Mitochondrial rewiring drives metabolic adaptation to NAD(H) shortage in triple negative breast cancer cells.

Nicotinamide phosphoribosyltransferase (NAMPT) is a key metabolic enzyme in NAD+ synthesis pathways and is found upregulated in several tumors, depicting NAD(H) lowering agents, like the NAMPT inhibitor FK866, as an appealing approach for anticancer therapy. Like other small molecules, FK866 triggers chemoresistance, observed in several cancer cellular models, which can prevent its clinical application. The molecular mechanisms sustaining the acquired of resistance to FK866 were studied in a model of triple negative breast cancer (MDA-MB-231 parental - PAR), exposed to increasing concentrations of the small molecule (MDA-MB-231 resistant - RES). RES cells are not sensitive to verapamil or cyclosporin A, excluding a potential role of increased efflux pumps activity as a mechanism of resistance. Similarly, the silencing of the enzyme Nicotinamide Riboside Kinase 1 (NMRK1) in RES cells does not increase FK866 toxicity, excluding this pathway as a compensatory mechanism of NAD+ production. Instead, Seahorse metabolic analysis revealed an increased mitochondrial spare respiratory capacity in RES cells. These cells presented a higher mitochondrial mass compared to the FK866-sensitive counterparts, as well as an increased consumption of pyruvate and succinate for energy production. Interestingly, co-treatment of PAR cells with FK866 and the mitochondrial pyruvate carrier (MPC) inhibitors UK5099 or rosiglitazone, as well as with the transient silencing of MPC2 but not of MPC1, induces a FK866-resistant phenotype. Taken together, these results unravel novel mechanisms of cell plasticity to counteract FK866 toxicity, that, besides the previously described LDHA dependency, rely on mitochondrial rewiring at functional and energetic levels.

HuR modulation counteracts lipopolysaccharide response in murine macrophages.

Lipopolysaccharide (LPS) exposure to macrophages induces an inflammatory response, which is regulated at the transcriptional and post-transcriptional levels. HuR (ELAVL1) is an RNA-binding protein that regulates cytokines and chemokines transcripts containing AU/U-rich elements (AREs) and mediates the LPS-induced response. Here, we show that small-molecule tanshinone mimics (TMs) inhibiting HuR-RNA interaction counteract LPS stimulus in macrophages. TMs exist in solution in keto-enolic tautomerism, and molecular dynamic calculations showed the ortho-quinone form inhibiting binding of HuR to mRNA targets. TM activity was lost in vitro by blocking the diphenolic reduced form as a diacetate, but resulted in prodrug-like activity in vivo. RNA and ribonucleoprotein immunoprecipitation sequencing revealed that LPS induces a strong coupling between differentially expressed genes and HuR-bound genes, and TMs reduced such interactions. TMs decreased the association of HuR with genes involved in chemotaxis and immune response, including Cxcl10, Il1b and Cd40, reducing their expression and protein secretion in primary murine bone marrow-derived macrophages and in an LPS-induced peritonitis model. Overall, TMs show anti-inflammatory properties in vivo and suggest HuR as a potential therapeutic target for inflammation-related diseases.

Identification and Characterization of an RRM-Containing, RNA Binding Protein in Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative pathogen, known to acquire resistance to antibiotics used in the clinic. The RNA-binding proteome of this bacterium is poorly characterized, in particular for what concerns the proteins containing RNA Recognition Motif (RRM). Here, we browsed the A. baumannii proteome for homologous proteins to the human HuR(ELAVL1), an RNA binding protein containing three RRMs. We identified a unique locus that we called AB-Elavl, coding for a protein with a single RRM with an average of 34% identity to the first HuR RRM. We also widen the research to the genomes of all the bacteria, finding 227 entries in 12 bacterial phyla. Notably we observed a partial evolutionary divergence between the RNP1 and RNP2 conserved regions present in the prokaryotes in comparison to the metazoan consensus sequence. We checked the expression at the transcript and protein level, cloned the gene and expressed the recombinant protein. The X-ray and NMR structural characterization of the recombinant AB-Elavl revealed that the protein maintained the typical ß1α1ß2ß3α2ß4 and three-dimensional organization of eukaryotic RRMs. The biochemical analyses showed that, although the RNP1 and RNP2 show differences, it can bind to AU-rich regions like the human HuR, but with less specificity and lower affinity. Therefore, we identified an RRM-containing RNA-binding protein actually expressed in A. baumannii.

