MFN2-driven mitochondria-to-nucleus tethering allows a non-canonical nuclear entry pathway of the mitochondrial pyruvate dehydrogenase complex.

The mitochondrial pyruvate dehydrogenase complex (PDC) translocates into the nucleus, facilitating histone acetylation by producing acetyl-CoA. We describe a noncanonical pathway for nuclear PDC (nPDC) import that does not involve nuclear pore complexes (NPCs). Mitochondria cluster around the nucleus in response to proliferative stimuli and tether onto the nuclear envelope (NE) via mitofusin-2 (MFN2)-enriched contact points. A decrease in nuclear MFN2 levels decreases mitochondria tethering and nPDC levels. Mitochondrial PDC crosses the NE and interacts with lamin A, forming a ring below the NE before crossing through the lamin layer into the nucleoplasm, in areas away from NPCs. Effective blockage of NPC trafficking does not decrease nPDC levels. The PDC-lamin interaction is maintained during cell division, when lamin depolymerizes and disassembles before reforming daughter nuclear envelopes, providing another pathway for nPDC entry during mitosis. Our work provides a different angle to understanding mitochondria-to-nucleus communication and nuclear metabolism.

Metabolic Enzymes Moonlighting in the Nucleus: Metabolic Regulation of Gene Transcription.

During evolution, cells acquired the ability to sense and adapt to varying environmental conditions, particularly in terms of fuel supply. Adaptation to fuel availability is crucial for major cell decisions and requires metabolic alterations and differential gene expression that are often epigenetically driven. A new mechanistic link between metabolic flux and regulation of gene expression is through moonlighting of metabolic enzymes in the nucleus. This facilitates delivery of membrane-impermeable or unstable metabolites to the nucleus, including key substrates for epigenetic mechanisms such as acetyl-CoA which is used in histone acetylation. This metabolism-epigenetics axis facilitates adaptation to a changing environment in normal (e.g., development, stem cell differentiation) and disease states (e.g., cancer), providing a potential novel therapeutic target.

Role for DNA damage signaling in pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is associated with sustained inflammation known to promote DNA damage. Despite these unfavorable environmental conditions, PAH pulmonary arterial smooth muscle cells (PASMCs) exhibit, in contrast to healthy PASMCs, a pro-proliferative and anti-apoptotic phenotype, sustained in time by the activation of miR-204, nuclear factor of activated T cells, and hypoxia-inducible factor 1-α. We hypothesized that PAH-PASMCs have increased the activation of poly(ADP-ribose) polymerase-1 (PARP-1), a critical enzyme implicated in DNA repair, allowing proliferation despite the presence of DNA-damaging insults, eventually leading to PAH. METHODS AND RESULTS: Human PAH distal pulmonary arteries and cultured PAH-PASMCs exhibit increased DNA damage markers (53BP1 and γ-H2AX) and an overexpression of PARP-1 (immunoblot and activity assay), in comparison with healthy tissues/cells. Healthy PASMCs treated with a clinically relevant dose of tumor necrosis factor-α harbored a similar phenotype, suggesting that inflammation induces DNA damage and PARP-1 activation in PAH. We also showed that PARP-1 activation accounts for miR-204 downregulation (quantitative reverse transcription polymerase chain reaction) and the subsequent activation of the transcription factors nuclear factor of activated T cells and hypoxia-inducible factor 1-α in PAH-PASMCs, previously shown to be critical for PAH in several models. These effects resulted in PASMC proliferation (Ki67, proliferating cell nuclear antigen, and WST1 assays) and resistance to apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling and Annexin V assays). In vivo, the clinically available PARP inhibitor ABT-888 reversed PAH in 2 experimental rat models (Sugen/hypoxia and monocrotaline). CONCLUSIONS: These results show for the first time that the DNA damage/PARP-1 signaling pathway is important for PAH development and provide a new therapeutic target for this deadly disease with high translational potential.

A reversible metabolic stress-sensitive regulation of CRMP2A orchestrates EMT/stemness and increases metastatic potential in cancer.

An epithelial-to-mesenchymal transition (EMT) phenotype with cancer stem cell-like properties is a critical feature of aggressive/metastatic tumors, but the mechanism(s) that promote it and its relation to metabolic stress remain unknown. Here we show that Collapsin Response Mediator Protein 2A (CRMP2A) is unexpectedly and reversibly induced in cancer cells in response to multiple metabolic stresses, including low glucose and hypoxia, and inhibits EMT/stemness. Loss of CRMP2A, when metabolic stress decreases (e.g., around blood vessels in vivo) or by gene deletion, induces extensive microtubule remodeling, increased glutamine utilization toward pyrimidine synthesis, and an EMT/stemness phenotype with increased migration, chemoresistance, tumor initiation capacity/growth, and metastatic potential. In a cohort of 27 prostate cancer patients with biopsies from primary tumors and distant metastases, CRMP2A expression decreases in the metastatic versus primary tumors. CRMP2A is an endogenous molecular brake on cancer EMT/stemness and its loss increases the aggressiveness and metastatic potential of tumors.

