Annarita Favia - Academia.edu (original) (raw)

Papers by Annarita Favia

Long noncoding RNAs (lncRNAs) are major regulators of physiological and disease-related gene expr... more Long noncoding RNAs (lncRNAs) are major regulators of physiological and disease-related gene expression, particularly in the central nervous system. Dysregulated lncRNA expression has been documented in several human cancers, and their tissue-specificity makes them attractive candidates as diagnostic/prognostic biomarkers and/or therapeutic agents. Here we show that linc-NeD125, which we previously characterized as a neuronal-induced lncRNA, is significantly overexpressed in Group 4 medulloblastomas (G4 MBs), the largest and least well characterized molecular MB subgroup. Mechanistically, linc-NeD125 is able to assemble the miRNA-induced silencing complex (miRISC) and to directly bind the microRNAs, miR-19a-3p, miR-19b-3p and miR-106a-5p. Functionally, linc-NeD125 acts as a competing endogenous RNA (ceRNA) that, sequestering the three miRNAs, leads to de-repression of their targets CDK6, MYCN, SNCAIP, and KDM6A, which are major driver genes of G4 MB. We also provide evidence that li...

A common catastrophic event in most human cancers is deregulation of MYC, a multifunctional trans... more A common catastrophic event in most human cancers is deregulation of MYC, a multifunctional transcription factor that controls gene expression in partnership with MAX and drives key biological mechanisms of the cell. Restraining its activity impairs cancer cell features and prevents tumor development, as shown by Omomyc - a 90 amino acid mini-protein interfering with MYC activity. MYC regulates many aspects of transcription by RNA polymerase II (RNAPII), such as activation, pause release, and elongation. That it may have a role in transcription termination as well is suggested by our finding of an interaction between MYC and the Protein Arginine Methyltransferase 5 (PRMT5), which catalyzes symmetrical dimethylation of RNAPII at the arginine residue R1810 (R1810me2s) allowing proper termination and splicing of transcripts. Here we show that MYC overexpression strongly increases R1810me2s, while the concomitant expression of Omomyc or a MYC-specific shRNA counteracts this capacity. Om...

Oncotarget, 2017

Long noncoding RNAs (lncRNAs) are major regulators of physiological and diseaserelated gene expre... more Long noncoding RNAs (lncRNAs) are major regulators of physiological and diseaserelated gene expression, particularly in the central nervous system. Dysregulated lncRNA expression has been documented in several human cancers, and their tissueand/or therapeutic agents. Here we show that linc-NeD125, which we previously subgroup. Mechanistically, linc-NeD125 is able to recruit the miRNA-induced silencing complex (miRISC) and to directly bind the microRNAs miR-19a-3p, miR-19b-3p and miR-106a-5p. Functionally, linc-NeD125 acts as a competing endogenous RNA (ceRNA) that, sequestering the three miRNAs, leads to de-repression of their targets CDK6, attenuates their proliferation, migration and invasion. system tumours and provides a novel molecular circuit underlying the enigmatic

e release of Ca2+ from intracellular stores is a key process involved in a variety of signaling p... more e release of Ca2+ from intracellular stores is a key process involved in a variety of signaling pathways. e second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca2+ from lysosomelike acidic stores but not from IP3-sensitive or ryanodine-sensitive Ca2+ stores in different cell types. To date, two receptors for NAADP have been discovered, namely TPC1 and TPC2. Interestingly, intracellular calcium dynamics plays a central role in angiogenesis of endothelial cells (EC) which is primarily triggered in response to growth factor such as vascular endothelial growth factor (VEGF). In addition to its well known role in the formation of new blood vessels, VEGF is also involved in a number of different EC functions such as proliferation, migration, regulation of vascular permeability and survival through the binding to two receptors (VEGFR1 and VEGFR2) both stimulating intracellular calcium mobilization. e aim of our study is to characterize the role of Ca2+ in VEGF-dependent signal transduction and to correlate its intracellular signaling to specific biological functions. Our data propose the involvement of NAADP in VEGFR2- but not VEGFR1-induced calcium mobilization. We show that both pharmacological inhibition of NAADP signaling with Ned-19 (a reported specific antagonist of NAADP) and the use of small interfering RNA (siRNA) targeting TPC1 and TPC2, significantly reduces VEGF-stimulated calcium release in Human Umbilical Vein Endothelial Cells (HUVEC). Our data suggest that both Ned-19 and silencing of TPC receptors by RNAi down regulates VEGF-stimulated ERK1/2 phosphorilation. Moreover, we observed that Ned-19 modulates Akt activation and inhibits VEGF-induced migration of EC. All together our data suggest that NAADP plays an important role in VEGF-induced Ca2+ signaling via tyrosine kinase receptor and open new strategies for studying EC functions

Scientific reports, Jan 6, 2016

A novel transduction pathway for the powerful angiogenic factor VEGF has been recently shown in e... more A novel transduction pathway for the powerful angiogenic factor VEGF has been recently shown in endothelial cells to operate through NAADP-controlled intracellular release of Ca(2+). In the present report the possible involvement of NAADP-controlled Ca(2+) signaling in tumor vascularization, growth and metastatic dissemination was investigated in a murine model of VEGF-secreting melanoma. Mice implanted with B16 melanoma cells were treated with NAADP inhibitor Ned-19 every second day for 4 weeks and tumor growth, vascularization and metastatization were evaluated. Control specimens developed well vascularized tumors and lung metastases, whereas in Ned-19-treated mice tumor growth and vascularization as well as lung metastases were strongly inhibited. In vitro experiments showed that Ned-19 treatment controls the growth of B16 cells in vitro, their migratory ability, adhesive properties and VEGFR2 expression, indicating NAADP involvement in intercellular autocrine signaling. To this ...

