational events in nuclear genes occurred in 56% of the cases, indicating that mtDNA mutations may precede the genetic instability of these genes canonically involved in progression from hyperplasia to tumor. Protein expression analysis revealed an increase in mitochondrial biogenesis and activation of oxidative stress response mechanisms in tumor tissues, but not in hyperplasia, in correlation with the occurrence of pathogenic mtDNA mutations. Our results point out an involvement of mtDNA mutations in EC progression and explain the increase in mitochondrial biogenesis of type I EC. Last, since mtDNA mutations occur after hyperplasia, their potential role in contributing to genetic instability may be envisioned.

The genetic and metabolic signature of oncocytic transformation implicates HIF1alpha destabilization.

We previously showed that disruptive complex I mutations in mitochondrial DNA are the main genetic hallmark of oncocytic tumors of the thyroid and kidney. We here report a high frequency of homoplasmic disruptive mutations in a large panel of oncocytic pituitary and head-and-neck tumors. The presence of such mutations implicates disassembly of respiratory complex I in vivo which in turn contributes to the inability of oncocytic tumors to stabilize HIF1alpha and to display pseudo-hypoxia. By utilizing transmitochondrial cytoplasmic hybrids (cybrids), we induced the shift to homoplasmy of a truncating mutation in the mitochondria-coded MTND1 gene. Such shift is associated with a profound metabolic impairment leading to the imbalance of alpha-ketoglutarate and succinate, the Krebs cycle metabolites which are the main responsible for HIF1alpha stabilization. We conclude that the main hallmarks of oncocytic transformation, namely the occurrence of homoplasmic disruptive mutations and complex I disassembly, may explain the benign nature of oncocytic neoplasms through lack of HIF1alpha stabilization.

MitoDrome: a database of Drosophila melanogaster nuclear genes encoding proteins targeted to the mitochondrion.

Mitochondria are organelles present in the cytoplasm of most eukaryotic cells; although they have their own DNA, the majority of the proteins necessary for a functional mitochondrion are coded by the nuclear DNA and only after transcription and translation they are imported in the mitochondrion as proteins. The primary role of the mitochondrion is electron transport and oxidative phosphorylation. Although it has been studied for a long time, the interest of researchers in mitochondria is still alive thanks to the discovery of mitochondrial role in apoptosis, aging and cancer. Aim of the MitoDrome database is to annotate the Drosophila melanogaster nuclear genes coding for mitochondrial proteins in order to contribute to the functional characterization of nuclear genes coding for mitochondrial proteins and to knowledge of gene diseases related to mitochondrial dysfunctions. Indeed D. melanogaster is one of the most studied organisms and a model for the Human genome. Data are derived from the comparison of Human mitochondrial proteins versus the Drosophila genome, ESTs and cDNA sequence data available in the FlyBase database. Links from the MitoDrome entries to the related homologous entries available in MitoNuC will be soon imple-mented. The MitoDrome database is available at http://bighost.area.ba.cnr.it/BIG/MitoDrome. Data are organised in a flat-file format and can be retrieved using the SRS system.