Cancer/Testis Antigen 55 is required for cancer cell proliferation and mitochondrial DNA maintenance.

Cancer/Testis Antigens (CTAs) represent a group of proteins whose expression under physiological conditions is restricted to testis but activated in many human cancers. Also, it was observed that co-expression of multiple CTAs worsens the patient prognosis. Five CTAs were reported acting in mitochondria and we recently reported 147 transcripts encoded by 67 CTAs encoding for proteins potentially targeted to mitochondria. Among them, we identified the two isoforms encoded by CT55 for whom the function is poorly understood. First, we found that patients with tumors expressing wild-type CT55 are associated with poor survival. Moreover, CT55 silencing decreases dramatically cell proliferation. Second, to investigate the role of CT55 on mitochondria, we first show that CT55 is localized to both mitochondria and endoplasmic reticulum (ER) due to the presence of an ambiguous N-terminal targeting signal. Then, we show that CT55 silencing decreases mtDNA copy number and delays mtDNA recovery after an acute depletion. Moreover, demethylation of CT55 promotor increases its expression, which in turn increases mtDNA copy number. Finally, we measured the mtDNA copy number in NCI-60 cell lines and screened for genes whose expression is strongly correlated to mtDNA amount. We identified CT55 as the second highest correlated hit. Also, we show that compared to siRNA scrambled control (siCtrl) treatment, CT55 specific siRNA (siCT55) treatment down-regulates aerobic respiration, indicating that CT55 sustains mitochondrial respiration. Altogether, these data show for first time that CT55 acts on mtDNA copy number, modulates mitochondrial activity to sustain cancer cell proliferation.

The Long Non-Coding RNA SAMMSON Is a Regulator of Chemosensitivity and Metabolic Orientation in MCF-7 Doxorubicin-Resistant Breast Cancer Cells.

Despite improvements in therapeutic strategies for treating breast cancers, tumor relapse and chemoresistance remain major issues in patient outcomes. Indeed, cancer cells display a metabolic plasticity allowing a quick adaptation to the tumoral microenvironment and to cellular stresses induced by chemotherapy. Recently, long non-coding RNA molecules (lncRNAs) have emerged as important regulators of cellular metabolic orientation. In the present study, we addressed the role of the long non-coding RNA molecule (lncRNA) SAMMSON on the metabolic reprogramming and chemoresistance of MCF-7 breast cancer cells resistant to doxorubicin (MCF-7dox). Our results showed an overexpression of SAMMSON in MCF-7dox compared to doxorubicin-sensitive cells (MCF-7). Silencing of SAMMSON expression by siRNA in MCF-7dox cells resulted in a metabolic rewiring with improvement of oxidative metabolism, decreased mitochondrial ROS production, increased mitochondrial replication, transcription and translation and an attenuation of chemoresistance. These results highlight the role of SAMMSON in the metabolic adaptations leading to the development of chemoresistance in breast cancer cells. Thus, targeting SAMMSON expression levels represents a promising therapeutic route to circumvent doxorubicin resistance in breast cancers.

Dominant ACO2 mutations are a frequent cause of isolated optic atrophy.

Biallelic mutations in ACO2, encoding the mitochondrial aconitase 2, have been identified in individuals with neurodegenerative syndromes, including infantile cerebellar retinal degeneration and recessive optic neuropathies (locus OPA9). By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, we identified 61 cases harbouring variants in ACO2, among whom 50 carried dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy. Analysis of the ophthalmological and clinical data revealed that recessive cases are affected more severely than dominant cases, while not significantly earlier. In addition, 27% of the recessive cases and 11% of the dominant cases manifested with extraocular features in addition to optic atrophy. In silico analyses of ACO2 variants predicted their deleterious impacts on ACO2 biophysical properties. Skin derived fibroblasts from patients harbouring dominant and recessive ACO2 mutations revealed a reduction of ACO2 abundance and enzymatic activity, and the impairment of the mitochondrial respiration using citrate and pyruvate as substrates, while the addition of other Krebs cycle intermediates restored a normal respiration, suggesting a possible short-cut adaptation of the tricarboxylic citric acid cycle. Analysis of the mitochondrial genome abundance disclosed a significant reduction of the mitochondrial DNA amount in all ACO2 fibroblasts. Overall, our data position ACO2 as the third most frequently mutated gene in autosomal inherited optic neuropathies, after OPA1 and WFS1, and emphasize the crucial involvement of the first steps of the Krebs cycle in the maintenance and survival of retinal ganglion cells.

