The chaperone TRAP1 as a modulator of the mitochondrial adaptations in cancer cells

Mitochondria can receive, integrate, and transmit a variety of signals to shape many biochemical activities of the cell. In the process of tumor onset and growth, mitochondria contribute to the capability of cells of escaping death insults, handling changes in ROS levels, rewiring metabolism, and reprograming gene expression. Therefore, mitochondria can tune the bioenergetic and anabolic needs of neoplastic cells in a rapid and flexible way, and these adaptations are required for cell survival and proliferation in the fluctuating environment of a rapidly growing tumor mass. The molecular bases of pro-neoplastic mitochondrial adaptations are complex and only partially understood. Recently, the mitochondrial molecular chaperone TRAP1 (tumor necrosis factor receptor associated protein 1) was identified as a key regulator of mitochondrial bioenergetics in tumor cells, with a profound impact on neoplastic growth. In this review, we analyze these findings and discuss the possibility that targeting TRAP1 constitutes a new antitumor approach

Metabolic Features of Neurofibromatosis Type 1-Associated Tumors

Rewiring cellular metabolism is a key hallmark of cancer. Multiple evidences show that alterations in various metabolic circuits directly contribute to the tumorigenic process at different levels (e.g. cancer initiation, metastasis, resistance). However, the characterization of the metabolic profile of Neurofibromatosis type 1 (NF1)-related neoplastic cells has been only partially elucidated both in benign neurofibromas and in malignant peripheral nerve sheath tumors (MPNSTs). Here, we illustrate the state of the art on the knowledge of the metabolic features of tumors related to NF1 and discuss their potential implications for the development of novel therapeutic perspectives

Rational Design of Allosteric and Selective Inhibitors of the Molecular Chaperone TRAP1

Summary: TRAP1 is the mitochondrial paralog of the heat shock protein 90 (HSP90) chaperone family. Its activity as an energy metabolism regulator has important implications in cancer, neurodegeneration, and ischemia. Selective inhibitors of TRAP1 could inform on its mechanisms of action and set the stage for targeted drug development, but their identification was hampered by the similarity among active sites in HSP90 homologs. We use a dynamics-based approach to identify a TRAP1 allosteric pocket distal to its active site that can host drug-like molecules, and we select small molecules with optimal stereochemical features to target the pocket. These leads inhibit TRAP1, but not HSP90, ATPase activity and revert TRAP1-dependent downregulation of succinate dehydrogenase activity in cancer cells and in zebrafish larvae. TRAP1 inhibitors are not toxic per se, but they abolish tumorigenic growth of neoplastic cells. Our results indicate that exploiting conformational dynamics can expand the chemical space of chaperone antagonists to TRAP1-specific inhibitors with wide therapeutic opportunities. : The molecular chaperone TRAP1 regulates energy metabolism, and its activity is relevant in cancer and degenerative diseases. Here, Sanchez-Martin et al. identify highly selective allosteric inhibitors of TRAP1. These compounds revert biochemical and pro-neoplastic effects of TRAP1 and could both enlighten its mode of action and disclose novel therapeutic strategies. Keywords: chaperone inhibitors, anticancer compound, molecular dynamics, allosteric ligands, TRAP1, HSP90, mitochondria, mitochondrial biology, zebrafish, cancer cells, neurofibrom

Metabolic reprogramming identifies the most aggressive lesions at early phases of hepatic carcinogenesis

Metabolic changes are associated with cancer, but whether they are just bystander effects of deregulated oncogenic signaling pathways or characterize early phases of tumorigenesis remains unclear. Here we show in a rat model of hepatocarcinogenesis that early preneoplastic foci and nodules that progress towards hepatocellular carcinoma (HCC) are characterized both by inhibition of oxidative phosphorylation (OXPHOS) and by enhanced glucose utilization to fuel the pentose phosphate pathway (PPP). These changes respectively require increased expression of the mitochondrial chaperone TRAP1 and of the transcription factor NRF2 that induces the expression of the rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase (G6PD), following miR-1 inhibition. Such metabolic rewiring exclusively identifies a subset of aggressive cytokeratin-19 positive preneoplastic hepatocytes and not slowly growing lesions. No such metabolic changes were observed during non-neoplastic liver regeneration occurring after two/third partial hepatectomy. TRAP1 silencing inhibited the colony forming ability of HCC cells while NRF2 silencing decreased G6PD expression and concomitantly increased miR-1; conversely, transfection with miR-1 mimic abolished G6PD expression. Finally, in human HCC patients increased G6PD expression levels correlates with grading, metastasis and poor prognosis. Our results demonstrate that the metabolic deregulation orchestrated by TRAP1 and NRF2 is an early event restricted to the more aggressive preneoplastic lesions

