Targeting cancer stem cell OXPHOS with tailored ruthenium complexes as a new anti-cancer strategy.

BACKGROUND: Previous studies by our group have shown that oxidative phosphorylation (OXPHOS) is the main pathway by which pancreatic cancer stem cells (CSCs) meet their energetic requirements; therefore, OXPHOS represents an Achille's heel of these highly tumorigenic cells. Unfortunately, therapies that target OXPHOS in CSCs are lacking. METHODS: The safety and anti-CSC activity of a ruthenium complex featuring bipyridine and terpyridine ligands and one coordination labile position (Ru1) were evaluated across primary pancreatic cancer cultures and in vivo, using 8 patient-derived xenografts (PDXs). RNAseq analysis followed by mitochondria-specific molecular assays were used to determine the mechanism of action. RESULTS: We show that Ru1 is capable of inhibiting CSC OXPHOS function in vitro, and more importantly, it presents excellent anti-cancer activity, with low toxicity, across a large panel of human pancreatic PDXs, as well as in colorectal cancer and osteosarcoma PDXs. Mechanistic studies suggest that this activity stems from Ru1 binding to the D-loop region of the mitochondrial DNA of CSCs, inhibiting OXPHOS complex-associated transcription, leading to reduced mitochondrial oxygen consumption, membrane potential, and ATP production, all of which are necessary for CSCs, which heavily depend on mitochondrial respiration. CONCLUSIONS: Overall, the coordination complex Ru1 represents not only an exciting new anti-cancer agent, but also a molecular tool to dissect the role of OXPHOS in CSCs. Results indicating that the compound is safe, non-toxic and highly effective in vivo are extremely exciting, and have allowed us to uncover unprecedented mechanistic possibilities to fight different cancer types based on targeting CSC OXPHOS.

Dysfunction of Drosophila mitochondrial carrier homolog (Mtch) alters apoptosis and disturbs development.

Mitochondrial carrier homologs 1 (MTCH1) and 2 (MTCH2) are orphan members of the mitochondrial transporter family SLC25. Human MTCH1 is also known as presenilin 1-associated protein, PSAP. MTCH2 is a receptor for tBid and is related to lipid metabolism. Both proteins have been recently described as protein insertases of the outer mitochondrial membrane. We have depleted Mtch in Drosophila and show here that mutant flies are unable to complete development, showing an excess of apoptosis during pupation; this observation was confirmed by RNAi in Schneider cells. These findings are contrary to what has been described in humans. We discuss the implications in view of recent reports concerning the function of these proteins.

Multicentric Standardization of Protocols for the Diagnosis of Human Mitochondrial Respiratory Chain Defects.

The quantification of mitochondrial respiratory chain (MRC) enzymatic activities is essential for diagnosis of a wide range of mitochondrial diseases, ranging from inherited defects to secondary dysfunctions. MRC lesion is frequently linked to extended cell damage through the generation of proton leak or oxidative stress, threatening organ viability and patient health. However, the intrinsic challenge of a methodological setup and the high variability in measuring MRC enzymatic activities represents a major obstacle for comparative analysis amongst institutions. To improve experimental and statistical robustness, seven Spanish centers with extensive experience in mitochondrial research and diagnosis joined to standardize common protocols for spectrophotometric MRC enzymatic measurements using minimum amounts of sample. Herein, we present the detailed protocols, reference ranges, tips and troubleshooting methods for experimental and analytical setups in different sample preparations and tissues that will allow an international standardization of common protocols for the diagnosis of MRC defects. Methodological standardization is a crucial step to obtain comparable reference ranges and international standards for laboratory assays to set the path for further diagnosis and research in the field of mitochondrial diseases.

Metformin reduces macrophage HIF1α-dependent proinflammatory signaling to restore brown adipocyte function in vitro.

