Monocyte-macrophage differentiation of acute myeloid leukemia cell lines by small molecules identified through interrogation of the Connectivity Map database

The transcription factor C/EBPα is required for granulocytic differentiation of normal myeloid progenitors and is frequently inactivated in acute myeloid leukemia (AML) cells. Ectopic expression of C/EBPα in AML cells suppresses proliferation and induces differentiation suggesting that restoring C/EBPα expression/activity in AML cells could be therapeutically useful. Unfortunately, current approaches of gene or protein delivery in leukemic cells are unsatisfactory. However, "drug repurposing" is becoming a very attractive strategy to identify potential new uses for existing drugs. In this study, we assessed the biological effects of candidate C/EBPα-mimetics identified by interrogation of the Connectivity Map database. We found that amantadine, an antiviral and anti-Parkinson agent, induced a monocyte-macrophage-like differentiation of HL60, U937, Kasumi-1 myeloid leukemia cell lines, as indicated by morphology and differentiation antigen expression, when used in combination with suboptimal concentration of all trans retinoic acid (ATRA) or Vit D3. The effect of amantadine depends, in part, on increased activity of the vitamin D receptor (VDR), since it induced VDR expression and amantadine-dependent monocyte-macrophage differentiation of HL60 cells was blocked by expression of dominant-negative VDR. These results reveal a new function for amantadine and support the concept that screening of the Connectivity Map database can identify small molecules that mimic the effect of transcription factors required for myelo-monocytic differentiation

Suppression of Invasion and Metastasis of Triple-Negative Breast Cancer Lines by Pharmacological or Genetic Inhibition of Slug Activity

AbstractMost triple-negative breast cancers (TNBCs) exhibit gene expression patterns associated with epithelial-to-mesenchymal transition (EMT), a feature that correlates with a propensity for metastatic spread. Overexpression of the EMT regulator Slug is detected in basal and mesenchymal-type TNBCs and is associated with reduced E-cadherin expression and aggressive disease. The effects of Slug depend, in part, on the interaction of its N-terminal SNAG repressor domain with the chromatin-modifying protein lysine demethylase 1 (LSD1); thus, we investigated whether tranylcypromine [also known as trans-2-phenylcyclopropylamine hydrochloride (PCPA) or Parnate], an inhibitor of LSD1 that blocks its interaction with Slug, suppresses the migration, invasion, and metastatic spread of TNBC cell lines. We show here that PCPA treatment induces the expression of E-cadherin and other epithelial markers and markedly suppresses migration and invasion of TNBC cell lines MDA-MB-231 and BT-549. These effects were phenocopied by Slug or LSD1 silencing. In two models of orthotopic breast cancer, PCPA treatment reduced local tumor growth and the number of lung metastases. In mice injected directly in the blood circulation with MDA-MB-231 cells, PCPA treatment or Slug silencing markedly inhibited bone metastases but had no effect on lung infiltration. Thus, blocking Slug activity may suppress the metastatic spread of TNBC and, perhaps, specifically inhibit homing/colonization to the bone

Elongation Factor 1 alpha interacts with phospho-Akt in breast cancer cells and regulates their proliferation, survival and motility

BACKGROUND: Akt/PKB is a serine/threonine kinase that has attracted much attention because of its central role in regulating cell proliferation, survival, motility and angiogenesis. Activation of Akt in breast cancer portends aggressive tumour behaviour, resistance to hormone-, chemo-, and radiotherapy-induced apoptosis and it is correlated with decreased overall survival. Recent studies have identified novel tumor-specific substrates of Akt that may provide new diagnostic and prognostic markers and serve as therapeutic targets. This study was undertaken to identify pAkt-interacting proteins and to assess their biological roles in breast cancer cells. RESULTS: We confirmed that one of the pAkt interacting proteins is the Elongation Factor EF1alpha. EF1alpha contains a putative Akt phosphorylation site, but is not phosphorylated by pAkt1 or pAkt2, suggesting that it may function as a modulator of pAkt activity. Indeed, downregulation of EF1alpha expression by siRNAs led to markedly decreased expression of pAkt1 and to less extent of pAkt2 and was associated with reduced proliferation, survival and invasion of HCC1937 cells. Proliferation and survival was further reduced by combining EF1alpha siRNAs with specific pAkt inhibitors whereas EF1alpha downregulation slightly attenuated the decreased invasion induced by Akt inhibitors. CONCLUSION: We show here that EF1alpha is a pAkt-interacting protein which regulates pAkt levels. Since EF1alpha is often overexpressed in breast cancer, the consequences of EF1alpha increased levels for proliferation, survival and invasion will likely depend on the relative concentration of Akt1 and Akt2

