Arginine methyltransferases critically regulate cellular homeostasis by modulating the functional outcome of their substrates. The protein arginine methyltransferase 5 (PRMT5) is an enzyme involved ...in growth and survival pathways promoting tumorigenesis. However, little is known about the biologic function of PRMT5 and its therapeutic potential in multiple myeloma (MM). In the present study, we identified and validated PRMT5 as a new therapeutic target in MM. PRMT5 is overexpressed in patient MM cells and associated with decreased progression-free survival and overall survival. Either genetic knockdown or pharmacological inhibition of PRMT5 with the inhibitor EPZ015666 significantly inhibited growth of both cell lines and patient MM cells. Furthermore, PRMT5 inhibition abrogated NF-κB signaling. Interestingly, mass spectrometry identified a tripartite motif-containing protein 21 TRIM21 as a new PRMT5-partner, and we delineated a TRIM21-dependent mechanism of NF-κB inhibition. Importantly, oral administration of EPZ015666 significantly decreased MM growth in a humanized murine model of MM. These data both demonstrate the oncogenic role and prognostic relevance of PRMT5 in MM pathogenesis, and provide the rationale for novel therapies targeting PRMT5 to improve patient outcome.
Phoenixin-14 (PNX) is a newly identified peptide co-expressed in the hypothalamus with the anorexic and cardioactive Nesfatin-1. Like Nesfatin-1, PNX is able to cross the blood–brain barrier and this ...suggests a role in peripheral modulation. Preliminary mass spectrography data indicate that, in addition to the hypothalamus, PNX is present in the mammalian heart. This study aimed to quantify PNX expression in the rat heart, and to evaluate whether the peptide influences the myocardial function under basal condition and in the presence of ischemia/reperfusion (I/R). By ELISA the presence of PNX was detected in both hypothalamus and heart. In plasma of normal, but not of obese rats, the peptide concentrations increased after meal. Exposure of the isolated and Langendorff perfused rat heart to exogenous PNX induces a reduction of contractility and relaxation, without effects on coronary pressure and heart rate. As revealed by immunoblotting, these effects were accompanied by an increase of Erk1/2, Akt and eNOS phosphorylation. PNX (EC
50
dose), administered after ischemia, induced post-conditioning-like cardioprotection. This was revealed by a smaller infarct size and a better systolic recovery with respect to those detected on hearts exposed to I/R alone. The peptide also activates the cardioprotective RISK and SAFE cascades and inhibits apoptosis. These effects were also observed in the heart of obese rats. Our data provide a first evidence on the peripheral activity of PNX and on its direct cardiomodulatory and cardioprotective role under both normal conditions and in the presence of metabolic disorders.
Interferon regulatory factor 4 (IRF4) is an attractive therapeutic target in multiple myeloma (MM). We here report that expression of IRF4 mRNA inversely correlates with microRNA (miR)-125b in MM ...patients. Moreover, we provide evidence that miR-125b is downregulated in TC2/3 molecular MM subgroups and in established cell lines. Importantly, constitutive expression of miR-125b-5p by lentiviral vectors or transfection with synthetic mimics impaired growth and survival of MM cells and overcame the protective role of bone marrow stromal cells in vitro. Apoptotic and autophagy-associated cell death were triggered in MM cells on miR-125b-5p ectopic expression. Importantly, we found that the anti-MM activity of miR-125b-5p was mediated via direct downregulation of IRF4 and its downstream effector BLIMP-1. Moreover, inhibition of IRF4 translated into downregulation of c-Myc, caspase-10 and cFlip, relevant IRF4-downstream effectors. Finally, in vivo intra-tumor or systemic delivery of formulated miR-125b-5p mimics against human MM xenografts in severe combined immunodeficient/non-obese diabetic mice induced significant anti-tumor activity and prolonged survival. Taken together, our findings provide evidence that miR-125b, differently from other hematologic malignancies, has tumor-suppressor activity in MM. Furthermore, our data provide proof-of-concept that synthetic miR-125b-5p mimics are promising anti-MM agents to be validated in early clinical trials.
