RICTOR/mTORC2 downregulation in BRAFV600E melanoma cells promotes resistance to BRAF/MEK inhibition.

BACKGROUND: The main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF-mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to assess in this context the role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit, regarded as an oncogenic driver in several tumor types, including MM. METHODS: After analyzing The Cancer Genome Atlas MM patients' database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAFV600E MM cell lines on their response to BRAF/MEKi. We performed proteomic screening to identify proteins modulated by changes in RICTOR expression, and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. RESULTS: Low RICTOR levels in BRAF-mutated MM correlate with a worse clinical outcome. Gene Set Enrichment Analysis of low-RICTOR tumors display gene signatures suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy production. RICTOR-deficient BRAFV600E cells are intrinsically tolerant to BRAF/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. Moreover, in drug-naïve cells we observed a decline in RICTOR expression shortly after BRAFi exposure. In RICTOR-depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, and their pharmacological inhibition restores sensitivity to BRAFi. CONCLUSIONS: Our work unveils an unforeseen tumor-suppressing role for mTORC2 in the early adaptation phase of BRAFV600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low RICTOR tumors. Importantly, our findings indicate that the evaluation of intra-tumor RICTOR levels has a prognostic value in metastatic melanoma and may help to guide therapeutic strategies in a personalized manner.

Iron supplementation is sufficient to rescue skeletal muscle mass and function in cancer cachexia.

Cachexia is a wasting syndrome characterized by devastating skeletal muscle atrophy that dramatically increases mortality in various diseases, most notably in cancer patients with a penetrance of up to 80%. Knowledge regarding the mechanism of cancer-induced cachexia remains very scarce, making cachexia an unmet medical need. In this study, we discovered strong alterations of iron metabolism in the skeletal muscle of both cancer patients and tumor-bearing mice, characterized by decreased iron availability in mitochondria. We found that modulation of iron levels directly influences myotube size in vitro and muscle mass in otherwise healthy mice. Furthermore, iron supplementation was sufficient to preserve both muscle function and mass, prolong survival in tumor-bearing mice, and even rescues strength in human subjects within an unexpectedly short time frame. Importantly, iron supplementation refuels mitochondrial oxidative metabolism and energy production. Overall, our findings provide new mechanistic insights in cancer-induced skeletal muscle wasting, and support targeting iron metabolism as a potential therapeutic option for muscle wasting diseases.

N-acetylaspartate release by glutaminolytic ovarian cancer cells sustains protumoral macrophages.

Glutaminolysis is known to correlate with ovarian cancer aggressiveness and invasion. However, how this affects the tumor microenvironment is elusive. Here, we show that ovarian cancer cells become addicted to extracellular glutamine when silenced for glutamine synthetase (GS), similar to naturally occurring GS-low, glutaminolysis-high ovarian cancer cells. Glutamine addiction elicits a crosstalk mechanism whereby cancer cells release N-acetylaspartate (NAA) which, through the inhibition of the NMDA receptor, and synergistically with IL-10, enforces GS expression in macrophages. In turn, GS-high macrophages acquire M2-like, tumorigenic features. Supporting this in␣vitro model, in silico data and the analysis of ascitic fluid isolated from ovarian cancer patients prove that an M2-like macrophage phenotype, IL-10 release, and NAA levels positively correlate with disease stage. Our study uncovers the unprecedented role of glutamine metabolism in modulating macrophage polarization in highly invasive ovarian cancer and highlights the anti-inflammatory, protumoral function of NAA.

SLC25A10 biallelic mutations in intractable epileptic encephalopathy with complex I deficiency.

Mitochondrial diseases are a plethora of inherited neuromuscular disorders sharing defects in mitochondrial respiration, but largely different from one another for genetic basis and pathogenic mechanism. Whole exome sequencing was performed in a familiar trio (trio-WES) with a child affected by severe epileptic encephalopathy associated with respiratory complex I deficiency and mitochondrial DNA depletion in skeletal muscle. By trio-WES we identified biallelic mutations in SLC25A10, a nuclear gene encoding a member of the mitochondrial carrier family. Genetic and functional analyses conducted on patient fibroblasts showed that SLC25A10 mutations are associated with reduction in RNA quantity and aberrant RNA splicing, and to absence of SLC25A10 protein and its transporting function. The yeast SLC25A10 ortholog knockout strain showed defects in mitochondrial respiration and mitochondrial DNA content, similarly to what observed in the patient skeletal muscle, and growth susceptibility to oxidative stress. Albeit patient fibroblasts were depleted in the main antioxidant molecules NADPH and glutathione, transport assays demonstrated that SLC25A10 is unable to transport glutathione. Here, we report the first recessive mutations of SLC25A10 associated to an inherited severe mitochondrial neurodegenerative disorder. We propose that SLC25A10 loss-of-function causes pathological disarrangements in respiratory-demanding conditions and oxidative stress vulnerability.

Cachexia, a Systemic Disease beyond Muscle Atrophy.

Cachexia is a complication of dismal prognosis, which often represents the last step of several chronic diseases. For this reason, the comprehension of the molecular drivers of such a condition is crucial for the development of management approaches. Importantly, cachexia is a syndrome affecting various organs, which often results in systemic complications. To date, the majority of the research on cachexia has been focused on skeletal muscle, muscle atrophy being a pivotal cause of weight loss and the major feature associated with the steep reduction in quality of life. Nevertheless, defining the impact of cachexia on other organs is essential to properly comprehend the complexity of such a condition and potentially develop novel therapeutic approaches.

The Crowded Crosstalk between Cancer Cells and Stromal Microenvironment in Gynecological Malignancies: Biological Pathways and Therapeutic Implication.

