Regulation of SRC-3 localization and dynamics by phosphorylation during ERα-dependent transcriptional activation

Transcription is controlled by promoter-selective transcriptional factors (TFs), which bind to cis-regulatory enhancers elements, termed hormone response elements (HREs), in a specific subset of genes. Regulation by these factors involves either the recruitment of coactivators or corepressors and direct interaction with the basal transcriptional machinery (1). Hormone-activated nuclear receptors (NRs) are well characterized transcriptional factors (2) that bind to the promoters of their target genes and recruit primary and secondary coactivator proteins which possess many enzymatic activities required for gene expression (1,3,4). In the present study, using single-cell high-resolution fluorescent microscopy and high throughput microscopy (HTM) coupled to computational imaging analysis, we investigated transcriptional regulation controlled by the estrogen receptor alpha (ERalpha), in terms of large scale chromatin remodeling and interaction with the associated coactivator SRC-3 (Steroid Receptor Coactivator-3), a member of p160 family (28) primary coactivators. ERalpha is a steroid-dependent transcriptional factor (16) that belongs to the NRs superfamily (2,3) and, in response to the hormone 17-ß estradiol (E2), regulates transcription of distinct target genes involved in development, puberty, and homeostasis (8,16). ERalpha spends most of its lifetime in the nucleus and undergoes a rapid (within minutes) intranuclear redistribution following the addition of either agonist or antagonist (17,18,19). We designed a HeLa cell line (PRL-HeLa), engineered with a chromosomeintegrated reporter gene array (PRL-array) containing multicopy hormone response-binding elements for ERalpha that are derived from the physiological enhancer/promoter region of the prolactin gene. Following GFP-ER transfection of PRL-HeLa cells, we were able to observe in situ ligand dependent (i) recruitment to the array of the receptor and associated coregulators, (ii) chromatin remodeling, and (iii) direct transcriptional readout of the reporter gene. Addition of E2 causes a visible opening (decondensation) of the PRL-array, colocalization of RNA Polymerase II, and transcriptional readout of the reporter gene, detected by mRNA FISH. On the contrary, when cells were treated with an ERalpha antagonist (Tamoxifen or ICI), a dramatic condensation of the PRL-array was observed, displacement of RNA Polymerase II, and complete decreasing in the transcriptional FISH signal. All p160 family coactivators (28) colocalize with ERalpha at the PRL-array. Steroid Receptor Coactivator-3 (SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160 family member and a known oncogenic protein (4,34). SRC-3 is regulated by a variety of posttranslational modifications, including methylation, phosphorylation, acetylation, ubiquitination and sumoylation (4,35). These events have been shown to be important for its interaction with other coactivator proteins and NRs and for its oncogenic potential (37,39). A number of extracellular signaling molecules, like steroid hormones, growth factors and cytokines, induce SRC-3 phosphorylation (40). These actions are mediated by a wide range of kinases, including extracellular-regulated kinase 1 and 2 (ERK1-2), c-Jun N-terminal kinase, p38 MAPK, and IkB kinases (IKKs) (41,42,43). Here, we report SRC-3 to be a nucleocytoplasmic shuttling protein, whose cellular localization is regulated by phosphorylation and interaction with ERalpha. Using a combination of high throughput and fluorescence microscopy, we show that both chemical inhibition (with U0126) and siRNA downregulation of the MAP/ERK1/2 kinase (MEK1/2) pathway induce a cytoplasmic shift in SRC-3 localization, whereas stimulation by EGF signaling enhances its nuclear localization by inducing phosphorylation at T24, S857, and S860, known partecipants in the regulation of SRC-3 activity (39). Accordingly, the cytoplasmic localization of a non-phosphorylatable SRC-3 mutant further supports these results. In the presence of ERalpha, U0126 also dramatically reduces: hormone-dependent colocalization of ERalpha and SRC-3 in the nucleus; formation of ER-SRC-3 coimmunoprecipitation complex in cell lysates; localization of SRC-3 at the ER-targeted prolactin promoter array (PRL-array) and transcriptional activity. Finally, we show that SRC-3 can also function as a cotransporter, facilitating the nuclear-cytoplasmic shuttling of estrogen receptor. While a wealth of studies have revealed the molecular functions of NRs and coregulators, there is a paucity of data on how these functions are spatiotemporally organized in the cellular context. Technically and conceptually, our findings have a new impact upon evaluating gene transcriptional control and mechanisms of action of gene regulators.