Cancer/Testis Antigens into mitochondria: a hub between spermatogenesis, tumorigenesis and mitochondrial physiology adaptation.

Cancer/Testis Antigens (CTAs) genes are expressed only during spermatogenesis and tumorigenesis. Both processes share common specific metabolic adaptation related to energy supply, with a glucose to lactate gradient, leading to changes in mitochondrial physiology paralleling CTAs expression. In this review, we address the role of CTAs in mitochondria (mitoCTAs), by reviewing all published data, and assessing the putative localization of CTAs by screening for the presence of a mitochondrial targeting sequence (MTS). We evidenced that among the 276 CTAs, five were already shown to interfere with mitochondrial activities and 67 display a potential MTS.

Phostine 3.1a as a pharmacological compound with antiangiogenic properties against diseases with excess vascularization.

Angiogenesis is a complex process leading to the growth of new blood vessels from existing vasculature, triggered by local proangiogenic factors such as VEGF. An excess of angiogenesis is a recurrent feature of various pathologic conditions such as tumor growth. Phostines are a family of synthetic glycomimetic compounds that exhibit anticancer properties, and the lead compound 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST 3.1a) shows antiglioblastoma properties both in vitro and in vivo. In the present study, we assessed the effect of PST 3.1a on angiogenesis and endothelial metabolism. In vitro, PST 3.1a (10 µM) inhibited all steps that regulate angiogenesis, including migration, proliferation, adhesion, and tube formation. In vivo, PST 3.1a reduced intersegmental vessel formation and vascularization of the subintestinal plexus in zebrafish embryos and also altered pathologic angiogenesis and glioblastoma progression in vivo. Mechanistically, PST 3.1a altered interaction of VEGF receptor 2 and glycosylation-regulating protein galectin-1, a key component regulating angiogenesis associated with tumor resistance. Thus, these data show that use of PST 3.1a is an innovative approach to target angiogenesis.-Bousseau, S., Marchand, M., Soleti, R., Vergori, L., Hilairet, G., Recoquillon, S., Le Mao, M., Gueguen, N., Khiati, S., Clarion, L., Bakalara, N., Martinez, M. C., Germain, S., Lenaers, G., Andriantsitohaina, R. Phostine 3.1a as a pharmacological compound with antiangiogenic properties against diseases with excess vascularization.

Alteration of Extracellular Nucleotide Metabolism in Pseudoxanthoma Elasticum.

Pseudoxanthoma elasticum (PXE) is a rare genetic condition primarily caused by hepatic ABCC6 transporter dysfunction. Most clinical manifestations of PXE are due to premature calcification of elastic fibers. However, the vascular impact of PXE is pleiotropic and remains ill defined. ABCC6 expression has recently been associated with cellular nucleotide export. We studied the impact of ABCC6 deficiency on blood levels of adenosine triphosphate and related metabolites and on soluble nucleotidase activities in PXE patients and Abcc6-/- mice. In addition, we investigated the expression of genes encoding ectocellular purinergic signaling proteins in mouse liver and aorta. Plasma adenosine triphosphate and pyrophosphate levels were significantly reduced in PXE patients and in Abcc6-/- mice, whereas adenosine concentration was not modified. Moreover, 5'-nucleotidase/CD73 activity was increased in the serum of PXE patients and Abcc6-/- mice. Consistent with alterations of purinergic signaling, the expression of genes involved in purine and phosphate transport/metabolism was dramatically modified in Abcc6-/- mouse aorta, with much less impact on the liver. ABCC6 deficiency causes impaired vascular homeostasis and tissue perfusion. Our findings suggest that these alterations are linked to changes in extracellular nucleotide metabolism that are remote from the liver. This opens new perspectives for the understanding of PXE pathophysiology.

Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2S short isoform.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.

Phostine PST3.1a Targets MGAT5 and Inhibits Glioblastoma-Initiating Cell Invasiveness and Proliferation.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor and accounts for a significant proportion of all primary brain tumors. Median survival after treatment is around 15 months. Remodeling of N-glycans by the N-acetylglucosamine glycosyltransferase (MGAT5) regulates tumoral development. Here, perturbation of MGAT5 enzymatic activity by the small-molecule inhibitor 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST3.1a) restrains GBM growth. In cell-based assays, it is demonstrated that PST3.1a alters the ß1,6-GlcNAc N-glycans of GBM-initiating cells (GIC) by inhibiting MGAT5 enzymatic activity, resulting in the inhibition of TGFßR and FAK signaling associated with doublecortin (DCX) upregulation and increase oligodendrocyte lineage transcription factor 2 (OLIG2) expression. PST3.1a thus affects microtubule and microfilament integrity of GBM stem cells, leading to the inhibition of GIC proliferation, migration, invasiveness, and clonogenic capacities. Orthotopic graft models of GIC revealed that PST3.1a treatment leads to a drastic reduction of invasive and proliferative capacity and to an increase in overall survival relative to standard temozolomide therapy. Finally, bioinformatics analyses exposed that PST3.1a cytotoxic activity is positively correlated with the expression of genes of the epithelial-mesenchymal transition (EMT), while the expression of mitochondrial genes correlated negatively with cell sensitivity to the compound. These data demonstrate the relevance of targeting MGAT5, with a novel anti-invasive chemotherapy, to limit glioblastoma stem cell invasion. Mol Cancer Res; 15(10); 1376-87. ©2017 AACR.

CLUH couples mitochondrial distribution to the energetic and metabolic status.

Mitochondrial dynamics and distribution are critical for supplying ATP in response to energy demand. CLUH is a protein involved in mitochondrial distribution whose dysfunction leads to mitochondrial clustering, the metabolic consequences of which remain unknown. To gain insight into the role of CLUH on mitochondrial energy production and cellular metabolism, we have generated CLUH-knockout cells using CRISPR/Cas9. Mitochondrial clustering was associated with a smaller cell size and with decreased abundance of respiratory complexes, resulting in oxidative phosphorylation (OXPHOS) defects. This energetic impairment was found to be due to the alteration of mitochondrial translation and to a metabolic shift towards glucose dependency. Metabolomic profiling by mass spectroscopy revealed an increase in the concentration of some amino acids, indicating a dysfunctional Krebs cycle, and increased palmitoylcarnitine concentration, indicating an alteration of fatty acid oxidation, and a dramatic decrease in the concentrations of phosphatidylcholine and sphingomyeline, consistent with the decreased cell size. Taken together, our study establishes a clear function for CLUH in coupling mitochondrial distribution to the control of cell energetic and metabolic status.

Lack of mitochondrial topoisomerase I (TOP1mt) impairs liver regeneration.

The liver has an exceptional replicative capacity following partial hepatectomy or chemical injuries. Cellular proliferation requires increased production of energy and essential metabolites, which critically depend on the mitochondria. To determine whether Top1mt, the vertebrate mitochondrial topoisomerase, is involved in this process, we studied liver regeneration after carbon tetrachloride (CCl4) administration. TOP1mt knockout (KO) mice showed a marked reduction in regeneration and hepatocyte proliferation. The hepatic mitochondrial DNA (mtDNA) failed to increase during recovery from CCl4 exposure. Reduced glutathione was also depleted, indicating increased reactive oxygen species (ROS). Steady-state levels of ATP, O2 consumption, mtDNA, and mitochondrial mass were also reduced in primary hepatocytes from CCl4-treated KO mice. To further test whether Top1mt acted by enabling mtDNA regeneration, we tested TOP1mt KO fibroblasts and human colon carcinoma HCT116 cells and measured mtDNA after 3-d treatment with ethidium bromide. Both types of TOP1mt knockout cells showed defective mtDNA regeneration following mtDNA depletion. Our study demonstrates that Top1mt is required for normal mtDNA homeostasis and for linking mtDNA expansion with hepatocyte proliferation.