Limonium duriusculum (de Girard) Kuntze Exhibits Anti-inflammatory Effect Via NF-κ B Pathway Modulation

HIGHLIGHTS L. duriusculum n-BuOH extract reduces inflammatory responses both in vitro and in vivo. L. duriusculum n-BuOH extract inhibits NF-κB-dependent transcriptional responses. L. duriusculum n-BuOH extract decreases the expression of TNF-α and IL-6 genes.

Abstract Limonium duriusculum is used in folk medicine to treat inflammatory disorders and has gained attention due to its richness in apigenin. The present investigation was performed to evaluate and confirm its anti-inflammatory properties, in cell lines and animal models. The potential anti-inflammatory properties of n-butanol (n-BuOH) extract of L. duriusculum (BEL) and isolated apigenins were examined on NF-κB transcriptional activity in TNFα- or LPS-stimulated cells, and on in vivo acute inflammatory models (carrageenan induced paw edema and peritonitis). BEL treatment was able to inhibit the activity of an NF-κB reporter gene in HCT116 cells both in the absence and in the presence of exogenous TNFα, used as NF-κB pathway inducer. This anti-inflammatory effect was even more potent compared to Apigenin (APG1) and was confirmed using monocyte-derived THP-1 cells treated with LPS to stimulate NF-κB-dependent transcription of IL-6 and TNFα mRNAs. Apigenin7-O-β-(6''-methylglucuronide) (APG2) was instead inactive both in HCT116 and THP-1 cells. BEL (oral, 200 mg/kg) led to paw swelling inhibition, vascular permeability and peritoneal leukocyte and PN migration diminution. Apigenins (intraperitoneal, APG1, APG2: 20 mg/kg) also evoked a significant anti-edema effect, early vascular permeability and leukocyte influx reduction. Collectively, this study demonstrates for the first time the effectiveness of L. duriusculum to inhibit NF-κB-dependent transcriptional responses in HCT116 and THP-1 cells. In vivo studies also established that L. duriusculum possesses a potential anti-inflammatory effect, confirm its traditional, empirical use, that could be attributed to its richness in apigenin.

Fasting-mimicking diet and hormone therapy induce breast cancer regression.

Approximately 75% of all breast cancers express the oestrogen and/or progesterone receptors. Endocrine therapy is usually effective in these hormone-receptor-positive tumours, but primary and acquired resistance limits its long-term benefit1,2. Here we show that in mouse models of hormone-receptor-positive breast cancer, periodic fasting or a fasting-mimicking diet3-5 enhances the activity of the endocrine therapeutics tamoxifen and fulvestrant by lowering circulating IGF1, insulin and leptin and by inhibiting AKT-mTOR signalling via upregulation of EGR1 and PTEN. When fulvestrant is combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic cycles of a fasting-mimicking diet promotes long-lasting tumour regression and reverts acquired resistance to drug treatment. Moreover, both fasting and a fasting-mimicking diet prevent tamoxifen-induced endometrial hyperplasia. In patients with hormone-receptor-positive breast cancer receiving oestrogen therapy, cycles of a fasting-mimicking diet cause metabolic changes analogous to those observed in mice, including reduced levels of insulin, leptin and IGF1, with the last two remaining low for extended periods. In mice, these long-lasting effects are associated with long-term anti-cancer activity. These results support further clinical studies of a fasting-mimicking diet as an adjuvant to oestrogen therapy in hormone-receptor-positive breast cancer.

Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood.