Scientific Reports, 2015

The c-Myc protein is dysregulated in many human cancers and its function has not been fully eluci... more The c-Myc protein is dysregulated in many human cancers and its function has not been fully elucitated yet. The c-Myc inhibitor Omomyc displays potent anticancer properties in animal models. It perturbs the c-Myc protein network, impairs c-Myc binding to the E-boxes, retaining transrepressive properties and inducing histone deacetylation. Here we have employed Omomyc to further analyse c-Myc activity at the epigenetic level. We show that both Myc and Omomyc stimulate histone H4 symmetric dimethylation of arginine (R) 3 (H4R3me2s), in human glioblastoma and HEK293T cells. Consistently, both associated with protein Arginine Methyltransferase 5 (PRMT5)-the catalyst of the reaction-and its co-factor Methylosome Protein 50 (MEP50). Confocal experiments showed that Omomyc co-localized with c-Myc, PRMT5 and H4R3me2s-enriched chromatin domains. Finally, interfering with PRMT5 activity impaired target gene activation by Myc whereas it restrained Omomyc-dependent repression. The identification of a histone-modifying complex associated with Omomyc represents the first demonstration of an active role of this miniprotein in modifying chromatin structure and adds new information regarding its action on c-Myc targets. More importantly, the observation that c-Myc may recruit PRMT5-MEP50, inducing H4R3 symmetric di-methylation, suggests previously unpredictable roles for c-Myc in gene expression regulation and new potential targets for therapy. Dysregulation of c-Myc-a basic helix-loop-helix/leucine zipper (bHLH-Zip) transcriptional regulator that controls a variety of normal cellular functions 1-is a major mechanism of tumorigenesis. Abnormal levels of Myc (c-, N-, L-Myc) proteins are strongly associated with a variety of human cancers. c-Mychereafter named Myc-dimerizes with another member of the same family, Max; the heterodimer binds DNA with maximum selectivity for the E-box sequence CACGTG. Myc also interacts with a large number of proteins and multicomponent complexes involved in the regulation of transcription and chromatin structure. Unlike the majority of transcription factors, it does not stimulate transcription initiation; rather

BioMed Research International, 2015

Angiopoietins are vascular factors essential for blood vessel assembly and correct organization a... more Angiopoietins are vascular factors essential for blood vessel assembly and correct organization and maturation. This study describes a novel calcium-dependent machinery activated through Angiopoietin-1/2-Tie receptor system in HUVECs monolayer. Both cytokines were found to elicit intracellular calcium mobilization. Targeting intracellular Ca2+signaling, antagonizing IP3with 2-APB or cADPR with 8Br-cADPR, was found to modulatein vitroangiogenic responses to Angiopoietins in a specific way. 2-APB and 8Br-cADPR impaired the phosphorylation of AKT and FAK induced by Ang-1 and Ang-2. On the other hand, phosphorylation of ERK1/2 and p38, as well as cell proliferation, was not affected by either inhibitor. The ability of ECs to migrate following Angs stimulation, evaluated by “scratch assay,” was reduced by either 2-APB or 8Br-cADPR following Ang-2 stimulation and only slightly affected by 2-APB in cells stimulated with Ang-1. These results identify a novel calcium-dependent machinery invo...

Journal of Cellular Biochemistry, 2013

Caveolin-1 (CAV1) is the principal structural component of caveolae which functions as scaffoldin... more Caveolin-1 (CAV1) is the principal structural component of caveolae which functions as scaffolding protein for the integration of a variety of signaling pathways. In this study, we investigated the involvement of CAV1 in endothelial cell (EC) functions and show that siRNA-induced CAV1 silencing in the human EC line EA.hy926 induces distinctive morphological changes, such as a marked increase in cell size and formation of stress fibers. Design-based stereology was employed in this work to make unbiased quantification of morphometric properties such as volume, length, and surface of CAV1 silenced versus control cells. In addition, we showed that downregulation of CAV1 affects cell cycle progression at G1/S phase transition most likely by perturbation of AKT signaling. With the aim to assess the contribution of CAV1 to typical biological processes of EC, we report here that CAV1 targeting affects cell migration and matrix metalloproteinases (MMPs) activity, and reduces angiogenesis in response to VEGF, in vitro. Taken together our data suggest that the proper expression of CAV1 is important not only for maintaining the appropriate morphology and size of ECs but it might represent a prospective molecular target for studying key biological mechanisms such as senescence and tumorigenesis.

Proceedings of the National Academy of Sciences, 2014

Significance The formation of new blood vessels (neoangiogenesis) accompanies tissue regeneration... more Significance The formation of new blood vessels (neoangiogenesis) accompanies tissue regeneration and healing, but is also crucial for tumor growth, hence understanding how capillaries are stimulated to grow in response to local cues is essential for the much sought-after aim of controlling this process. We have elucidated a Ca 2+ signaling pathway involving NAADP, TPCs, and lysosomal Ca 2+ release activated in vascular endothelial cells by VEGF, the main angiogenic growth factor, and we show that the angiogenic response can be abolished, in cultured cells and in vivo, by inhibiting components of this signaling cascade. The specificity of this pathway in terms of VEGF receptor subtype, intracellular messengers, target channels and Ca 2+ storage organelles, offers new targets for novel antiangiogenic therapeutic strategies.

Additional file 2: Figure S2. Representative examples of asexual parasites. ER: Early rings; LR: ... more Additional file 2: Figure S2. Representative examples of asexual parasites. ER: Early rings; LR: Late rings; ET: Early trophozoites; LT: Late trophozoites; ES: Early schizonts; LS: Late schizonts. Scale bar: 5 μm.

Additional file 1: Figure S1. Ned-19 induces morphological changes in the structure of late asexu... more Additional file 1: Figure S1. Ned-19 induces morphological changes in the structure of late asexual stages of P. falciparum. Late stages parasites were incubated for 6 h in the presence or absence of 125 μM Ned-19 and processed to examine their morphology. A) Samples were Giemsa stained and examined for conspicuous morphological alterations. Representative untreated and treated parasites are shown. Scale bar: 5 μm. B) Electron Micrographs showing representative DMSO and Ned-19-treated parasites. R: Rhoptry. N: Nucleus. Scale bar: 0.5 μm.

