Cytosolic and mitochondrial translation elongation are coordinated through the molecular chaperone TRAP1 for the synthesis and import of mitochondrial proteins.

A complex interplay between mRNA translation and cellular respiration has been recently unveiled, but its regulation in humans is poorly characterized in either health or disease. Cancer cells radically reshape both biosynthetic and bioenergetic pathways to sustain their aberrant growth rates. In this regard, we have shown that the molecular chaperone TRAP1 not only regulates the activity of respiratory complexes, behaving alternatively as an oncogene or a tumor suppressor, but also plays a concomitant moonlighting function in mRNA translation regulation. Herein, we identify the molecular mechanisms involved, showing that TRAP1 (1) binds both mitochondrial and cytosolic ribosomes, as well as translation elongation factors; (2) slows down translation elongation rate; and (3) favors localized translation in the proximity of mitochondria. We also provide evidence that TRAP1 is coexpressed in human tissues with the mitochondrial translational machinery, which is responsible for the synthesis of respiratory complex proteins. Altogether, our results show an unprecedented level of complexity in the regulation of cancer cell metabolism, strongly suggesting the existence of a tight feedback loop between protein synthesis and energy metabolism, based on the demonstration that a single molecular chaperone plays a role in both mitochondrial and cytosolic translation, as well as in mitochondrial respiration.

Cytosolic and mitochondrial translation elongation are coordinated through the molecular chaperone TRAP1 for the synthesis and import of mitochondrial proteins.

A complex interplay between mRNA translation and cellular respiration has been recently unveiled, but its regulation in humans is poorly characterized in either health or disease. Cancer cells radically reshape both biosynthetic and bioenergetic pathways to sustain their aberrant growth rates. In this regard, we have shown that the molecular chaperone TRAP1 not only regulates the activity of respiratory complexes, behaving alternatively as an oncogene or a tumor suppressor, but also plays a concomitant moonlighting function in mRNA translation regulation. Herein we identify the molecular mechanisms involved, demonstrating that TRAP1: i) binds both mitochondrial and cytosolic ribosomes as well as translation elongation factors, ii) slows down translation elongation rate, and iii) favors localized translation in the proximity of mitochondria. We also provide evidence that TRAP1 is coexpressed in human tissues with the mitochondrial translational machinery, which is responsible for the synthesis of respiratory complex proteins. Altogether, our results show an unprecedented level of complexity in the regulation of cancer cell metabolism, strongly suggesting the existence of a tight feedback loop between protein synthesis and energy metabolism, based on the demonstration that a single molecular chaperone plays a role in both mitochondrial and cytosolic translation, as well as in mitochondrial respiration.

Regulation of mitochondrial complex III activity and assembly by TRAP1 in cancer cells.

BACKGROUND: Metabolic reprogramming is an important issue in tumor biology. A recently-identified actor in this regard is the molecular chaperone TRAP1, that is considered an oncogene in several cancers for its high expression but an oncosuppressor in others with predominant oxidative metabolism. TRAP1 is mainly localized in mitochondria, where it interacts with respiratory complexes, although alternative localizations have been described, particularly on the endoplasmic reticulum, where it interacts with the translational machinery with relevant roles in protein synthesis regulation. RESULTS: Herein we show that, inside mitochondria, TRAP1 binds the complex III core component UQCRC2 and regulates complex III activity. This decreases respiration rate during basal conditions but allows sustained oxidative phosphorylation when glucose is limiting, a condition in which the direct TRAP1-UQCRC2 binding is disrupted, but not TRAP1-complex III binding. Interestingly, several complex III components and assembly factors show an inverse correlation with survival and response to platinum-based therapy in high grade serous ovarian cancers, where TRAP1 inversely correlates with stage and grade and directly correlates with survival. Accordingly, drug-resistant ovarian cancer cells show high levels of complex III components and high sensitivity to complex III inhibitory drug antimycin A. CONCLUSIONS: These results shed new light on the molecular mechanisms involved in TRAP1-dependent regulation of cancer cell metabolism and point out a potential novel target for metabolic therapy in ovarian cancer.