BACKGROUND: Extracellular vesicles (EVs) are secreted membranous particles intensively studied for their potential cargo of diagnostic markers. Efficient and cost-effective isolation methods need to be established for the reproducible and high-throughput study of EVs in the clinical practice. METHODS: We designed the nickel-based isolation (NBI) to rapidly isolate EVs and combined it with newly-designed amplified luminescent proximity homogeneous assay or digital PCR to detect biomarkers of clinical utility. FINDINGS: From plasma of 46 healthy donors, we systematically recovered small EV (~250 nm of mean diameter; ~3 × 1010/ml) and large EV (~560 nm of mean diameter; ~5 × 108/ml) lineages ranging from 50 to 700 nm, which displayed hematopoietic/endothelial cell markers that were also used in spike-in experiments using EVs from tumor cell lines. In retrospective studies, we detected picomolar concentrations of prostate-specific membrane antigen (PSMA) in fractions of EVs isolated from the plasma of prostate cancer patients, discriminating them from control subjects. Directly from oil-encapsulated EVs for digital PCR, we identified somatic BRAF and KRAS mutations circulating in the plasma of metastatic colorectal cancer (CRC) patients, matching 100% of concordance with tissue diagnostics. Importantly, with higher sensitivity and specificity compared with immuno-isolated EVs, we revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies. INTERPRETATION: We propose NBI-combined approaches as simple, fast, and robust strategies to probe the tumor heterogeneity and contribute to the development of EV-based liquid biopsy studies. FUND: Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio Trento e Rovereto (CARITRO), and the Italian Ministero Istruzione, Università e Ricerca (Miur).

Novel Compounds Targeting the RNA-Binding Protein HuR. Structure-Based Design, Synthesis, and Interaction Studies.

The key role of RNA-binding proteins (RBPs) in regulating post-transcriptional processes and their involvement in several pathologies (i.e., cancer and neurodegeneration) have highlighted their potential as therapeutic targets. In this scenario, Embryonic Lethal Abnormal Vision (ELAV) or Hu proteins and their complexes with target mRNAs have been gaining growing attention. Compounds able to modulate the complex stability could constitute an innovative pharmacological strategy for the treatment of numerous diseases. Nevertheless, medicinal-chemistry efforts aimed at developing such compounds are still at an early stage. As part of our ongoing research in this field, we hereby present the rational design and synthesis of structurally novel HuR ligands, potentially acting as HuR-RNA interferers. The following assessment of the structural features of their interaction with HuR, combining saturation-transfer difference NMR and in silico studies, provides a guide for further research on the development of new effective interfering compounds of the HuR-RNA complex.

Screening Approaches for Targeting Ribonucleoprotein Complexes: A New Dimension for Drug Discovery.

RNA-binding proteins (RBPs) are pleiotropic factors that control the processing and functional compartmentalization of transcripts by binding primarily to mRNA untranslated regions (UTRs). The competitive and/or cooperative interplay between RBPs and an array of coding and noncoding RNAs (ncRNAs) determines the posttranscriptional control of gene expression, influencing protein production. Recently, a variety of well-recognized and noncanonical RBP domains have been revealed by modern system-wide analyses, underlying an evolving classification of ribonucleoproteins (RNPs) and their importance in governing physiological RNA metabolism. The possibility of targeting selected RNA-protein interactions with small molecules is now expanding the concept of protein "druggability," with new implications for medicinal chemistry and for a deeper characterization of the mechanism of action of bioactive compounds. Here, taking SF3B1, HuR, LIN28, and Musashi proteins as paradigmatic case studies, we review the strategies applied for targeting RBPs, with emphasis on the technological advancements to study protein-RNA interactions and on the requirements of appropriate validation strategies to parallel high-throughput screening (HTS) efforts.

Exploration of ligand binding modes towards the identification of compounds targeting HuR: a combined STD-NMR and Molecular Modelling approach.

Post-transcriptional processes have been recognised as pivotal in the control of gene expression, and impairments in RNA processing are reported in several pathologies (i.e., cancer and neurodegeneration). Focusing on RNA-binding proteins (RBPs), the involvement of Embryonic Lethal Abnormal Vision (ELAV) or Hu proteins and their complexes with target mRNAs in the aetiology of various dysfunctions, has suggested the great potential of compounds able to interfere with the complex stability as an innovative pharmacological strategy for the treatment of numerous diseases. Here, we present a rational follow-up investigation of the interaction between ELAV isoform HuR and structurally-related compounds (i.e., flavonoids and coumarins), naturally decorated with different functional groups, by means of STD-NMR and Molecular Modelling. Our results represent the foundation for the development of potent and selective ligands able to interfere with ELAV-RNA complexes.