Italian journal of anatomy and embryology, 2015

We have recently identified a novel transduction pathway through which Vascular Endothelial Growt... more We have recently identified a novel transduction pathway through which Vascular Endothelial Growth Factor (VEGF) controls experimentally induced neoangiogenesis, specifically involving endothelial VEGF receptor subtype 2 and the release of intracellular calcium from NAADP (Nicotinic Acid Adenosine Dinucleotide Phosphate) responsive acidic stores (1). We have now extended this research to an in vivo model of tumor angiogenesis and show that the pharmacologic NAADP inhibitor Ned-19 (2) impairs the vascularization, growth and metastatic spreading of the very aggressive VEGF producing murine tumor, B16 melanoma. In parallel in vitro experiments, we tested whether Ned-19 could directly affect the production of VEGF by the tumor cells, and found that treatment of B16 cells with Ned-19 unexpectedly results in increased VEGF release. These observations indicate that in our model 1) tumor angiogenesis is impaired by Ned-19 even in the presence of increased exposure to VEGF and 2) that NAADP ...

Two Pore Channels (TPCs) are an emerging family of intracellular channels, expressed on acidic co... more Two Pore Channels (TPCs) are an emerging family of intracellular channels, expressed on acidic compartments, which mediate calcium signaling evoked by NAADP. In particular, we demonstrated that TPC2 isoform has a main role in angiogenesis (Favia et al. PNAS 2014 Nov 4;111(44):E4706-15). TPC2 inhibition is emerging as a key therapeutic step in a range of important pathological conditions including the progression and metastatic potential of cancer, Parkinson’s disease, and Ebola virus infection. We introduce naringenin, a natural flavonoid, as a novel TPC2 inhibitor as shown by electrophysiological evidence in a heterologous system, i.e. Arabidopsis vacuoles lacking endogenous TPCs. In view of the control exerted by TPC2 on intracellular calcium signaling and angiogenesis, we demonstrate that naringenin dampens intracellular calcium responses of human endothelial cells stimulated with VEGF, histamine or NAADP-AM, but not with ATP or Angiopoietin-1. The ability of naringenin to impair...

Scientific Reports

Protein Arginine (R) methylation is the most common post-translational methylation in mammalian c... more Protein Arginine (R) methylation is the most common post-translational methylation in mammalian cells. Protein Arginine Methyltransferases (PRMT) 1 and 5 dimethylate their substrates on R residues, asymmetrically and symmetrically, respectively. They are ubiquitously expressed and play fundamental roles in tumour malignancies, including glioblastoma multiforme (GBM) which presents largely deregulated Myc activity. Previously, we demonstrated that PRMT5 associates with Myc in GBM cells, modulating, at least in part, its transcriptional properties. Here we show that Myc/PRMT5 protein complex includes PRMT1, in both HEK293T and glioblastoma stem cells (GSCs). We demonstrate that Myc is both asymmetrically and symmetrically dimethylated by PRMT1 and PRMT5, respectively, and that these modifications differentially regulate its stability. Moreover, we show that the ratio between symmetrically and asymmetrically dimethylated Myc changes in GSCs grown in stem versus differentiating conditio...

Malaria Journal

Background: Although malaria is a preventable and curable human disease, millions of people risk ... more Background: Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca 2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. Results: This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca 2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca 2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca 2+ levels. Conclusions: This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca 2+ oscillations suggests a potential role of NAADP in regulating Ca 2+ signalling of P. falciparum.

Vascular endothelial growth factor (VEGF) and its transmembrane receptors VEGFR1 and VEGFR2 play ... more Vascular endothelial growth factor (VEGF) and its transmembrane receptors VEGFR1 and VEGFR2 play a key role in controlling both physiological and pathological angiogenesis, including vascularization of solid tumours. We have identified a novel and crucial signalling mechanism through which activation of VEGFR2 in human endothelial cells (HUVEC) selectively triggers the intracellular release of calcium from acidic compartments, operated by the second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). Live imaging of calcium fluxes in cells treated with VEGF in the presence of specific inhibitors have shown that 1) VEGF-activated calcium stores are different from IP3 and ryanodine sensitive compartments and are of acidic nature, which strongly indicates the involvement of NAADP signalling 2) NAADP inhibition by its specific antagonist Ned-19 abolishes VEGF-induced calcium response. This inhibition is accompanied by impaired phosphorylation of downstream targets ERK1/2, Akt, eNOS, JNK (but not p38) and results in significant reduction of cell proliferation, migration and capillary-like tube formation in vitro. Interestingly, when the angiogenic response to VEGF was assayed in vivo utilizing Matrigel plugs subcutaneously implanted in mice, Ned-19 was found to dramatically inhibit VEGF-induced angiogenesis. Altogether our data showing that NAADP plays a key role in the control of VEGF-induced angiogenesis could potentially contribute to identify new targets for antiangiogenic therapeutic strategies, a goal to which much scientific effort has long been devoted but still awaiting ultimate success.

