Alike but Different: RAF Paralogs and Their Signaling Outputs.

RAF links RAS, one of the most potent human oncogenes, to its effector ERK and to proliferation. This role is evolutionarily conserved, but while simpler multicellular organisms express one RAF, mammals have three. This Minireview highlights common and divergent features of RAF paralogs, their signaling outputs, and roles in tumorigenesis.

[Integrated and structured clinical networks. A new model of pro-active management of chronicity and sustainability]. / Reti cliniche Integrate e Strutturate. Un nuovo modello di gestione proattiva della cronicità e per la sostenibilità.

The population ageing and the increase of the prevalence of chronicity and multimorbidity, require a multi-dimensional and long-term care system, overcaming the current vision "hospital-centered" toword a structured model, able to network services. The new organisational systemic model, named "Integrated and Structured Clinical Network", developed by a experimentation conducted in an Local Health Unit, in Tuscany, has highlighted very relevant results both for the health of the citizens taken in care, redusing the need for hospitalization, the demand for heavy diagnostics (and waiting times ), the access to the Emergency Room and the final costs of care pathways, largely the result of avoidable hospitalization! The project has been developed with the purpose of create a proactive medicine model to managing chronicity, complexity and fragility, in accordance with aims of "Population health management" and with Chronicity National Plan. The organizzational requirements of this new chronicity management model are rappresented by: - Estabilishment of multi-professional team - Multi-dimensional evaluation of clinical and social assistance needs - For each patient, definition of personalized "pro-active" PDTAs - Identification, in every AFT (Territorial Functional Aggregation ), of "expert" general practioners and provision of first-level diagnostic technologies - Identification of reference specialists - Structured reorganization of "Community of Practice" between primary care physicians and referral specialists - Design of an enabling information system to exchange of socio-health data and for the teleconsultation, telemedicine, remote control.

Dehydroepiandrosterone triggers autophagic cell death in human hepatoma cell line HepG2 via JNK-mediated p62/SQSTM1 expression.

Autophagy is a catabolic process that cancer cells usually exploit during stress conditions to provide energy by recycling organelles and proteins. Beyond its prosurvival role, it is well accepted that occurrence of autophagy is often associated with a particular type of programmed cell death known as autophagic cell death (ACD). Dehydroepiandrosterone (DHEA) is an endogenous hormone showing anticancer properties even if the underlying mechanisms are not fully clear yet. Here, we provide evidence that DHEA induces ACD in human hepatoma cell line, HepG2. Indeed, autophagy inhibitors (i.e. 3-methyladenine or Atg5 siRNA) significantly reduced the percentage of dead cells. DHEA induces p62-dependent autophagy, which turns detrimental and brings about death. DHEA stimulates reactive oxygen species-independent jun N-terminal kinase (JNK) phosphoactivation and the treatment with JNK inhibitor reduces p62 mRNA levels, as well as DHEA-induced ACD. The transcription factor nuclear factor (erythroid-derived-2)-like-2 (Nrf2) constitutes the link between JNK and p62 since its migration to the nucleus is suppressed by JNK inhibitor and its inhibition through a dominant negative Nrf2 plasmid transfection decreases p62 protein levels. Overall, our data indicate that DHEA induces ACD in HepG2 via a JNK-Nrf2-p62 axis. Thus, DHEA could represent a new appealing drug for eliminating tumor cells through autophagy particularly in apoptosis-resistant cases.

Managing lipid metabolism in proliferating cells: new perspective for metformin usage in cancer therapy.

Cancer cells metabolically adapt to undergo cellular proliferation. Lipids, besides their well-known role as energy storage, represent the major building blocks for the synthesis of neo-generated membranes. There is increasing evidence that cancer cells show specific alterations in different aspects of lipid metabolism. The changes of expression and activity of lipid metabolising enzymes are directly regulated by the activity of oncogenic signals. The dependence of tumour cells on the deregulated lipid metabolism suggests that proteins involved in this process could be excellent chemotherapeutic targets for cancer treatment. Due to its rare side effects in non-cancerous cells, metformin has been recently revaluated as a potential anti-tumourigenic drug, which negatively affects lipid biosynthetic pathways. In this review we summarised the emerging molecular events linking the anti-proliferative effect of metformin with lipid metabolism in cancer cells.