Genotypic variation in Actinidia deliciosa fruit size and carbohydrate content

In a global and increasingly competitive fresh produce market, more attention is being given to fruit quality traits and consumer satisfaction. Kiwifruit occupies a niche position in the worldwide market, when compared to apples, oranges or bananas. It is a fruit with extraordinarily good nutritional traits, and its benefits to human health have been widely described. Until recently, international trade in kiwifruit was restricted to a single cultivar, but different types of kiwifruit are now becoming available in the market. Effective programmes of kiwifruit improvement start by considering the requirements of consumers, and recent surveys indicate that sweeter fruit with better flavour are generally preferred. There is a strong correlation between at-harvest dry matter and starch content, and soluble solid concentration and flavour when fruit are eating ripe. This suggests that carbon accumulation strongly influences the development of kiwifruit taste. The overall aim of the present study was to determine what factors affect carbon accumulation during Actinidia deliciosa berry development. One way of doing this is by comparing kiwifruit genotypes that differ greatly in their ability to accumulate dry matter in their fruit. Starch is the major component of dry matter content. It was hypothesized that genotypes were different in sink strength. Sink strength, by definition, is the effect of sink size and sink activity. Chapter 1 reviews fruit growth, kiwifruit growth and development and carbon metabolism. Chapter 2 describes the materials and methods used. Chapter 3, 4, 5 and 6 describes different types of experimental work. Chapter 7 contains the final discussions and the conclusions Three Actinidia deliciosa breeding populations were analysed in detail to confirm that observed differences in dry matter content were genetically determined. Fruit of the different genotypes differed in dry matter content mainly because of differences in starch concentrations and dry weight accumulation rates, irrespective of fruit size. More detailed experiments were therefore carried out on genotypes which varied most in fruit starch concentrations to determine why sink strengths were so different. The kiwifruit berry comprises three tissues which differ in dry matter content. It was initially hypothesised that observed differences in starch content could be due to a larger proportion of one or other of these tissues, for example, of the central core which is highest in dry matter content. The study results showed that this was not the case. Sink size, intended as cell number or cell size, was then investigated. The outer pericarp makes up about 60% of berry weight in ‘Hayward’ kiwifruit. The outer pericarp contains two types of parenchyma cells: large cells with low starch concentration, and small cells with high starch concentration. Large cell, small cell and total cell densities in the outer pericarp were shown to be not correlated with either dry matter content or fruit size but further investigation of volume proportion among cell types seemed justified. It was then shown that genotypes with fruit having higher dry matter contents also had a higher proportion of small cells. However, the higher proportion of small cell volume could only explain half of the observed differences in starch content. So, sink activity, intended as sucrose to starch metabolism, was investigated. In transiently starch storing sinks, such as tomato fruit and potato tubers, a pivotal role in carbon metabolism has been attributed to sucrose cleaving enzymes (mainly sucrose synthase and cell wall invertase) and to ADP-glucose pyrophosphorylase (the committed step in starch synthesis). Studies on tomato and potato genotypes differing in starch content or in final fruit soluble solid concentrations have demonstrated a strong link with either sucrose synthase or ADP-glucose pyrophosphorylase, at both enzyme activity and gene expression levels, depending on the case. Little is known about sucrose cleaving enzyme and ADP-glucose pyrophosphorylase isoforms. The HortResearch Actinidia EST database was then screened to identify sequences putatively encoding for sucrose synthase, invertase and ADP-glucose pyrophosphorylase isoforms and specific primers were designed. Sucrose synthase, invertase and ADP-glucose pyrophosphorylase isoform transcript levels were anlayzed throughout fruit development of a selection of four genotypes (two high dry matter and two low dry matter). High dry matter genotypes showed higher amounts of sucrose synthase transcripts (SUS1, SUS2 or both) and higher ADP-glucose pyrophosphorylase (AGPL4, large subunit 4) gene expression, mainly early in fruit development. SUS1- like gene expression has been linked with starch biosynthesis in several crop (tomato, potato and maize). An enhancement of its transcript level early in fruit development of high dry matter genotypes means that more activated glucose (UDP-glucose) is available for starch synthesis. This can be then correlated to the higher starch observed since soon after the onset of net starch accumulation. The higher expression level of AGPL4 observed in high dry matter genotypes suggests an involvement of this subunit in drive carbon flux into starch. Changes in both enzymes (SUSY and AGPse) are then responsible of higher starch concentrations. Low dry matter genotypes showed generally higher vacuolar invertase gene expression (and also enzyme activity), early in fruit development. This alternative cleavage strategy can possibly contribute to energy loss, in that invertases’ products are not adenylated, and further reactions and transport are needed to convert carbon into starch. Although these elements match well with observed differences in starch contents, other factors could be involved in carbon metabolism control. From the microarray experiment, in fact, several kinases and transcription factors have been found to be differentially expressed. Sink strength is known to be modified by application of regulators. In ‘Hayward’ kiwifruit, the synthetic cytokinin CPPU (N-(2-Chloro-4-Pyridyl)-N-Phenylurea) promotes a dramatic increase in fruit size, whereas dry matter content decreases. The behaviour of CPPU-treated ‘Hayward’ kiwifruit was similar to that of fruit from low dry matter genotypes: dry matter and starch concentrations were lower. However, the CPPU effect was strongly source limited, whereas in genotype variation it was not. Moreover, CPPU-treated fruit gene expression (at sucrose cleavage and AGPase levels) was similar to that in high dry matter genotypes. It was therefore concluded that CPPU promotes both sink size and sink activity, but at different “speeds” and this ends in the observed decrease in dry matter content and starch concentration. The lower “speed” in sink activity is probably due to a differential partitioning of activated glucose between starch storage and cell wall synthesis to sustain cell expansion. Starch is the main carbohydrate accumulated in growing Actinidia deliciosa fruit. Results obtained in the present study suggest that sucrose synthase and AGPase enzymes contribute to sucrose to starch conversion, and differences in their gene expression levels, mainly early in fruit development, strongly affect the rate at which starch is therefore accumulated. This results are interesting in that starch and Actinidia deliciosa fruit quality are tightly connected.

