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MIRUMIR: an online tool to test microRNAs as biomarkers to predict survival in cancer using multiple clinical data sets

Insulin-like growth factors (IGFs) regulate the autocrine/paracrine growth of neuroblastomas. The IGFs bind to specific binding proteins (IGFBPs) which modulate their biological activity. We investigated, by Western ligand blotting (WLB), the presence of IGFBPs and their possible modulation by retinoic acid (RA), IGF-I, IGF-II and truncated Des(1-3)IGF-I in conditioned medium (CM) of the human neuroblastoma SK-N-BE(2) cell line. We demonstrated the presence of two IGFBPs, with MW 37 kDa and 25 kDa. Following immunoprecipitation, they turned out to be IGFBP-2 and -4, respectively. The RA-induced differentiation in SK-N-BE(2) cells was accompanied by a marked reduction of the intensity of both IGFBP bands after 48 h (32% and 24% of control, respectively) and 72 h (2% and 0% of control, respectively) incubation. The addition of exogenous IGFs, which did not induce cell differentiation, did not change the IGFBP pattern significantly, except for the truncated form of IGF-I, which induced a marked decrease in both the 37 kDa and 25 kDa bands after 72 h incubation (45% and 18% of control, respectively). These findings suggest that IGFBPs have a role in RA-induced differentiation in human neuroblastoma cells.

The Hippo tumor suppressor pathway regulates cellular proliferation and survival, thus has profound effects on normal cell fate and tumorigenesis. WW domain-containing proteins regulate diverse biological processes through interaction with proline-tyrosine (PPxY) containing targets. Here, we showed that the E3 ligase ITCH regulates stability of LATS1, a key components of the Hippo pathway through protein-protein interaction of the PPxY motifs of LATS1 and the WW domains of ITCH by showing both endogenous and exogenous LATS1 and ITCH co-immunoprecipitation and that a double mutation in LATS1 PPxY motifs abrogates ITCH-LATS1 interaction. Wild type, but not mutant, ITCH catalyzed ubiquitination of LATS1 and stimulated its proteosomal degradation. Activation of the Hippo pathway, either by overexpressing MST2 or culturing cells at high density, induced ITCH and subsequent degradation of LATS1. Modulation of LATS1 levels via ITCH overexpression or Knockdown was associated with status of YAP phosphorylation, its subcellular localization and subsequently its transcription co-activation function. While ITCH knockdown induced epithelial-mesenchymal transition (EMT) phenotypes and tumorigenicity, ITCH overexpression induced these phenotypes. These findings provide evidence of a novel functional link between ITCH and Hippo pathway, underscoring a critical role for this association in tumorogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-126. doi:10.1158/1538-7445.AM2011-LB-126

Insulin-like growth factor 2 (IGF-2) is the major autocrine growth factor for neuroblastoma. IGF-2 mRNA can just be detected in SK-N-BE(2) cell line; higher levels are present in two clones derived from it [BE(2)-C; BE(2)-M17]. IGF-2 mRNA is increased by retinoic acid (RA) only in the clones. IGF-2 expression/induction is more marked in BE(2)-M17, which shows more RA-resistance (evaluated as growth inhibition, neurite outgrowth and induction of programmed cell death). Under RA exposure, the parental line shows a more pronounced growth inhibition, neurite outgrowth and programmed cell death, as compared to its clones. BE(2)-C cells also express type 1 IGF receptor mRNA, though with a different time course than for expression of IGF-2. The data suggest that IGF-2 expression is correlated with growth, and may counteract the growth retardation, neurite outgrowth and programmed cell death effects of retinoic acid. Therefore the autocrine pattern of IGF-2 production by neuroblastoma cells may promote RA-resistance.

The human glioblastoma cell line LI showed morphological features typical of its neuroectodermal origin. Cells were positive by immunofluorescence to GFAP, MHC class II, and L1 determinants. Cytogenetic analysis showed the presence of a modal chromosome number of 63, ranging from 58 to 69 chromosomes (DNA index was 1.6). Northern blot analysis demonstrated the presence of mRNA transcripts specific for transglutaminase C (type II or "tissue"), growth-hormone releasing-hormone (GHRH), insulin-like growth factor II (IGF-II), and proopiomelanocortin (POMC). The GHRH mRNA was present in two different sizes, one similar to the normal hypothalamic species of 0.75 kb, whilst the second species was a large transcript of approximately 10 kb size. Treatment with 5 microM retinoic acid or 5 mM alpha-difluoromethylornithine for 5 days sharply reduced the growth rate and also induced modulation of the ultrastructure and antigenic profile. This cell line may be useful to study glial differentiation and the relationship of GHRH, IGF-II and POMC expression with differentiation in neuroectodermal tumours.

There are 'roadblocks' at several points in the signalling pathways that control programmed cell death. When the cell needs to die, these roadblocks must be removed, and a vertebrate protein that removes one such block has now been identified.

The incorporation and redistribution of [1-14C]arachidonic acid in SK-N-BE human neuroblastoma cell phospholipids were investigated. By continuous labelling in serum-enriched medium, a rapid radioactivity incorporation into phosphatidylcholine (PtdCho), phosphatidylinositol, and phosphatidylserine was observed; initially, phosphatidylethanolamine (PtdEtn) was poorly labelled, but at later stages it displayed the highest level of arachidonic acid incorporation, in comparison with other phospholipid classes. Labelling of triacylglycerols was also observed. When cells were pulse-labelled with [1-14C]arachidonic acid and then reincubated in label-free medium, a decrease of the radioactivity in triacylglycerols was observed initially, paralleled by an increase of phospholipid labelling; thereafter, arachidonic acid redistribution was consistent with a net decrease of the radioactivity associated with PtdCho acid-stable forms (i.e., diacyl plus alkylacyl forms), concomitantly with a net labelling increase of both acid-stable PtdEtn and alkenylacyl-PtdEtn. Data indicate the following: (a) neuroblastoma cells incorporate arachidonic acid into phospholipids through complex kinetics involving transfer of the fatty acid from acid-stable PtdCho to both alkenylacyl-PtdEtn and acid-stable PtdEtn; and (b) triacylglycerols act as storage molecules for arachidonic acid which is subsequently incorporated into phospholipids. The possibility that arachidonic acid transfer to PtdEtn subclasses is driven by distinct mechanisms is discussed.