Cancer cell metabolic plasticity allows resistance to NAMPT inhibition but invariably induces dependence on LDHA.

BACKGROUND: Inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis from nicotinamide, exhibit anticancer effects in preclinical models. However, continuous exposure to NAMPT inhibitors, such as FK866, can induce acquired resistance. METHODS: We developed FK866-resistant CCRF-CEM (T cell acute lymphoblastic leukemia) and MDA MB231 (breast cancer) models, and by exploiting an integrated approach based on genetic, biochemical, and genome wide analyses, we annotated the drug resistance mechanisms. RESULTS: Acquired resistance to FK866 was independent of NAMPT mutations but rather was based on a shift towards a glycolytic metabolism and on lactate dehydrogenase A (LDHA) activity. In addition, resistant CCRF-CEM cells, which exhibit high quinolinate phosphoribosyltransferase (QPRT) activity, also exploited amino acid catabolism as an alternative source for NAD+ production, becoming addicted to tryptophan and glutamine and sensitive to treatment with the amino acid transport inhibitor JPH203 and with l-asparaginase, which affects glutamine exploitation. Vice versa, in line with their low QPRT expression, FK866-resistant MDA MB231 did not rely on amino acids for their resistance phenotype. CONCLUSIONS: Our study identifies novel mechanisms of resistance to NAMPT inhibition, which may be useful to design more rational strategies for targeting cancer metabolism.

Interfering with HuR-RNA Interaction: Design, Synthesis and Biological Characterization of Tanshinone Mimics as Novel, Effective HuR Inhibitors.

The human antigen R (HuR) is an RNA-binding protein known to modulate the expression of target mRNA coding for proteins involved in inflammation, tumorigenesis, and stress responses and is a valuable drug target. We previously found that dihydrotanshinone-I (DHTS, 1) prevents the association of HuR with its RNA substrate, thus imparing its function. Herein, inspired by DHTS structure, we designed and synthesized an array of ortho-quinones (tanshinone mimics) using a function-oriented synthetic approach. Among others, compound 6a and 6n turned out to be more effective than 1, showing a nanomolar Ki and disrupting HuR binding to RNA in cells. A combined approach of NMR titration and molecular dynamics (MD) simulations suggests that 6a stabilizes HuR in a peculiar closed conformation, which is incompatible with RNA binding. Alpha screen and RNA-electrophoretic mobility shift assays (REMSA) data on newly synthesized compounds allowed, for the first time, the generation of structure activity relationships (SARs), thus providing a solid background for the generation of highly effective HuR disruptors.

Regulation of HuR structure and function by dihydrotanshinone-I.

The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.

The Ribonucleic Complex HuR-MALAT1 Represses CD133 Expression and Suppresses Epithelial-Mesenchymal Transition in Breast Cancer.

Epithelial-to-mesenchymal transition (EMT) is a core process underlying cell movement during embryonic development and morphogenesis. Cancer cells hijack this developmental program to execute a multi-step cascade, leading to tumorigenesis and metastasis. CD133 (PROM1), a marker of cancer stem cells, has been shown to facilitate EMT in various cancers, but the regulatory networks controlling CD133 gene expression and function in cancer remain incompletely delineated. In this study, we show that a ribonucleoprotein complex including the long noncoding RNA MALAT1 and the RNA-binding protein HuR (ELAVL1) binds the CD133 promoter region to regulate its expression. In luminal nonmetastatic MCF-7 breast cancer cells, HuR silencing was sufficient to upregulate N-cadherin (CDH2) and CD133 along with a migratory and mesenchymal-like phenotype. Furthermore, we found that in the basal-like metastatic cell line MDA-MB-231 and primary triple-negative breast cancer tumor cells, the repressor complex was absent from the CD133-regulatory region, but was present in the MCF-7 and primary ER+ tumor cells. The absence of the complex from basal-like cells was attributed to diminished expression of MALAT1, which, when overexpressed, dampened CD133 levels. In conclusion, our findings suggest that the failure of a repressive complex to form or stabilize in breast cancer promotes CD133 upregulation and an EMT-like program, providing new mechanistic insights underlying the control of prometastatic processes. Cancer Res; 76(9); 2626-36. ©2016 AACR.