Identificazione delle Lamine di tipo A come nuovi substrati della protein chinasi Akt/PKB

Akt (also called PKB) is a 63 kDa serine/threonine kinase involved in promotion of cell survival, proliferation a nd metabolic responses downstream the phosphoinositide-3-kinase (PI 3-kinase) signaling pathway. In resting cells, Akt is a predominantly cytosolic enzyme; however generation of PI 3-kinase lipid products recruits Akt to the plasma membrane, resulting in a conformational change which confers full enzymatic activity through the phosphorylation of the membrane-bound protein at two residues, Thr308, and Ser473. Activated Akt redistributes to cytoplasm and nucleus, where phosphorylation of specific substrates occurs. Both the presence and the activity of Akt in the nucleus have been described. An interesting mechanism that mediates nuclear translocation of Akt has been described in human mature T-cell leukemia: the product of TCL1 gene, Tcl1, interacts with the PH domain of phosphorylated Akt, thus driving Akt to the nucleus. In this context, Tcl1 may act as a direct transporter of Akt or may contribute to the formation of a complex that promotes the transport of active Akt to the nucleus, where it can phosphorylate nuclear substrates. A well described nuclear substrate if Foxo. IGF-1 triggers phosphorylation of Foxo by Akt inside the nucleus, where phospho-Foxo associates to 14.3.3 proteins that, in turn, promote its export to the cytoplasm where it is sequestered. Remarkably, Foxo phosphorylation by Akt has been shown to be a crucial event in Akt-dependent myogenesis. However, most Akt nuclear substrates have so far remained elusive, as well as nuclear Akt functions. This lack of information prompted us to undertake a search of substrates of Akt in the nucleus, by the combined use of 2D-separation/mass spectrometry and anti-Akt-phosphosubstrate antibody. This study presents evidence of A-type lamins as novel nuclear substrates of Akt. Lamins are type V intermediate filaments proteins found in the nucleus of higher eukaryotes where, together with lamin-binding proteins, they form the lamina at the nuclear envelope, providing mechanical stability for the nuclear membrane. By coimmunoprecipitation, it is demonstrated here that endogenous lamin A and Akt interact, and that A-type lamins are phosphorylated by Akt both in vitro and in vivo. Moreover, by phosphoaminoacid analysis and mutagenesis, it is further demonstrated that Akt phosphorylates lamin A at Ser404, and, more importantly, that while lamin A/C phosphorylation is stable throughout the cell cycle, phosphorylation of the precursor prelamin A becomes detectable as cells enter the G2 phase, picking at G2/M. This study also shows that lamin phosphorylation by Akt creates a binding site for 14.3.3 adaptors which, in turn, promote prelamin A degradation. While this mechanism is in agreement with a general role of Akt in the regulation of a subset of its substrates, opposite to what has been described, degradation is not mediated through a ubiquitination and proteasomal mechanism but through a lysosomal pathway, as indicated by the reverting action of the lysosomal inhibitor cloroquine. Phosphorylation is a key event in the mitotic breakdown of the nuclear lamina. However, the kinases and the precise sites of phosphorylation are scarcely known. Therefore, these results represent an important breakthrough in this very significant but understudied area. The phosphorylation of the precursor protein prelamin A and its subsequent degradation at G2/M, when both the nuclear envelop and the nuclear lamina disassemble, can be view as part of a mechanism to dispose off the precursor that is not needed in this precise context. The recently reported finding that patients affected by Emery-Dreifuss muscular dystrophy carry a mutation at Arg 401, in the Akt phosphorylation motif, open new perspective that warrant further investigation in this very important field.