The tumor microenvironment plays a pillar role in the progression and the distance dissemination of cancer cells in the main malignancies affecting women-epithelial ovarian cancer, endometrial cancer and cervical cancer. Their milieu acquires specific properties thanks to intense crosstalk between stromal and cancer cells, leading to a vicious circle. Fibroblasts, pericytes, lymphocytes and tumor associated-macrophages orchestrate most of the biological pathways. In epithelial ovarian cancer, high rates of activated pericytes determine a poorer prognosis, defining a common signature promoting ovarian cancer proliferation, local invasion and distant spread. Mesenchymal cells also release chemokines and cytokines under hormonal influence, such as estrogens that drive most of the endometrial cancers. Interestingly, the architecture of the cervical cancer milieu is shaped by the synergy of high-risk Human Papilloma Virus oncoproteins and the activity of stromal estrogen receptor α. Lymphocytes represent a shield against cancer cells but some cell subpopulation could lead to immunosuppression, tumor growth and dissemination. Cytotoxic tumor infiltrating lymphocytes can be eluded by over-adapted cancer cells in a scenario of immune-tolerance driven by T-regulatory cells. Therefore, the tumor microenvironment has a high translational potential offering many targets for biological and immunological therapies.

Monoamine oxidase-dependent histamine catabolism accounts for post-ischemic cardiac redox imbalance and injury.

Monoamine oxidase (MAO), a mitochondrial enzyme that oxidizes biogenic amines generating hydrogen peroxide, is a major source of oxidative stress in cardiac injury. However, the molecular mechanisms underlying its overactivation in pathological conditions are still poorly characterized. Here, we investigated whether the enhanced MAO-dependent hydrogen peroxide production can be due to increased substrate availability using a metabolomic profiling method. We identified N1-methylhistamine -the main catabolite of histamine- as an important substrate fueling MAO in Langendorff mouse hearts, directly perfused with a buffer containing hydrogen peroxide or subjected to ischemia/reperfusion protocol. Indeed, when these hearts were pretreated with the MAO inhibitor pargyline we observed N1-methylhistamine accumulation along with reduced oxidative stress. Next, we showed that synaptic terminals are the major source of N1-methylhistamine. Indeed, in vivo sympathectomy caused a decrease of N1-methylhistamine levels, which was associated with a marked protection in post-ischemic reperfused hearts. As far as the mechanism is concerned, we demonstrate that exogenous histamine is transported into isolated cardiomyocytes and triggers a rise in the levels of reactive oxygen species (ROS). Once again, pargyline pretreatment induced intracellular accumulation of N1-methylhistamine along with decrease in ROS levels. These findings uncover a receptor-independent mechanism for histamine in cardiomyocytes. In summary, our study reveals a novel and important pathophysiological causative link between MAO activation and histamine availability during pathophysiological conditions such as oxidative stress/cardiac injury.

Mitochondrial carriers in inflammation induced by bacterial endotoxin and cytokines.

Significant metabolic changes occur in the shift from resting to activated cellular status in inflammation. Thus, changes in expression of a large number of genes and extensive metabolic reprogramming gives rise to acquisition of new functions (e.g. production of cytokines, intermediates for biosynthesis, lipid mediators, PGE, ROS and NO). In this context, mitochondrial carriers, which catalyse the transport of solute across mitochondrial membrane, change their expression to transport mitochondrially produced molecules, among which citrate and succinate, to be used as intracellular signalling molecules in inflammation. This review summarises the mitochondrial carriers studied so far that are, directly or indirectly, involved in inflammation.

Acetylation of human mitochondrial citrate carrier modulates mitochondrial citrate/malate exchange activity to sustain NADPH production during macrophage activation.

The mitochondrial citrate-malate exchanger (CIC), a known target of acetylation, is up-regulated in activated immune cells and plays a key role in the production of inflammatory mediators. However, the role of acetylation in CIC activity is elusive. We show that CIC is acetylated in activated primary human macrophages and U937 cells and the level of acetylation is higher in glucose-deprived compared to normal glucose medium. Acetylation enhances CIC transport activity, leading to a higher citrate efflux from mitochondria in exchange with malate. Cytosolic citrate levels do not increase upon activation of cells grown in deprived compared to normal glucose media, indicating that citrate, transported from mitochondria at higher rates from acetylated CIC, is consumed at higher rates. Malate levels in the cytosol are lower in activated cells grown in glucose-deprived compared to normal glucose medium, indicating that this TCA intermediate is rapidly recycled back into the cytosol where it is used by the malic enzyme. Additionally, in activated cells CIC inhibition increases the NADP+/NADPH ratio in glucose-deprived cells; this ratio is unchanged in glucose-rich grown cells due to the activity of the pentose phosphate pathway. Consistently, the NADPH-producing isocitrate dehydrogenase level is higher in activated glucose-deprived as compared to glucose rich cells. These results demonstrate that, in the absence of glucose, activated macrophages increase CIC acetylation to enhance citrate efflux from mitochondria not only to produce inflammatory mediators but also to meet the NADPH demand through the actions of isocitrate dehydrogenase and malic enzyme.

The contribution of the citrate pathway to oxidative stress in Down syndrome.

Inflammatory conditions and oxidative stress have a crucial role in Down syndrome (DS). Emerging studies have also reported an altered lipid profile in the early stages of DS. Our previous works demonstrate that citrate pathway activation is required for oxygen radical production during inflammation. Here, we find up-regulation of the citrate pathway and down-regulation of carnitine/acylcarnitine carrier and carnitine palmitoyl-transferase 1 genes in cells from children with DS. Interestingly, when the citrate pathway is inhibited, we observe a reduction in oxygen radicals as well as in lipid peroxidation levels. Our preliminary findings provide evidence for a citrate pathway dysregulation, which could be related to some phenotypic traits of people with DS.