Impaired degradation of YAP1 and IL6ST by chaperone-mediated autophagy promotes proliferation and migration of normal and hepatocellular carcinoma cells.

Impaired degradation of the transcriptional coactivator YAP1 and IL6ST (interleukin 6 cytokine family signal transducer), two proteins deregulated in liver cancer, has been shown to promote tumor growth. Here, we demonstrate that YAP1 and IL6ST are novel substrates of chaperone-mediated autophagy (CMA) in human hepatocellular carcinoma (HCC) and hepatocyte cell lines. Knockdown of the lysosomal CMA receptor LAMP2A increases protein levels of YAP1 and IL6ST, without changes in mRNA expression. Additionally, both proteins show KFERQ-dependent binding to the CMA chaperone HSPA8 and accumulate into isolated lysosomes after stimulation of CMA by prolonged starvation. We further show that LAMP2A downregulation promotes the proliferation and migration in HCC cells and a human hepatocyte cell line, and that it does so in a YAP1- and IL6ST-dependent manner. Finally, LAMP2A expression is downregulated, and YAP1 and IL6ST expression is upregulated, in human HCC biopsies. Taken together, our work reveals a novel mechanism that controls the turnover of two cancer-relevant proteins and suggests a tumor suppressor function of CMA in the liver, advocating for the exploitation of CMA activity for diagnostic and therapeutic purposes.Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; ATG7: autophagy related 7; CMA: chaperone-mediated autophagy; eMI: endosomal microautophagy; HCC: hepatocellular carcinoma; HSPA8: heat shock protein family A (Hsp70) member 8; IL6ST: interleukin 6 cytokine family signal transducer; JAK: Janus kinase; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MAPK8: mitogen-activated protein kinase 8; P6: pyridine 6; SQSTM1: sequestosome 1; TUBA: tubulin alpha; VDAC1: voltage dependent anion channel 1; VP: verteporfin; YAP1: Yes1 associated transcriptional regulator.

Hindering NAT8L expression in hepatocellular carcinoma increases cytosolic aspartate delivery that fosters pentose phosphate pathway and purine biosynthesis promoting cell proliferation.

N-acetylaspartate (NAA) is synthesized by the mitochondrial enzyme NAT8L, which uses acetyl-CoA and aspartate as substrates. These metabolites are fundamental for bioenergetics and anabolic requirements of highly proliferating cells, thus, NAT8L modulation may impinge on the metabolic reprogramming of cancer cells. Specifically, aspartate represents a limiting amino acid for nucleotide synthesis in cancer. Here, the expression of the NAT8L enzyme was modulated to verify how it impacts the metabolic adaptations and proliferative capacity of hepatocellular carcinoma. We demonstrated that NAT8L downregulation is associated with increased proliferation of hepatocellular carcinoma cells and immortalized hepatocytes. The overexpression of NAT8L instead decreased cell growth. The pro-tumoral effect of NAT8L silencing depended on glutamine oxidation and the rewiring of glucose metabolism. Mechanistically, NAT8L downregulation triggers aspartate outflow from mitochondria via the exporter SLC25A13 to promote glucose flux into the pentose phosphate pathway, boosting purine biosynthesis. These results were corroborated by the analyses of human and mouse hepatocellular carcinoma samples revealing a decrease in NAT8L expression compared to adjacent non-tumoral tissues. Overall, this work demonstrates that NAT8L expression in liver cells limits the cytosolic availability of aspartate necessary for enhancing the pentose phosphate pathway and purine biosynthesis, counteracting cell proliferation.

Targeting aerobic glycolysis: 3-bromopyruvate as a promising anticancer drug.

The Warburg effect refers to the phenomenon whereby cancer cells avidly take up glucose and produce lactic acid under aerobic conditions. Although the molecular mechanisms underlying tumor reliance on glycolysis remains not completely clear, its inhibition opens feasible therapeutic windows for cancer treatment. Indeed, several small molecules have emerged by combinatorial studies exhibiting promising anticancer activity both in vitro and in vivo, as a single agent or in combination with other therapeutic modalities. Therefore, besides reviewing the alterations of glycolysis that occur with malignant transformation, this manuscript aims at recapitulating the most effective pharmacological therapeutics of its targeting. In particular, we describe the principal mechanisms of action and the main targets of 3-bromopyruvate, an alkylating agent with impressive antitumor effects in several models of animal tumors. Moreover, we discuss the chemo-potentiating strategies that would make unparalleled the putative therapeutic efficacy of its use in clinical settings.