Transglutaminase 2 (TG2 or TGM2) is a multi-functional enzyme which catalyzes transamidation reactions or acts as a G-protein in intracellular signalling. Tgm2-/- Mice lacking TG2 activity are glucose intolerant and show impairment of insulin secretion, suggesting an important physiological role for TG2 in the pancreatic beta cell. We have previously described a TGM2 heterozygous missense mutation ((c.998A>G, p.N333S) in a 14 year-old patient with insulin-treated diabetes and in his diabetic father. The aim of this study was to further investigate the role of TG2 in early-onset type 2 diabetes. We analysed the TGM2 gene in 205 patients with clinically defined Maturity Onset Diabetes of the Young (MODY) or early-onset type 2 diabetes. We found two novel heterozygous mutations (c.989T>G, p.M330R; c.992T>A, p.I331N), which were not detected in 300 normoglycemic controls. All mutations were in residues which are located close to the catalytic site and impaired transamidating activity in vitro. Gene expression of TGM family genes and localization of TG2 in normal human pancreas indicated that TG2 is the only transglutaminase significantly expressed in human pancreatic islet cells. We conclude that reduced TG2 activity can contribute to disorders of glucose metabolism possibly via an impairment of insulin secretion.

The 1-p-(3-methyltriazeno) benzoic acid potassium salt (MTBA) is a triazeno analogue of dacarbazine, an antineoplastic agent capable of mediating the appearance of new antigenic specificities on cancer cells in mice, a phenomenon described as 'chemical xenogenization' (CX). Recently we reported the clastogenic potential of MTBA on human lymphocytes. Since sister-chromatid exchange (SCE) assay is more sensitive than clastogenic tests, at least at low drug concentrations, we assessed SCE frequencies induced by MTBA on human lymphocytes stimulated by PHA. Drug treatment at 2-500 micrograms/ml was performed in vitro prior to or after PHA addition. SCE values increased significantly in a dose-dependent manner up to 200 micrograms/ml. However, SCE frequencies, as well as chromosome breaks, did not increase dramatically. These data indicate that MTBA concentrations used for CX do not cause severe cytogenetic damage to immune cells at least in vitro.

Epidermal keratinocytes undergo differentiation in response to several stimuli to form the cornified envelope, a structure that contributes to the barrier function of skin. Although differentiation has been extensively analyzed, the precise role of vitamin C during this process is still not defined. Ascorbic acid, besides acting as a radical scavenger, has been shown to promote mesenchymal differentiation. In this study, we found that keratinocytes grown in ascorbate-supplemented medium developed a differentiated phenotype, as demonstrated by enhanced expression of marker genes and increase in cornified envelope content. The pro-differentiating effects of ascorbate were mediated by the protein-kinase-C-dependent induction of activating protein 1 DNA binding activity; indeed, down-modulation of protein kinase C activity abolished differentiation triggered by ascorbic acid. Although vitamin C appeared to regulate the same signaling pathway modulated by calcium, a classical in vitro inducer of epidermal differentiation, nonetheless terminally differentiated keratinocytes exhibited different ascorbate homeostasis and cellular antioxidant status. Indeed, we found that, unlike calcium, differentiation promoted by ascorbate was accompanied by (i) an enhanced ascorbate transport, due to overexpression of specific transporters, (ii) a great efficiency of dehydroascorbate uptake, and (iii) an increase in glutathione content with respect to proliferating cells. Ascorbic acid may be useful to promote epidermal differentiation, avoiding depletion of hydrophilic antioxidant stores.

Apoptotic cells show morphological modifications which occur as the result of complex molecular mechanisms involving several proteins including `tissue' transglutaminase (tTG). Although tTG was originally thought to be responsible for the protein crosslinks which prevent the leakage of intracellular components, thereby reducing inflammation and autoimmunity, recent evidence indicates that tTG is a multifunctional enzyme involved in the complex upstream regulation of the apoptotic machinery: (i) it functions as a GTP-binding protein to transduce signals; (ii) it binds/crosslinks only specific cytosolic and nuclear substrates, suggesting highly specific actions, e.g. on intermediate filaments and in cell cycle control; (iii) it is finely tuned by Ca2+, GTP, S-nitrosylation, polyamines. In light of these recent discoveries, the role of tTG in the regulation of the crucial balance between survival and death is clearly complex.

The involvement of the homologous to E6-AP carboxyl terminus (HECT)-type E3s in crucial signaling pathways implicated in tumorigenesis is presently an area of intense research and extensive scientific interest. This review highlights recent discoveries on the ubiquitin-mediated degradation of crucial tumor suppressor molecules catalyzed by the HECT-type E3s. By providing a portrait of their protein targets, we intend to link the substrate specificity of HECT-type E3s with their contribution to tumorigenesis. Moreover, we discuss the relevance of targeting the HECT E3s, through the development of small-molecule inhibitors, as an anticancer therapeutic strategy.