The p53 Codon 72 Pro/Pro Genotype Identifies Poor-Prognosis Neuroblastoma Patients: Correlation with Reduced Apoptosis and Enhanced Senescence by the p53-72P Isoform.

The p53 gene is rarely mutated in neuroblastoma, but codon 72 polymorphism that modulates its proapoptotic activity might influence cancer risk and clinical outcome. We investigated whether this polymorphism affects neuroblastoma risk and disease outcome and assessed the biologic effects of the p53-72R and p53-72P isoforms in p53-null cells. Comparison of 288 healthy subjects and 286 neuroblastoma patients revealed that the p53-72 polymorphism had no significant impact on the risk of developing neuroblastoma; however, patients with the Pro/Pro genotype had a shorter survival than those with the Arg/Arg or the Arg/Pro genotypes even in the stage 3 and 4 subgroup without MYCN amplification. By Cox regression analysis, the p53 Pro/Pro genotype seems to be an independent marker of poor prognosis (hazard ratio = 2.74; 95% confidence interval = 1.14\u20136.55, P = .014) together with clinical stage, MYCN status, and age at diagnosis. In vitro, p53-72P was less effective than p53-72R in inducing apoptosis and inhibiting survival of p53-null LAN-1 cells treated with etoposide, topotecan, or ionizing radiation but not taxol. By contrast, p53-72P was more effective in promoting p21-dependent accelerated senescence, alone or in the presence of etoposide. Thus, the p53-72 Pro/Pro genotype might be a marker of poor outcome independent of MYCN amplification, possibly improving risk stratification. Moreover, the lower apoptosis and the enhanced accelerated senescence by the p53-72P isoform in response to DNA damage suggest that patients with neuroblastoma with the p53-72 Pro/Pro genotype may benefit from therapeutic protocols that do not rely only on cytotoxic drugs that function, in part, through p53 activation

The RNA Binding Protein SAM68 Transiently Localizes in the Chromatoid Body of Male Germ Cells and Influences Expression of Select MicroRNAs

The chromatoid body (CB) is a unique structure of male germ cells composed of thin filaments that condense into a perinuclear organelle after meiosis. Due to the presence of proteins involved in different steps of RNA metabolism and of different classes of RNAs, including microRNAs (miRNAs), the CB has been recently suggested to function as an RNA processing centre. Herein, we show that the RNA binding protein SAM68 transiently localizes in the CB, in concomitance with the meiotic divisions of mouse spermatocytes. Precise staging of the seminiferous tubules and co-localization studies with MVH and MILI, two well recognized CB markers, documented that SAM68 transiently associates with the CB in secondary spermatocytes and early round spermatids. Furthermore, although SAM68 co-immunoprecipitated with MVH in secondary spermatocytes, its ablation did not affect the proper localization of MVH in the CB. On the other hand, ablation of the CB constitutive component MIWI did not impair association of SAM68 with the CB. Isolation of CBs from Sam68 wild type and knockout mouse testes and comparison of their protein content by mass spectrometry indicated that Sam68 ablation did not cause overall alterations in the CB proteome. Lastly, we found that SAM68 interacts with DROSHA and DICER in secondary spermatocytes and early round spermatids and that a subset of miRNAs were altered in Sam68−/−germ cells. These results suggest a novel role for SAM68 in the miRNA pathway during spermatogenesis