A xCT role in tumour-associated ferroptosis shed light on novel therapeutic options.

Solute carrier family 7 member 11 (SLC7A11; also known as xCT), a key component of the cystine/glutamate antiporter, is essential for the maintenance of cellular redox status and the regulation of tumor-associated ferroptosis. Accumulating evidence has demonstrated that xCT overexpression, resulting from different oncogenic and tumor suppressor signaling, promotes tumor progression and multidrug resistance partially via suppressing ferroptosis. In addition, recent studies have highlighted the role of xCT in regulating the metabolic flexibility in cancer cells. In this review, the xCT activities in intracellular redox balance and in ferroptotic cell death have been summarized. Moreover, the role of xCT in promoting tumor development, drug resistance, and nutrient dependency in cancer cells has been explored. Finally, different therapeutic strategies, xCT-based, for anti-cancer treatments have been discussed.

Decreased Levels of GSH Are Associated with Platinum Resistance in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is the most common and aggressive OC histotype. Although initially sensitive to standard platinum-based chemotherapy, most HGSOC patients relapse and become chemoresistant. We have previously demonstrated that platinum resistance is driven by a metabolic shift toward oxidative phosphorylation via activation of an inflammatory response, accompanied by reduced cholesterol biosynthesis and increased uptake of exogenous cholesterol. To better understand metabolic remodeling in OC, herein we performed an untargeted metabolomic analysis, which surprisingly showed decreased reduced glutathione (GSH) levels in resistant cells. Accordingly, we found reduced levels of enzymes involved in GSH synthesis and recycling, and compensatory increased expression of thioredoxin reductase. Cisplatin treatment caused an increase of reduced GSH, possibly due to direct binding hindering its oxidation, and consequent accumulation of reactive oxygen species. Notably, expression of the cysteine-glutamate antiporter xCT, which is crucial for GSH synthesis, directly correlates with post-progression survival of HGSOC patients, and is significantly reduced in patients not responding to platinum-based therapy. Overall, our data suggest that cisplatin treatment could positively select cancer cells which are independent from GSH for the maintenance of redox balance, and thus less sensitive to cisplatin-induced oxidative stress, opening new scenarios for the GSH pathway as a therapeutic target in HGSOC.

Targeting Mitochondrial Protein Expression as a Future Approach for Cancer Therapy.

Extensive metabolic remodeling is a fundamental feature of cancer cells. Although early reports attributed such remodeling to a loss of mitochondrial functions, it is now clear that mitochondria play central roles in cancer development and progression, from energy production to synthesis of macromolecules, from redox modulation to regulation of cell death. Biosynthetic pathways are also heavily affected by the metabolic rewiring, with protein synthesis dysregulation at the hearth of cellular transformation. Accumulating evidence in multiple organisms shows that the metabolic functions of mitochondria are tightly connected to protein synthesis, being assembly and activity of respiratory complexes highly dependent on de novo synthesis of their components. In turn, protein synthesis within the organelle is tightly connected with the cytosolic process. This implies an entire network of interactions and fine-tuned regulations that build up a completely under-estimated level of complexity. We are now only preliminarily beginning to reconstitute such regulatory level in human cells, and to perceive its role in diseases. Indeed, disruption or alterations of these connections trigger conditions of proteotoxic and energetic stress that could be potentially exploited for therapeutic purposes. In this review, we summarize the available literature on the coordinated regulation of mitochondrial and cytosolic mRNA translation, and their effects on the integrity of the mitochondrial proteome and functions. Finally, we highlight the potential held by this topic for future research directions and for the development of innovative therapeutic approaches.

Different Impacts of MucR Binding to the babR and virB Promoters on Gene Expression in Brucella abortus 2308.