ROS-mediated activation of p38 protects hepatocellular carcinoma cells from caspase-independent death elicited by lysosomal damage.

Hepatocellular carcinoma (HCC) is one of the most common and deadliest cancer in the world. Despite this, few effective drugs are available for its treatment, in part due to the development of resistance, and surgical resection remains the most valuable option, when applicable. Upregulation of anti-apoptotic proteins, downregulation of pro-apoptotic factors and the acquisition of mutations in signaling pathways leading to caspase activation are a few examples of mechanisms that allow cancer cells to evade caspase-dependent apoptosis and continue to grow. The identification of drugs triggering the activation of caspase-independent death may therefore be an effective strategy to circumvent resistance and kill cancer cells. Here, we show that the lysosome damaging compound glycyl-l-phenylalanine 2-naphthylamide (GPN) induces cell death by a caspase-independent mechanism in HCC cell lines. Additionally, we identify the MAPK p38 as a novel mediator of the lysosomal stress response. Indeed, a ROS-dependent activation of p38 occurs in response to lysosomal damage, promoting the recovery of lysosomal integrity. As a consequence, pharmacological or genetic inhibition of p38 increases cell death elicited by GPN. Our findings identify p38 as a potential target to potentiate the cytotoxic effects of lysosomal damage and induce caspase-independent cell death in HCC cells, laying the ground for future evaluation of the efficacy of combination therapy.

Inhibition of JNK increases the sensitivity of hepatocellular carcinoma cells to lysosomotropic drugs via LAMP2A destabilization.

Alteration of lysosomal homeostasis is common in cancer cells, which often feature an enlarged and peripheral distributed lysosomal compartment and the overexpression of cathepsins. These alterations accelerate the production of building blocks for the de novo synthesis of macromolecules and contribute to the degradation of the extracellular matrix, thus contributing to tumor growth and invasion. At the same time, they make lysosomes more fragile and more prone to lysosomal membrane permeabilization, a condition that can cause the release of proteases into the cytosol and the activation of cell death. Therefore, lysosomes represent a weak spot of cancer cells that can be targeted for therapeutic purposes. Here, we identify a novel role of the kinase JNK as keeper of lysosomal stability in hepatocellular carcinoma cells. JNK inhibition reduces the stability of LAMP2A, a lysosomal membrane protein responsible for the stability of the lysosomal membrane, promoting its degradation by the proteasome. LAMP2A decrease enhances the lysosomal damage induced by lysosomotropic agents, ultimately leading to cell death. The effect is cancer-specific, as JNK inhibition does not decrease LAMP2A in non-tumoral liver cells and does not alter their sensitivity to lysosomotropic drugs. Our finding on the new role of JNK as cancer-specific keeper of lysosomal homeostasis lays the ground for future evaluation of the efficacy of the combination of JNK inhibition and lysosomotropic agents as a potential therapeutic strategy in hepatocellular carcinoma.

Extracellular vesicles in endothelial cells: from mediators of cell-to-cell communication to cargo delivery tools.

Extracellular vesicles (EVs) are nanosized vesicles released from most cell types that play a key role in cell-to-cell communication by carrying DNA, non-coding RNAs, proteins and lipids out of cells. The composition of EVs depends on the cell or tissue of origin and changes according to their pathophysiological conditions, making EVs a potential circulating biomarker of disease. Additionally, the natural tropism of EVs for specific organs and cells has raised the interest in their use as delivery vehicles. In this review, we provide an overview of EV biogenesis, isolation and characterization. We also discuss EVs in the context of endothelial pathophysiology, summarizing the current knowledge about their role in cell communication in quiescent and activated endothelial cells. In the last part, we describe the potential use of EVs as delivery vehicles of bioactive compounds and the current strategies to load exogenous cargo and to functionalize EVs to drive them to a specific tissue.