Palmoplantar keratodermas are a group of heterogeneous diseases characterized by thickening, and marked hyperkeratosis, of the epidermis of the palms and soles. Palmoplantar keratodermas can be divided into four major classes: diffuse, focal, punctate, and palmoplantar ectodermal dysplasias. All forms are genetic diseases inherited as autosomal dominant disorders. We studied a patient exhibiting a localized thickening of the skin in parts of the right palm and the right sole, following Blaschko's lines, that does not fit into any classes already described. We sequenced the keratin 16 cDNA derived from skin biopsy material from affected and non affected palms. The keratin 16 cDNA sequence from lesional epidermis showed a 12 base pair deletion (309–320del), which deletes codons 104–107. The mutation is predicted to delete four amino acids, GGFA, from the V1 domain of the keratin 16 polypeptide, close to the 1A domain. Full-length keratin 16 cDNA sequence derived from the unaffected palm was completely normal, consistent with a postzygotic mutation as is suggested by the mosaicism observed. We defined this new clinical entity, ‘‘unilateral palmoplantar verrucous nevus'', rather than localized or focal epidermolytic palmoplantar keratodermas, as the lesions are present only on one side of the body and follow Blaschko's lines. This study is a report of a mosaic mutation in keratin 16 and also the association of a mutation in the V1 domain of a type I keratin associated with a human disease.

Retinoic acid modulates growth and induces differentiation and apoptosis of neuroblastoma cells in vitro, with the all-trans and 9-cis isomers having different biological properties. Transcriptional activation in response to retinoic acid isomers is mediated by retinoic acid receptors and retinoid X receptors. The differential expression of co-activators and co-repressors which preferentially interact with retinoic acid receptors or retinoid X receptors may be a mechanism leading to different cellular responses to 9-cis and all-trans retinoic acid. To test this hypothesis, we have studied the expression of the nuclear receptor co-regulators TIF1alpha, TIF1beta, SUG1 and SMRT in the N-type and S-type neuroblastoma cell lines SH SY 5Y and SH S EP. Transcripts for all four co-regulators were expressed in these neuroblastoma cells. The expression of TIF1alpha, TIF1beta and SUG1 did not change in response to retinoic acid; however, SMRT was induced in both neuroblastoma cell lines, but particularly by all-trans retinoic acid in SH S EP cells. An additional co-activator, Trip3, was isolated by differential mRNA display and shown to be preferentially induced by 9-cis retinoic acid in SH SY 5Y and SH S EP cells. These data suggest that retinoic acid isomer-specific induction of nuclear receptor co-regulators may determine, in part, the differential biological effects of retinoic acid isomers.

All known members of the p53 gene family, including the two homologs p73 and p63, have multiple biological functions. In neurons, p53 and p73 are known to regulate cell death in the developing and adult nervous system. A report by Jacobs et al. in this issue of Neuron shows that the more ancestral member of this gene family, p63, is an essential proapoptotic protein during neuronal development.

The triazene derivative 1-p-(3-methyltriazeno)benzoic acid potassium salt (MTBA) shows pharmacological properties similar to those of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC, trade name dacarbazine), which is known to induce antigenic modulation in tumor cells (xenogenization) and is currently used in cancer therapy. Mutagenic, teratogenic and cancerogenic properties of triazene derivatives have been demonstrated but there is no report on their possible clastogenicity. We describe here the in vitro cytogenetic effects of MTBA on human peripheral blood lymphocytes. The drug was tested at different culture times in a range of concentrations from 2 to 500 micrograms/ml. MTBA caused a dose-dependent increase in the frequency of chromosomal breaks. Different blood donors showed different sensitivity to the treatment. Cell proliferation, as evaluated by [3H]thymidine incorporation, was inhibited at the highest concentrations of the drug. These data might be relevant for comparison with in vivo effects of the drug in clinical trials and to investigate the possible relations between xenogenization induced by MTBA and its genetic and cytogenetic effects in human lymphocytes.

A 15 month old boy with a stage IV right suprarenal gland neuroblastoma showed a number of raised biochemical parameters, whilst catecholamines and skeletal survey were normal. Treatment with peptichemio failed to give a clinical response. Histological evidence of neuroblastoma infiltration in the bone marrow aspirate was absent. Immunofluorescence on sedimented cells was negative using antibody UJ223.8, PI153/3 and H11; only UJ308 and to a lesser extent UJ13A gave positive results. After 21 days, however, the same cells in culture showed highly differentiated dendritic processes. Thirty-seven percent metaphases from bone marrow aspirate showed the following karyotype 45XY, del (1) (p32), and two markers. Mar1 = der (2) t (2; 2) (2qter----2q14::2p24----2qter). Mar2 = der (15) t (15; 2) (15qter----15p11::2p11----2pter). Treatment with methotrexate reduced the aberrant mitoses rate to 2%. N-myc in situ hybridisation showed significant signal on both markers confirming the cytogenetic interpretation. Peripheral blood lymphocytes at 72 h showed a higher level of breaks per cell than control. After treatment with aphidicolin (APC) or methotrexate (MTX) for the last 24 h, to induce fragile sites, the incidence of breaks per cells was increased. Moreover 11.4% of APC-induced breaks were in 1p31-32 (mean of normal controls = 2.3%). The mother presented an increased sensitivity to the inducibility of fragile sites, while the father's lymphocytes showed values within the control range. The genetic changes produced by the abnormalities on chromosomes 1 and 2 might be related to tumour progression. Furthermore this is the first description of correlation between a high frequency of fragile site 1p31-32 induced by APC in the patient's lymphocytes and deletion of 1p32 in tumour cells. The interpretation of these findings and of other similar correlations needs further study. Images Figure 1 Figure 2

The promyelocytic leukaemia gene (Pml) is a tumor suppressor identified in acute promyelocytic leukaemia (APL), where it is fused to RAR alpha gene as a result of the chromosomal translocation t(15;17). Pml encodes both nuclear and cytoplasmic isoforms. While nuclear PML has been intensively investigated, cytoplasmic PML proteins are less characterized. PML nuclear isoforms (nPML) are the essential components of subnuclear structures referred to as PML nuclear bodies (PML-NB). In response to cellular insults such as DNA damage and oncogenic activation, nPML modulates p53 activity through CBP-mediated acetylation and activates its pro-apoptotic and growth suppressive functions. Two missense mutations resulting in truncated PML cytoplasmic proteins (Mut PML) have been identified in aggressive APL cases. Here we report that cytoplasmic PML is able to induce the relocation of nPML to the cytoplasm, thus reducing the number of PML-NBs. Remarkably, Mut PML inhibits p53 transcriptional, growth suppressive, and apoptotic functions, thus suggesting that cytoplasmic expression of PML has an impact on survival through inhibition of nuclear PML. Overall our findings shed new light on the role of PML cytoplasmic proteins in the regulation of p53.

