TFEB inhibition induces melanoma shut-down by blocking the cell cycle and rewiring metabolism.

Melanomas are characterised by accelerated cell proliferation and metabolic reprogramming resulting from the contemporary dysregulation of the MAPK pathway, glycolysis and the tricarboxylic acid (TCA) cycle. Here, we suggest that the oncogenic transcription factor EB (TFEB), a key regulator of lysosomal biogenesis and function, controls melanoma tumour growth through a transcriptional programme targeting ERK1/2 activity and glucose, glutamine and cholesterol metabolism. Mechanistically, TFEB binds and negatively regulates the promoter of DUSP-1, which dephosphorylates ERK1/2. In melanoma cells, TFEB silencing correlates with ERK1/2 dephosphorylation at the activation-related p-Thr185 and p-Tyr187 residues. The decreased ERK1/2 activity synergises with TFEB control of CDK4 expression, resulting in cell proliferation blockade. Simultaneously, TFEB rewires metabolism, influencing glycolysis, glucose and glutamine uptake, and cholesterol synthesis. In TFEB-silenced melanoma cells, cholesterol synthesis is impaired, and the uptake of glucose and glutamine is inhibited, leading to a reduction in glycolysis, glutaminolysis and oxidative phosphorylation. Moreover, the reduction in TFEB level induces reverses TCA cycle, leading to fatty acid production. A syngeneic BRAFV600E melanoma model recapitulated the in vitro study results, showing that TFEB silencing sustains the reduction in tumour growth, increase in DUSP-1 level and inhibition of ERK1/2 action, suggesting a pivotal role for TFEB in maintaining proliferative melanoma cell behaviour and the operational metabolic pathways necessary for meeting the high energy demands of melanoma cells.

Consensus clustering methodology to improve molecular stratification of non-small cell lung cancer.

Recent advances in machine learning research, combined with the reduced sequencing costs enabled by modern next-generation sequencing, paved the way to the implementation of precision medicine through routine multi-omics molecular profiling of tumours. Thus, there is an emerging need of reliable models exploiting such data to retrieve clinically useful information. Here, we introduce an original consensus clustering approach, overcoming the intrinsic instability of common clustering methods based on molecular data. This approach is applied to the case of non-small cell lung cancer (NSCLC), integrating data of an ongoing clinical study (PROMOLE) with those made available by The Cancer Genome Atlas, to define a molecular-based stratification of the patients beyond, but still preserving, histological subtyping. The resulting subgroups are biologically characterized by well-defined mutational and gene-expression profiles and are significantly related to disease-free survival (DFS). Interestingly, it was observed that (1) cluster B, characterized by a short DFS, is enriched in KEAP1 and SKP2 mutations, that makes it an ideal candidate for further studies with inhibitors, and (2) over- and under-representation of inflammation and immune systems pathways in squamous-cell carcinomas subgroups could be potentially exploited to stratify patients treated with immunotherapy.

Lenalidomide normalizes tumor vessels in colorectal cancer improving chemotherapy activity.

BACKGROUND: Angiogenesis inhibition is a promising approach for treating metastatic colorectal cancer (mCRC). Recent evidences support the seemingly counterintuitive ability of certain antiangiogenic drugs to promote normalization of residual tumor vessels with important clinical implications. Lenalidomide is an oral drug with immune-modulatory and anti-angiogenic activity against selected hematologic malignancies but as yet little is known regarding its effectiveness for solid tumors. The aim of this study was to determine whether lenalidomide can normalize colorectal cancer neo-vessels in vivo, thus reducing tumor hypoxia and improving the benefit of chemotherapy. METHODS: We set up a tumorgraft model with NOD/SCID mice implanted with a patient-derived colorectal cancer liver metastasis. The mice were treated with oral lenalidomide (50 mg/Kg/day for 28 days), intraperitoneal 5-fluorouracil (5FU) (20 mg/Kg twice weekly for 3 weeks), combination (combo) of lenalidomide and 5FU or irrelevant vehicle. We assessed tumor vessel density (CD146), pericyte coverage (NG2; alphaSMA), in vivo perfusion capability of residual vessels (lectin distribution essay), hypoxic areas (HP2-100 Hypoxyprobe) and antitumor activity in vivo and in vitro. RESULTS: Treatment with lenalidomide reduced tumor vessel density (p = 0.0001) and enhanced mature pericyte coverage of residual vessels (p = 0.002). Perfusion capability of tumor vessels was enhanced in mice treated with lenalidomide compared to controls (p = 0.004). Accordingly, lenalidomide reduced hypoxic tumor areas (p = 0.002) and enhanced the antitumor activity of 5FU in vivo. The combo treatment delayed tumor growth (p = 0.01) and significantly reduced the Ki67 index (p = 0.0002). Lenalidomide alone did not demonstrate antitumor activity compared to untreated controls in vivo or against 4 different mCRC cell lines in vitro. CONCLUSIONS: We provide the first evidence of tumor vessel normalization and hypoxia reduction induced by lenalidomide in mCRC in vivo. This effect, seemingly counterintuitive for an antiangiogenic compound, translates into indirect antitumor activity thus enhancing the therapeutic index of chemotherapy. Our findings suggest that further research should be carried out on synergism between lenalidomide and conventional therapies for treating solid tumors that might benefit from tumor vasculature normalization.