Therapeutic potential of metformin in obese/diabetic patients has been associated to its ability to combat insulin resistance. However, it remains largely unknown the signaling pathways involved and whether some cell types are particularly relevant for its beneficial effects. M1-activation of macrophages by bacterial lipopolysaccharide (LPS) promotes a paracrine activation of hypoxia-inducible factor-1α (HIF1α) in brown adipocytes which reduces insulin signaling and glucose uptake, as well as ß-adrenergic sensitivity. Addition of metformin to M1-polarized macrophages blunted these signs of brown adipocyte dysfunction. At the molecular level, metformin inhibits an inflammatory program executed by HIF1α in macrophages by inducing its degradation through the inhibition of mitochondrial complex I activity, thereby reducing oxygen consumption in a reactive oxygen species (ROS)-independent manner. In obese mice, metformin reduced inflammatory features in brown adipose tissue (BAT) such as macrophage infiltration, proinflammatory signaling and gene expression, and restored the response to cold exposure. In conclusion, the impact of metformin on macrophages by suppressing a HIF1α-dependent proinflammatory program is likely responsible for a secondary beneficial effect on insulin-mediated glucose uptake and ß-adrenergic responses in brown adipocytes.

Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells.

Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7-9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies.

c-Src functionality controls self-renewal and glucose metabolism in MCF7 breast cancer stem cells.

Deregulation of Src kinases is associated with cancer. We previously showed that SrcDN conditional expression in MCF7 cells reduces tumorigenesis and causes tumor regression in mice. However, it remained unclear whether SrcDN affected breast cancer stem cell functionality or it reduced tumor mass. Here, we address this question by isolating an enriched population of Breast Cancer Stem Cells (BCSCs) from MCF7 cells with inducible expression of SrcDN. Induction of SrcDN inhibited self-renewal, and stem-cell marker expression (Nanog, Oct3-4, ALDH1, CD44). Quantitative proteomic analyses of mammospheres from MCF7-Tet-On-SrcDN cells (data are available via ProteomeXchange with identifier PXD017789, project DOI: 10.6019/PXD017789) and subsequent GSEA showed that SrcDN expression inhibited glycolysis. Indeed, induction of SrcDN inhibited expression and activity of hexokinase, pyruvate kinase and lactate dehydrogenase, resulting in diminished glucose consumption and lactate production, which restricted Warburg effect. Thus, c-Src functionality is important for breast cancer stem cell maintenance and renewal, and stem cell transcription factor expression, effects linked to glucose metabolism reduction.

ISG15 and ISGylation is required for pancreatic cancer stem cell mitophagy and metabolic plasticity.

Pancreatic cancer stem cells (PaCSCs) drive pancreatic cancer tumorigenesis, chemoresistance and metastasis. While eliminating this subpopulation of cells would theoretically result in tumor eradication, PaCSCs are extremely plastic and can successfully adapt to targeted therapies. In this study, we demonstrate that PaCSCs increase expression of interferon-stimulated gene 15 (ISG15) and protein ISGylation, which are essential for maintaining their metabolic plasticity. CRISPR-mediated ISG15 genomic editing reduces overall ISGylation, impairing PaCSCs self-renewal and their in vivo tumorigenic capacity. At the molecular level, ISG15 loss results in decreased mitochondrial ISGylation concomitant with increased accumulation of dysfunctional mitochondria, reduced oxidative phosphorylation (OXPHOS) and impaired mitophagy. Importantly, disruption in mitochondrial metabolism affects PaCSC metabolic plasticity, making them susceptible to prolonged inhibition with metformin in vivo. Thus, ISGylation is critical for optimal and efficient OXPHOS by ensuring the recycling of dysfunctional mitochondria, and when absent, a dysregulation in mitophagy occurs that negatively impacts PaCSC stemness.

Metabolic labeling of RNA uncovers the contribution of transcription and decay rates on hypoxia-induced changes in RNA levels.

Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. However, most transcriptomic studies do not distinguish the relative contribution of transcription, RNA processing, and RNA degradation processes to cellular homeostasis. Here we used metabolic labeling followed by massive parallel sequencing of newly transcribed and preexisting RNA fractions to simultaneously analyze RNA synthesis and decay in primary endothelial cells exposed to low oxygen tension. We found that changes in transcription rates induced by hypoxia are the major determinant of changes in RNA levels. However, degradation rates also had a significant contribution, accounting for 24% of the observed variability in total mRNA. In addition, our results indicated that hypoxia led to a reduction of the overall mRNA stability from a median half-life in normoxia of 8.7 h, to 5.7 h in hypoxia. Analysis of RNA content per cell confirmed a decrease of both mRNA and total RNA in hypoxic samples and that this effect is dependent on the EGLN/HIF/TSC2 axis. This effect could potentially contribute to fundamental global responses such as inhibition of translation in hypoxia. In summary, our study provides a quantitative analysis of the contribution of RNA synthesis and stability to the transcriptional response to hypoxia and uncovers an unexpected effect on the latter.