Epidermolytic PalmoPlantar keratoderma (EPPK) Vörner-type is an autosomal dominantly inherited skin disease, characterized by severe thickening of the palms and soles, caused by mutations in the keratin K9 (KRT9) gene. To date, a number of KRT9 mutations have been detected, most of which affect the highly conserved 1A region of the central alpha-helical domain, important for keratin heterodimerization. The most common mutation is the substitution of the arginine in position 163 with a tryptophan (R163W), which has been reported in North American, European, and Japanese populations. In a small number of cases, EPPK is associated with knuckle pad keratosis, but no correlation between this additional phenotype and a specific mutation has been found. Moreover, K9 is not normally expressed in knuckle skin, raising the question of the pathogenic mechanism leading to this additional phenotype. Here we show that in a family affected by EPPK and knuckle pad keratosis, carrying the R163W substitution, wild type (wt) and mutated K9 are strongly expressed in knuckle pads. These results suggest that the knuckle pad phenotype is due to ectopical expression of K9.

Nitric oxide (NO), peroxynitrite, and 4-hydroxynonenal (HNE) may be involved in the pathological demise of cells via apoptosis. Apoptosis induced by these agents is inhibited by Bcl-2, suggesting the involvement of mitochondria in the death pathway. In vitro, NO, peroxynitrite and HNE can cause direct permeabilization of mitochondrial membranes, and this effect is inhibited by cyclosporin A, indicating involvement of the permeability transition pore complex (PTPC) in the permeabilization event. NO, peroxynitrite and HNE also permeabilize proteoliposomes containing the adenine nucleotide translocator (ANT), one of the key components of the PTPC, yet have no or little effects on protein-free control liposomes. ANT-dependent, NO-, peroxynitrite- or HNE-induced permeabilization is at least partially inhibited by recombinant Bcl-2 protein, as well as the antioxidants trolox and butylated hydroxytoluene. In vitro, none of the tested agents (NO, peroxynitrite, HNE, and tert-butylhydroperoxide) causes preferential carbonylation HNE adduction, or nitrotyrosylation of ANT. However, all these agents induced ANT to undergo thiol oxidation/derivatization. Peroxynitrite and HNE also caused significant lipid peroxidation, which was antagonized by butylated hydroxytoluene but not by recombinant Bcl-2. Transfection-enforced expression of vMIA, a viral apoptosis inhibitor specifically targeted to ANT, largely reduces the mitochondrial and nuclear signs of apoptosis induced by NO, peroxynitrite and HNE in intact cells. Taken together these data suggest that NO, peroxynitrite, and HNE may directly act on ANT to induce mitochondrial membrane permeabilization and apoptosis.

OTX proteins, homologs of the Drosophila orthodenticle (Otd), are important for the morphogenesis of the neuroectoderm, and for the central nervous system formation. OTX1 and OTX2 are important for the cochlea and macula development, indeed when OTX1 is knocked down, these organs undergo developmental failure. Moreover OTX2 transfection revert this effect in OTX1(-/-) mice. The TA isoform of TP63, involved in Notch regulation pathway, has a critical function in the cochlear neuroepithelium differentiation. TAp63 positively regulates Hes5 and Atoh1 transcription. This pathway has been also demonstrated in p63(-/-) mice, and in patients p63 mutated, affected by Ectodermal Dysplasia (ED, OMIM 129810). These patients are affected by mild sensorineural deafness, most likely related to the mutation in p63 gene impairing the Notch pathway. We demonstrated the role of OTX2 on TAp63 regulation necessary for the correct formation of macular neuroepithelium and we confirmed the impairment of vestibular function caused by p63 mutations. Although the abnormalities found in our patient were still at a subclinical extent, aging could exacerbate this impairment and cause a decrease in quality of life.

p63 is a transcriptional factor belonging to p53 family of genes. Beside the role in cancer, partially shared with p53 and the other member p73, p63 also plays exclusive roles in development and homeostasis of ectodermal/epidermal-related organs. Here we show that p63 transcriptionally controls the expression of the matrix metallopeptidase 13 (MMP13). p63 binds a p53-like responsive element in the human promoter of MMP13, thus promoting the activation of its transcription. The catalytic activity of MMP13 is required in high invasion capacity of metastatic cancer cells, however, although p63 and MMP13 expression correlates in cancer patients, their co-expression does not predict cancer patient survival. Our results demonstrate that p63 directly controls MMP13 expression. published

Prostate cancers show a slow progression from a local lesion (primary tumor) to a metastatic and hormone-resistant phenotype. After an initial step of hyperplasia, in a high percentage of cases a neoplastic transformation event occurs that, less frequently, is followed by epithelial to mesenchymal transition and invasion of healthy tissues (usually bones). MicroRNA-203 (miR-203) is a tumor suppressor microRNA often silenced in different malignancies. Here, we show that miR-203 is downregulated in clinical primary prostatic tumors compared to normal prostate tissue, and in metastatic prostate cancer cell lines compared to normal epithelial prostatic cells. Overexpression of miR-203 in brain or bone metastatic prostate cell lines (DU145 and PC3) is sufficient to induce a mesenchymal to epithelial transition with inhibition of cell proliferation, migration and invasiveness. We have identified CKAP2, LASP1, BIRC5, WASF1, ASAP1 and RUNX2 as new miR-203 direct target mRNAs involved in these events. Therefore, miR-203 could be a potentially new prognostic marker and therapeutic target in metastatic prostate cancer.