The protein MucR from Brucella abortus has been described as a transcriptional regulator of many virulence genes. It is a member of the Ros/MucR family comprising proteins that control the expression of genes important for the successful interaction of α-proteobacteria with their eukaryotic hosts. Despite clear evidence of the role of MucR in repressing virulence genes, no study has been carried out so far demonstrating the direct interaction of this protein with the promoter of its target gene babR encoding a LuxR-like regulator repressing virB genes. In this study, we show for the first time the ability of MucR to bind the promoter of babR in electrophoretic mobility shift assays demonstrating a direct role of MucR in repressing this gene. Furthermore, we demonstrate that MucR can bind the virB gene promoter. Analyses by RT-qPCR showed no significant differences in the expression level of virB genes in Brucella abortus CC092 lacking MucR compared to the wild-type Brucella abortus strain, indicating that MucR binding to the virB promoter has little impact on virB gene expression in B. abortus 2308. The MucR modality to bind the two promoters analyzed supports our previous hypothesis that this is a histone-like protein never found before in Brucella.

Cholesterol Homeostasis Modulates Platinum Sensitivity in Human Ovarian Cancer.

Despite initial chemotherapy response, ovarian cancer is the deadliest gynecologic cancer, due to frequent relapse and onset of drug resistance. To date, there is no affordable diagnostic/prognostic biomarker for early detection of the disease. However, it has been recently shown that high grade serous ovarian cancers show peculiar oxidative metabolism, which is in turn responsible for inflammatory response and drug resistance. The molecular chaperone TRAP1 plays pivotal roles in such metabolic adaptations, due to the involvement in the regulation of mitochondrial respiration. Here, we show that platinum-resistant ovarian cancer cells also show reduced cholesterol biosynthesis, and mostly rely on the uptake of exogenous cholesterol for their needs. Expression of FDPS and OSC, enzymes involved in cholesterol synthesis, are decreased both in drug-resistant cells and upon TRAP1 silencing, whereas the expression of LDL receptor, the main mediator of extracellular cholesterol uptake, is increased. Strikingly, treatment with statins to inhibit cholesterol synthesis reduces cisplatin-induced apoptosis, whereas silencing of LIPG, an enzyme involved in lipid metabolism, or withdrawal of lipids from the culture medium, increases sensitivity to the drug. These results suggest caveats for the use of statins in ovarian cancer patients and highlights the importance of lipid metabolism in ovarian cancer treatment.

Modulation of Mitochondrial Metabolic Reprogramming and Oxidative Stress to Overcome Chemoresistance in Cancer.

Metabolic reprogramming, carried out by cancer cells to rapidly adapt to stress such as hypoxia and limited nutrient conditions, is an emerging concepts in tumor biology, and is now recognized as one of the hallmarks of cancer. In contrast with conventional views, based on the classical Warburg effect, these metabolic alterations require fully functional mitochondria and finely-tuned regulations of their activity. In turn, the reciprocal regulation of the metabolic adaptations of cancer cells and the microenvironment critically influence disease progression and response to therapy. This is also realized through the function of specific stress-adaptive proteins, which are able to relieve oxidative stress, inhibit apoptosis, and facilitate the switch between metabolic pathways. Among these, the molecular chaperone tumor necrosis factor receptor associated protein 1 (TRAP1), the most abundant heat shock protein 90 (HSP90) family member in mitochondria, is particularly relevant because of its role as an oncogene or a tumor suppressor, depending on the metabolic features of the specific tumor. This review highlights the interplay between metabolic reprogramming and cancer progression, and the role of mitochondrial activity and oxidative stress in this setting, examining the possibility of targeting pathways of energy metabolism as a therapeutic strategy to overcome drug resistance, with particular emphasis on natural compounds and inhibitors of mitochondrial HSP90s.

Identification of Novel Biomarkers of Homologous Recombination Defect in DNA Repair to Predict Sensitivity of Prostate Cancer Cells to PARP-Inhibitors.