Dendritic cells (DCs) have a key role in regulating tumor immunity, tumor cell growth and drug resistance. We hypothesized that multiple myeloma (MM) cells might recruit and reprogram DCs to a ...tumor-permissive phenotype by changes within their microRNA (miRNA) network. By analyzing six different miRNA-profiling data sets, miR-29b was identified as the only miRNA upregulated in normal mature DCs and significantly downregulated in tumor-associated DCs. This finding was validated in primary DCs co-cultured in vitro with MM cell lines and in primary bone marrow DCs from MM patients. In DCs co-cultured with MM cells, enforced expression of miR-29b counteracted pro-inflammatory pathways, including signal transducer and activator of transcription 3 and nuclear factor-κB, and cytokine/chemokine signaling networks, which correlated with patients' adverse prognosis and development of bone disease. Moreover, miR-29b downregulated interleukin-23 in vitro and in the SCID-synth-hu in vivo model, and antagonized a Th17 inflammatory response. All together, these effects translated into strong anti-proliferative activity and reduction of genomic instability of MM cells. Our study demonstrates that MM reprograms the DCs functional phenotype by downregulating miR-29b whose reconstitution impairs DCs ability to sustain MM cell growth and survival. These results underscore miR-29b as an innovative and attractive candidate for miRNA-based immune therapy of MM.
MicroRNAs (miRNAs) recently emerged with a key role in multiple myeloma (MM) pathophysiology and are considered important regulators of MM cell growth and survival. Since miRNAs can act either as ...oncogenes or tumour suppressors, the potential of targeting the miRNA network arises as a novel therapeutic approach for human cancer. Potential strategies based on miRNA therapeutics basically rely on miRNA inhibition or miRNA replacement approaches and take benefit respectively from the use of antagomirs or synthetic miRNAs as well as from lipid-based nanoparticles which allow an efficient miRNA-delivery. The availability of experimental in vivo platforms which recapitulate the growth of MM cells within the specific human bone marrow microenvironment in immunocompromised mice (SCID-hu and SCID-synth-hu) provides powerful systems for development of miRNA-based therapeutics in MM. Preliminary findings on the anti-MM activity of synthetic miRNAs in such experimental models offer a proof-of-principle that miRNA therapeutics is a promising opportunity for this still incurable disease representing the rationale for a new venue of investigation in this specific field.
Nesfatin-1 is an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide. It controls feeding behavior, water intake, and glucose homeostasis. If intracerebrally administered, it induces ...hypertension, thus suggesting a role in central cardiovascular control. However, it is not known whether it is able to directly control heart performance. We aimed to verify the hypothesis that, as in the case of other hypothalamic satiety peptides, Nesfatin-1 acts as a peripheral cardiac modulator. By western blotting and QT-PCR, we identified the presence of both Nesfatin-1 protein and NUCB2 mRNA in rat cardiac extracts. On isolated and Langendorff-perfused rat heart preparations, we found that exogenous Nesfatin-1 depresses contractility and relaxation without affecting coronary motility. These effects did not involve Nitric oxide, but recruited the particulate guanylate cyclase (pGC) known as natriuretic peptide receptor A (NPR-A), protein kinase G (PKG) and extracellular signal-regulated kinases1/2 (ERK1/2). Co-immunoprecipitation and bioinformatic analyses supported an interaction between Nesfatin-1 and NPR-A. Lastly, we preliminarily observed, through post-conditioning experiments, that Nesfatin-1 protects against ischemia/reperfusion (I/R) injury by reducing infarct size, lactate dehydrogenase release, and postischemic contracture. This protection involves multiple prosurvival kinases such as PKCε, ERK1/2, signal transducer and activator of transcription 3, and mitochondrial KATP channels. It also ameliorates contractility recovery. Our data indicate that: (1) the heart expresses Nesfatin-1, (2) Nesfatin-1 directly affects myocardial performance, possibly involving pGC-linked NPR-A, the pGC/PKG pathway, and ERK1/2, (3) the peptide protects the heart against I/R injury. Results pave the way to include Nesfatin-1 in the neuroendocrine modulators of the cardiac function, also encouraging the clarification of its clinical potential in the presence of nutrition-dependent physio-pathologic cardiovascular diseases.
Mouse models of multiple myeloma (MM) are basic tools for translational research and play a fundamental role in the development of new therapeutics against plasma cell malignancies. All available ...models, including transplantable murine tumors in syngenic mice, xenografts of established human cell lines in immunocompromised mice and transgenic models that mirror specific steps of MM pathogenesis, have demonstrated some weaknesses in predicting clinical results, particularly for new drugs targeting the human bone marrow microenvironment (huBMM). The recent interest to models recapitulating the in vivo growth of primary MM cells in a human (SCID-hu) or humanized (SCID-synth-hu) host recipient has provided powerful platforms for the investigation of new compounds targeting MM and/or its huBMM. Here, we review and discuss strengths and weaknesses of the key in vivo models that are currently utilized in the MM preclinical investigation.