SNPs for Genes Encoding the Mitochondrial Proteins Sirtuin3 and Uncoupling Protein 2 Are Associated With Disease Severity, Type 2 Diabetes, and Outcomes in Patients With Pulmonary Arterial Hypertension and This Is Recapitulated in a New Mouse Model Lacking Both Genes.

Background Isolated loss-of-function single nucleotide polymorphisms (SNPs) for SIRT3 (a mitochondrial deacetylase) and UCP2 (an atypical uncoupling protein enabling mitochondrial calcium entry) have been associated with both pulmonary arterial hypertension (PAH) and insulin resistance, but their collective role in animal models and patients is unknown. Methods and Results In a prospective cohort of patients with PAH (n=60), we measured SNPs for both SIRT3 and UCP2, along with several clinical features (including invasive hemodynamic data) and outcomes. We found SIRT3 and UCP2 SNPs often both in the same patient in a homozygous or heterozygous manner, correlating positively with PAH severity and associated with the presence of type 2 diabetes and 10-year outcomes (death and transplantation). To explore this mechanistically, we generated double knockout mice for Sirt3 and Ucp2 and found increasing severity of PAH (mean pulmonary artery pressure, right ventricular hypertrophy/dilatation and extensive vascular remodeling, including inflammatory plexogenic lesions, in a gene dose-dependent manner), along with insulin resistance, compared with wild-type mice. The suppressed mitochondrial function (decreased respiration, increased mitochondrial membrane potential) in the double knockout pulmonary artery smooth muscle cells was associated with apoptosis resistance and increased proliferation, compared with wild-type mice. Conclusions Our work supports the metabolic theory of PAH and shows that these mice exhibit spontaneous severe PAH (without environmental or chemical triggers) that mimics human PAH and may explain the findings in our patient cohort. Our study offers a new mouse model of PAH, with several features of human disease that are typically absent in other PAH mouse models.

Interaction with p53 explains a pro-proliferative function for VHL in cancer.

The von Hippel-Lindau (VHL) protein binds and degrades hypoxia-inducible factors (HIF) hydroxylated by prolyl-hydroxylases under normoxia. Although originally described as a tumor suppressor, there is growing evidence that VHL may paradoxically promote tumor growth. The significance of its described interactions with many other proteins remains unclear. We found that VHL interacts with p53, preventing its tetramerization, promoter binding and expression of its target genes p21, PUMA, and Bax. VHL limited the decrease in proliferation and increase in apoptosis caused by p53 activation, independent of prolyl-hydroxylation and HIF activity, and its presence in tumors caused a resistance to p53-inducing chemotherapy in vivo. We propose that VHL has both anti-tumor function, via HIF degradation, and a new pro-tumor function via p53 target (p21, PUMA, Bax) inhibition. Because p53 plays a critical role in tumor biology, is activated by many chemotherapies, and because VHL levels vary among different tumors and its function can even be lost by mutations in some tumors, our results have important clinical applications. KEY MESSAGES: VHL and p53 physically interact and VHL inhibits p53 activity by limiting the formation of p53 tetramers. VHL attenuates the expression of p53 target genes in response to p53 stimuli. The inhibition of p53 by VHL is independent of HIF and prolyl-hydroxylation.

Metabolic Modulation of Clear-cell Renal Cell Carcinoma with Dichloroacetate, an Inhibitor of Pyruvate Dehydrogenase Kinase.