One of the most common malignancies in men is prostate cancer, for which androgen deprivation is the standard therapy. However, prostate cancer cells become insensitive to anti-androgen treatment and proceed to a castration-resistant state with limited therapeutic options. Therefore, besides the androgen deprivation approach, novel biomarkers are urgently required for specific targeting in this deadly disease. Recently, germline or somatic mutations in the homologous recombination (HR) DNA repair genes have been identified in at least 20-25% of metastatic castration-resistant prostate cancers (mCRPC). Defects in genes involved in HR DNA repair can sensitize cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors, a class of drugs already approved by the Food and Drug Administration (FDA) for breast and ovarian cancer carrying germline mutations in BRCA1/2 genes. For advanced prostate cancer carrying Breast cancer1/2 (BRCA1/2) or ataxia telengiectasia mutated (ATM) mutations, preclinical studies and clinical trials support the use of PARP-inhibitors, which received breakthrough therapy designation by the FDA. Based on these assumptions, several trials including DNA damage response and repair (DDR) targeting have been launched and are ongoing for prostate cancer. Here, we review the state-of-the-art potential biomarkers that could be predictive of cancer cell synthetic lethality with PARP inhibitors. The identification of key molecules that are affected in prostate cancer could be assayed in future clinical studies to better stratify prostate cancer patients who might benefit from target therapy.

CCDC6 and USP7 expression levels suggest novel treatment options in high-grade urothelial bladder cancer.

BACKGROUND: The muscle invasive form of urothelial bladder cancer (UBC) is a deadly disease. Currently, the therapeutic approach of UBC is mostly based on surgery and standard chemotherapy. Biomarkers to establish appropriate drugs usage are missing. Deficiency of the tumor suppressor CCDC6 determines PARP-inhibitor sensitivity. The CCDC6 levels are modulated by the deubiquitinase USP7. In this work we scored CCDC6 and USP7 expression levels in primary UBC and we evaluated the expression levels of CCDC6 in correlation with the effects of the PARP-inhibitors combined with the USP7 inhibitor, P5091, in vitro. Since PARP-inhibitors could be enhanced by conventional chemotherapy or DNA damage inducers, we tested the new agent RRx-001, able to induce DNA damage, to prove the benefit of combined treatments in bladder cancer cells. METHODS: The J82, T24, 5637 and KU-19-19 bladder cancer cells were exposed to USP7 inhibitor P5091 in presence of cycloheximide to analyse the CCDC6 stability. Upon the CCDC6 degradation induced by P5091, the cells sensitivity to PARP-inhibitor was evaluated by cell viability assays. The ability of the DNA damage inducer RRx-001 to modulate CCDC6 protein levels and H2AX phosphorylation was detected at immunoblot. The combination of USP7 inhibitor plus RRx-001 enhanced the PARP-inhibitor sensitivity, as evaluated by cell viability assays. The results of the scores and correlation of CCDC6 and USP7 expression levels obtained by UBC primary biopsies staining were used to cluster patients by a K-mean cluster analysis. RESULTS: P5091 determining CCDC6 degradation promoted bladder cancer cells sensitivity to PARP-inhibitor drugs. RRx-001, by inducing DNA damage, enhanced the effects of the combined treatment. The immunohistochemical staining of both CCDC6 and USP7 proteins allowed to cluster the high grade (G3) UBC patients, on the basis of CCDC6 expression levels. CONCLUSIONS: In high grade UBC the identification of two clusters of patients based on CCDC6 and USP7 expession can possibly indicate the use of PARP-inhibitor drugs, in combination with USP7 inhibitor in addition to the DNA damage inducer RRx-001, that also acts as an immunomodulatory agent, offering novel therapeutic strategy for personalized medicine in bladder cancer patients.

New combinatorial strategies to improve the PARP inhibitors efficacy in the urothelial bladder Cancer treatment.