Correction: Enhanced tumorigenicity by mitochondrial DNA mild mutations.

[This corrects the article DOI: 10.18632/oncotarget.3698.].

Human COA3 Is an Oligomeric Highly Flexible Protein in Solution.

The assembly of the protein complex of cytochrome c oxidase (COX), which participates in the mitochondrial respiratory chain, requires a large number of accessory proteins (the so-called assembly factors). Human COX assembly factor 3 (hCOA3), also known as MITRAC12 or coiled-coil domain-containing protein 56 (CCDC56), interacts with the first subunit protein of COX to form its catalytic core and promotes its assemblage with the other units. Therefore, hCOA3 is involved in COX biogenesis in humans and can be exploited as a drug target in patients with mitochondrial dysfunctions. However, to be considered a molecular target, its structure and conformational stability must first be elucidated. We have embarked on the description of such features by using spectroscopic and hydrodynamic techniques, in aqueous solution and in the presence of detergents, together with computational methods. Our results show that hCOA3 is an oligomeric protein, forming aggregates of different molecular masses in aqueous solution. Moreover, on the basis of fluorescence and circular dichroism results, the protein has (i) its unique tryptophan partially shielded from solvent and (ii) a relatively high percentage of secondary structure. However, this structure is highly flexible and does not involve hydrogen bonding. Experiments in the presence of detergents suggest a slightly higher content of nonrigid helical structure. Theoretical results, based on studies of the primary structure of the protein, further support the idea that hCOA3 is a disordered protein. We suggest that the flexibility of hCOA3 is crucial for its interaction with other proteins to favor mitochondrial protein translocation and assembly of proteins involved in the respiratory chain.

Enhanced tumorigenicity by mitochondrial DNA mild mutations.

To understand how mitochondria are involved in malignant transformation we have generated a collection of transmitochondrial cybrid cell lines on the same nuclear background (143B) but with mutant mitochondrial DNA (mtDNA) variants with different degrees of pathogenicity. These include the severe mutation in the tRNALys gene, m.8363G>A, and the three milder yet prevalent Leber's hereditary optic neuropathy (LHON) mutations in the MT-ND1 (m.3460G>A), MT-ND4 (m.11778G>A) and MT-ND6 (m.14484T>C) mitochondrial genes. We found that 143B ρ0 cells devoid of mtDNA and cybrids harboring wild type mtDNA or that causing severe mitochondrial dysfunction do not produce tumors when injected in nude mice. By contrast cybrids containing mild mutant mtDNAs exhibit different tumorigenic capacities, depending on OXPHOS dysfunction.The differences in tumorigenicity correlate with an enhanced resistance to apoptosis and high levels of NOX expression. However, the final capacity of the different cybrid cell lines to generate tumors is most likely a consequence of a complex array of pro-oncogenic and anti-oncogenic factors associated with mitochondrial dysfunction.Our results demonstrate the essential role of mtDNA in tumorigenesis and explain the numerous and varied mtDNA mutations found in human tumors, most of which give rise to mild mitochondrial dysfunction.

The pathogenicity scoring system for mitochondrial tRNA mutations revisited.

Confirming the pathogenicity of mitochondrial tRNA point mutations is one of the classical challenges in the field of mitochondrial medicine. In addition to genetic and functional studies, the evaluation of a genetic change using a pathogenicity scoring system is extremely useful to discriminate between disease-causing mutations from neutral polymorphisms. The pathogenicity scoring system is very robust for confirming pathogenicity, especially of mutations that show impaired activity in functional studies. However, mutations giving normal results using the same functional approaches are disregarded, and this compromises the power of the system to rule out pathogenicity. We propose to include a new criterion in the pathogenicity scoring systems regarding mutations which fail to show any mitochondrial defect in functional studies. To evaluate this proposal we characterized two mutations, m.8296A>G and m.8347A>G, in the mitochondrial tRNA(L) (ys) gene (MT-TK) using trans-mitochondrial cybrid analysis. m.8347A>G mutation severely impairs oxidative phosphorylation, suggesting that it is highly pathogenic. By contrast, the behavior of cybrids homoplasmic for the m.8296A>G mutation is similar to cybrids containing wild-type mitochondrial DNA (mtDNA). The results indicate that including not only positive but also negative outcomes of functional studies in the scoring system is critical for facilitating the diagnosis of this complex group of diseases.