Mitochondrial dysfunction in a cell model of thyroid oncocytoma

The role of mitochondrial dysfunction in cancer has long been a subject of great interest. In this study, such dysfunction has been examined with regards to thyroid oncocytoma, a rare form of cancer, accounting for less than 5% of all thyroid cancers. A peculiar characteristic of thyroid oncocytic cells is the presence of an abnormally large number of mitochondria in the cytoplasm. Such mitochondrial hyperplasia has also been observed in cells derived from patients suffering from mitochondrial encephalomyopathies, where mutations in the mitochondrial DNA(mtDNA) encoding the respiratory complexes result in oxidative phosphorylation dysfunction. An increase in the number of mitochondria occurs in the latter in order to compensate for the respiratory deficiency. This fact spurred the investigation into the presence of analogous mutations in thyroid oncocytic cells. In this study, the only available cell model of thyroid oncocytoma was utilised, the XTC-1 cell line, established from an oncocytic thyroid metastasis to the breast. In order to assess the energetic efficiency of these cells, they were incubated in a medium lacking glucose and supplemented instead with galactose. When subjected to such conditions, glycolysis is effectively inhibited and the cells are forced to use the mitochondria for energy production. Cell viability experiments revealed that XTC-1 cells were unable to survive in galactose medium. This was in marked contrast to the TPC-1 control cell line, a thyroid tumour cell line which does not display the oncocytic phenotype. In agreement with these findings, subsequent experiments assessing the levels of cellular ATP over incubation time in galactose medium, showed a drastic and continual decrease in ATP levels only in the XTC-1 cell line. Furthermore, experiments on digitonin-permeabilised cells revealed that the respiratory dysfunction in the latter was due to a defect in complex I of the respiratory chain. Subsequent experiments using cybrids demonstrated that this defect could be attributed to the mitochondrially-encoded subunits of complex I as opposed to the nuclearencoded subunits. Confirmation came with mtDNA sequencing, which detected the presence of a novel mutation in the ND1 subunit of complex I. In addition, a mutation in the cytochrome b subunit of complex III of the respiratory chain was detected. The fact that XTC-1 cells are unable to survive when incubated in galactose medium is consistent with the fact that many cancers are largely dependent on glycolysis for energy production. Indeed, numerous studies have shown that glycolytic inhibitors are able to induce apoptosis in various cancer cell lines. Subsequent experiments were therefore performed in order to identify the mode of XTC-1 cell death when subjected to the metabolic stress imposed by the forced use of the mitochondria for energy production. Cell shrinkage and mitochondrial fragmentation were observed in the dying cells, which would indicate an apoptotic type of cell death. Analysis of additional parameters however revealed a lack of both DNA fragmentation and caspase activation, thus excluding a classical apoptotic type of cell death. Interestingly, cleavage of the actin component of the cytoskeleton was observed, implicating the action of proteases in this mode of cell demise. However, experiments employing protease inhibitors failed to identify the specific protease involved. It has been reported in the literature that overexpression of Bcl-2 is able to rescue cells presenting a respiratory deficiency. As the XTC-1 cell line is not only respiration-deficient but also exhibits a marked decrease in Bcl-2 expression, it is a perfect model with which to study the relationship between Bcl-2 and oxidative phosphorylation in respiratory-deficient cells. Contrary to the reported literature studies on various cell lines harbouring defects in the respiratory chain, Bcl-2 overexpression was not shown to increase cell survival or rescue the energetic dysfunction in XTC-1 cells. Interestingly however, it had a noticeable impact on cell adhesion and morphology. Whereas XTC-1 cells shrank and detached from the growth surface under conditions of metabolic stress, Bcl-2-overexpressing XTC-1 cells appeared much healthier and were up to 45% more adherent. The target of Bcl-2 in this setting appeared to be the actin cytoskeleton, as the cleavage observed in XTC-1 cells expressing only endogenous levels of Bcl-2, was inhibited in Bcl-2-overexpressing cells. Thus, although unable to rescue XTC-1 cells in terms of cell viability, Bcl-2 is somehow able to stabilise the cytoskeleton, resulting in modifications in cell morphology and adhesion. The mitochondrial respiratory deficiency observed in cancer cells is thought not only to cause an increased dependency on glycolysis but it is also thought to blunt cellular responses to anticancer agents. The effects of several therapeutic agents were thus assessed for their death-inducing ability in XTC-1 cells. Cell viability experiments clearly showed that the cells were more resistant to stimuli which generate reactive oxygen species (tert-butylhydroperoxide) and to mitochondrial calcium-mediated apoptotic stimuli (C6-ceramide), as opposed to stimuli inflicting DNA damage (cisplatin) and damage to protein kinases(staurosporine). Various studies in the literature have reported that the peroxisome proliferator-activated receptor-coactivator 1(PGC-1α), which plays a fundamental role in mitochondrial biogenesis, is also involved in protecting cells against apoptosis caused by the former two types of stimuli. In accordance with these observations, real-time PCR experiments showed that XTC-1 cells express higher mRNA levels of this coactivator than do the control cells, implicating its importance in drug resistance. In conclusion, this study has revealed that XTC-1 cells, like many cancer cell lines, are characterised by a reduced energetic efficiency due to mitochondrial dysfunction. Said dysfunction has been attributed to mutations in respiratory genes encoded by the mitochondrial genome. Although the mechanism of cell demise in conditions of metabolic stress is unclear, the potential of targeting thyroid oncocytic cancers using glycolytic inhibitors has been illustrated. In addition, the discovery of mtDNA mutations in XTC-1 cells has enabled the use of this cell line as a model with which to study the relationship between Bcl-2 overexpression and oxidative phosphorylation in cells harbouring mtDNA mutations and also to investigate the significance of such mutations in establishing resistance to apoptotic stimuli.

