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The c-MYC oncoprotein is a DNA binding transcription factor that enhances the expression of many active genes. c-MYC transcriptional signatures vary according to the transcriptional program defined in each cell type during differentiation. Little is known on the involvement of c-MYC in regulation of gene expression programs that are induced by extracellular cues such as a changing microenvironment. Here we demonstrate that inhibition of c-MYC in glioblastoma multiforme cells blunts hypoxia-dependent glycolytic reprogramming and mitochondria fragmentation in hypoxia. This happens because c-MYC inhibition alters the cell transcriptional response to hypoxia and finely tunes the expression of a subset of Hypoxia Inducible Factor 1-regulated genes. We also show that genes whose expression in hypoxia is affected by c-MYC inhibition are able to distinguish the Proneural subtype of glioblastoma multiforme, thus potentially providing a molecular signature for this class of tumors that are the least tractable among glioblastomas., Competing Interests: The authors declare no conflicts of interest.

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.

In the present study we demonstrated that TLQP-21, a biologically active peptide derived from the processing of the larger pro-VGF granin, plays a role in mammotrophic cell differentiation. We used an established in vitro model, the GH3 cell line, which upon treatment with epidermal growth factor develops a mammotrophic phenotype consisting of induction of prolactin expression and secretion, and inhibition of growth hormone. Here we determined for the first time that during mammotrophic differentiation, epidermal growth factor also induces Vgf gene expression and increases VGF protein precursor processing and peptide secretion. After this initial observation we set out to determine the specific role of the VGF encoded TLQP-21 peptide on this model. TLQP-21 induced a trophic effect on GH3 cells and increased prolactin expression and its own gene transcription without affecting growth hormone expression. TLQP-21 was also able to induce a significant rise of cytoplasmic calcium, as measured by Fura2AM, due to the release from a thapsigargin-sensitive store. TLQP-21-dependent rise in cytoplasmic calcium was, at least in part, dependent on the activation of phospholipase followed by phosphorylation of PKC and ERK. Taken together, the present results demonstrate that TLQP-21 contributes to differentiation of the GH3 cell line toward a mammotrophic phenotype and suggest that it may exert a neuroendocrine role in vivo on lactotroph cells in the pituitary gland., (Copyright © 2012 S. Karger AG, Basel.)

The peptides encoded by the VGF gene are gaining biomedical interest and are increasingly being scrutinized as biomarkers for human disease. An endocrine/neuromodulatory role for VGF peptides has been suggested but never demonstrated. Furthermore, no study has demonstrated so far the existence of a receptor-mediated mechanism for any VGF peptide. In the present study, we provide a comprehensive in vitro, ex vivo and in vivo identification of a novel pro-lipolytic pathway mediated by the TLQP-21 peptide. We show for the first time that VGF-immunoreactivity is present within sympathetic fibres in the WAT (white adipose tissue) but not in the adipocytes. Furthermore, we identified a saturable receptor-binding activity for the TLQP-21 peptide. The maximum binding capacity for TLQP-21 was higher in the WAT as compared with other tissues, and selectively up-regulated in the adipose tissue of obese mice. TLQP-21 increases lipolysis in murine adipocytes via a mechanism encompassing the activation of noradrenaline/β-adrenergic receptors pathways and dose-dependently decreases adipocytes diameters in two models of obesity. In conclusion, we demonstrated a novel and previously uncharacterized peripheral lipolytic pathway encompassing the VGF peptide TLQP-21. Targeting the sympathetic nerve-adipocytes interaction might prove to be a novel approach for the treatment of obesity-associated metabolic complications.

Vgf, is a neuro-endocrine specific gene encoding for a large protein precursor of different peptides. A role for VGF in pain modulation has been suggested from immunohistochemical studies showing VGF mRNA widely expressed in primary sensory neurons. In this study, the presence of VGF on the primary sensory afferents in mice was confirmed by showing its immunostaining in cultured neurons of dorsal root ganglia in secretory granule varicosities colocalized with Substance P. Moreover, the functional role of a C-terminal internal VGF-derived peptide, i.e. TLQP-21, was assessed by investigating its peripheral (1, 2, 4, 8mM) and central (1, 2, 4 mM) effects on inflammatory pain in the formalin test. A significant increase of pain-related licking response following peripheral injection of TLQP-21 (4 and 8mM) was observed in the second inflammatory phase of the test. In addition, an increase in licking response was detected when 4 mM of the peptide was injected alone without formalin. On the other hand, the central administration of TLQP-21 induced an U-shaped curve, with the dose of 2 mM being analgesic during the second phase. This study shows for the first time that a VGF-derived peptide may be involved in inflammatory pain in vivo and demonstrates a different action for TLQP21 at the peripheral and central levels of the nociceptive pathways.

Different VGF peptides derived from Vgf, originally identified as a nerve growth factor responsive gene, have been detected in neurons within the central and peripheral nervous system and in various endocrine cells. In the current study, we have evaluated the ability of TLQP-21, a VGF-derived peptide, to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death. We demonstrated that TLQP-21 increased survival of CGCs by decreasing the degree of apoptosis as assessed by cell viability and DNA fragmentation. Moreover, TLQP-21 significantly activated extracellular signal-regulated kinase 1/2, serine/threonine protein kinase, and c-jun N-terminal kinase phosphorylation, while decreased the extent of protein kinase C phosphorylation, as demonstrated by western blot analysis. In addition, TLQP-21 induced significant increase in intracellular calcium (as measured by fura-2AM) in about 60% of the recorded neurons. Taken together, the present results demonstrate that TLQP-21 promotes the survival of CGCs via pathways involving, within few minutes, modulation of kinases associated with CGCs survival, and by increasing intracellular calcium which can contribute to the neuroprotective effect of the peptide.