Class 3 semaphorins: physiological vascular normalizing agents for anti-cancer therapy.

Findings from preclinical and clinical studies show that vascular normalization represents a novel strategy to enhance the efficacy of and overcome the acquired resistance to anti-angiogenic therapies in cancer. Several mechanisms of tumour vessel normalization have been revealed. Amongst them, secreted class 3 semaphorins (Sema3), which regulate axon guidance and angiogenesis, have been recently identified as novel vascular normalizing agents that inhibit metastatic dissemination by restoring vascular function. Here, we discuss the different biological functions and mechanisms of action of Sema3 in the context of tumour vascular normalization, and their impact on the different cellular components of the tumour microenvironment.

Symmetry breaking mechanism for epithelial cell polarization.

In multicellular organisms, epithelial cells form layers separating compartments responsible for different physiological functions. At the early stage of epithelial layer formation, each cell of an aggregate defines an inner and an outer side by breaking the symmetry of its initial state, in a process known as epithelial polarization. By integrating recent biochemical and biophysical data with stochastic simulations of the relevant reaction-diffusion system, we provide evidence that epithelial cell polarization is a chemical phase-separation process induced by a local bistability in the signaling network at the level of the cell membrane. The early symmetry breaking event triggering phase separation is induced by adhesion-dependent mechanical forces localized in the point of convergence of cell surfaces when a threshold number of confluent cells is reached. The generality of the emerging phase-separation scenario is likely common to many processes of cell polarity formation.

Tumor necrosis factor-alpha in airway secretions from cystic fibrosis patients upregulate endothelial adhesion molecules and induce airway epithelial cell apoptosis: implications for cystic fibrosis lung disease.

Airway inflammation plays a crucial role in lung damage in cystic fibrosis (CF) and is characterized by a persistent influx of neutrophils into the airways. We hypothesized that the high levels of inflammatory products that accumulate in the microenvironment of the CF lung contribute to induce the persistent neutrophil recruitment and the airway epithelial damage. Thus, we evaluated the in vitro effect of sputum sol phase (SSP) from CF patients on a) adhesion molecule expression by human microvascular endothelial cells (HMECs) and b) apoptosis of human bronchial epithelial cells (HBECs), both wild-type and CFTR-defective. SSP was obtained from 7 clinically stable adult CF patients and 8 patients with an acute exacerbation. HMECs and HBECs were cultured in the absence or presence of SSP. Cell adhesion molecule expression was assessed by flow cytometry and cell death by the detection of histone-associated DNA fragments, caspase activation, and cytochrome c release. SSP obtained from CF patients, especially at the time of an acute exacerbation, induced a) an upregulation of endothelial adhesion molecules on cultured HMECs that was associated with an increase of neutrophil adhesion to these cells, and was mediated at least in part by TNF-alpha and IL-1 and b) apoptosis of airway epithelial cells, mainly activated by TNF- alpha pathway. These results suggest that the high concentrations of inflammatory mediators in CF airways contribute both to the chronic neutrophil influx and the airway damage, and support the crucial role of early anti-inflammatory treatment in the disease.

Diacylglycerol kinase-alpha phosphorylation by Src on Y335 is required for activation, membrane recruitment and Hgf-induced cell motility.