Trasduzione del segnale e poliamine nell'apoptosi di cellule cardiache

Introduction: Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy and heart failure, thus representing a potential therapeutic target. Apoptosis of cardiac cells can be induced experimentally by several stimuli including hypoxia, serum withdrawal or combination of both. Several lines of research suggest that neurohormonal mechanisms play a central role in the progression of heart failure. In particular, excessive activation of the sympathetic nervous system or the renin-angiotensin-aldosterone system is known to have deleterious effects on the heart. Recent studies report that norepinephrine (NE), the primary transmitter of sympathetic nervous system, and aldosterone (ALD), which is actively produced in failing human heart, are able to induce apoptosis of rat cardiomyocytes. Polyamines are biogenic amines involved in many cellular processes, including apoptosis. Actually it appears that these molecules can act as promoting, modulating or protective agents in apoptosis depending on apoptotic stimulus and cellular model. We have studied the involvement of polyamines in the apoptosis of cardiac cells induced in a model of simulated ischemia and following treatment with NE or ALD. Methods: H9c2 cardiomyoblasts were exposed to a condition of simulated ischemia, consisting of hypoxia plus serum deprivation. Cardiomyocyte cultures were prepared from 1-3 day-old neonatal Wistar rat hearts. Polyamine depletion was obtained by culturing the cells in the presence of α-difluoromethylornithine (DFMO). Polyamines were separated and quantified in acidic cellular extracts by HPLC after derivatization with dansyl chloride. Caspase activity was measured by the cleavage of the fluorogenic peptide substrate. Ornithine decarboxylase (ODC) activity was measured by estimation of the release of 14C-CO2 from 14C-ornithine. DNA fragmentation was visualized by the method of terminal transferase-mediated dUTP nick end-labeling (TUNEL), and DNA laddering on agarose gel electophoresis. Cytochrome c was detected by immunoflorescent staining. Activation of signal transduction pathways was investigated by western blotting. Results: The results indicate that simulated ischemia, NE and ALD cause an early induction of the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, followed by a later increase of caspase activity, a family of proteases that execute the death program and induce cell death. This effect was prevented in the presence of DFMO, an irreversible inhibitor of ODC, thus suggesting that polyamines are involved in the execution of the death program activated by these stimuli. In H9c2 cells DFMO inhibits several molecular events related to apoptosis that follow simulated ischemia, such as the release of cytochrome c from mitochondria, down-regulation of Bcl-xL, and DNA fragmentation. The anti-apoptotic protein survivin is down-regulated after ALD or NE treatement and polyamine depletion obtained by DFMO partially opposes survivin decrease. Moreover, a study of key signal transduction pathways governing cell death and survival, revealed an involvement of AMP activated protein kinase (AMPK) and AKT kinase, in the modulation by polyamines of the response of cardiomyocytes to NE. In fact polyamine depleted cells show an altered pattern of AMPK and AKT activation that may contrast apoptosis and appears to result from a differential effect on the specific phosphatases that dephosphorylate and switch off these signaling proteins. Conclusions: These results indicate that polyamines are involved in the execution of the death program activated in cardiac cells by heart failure-related stimuli, like ischemia, ALD and NE, and suggest that their apoptosis facilitating action is mediated by a network of specific phosphatases and kinases.

Transcriptional functions of DNA Topoisomerases at a genome-wide scale and a single gene levels

The DNA topology is an important modifier of DNA functions. Torsional stress is generated when right handed DNA is either over- or underwound, producing structural deformations which drive or are driven by processes such as replication, transcription, recombination and repair. DNA topoisomerases are molecular machines that regulate the topological state of the DNA in the cell. These enzymes accomplish this task by either passing one strand of the DNA through a break in the opposing strand or by passing a region of the duplex from the same or a different molecule through a double-stranded cut generated in the DNA. Because of their ability to cut one or two strands of DNA they are also target for some of the most successful anticancer drugs used in standard combination therapies of human cancers. An effective anticancer drug is Camptothecin (CPT) that specifically targets DNA topoisomerase 1 (TOP 1). The research project of the present thesis has been focused on the role of human TOP 1 during transcription and on the transcriptional consequences associated with TOP 1 inhibition by CPT in human cell lines. Previous findings demonstrate that TOP 1 inhibition by CPT perturbs RNA polymerase (RNAP II) density at promoters and along transcribed genes suggesting an involvement of TOP 1 in RNAP II promoter proximal pausing site. Within the transcription cycle, promoter pausing is a fundamental step the importance of which has been well established as a means of coupling elongation to RNA maturation. By measuring nascent RNA transcripts bound to chromatin, we demonstrated that TOP 1 inhibition by CPT can enhance RNAP II escape from promoter proximal pausing site of the human Hypoxia Inducible Factor 1 (HIF-1) and c-MYC genes in a dose dependent manner. This effect is dependent from Cdk7/Cdk9 activities since it can be reversed by the kinases inhibitor DRB. Since CPT affects RNAP II by promoting the hyperphosphorylation of its Rpb1 subunit the findings suggest that TOP 1inhibition by CPT may increase the activity of Cdks which in turn phosphorylate the Rpb1 subunit of RNAP II enhancing its escape from pausing. Interestingly, the transcriptional consequences of CPT induced topological stress are wider than expected. CPT increased co-transcriptional splicing of exon1 and 2 and markedly affected alternative splicing at exon 11. Surprisingly despite its well-established transcription inhibitory activity, CPT can trigger the production of a novel long RNA (5’aHIF-1) antisense to the human HIF-1 mRNA and a known antisense RNA at the 3’ end of the gene, while decreasing mRNA levels. The effects require TOP 1 and are independent from CPT induced DNA damage. Thus, when the supercoiling imbalance promoted by CPT occurs at promoter, it may trigger deregulation of the RNAP II pausing, increased chromatin accessibility and activation/derepression of antisense transcripts in a Cdks dependent manner. A changed balance of antisense transcripts and mRNAs may regulate the activity of HIF-1 and contribute to the control of tumor progression After focusing our TOP 1 investigations at a single gene level, we have extended the study to the whole genome by developing the “Topo-Seq” approach which generates a map of genome-wide distribution of sites of TOP 1 activity sites in human cells. The preliminary data revealed that TOP 1 preferentially localizes at intragenic regions and in particular at 5’ and 3’ ends of genes. Surprisingly upon TOP 1 downregulation, which impairs protein expression by 80%, TOP 1 molecules are mostly localized around 3’ ends of genes, thus suggesting that its activity is essential at these regions and can be compensate at 5’ ends. The developed procedure is a pioneer tool for the detection of TOP 1 cleavage sites across the genome and can open the way to further investigations of the enzyme roles in different nuclear processes.