The role of mitochondria in shaping the oncogenic signaling

Cancer cells are endowed with the capability to reprogram cell metabolism in order to support neoplastic growth and cell death escape. A key role in this process is played by signaling pathways, mainly cascades regulated by kinases. Integration of survival and death stimuli occurs on mitochondria, where many of these signals converge to the regulation of a channel, the permeability transition pore (PTP), that triggers mitochondrial depolarization and release of pro-apoptotic factors from mitochondria. PTP opening commits cells to death, and it is regulated by a variety of factors. Here I have studied how signal transduction pathways affect cell metabolism, particularly mitochondrial bioenergetics, which could eventually lead to modulation of PTP opening. In the first part of my work, I have explored the presence of the PTP in cells depleted of mitochondrial DNA (ρ0 cells) which lack respiration and constitute a model for the analysis of mitochondrial involvement in several pathological conditions, among which cancer. I have observed that these cells are indeed equipped with a functioning PTP, whose regulatory mechanisms are similar to those observed in cancer cells. In detail, inhibition of PTP opening is a survival mechanism achieved by two different mechanisms: the first one is the mitochondrial binding of the glycolytic enzyme hexokinase (HK) II, which is up-regulated in ρ0 cells; the second one relies on the hyper-activation of the ERK-GSK3 signaling pathway that converge on mitochondria where it maintains the PTP regulator cyclophilin D (CyP-D) in the dephosphorylated form. I observed that mitochondria of ρ0 cells maintain a membrane potential which is readily dissipated after displacement of HK II from the mitochondrial surface by treatment with either the drug clotrimazole or with a cell-permeant HK II peptide, or by keeping ρ0 cells in serum and glucose starvation. The PTP inhibitor cyclosporin A (CsA) is able to decrease the mitochondrial depolarization induced by either HK II displacement or by nutrient depletion. Moreover, glucose and serum deprivation causes concomitant ERK1/2 inhibition and GSK3α/β activation with the ensuing phosphorylation of CyP-D and PTP opening. Indeed, GSK3α/β inhibition with indirubin-3′-oxime decreases PTP-induced cell death in ρ0 cells following nutrient ablation. In the second part of my work, I have focused my attention on a tumor predisposition syndrome, called Neurofibromatosis type 1 (NF1), caused by loss of function of neurofibromin, which acts as a negative regulator of the Ras signaling cascades. Here I have investigated whether the Ras hyper-activation induced by neurofibromin ablation can affect mitochondria bioenergetics, thus contributing to the metabolic rewiring of NF1 tumors. I observed that the absence of neurofibromin confers to mouse embryonic fibroblasts (MEFs) the capability to form colonies in an in vitro tumorigenesis assay, and that the use of an ERK inhibitor completely abrogates colony formation. Moreover, I injected wild-type and Nf1-/- MEFs subcutaneously into nude mice in order to test the capability of the cells to form tumors in vivo. I observed that the absence of Nf1 leads to the growth of a tumor mass within a month, whereas no tumor can be formed in the same time frame by wild-type MEFs. I started the analysis of mitochondrial bioenergetics by measurements of oxygen consumption rate (OCR) performed with an extracellular flux analyzer on adherent cell monolayers. Nf1-/- MEFs display a lower OCR than wild-type MEFs both in basal condition and upon treatment with a low concentration of an uncoupler, which stimulates respiration maximally. Moreover, I found that the fraction of oxygen consumption coupled to ATP production by mitochondrial ATP synthase is lower in cells without Nf1, suggesting that ATP requirements are mainly supplied by glycolysis. Accordingly, upon glucose starvation or inhibition of glycolysis with 5-thioglucose Nf1-/- cells display a stronger decrease in ATP levels compared to wild-type cells. The different OCRs between wild-type and Nf1-/- cells are not