A New Insight into MYC Action: Control of RNA Polymerase II Methylation and Transcription Termination

MYC oncoprotein deregulation is a common catastrophic event in human cancer and limiting its activity restrains tumor development and maintenance, as clearly shown via Omomyc, an MYC-interfering 90 amino acid mini-protein. MYC is a multifunctional transcription factor that regulates many aspects of transcription by RNA polymerase II (RNAPII), such as transcription activation, pause release, and elongation. MYC directly associates with Protein Arginine Methyltransferase 5 (PRMT5), a protein that methylates a variety of targets, including RNAPII at the arginine residue R1810 (R1810me2s), crucial for proper transcription termination and splicing of transcripts. Therefore, we asked whether MYC controls termination as well, by affecting R1810me2S. We show that MYC overexpression strongly increases R1810me2s, while Omomyc, an MYC shRNA, or a PRMT5 inhibitor and siRNA counteract this phenomenon. Omomyc also impairs Serine 2 phosphorylation in the RNAPII carboxyterminal domain, a modification that sustains transcription elongation. ChIP-seq experiments show that Omomyc replaces MYC and reshapes RNAPII distribution, increasing occupancy at promoter and termination sites. It is unclear how this may affect gene expression. Transcriptomic analysis shows that transcripts pivotal to key signaling pathways are both up- or down-regulated by Omomyc, whereas genes directly controlled by MYC and belonging to a specific signature are strongly down-regulated. Overall, our data point to an MYC/PRMT5/RNAPII axis that controls termination via RNAPII symmetrical dimethylation and contributes to rewiring the expression of genes altered by MYC overexpression in cancer cells. It remains to be clarified which role this may have in tumor development

Regulation of Angiogenic Functions by Angiopoietins through Calcium-Dependent Signaling Pathways

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 IP3 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 IP3- and cADPR-induced Ca2+ release specifically regulates distinct Angs-mediated angiogenic steps

TARGETING OF NAADP-MEDIATED CALCIUM SIGNALING AFFECTS VEGF-INDUCED ANGIOGENESIS

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

The neuronal long noncoding RNA linc-NeD125 controls the expression of Group 4 medulloblastoma driver genes, acting as a microRNA sponge

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 linc-NeD125 ectopic expression in cells of the aggressive Group 3 MB attenuates their proliferation, migration and invasion. This study unveils the first lncRNA-based ceRNA network in central nervous system tumours and provides a novel molecular circuit underlying the enigmatic Group 4 medulloblastoma. Its features make linc-NeD125 as a potential target for MB therapy

Naringenin Impairs Two-Pore Channel 2 Activity And Inhibits VEGF-Induced Angiogenesis

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\u2019s 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

Linc-NeD125 establishes a ceRNA network in Group 4 Medulloblastoma

Long noncoding RNAs (lncRNAs) are regarded as crucial regulators of cellular processes in Eukaryotes. About 40% of currently characterized lncRNAs are specifically expressed in central nervous system, where they are involved in critical neural functions. Consistently, lncRNA aberrant expression is associated to neurological disorders. We recently identified a novel human lncRNA, linc-NeD125, that is induced in response to neuronal differentiation stimulus both in tumour cell lines and in embryonic stem cells. Notably, linc-Ned125 is significantly upregulated in a specific and still largely uncharacterized subgroup (Group 4) of Medulloblastoma (MB), the most common malignant paediatric brain tumour. Combining mechanistic and functional studies, we unveiled a novel lncRNA-mediated miRNA sponge regulatory network, in which the cross-talk among linc-NeD125, microRNAs and four Group 4 MB driver gene transcripts may significantly contribute to Group 4 MB cancerogenesis