Coiled coil domain-containing protein 56 (CCDC56) is a novel mitochondrial protein essential for cytochrome c oxidase function.

In Drosophila melanogaster, the mitochondrial transcription factor B1 (d-mtTFB1) transcript contains in its 5'-untranslated region a conserved upstream open reading frame denoted as CG42630 in FlyBase. We demonstrate that CG42630 encodes a novel protein, the coiled coil domain-containing protein 56 (CCDC56), conserved in metazoans. We show that Drosophila CCDC56 protein localizes to mitochondria and contains 87 amino acids in flies and 106 in humans with the two proteins sharing 42% amino acid identity. We show by rapid amplification of cDNA ends and Northern blotting that Drosophila CCDC56 protein and mtTFB1 are encoded on a bona fide bicistronic transcript. We report the generation and characterization of two ccdc56 knock-out lines in Drosophila carrying the ccdc56(D6) and ccdc56(D11) alleles. Lack of the CCDC56 protein in flies induces a developmental delay and 100% lethality by arrest of larval development at the third instar. ccdc56 knock-out larvae show a significant decrease in the level of fully assembled cytochrome c oxidase (COX) and in its activity, suggesting a defect in complex assembly; the activity of the other oxidative phosphorylation complexes remained either unaffected or increased in the ccdc56 knock-out larvae. The lethal phenotype and the decrease in COX were partially rescued by reintroduction of a wild-type UAS-ccdc56 transgene. These results indicate an important role for CCDC56 in the oxidative phosphorylation system and in particular in COX function required for proper development in D. melanogaster. We propose CCDC56 as a candidate factor required for COX biogenesis/assembly.

Mitochondrial transcription factor B2 is essential for metabolic function in Drosophila melanogaster development.

Characterization of the basal transcription machinery of mitochondrial DNA (mtDNA) is critical to understand mitochondrial pathophysiology. In mammalian in vitro systems, mtDNA transcription requires mtRNA polymerase, transcription factor A (TFAM), and either transcription factor B1 (TFB1M) or B2 (TFB2M). We have silenced the expression of TFB2M by RNA interference in Drosophila melanogaster. RNA interference knockdown of TF2BM causes lethality by arrest of larval development. Molecular analysis demonstrates that TF2BM is essential for mtDNA transcription during Drosophila development and is not redundant with TFB1M. The impairment of mtDNA transcription causes a dramatic decrease in oxidative phosphorylation and mitochondrial ATP synthesis in the long-lived larvae, and a metabolic shift to glycolysis, which partially restores ATP levels and elicits a compensatory response at the nuclear level that increases mitochondrial mass. At the cellular level, the mitochondrial dysfunction induced by TFB2M knockdown causes a severe reduction in cell proliferation without affecting cell growth, and increases the level of apoptosis. In contrast, cell differentiation and morphogenesis are largely unaffected. Our data demonstrate the essential role of TFB2M in mtDNA transcription in a multicellular organism, and reveal the complex cellular, biochemical, and molecular responses induced by impairment of oxidative phosphorylation during Drosophila development.

Drosophila melanogaster as a model system to study mitochondrial biology.

Mitochondria play an essential role in cellular homeostasis. Although in the last few decades our knowledge of mitochondria has increased substantially, the mechanisms involved in the control of mitochondrial biogenesis remain largely unknown. The powerful genetics of Drosophila combined with a wealth of available cell and molecular biology techniques, make this organism an excellent system to study mitochondria. In this chapter we will review briefly the opportunities that Drosophila offers as a model system and describe in detail how to purify mitochondria from Drosophila and to perform the analysis of developmental gene expression using in situ hybridization.

Modeling human mitochondrial diseases in flies.