Diacylglycerol (DAG) kinases (Dgk), which phosphorylate DAG to generate phosphatidic acid, act as either positive or negative key regulators of cell signaling. We previously showed that Src mediates growth factors-induced activation of Dgk-alpha, whose activity is required for cell motility, proliferation and angiogenesis. Here, we demonstrate that both hepatocytes growth factor (HGF) stimulation and v-Src transformation induce tyrosine phosphorylation of Dgk-alpha on Y335, through a mechanism requiring its proline-rich C-terminal sequence. Moreover, we show that both proline-rich sequence and phosphorylation of Y335 of Dgk-alpha mediate: (i) its enzymatic activation, (ii) its ability to interact respectively with SH3 and SH2 domains of Src, (iii) its recruitment to the membrane. In addition, we show that phosphorylation of Dgk-alpha on Y335 is required for HGF-induced motility, while its constitutive recruitment at the membrane by myristylation is sufficient to trigger spontaneous motility in absence of HGF. Providing the first evidence that tyrosine phosphorylation of Dgk-alpha is required for growth-factors-induced activation and membrane recruitment, these findings underscore its relevance as a rheostat, whose activation is a threshold to elicit growth factors-induced migratory signaling.

Percolation, morphogenesis, and burgers dynamics in blood vessels formation.

Experiments of in vitro formation of blood vessels show that cells randomly spread on a gel matrix autonomously organize to form a connected vascular network. We propose a simple model which reproduces many features of the biological system. We show that both the model and the real system exhibit a fractal behavior at small scales, due to the process of migration and dynamical aggregation, followed at large scale by a random percolation behavior due to the coalescence of aggregates. The results are in good agreement with the analysis performed on the experimental data.

Recombinant AAV vector encoding human VEGF165 enhances wound healing.

Delivery of therapeutic genes represents an appealing possibility to accelerate healing of wounds that are otherwise difficult to treat, such as those in patients with metabolic disorders or infections. Experimental evidence indicates that in such conditions potentiation of neo-angiogenesis at the wound site might represent an important therapeutic target. Here we explore the efficacy of gene therapy of wound healing with an adeno-associated virus (AAV) vector expressing the 165 amino acid isoform of vascular endothelial growth factor-A (VEGF-A). By gene marker studies, we found that AAV vectors are highly efficient for gene transfer to the rat skin, displaying an exquisite tropism for the panniculus carnosus. Gene expression from these vectors is sustained and persistent over time. Delivery of VEGF165 to full thickness excisional wounds in rats resulted in remarkable induction of new vessel formation, with consequent reduction of the healing time. Histological examination of treated wounds revealed accelerated remodeling of epidermis and dermis, with formation of a thick granular layer, containing numerous newly formed capillaries, as well as vessels of larger size. These data underline the importance of neo-angiogenesis in the healing process and indicate that VEGF gene transfer might represent a novel approach to treat wound healing disorders.

Cytoadherence of Plasmodium falciparum-infected erythrocytes is mediated by a redox-dependent conformational fraction of CD36.

The adherence of Plasmodium falciparum-infected RBC (IRBC) to postcapillary venular endothelium is an important determinant of the pathogenesis of severe malaria complications. Cytoadherence of IRBC to endothelial cells involves specific receptor/ligand interactions. The glycoprotein CD36 expressed on endothelial cells is the major receptor involved in this interaction. Treatment of CD36-expressing cells with reducing agents, such as DTT and N-acetylcysteine, was followed by CD36 conformational change monitorable by the appearance of the Mo91 mAb epitope. Only a fraction of the surface expressed CD36 molecules became Mo91 positive, suggesting the presence of two subpopulations of molecules with different sensitivities to reduction. The Mo91 epitope has been localized on a peptide (residues 260-279) of the C-terminal, cysteine-rich region of CD36. Treatment with reducing agents inhibited the CD36-dependent cytoadherence of IRBC to CD36-expressing cells and dissolved pre-existent CD36-mediated IRBC/CD36-expressing cell aggregates. CD36 reduction did not impair the functionality of CD36, since the reactivity of other anti-CD36 mAbs as well as the binding of oxidized low density lipoprotein, a CD36 ligand, were maintained. The modifications induced by reduction were reversible. After 14 h CD36 was reoxidized, the cells did not express the Mo91 epitope, and cytoadherence to IRBC was restored. The results indicate that IRBCs bind only to a redox-modulated fraction of CD36 molecules expressed on the cell surface. The present data indicate the therapeutic potential of reducing agents, such as the nontoxic drug N-acetylcysteine, to prevent or treat malaria complications due to IRBC cytoadhesion.