Our research introduces the natural flavonoid naringenin as a novel inhibitor of an emerging clas... more Our research introduces the natural flavonoid naringenin as a novel inhibitor of an emerging class of intracellular channels, Two-Pore Channel 2 (TPC2), as shown by electrophysiological evidence in a heterologous system, i.e. Arabidopsis vacuoles lacking endogenous TPCs. In view of the control exerted by TPC2 on intracellular calcium signaling, we demonstrated that naringenin dampens intracellular calcium responses of human endothelial cells stimulated with VEGF, histamine or NAADP-AM, but not with ATP or Angiopoietin-1 (negative controls). The ability of naringenin to impair TPC2-dependent biological activities was further explored in an established in vivo model, in which VEGF-containing matrigel plugs implanted in mice failed to be vascularized in the presence of naringenin. Overall, the present data suggest that naringenin inhibition of TPC2 activity and the observed inhibition of angiogenic response to VEGF are linked by impaired intracellular calcium signaling. TPC2 inhibition is emerging as a key therapeutic step in a range of important pathological conditions including the progression and metastatic potential of melanoma, Parkinson's disease, and Ebola virus infection. The identification of naringenin as an inhibitor of TPC2-mediated signaling provides a novel and potentially relevant tool for the advancement of this field of research. Naringenin (Nar) (5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one) is one of the main flavonoids present in the human diet. Epidemiological studies have demonstrated that the consumption of vegetables and fruit with a high Nar content, such as citruses and tomatoes or their food products, is associated with a reduced incidence of metabolic and chronic-degenerative diseases 1, 2. Following this kind of evidence, several in vitro studies have shown that Nar interacts with various cellular pathways, highlighting its potential as an antioxidant, antinflammatory, chemopreventive and antidegenerative agent 3 ; moreover, Nar was shown to exert anti-angiogenic effects in the avian chorio-allantoid membrane model 4 while this manuscript was being prepared. Naringenin lowers the plasma and hepatic cholesterol concentrations by suppressing HMG-CoA reductase and ACAT in rats fed a high-cholesterol diet 5. Pre-clinical studies have revealed the potential of Nar and of its precursor naringin in the treatment of metabolic and cardiovascular disorders, including hyperlipidemia, hypertension, cardiac toxicity, hyperglycemia and diabetes, hepatic steatosis and atherosclerosis 2 ; its therapeutic use to combat various kinds of cancer has also been envisaged 3. Indeed, Nar exerts chemopreventive and anticancer activity by blocking the progression and the formation of metastasis in various experimental models of oral 6 , melanoma 7 , breast 8, 9 , colon 10 , lung 11 and liver 12 cancer. It acts by upregulating many different cell survival proteins or arresting the cell cycle, by inducing p53-dependent apoptosis or inhibiting inflammatory processes and, in some cases, by exploiting all of these mechanisms 13-15. Despite the wealth of evidence on Nar efficacy, a comprehensive view of its mechanism of action is still lacking, as is the experimental evidence needed to bridge the gap between multiple cellular targets

A novel transduction pathway for the powerful angiogenic factor VEGF has been recently shown in e... more A novel transduction pathway for the powerful angiogenic factor VEGF has been recently shown in endothelial cells to operate through NAADP-controlled intracellular release of Ca 2+. In the present report the possible involvement of NAADP-controlled Ca 2+ signaling in tumor vascularization, growth and metastatic dissemination was investigated in a murine model of VEGF-secreting melanoma. Mice implanted with B16 melanoma cells were treated with NAADP inhibitor Ned-19 every second day for 4 weeks and tumor growth, vascularization and metastatization were evaluated. Control specimens developed well vascularized tumors and lung metastases, whereas in Ned-19-treated mice tumor growth and vascularization as well as lung metastases were strongly inhibited. In vitro experiments showed that Ned-19 treatment controls the growth of B16 cells in vitro, their migratory ability, adhesive properties and VEGFR2 expression, indicating NAADP involvement in intercellular autocrine signaling. To this regard, Ca 2+ imaging experiments showed that the response of B16 cells to VEGF stimulation is NAADP-dependent. The whole of these observations indicate that NAADP-controlled Ca 2+ signaling can be relevant not only for neoangiogenesis but also for direct control of tumor cells. Vascular endothelial growth factor (VEGF) and its receptor VEGFR2 play a pivotal role in stimulating angiogen-esis, including vascularization of solid tumors 1. Angiogenesis is an important step in the outgrowth of tumors 2 controlling their dissemination, progression and metastasis. Therapeutic targeting of angiogenesis is therefore an important challenge, still in need of basic research. In particular, no clinical tool is at present successful in inhibiting the long-term response to VEGF, the most important angiogenic factor overexpressed by the vast majority of human cancers 3. Owing to the central role of VEGF, the first angiogenic inhibitors designed for clinical use were targeted against VEGF and its tyrosine kinase receptor 4. VEGF neutralizing antibodies and some multi-target tyrosine kinase inhibitors have been approved for clinical use, and many more are being evaluated in clinical trials 5. Although these therapies provide improvement in progression-free or overall survival, they are nonetheless met with drug tolerance and side effects, attesting to the complexity of VEGF signaling and tumor angiogenic cascade. Anti-VEGF treatments can induce adverse effects, possibly due to the blockade of the entire complex of VEGF signaling pathways resulting in 'off-target' effects 6–9. These findings suggest that the challenge represented by therapeutic targeting of angiogenesis in advanced cancer is an interesting aim to further pursue through better characterization of the intracellular pathways involved in the response to VEGF. Melanoma is one of the most aggressive and highly metastatic cancers. It is known that VEGF levels are associated with melanoma progression 10,11 and are used as prognostic indicators 12 , but the molecular mechanisms regulating the interaction between tumor and endothelial cells (ECs) and their role in cancer cell extravasation and

Angiopoietins are vascular factors essential for blood vessel assembly and correct organization a... more Angiopoietins are vascular factors essential for blood vessel assembly and correct organization and maturation. This study describes a novel calcium-dependent machinery activated through Angiopoietin-1/2-Tie receptor system in HUVECs monolayer. Both cytokines were found to elicit intracellular calcium mobilization. Targeting intracellular Ca 2+ signaling, antagonizing IP 3 with 2-APB or cADPR with 8Br-cADPR, was found to modulate in vitro angiogenic responses to Angiopoietins in a specific way. 2-APB and 8Br-cADPR impaired the phosphorylation of AKT and FAK induced by Ang-1 and Ang-2. On the other hand, phosphorylation of ERK1/2 and p38, as well as cell proliferation, was not affected by either inhibitor. The ability of ECs to migrate following Angs stimulation, evaluated by " scratch assay, " was reduced by either 2-APB or 8Br-cADPR following Ang-2 stimulation and only slightly affected by 2-APB in cells stimulated with Ang-1. These results identify a novel calcium-dependent machinery involved in the complex interplay regulating angiogenic processes showing that IP 3-and cADPR-induced Ca 2+ release specifically regulates distinct Angs-mediated angiogenic steps.