Ruolo dei recettori tirosinchinasici in oncologia animale

Disregolazioni dei recettori tirosinchinasici (RTK) sono di frequente riscontro nei tumori dell’uomo e in molti casi sono indicatori biologici che permettono di definire in maniera più accurata la prognosi dei pazienti. Possono rappresentare inoltre marker predittivi per la risposta a terapie antitumorali con farmaci a bersaglio molecolare. Numerosi inibitori tirosinchinasici (TKI) sono attualmente in corso di studio o già disponibili per l’utilizzo in oncologia umana, e molti di questi hanno dimostrato una significativa efficacia utilizzati singolarmente o in combinazione a terapie convenzionali. Studi recenti indicano che un quadro analogo di disregolazione dei recettori tirosinchinasici è presente anche nelle neoplasie dei piccoli animali, e ne suggeriscono in molti casi un’implicazione prognostica. Gli inibitori tirosinchinasi sono da poco entrati nell’arena dell’oncologia veterinaria, ma i primi risultati lasciano supporre che siano destinati ad essere integrati definitivamente nei protocolli terapeutici standard. La tesi consiste in una parte introduttiva in cui sono trattate le principali funzioni biologiche dei recettori tirosinchinasici, la loro struttura e il loro ruolo nell’oncogenesi e nella progressione tumorale in medicina umana e veterinaria. Si affrontano inoltre le principali metodiche di laboratorio per l’analisi molecolare in oncologia e i meccanismi d’azione dei farmaci inibitori tirosinchinasici, con un cenno ai prodotti maggiormente utilizzati e alle loro indicazioni. Segue la presentazione e la discussione dei risultati di quattro studi relativi alla valutazione delle disregolazioni del recettore tirosinchinasico Kit (espressione aberrante e mutazioni genomiche) nel mastocitoma cutaneo del gatto e del recettore del fattore di crescita epidermico (EGFR) nel carcinoma squamocellulare cutaneo del gatto e nei tumori polmonari primitivi del cane, con particolare attenzione al loro ruolo prognostico.

Analisi preclinica di nuove strategie terapeutiche basate sull'inibizione di protein-chinasi nell'osteosarcoma.

Scopo: L’obiettivo del presente programma di studio è stato quello di identificare e validare nuovi possibili bersagli terapeutici per l’osteosarcoma (OS) partendo dall’analisi del chinoma umano. Risultati: L’analisi del profilo di espressione genica ottenuta su 21 campioni clinici di OS ad alto grado di malignità ha permesso di selezionare le seguenti chinasi di possibile rilevanza biologica per l’OS: AURK-A, AURK-B, CDK2, PIK3CA, PLK-1. Le chinasi selezionate sono state validate tramite RNA interference. Successivamente è stata valutata l’efficacia dei relativi inibitori specifici: VX-680 e ZM-447439 inibitori delle Aurora-chinasi, Roscovitina di CDK2 e NMS1 di PLK-1, già inclusi in studi clinici. In termini d’inibizione della crescita cellulare le linee sono risultate maggiomente sensibili ai farmaci VX-680 e NMS1. E’ stata osservata una minor sensibilità ai farmaci VX-680, ZM447439 e NMS1 nelle linee doxorubicina(DX)-resistenti (caratterizzate da elevati livelli di espressione di ABCB1), indicando questi farmaci come potenziali substrati di ABCB1. La Roscovitina, nonostante i valori di IC50 elevati, non sembrerebbe substrato di ABCB1. La validazione preclinica di VX-680 e ZM447439 è stata completata. La forte inibizione della crescita è causata da endoreduplicazione per mancata citodieresi con conseguente formazione di una popolazione iperploide e apoptosi. Inoltre, VX-680 inibisce la motilità e la capacità di formare colonie. Esperimenti di associazione farmacologica mostrano che VX-680 interagisce positivamente con tutti i chemioterapici convenzionali impiegati nel trattamento dell’OS. NMS-1 produce interazioni positive con la DX in linee cellulari DX-resistenti, probabilmente grazie all’effetto revertante esercitato su ABCB1. La Roscovitina produce interazioni positive con CDDP e DX nelle varianti resistenti, effetto probbilmente dovuto al ruolo di CDK2 nei meccanismi di riparo del DNA. Conclusioni: L’analisi in vitro dell’attività degli inibitori ha permesso di identificare VX-680 come nuovo farmaco di potenziale interesse clinico, soprattutto in virtù delle sue interazioni sinergiche con i chemioterapici di uso convenzionale nel trattamento dell’osteosarcoma.