Lipid Catabolism and ROS in Cancer: A Bidirectional Liaison.

Although cancer cell metabolism was mainly considered to rely on glycolysis, with the concomitant impairment of mitochondrial metabolism, it has recently been demonstrated that several tumor types are sustained by oxidative phosphorylation (OXPHOS). In this context, endogenous fatty acids (FAs) deriving from lipolysis or lipophagy are oxidised into the mitochondrion, and are used as a source of energy through OXPHOS. Because the electron transport chain is the main source of ROS, cancer cells relying on fatty acid oxidation (FAO) need to be equipped with antioxidant systems that maintain the ROS levels under the death threshold. In those conditions, ROS can act as second messengers, favouring proliferation and survival. Herein, we highlight the different responses that tumor cells adopt when lipid catabolism is augmented, taking into account the different ROS fates. Many papers have demonstrated that the pro- or anti-tumoral roles of endogenous FA usage are hugely dependent on the tumor type, and on the capacity of cancer cells to maintain redox homeostasis. In light of this, clinical studies have taken advantage of the boosting of lipid catabolism to increase the efficacy of tumor therapy, whereas, in other contexts, antioxidant compounds are useful to reduce the pro-survival effects of ROS deriving from FAO.

Overcoming Barriers to the Effective Management of Severe Asthma in Italy.

INTRODUCTION: People with severe asthma (SA) often have poor disease control and quality of life, and are at high risk of exacerbations, lung function decline and asthma-related death. The present expert opinion article aimed to identify unmet needs in the management of SA in Italy, and propose possible solutions to address these needs. METHODS: At five multidisciplinary events in Italy, attendees identified factors that interfered with the effective management of SA and suggested how these barriers could be overcome. A core group of 12 Italian experts (pulmonologists, general practitioners, allergists, payers and patients) identified the main issues and proposed possible solutions based on the results from the meetings and relevant articles from the literature. RESULTS AND CONCLUSIONS: We reviewed the gap between real-world practice and guidelines, oral corticosteroid overuse, SA-related mortality, and barriers to effective SA treatment. Common themes were lack of awareness about SA among both patients and clinicians, and lack of networking/information exchange between those involved in the treatment of SA. Participants agreed on the need to implement patient education and create multidisciplinary groups of specialists to improve SA management through multidisciplinary educational initiatives, meetings with local experts, development of a flow chart for referral/connection with local experts and specialized centers. Clinical instruments that might help specialists improve SA management included referral networks, integrated care pathways, phenotyping and treatment algorithms, exacerbation tracking, and examination of electronic medical records for patients with uncontrolled asthma. The following actions need to be implemented in Italy: i) maximize the use of advanced therapies, eg, biologics; ii) increase/improve education for physicians and patients; iii) improve multidisciplinary communication and care coordination; iv) introduce regional and local protocols for SA diagnosis and treatment; and v) change the structure of healthcare services to reduce specialist waiting times and facilitate access to biologic therapies.

Targeting Glutathione Metabolism: Partner in Crime in Anticancer Therapy.

Glutathione (GSH) is the predominant low-molecular-weight antioxidant with a ubiquitous distribution inside the cell. The steady-state level of cellular GSH is dependent on the balance between synthesis, hydrolysis, recycling of glutathione disulphide (GSSG) as well as cellular extrusion of reduced, oxidized, or conjugated-forms. The augmented oxidative stress typical of cancer cells is accompanied by an increase of glutathione levels that confers them growth advantage and resistance to a number of chemotherapeutic agents. Targeting glutathione metabolism has been widely investigated for cancer treatment although GSH depletion as single therapeutic strategy has resulted largely ineffective if compared with combinatorial approaches. In this review, we circumstantiate the role of glutathione in tumour development and progression focusing on how interfering with different steps of glutathione metabolism can be exploited for therapeutic purposes. A dedicated section on synthetic lethal interactions with GSH modulators will highlight the promising option of harnessing glutathione metabolism for patient-directed therapy in cancer.