Cajal Bodies are one of many specialised organelles contained in the eukaryotic cell nucleus, and are involved in a number of functions, including regulation of replication-dependent histone gene transcription. In normal diploid cells their number varies between 0 and 4 depending on the cell cycle phase, although in cancer cell lines their number is extremely variable and it has been suggested that it correlates with cell ploidy. Here we show that in mammalian cells, as in Drosophila, two distinct though functionally related bodies exist: a histone gene locus body and a Cajal Body. The first one can be detected using FLASH or NPAT as markers while the second is labelled using antibodies against Coilin. Only the number of FLASH/NPAT histone gene locus bodies correlates with ploidy and only these organelles appear to be regulated during the cell cycle. Finally, we show that the two organelles completely co-localize during the S phase of the cell cycle.

P73 is a member of the p53 transcription factors family with a prominent role in neurobiology, affecting brain development as well as controlling neuronal survival. Accordingly, p73 has been identified as key player in many age-related neurodegenerative diseases, such as Alzheimer's disease, neuroAIDS and Niemann-Pick type C disease. Here we investigate possible correlations of p73 with Parkinson disease. Tyrosine hydroxylase is a crucial player in Parkinson disease being the enzyme necessary for dopamine synthesis. In this work we show that levels of tyrosine hydroxylase can be influenced by p73. We also demonstrate that p73 can protect against tyrosine hydroxylase depletion in an in vitro model of Parkinson disease.

The major drug discovery efforts in oncology have been concentrated on the development of selective molecules that are supposed to act specifically on one anticancer mechanism by modulating a single or several closely related drug targets. However, a bird's eye view on data from multiple available bioassays implies that most approved anticancer agents do, in fact, target many more proteins with different functions. Here we will review and systematize currently available information on the targets of several anticancer drugs along with revision of their potential mechanisms of action. Polypharmacology of the current antineoplastic agents suggests that drug clinical efficacy in oncology can be achieved only via modulation of multiple cellular mechanisms.

Molecular scanning of human IRS-1 gene revealed a common polymorphism causing Gly--Arg972 change. Diabetic and pre-diabetic carriers of Arg972 IRS-1 are characterized by low fasting levels of insulin and C-peptide. To investigate directly whether the Arg 972 IRS-1 affects human islet cells survival, we took advantage of the unique opportunity to analyze pancreatic islets isolated from three donors heterozygous for the Arg972 and six donors carrying wild-type IRS-1. Islets from carriers of Arg972 IRS-1 showed a two-fold increase in the number of apoptotic cells as compared with wild-type. IRS-1-associated PI3-kinase activity was decreased in islets from carriers of Arg972 IRS-1. Same results were reproduced in RIN rat b-cell lines stably expressing wild-type IRS-1 or Arg972 IRS-1. Using these cells, we characterized the downstream pathway by which Arg972 IRS-1 impairs b-cell survival. RIN-Arg972 cells exhibited a marked impairment in the sequential activation of PI3-kinase, Akt, and BAD as compared with RI N-WT. Impaired BAD phosphorylation resulted in increased binding to Bcl-XL instead of 14-3-3 protein, thus sequestering the Bcl-XL antiapoptotic protein to promote survival. Both caspase-9 and caspase-3 activities were increased in RIN-Arg972 cells. The results show that the common Arg972 polymorphism in IRS-1 impairs human b-cell survival and causes resistance to antiapoptotic effects of insulin by affecting the PI3-kinase/Akt survival pathway. These findings establish an important role for the insulin signaling in human b-cell survival and suggest that genetic defects in early steps of insulin signaling may contribute to b-cell failure.

Metabolic adaptation has emerged as a hallmark of cancer and a promising therapeutic target, as rapidly proliferating cancer cells adapt their metabolism increasing nutrient uptake and reorganizing metabolic fluxes to support biosynthesis. The transcription factor p73 belongs to the p53-family and regulates tumorigenesis via its two N-terminal isoforms, with (TAp73) or without (ΔNp73) a transactivation domain. TAp73 acts as tumor suppressor, at least partially through induction of cell cycle arrest and apoptosis and through regulation of genomic stability. Here, we sought to investigate whether TAp73 also affects metabolic profiling of cancer cells. Using high throughput metabolomics, we unveil a thorough and unexpected role for TAp73 in promoting Warburg effect and cellular metabolism. TAp73-expressing cells show increased rate of glycolysis, higher amino acid uptake and increased levels and biosynthesis of acetyl-CoA. Moreover, we report an extensive TAp73-mediated upregulation of several anabolic pathways including polyamine and synthesis of membrane phospholipids. TAp73 expression also increases cellular methyl-donor S-adenosylmethionine (SAM), possibly influencing methylation and epigenetics, and promotes arginine metabolism, suggestive of a role in extracellular matrix (ECM) modeling. In summary, our data indicate that TAp73 regulates multiple metabolic pathways that impinge on numerous cellular functions, but that, overall, converge to sustain cell growth and proliferation.

The transcription factor p73 belongs to the p53 family of tumour suppressors and similar to other family members, transcribed as different isoforms with opposing pro- and anti-apoptotic functions. Unlike p53, p73 mutations are extremely rare in cancers. Instead, the pro-apoptotic activities of transcriptionally active p73 isoforms are commonly inhibited by over-expression of the dominant negative p73 isoforms. Therefore the relative ratio of different p73 isoforms is critical for the cellular response to a chemotherapeutic agent. Here, we analysed the expression of N-terminal p73 isoforms in cell lines and mouse tissues. Our data showed that the transcriptionally competent TAp73 isoform is abundantly expressed in cancer cell lines compared to the dominant negative ΔNp73 isoform. Interestingly, we detected higher levels of ΔNp73 in some mouse tissues, suggesting that ΔNp73 may have a physiological role in these tissues.