BACKGROUND: Clear-cell renal cell carcinoma (ccRCC) exhibits suppressed mitochondrial function and preferential use of glycolysis even in normoxia, promoting proliferation and suppressing apoptosis. ccRCC resistance to therapy is driven by constitutive hypoxia-inducible factor (HIF) expression due to genetic loss of von Hippel-Lindau factor. In addition to promoting angiogenesis, HIF suppresses mitochondrial function by inducing pyruvate dehydrogenase kinase (PDK), a gatekeeping enzyme for mitochondrial glucose oxidation. OBJECTIVE: To reverse mitochondrial suppression of ccRCC using the PDK inhibitor dichloroacetate (DCA). DESIGN, SETTING, AND PARTICIPANTS: Radical nephrectomy specimens from patients with ccRCC were assessed for PDK expression. The 786-O ccRCC line and two animal models (chicken in ovo and murine xenografts) were used for mechanistic studies. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Mitochondrial function, proliferation, apoptosis, HIF transcriptional activity, angiogenesis, and tumor size were measured in vitro and in vivo. Independent-sample t-tests and analysis of variance were used for statistical analyses. RESULTS: PDK was elevated in 786-O cells and in ccRCC compared to normal kidney tissue from the same patient. DCA reactivated mitochondrial function (increased respiration, Krebs cycle metabolites such as α-ketoglutarate [cofactor of factor inhibiting HIF], and mitochondrial reactive oxygen species), increased p53 activity and apoptosis, and decreased proliferation in 786-O cells. DCA reduced HIF transcriptional activity in an FIH-dependent manner, inhibiting angiogenesis in vitro. DCA reduced tumor size and angiogenesis in vivo in both animal models. CONCLUSIONS: DCA can reverse the mitochondrial suppression of ccRCC and decrease HIF transcriptional activity, bypassing its constitutive expression. Its previous clinical use in humans makes it an attractive candidate for translation to ccRCC patients. PATIENT SUMMARY: We show that an energy-boosting drug decreases tumor growth and tumor blood vessels in animals carrying human kidney cancer cells. This generic drug has been used in patients for other conditions and thus could be tested in kidney cancer that remains incurable.

Sirtuin 3 deficiency is associated with inhibited mitochondrial function and pulmonary arterial hypertension in rodents and humans.

Suppression of mitochondrial function promoting proliferation and apoptosis suppression has been described in the pulmonary arteries and extrapulmonary tissues in pulmonary arterial hypertension (PAH), but the cause of this metabolic remodeling is unknown. Mice lacking sirtuin 3 (SIRT3), a mitochondrial deacetylase, have increased acetylation and inhibition of many mitochondrial enzymes and complexes, suppressing mitochondrial function. Sirt3KO mice develop spontaneous PAH, exhibiting previously described molecular features of PAH pulmonary artery smooth muscle cells (PASMC). In human PAH PASMC and rats with PAH, SIRT3 is downregulated, and its normalization with adenovirus gene therapy reverses the disease phenotype. A loss-of-function SIRT3 polymorphism, linked to metabolic syndrome, is associated with PAH in an unbiased cohort of 162 patients and controls. If confirmed in large patient cohorts, these findings may facilitate biomarker and therapeutic discovery programs in PAH.

A metabolic remodeling in right ventricular hypertrophy is associated with decreased angiogenesis and a transition from a compensated to a decompensated state in pulmonary hypertension.

UNLABELLED: Right ventricular (RV) failure is an important clinical problem with no available therapies, largely because its molecular mechanisms are unknown. Mitochondrial remodeling resulting to a metabolic shift toward glycolysis has been described in RV hypertrophy (RVH), but it is unknown whether this is beneficial or detrimental. While clinically RV failure follows a period of compensation, the transition from a compensated (cRVH) to a decompensated hypertrophied RV (dRVH) is not studied in animal models. We modeled the natural history of RVH and failure in the monocrotaline rat model of pulmonary hypertension by serially assessing clinically relevant parameters in the same animal. We defined dRVH as the stage in which RV systolic pressure started decreasing, along with the cardiac output, while the RV continued to remodel. dRVH was characterized by ascites, weight loss, and high mortality, compared to cRVH. A cRVH myocardium had hyperpolarized mitochondria and low production of mitochondria-derived reactive oxygen species (mROS), activated hypoxia-inducible factor 1α (HIF1α), and increased levels of glucose transporter 1, vascular endothelial growth factor, and stromal-derived factor 1, promoting increased glucose uptake (measured by positron emission tomography-computed tomography) and angiogenesis measured by lectin imaging in vivo. The transition to dRVH was marked by a sharp rise in mROS, inhibition of HIF1α, and activation of p53, both of which contributed to down-regulation of pyruvate dehydrogenase kinase and decreased glucose uptake. This transition was also associated with a sharp decrease in angiogenic factors and angiogenesis. We show that the previously described metabolic shift, promoting HIF1α activation and angiogenesis, is not sustained during the progression of RV failure. The loss of this beneficial remodeling may be triggered by a rise in mROS resulting in HIF1α inhibition and suppressed angiogenesis. The resultant ischemia may contribute to the rapid deterioration of RV function upon entrance to a decompensation phase. The use of clinical criteria and techniques to define and study dRVH facilitates clinical translation of our findings with direct implications for RV therapeutic and biomarker discovery programs. KEY MESSAGE: Decreased RV angiogenesis marks the transition from a cRVH to a dRVH. The RVs in cRVH animals are associated with decreased mROS and increased HIF1α activity compared to dRVH. The RVs in cRVH animals have increased GLUT1 levels and increased glucose uptake compared to the dRVH.