Involvement of a serine protease, but not of neutrophil elastase, in tumor necrosis factor-induced lethal hepatitis and induction of platelet-activating factor.

BACKGROUND/AIMS: Tumor necrosis factor (TNF) plays an essential role in several types of acute and chronic hepatitis. Our aims in the present study were to elucidate the mechanism by which TNF leads to acute lethal hepatitis, thereby focusing on the role of serine proteases and platelet-activating factor (PAF). METHODS: All experiments were performed in a model of acute hepatitis, induced by TNF in combination with D-(+)-galactosamine (GalN). Neutrophil elastase (NE)-deficient mice, generated by gene targeting were used in the studies. RESULTS: We found that a serine protease plays an essential mediating role in the in vivo TNF effect as alpha1-antitrypsin (alpha1-AT), soybean trypsin inhibitor (STI) and turkey trypsin inhibitor (TTI), confer complete protection. alpha1-AT and TTI, but not STI, reduce PAF blood levels, induced by TNF/GalN, which is compatible with an elastase-like serine protease involvement in PAF synthesis. In our search for relevant serine proteases we believed that NE was an excellent candidate protease. However, we found that TNF/GalN-induced lethality is not attenuated in mice deficient in NE. CONCLUSIONS: The data suggest that TNF-induced lethal hepatitis is accompanied by increases in circulating PAF and plasma clotting time, and mediated by a serine protease, but not by NE.

Dynamic modules and heterogeneity of function: a lesson from tyrosine kinase receptors in endothelial cells.

An important unresolved issue related to tyrosine kinase receptor signaling pathways is the lack of specificity of the molecular effectors involved. The specificity of the biological responses that are nevertheless elicited may be explained by differences in activation thresholds, as well as by temporal (transient versus sustained) and topographical aspects of receptor activation. On the basis of recent lessons from endothelial cells, we argue that an additional strategy can be adopted to generate specificity, i.e. tyrosine kinase receptors may form distinct signaling modules with other transmembrane proteins, such as adhesive receptors, to elicit different biological programs in stimulated cells.

Interactions between endothelial cells and HIV-1.

Endothelial cells (EC) participate in inflammatory and immune reactions by producing and responding to soluble mediators. Human immunodeficiency virus (HIV)-1 profoundly alters the features of EC. In some anatomical districts, they are infected by the virus and may represent a relevant reservoir. During lymphomononuclear cell diapedesis, EC activate virus replication in crossing cells. Direct or indirect damage of EC is particularly relevant in central nervous system, where blood-brain barrier perturbation is pivotal in neuronal degeneration. The observed alterations of EC adhesive properties contribute in altered leukocyte traffic from blood to lymphoid organs and tissues and play a role in the onset of immune surveillance alteration. These alterations of EC functions are relevant for the general vasculopathy, which marks the acquired immunodeficiency syndrome, and in particular are instrumental in the pathogenesis of Kaposi's sarcoma. Here we discuss the biological and molecular activation of EC in HIV-1 infection that represents the basis to understand the pathogenesis of HIV-1 associated vascular diseases.

Expression of angiopoietin-1 in human glioblastomas regulates tumor-induced angiogenesis: in vivo and in vitro studies.

To define a role for the angiopoietin/Tie2 system in astrocytoma angiogenesis, we examined the expression of angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) in these tumors by immunohistochemistry and in situ hybridization. Furthermore, we studied in vitro the effects elicited by glioblastoma cell-secreted Ang1 or by recombinant Ang1 on functions of endothelial cells (ECs). Our observations of astrocytomas show that a stage-specific induction of angiopoietins occurs and is correlated with angiogenic phases of different intensity. Ang1 expression was found in a few astrocytes scattered in the tumor at all stages of astrocytoma progression. In blood vessels, Ang1 mRNA increased progressively in high-grade glioblastomas, in which the number of vessels was higher than in low-grade tumors. Ang2 was detected in tumor cells and in ECs in high-grade astrocytomas, whereas its expression was negligible in low-grade tumors. Coculture of glioblastoma cell lines producing Ang1 with endothelium demonstrated a key role of this ligand in the control of EC network organization. We found that recombinant Ang1 in vitro induces EC spreading and reorganization of the cell monolayer into cordlike structures. These results suggest that Ang1 directly acts on ECs by modulating cell-cell and cell-matrix associations and promoting the differentiation phase of angiogenesis.