related to differences in mitochondrial mass or membrane potential. In order to establish whether a modulation of respiratory chain activity could account for the observed OCR differences, I analyzed the expression level of the respiratory chain complexes. Complex I (NADH dehydrogenase) is down-regulated by Nf1 ablation, as a lower level of some of its subunits and of the assembled complex I was detected. Furthermore, the enzymatic activity of complex I is expectedly lower in Nf1-/- cells. Interestingly, administration of an ERK inhibitor increases the expression of complex I subunits resulting in its augmented assembly and activity; remarkably, this effect is more pronounced in Nf1-/- MEFs than in wild-type cells. I also observed down-regulation of complex II (succinate dehydrogenase) activity in knock-out cells compared to wild-type MEFs. Interestingly, I found that complex II interacts with the mitochondrial chaperone TRAP1 and the kinase ERK, a fraction of which is mitochondrial; these interactions are stronger in Nf1-/- than in wild-type cells, as assessed by blue native gel analysis. However, silencing of TRAP1 does not change the activity of complex II; in accordance to this, the OCR of cells in which TRAP1 has been down-regulated does not vary, yet modulation of TRAP1 levels affects maximal respiration of Nf1-/- cells. Interestingly, silencing of TRAP1 in Nf1-deficient MEFs compromises the tumorigenic properties of these cells. Taken together, these observations suggest that the absence of neurofibromin leads to a more glycolytic metabolism by down-modulating the activity of respiratory chain complexes. The metabolic switch observed upon Nf1 deletion is a typical marker of cancer cells that favors tumor progression. We hypothesize that Ras-ERK signaling is upstream to the regulation of mitochondrial bioenergetics, and that the metabolic changes prompted by Ras-ERK activation can contribute to the transformed phenotype that we observe in Nf1-/- MEFs.Le cellule tumorali hanno la capacità di riprogrammare il metabolismo al fine di sostenere la crescita cellulare e di sfuggire ai segnali di morte. Un ruolo centrale in questo processo è a carico delle vie di segnale, principalmente regolate da chinasi. L’integrazione degli stimoli di morte e di sopravvivenza avviene nei mitocondri, dove molti di questi segnali convergono sulla regolazione di un canale, il poro di transizione di permeabilità (PTP), che è responsabile della depolarizzazione mitocondriale con il conseguente rilascio di fattori pro-apoptotici dall’organello. L’apertura del PTP porta le cellule alla morte ed è regolato da un’ampia varietà di fattori. In questo lavoro ho studiato come i meccanismi di trasduzione del segnale impattano sul metabolismo cellulare, in particolare sulla bioenergetica mitocondriale, giungendo quindi ad una modulazione dell’apertura del PTP. Nella prima parte del mio lavoro ho indagato la presenza del PTP in cellule depletate del DNA mitocondriale (cellule ρ0) che presentano una totale assenza della respirazione e costituiscono un buon modello per l’analisi del coinvolgimento dei mitocondri in molte condizioni patologiche tra cui il cancro. Ho osservato che queste cellule possiedono un PTP funzionale, i cui meccanismi di regolazione sono simili a quelli presenti nelle cellule tumorali. Nel dettaglio, l’inibizione dell’apertura del PTP è un meccanismo di sopravvivenza raggiunto attraverso due meccanismi: il primo riguarda la localizzazione mitocondriale dell’enzima glicolitico esochinasi (HK) II, che è over-espresso nelle cellule ρ0; il secondo meccanismo è basato sull’iper-attivazione della via di segnale ERK-GSK3 che converge sui mitocondri dove mantiene il regolatore del poro, la ciclofilina D (CyP-D), nello stato defosforilato e quindi inattivo. Ho osservato che i mitocondri delle cellule ρ0 mantengono un potenziale di membrane che è dissipato in seguito alla dislocazione del HK II dalla superficie mitocondriale attraverso trattamento con clotrimazolo o tramite l’uso di un peptide che permea le membrane, o, in alternativa, a seguito di deplezione di siero e glucosio. L’inibitore del poro, la ciclosporina A (CsA), è in grado di diminuire la depolarizzazione mitocondriale indotta dalla dislocazione del HK II o dalla deplezione di nutrienti. Inoltre, la deplezione di siero e glucosio causa una concomitante inibizione di ERK1/2, un’attivazione di GSK3α/β e una conseguente fosforilazione di CyP-D con apertura del PTP. Infatti, inibendo GSK3α/β con l’indirubina, la morte cellulare causata dall’apertura del poro in seguito a deprivazione di nutrienti è diminuita. Nella seconda parte del mio lavoro mi sono focalizzata su una sindrome che predispone i pazienti all’insorgenza di tumori, la Neurofibromatosi di tipo 1, causata da perdita di funzione della neurofibromina, un regolatore negativo della via di segnale dominata da Ras. Ho studiato la possibilità che l’iper-attivazione della via di Ras dovuta all’inattivazione della neurofibromina possa modificare la bioenergetica mitocondriale, contribuendo così a cambiamenti metabolici in tumori di pazienti con NF1. Ho osservato che l’assenza della neurofibromina in fibroblasti embrionali di topo (MEFs) conferisce la capacità di formare colonie in un saggio tumorigenico in vitro, mentre il trattamento con un inibitore di ERK inibisce la formazione delle colonie in modo significativo. Inoltre, ho iniettato queste cellule sotto cute in topi nudi al fine di testare il loro potenziale tumorigenico in vivo. Ho osservato che l’assenza di neurofibromina porta alla crescita di una massa tumorale nell’arco di un mese dall’iniezione, mentre non si osservano masse tumorali nel caso di cellule wild-type. Ho iniziato l’analisi del profilo bioenergetico mitocondriale misurando il consumo di ossigeno in cellule adese ed ho osservato che cellule senza neurofibromina presentano un diminuito tasso di respirazione basale, oltre che una minore respirazione massima raggiunta dopo trattamento con basse concentrazioni di un disaccoppiante. Inoltre, la frazione di respirazione accoppiata alla produzione di ATP da parte dell’ATP sintasi è minore nelle cellule senza neurofibromina, suggerendo che le richieste energetiche sono principalmente a carico della glicolisi. In accordo a questi dati, in seguito a deplezione di glucosio o all'inibizione della glicolisi con 5-tioglucosio le cellule assenti della neurofibromina presentano un maggiore calo nei livelli di ATP rispetto a cellule wild-type. Le differenze del consumo di ossigeno, inoltre, non sono dovute a cambiamenti nella massa mitocondriale o nel potenziale di membrana. Al fine di determinare se possibili differenze nell’attività della catena respiratoria siano la causa di una diversa respirazione, ho analizzato l’espressione proteica di diverse subunità dei complessi respiratori. Il complesso I (NADH deidrogenasi) è diminuito in seguito alla deplezione della neurofibromina, così come la sua attività. Inoltre, la repressione farmacologica di ERK in cellule knock-out è in grado di aumentare specificatamente l’espressione delle subunità del complesso I, e quindi la sua attività; al contrario, questo effetto è meno forte nelle cellule wild-type. Ho osservato anche una diminuita attività del complesso II (succinato deidrogenasi) nelle cellule knock-out. Inoltre, ho trovato un’interazione tra il complesso II, lo sciaperone mitocondriale TRAP1 e la chinasi ERK, una frazione della quale è stata trovata nei mitocondri; queste interazioni sono molto più forti nelle cellule knock-out rispetto a quelle wild-type. Tuttavia, il silenziamento di TRAP1 non causa una variazione dell’attività del complesso II, bensì un’aumentata respirazione massima nelle cellule senza neurofibromina. Inoltre, la diminuita espressione di TRAP1 causa la perdita del potenziale tumorigenico delle cellule knock-out. Questi risultati suggeriscono che l’assenza della neurofibromina porta ad un fenotipo glicolitico con una diminuzione della respirazione mitocondriale. Questo cambiamento metabolico è tipico delle cellule tumorali poiché favorisce la progressione tumorale. Abbiamo quindi ipotetizzato che i cambiamenti metabolici causati dall’iper-attivazione della via di segnale Ras-ERK possano contribuire al fenotipo trasformante che abbiamo osservato nelle cellule senza neurofibromina