Human mitochondrial diseases are associated with a wide range of clinical symptoms, and those that result from mutations in mitochondrial DNA affect at least 1 in 8500 individuals. The development of animal models that reproduce the variety of symptoms associated with this group of complex human disorders is a major focus of current research. Drosophila represents an attractive model, in large part because of its short life cycle, the availability of a number of powerful techniques to alter gene structure and regulation, and the presence of orthologs of many human disease genes. We describe here Drosophila models of mitochondrial DNA depletion, deafness, encephalopathy, Freidreich's ataxia, and diseases due to mitochondrial DNA mutations. We also describe several genetic approaches for gene manipulation in flies, including the recently developed method of targeted mutagenesis by recombinational knock-in.

Comparative analysis of the pathogenic mechanisms associated with the G8363A and A8296G mutations in the mitochondrial tRNA(Lys) gene.

Two mutations (G8363A and A8296G) in the mtDNA (mitochondrial DNA) tRNA(Lys) gene have been associated with severe mitochondrial diseases in a number of reports. Their functional significance, however, remains unknown. We have already shown that homoplasmic cybrids harbouring the A8296G mutation display normal oxidative phosphorylation, although the possibility of a subtle change in mitochondrial respiratory capacity remains an open issue. We have now investigated the pathogenic mechanism of another mutation in the tRNA(Lys) gene (G8363A) by repopulating an mtDNA-less human osteosarcoma cell line with mitochondria harbouring either this genetic variant alone or an unusual combination of the two mutations (A8296G+G8363A). Cybrids homoplasmic for the single G8363A or the A8296G+G8363A mutations have defective respiratory-chain enzyme activities and low oxygen consumption, indicating a severe impairment of the oxidative phosphorylation system. Generation of G8363A cybrids within a wild-type or the A8296G mtDNA genetic backgrounds resulted in an important alteration in the conformation of the tRNA(Lys), not affecting tRNA steady-state levels. Moreover, mutant cybrids have an important decrease in the proportion of amino-acylated tRNA(Lys) and, consequently, mitochondrial protein synthesis is greatly decreased. Our results demonstrate that the pathogenicity of the G8363A mutation is due to a change in the conformation of the tRNA that severely impairs aminoacylation in the absence of changes in tRNA stability. The only effect detected in the A8296G mutation is a moderate decrease in the aminoacylation capacity, which does not affect mitochondrial protein biosynthesis.

Expression of the Drosophila melanogaster ATP synthase alpha subunit gene is regulated by a transcriptional element containing GAF and Adf-1 binding sites.

Mitochondrial biogenesis is a complex and highly regulated process that requires the controlled expression of hundreds of genes encoded in two separated genomes, namely the nuclear and mitochondrial genomes. To identify regulatory proteins involved in the transcriptional control of key nuclear-encoded mitochondrial genes, we have performed a detailed analysis of the promoter region of the alpha subunit of the Drosophila melanogaster F1F0 ATP synthase complex. Using transient transfection assays, we have identified a 56 bp cis-acting proximal regulatory region that contains binding sites for the GAGA factor and the alcohol dehydrogenase distal factor 1. In vitro mutagenesis revealed that both sites are functional, and phylogenetic footprinting showed that they are conserved in other Drosophila species and in Anopheles gambiae. The 56 bp region has regulatory enhancer properties and strongly activates heterologous promoters in an orientation-independent manner. In addition, Northern blot and RT-PCR analysis identified two alpha-F1-ATPase mRNAs that differ in the length of the 3' untranslated region due to the selection of alternative polyadenylation sites.

The A8296G mtDNA mutation associated with several mitochondrial diseases does not cause mitochondrial dysfunction in cybrid cell lines.

Transmitochondrial cybrid cell lines homoplasmic for the A8296G mtDNA transition, a mutation associated with several mitochondrial diseases, have a normal oxidative phosphorylation function, as shown by oxygen consumption, lactate production, respiratory enzyme activities, and growth using galactose as the only source of energy. The synthesis of mitochondrial proteins is also similar in mutant and wild-type cybrids. Our results suggest that the A8296G mutation is a polymorphism and reinforce the necessity of performing functional studies to assess the pathogenicity of mtDNA mutations.