Poisoning of mitochondrial topoisomerase I by lamellarin D.

Lamellarin D (Lam-D) is a hexacyclic pyrole alkaloid isolated from marine invertebrates, whose biologic properties have been attributed to mitochondrial targeting. Mitochondria contain their own DNA (mtDNA), and the only specific mitochondrial topoisomerase in vertebrates is mitochondrial topoisomerase I (Top1mt). Here, we show that Top1mt is a direct mitochondrial target of Lam-D. In vitro Lam-D traps Top1mt and induces Top1mt cleavage complexes (Top1mtcc). Using single-molecule analyses, we also show that Lam-D slows down supercoil relaxation of Top1mt and strongly inhibits Top1mt religation in contrast to the inefficacy of camptothecin on Top1mt. In living cells, we show that Lam-D accumulates rapidly inside mitochondria, induces cellular Top1mtcc, and leads to mtDNA damage. This study provides evidence that Top1mt is a direct mitochondrial target of Lam-D and suggests that developing Top1mt inhibitors represents a novel strategy for targeting mitochondrial DNA.

Increased negative supercoiling of mtDNA in TOP1mt knockout mice and presence of topoisomerases IIα and IIß in vertebrate mitochondria.

Topoisomerases are critical for replication, DNA packing and repair, as well as for transcription by allowing changes in DNA topology. Cellular DNA is present both in nuclei and mitochondria, and mitochondrial topoisomerase I (Top1mt) is the only DNA topoisomerase specific for mitochondria in vertebrates. Here, we report in detail the generation of TOP1mt knockout mice, and demonstrate that mitochondrial DNA (mtDNA) displays increased negative supercoiling in TOP1mt knockout cells and murine tissues. This finding suggested imbalanced topoisomerase activity in the absence of Top1mt and the activity of other topoisomerases in mitochondria. Accordingly, we found that both Top2α and Top2ß are present and active in mouse and human mitochondria. The presence of Top2α-DNA complexes in the mtDNA D-loop region, at the sites where both ends of 7S DNA are positioned, suggests a structural role for Top2 in addition to its classical topoisomerase activities.

Mitochondrial topoisomerase I (top1mt) is a novel limiting factor of doxorubicin cardiotoxicity.

PURPOSE: Doxorubicin is one of the most effective chemotherapeutic agents. However, up to 30% of the patients treated with doxorubicin suffer from congestive heart failure. The mechanism of doxorubicin cardiotoxicity is likely multifactorial and most importantly, the genetic factors predisposing to doxorubicin cardiotoxicity are unknown. On the basis of the fact that mtDNA lesions and mitochondrial dysfunctions have been found in human hearts exposed to doxorubicin and that mitochondrial topoisomerase 1 (Top1mt) specifically controls mtDNA homeostasis, we hypothesized that Top1mt knockout (KO) mice might exhibit hypersensitivity to doxorubicin. EXPERIMENTAL DESIGN: Wild-type (WT) and KO Top1mt mice were treated once a week with 4 mg/kg doxorubicin for 8 weeks. Heart tissues were analyzed one week after the last treatment. RESULTS: Genetic inactivation of Top1mt in mice accentuates mtDNA copy number loss and mtDNA damage in heart tissue following doxorubicin treatment. Top1mt KO mice also fail to maintain respiratory chain protein production and mitochondrial cristae ultrastructure organization. These mitochondrial defects result in decreased O2 consumption, increased reactive oxygen species production, and enhanced heart muscle damage in animals treated with doxorubicin. Accordingly, Top1mt KO mice die within 45 days after the last doxorubicin injection, whereas the WT mice survive. CONCLUSIONS: Our results provide evidence that Top1mt, which is conserved across vertebrates, is critical for cardiac tolerance to doxorubicin and adaptive response to doxorubicin cardiotoxicity. They also suggest the potential of Top1mt single-nucleotide polymorphisms testing to investigate patient susceptibility to doxorubicin-induced cardiotoxicity.