IL-12 inhibition of endothelial cell functions and angiogenesis depends on lymphocyte-endothelial cell cross-talk.

In vivo IL-12-dependent tumor inhibition rests on the ability of IL-12 to activate a CD8-mediated cytotoxicity, inhibit angiogenesis, and cause vascular injury. Although in vivo studies have shown that such inhibition stems from complex interactions of immune cells and the production of IFN-gamma and other downstream angiostatic chemokines, the mechanisms involved are still poorly defined. Here we show that IL-12 activates an anti-angiogenic program in Con A-activated mouse spleen cells (activated spc) or human PBMC (activated PBMC). The soluble factors they release in its presence arrest the cycle of endothelial cells (EC), inhibit in vitro angiogenesis, negatively modulate the production of matrix metalloproteinase-9, and the ability of EC to adhere to vitronectin and up-regulate ICAM-1 and VCAM-1 expression. These effects do not require direct cell-cell contact, yet result from continuous interaction between activated lymphoid cells and EC. We used neutralizing Abs to show that the IFN-inducible protein-10 and monokine-induced by IFN-gamma chemokines are pivotal in inducing these effects. Experiments with nu/nu mice, nonobese diabetic-SCID mice, or activated spc enriched in specific cell subpopulations demonstrated that CD4(+), CD8(+), and NK cells are all needed to mediate the full anti-angiogenetic effect of IL-12.

Tat-induced platelet-activating factor synthesis contributes to the angiogenic effect of HIV-1 Tat.

This study shows that human umbilical cord vein-derived endothelial cells (HUVEC) stimulated with HIV-1 Tat synthesized platelet-activating factor (PAF), a phospholipid mediator of inflammation that possesses angiogenic properties. The synthesis of PAF by HUVEC stimulated with Tat was dose and time dependent. Moreover, in vitro experiments were performed to evaluate whether migration of HUVEC induced by Tat was dependent on the synthesis of PAF. It was found that the cell motility induced by Tat was inhibited by WEB 2170, a specific PAF receptor antagonist. In vivo, the neoangiogenesis induced by Tat was also inhibited by WEB 2170 in a murine model, in which matrigel subcutaneously injected was used as substratum for angiogenesis. These results suggest that the synthesis of PAF by endothelial cells mediates, at least in part, the angiogenic activity of Tat by promoting the endothelial cell migration.

HIV-1 Tat protein stimulates in vivo vascular permeability and lymphomononuclear cell recruitment.

HIV-1 Tat protein released by infected cells is a chemotactic molecule for leukocytes and induces a proinflammatory program in endothelial cells (EC) by activating vascular endothelial growth factor (VEGF) receptors expressed on both cell types. Its potential role in causing vascular permeability and leukocyte recruitment was studied in vivo following its s.c. injection in mice. Tat caused a dose-dependent early (15 min) and late (6 h) wave of permeability that were inhibited by a neutralizing Ab anti-VEGF receptor type 2. Tissue infiltration of lymphomononuclear cells, mainly monocytes (76%), was evident at 6 h and persisted up to 24 h. WEB2170, a platelet activating factor (PAF) receptor antagonist, reduced the early leakage by 70-80%, but only slightly inhibited the late wave and cell recruitment. In vitro, Tat induced a dose-dependent flux of albumin through the EC monolayer that was inhibited by Ab anti-vascular VEGF receptor type 2 and WEB2170, and PAF synthesis in EC that was blocked by the Ab anti-VEGF receptor type 2. Lastly, an anti-monocyte chemotactic peptide-1 (MCP-1) Ab significantly reduced the lymphomononuclear infiltration elicited by Tat. In vitro, Tat induced a dose-dependent production of MCP-1 by EC after a 24-h stimulation. These results highlighted the role of PAF and MCP-1 as secondary mediators in the onset of lymphomononuclear cell recruitment in tissues triggered by Tat.