The role of mitochondria in shaping the oncogenic signaling

Cancer cells are endowed with the capability to reprogram cell metabolism in order to support neoplastic growth and cell death escape. A key role in this process is played by signaling pathways, mainly cascades regulated by kinases. Integration of survival and death stimuli occurs on mitochondria, where many of these signals converge to the regulation of a channel, the permeability transition pore (PTP), that triggers mitochondrial depolarization and release of pro-apoptotic factors from mitochondria. PTP opening commits cells to death, and it is regulated by a variety of factors. Here I have studied how signal transduction pathways affect cell metabolism, particularly mitochondrial bioenergetics, which could eventually lead to modulation of PTP opening. In the first part of my work, I have explored the presence of the PTP in cells depleted of mitochondrial DNA (ρ0 cells) which lack respiration and constitute a model for the analysis of mitochondrial involvement in several pathological conditions, among which cancer. I have observed that these cells are indeed equipped with a functioning PTP, whose regulatory mechanisms are similar to those observed in cancer cells. In detail, inhibition of PTP opening is a survival mechanism achieved by two different mechanisms: the first one is the mitochondrial binding of the glycolytic enzyme hexokinase (HK) II, which is up-regulated in ρ0 cells; the second one relies on the hyper-activation of the ERK-GSK3 signaling pathway that converge on mitochondria where it maintains the PTP regulator cyclophilin D (CyP-D) in the dephosphorylated form. I observed that mitochondria of ρ0 cells maintain a membrane potential which is readily dissipated after displacement of HK II from the mitochondrial surface by treatment with either the drug clotrimazole or with a cell-permeant HK II peptide, or by keeping ρ0 cells in serum and glucose starvation. The PTP inhibitor cyclosporin A (CsA) is able to decrease the mitochondrial depolarization induced by either HK II displacement or by nutrient depletion. Moreover, glucose and serum deprivation causes concomitant ERK1/2 inhibition and GSK3α/β activation with the ensuing phosphorylation of CyP-D and PTP opening. Indeed, GSK3α/β inhibition with indirubin-3′-oxime decreases PTP-induced cell death in ρ0 cells following nutrient ablation. In the second part of my work, I have focused my attention on a tumor predisposition syndrome, called Neurofibromatosis type 1 (NF1), caused by loss of function of neurofibromin, which acts as a negative regulator of the Ras signaling cascades. Here I have investigated whether the Ras hyper-activation induced by neurofibromin ablation can affect mitochondria bioenergetics, thus contributing to the metabolic rewiring of NF1 tumors. I observed that the absence of neurofibromin confers to mouse embryonic fibroblasts (MEFs) the capability to form colonies in an in vitro tumorigenesis assay, and that the use of an ERK inhibitor completely abrogates colony formation. Moreover, I injected wild-type and Nf1-/- MEFs subcutaneously into nude mice in order to test the capability of the cells to form tumors in vivo. I observed that the absence of Nf1 leads to the growth of a tumor mass within a month, whereas no tumor can be formed in the same time frame by wild-type MEFs. I started the analysis of mitochondrial bioenergetics by measurements of oxygen consumption rate (OCR) performed with an extracellular flux analyzer on adherent cell monolayers. Nf1-/- MEFs display a lower OCR than wild-type MEFs both in basal condition and upon treatment with a low concentration of an uncoupler, which stimulates respiration maximally. Moreover, I found that the fraction of oxygen consumption coupled to ATP production by mitochondrial ATP synthase is lower in cells without Nf1, suggesting that ATP requirements are mainly supplied by glycolysis. Accordingly, upon glucose starvation or inhibition of glycolysis with 5-thioglucose Nf1-/- cells display a stronger decrease in ATP levels compared to wild-type cells. The different OCRs between wild-type and Nf1-/- cells are not