The GSK3ß inhibitor BIS I reverts YAP-dependent EMT signature in PDAC cell lines by decreasing SMADs expression level.

The Yes-associated protein, YAP, is a transcriptional co-activator, mediating the Epithelial to Mesenchymal Transition program in pancreatic ductal adenocarcinoma (PDAC). With the aim to identify compounds that can specifically modulate YAP functionality in PDAC cell lines, we performed a small scale, drug-based screening experiment using YAP cell localization as the read-out. We identified erlotinib as an inducer of YAP cytoplasmic localization, an inhibitor of the TEA luciferase reporter system and the expression of the bona fide YAP target gene, Connective Tissue Growth Factor CTGF. On the other hand, BIS I, an inhibitor of PKCδ and GSK3ß, caused YAP accumulation into the nucleus. Activation of ß-catenin reporter and interfering experiments show that inhibition of the PKCδ/GSK3ß pathway triggers YAP nuclear accumulation inducing YAP/TEAD transcriptional response. Inhibition of GSK3ß by BIS I reduced the expression levels of SMADs protein and reduced YAP contribution to EMT. Notably, BIS I reduced proliferation, migration and clonogenicity of PDAC cells in vitro, phenocopying YAP genetic down-regulation. As shown by chromatin immunoprecipitation experiments and YAP over-expressing rescue experiments, BIS I reverted YAP-dependent EMT program by modulating the expression of the YAP target genes E-cadherin, vimentin, CTGF and of the newly identified target, CD133. In conclusion, we identified two different molecules, erlotinib and BIS I, modulating YAP functionality although via different mechanisms of action, with the second one specifically inhibiting the YAP-dependent EMT program in PDAC cell lines.

Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function.

Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki = 3.74 ± 1.63 nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells.

EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD(+) biosynthesis from nicotinamide, is one of the major factors regulating cancer cells metabolism and is considered a promising target for treating cancer. The prototypical NAMPT inhibitor FK866 effectively lowers NAD(+) levels in cancer cells, reducing the activity of NAD(+)-dependent enzymes, lowering intracellular ATP, and promoting cell death. RESULTS: We show that FK866 induces a translational arrest in leukemia cells through inhibition of MTOR/4EBP1 signaling and of the initiation factors EIF4E and EIF2A. Specifically, treatment with FK866 is shown to induce 5'AMP-activated protein kinase (AMPK) activation, which, together with EIF2A phosphorylation, is responsible for the inhibition of protein synthesis. Notably, such an effect was also observed in patients' derived primary leukemia cells including T-cell Acute Lymphoblastic Leukemia. Jurkat cells in which AMPK or LKB1 expression was silenced or in which a non-phosphorylatable EIF2A mutant was ectopically expressed showed enhanced sensitivity to the NAMPT inhibitor, confirming a key role for the LKB1-AMPK-EIF2A axis in cell fate determination in response to energetic stress via NAD(+) depletion. CONCLUSIONS: We identified EIF2A phosphorylation as a novel early molecular event occurring in response to NAMPT inhibition and mediating protein synthesis arrest. In addition, our data suggest that tumors exhibiting an impaired LBK1- AMPK- EIF2A response may be especially susceptible to NAMPT inhibitors and thus become an elective indication for this type of agents.

APO866 Increases Antitumor Activity of Cyclosporin-A by Inducing Mitochondrial and Endoplasmic Reticulum Stress in Leukemia Cells.