Long noncoding RNAs (lncRNAs) are major regulators of physiological and disease-related gene expr... more Long noncoding RNAs (lncRNAs) are major regulators of physiological and disease-related gene expression, particularly in the central nervous system. Dysregulated lncRNA expression has been documented in several human cancers, and their tissue-specificity makes them attractive candidates as diagnostic/prognostic biomarkers and/or therapeutic agents. Here we show that linc-NeD125, which we previously characterized as a neuronal-induced lncRNA, is significantly overexpressed in Group 4 medulloblastomas (G4 MBs), the largest and least well characterized molecular MB subgroup. Mechanistically, linc-NeD125 is able to assemble the miRNA-induced silencing complex (miRISC) and to directly bind the microRNAs, miR-19a-3p, miR-19b-3p and miR-106a-5p. Functionally, linc-NeD125 acts as a competing endogenous RNA (ceRNA) that, sequestering the three miRNAs, leads to de-repression of their targets CDK6, MYCN, SNCAIP, and KDM6A, which are major driver genes of G4 MB. We also provide evidence that li...

A common catastrophic event in most human cancers is deregulation of MYC, a multifunctional trans... more A common catastrophic event in most human cancers is deregulation of MYC, a multifunctional transcription factor that controls gene expression in partnership with MAX and drives key biological mechanisms of the cell. Restraining its activity impairs cancer cell features and prevents tumor development, as shown by Omomyc - a 90 amino acid mini-protein interfering with MYC activity. MYC regulates many aspects of transcription by RNA polymerase II (RNAPII), such as activation, pause release, and elongation. That it may have a role in transcription termination as well is suggested by our finding of an interaction between MYC and the Protein Arginine Methyltransferase 5 (PRMT5), which catalyzes symmetrical dimethylation of RNAPII at the arginine residue R1810 (R1810me2s) allowing proper termination and splicing of transcripts. Here we show that MYC overexpression strongly increases R1810me2s, while the concomitant expression of Omomyc or a MYC-specific shRNA counteracts this capacity. Om...

Oncotarget, 2017

Long noncoding RNAs (lncRNAs) are major regulators of physiological and diseaserelated gene expre... more Long noncoding RNAs (lncRNAs) are major regulators of physiological and diseaserelated gene expression, particularly in the central nervous system. Dysregulated lncRNA expression has been documented in several human cancers, and their tissueand/or therapeutic agents. Here we show that linc-NeD125, which we previously subgroup. Mechanistically, linc-NeD125 is able to recruit the miRNA-induced silencing complex (miRISC) and to directly bind the microRNAs miR-19a-3p, miR-19b-3p and miR-106a-5p. Functionally, linc-NeD125 acts as a competing endogenous RNA (ceRNA) that, sequestering the three miRNAs, leads to de-repression of their targets CDK6, attenuates their proliferation, migration and invasion. system tumours and provides a novel molecular circuit underlying the enigmatic

e release of Ca2+ from intracellular stores is a key process involved in a variety of signaling p... more e release of Ca2+ from intracellular stores is a key process involved in a variety of signaling pathways. e second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca2+ from lysosomelike acidic stores but not from IP3-sensitive or ryanodine-sensitive Ca2+ stores in different cell types. To date, two receptors for NAADP have been discovered, namely TPC1 and TPC2. Interestingly, intracellular calcium dynamics plays a central role in angiogenesis of endothelial cells (EC) which is primarily triggered in response to growth factor such as vascular endothelial growth factor (VEGF). In addition to its well known role in the formation of new blood vessels, VEGF is also involved in a number of different EC functions such as proliferation, migration, regulation of vascular permeability and survival through the binding to two receptors (VEGFR1 and VEGFR2) both stimulating intracellular calcium mobilization. e aim of our study is to characterize the role of Ca2+ in VEGF-dependent signal transduction and to correlate its intracellular signaling to specific biological functions. Our data propose the involvement of NAADP in VEGFR2- but not VEGFR1-induced calcium mobilization. We show that both pharmacological inhibition of NAADP signaling with Ned-19 (a reported specific antagonist of NAADP) and the use of small interfering RNA (siRNA) targeting TPC1 and TPC2, significantly reduces VEGF-stimulated calcium release in Human Umbilical Vein Endothelial Cells (HUVEC). Our data suggest that both Ned-19 and silencing of TPC receptors by RNAi down regulates VEGF-stimulated ERK1/2 phosphorilation. Moreover, we observed that Ned-19 modulates Akt activation and inhibits VEGF-induced migration of EC. All together our data suggest that NAADP plays an important role in VEGF-induced Ca2+ signaling via tyrosine kinase receptor and open new strategies for studying EC functions

Scientific reports, Jan 6, 2016

A novel transduction pathway for the powerful angiogenic factor VEGF has been recently shown in e... more A novel transduction pathway for the powerful angiogenic factor VEGF has been recently shown in endothelial cells to operate through NAADP-controlled intracellular release of Ca(2+). In the present report the possible involvement of NAADP-controlled Ca(2+) signaling in tumor vascularization, growth and metastatic dissemination was investigated in a murine model of VEGF-secreting melanoma. Mice implanted with B16 melanoma cells were treated with NAADP inhibitor Ned-19 every second day for 4 weeks and tumor growth, vascularization and metastatization were evaluated. Control specimens developed well vascularized tumors and lung metastases, whereas in Ned-19-treated mice tumor growth and vascularization as well as lung metastases were strongly inhibited. In vitro experiments showed that Ned-19 treatment controls the growth of B16 cells in vitro, their migratory ability, adhesive properties and VEGFR2 expression, indicating NAADP involvement in intercellular autocrine signaling. To this ...

