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Cell migration/adhesion assays on HuR KD cells and quality controls on the their xenotransplants.

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.

Rescue of phenotype by CD133 silencing in HuR KD cells and assessment of HuR binding to CD133 mRNA.

Quality controls on HuR and MALAT1 silencing

Evaluation of CHART protein fraction.

HuR expression ablation does not induce anoikis.

Clinical characteristics of the tumor specimens form which primary cancer stem cells were isolated.

Primers used in PCR reactions. Gene silencing and over-expression tools.

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 (ΔΨm), 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 ΔΨm 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. Clin Cancer Res; 21(17); 3934–45. ©2015 AACR.

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Supplemental Figures 1-8. Figure 1S: A) Nampt locus (human chromosome 7q22.3) copy number heatmap for lines included in the Cancer Cell Line Encyclopedia. B) mRNA Nampt expression plotted vs. DNA copy number (log2 ratio). Figure 2S: effect of APO866 combined plus FK506 on Mec.1 cell lines. Figure 3S: intracellular APO866 determination in primary B-CLL cell extract. Figure 4S: anti-tumor activity of APO866 combined with Pg-p inhibitors (CsA, Verapamil and Nilotinib) on primary B-CLL and AML cells. Figure 5S: effects of drugs combination on healthy PBPCs (A) and PBMCs (B) cells. Figure 6S: intracellular NAD+(H) and ATP levels measured in AML cells following drugs combination. Figure 7S: relative expression of WBs showed in Figure 6B. Figure 8S: effects of NA addition on anti-tumor activity of APO866 combined with PgP inhibitors and schematic representation of proposed model.

Lipopolysaccharide exposure to macrophages induces an inflammatory response that is heavily regulated at the transcriptional and post-transcriptional levels. HuR (ELAVL1) is an RNA binding protein that binds and regulates the maturation and half-life of AU/U rich elements (ARE) containing cytokines and chemokines transcripts, mediating the LPS-induced response. Here we investigated how and to what extent small molecule tanshinone mimics (TMs) inhibiting HuR-RNA interaction counteract LPS stimulus in macrophages. We show TMs exist in solution in keto-enolic tautomerism and that, by molecular dynamic calculations, the orto quinone form is the bioactive species interacting with HuR and inhibiting its binding mode vs mRNA targets. A chemical blockage of the diphenolic, reduced form as a diacetate caused the loss of activity of TMsin vitrobut resulted to prodrug-like activityin vivo. The murine macrophage cell line RAW264.7 was treated with LPS and TMs, and the modulation of cellular LPS-induced response was monitored by RNA and Ribonucleoprotein immunoprecipitation sequencing. Correlation analyses indicated that LPS induced a strong coupling between differentially expressed genes and HuR-bound genes, and that TMs reduced such interactions. Functional annotation addressed a specific set of genes involved in chemotaxis and immune response, such asCxcl10, Il1b, Cd40, andFas, with a decreased association with HuR, a reduction of their expression and protein secretion. The same effect was observed in primary murine bone marrow-derived macrophages, andin vivoin an LPS induced peritonitis model, in which the serum level of Cxcl10 and Il1b was strongly reduced, endowing TMs such asTM7noxwith remarkable anti-inflammatory propertiesin vivo.

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.

RNA-binding proteins (RBPs) have been widely recognized for their pivotal role in theregulation of post-transcriptional processes. [...]

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.