related to differences in mitochondrial mass or membrane potential. In order to establish whether a modulation of respiratory chain activity could account for the observed OCR differences, I analyzed the expression level of the respiratory chain complexes. Complex I (NADH dehydrogenase) is down-regulated by Nf1 ablation, as a lower level of some of its subunits and of the assembled complex I was detected. Furthermore, the enzymatic activity of complex I is expectedly lower in Nf1-/- cells. Interestingly, administration of an ERK inhibitor increases the expression of complex I subunits resulting in its augmented assembly and activity; remarkably, this effect is more pronounced in Nf1-/- MEFs than in wild-type cells. I also observed down-regulation of complex II (succinate dehydrogenase) activity in knock-out cells compared to wild-type MEFs. Interestingly, I found that complex II interacts with the mitochondrial chaperone TRAP1 and the kinase ERK, a fraction of which is mitochondrial; these interactions are stronger in Nf1-/- than in wild-type cells, as assessed by blue native gel analysis. However, silencing of TRAP1 does not change the activity of complex II; in accordance to this, the OCR of cells in which TRAP1 has been down-regulated does not vary, yet modulation of TRAP1 levels affects maximal respiration of Nf1-/- cells. Interestingly, silencing of TRAP1 in Nf1-deficient MEFs compromises the tumorigenic properties of these cells. Taken together, these observations suggest that the absence of neurofibromin leads to a more glycolytic metabolism by down-modulating the activity of respiratory chain complexes. The metabolic switch observed upon Nf1 deletion is a typical marker of cancer cells that favors tumor progression. We hypothesize that Ras-ERK signaling is upstream to the regulation of mitochondrial bioenergetics, and that the metabolic changes prompted by Ras-ERK activation can contribute to the transformed phenotype that we observe in Nf1-/- MEFs.Le cellule tumorali hanno la capacità di riprogrammare il metabolismo al fine di sostenere la crescita cellulare e di sfuggire ai segnali di morte. Un ruolo centrale in questo processo è a carico delle vie di segnale, principalmente regolate da chinasi. L’integrazione degli stimoli di morte e di sopravvivenza avviene nei mitocondri, dove molti di questi segnali convergono sulla regolazione di un canale, il poro di transizione di permeabilità (PTP), che è responsabile della depolarizzazione mitocondriale con il conseguente rilascio di fattori pro-apoptotici dall’organello. L’apertura del PTP porta le cellule alla morte ed è regolato da un’ampia varietà di fattori. In questo lavoro ho studiato come i meccanismi di trasduzione del segnale impattano sul metabolismo cellulare, in particolare sulla bioenergetica mitocondriale, giungendo quindi ad una modulazione dell’apertura del PTP. Nella prima parte del mio lavoro ho indagato la presenza del PTP in cellule depletate del DNA mitocondriale (cellule ρ0) che presentano una totale assenza della respirazione e costituiscono un buon modello per l’analisi del coinvolgimento dei mitocondri in molte condizioni patologiche tra cui il cancro. Ho osservato che queste cellule possiedono un PTP funzionale, i cui meccanismi di regolazione sono simili a quelli presenti nelle cellule tumorali. Nel dettaglio, l’inibizione dell’apertura del PTP è un meccanismo di sopravvivenza raggiunto attraverso due meccanismi: il primo riguarda la localizzazione mitocondriale dell’enzima glicolitico esochinasi (HK) II, che è over-espresso nelle cellule ρ0; il secondo meccanismo è basato sull’iper-attivazione della via di segnale ERK-GSK3 che converge sui mitocondri dove mantiene il regolatore del poro, la ciclofilina D (CyP-D), nello stato defosforilato e quindi inattivo. Ho osservato che i mitocondri delle cellule ρ0 mantengono un potenziale di membrane che è dissipato in seguito alla dislocazione del HK II dalla superficie mitocondriale attraverso trattamento con clotrimazolo o tramite l’uso di un peptide che permea le membrane, o, in alternativa, a seguito di deplezione di siero e glucosio. L’inibitore del poro, la ciclosporina A (CsA), è in grado di diminuire la depolarizzazione mitocondriale indotta dalla dislocazione del HK II o dalla deplezione di nutrienti. Inoltre, la deplezione di siero e glucosio causa una concomitante inibizione di ERK1/2, un’attivazione di GSK3α/β e una conseguente fosforilazione di CyP-D con apertura del PTP. Infatti, inibendo GSK3α/β con l’indirubina, la morte cellulare causata dall’apertura del poro in seguito a deprivazione di nutrienti è diminuita. Nella seconda parte del mio lavoro mi sono focalizzata su una sindrome che predispone i pazienti all’insorgenza di tumori, la Neurofibromatosi di tipo 1, causata da perdita di funzione della neurofibromina, un regolatore negativo della via di segnale dominata da Ras. Ho studiato la possibilità che l’iper-attivazione della via di Ras dovuta all’inattivazione della neurofibromina possa modificare la bioenergetica mitocondriale, contribuendo così a cambiamenti metabolici in tumori di pazienti con NF1. Ho osservato che l’assenza della neurofibromina in fibroblasti embrionali di topo (MEFs) conferisce la capacità di formare colonie in un saggio tumorigenico in vitro, mentre il trattamento con un inibitore di ERK inibisce la formazione delle colonie in modo significativo. Inoltre, ho iniettato queste cellule sotto cute in topi nudi al fine di testare il loro potenziale tumorigenico in vivo. Ho osservato che l’assenza di neurofibromina porta alla crescita di una massa tumorale nell’arco di un mese dall’iniezione, mentre non si osservano masse tumorali nel caso di cellule wild-type. Ho iniziato l’analisi del profilo bioenergetico mitocondriale misurando il consumo di ossigeno in cellule adese ed ho osservato che cellule senza neurofibromina presentano un diminuito tasso di respirazione basale, oltre che una minore respirazione massima raggiunta dopo trattamento con basse concentrazioni di un disaccoppiante. Inoltre, la frazione di respirazione accoppiata alla produzione di ATP da parte dell’ATP sintasi è minore nelle cellule senza neurofibromina, suggerendo che le richieste energetiche sono principalmente a carico della glicolisi. In accordo a questi dati, in seguito a deplezione di glucosio o all'inibizione della glicolisi con 5-tioglucosio le cellule assenti della neurofibromina presentano un maggiore calo nei livelli di ATP rispetto a cellule wild-type. Le differenze del consumo di ossigeno, inoltre, non sono dovute a cambiamenti nella massa mitocondriale o nel potenziale di membrana. Al fine di determinare se possibili differenze nell’attività della catena respiratoria siano la causa di una diversa respirazione, ho analizzato l’espressione proteica di diverse subunità dei complessi respiratori. Il complesso I (NADH deidrogenasi) è diminuito in seguito alla deplezione della neurofibromina, così come la sua attività. Inoltre, la repressione farmacologica di ERK in cellule knock-out è in grado di aumentare specificatamente l’espressione delle subunità del complesso I, e quindi la sua attività; al contrario, questo effetto è meno forte nelle cellule wild-type. Ho osservato anche una diminuita attività del complesso II (succinato deidrogenasi) nelle cellule knock-out. Inoltre, ho trovato un’interazione tra il complesso II, lo sciaperone mitocondriale TRAP1 e la chinasi ERK, una frazione della quale è stata trovata nei mitocondri; queste interazioni sono molto più forti nelle cellule knock-out rispetto a quelle wild-type. Tuttavia, il silenziamento di TRAP1 non causa una variazione dell’attività del complesso II, bensì un’aumentata respirazione massima nelle cellule senza neurofibromina. Inoltre, la diminuita espressione di TRAP1 causa la perdita del potenziale tumorigenico delle cellule knock-out. Questi risultati suggeriscono che l’assenza della neurofibromina porta ad un fenotipo glicolitico con una diminuzione della respirazione mitocondriale. Questo cambiamento metabolico è tipico delle cellule tumorali poiché favorisce la progressione tumorale. Abbiamo quindi ipotetizzato che i cambiamenti metabolici causati dall’iper-attivazione della via di segnale Ras-ERK possano contribuire al fenotipo trasformante che abbiamo osservato nelle cellule senza neurofibromina