Scientific Reports, 2015

The c-Myc protein is dysregulated in many human cancers and its function has not been fully eluci... more The c-Myc protein is dysregulated in many human cancers and its function has not been fully elucitated yet. The c-Myc inhibitor Omomyc displays potent anticancer properties in animal models. It perturbs the c-Myc protein network, impairs c-Myc binding to the E-boxes, retaining transrepressive properties and inducing histone deacetylation. Here we have employed Omomyc to further analyse c-Myc activity at the epigenetic level. We show that both Myc and Omomyc stimulate histone H4 symmetric dimethylation of arginine (R) 3 (H4R3me2s), in human glioblastoma and HEK293T cells. Consistently, both associated with protein Arginine Methyltransferase 5 (PRMT5)-the catalyst of the reaction-and its co-factor Methylosome Protein 50 (MEP50). Confocal experiments showed that Omomyc co-localized with c-Myc, PRMT5 and H4R3me2s-enriched chromatin domains. Finally, interfering with PRMT5 activity impaired target gene activation by Myc whereas it restrained Omomyc-dependent repression. The identification of a histone-modifying complex associated with Omomyc represents the first demonstration of an active role of this miniprotein in modifying chromatin structure and adds new information regarding its action on c-Myc targets. More importantly, the observation that c-Myc may recruit PRMT5-MEP50, inducing H4R3 symmetric di-methylation, suggests previously unpredictable roles for c-Myc in gene expression regulation and new potential targets for therapy. Dysregulation of c-Myc-a basic helix-loop-helix/leucine zipper (bHLH-Zip) transcriptional regulator that controls a variety of normal cellular functions 1-is a major mechanism of tumorigenesis. Abnormal levels of Myc (c-, N-, L-Myc) proteins are strongly associated with a variety of human cancers. c-Mychereafter named Myc-dimerizes with another member of the same family, Max; the heterodimer binds DNA with maximum selectivity for the E-box sequence CACGTG. Myc also interacts with a large number of proteins and multicomponent complexes involved in the regulation of transcription and chromatin structure. Unlike the majority of transcription factors, it does not stimulate transcription initiation; rather

BioMed Research International, 2015

Angiopoietins are vascular factors essential for blood vessel assembly and correct organization a... more Angiopoietins are vascular factors essential for blood vessel assembly and correct organization and maturation. This study describes a novel calcium-dependent machinery activated through Angiopoietin-1/2-Tie receptor system in HUVECs monolayer. Both cytokines were found to elicit intracellular calcium mobilization. Targeting intracellular Ca2+signaling, antagonizing IP3with 2-APB or cADPR with 8Br-cADPR, was found to modulatein vitroangiogenic responses to Angiopoietins in a specific way. 2-APB and 8Br-cADPR impaired the phosphorylation of AKT and FAK induced by Ang-1 and Ang-2. On the other hand, phosphorylation of ERK1/2 and p38, as well as cell proliferation, was not affected by either inhibitor. The ability of ECs to migrate following Angs stimulation, evaluated by “scratch assay,” was reduced by either 2-APB or 8Br-cADPR following Ang-2 stimulation and only slightly affected by 2-APB in cells stimulated with Ang-1. These results identify a novel calcium-dependent machinery invo...

Journal of Cellular Biochemistry, 2013

Caveolin-1 (CAV1) is the principal structural component of caveolae which functions as scaffoldin... more Caveolin-1 (CAV1) is the principal structural component of caveolae which functions as scaffolding protein for the integration of a variety of signaling pathways. In this study, we investigated the involvement of CAV1 in endothelial cell (EC) functions and show that siRNA-induced CAV1 silencing in the human EC line EA.hy926 induces distinctive morphological changes, such as a marked increase in cell size and formation of stress fibers. Design-based stereology was employed in this work to make unbiased quantification of morphometric properties such as volume, length, and surface of CAV1 silenced versus control cells. In addition, we showed that downregulation of CAV1 affects cell cycle progression at G1/S phase transition most likely by perturbation of AKT signaling. With the aim to assess the contribution of CAV1 to typical biological processes of EC, we report here that CAV1 targeting affects cell migration and matrix metalloproteinases (MMPs) activity, and reduces angiogenesis in response to VEGF, in vitro. Taken together our data suggest that the proper expression of CAV1 is important not only for maintaining the appropriate morphology and size of ECs but it might represent a prospective molecular target for studying key biological mechanisms such as senescence and tumorigenesis.

Proceedings of the National Academy of Sciences, 2014

Significance The formation of new blood vessels (neoangiogenesis) accompanies tissue regeneration... more Significance The formation of new blood vessels (neoangiogenesis) accompanies tissue regeneration and healing, but is also crucial for tumor growth, hence understanding how capillaries are stimulated to grow in response to local cues is essential for the much sought-after aim of controlling this process. We have elucidated a Ca 2+ signaling pathway involving NAADP, TPCs, and lysosomal Ca 2+ release activated in vascular endothelial cells by VEGF, the main angiogenic growth factor, and we show that the angiogenic response can be abolished, in cultured cells and in vivo, by inhibiting components of this signaling cascade. The specificity of this pathway in terms of VEGF receptor subtype, intracellular messengers, target channels and Ca 2+ storage organelles, offers new targets for novel antiangiogenic therapeutic strategies.

Additional file 2: Figure S2. Representative examples of asexual parasites. ER: Early rings; LR: ... more Additional file 2: Figure S2. Representative examples of asexual parasites. ER: Early rings; LR: Late rings; ET: Early trophozoites; LT: Late trophozoites; ES: Early schizonts; LS: Late schizonts. Scale bar: 5 μm.

Additional file 1: Figure S1. Ned-19 induces morphological changes in the structure of late asexu... more Additional file 1: Figure S1. Ned-19 induces morphological changes in the structure of late asexual stages of P. falciparum. Late stages parasites were incubated for 6 h in the presence or absence of 125 μM Ned-19 and processed to examine their morphology. A) Samples were Giemsa stained and examined for conspicuous morphological alterations. Representative untreated and treated parasites are shown. Scale bar: 5 μm. B) Electron Micrographs showing representative DMSO and Ned-19-treated parasites. R: Rhoptry. N: Nucleus. Scale bar: 0.5 μm.