BACKGROUND: Novel therapeutic strategies are urgently needed for the treatment of metastatic Urothelial Bladder Cancer. DNA damaging repair (DDR) targeting has been introduced in cinical trials for bladder cancer patients that carry alterations in homologous DNA repair genes, letting to envisage susceptibility to the Poly (adenosine diphosphate [ADP]) ribose polymerase (PARP) inhibitors. MAIN BODY: PARP inhibition, by amplifying the DNA damage, augments the mutational burden and promotes the immune priming of the tumor by increasing the neoantigen exposure and determining upregulation of programmed death ligand 1 (PD-L1) expression. Thus, the combination of PARP-inhibition and the PD/PD-L1 targeting may represent a compelling strategy to treat bladder cancer and has been introduced in recent clinical trials. The targeting of DDR has been also used in combination with epigenetic drugs able to modulate the expression of genes involved in DDR, and also able to act as immunomodulator agents suggesting their use in combination with immune-checkpoint inhibitors. CONCLUSION: In conclusion, it may be envisaged the combination of three classes of drugs to treat bladder cancer, by targeting the DDR process in a tumor context of DDR defect, together with epigenetic agents and immune-checkpoint inhibitors, whose association may amplify the effects and reduce the doses and the toxicity of each single drug.

Complete Genome Sequencing of 10 Brucella abortus Biovar 3 Strains Isolated from Water Buffalo.

Brucellosis is a zoonotic disease that affects both humans and animals. Its distribution is global, concentrated in the Mediterranean area, India, Central Asia, and Latin America. Here, we present a complete genome assembly of 10 Brucella abortus strains isolated from water buffaloes farmed in the Campania region of Italy.

Bacterial components induce cytokine and intercellular adhesion molecules-1 and activate transcription factors in dermal fibroblasts.

This study investigated the effect of various structural components of Gram-positive (lipotheichoic acid and protein A) and Gram-negative (porins and lipopolysaccharide) bacteria on human dermal fibroblasts. Fibroblasts are important effector cells which have a potential role in augmenting the inflammatory response in various diseases. In this study we present a profile of TNF-alpha, IL-6 and IL-8, the expression of intercellular adhesion molecules (ICAM-1) and the activation of transcriptional nuclear factor NF-kB and AP-1 in human dermal fibroblasts stimulated by bacterial surface components. Compared to the controls, increased ICAM-1, IL-6 and IL-8 gene expression after stimulation of LPS and porins at 2 and 4 h was more evident than that obtained following stimulation of LTA and PA. Gene expression was also associated with the production of cytokine proteins in culture supernatants. TNF-alpha gene expression remained undetectable. Moreover, LPS and porin treatments determined IkBalpha phosphorylation and degradation in human dermal fibroblasts and the subsequent activation of nuclear factors NF-kB and AP-1. These data suggest the importance of such stimuli in the first step of the inflammatory process, as well as the important role played by fibroblasts in skin inflammatory disease.

Cytokine pattern in blister fluid and sera of patients with pemphigus.

BACKGROUND: Pemphigus is a chronic auto-immune blistering disease with four main variants, i.e. pemphigus vulgaris (PV), foliaceus (PF), erythematosus (PE) and vegetans. The common histological feature of this disease is acantholysis. OBJECTIVE: The aim of this study was to compare levels of some cytokines in blister fluid and sera of patients with pemphigus, using as control blister fluid of patients with bullous pemphigoid (BP) and bullous contact dermatitis (BCD). METHODS: Using an immuno-enzymatic assay (ELISA), we tested 16 sera and 6 blister fluids of patients with various forms of pemphigus (13 with PV, 1 with PF, 2 with PE), the sera of 16 healthy control subjects, 5 blister fluids of patients with BP and 5 blister fluids of patients with BCD, for the presence of some cytokines (IL-10, IL-8 and IFN-gamma). Intercellular antibodies were searched for and titred; desmoglein 1 and 3 antibody levels were independently evaluated to compare them with the severity of both cutaneous and oral involvement. RESULTS: The levels of IL-10 in the sera of patients with pemphigus were below the detection limits. IL-8 was significantly increased only in 4 samples of sera from pemphigus patients compared with controls, while IFN-gamma was detected at low levels in almost all patients compared with sera of controls. The cytokine levels in blister fluid of patients with pemphigus were significantly higher than in the sera. There was a difference between the expression of cytokines in blister fluid of control patients with BP and BCD compared with those of pemphigus patients. CONCLUSION: This report discusses the anti-inflammatory role played by IL-10 in the chronic form of pemphigus and the hypothesis of a possible role of IL-8 in neutrophil and lymphocyte-monocyte recruitment.