Study of an intrinsically safe infrastructure for training and research on nuclear technologies

Within European Partitioning & Transmutation research programs, infrastructures specifically dedicated to the study of fundamental reactor physics and engineering parameters of future fast-neutron-based reactors are very important, being some of these features not available in present zero-power prototypes. This presentation will illustrate the conceptual design of an Accelerator-Driven System with high safety standards, but ample flexibility for measurements. The design assumes as base option a 70MeV, 0.75mA proton cyclotron, as the one which will be installed at the INFN National Laboratory in Legnaro, Italy and a Beryllium target, with Helium gas as core coolant. Safety is guaranteed by limiting the thermal power to 200 kW, with a neutron multiplication coefficient around 0.94, loading the core with fuel containing Uranium enriched at 20% inserted in a solid-lead diffuser. The small decay heat can be passively removed by thermal radiation from the vessel. Such a system could be used to study, among others, some specific aspects of neutron diffusion in lead, beam-core coupling, target cooling and could serve as a training facility

Combined therapy with RAD001 e BEZ235 overcomes resistance of PET immortalized cell lines to mTOR inhibition

Pancreatic endocrine tumors (PETs) are characterised by an indolent behaviour in terms of tumor growth. However, most patients display metastasis at diagnosis and no cure is currently available. Since the PI3K/AKT/mTOR axis is deregulated in PETs, the mTOR inhibitor RAD001 represents the first line treatment. Nevertheless, some patients do not respond to treatments and most acquire resistance. Inhibition of mTOR leads to feedback re-activation of PI3K activity, which may promote resistance to RAD001. Thus, PI3K represents a novel potential target for PETs. We tested the impact of three novel PI3K inhibitors (BEZ235, BKM120 and BYL719) on proliferation of PET cells that are responsive (BON-1) or unresponsive (QGP-1) to RAD001. BEZ235 was the most efficient in inhibiting proliferation in PET cells. Furthermore, combined treatment with BEZ235 and RAD001 exhibited synergic effects and was also effective in BON-1 that acquired resistance to RAD001 (BON-1 RR). Analysis of PI3K/AKT/mTOR pathway showed that RAD001 and BEZ235 only partially inhibited mTOR-dependent phosphorylation of 4EBP1. By contrast, combined therapy with the two inhibitors strongly inhibited phosphorylation of 4EBP1, assembly of the translational initiation complex and protein synthesis. Thus, combined treatment with BEZ235 may represent suitable therapy to counteract primary and acquired resistance to RAD001 in PETs

Combined therapy with RAD001 e BEZ235 overcomes resistance of PET immortalized cell lines to mTOR inhibition

Pancreatic endocrine tumors (PETs) are characterised by an indolent behaviour in terms of tumor growth. However, most patients display metastasis at diagnosis and no cure is currently available. Since the PI3K/AKT/mTOR axis is deregulated in PETs, the mTOR inhibitor RAD001 represents the first line treatment. Nevertheless, some patients do not respond to treatments and most acquire resistance. Inhibition of mTOR leads to feedback re-activation of PI3K activity, which may promote resistance to RAD001. Thus, PI3K represents a novel potential target for PETs. We tested the impact of three novel PI3K inhibitors (BEZ235, BKM120 and BYL719) on proliferation of PET cells that are responsive (BON-1) or unresponsive (QGP-1) to RAD001. BEZ235 was the most efficient in inhibiting proliferation in PET cells. Furthermore, combined treatment with BEZ235 and RAD001 exhibited synergic effects and was also effective in BON-1 that acquired resistance to RAD001 (BON-1 RR). Analysis of PI3K/AKT/mTOR pathway showed that RAD001 and BEZ235 only partially inhibited mTOR-dependent phosphorylation of 4EBP1. By contrast, combined therapy with the two inhibitors strongly inhibited phosphorylation of 4EBP1, assembly of the translational initiation complex and protein synthesis. Thus, combined treatment with BEZ235 may represent suitable therapy to counteract primary and acquired resistance to RAD001 in PETs