A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths, but its molecular heterogeneity hampers the design of targeted therapies. Currently, the only therapeutic option for advanced HCC is Sorafenib, an inhibitor whose targets include RAF. Unexpectedly, RAF1 expression is reduced in human HCC samples. Modelling RAF1 downregulation by RNAi increases the proliferation of human HCC lines in xenografts and in culture; furthermore, RAF1 ablation promotes chemical hepatocarcinogenesis and the proliferation of cultured (pre)malignant mouse hepatocytes. The phenotypes depend on increased YAP1 expression and STAT3 activation, observed in cultured RAF1-deficient cells, in HCC xenografts, and in autochthonous liver tumours. Thus RAF1, although essential for the development of skin and lung tumours, is a negative regulator of hepatocarcinogenesis. This unexpected finding highlights the contribution of the cellular/tissue environment in determining the function of a protein, and underscores the importance of understanding the molecular context of a disease to inform therapy design.

Mitochondrial dysfunctions in cancer: genetic defects and oncogenic signaling impinging on TCA cycle activity.

The tricarboxylic acid (TCA) cycle is a central route for oxidative metabolism. Besides being responsible for the production of NADH and FADH2, which fuel the mitochondrial electron transport chain to generate ATP, the TCA cycle is also a robust source of metabolic intermediates required for anabolic reactions. This is particularly important for highly proliferating cells, like tumour cells, which require a continuous supply of precursors for the synthesis of lipids, proteins and nucleic acids. A number of mutations among the TCA cycle enzymes have been discovered and their association with some tumour types has been established. In this review we summarise the current knowledge regarding alterations of the TCA cycle in tumours, with particular attention to the three germline mutations of the enzymes succinate dehydrogenase, fumarate hydratase and isocitrate dehydrogenase, which are involved in the pathogenesis of tumours, and to the aberrant regulation of TCA cycle components that are under the control of oncogenes and tumour suppressors.

MAPK14/p38α-dependent modulation of glucose metabolism affects ROS levels and autophagy during starvation.

Increased glycolytic flux is a common feature of many cancer cells, which have adapted their metabolism to maximize glucose incorporation and catabolism to generate ATP and substrates for biosynthetic reactions. Indeed, glycolysis allows a rapid production of ATP and provides metabolic intermediates required for cancer cells growth. Moreover, it makes cancer cells less sensitive to fluctuations of oxygen tension, a condition usually occurring in a newly established tumor environment. Here, we provide evidence for a dual role of MAPK14 in driving a rearrangement of glucose metabolism that contributes to limiting reactive oxygen species (ROS) production and autophagy activation in condition of nutrient deprivation. We demonstrate that MAPK14 is phosphoactivated during nutrient deprivation and affects glucose metabolism at 2 different levels: on the one hand, it increases SLC2A3 mRNA and protein levels, resulting in a higher incorporation of glucose within the cell. This event involves the MAPK14-mediated enhancement of HIF1A protein stability. On the other hand, MAPK14 mediates a metabolic shift from glycolysis to the pentose phosphate pathway (PPP) through the modulation of PFKFB3 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase 3) degradation by the proteasome. This event requires the presence of 2 distinct degradation sequences, KEN box and DSG motif Ser273, which are recognized by 2 different E3 ligase complexes. The mutation of either motif increases PFKFB3 resistance to starvation-induced degradation. The MAPK14-driven metabolic reprogramming sustains the production of NADPH, an important cofactor for many reduction reactions and for the maintenance of the proper intracellular redox environment, resulting in reduced levels of ROS. The final effect is a reduced activation of autophagy and an increased resistance to nutrient deprivation.

Mitochondrial Stress Signalling: HTRA2 and Parkinson's Disease.

Mitochondria are cellular energy generators whose activity requires a continuous supply of oxygen. Recent genetic analysis has suggested that defects in mitochondrial quality control may be key factors in the development of Parkinson's disease (PD). Mitochondria have a crucial role in supplying energy to the brain, and their deterioration can affect the function and viability of neurons, contributing to neurodegeneration. These organelles can sow the seeds of their own demise because they generate damaging oxygen-free radicals as a byproduct of their intrinsic physiological functions. Mitochondria have therefore evolved specific molecular quality control mechanisms to compensate for the action of damaging agents such as oxygen-free radicals. PTEN-induced putative kinase 1 (PINK1) and high-temperature-regulated A2 (HTRA2), a mitochondrial protease, have recently been proposed to be key modulators of mitochondrial molecular quality control. Here, we review some of the most recent advances in our understanding of mitochondria stress-control pathways, focusing on how signalling by the p38 stress kinase pathway may regulate mitochondrial stress by modulating the activity of HTRA2 via PINK1 and cyclin-dependent kinase 5 (CDK5). We also propose how defects in this pathway may contribute to PD.