Italian journal of anatomy and embryology, 2015

We have recently identified a novel transduction pathway through which Vascular Endothelial Growt... more We have recently identified a novel transduction pathway through which Vascular Endothelial Growth Factor (VEGF) controls experimentally induced neoangiogenesis, specifically involving endothelial VEGF receptor subtype 2 and the release of intracellular calcium from NAADP (Nicotinic Acid Adenosine Dinucleotide Phosphate) responsive acidic stores (1). We have now extended this research to an in vivo model of tumor angiogenesis and show that the pharmacologic NAADP inhibitor Ned-19 (2) impairs the vascularization, growth and metastatic spreading of the very aggressive VEGF producing murine tumor, B16 melanoma. In parallel in vitro experiments, we tested whether Ned-19 could directly affect the production of VEGF by the tumor cells, and found that treatment of B16 cells with Ned-19 unexpectedly results in increased VEGF release. These observations indicate that in our model 1) tumor angiogenesis is impaired by Ned-19 even in the presence of increased exposure to VEGF and 2) that NAADP ...

Two Pore Channels (TPCs) are an emerging family of intracellular channels, expressed on acidic co... more Two Pore Channels (TPCs) are an emerging family of intracellular channels, expressed on acidic compartments, which mediate calcium signaling evoked by NAADP. In particular, we demonstrated that TPC2 isoform has a main role in angiogenesis (Favia et al. PNAS 2014 Nov 4;111(44):E4706-15). TPC2 inhibition is emerging as a key therapeutic step in a range of important pathological conditions including the progression and metastatic potential of cancer, Parkinson’s disease, and Ebola virus infection. We introduce naringenin, a natural flavonoid, as a novel TPC2 inhibitor as shown by electrophysiological evidence in a heterologous system, i.e. Arabidopsis vacuoles lacking endogenous TPCs. In view of the control exerted by TPC2 on intracellular calcium signaling and angiogenesis, we demonstrate that naringenin dampens intracellular calcium responses of human endothelial cells stimulated with VEGF, histamine or NAADP-AM, but not with ATP or Angiopoietin-1. The ability of naringenin to impair...

Scientific Reports

Protein Arginine (R) methylation is the most common post-translational methylation in mammalian c... more Protein Arginine (R) methylation is the most common post-translational methylation in mammalian cells. Protein Arginine Methyltransferases (PRMT) 1 and 5 dimethylate their substrates on R residues, asymmetrically and symmetrically, respectively. They are ubiquitously expressed and play fundamental roles in tumour malignancies, including glioblastoma multiforme (GBM) which presents largely deregulated Myc activity. Previously, we demonstrated that PRMT5 associates with Myc in GBM cells, modulating, at least in part, its transcriptional properties. Here we show that Myc/PRMT5 protein complex includes PRMT1, in both HEK293T and glioblastoma stem cells (GSCs). We demonstrate that Myc is both asymmetrically and symmetrically dimethylated by PRMT1 and PRMT5, respectively, and that these modifications differentially regulate its stability. Moreover, we show that the ratio between symmetrically and asymmetrically dimethylated Myc changes in GSCs grown in stem versus differentiating conditio...

Malaria Journal

Background: Although malaria is a preventable and curable human disease, millions of people risk ... more Background: Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca 2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. Results: This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca 2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca 2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca 2+ levels. Conclusions: This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca 2+ oscillations suggests a potential role of NAADP in regulating Ca 2+ signalling of P. falciparum.

Vascular endothelial growth factor (VEGF) and its transmembrane receptors VEGFR1 and VEGFR2 play ... more Vascular endothelial growth factor (VEGF) and its transmembrane receptors VEGFR1 and VEGFR2 play a key role in controlling both physiological and pathological angiogenesis, including vascularization of solid tumours. We have identified a novel and crucial signalling mechanism through which activation of VEGFR2 in human endothelial cells (HUVEC) selectively triggers the intracellular release of calcium from acidic compartments, operated by the second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). Live imaging of calcium fluxes in cells treated with VEGF in the presence of specific inhibitors have shown that 1) VEGF-activated calcium stores are different from IP3 and ryanodine sensitive compartments and are of acidic nature, which strongly indicates the involvement of NAADP signalling 2) NAADP inhibition by its specific antagonist Ned-19 abolishes VEGF-induced calcium response. This inhibition is accompanied by impaired phosphorylation of downstream targets ERK1/2, Akt, eNOS, JNK (but not p38) and results in significant reduction of cell proliferation, migration and capillary-like tube formation in vitro. Interestingly, when the angiogenic response to VEGF was assayed in vivo utilizing Matrigel plugs subcutaneously implanted in mice, Ned-19 was found to dramatically inhibit VEGF-induced angiogenesis. Altogether our data showing that NAADP plays a key role in the control of VEGF-induced angiogenesis could potentially contribute to identify new targets for antiangiogenic therapeutic strategies, a goal to which much scientific effort has long been devoted but still awaiting ultimate success.