In this report, we show that the overexpression of tissue transglutaminase (tTG) in the human neuroblastoma cell line SK-N-BE(2) renders these neural crest-derived cells highly susceptible to death by apoptosis. Cells transfected with a full-length tTG cDNA, under the control of a constitutive promoter, show a drastic reduction in proliferative capacity paralleled by a large increase in cell death rate. The dying tTG-transfected cells exhibit both cytoplasmic and nuclear changes characteristic of cells undergoing apoptosis. The tTG-transfected cells express high Bcl-2 protein levels as well as phenotypic neural cell adhesion molecule markers (NCAM and neurofilaments) of cells differentiating along the neuronal pathway. In keeping with these findings, transfection of neuroblastoma cells with an expression vector containing segments of the human tTG cDNA in antisense orientation resulted in a pronounced decrease of both spontaneous and retinoic acid (RA)-induced apoptosis. We also present evidence that (i) the apoptotic program of these neuroectodermal cells is strictly regulated by RA and (ii) cell death by apoptosis in the human neuroblastoma SK-N-BE(2) cells preferentially occurs in the substrate-adherent phenotype. For the first time, we report here a direct effect of tTG in the phenotypic maturation toward apoptosis. These results indicate that the tTG-dependent irreversible cross-linking of intracellular protein represents an important biochemical event in the induction of the structural changes featuring cells dying by apoptosis.

I am not afraid of death—I am privileged to have been able to work for so long. If I die tomorrow or in a year, it is the same. It is the message you leave behind you that counts, and the young scientists who carry on your work. —Rita Levi-MontalciniOn December 30, 2012, Rita Levi-Montalcini passed away at her home in Rome at age 103. Rita was a model and inspiration for the younger generation: not simply because of her scientific and political achievements but equally for her warm personality, exemplary life, and boundless enthusiasm.Rita, along with her twin sister Paola, was born into an intellectual and highly cultured Turinese Jewish family on April 22, 1909—the two youngest of four children.A polymath, postenlightenment ethos flowed from their parents to each child, creating an ambience where intellectual pursuits were highly appreciated and avidly pursued. Adamo Levi, her father, was an engineer and talented mathematician, and her mother Adele Montalcini, renowned for her personal charm, was a gifted painter. Her oldest brother, Gino, was among Italy’s best-known architects and a professor at Turin University. Anna was a keen “child of the Renaissance” immersed in literature and the humanities, whereas Paola, from early childhood, displayed extraordinary artistic talents, especially, like her mother, as a painter.After completing grammar school, the young Rita felt unable to adjust to the conventional feminine role (expected by her father) and embarked instead on a career in medical research. As a result, she studied at Turin University under Giuseppe Levi, the then Professor of Histology, who also tutored Salvador Luria and Renato Dulbecco, two other future Nobel laureates. All three became close friends, as well as colleagues.In 1936, Rita graduated in Medicine and Surgery and began her specialization in neuropsychiatry—fortuitously two years prior to publication of the antisemitic “A manifesto for defending our race” by a coterie of Fascist scientists and intellectuals (first published on July 15, 1938, it led to the formulation of Act n. 1024 of July 13, 1939 XVII, promoted by the antisemitic area of the National Fascist Party). Rita spent a short sabbatical in Brussels in 1940 to improve her neuroscience skills and returned to Turin shortly before the German army invaded Belgium. This was a very difficult time, and Rita pursued her research activity from a small laboratory that she built at home, continuing her collaboration with Giuseppe Levi until her family was forced to flee a heavily bombed Turin for Florence in 1943. During 1943 and 1944, she established close contacts with partisans through friends and members of the antifascist coalition known as the Partito d’Azione (1942–1947, important during the war as well as in the early postwar years and leading to the “Assemblea Costituente” that prepared the new Italian Constitution). Subsequently, she worked as a medical doctor assigned to a refugee camp, where epidemics and infectious disease daily undermined the health of already weak and malnourished war victims.In opting to specialize in neuropsychiatry, Rita was inspired by the Viktor Hamburger’s early scientific work on the effects of limb extirpation in chick embryos. Later, in 1947, Rita was able to join Hamburger’s laboratory at Washington University in St. Louis, MO, where in 1956 she became an Associate Professor and in 1958 became a Full Professor, a position that she held until her retirement in 1977. During this period, she also became a member of the Accademia dei Lincei (Italy’s leading Academic Society founded in 1603), of the Pontifical Academy of Sciences (Vatican), of the Royal Society, and of the National Academy of Sciences of the USA.During these years in St Louis, she collaborated with Stanley Cohen and, in the early 1950s, found that mouse tumor cells release a factor that causes rapid growth of nerve cells both in vitro and in vivo. Today, it comes as little surprise that this was nerve growth factor (NGF). Together with Cohen, Rita shared the Nobel Prize in Physiology for Medicine in 1986 for this discovery. NGF as a neuronal survival factor was identified well before many other endogenous cell survival ligands, and consequently, this discovery became the template for understanding fundamental mechanisms in biology.NGF has the potential to improve clinical therapy for the increasing burden of neurodegenerative disease; indeed, it is now at an advanced stage of optimization of formulation and delivery. Rita contributed significantly to this, partly by improving large-scale production of recombinant human NGF (rhNGF), as well as through a small-scale human study that demonstrated how NGF therapy can reduce retinal-ganglion cell loss in patients with glaucoma.In 1962, Rita established a research unit in Rome, dividing her time between there and St. Louis. In Rome, she established the Institute of Cell Biology at the Italian National Research Council (CNR). Additionally, in 2002, to further develop basic and applied research on NGF and neurodegenerative diseases, as well as to attract world-class scientists, she created the European Brain Research Institute (EBRI), also in Rome, where I had the honor to serve as Scientific Director. There, I had the great pleasure of closely interacting with Rita and admiring her curiosity, her desire to keep abreast of knowledge, and her ability to disseminate enthusiasm and perseverance among the young scientists—characteristics that persisted to her very last day. Indeed, her last papers were published only a few months ago. In simple words, Rita was a model for scientists and a model for life. Research at EBRI covers a wide range of neurodegenerative pathologies, as reflected in the content of the proceedings published on the occasion of her 100th and 102nd birthdays, and I hope the Institute will continue to diffuse her ideas for forthcoming generations. The Israel Hospital in Rome will also shortly adopt her name.Beyond medicine, Rita championed ethics and women’s rights throughout her adult life. In recent years, the Rita Levi-Montalcini Foundation has supported education for thousands of African women, and Rita herself has published more than 20 books disseminating her beliefs as standards for the young. Rita was among her country’s most popular women.In 2001, she was nominated a Senator-for-Life by Italy’s President Carlo Azeglio Ciampi and was a firm supporter of the center-left Romano Prodi government, especially during its difficult times. Then, Rita was determined to be present up to 10 hours in the Chamber, participating in the discussions and on occasion providing the crucial single vote of majority.In the words of Aaron Ciechanover, “Rita was ‘one of a kind’ in the deepest and broadest sense of these words, very close to me in person. Hard to imagine that this thin, frail, and small Jewish woman—who was a true lady, the ultimate in fine taste—carved her way during days when women were hardly seen in universities, in Fascist Italy, to peaks of science and humanity, where the air is thin and only a few, much stronger than her can survive. She probably had attributes we may never be able to unveil. I will miss her sorely.”She will always live in our hearts.Rita Levi-MontalciniView Large Image | View Hi-Res Image | Download PowerPoint Slide