Airway Basal Cell Vascular Endothelial Growth Factor-mediated Cross-Talk Regulates Endothelial Cell Dependent Growth Support of Human Airway Basal Cells

The human airway epithelium is a pseudostratified heterogenous layer comprised of cili-ated, secretory, intermediate and basal cells. As the stem/progenitor population of the airway epi-thelium, airway basal cells differentiate into ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. Transcriptome analysis of airway basal cells revealed high expression of vascular endothelial growth factor A (VEGFA), a gene not typically associated with the function of this cell type. Using cultures of primary human airway basal cells, we demonstrate that basal cells express all of the 3 major isoforms of VEGFA (121, 165 and 189) but lack functional expression of the classical VEGFA receptors VEGFR1 and VEGFR2. The VEGFA is actively secreted by basal cells and while it appears to have no direct autocrine function on basal cell growth and proliferation, it functions in a paracrine manner to activate MAPK signaling cascades in endothelium via VEGFR2 dependent signaling pathways. Using a cytokine- and serum-free co-culture system of primary human airway basal cells and human endothelial cells revealed that basal cell secreted VEGFA activated endothelium to ex-press mediators that, in turn, stimulate and support basal cell proliferation and growth. These data demonstrate novel VEGFA mediated cross-talk between airway basal cells and endothe-lium, the purpose of which is to modulate endothelial activation and in turn stimulate and sustain basal cell growth.

Attenuation of Eph Receptor Kinase Activation in Cancer Cells by Coexpressed Ephrin Ligands

The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting “in trans” with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral “cis” associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis-inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans (Falivelli et al. 2013).

The constitutive activation of the DNA damage response pathway is a novel therapeutic target in aggressive B-cell lymphoma

The recent finding that MYC-driven cancers are sensitive to inhibition of the DNA damage response (DDR) pathway, prompted us to investigate the role of DDR pathway as therapeutic target in diffuse large B-cell lymphoma (DLBCL), which frequently overexpresses the MYC oncogene. In a preliminary immunohistochemical study conducted on 99 consecutive DLBCL patients, we found that about half of DLBCLs showed constitutive expression of the phosphorylated forms of checkpoint kinases (CHK) and CDC25c, markers of DDR activation, and of phosphorylated histone H2AX (γH2AX), marker of DNA damage and genomic instability. Constitutive γH2AX expression correlated with c-MYC levels and DDR activation, and defined a subset of tumors characterised by poor outcome. Next, we used the CHK inhibitor PF-0477736 as a tool to investigate whether the inhibition of the DDR pathway might represent a novel therapeutic approach in DLBCL. Submicromolar concentrations of PF-0477736 hindered proliferation in DLBCL cell lines with activated DDR pathway. These results were fully recapitulated with a different CHK inhibitor (AZD-7762). Inhibition of checkpoint kinases induced rapid DNA damage accumulation and apoptosis in DLBCL cell lines and primary cells. These data suggest that pharmacologic inhibition of DDR through targeting of CHK kinases may represent a novel therapeutic strategy in DLBCL. The second part of this work is the clinical, molecular and functional description of a paradigmatic case of primary refractory Burkitt lymphoma characterized by spatial intratumor heterogeneity for the TP53 mutational status, high expression levels of genomic instability and DDR activation markers, primary resistance to chemotherapy and exquisite sensitivity to DDR inhibitors.