Induction of the permeability transition pore in cells depleted of mitochondrial DNA

AbstractRespiratory complexes are believed to play a role in the function of the mitochondrial permeability transition pore (PTP), whose dysregulation affects the process of cell death and is involved in a variety of diseases, including cancer and degenerative disorders. We investigated here the PTP in cells devoid of mitochondrial DNA (ρ0 cells), which lack respiration and constitute a model for the analysis of mitochondrial involvement in several pathological conditions. We observed that mitochondria of ρ0 cells maintain a membrane potential and that this is readily dissipated after displacement of hexokinase (HK) II from the mitochondrial surface by treatment with either the drug clotrimazole or with a cell-permeant HK II peptide, or by placing ρ0 cells in a medium without serum and glucose. The PTP inhibitor cyclosporin A (CsA) could decrease the mitochondrial depolarization induced by either HK II displacement or by nutrient depletion. We also found that a fraction of the kinases ERK1/2 and GSK3α/β is located in the mitochondrial matrix of ρ0 cells, and that glucose and serum deprivation caused concomitant ERK1/2 inhibition and GSK3α/β activation with the ensuing phosphorylation of cyclophilin D, the mitochondrial target of CsA. GSK3α/β inhibition with indirubin-3′-oxime decreased PTP-induced cell death in ρ0 cells following nutrient ablation. These findings indicate that ρ0 cells are equipped with a functioning PTP, whose regulatory mechanisms are similar to those observed in cancer cells, and suggest that escape from PTP opening is a survival factor in this model of mitochondrial diseases. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012)

Metabolic Plasticity of Tumor Cell Mitochondria

Mitochondria are dynamic organelles that exchange a multiplicity of signals with other cell compartments, in order to finely adjust key biological routines to the fluctuating metabolic needs of the cell. During neoplastic transformation, cells must provide an adequate supply of the anabolic building blocks required to meet a relentless proliferation pressure. This can occur in conditions of inconstant blood perfusion leading to variations in oxygen and nutrient levels. Mitochondria afford the bioenergetic plasticity that allows tumor cells to adapt and thrive in this ever changing and often unfavorable environment. Here we analyse how mitochondria orchestrate the profound metabolic rewiring required for neoplastic growth

Contribution of the CK2 Catalytic Isoforms \u3b1 and \u3b1\u2019 to the Glycolytic Phenotype of Tumor Cells

CK2 is a Ser/Thr protein kinase overexpressed in many cancers. It is usually present in cells as a tetrameric enzyme, composed of two catalytic (α or α’) and two regulatory (β) subunits, but it is active also in its monomeric form, and the specific role of the different isoforms is largely unknown. CK2 phosphorylates several substrates related to the uncontrolled proliferation, motility, and survival of cancer cells. As a consequence, tumor cells are addicted to CK2, relying on its activity more than healthy cells for their life, and exploiting it for developing multiple oncological hallmarks. However, little is known about CK2 contribution to the metabolic rewiring of cancer cells. With this study we aimed at shedding some light on it, especially focusing on the CK2 role in the glycolytic onco-phenotype. By analyzing neuroblastoma and osteosarcoma cell lines depleted of either one (α) or the other (α’) CK2 catalytic subunit, we also aimed at disclosing possible pro-tumor functions which are specific of a CK2 isoform. Our results suggest that both CK2 α and α’ contribute to cell proliferation, survival and tumorigenicity. The analyzed metabolic features disclosed a role of CK2 in tumor metabolism, and suggest prominent functions for CK2 α isoform. Results were also confirmed by CK2 pharmacological inhibition. Overall, our study provides new information on the mechanism of cancer cells addiction to CK2 and on its isoform-specific functions, with fundamental implications for improving future therapeutic strategies based on CK2 targeting