International audience; Luteoviruses, that is, members of the Luteoviridae family, are plant viruses that can infect a wide range of host plants, including many important crops such as cereals, cucurbits, legumes, potato, sugarbeet and sugar cane. Their icosahedral virions contain a single (+) ribonucleic acid genome in a capsid composed of two structural proteins. They are limited to phloem cells in host plants and are only transmitted by aphids in a circulative and nonpropagative mode with high specificity. Growing data are accumulating on plant–luteovirus relationships and more particularly on the mechanism developed by the virus to overcome plant defence. The tight interaction between luteoviruses and their aphid vector has also been extensively studied. Information from these studies together with a better understanding of the epidemiology of luteoviruses will help to combat their detrimental effects on crops.

Thèse en co-tutelle : Universita degli Studi di Roma "Tor Vergata" et Université Pierre & Marie Curie (UParis); In our search for the underlying causes of cancer, TP53 is the most intensively studied gene. p53 plays a central role for balancing the antagonistic processes of proliferation and apoptosis. As a sequence-specific transcription factor, p53 regulates the expression of genes involved in cell cycle arrest and apoptosis in response to genotoxic damage or cellular stress. Failure of p53 function consequently leads to uncontrolled cell growth, a defining feature of cancer cells. Given the importance of p53 as a tumor suppressor, it is therefore no wonder that p53 is the most frequent site of genetic alterations found in human cancers. The recent discovery of two TP53-related genes, TP73 and TP63 with striking sequence homology, was therefore a big surprise, raising the possibility that other tumor suppressors exist which share the power of p53 in preventing cancer formation. The three members of the p53 family share significant homology both at the genomic and at the protein level. The highest level of identity is reached in the DBD (DNA-Binding Domain), suggesting that they can bind to the same DNA sequence and transactivate the same promoters. In fact, p73 and p63 are able to activate some p53 targets and to induce apoptosis, but they appear more and more different from their relative. The study of the respective knock-out mice gives a good illustration of these differences : while p53-null mice develop normally but present spontaneous tumors, the p73 and p63-null mice present severe developmental troubles but no spontaneous tumors, indicating that they may have more complex functions. Conversely to p53, p73 and p63 contain additional C-terminal extensions. In both proteins, these extensions show alternative splicing, which results in at least six C-terminal variants for p73 and three for p63. These isoforms have different transcription and biological properties, and their expression patterns change among normal tissues. Moreover, the α variants of p73 and p63 have close to their C terminus a SAM (Sterile Alpha Motif) domain, which is thought to be responsible for regulating p53-like functions, and is implicated in various human syndromes where p63 is mutated. In addition to the C-terminal variants aminoterminous truncated variants of p73 and p63 exist : ΔNp73 and ΔΝp63. These N-terminally truncated isoforms lack the transactivation domain (TA), which is coded by the first 3 exons, and derive from the use of an alternative promoter (P2) located in intron 3 and an additional exon (exon 3'). While TAp73 isoforms work as transcription factors and can induce irreversible cell cycle arrest and apoptosis like p53, the ΔNp73 isoforms that lack the transactivation domain are incapable of directly inducing gene expression and do not induce growth arrest or cell death. However, the ΔNp73 forms have a very important regulatory role, since they exert a dominant negative effect on p53 and TAp73 by blocking their transactivation activity, and hence their ability to induce apoptosis. The relative levels of expression of the ΔNp73 isoforms can therefore determine the function of both TAp73 and p53. It is most interesting that the ΔNp73 promoter (P2) contains a very efficient p53/p73 responsive element and consequently, p53 and TAp73 efficiently induce ΔNp73 expression. Moreover, upon strong DNA damage, induced by UV irradiation or drug treatment, ΔNp73 is rapidly degraded, releasing the block exerted on p53 and TAp73 and thus allowing cell cycle arrest and apoptosis to proceed. Hence, ΔNp73 is part of a dominant negative feedback loop that regulates the function of both p53 and TAp73 and this regulation can be overcome in case of strong DNA damage.; Les cellules d’un organisme subissent chaque jour des stress dus à l’environnement (rayons UV, agents chimiques, métaux lourds) pouvant conduire à des lésions du patrimoine génétique ou à un déséquilibre de l’état RedOx. De nombreux systèmes cellulaires permettent tout d’abord d’identifier le dommage puis d’induire éventuellement la réparation de l’ADN ou la mort de la cellule si le dommage subi est irréversible. Une