Efficient delivery of therapeutic small nucleic acids to prostate cancer cells using ketal nucleoside lipid nanoparticles.

A novel nucleoside lipid derived from dioleyl ketal was synthesized from uridine in three steps starting from dioleyl ketone. Electronic microscopy studies show that Ketals Nucleoside Lipids (KNL) self-assemble to form liposome-like structures in aqueous solutions. KNL is able to bind siRNA as demonstrated by electrophoresis experiment and standard ethidium bromide fluorescence displacement assay. Transfection assays of stable hepatic cell lines HupIRF, carrying a luciferase reporter gene demonstrate that KNL is able to transfect siRNA and exhibits protein knockdown more efficiently than its diester analog (DOTAU) and lipofectamine. Herein, we also report that KNLs are suitable transfecting reagents for the development of novel therapeutic approaches involving either siRNA or antisense oligonucleotide against human prostate cancer PC-3 cells resistant to chemotherapy.

Nucleoside-lipid-based nanoparticles for cisplatin delivery.

The use of delivery vehicles to selectively transport anticancer agents to tumors is very attractive to address both toxicity and efficacy issues. We report a novel approach based on hybrid nucleoside-lipids allowing the efficient encapsulation and delivery of cisplatin. We demonstrate that the nucleoside polar heads guide the self-assembly of the aggregates into highly loaded and stable nanoparticles. The nanoparticles, which are efficient vehicles for the delivery of cisplatin into different sensitive and resistant cancer cell lines, can overcome the disadvantages and limitations of drug delivery systems previously reported.

Reduction-triggered delivery using nucleoside-lipid based carriers possessing a cleavable PEG coating.

A new non-ionic nucleoside based lipid (DOU-SS-PEG(2000), 5'-PEG(2000)-2',3'-dioleoyluridine) featuring uridine (U) as nucleoside and 2',3'-dioleyl (DO), as lipid moieties and a poly(ethylene glycol) (PEG) thiolytic cleavable spacer for in vitro delivery of drugs is described. The PEG detachable nucleotide lipid (DOU-SS-PEG(2000)) was prepared via a convergent synthesis starting from HS-PEG-OMe and uridine. The reduction-triggered delivery using the PEG detachable nucleoside lipid DOU-SS-PEG(2000) was evaluated on both liposomal and micellar objects. The liposomes were prepared from of a mixture of DOTAU (N-[5'-(2',3'-dioleoyl)uridine]-N',N',N'-trimethylammonium tosylate), the PEG detachable nucleoside lipid DOU-SS-PEG(2000) and DOPE-rhodamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl ammonium salt) (60/40/0.1), whereas a mixture of 99.9% of DOU-SS-PEG(2000) and 0.1% of DOPE-rhodamine was used to prepare micelles. In addition, the supramolecular systems underwent a reduction-induced morphology transition from a micellar to vesicular states, which was characterized by DLS, zeta potential and TEM. The disulfide bond of the PEG chain was cleaved, by adding a reducing agent such as dithiothréitol (DTT), to expose the cationic surface of the liposome. The internalization of the resulting liposomes was facilitated as shown by the enhanced fluorescence signal observed in ovarian cancer cells (SKOV3) compared to the pegylated liposome. Likewise, when DTT was added to the mixture of cells incubated in the presence of DOU-SS-PEG(2000)/DOPE-rhodamine micelle, the fluorescence was observed in almost 100% of the SKOV3 cells.