Objective: Phoenixin (PNX) is a peptide identified in 2013. It is mainly expressed in the hypothalamus in which it shares a distribution patter comparable to nesfatin-1, a peptide with anorexic and ...cardiovascular activities. It is able to cross the blood-brain barrier and this suggests that it elicits peripheral functions. Preliminary mass spectrography data indicate that PNX is present in the mammalian heart. This study aimed to quantity PNX in heart and plasma in of normo-weight and obese rats, and to evaluate if the peptide influences the myocardial function under basal condition and in the presence of ischemia/reperfusion (I/R). Methods: By EL1SA, PNX was detected in both hypothalamus and heart Results: In the plasma, PNX levels increased during the postprandial phase in normal but not in obese rats. On the isolated and Langendorff perfused rat heart exogenous PNX determined negative inotropism and lusitropism. Western Blotting of cardiac extracts revealed that Erkl/2, Akt and eNOS phosphorylation increased after exposure to increasing concentrations of PNX. PNX (EC50 dose), administered in post-conditioning, induced a better systolic recovery and a smaller infarct size with respect to hearts exposed to l/R alone. PNX-dependent cardioprotection was mediated by RISK and SAFE cascades and apoptosis inhibition. Preliminary data obtained in obese rats showed that PNX post-conditioning cardioprotection is obtained only at a concentration (1 nM) that is higher than that required in normo-weight rats. Conclusions: This study represents a first evidence on the involvement of PNX in cardiac modulation, particularly in relation to normal and pathological conditions.
The richly structured neuroendocrine control of the heart in health and disease requires, in addition to the autonomic nervous outflow, the essential contribute of various and often interacting ...humoral peptides (e.g. natriuretic peptides, Chromogranin-A-derived fragments, etc). In many cases, these molecules also influence the activity of other organ systems, including the gastrointestinal apparatus, in which they control mucosal function as well as motility and secretion. Interestingly, by acting centrally, some of these peptides also regulate satiety and appetite, thus forming an interesting link between cardiac and gastrointestinal function, and the feeding pattern. Prolonged inhibition and/or activation of these peptide pathways frequently results in severe and long-lasting dysfunctions, including cardiovascular diseases associated to alimentary disorders (e.g. obesity). Notably, their multifarious actions and mutual interactions make them excellent candidates for long-term resetting of both cardiac, gastrointestinal and nutrition homeostasis. Here we will provide only few examples taken from the quickly evolving scenario, with the purpose to provide indications concerning the complex circuits generated by multilevel signalling peptides, which contributes to orchestrate the association between cardiovascular, gastrointestinal and alimentary functions. This will highlight not only the complexity of the cardiovascular and GI regulatory networks, but also aspects of integration between feeding stimulating peptides and the other neuroendocrine systems affecting the heart and the GI tract.
Abstract Background and aims The anorexigenic glucagon-like peptide (GLP)-2 is produced by intestinal L cells and released in response to food intake. It affects intestinal function involving ...G-protein-coupled receptors. To verify whether GLP-2 acts as a cardiac modulator in mammals, we analysed, in the rat heart, the expression of GLP-2 receptors and the myocardial and coronary responses to GLP-2. Methods and results GLP-2 receptors were detected on ventricular extracts by quantitative real-time polymerase chain reaction (Q-RT-PCR) and Western blotting. Cardiac GLP-2 effects were analysed on Langendorff perfused hearts. Intracellular GLP-2 signalling was investigated on Langendorff perfused hearts and by Western blotting and enzyme-linked immunosorbent assay (ELISA) on ventricular extracts. By immunoblotting and Q-RT-PCR, we revealed the expression of ventricular GLP-2 receptors. Perfusion analyses showed that GLP-2 induces positive inotropism at low concentration (10–12 mol l−1 ), and negative inotropism and lusitropism from 10 to 10 mol l−1 . It dose-dependently constricts coronaries. The negative effects of GLP-2 were independent from GLP-1 receptors, being unaffected by exendin-3 (9–39) amide. GLP-2-dependent negative action involves Gi/o proteins, associates with a reduction of intracellular cyclic adenosine monophosphate (cAMP), an increase in extracellular signal regulated kinases 1 and 2 (ERK1/2) and a decrease in phospholamban phosphorylation, but is independent from endothelial nitric oxide synthase (eNOS) and protein kinase G (PKG). Finally, GLP-2 competitively antagonised β-adrenergic stimulation. Conclusions For the first time, to our knowledge, we found that: (1) the rat heart expresses functional GLP-2 receptors; (2) GLP-2 acts on both myocardium and coronaries, negatively modulating both basal and β-adrenergic stimulated cardiac performance; and (3) GLP-2 effects are mediated by G-proteins and involve ERK1/2.
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