Glutathione participates in the modulation of starvation-induced autophagy in carcinoma cells.

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, GSH) is the most abundant low molecular weight, thiol-containing compound within the cells and has a primary role in the antioxidant defense and intracellular signaling. Here we demonstrated that nutrient deprivation led to a significant decrease of intracellular GSH levels in three different carcinoma cell lines. This phenomenon was dependent on ABCC1-mediated GSH extrusion, along with GCL inhibition and, to a minor extent, the formation of GSH-protein mixed disulfides that synergistically contributed to the modulation of autophagy by shifting the intracellular redox state toward more oxidizing conditions. Modulation of intracellular GSH by inhibiting its de novo synthesis through incubation with buthionine sulfoximine, or by maintaining its levels through GSH ethyl ester, affected the oxidation of protein thiols, such as PRDXs and consequently the kinetics of autophagy activation. We also demonstrated that thiol-oxidizing or -alkylating agents, such as diamide and diethyl maleate activated autophagy, corroborating the evidence that changes in thiol redox state contributed to the occurrence of autophagy.

Under the ROS thiol network is the principal suspect for autophagy commitment.

Low molecular weight and protein sulphydryls undergo reactive oxygen species (ROS)-mediated oxidation. However, in contrast to the irreversible damages that oxidative conditions yield on biomolecules, the oxidation of reactive cysteines frequently results in reversible modifications, which represent the prototype of the molecular mechanisms underlying redox signaling. Many proteins involved in a wide range of cellular processes have been classified as "redoxsensitive," thereby modulating their function/activity dependent on the redox state of their critical thiols. Growing evidence from the past few years supports the idea that ROS production also correlates with the occurrence of autophagy. Nonetheless, the cysteine protease Atg4 remains the sole example of a protein whose redox regulation has been completely characterized and demonstrated to be necessary for the progression of autophagy. The principal aim of this commentary is to draw attention to the remarkable number of proteins that can fit the double role of: (i) being involved in autophagy, especially in autophagosome formation and (ii) sensing alterations of the cellular redox state by means of reactive cysteine residues. We will also attempt to provide a hypothetical model to explain the possible functional role of thiols in the occurrence of autophagy and outline a network of redox reactions likely concurring to allow the correct initiation and completion of autophagosomes.

Carcinoma cells activate AMP-activated protein kinase-dependent autophagy as survival response to kaempferol-mediated energetic impairment.

Kaempferol, a dietary cancer chemopreventive polyphenol, has been reported to trigger apoptosis in several tumor histotypes, but the mechanism underlying this phenomenon is not fully understood. Here, we demonstrate that in HeLa cells, kaempferol induces energetic failure due to inhibition of both glucose uptake and Complex I of the mitochondrial respiratory chain. As adaptive response, cells activate autophagy, the occurrence of which was established cytofluorometrically, upon acridine orange staining, and immunochemically, by following the increase of the autolysosome-associated form of the microtubule-associated protein light chain 3 (LC3-II). Autophagy is an early and reversible process occurring as survival mechanisms against apoptosis. Indeed, chemical inhibition of autophagy, by incubations with monensin, wortmannin, 3-methyladenine, or by silencing Atg5, significantly increases the extent of apoptosis, which takes place via the mitochondrial pathway, and shortens the time in which the apoptotic markers are detectable. We also demonstrate that autophagy depends on the early activation of the AMP-activated protein kinase (AMPK)/mTOR-mediated pathway. The overexpression of dominant negative AMPK results in a decrease of autophagic cells, a decrement of LC3-II levels, and a significant increase of apoptosis. Experiments performed with another carcinoma cell line yielded the same results, suggesting for kaempferol a unique mechanism of action.