Prevention by delay: nonspecific immunity elicited by IL-12 hinders Her-2/neu mammary carcinogenesis in transgenic mice.

As a natural consequence of the expression of the activated transforming rat Her-2/neu oncogene all mammary glands of female transgenic BALB/c (BALB-neuT) mice develop atypical epithelial hyperplasia which progresses to invasive carcinoma. A lobular carcinoma is palpable in all mammary glands of 33-week-old BALB-neuT mice. This progression is markedly delayed by systemic administration of IL-12. In a series of studies the best administration schedule, the lowest dose and the most effective administration time have been defined. The cellular and molecular mechanisms resulting in the delay of carcinogenesis have been established. By means of a series of downstream mediators IL-12 inhibits the angiogenic burst that goes along with the passage from preneoplastic to neoplastic and invasive lesions; it also recruits lymphoid cells in the mammary pad and activates their cytotoxicity towards neoplastic cells and newly formed vessels; and furthermore, it induces lymphoid cells to trigger antiangiogenic activities in neoplastic epithelial cells. Effective, low-dose and non-toxic IL-12 treatments may thus be envisaged as a possible option in the management of preneoplastic mammary lesions and in mammary cancer prevention.

Human immunodeficiency virus transactivator protein (Tat) stimulates chemotaxis, calcium mobilization, and activation of human polymorphonuclear leukocytes: implications for Tat-mediated pathogenesis.

The extracellular activities of the human immunodeficiency virus (HIV) transactivator protein (Tat) include induction of angiogenesis and stimulation of monocyte migration. Here it is shown that polymorphonuclear leukocytes (PMNL), mostly neutrophils, rapidly invade in response to Tat in vivo and initiate the formation of new vessels. In vitro, Tat was chemotactic for PMNL and induced calcium (Ca(2+)) mobilization. Tat proteins with inactivating substitutions in the arginine-glycine-aspartic acid or basic domain were still active in inducing PMNL migration, whereas Tat peptides mapped the migration and Ca(2+) mobilization activity to a cysteine-rich core domain, previously described as a Tat "chemokine-like" region (peptide CysL(24-51)). Tat and the CysL(24-51) peptide also induced PMNL superoxide production and the release of the angiogenic factors interleukin-8 and vascular endothelial growth factor from PMNL. CysL(24-51) did not induce endothelial cell migration but was angiogenic in vivo. These data indicate that the Tat activity on PMNL is mediated by its chemokine-like region and that PMNL recruitment by Tat is linked to angiogenesis.

Cu(II) and Zn(II) complexes with hyaluronic acid and its sulphated derivative. Effect on the motility of vascular endothelial cells.

With the aim of improving the compatibility of biomaterials to be used for the construction of cardiovascular prosthesis, we have designed bioactive macromolecules resulting from chemical modifications of hyaluronic acid (Hyal). The stability constants of Cu(II) and Zn(II) complexes with the sulphated derivative of hyaluronic acid (HyalS3.5) were evaluated. Two different complexes have been found for each metal ion, CuL, Cu(OH)2L and ZnL, Zn(OH)2L (L means the disaccharide unit of the ligands) in aqueous solution at 37 degrees C. The dihydroxo Cu(II) complex was present in high percentage at pH=7.4. On the contrary, the Zn(II) ion was present with a relatively low percentage of both complexes. The ability to stimulate endothelial cell adhesion and migration was evaluated for Hyal, HyalS3.5 and their complexes with Cu(II) and Zn(II) ions. The results revealed that Hyal and [Cu(OH)2HyalS3.5](4.5)- induced cell adhesion, while [ZnHyalS3.5](2.5)- and [Zn(OH)2HyalS3.5](4.5)- inhibited the process. The chemotactic activity of increasing concentrations of the above complexes was also evaluated, demonstrating that [Cu(OH)2HyalS3.5](4.5)- complex at 1 microM concentration was the most active in inducing cell migration. These results have been also strengthened by analysing adherent cell migration in agarose. In conclusion, sulphated hyaluronic acid coordinated to Cu(II) seems to be a promising matrix molecule for the construction of cardiovascular prosthesis.