cellule cancéreuse est le résultat d’échecs cumulés des systèmes de contrôle intra et extra-cellulaires et de mort programmée. Identifiée en 1979, la protéine p53 est un facteur de transcription muté dans 50% des cancers. Elle joue un rôle central dans la régulation de la prolifération cellulaire, de la réparation de l’ADN et de l’apoptose après stress, génotoxique ou non. Etonnamment, p53 semblait jouer seule ce rôle prépondérant qui lui a valu la dénomination de "gardienne du génome", et, pendant 20 ans, toutes tentatives pour caractériser d'éventuels homologues sont restées vaines. En 1997, p73, un homologue de p53, fut identifiée dans la bande p36 du chromosome 1, une région dont la délétion est souvent associée à de nombreux neuroblastomes. La caractérisation de p73 fut accueillie avec enthousiasme et sa grande homologie avec p53 semblait pouvoir expliquer les 50% de cancers présentant une p53 non mutée. L'année suivante, un deuxième homologue fut identifié et caractérisé: p63. Les trois membres de la famille p53 présentent une grande homologie, notamment dans le domaine de liaison à l'ADN : p73 et p63 sont en effet capables d'activer l'expression de nombreux gènes cibles de p53 et d'induire l'apoptose ou de bloquer le cycle cellulaire. Toutefois, 6 ans après leur découverte, p73 et p63 semblent de plus en plus différents de leur "parente" p53. La génération et l'étude de souris déficientes pour les membres de la famille illustrent ces différences : si les souris manquant p53 atteignent normalement l'âge adulte et développent spontanément des tumeurs, les souris manquant p73 ou p63 présentent de graves troubles du développement embryonnaire, indiquant un rôle majeur dans la différenciation cellulaire. Si p73 et p63 présentent une structure globale comparable à p53 (un domaine de transactivation, un domaine de liaison à l’ADN et un domaine d’oligomérisation, impliqué dans la tétramérisation de la protéine nécessaire à son activité transcriptionnelle), elles possèdent en effet un prolongement du domaine C-terminal, absent de la séquence de p53, et qui semble impliqué dans leurs propriétés propres. La maturation des transcrits de p73 et p63 donne lieu à différents splicing variants (6 pour p73 et au moins 3 pour p63) en C-terminal, dont les fonctions transcriptionnelles et le pattern d'expression sont différents. De plus, les formes les plus longues des deux protéines présentent un domaine SAM (Sterile Alpha Motif), commun à de nombreuses protéines impliquées dans le développement. Ce domaine est souvent muté dans des syndromes humains impliquant p63, ce qui laisse présager un rôle important dans la régulation de l'activité de p63 et p73. Par ailleurs, des formes tronquées en N-terminal ont été décrites dans la souris : ces formes sont nommées ΔΝ, elles ne possèdent pas le domaine de transactivation, au contraire des formes longues (TA). Ainsi, ΔΝp73 et ΔΝp63 agissent dans la souris comme dominants négatifs des fonctions pro-apoptotiques de TAp73 et TAp63. Il a été établi par la suite que ΔΝp73 et ΔΝp63 sont transcrites à partir d'un second promoteur, localisé dans le troisième intron de la forme longue. L'étude présentée ici décrit la première caractérisation de la forme ΔΝp73 humaine, la régulation de son expression et de son activité. De même que ΔΝp73 murine, la forme humaine inhibe les fonctions pro-apoptotiques de TAp73 et p53, via interactions protéines/protéines ou compétition pour les sites de liaison sur les promoteurs cibles. De plus, la présence d’un élément de réponse à p53 situé dans le promoteur de ΔΝp73 caractérise une boucle de régulation négative qui s’ajoute à la boucle de régulation MDM2/p53. ΔΝp73 agissant comme un oncogène, il semble donc que le ratio ΔΝ/TA ou ΔΝ/p53 soit fondamental à l’équilibre cellulaire et que sa dérégulation puisse être impliquée dans la formation tumorale, ce ratio pouvant être contrôlé au niveau transcriptionnel ou post-traductionel de la protéine ΔΝp73. L’étude du promoteur de ΔΝp73 a mis en évidence de nombreux éléments de réponse à différents facteurs de transcription. De récents travaux ayant associé une augmentation de ΔΝp73 à certains types de neuroblastomes, nous nous sommes particulièrement intéressés à N-Myc, facteur de transcription également amplifié dans certains neuroblastomes. Nous n’avons pu toutefois mettre en évidence une activation transcriptionnelle directe de ΔΝp73 de la part de N-Myc, mais d’autres éléments de réponse restent encore à caractériser, notamment NFκB et p300, tous deux impliqués dans la régulation de l’apoptose. Par ailleurs, l’étude de modifications post-traductionnelles de ΔΝp73 a mis en évidence une rapide dégradation de la protéine après dommages à l’ADN induits par rayons Ultra-Violets ou traitement par drogues, libérant ainsi p53 et TAp73 de son inhibition et permettant ainsi l’apoptose ou l’arrêt du cycle cellulaire. De plus, notre étude a mis en évidence une très brève hémie-vie de la forme ΔΝ par rapport aux formes contenant le domaine de transactivation. Ainsi, le ratio ΔΝ/formes longues pourrait également dépendre d’une fine régulation de la dégradation des deux protéines.