Caratterizzazione e ruolo di PKCε e PKCδ in modelli di differenziamento megacariocitario normale e patologico

La PKCε e la PKCδ, chinasi ubiquitariamente distribuite e ad azione pleiotropica, sono implicate del differenziamento, sopravvivenza e proliferazione cellulare. Esse sono coinvolte nel processo differenziativo delle cellule staminali ematopoietiche e in fenomeni patologici associati al compartimento sanguigno. In questa tesi sono presentati i risultati riguardanti lo studio in vitro del ruolo di PKCε e PKCδ nel contesto del differenziamento megacariocitario, in particolare si caratterizza l’espressione e la funzione di queste chinasi nel modello umano e nel modello murino di Megacariocitopoiesi, normale e patologica. Confrontando le cinetiche dei due modelli presi in analisi nello studio è stato possibile osservare come in entrambi PKCε e PKCδ dimostrino avere una chiara e specifica modulazione nel progredire del processo differenziativo. Questi dati, se confrontati, permettono di affermare che PKCε e PKCδ presentano un pattern di espressione opposto e, nel modello umano rispetto a quello murino, reciproco: nell’uomo i livelli di PKCε devono essere down-modulati, mentre nel topo, al contrario, i livelli della chinasi risultano up-modulati durante lo stesso processo. Analogamente, le CD34+ in differenziazione presentano una costante e maggiore espressione di PKCδ durante la maturazione MK, mentre nel modello murino tale proteina risulta down-modulata nella fase più tardiva di formazione della piastrina. Le chinasi mostrano in oltre di agire, nei due modelli, attraverso pathways distinti e cioè RhoA nel topo e Bcl-xL nell’uomo. È stato inoltre verificato che l’aberrante differenziamento MK osservato nella mielofibrosi primaria (PMF), è associato a difetti di espressione di PKCε e di Bcl-xL e che una forzata down-modulazione di PKCε porta ad un ripristino di un normale livello di espressione di Bcl-xL così come della popolazione di megacariociti formanti propiastrine. I dati ottenuti indicano quindi che PKCε e PKCδ svolgono un ruolo importante nel corretto differenziamento MK e che PKCε potrebbe essere un potenziale nuovo target terapeutico nelle PMF.

Study of PI3K/Akt signaling pathway as potential molecular target for T-cell acute lymphoblastic leukemia (T-ALL) treatment: pan-inhibition of PI3K catalitic isoforms as better therapeutic approach

Class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases consisting of a regulatory subunit and one of four catalytic subunits (p110α, p110β, p110γ or p110δ). p110γ/p110δ PI3Ks are highly enriched in leukocytes. In general, PI3Ks regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Their activity is tightly regulated by the phosphatase and tensin homolog (PTEN) lipid phosphatase. PI3Ks are widely implicated in human cancers, and in particular are upregulated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to loss of PTEN function. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. At present different compounds which target single or multiple PI3K isoforms have entered clinical trials. In the present research, it has been analyzed the therapeutic potential of the pan-PI3K inhibitor BKM120, an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T-lymphoblasts. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. BKM120 efficacy was confirmed in in vivo studies to a subcutaneous xenotransplant model of human T-ALL. Because it is still unclear which agents among isoform-specific or pan inhibitors can achieve the greater efficacy, further analyses have been conducted to investigate the effects of PI3K inhibition, in order to elucidate the mechanisms responsible for the proliferative impairment of T-ALL. Overall, these results indicated that BKM120 may be an efficient treatment for T-ALLs that have aberrant up-regulation of the PI3K signaling pathway and strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in T-ALL treatment.

Identification of novel genetic alterations in pediatric cytogenetically normal acute myeloid leukemia by next-generation sequencing

Pediatric acute myeloid leukemia (AML) is a molecularly heterogeneous disease that arises from genetic alterations in pathways that regulate self-renewal and myeloid differentiation. While the majority of patients carry recurrent chromosomal translocations, almost 20% of childhood AML do not show any recognizable cytogenetic alteration and are defined as cytogenetically normal (CN)-AML. CN-AML patients have always showed a great variability in response to therapy and overall outcome, underlining the presence of unknown genetic changes, not detectable by conventional analyses, but relevant for pathogenesis, and outcome of AML. The development of novel genome-wide techniques such as next-generation sequencing, have tremendously improved our ability to interrogate the cancer genome. Based on this background, the aim of this research study was to investigate the mutational landscape of pediatric CN-AML patients negative for all the currently known somatic mutations reported in AML through whole-transcriptome sequencing (RNA-seq). RNA-seq performed on diagnostic leukemic blasts from 19 pediatric CN-AML cases revealed a considerable incidence of cryptic chromosomal rearrangements, with the identification of 21 putative fusion genes. Several of the fusion genes that were identified in this study are recurrent and might have a prognostic and/or therapeutic relevance. A paradigm of that is the CBFA2T3-GLIS2 fusion, which has been demonstrated to be a common alteration in pediatric CN-AML, predicting poor outcome. Important findings have been also obtained in the identification of novel therapeutic targets. On one side, the identification of NUP98-JARID1A fusion suggests the use of disulfiram; on the other, here we describe alteration-activating tyrosine kinases, providing functional data supporting the use of tyrosine kinase inhibitors to specifically inhibit leukemia cells. This study provides new insights in the knowledge of genetic alterations underlying pediatric AML, defines novel prognostic markers and putative therapeutic targets, and prospectively ensures a correct risk stratification and risk-adapted therapy also for the “all-neg” AML subgroup.