Breast cancer (BC) is the most common malignancy with 1.7 million new diagnoses/year and is responsible for more than 450,000 yearly deaths worldwide. Two thirds of BC express the estrogen receptor (ER) and/or progesterone receptor and are referred to as hormone receptor-positive (HR+) BC. Endocrine therapy (ET) is usually active in these tumors, although drug resistance and side effects limit its benefit. Growth factor signaling through the PI3K/AKT/mammalian target of rapamycin (mTOR) and MAP kinase axes enhances ER activity and is a key mechanism underlying endocrine resistance. Water-only fasting (fasting) or plant-based, low-calorie, carbohydrate- and protein-restricted fasting-mimicking diets (FMDs) reduce circulating growth factors, such as insulin and IGF1 Therefore, we hypothesized that these dietary interventions could be used to enhance the activity of ET and delay the occurrence of resistance. For our in vitro experiments we used the HR+ BC cell lines, MCF7, T47D, and ZR-75-1, as well as metastases-derived organoids from patients with HR+ BC. Our in vivo experiments in mouse xenografts of human BC cell lines, were conducted in six-to-eight-week old female NOD SCID or athymic Nude-FoxN1 mice treated with ET w/ or w/o 48-72 hours of fasting/FMD. We monitored tumor growth and mouse survival and collected tumor masses and blood to detect circulating levels of several growth factors, adipokines and cytokines. In vivo add back experiments with fasting-reduced factors were done with IGF1, insulin and leptin. Circulating growth factors and adipo-cytokines were also detected in blood samples from 36 patients with HR+ BC, who were enrolled in either one of two clinical trials (NCT03595540 and NCT03340935) assessing safety and feasibility of periodic FMD in cancer patients. Patient nutritional status and response to treatment were also monitored in our clinical trials.We found that in HR+ BC models, periodic fasting or FMD enhanced tamoxifen and fulvestrant activity by lowering circulating IGF1, insulin, and leptin levels and by blocking AKT-mTOR signaling via EGR1 and PTEN upregulation. When fulvestrant was combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic FMD cycles promoted long-lasting tumour regressions and reverted acquired resistance to this regime. Moreover, both fasting and FMD prevented tamoxifen-induced endometrial hyperplasia. In HR+ BC patients receiving ET, FMD cycles caused metabolic changes analogous to those observed in mice, including reduced leptin and IGF1 levels, which were found to remain low for extended periods. In mice, these long-lasting effects were associated with carryover anticancer activity. Overall, our results provide the rationale for conducting further clinical studies of fasting-based dietary strategies as an adjuvant to ET w/ or w/o CDK4/6 inhibitors in patients with HR+ BC. Citation Format: Irene Caffa, Vanessa Spagnolo, Pamela Becherini, Francesca Valdemarin, Claudio Vernieri, Min Wei, Sebastian Brandhorst, Chiara Zucal, Else Driehuis, Lorenzo Ferrando, Luca Mastracci, Michele Cilli, Francesco Piacente, Anna Laura Cremonini, Mario Passalacqua, Valerio Vellone, Gabriele Zoppoli, Michele Cea, Giulia Salvadori, Salvatore Cortellino, Hans Clevers, Filippo De Braud, Alessandro Provenzani, Valter D. Longo, Alessio Nencioni. Fasting-mimicking diet and hormone therapy modulates metabolic factors to promote breast cancer regression and reduce side effects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT075.

[Image: see text] 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.

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.

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, Universita e Ricerca (Miur).

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.

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.

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. Electronic supplementary material The online version of this article (10.1186/s40170-018-0174-7) contains supplementary material, which is available to authorized users.

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.

Using bioinformatic methods we have detected the genes of 40 keratin-associated beta-proteins (KAbetaPs) (beta-keratins) from the first available draft genome sequence of a reptile, the lizard Anolis carolinensis (Broad Institute, Boston). All genes are clustered in a single but not yet identified chromosomal locus, and contain a single intron of variable length. 5'-RACE and RT-PCR analyses using RNA from different epidermal regions show tissue-specific expression of different transcripts. These results were confirmed from the analysis of the A. carolinensis EST libraries (Broad Institute). Most deduced proteins are 12-16 kDa with a pI of 7.5-8.5. Two genes encoding putative proteins of 40 and 45 kDa are also present. Despite variability in amino acid sequences, four main subfamilies can be described. The largest subfamily includes proteins high in glycine, a small subfamily contains proteins high in cysteine, a third large subfamily contains proteins high in cysteine and glycine, and the fourth, smallest subfamily comprises proteins low in cysteine and glycine. An inner region of high amino acid identity is the most constant characteristic of these proteins and maps to a region with two to three close beta-folds in the proteins. This beta-fold region is responsible for the formation of filaments of the corneous material in all types of scales in this species. Phylogenetic analysis shows that A. carolinensis KAbetaPs are more similar to those of other lepidosaurians (snake, lizard, and gecko lizard) than to those of archosaurians (chick and crocodile) and turtles.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1845-1) contains supplementary material, which is available to authorized users.

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 (ΔΨm), 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 ΔΨm 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. Clin Cancer Res; 21(17); 3934–45. ©2015 AACR.

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.

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.