PURPOSE: The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, APO866, has been previously shown to have antileukemic activity in preclinical models, but its cytotoxicity in primary leukemia cells is frequently limited. The success of current antileukemic treatments is reduced by the occurrence of multidrug resistance, which, in turn, is mediated by membrane transport proteins, such as P-glycoprotein-1 (Pgp). Here, we evaluated the antileukemic effects of APO866 in combination with Pgp inhibitors and studied the mechanisms underlying the interaction between these two types of agents. EXPERIMENTAL DESIGN: The effects of APO866 with or without Pgp inhibitors were tested on the viability of leukemia cell lines, primary leukemia cells (AML, n = 6; B-CLL, n = 19), and healthy leukocytes. Intracellular nicotinamide adenine dinucleotide (NAD(+)) and ATP levels, mitochondrial transmembrane potential (ΔΨ(m)), markers of apoptosis and of endoplasmic reticulum (ER) stress were evaluated. RESULTS: The combination of APO866 with Pgp inhibitors resulted in a synergistic cytotoxic effect in leukemia cells, while sparing normal CD34(+) progenitor cells and peripheral blood mononuclear cells. Combining Pgp inhibitors with APO866 led to increased intracellular APO866 levels, compounded NAD(+) and ATP shortage, and induced ΔΨ(m) dissipation. Notably, APO866, Pgp inhibitors and, to a much higher extent, their combination induced ER stress and ER stress inhibition strongly reduced the activity of these treatments. CONCLUSIONS: APO866 and Pgp inhibitors show a strong synergistic cooperation in leukemia cells, including acute myelogenous leukemia (AML) and B-cell chronic lymphocytic leukemia (B-CLL) samples. Further evaluations of the combination of these agents in clinical setting should be considered.

Targeting the multifaceted HuR protein, benefits and caveats.

The RNA-binding protein (RBP) HuR is one of the most widely studied regulators of the eukaryotic posttranscriptional gene expression and it plays a physiological role in mediating the cellular response to apoptotic, proliferating and survival stimuli. Following physiological or stress stimuli, HuR protein binds to Adenylate-Urydinilate rich elements (AREs) generally contained in the 3'UTR of transcripts, then it shuttles from the nucleus to the cytoplasm and regulates the half-life and/or translation of cargo mRNAs. Derangements in sub-cellular localization and expression of HuR have been associated with the pathophysiology of many diseases and this protein has been proposed as a potential drug target. Recent findings also re-evaluated HuR as a splicing and polyadenylation factor, expanding its spectrum of functional activity up to the maturation of pre-mRNAs. In this review, we generate a comprehensive picture of HuR functionality to discuss the implications of considering HuR as pharmacological target and the detrimental or positive impact that can be expected upon its modulation. Firstly, we focus on the recent findings about the mechanistic role of HuR in the nucleus and in the regulation of long non coding RNAs; then we describe the animal models and the clinical association and significance in cancer; finally, we have reviewed the pharmacological tools that influence HuR's post-transcriptional control and the efforts made to identify specific HuR inhibitors.

Runx-2 gene expression is associated with age-related changes of bone mineral density in the healthy young-adult population.

Bone mineral density (BMD) and peak bone mass (PBM) are important determinants of skeletal resistance. The development of bone densitometry improved the possibility of studying BMD and the influence of genetic and environmental factors on bone. Heredity factors are important for BMD, and Runx-2 is accepted as a regulator of osteoblasts and bone formation. The aim of our study was to evaluate the behavior of Runx-2 during skeletal maturity in the healthy young-adult population. We analyzed spine and hip BMD in 153 volunteers, 98 women and 55 men, using dual-energy X-ray absorptiometry. In a subgroup of these volunteers, a sample of peripheral blood was taken to perform gene expression analysis of Runx-2 both in peripheral mesenchymal stem cells (MSCs; 28 subjects) and in peripheral mononuclear cells (PBMCs; 140 subjects). In our work BMD was comparable in both genders after puberty, then became higher in men than women during the third and fourth decades. PBM was achieved in the third decade in women and in the fourth in men. More interestingly, Runx-2 gene expression highly correlated with BMD in both genders. MSCs and PBMCs showed the same gene expression profile of Runx-2. In conclusion, PBM is reached earlier in females, BMD becomes higher in males later in life, and BMD and PBM are strictly associated with Runx-2. In addition, PBMC should be considered an important source for gene expression analysis in bone diseases.