Our research introduces the natural flavonoid naringenin as a novel inhibitor of an emerging clas... more Our research introduces the natural flavonoid naringenin as a novel inhibitor of an emerging class of intracellular channels, Two-Pore Channel 2 (TPC2), as shown by electrophysiological evidence in a heterologous system, i.e. Arabidopsis vacuoles lacking endogenous TPCs. In view of the control exerted by TPC2 on intracellular calcium signaling, we demonstrated that naringenin dampens intracellular calcium responses of human endothelial cells stimulated with VEGF, histamine or NAADP-AM, but not with ATP or Angiopoietin-1 (negative controls). The ability of naringenin to impair TPC2-dependent biological activities was further explored in an established in vivo model, in which VEGF-containing matrigel plugs implanted in mice failed to be vascularized in the presence of naringenin. Overall, the present data suggest that naringenin inhibition of TPC2 activity and the observed inhibition of angiogenic response to VEGF are linked by impaired intracellular calcium signaling. TPC2 inhibition is emerging as a key therapeutic step in a range of important pathological conditions including the progression and metastatic potential of melanoma, Parkinson's disease, and Ebola virus infection. The identification of naringenin as an inhibitor of TPC2-mediated signaling provides a novel and potentially relevant tool for the advancement of this field of research. Naringenin (Nar) (5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one) is one of the main flavonoids present in the human diet. Epidemiological studies have demonstrated that the consumption of vegetables and fruit with a high Nar content, such as citruses and tomatoes or their food products, is associated with a reduced incidence of metabolic and chronic-degenerative diseases 1, 2. Following this kind of evidence, several in vitro studies have shown that Nar interacts with various cellular pathways, highlighting its potential as an antioxidant, antinflammatory, chemopreventive and antidegenerative agent 3 ; moreover, Nar was shown to exert anti-angiogenic effects in the avian chorio-allantoid membrane model 4 while this manuscript was being prepared. Naringenin lowers the plasma and hepatic cholesterol concentrations by suppressing HMG-CoA reductase and ACAT in rats fed a high-cholesterol diet 5. Pre-clinical studies have revealed the potential of Nar and of its precursor naringin in the treatment of metabolic and cardiovascular disorders, including hyperlipidemia, hypertension, cardiac toxicity, hyperglycemia and diabetes, hepatic steatosis and atherosclerosis 2 ; its therapeutic use to combat various kinds of cancer has also been envisaged 3. Indeed, Nar exerts chemopreventive and anticancer activity by blocking the progression and the formation of metastasis in various experimental models of oral 6 , melanoma 7 , breast 8, 9 , colon 10 , lung 11 and liver 12 cancer. It acts by upregulating many different cell survival proteins or arresting the cell cycle, by inducing p53-dependent apoptosis or inhibiting inflammatory processes and, in some cases, by exploiting all of these mechanisms 13-15. Despite the wealth of evidence on Nar efficacy, a comprehensive view of its mechanism of action is still lacking, as is the experimental evidence needed to bridge the gap between multiple cellular targets

A novel transduction pathway for the powerful angiogenic factor VEGF has been recently shown in e... more A novel transduction pathway for the powerful angiogenic factor VEGF has been recently shown in endothelial cells to operate through NAADP-controlled intracellular release of Ca 2+. In the present report the possible involvement of NAADP-controlled Ca 2+ signaling in tumor vascularization, growth and metastatic dissemination was investigated in a murine model of VEGF-secreting melanoma. Mice implanted with B16 melanoma cells were treated with NAADP inhibitor Ned-19 every second day for 4 weeks and tumor growth, vascularization and metastatization were evaluated. Control specimens developed well vascularized tumors and lung metastases, whereas in Ned-19-treated mice tumor growth and vascularization as well as lung metastases were strongly inhibited. In vitro experiments showed that Ned-19 treatment controls the growth of B16 cells in vitro, their migratory ability, adhesive properties and VEGFR2 expression, indicating NAADP involvement in intercellular autocrine signaling. To this regard, Ca 2+ imaging experiments showed that the response of B16 cells to VEGF stimulation is NAADP-dependent. The whole of these observations indicate that NAADP-controlled Ca 2+ signaling can be relevant not only for neoangiogenesis but also for direct control of tumor cells. Vascular endothelial growth factor (VEGF) and its receptor VEGFR2 play a pivotal role in stimulating angiogen-esis, including vascularization of solid tumors 1. Angiogenesis is an important step in the outgrowth of tumors 2 controlling their dissemination, progression and metastasis. Therapeutic targeting of angiogenesis is therefore an important challenge, still in need of basic research. In particular, no clinical tool is at present successful in inhibiting the long-term response to VEGF, the most important angiogenic factor overexpressed by the vast majority of human cancers 3. Owing to the central role of VEGF, the first angiogenic inhibitors designed for clinical use were targeted against VEGF and its tyrosine kinase receptor 4. VEGF neutralizing antibodies and some multi-target tyrosine kinase inhibitors have been approved for clinical use, and many more are being evaluated in clinical trials 5. Although these therapies provide improvement in progression-free or overall survival, they are nonetheless met with drug tolerance and side effects, attesting to the complexity of VEGF signaling and tumor angiogenic cascade. Anti-VEGF treatments can induce adverse effects, possibly due to the blockade of the entire complex of VEGF signaling pathways resulting in 'off-target' effects 6–9. These findings suggest that the challenge represented by therapeutic targeting of angiogenesis in advanced cancer is an interesting aim to further pursue through better characterization of the intracellular pathways involved in the response to VEGF. Melanoma is one of the most aggressive and highly metastatic cancers. It is known that VEGF levels are associated with melanoma progression 10,11 and are used as prognostic indicators 12 , but the molecular mechanisms regulating the interaction between tumor and endothelial cells (ECs) and their role in cancer cell extravasation and

Angiopoietins are vascular factors essential for blood vessel assembly and correct organization a... more Angiopoietins are vascular factors essential for blood vessel assembly and correct organization and maturation. This study describes a novel calcium-dependent machinery activated through Angiopoietin-1/2-Tie receptor system in HUVECs monolayer. Both cytokines were found to elicit intracellular calcium mobilization. Targeting intracellular Ca 2+ signaling, antagonizing IP 3 with 2-APB or cADPR with 8Br-cADPR, was found to modulate in vitro angiogenic responses to Angiopoietins in a specific way. 2-APB and 8Br-cADPR impaired the phosphorylation of AKT and FAK induced by Ang-1 and Ang-2. On the other hand, phosphorylation of ERK1/2 and p38, as well as cell proliferation, was not affected by either inhibitor. The ability of ECs to migrate following Angs stimulation, evaluated by " scratch assay, " was reduced by either 2-APB or 8Br-cADPR following Ang-2 stimulation and only slightly affected by 2-APB in cells stimulated with Ang-1. These results identify a novel calcium-dependent machinery involved in the complex interplay regulating angiogenic processes showing that IP 3-and cADPR-induced Ca 2+ release specifically regulates distinct Angs-mediated angiogenic steps.