Ribosome Biogenesis and cell cycle regulation: Effect of RNA Polymerase III inhibition

In cycling cells positive stimuli like nutrient, growth factors and mitogens increase ribosome biogenesis rate and protein synthesis to ensure both growth and proliferation. In contrast, under stress situation, proliferating cells negatively modulate ribosome production to reduce protein synthesis and block cell cycle progression. The main strategy used by cycling cell to coordinate cell proliferation and ribosome biogenesis is to share regulatory elements, which participate directly in ribosome production and in cell cycle regulation. In fact, there is evidence that stimulation or inhibition of cell proliferation exerts direct effect on activity of the RNA polymerases controlling the ribosome biogenesis, while several alterations in normal ribosome biogenesis cause changes of the expression and the activity of the tumor suppressor p53, the main effector of cell cycle progression inhibition. The available data on the cross-talk between ribosome biogenesis and cell proliferation have been until now obtained in experimental model in which changes in ribosome biogenesis were obtained either by reducing the activity of the RNA polymerase I or by down-regulating the expression of the ribosomal proteins. The molecular pathways involved in the relationship between the effect of the inhibition of RNA polymerase III (Pol III) activity and cell cycle progression have been not yet investigated. In eukaryotes, RNA Polymerase III is responsible for transcription of factors involved both in ribosome assembly (5S rRNA) and rRNA processing (RNAse P and MRP).Thus, the aim of this study is characterize the effects of the down-regulation of RNA Polymerase III activity, or the specific depletion of 5S rRNA. The results that will be obtained might lead to a deeper understanding of the molecular pathway that controls the coordination between ribosome biogenesis and cell cycle, and might give useful information about the possibility to target RNA Polymerase III for cancer treatment.

New DAG-dependent mechanisms modulate cell cycle progression

Through the years, several studies reported the involvement of nuclear lipid signalling as highly connected with cell cycle progression. Indeed, nuclear Phosphatidylinositol-4,5-Biphosphate (PIP2) hydrolisis mediated by Phospholipases C (PLC), which leads to production of the second messengers Diacylglycerol (DAG) and Inositol-1,4,5-Triphosphate (IP3), is a fundamental event for both G1/S and G2/M checkpoints. In particular, we found that nuclear DAG production was mediated by PLCbeta1, enzyme mainly localized in the nucleus of K562 human erythroleukemia cells. This event triggered the activation and nuclear translocation of PKCalpha, which, in turn, resulted able to affect cell cycle via modulation of Cyclin D3 and Cyclin B1, two important enzymes for G1/S transition and G2/M progression respectively.

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.

Inibizione selettiva del gene MYCN mediante PNA (acidi peptido nucleici) anti-gene nel rabdomiosarcoma umano

MYCN oncogene amplification/expression is a feature of many childhood tumors, and some adult tumors, and it is associated with poor prognosis. While MYC expression is ubiquitary, MYCN has a restricted expression after birth and it is an ideal target for an effective therapy. PNAs belong to the latest class of nucleic acid-based therapeutics, and they can bind chromosomal DNA and block gene transcription (anti-gene activity). We have developed an anti-gene PNA that targets specifically the MYCN gene to block its transcription. We report for the first time MYCN targeted inhibition in Rhabdomyosarcoma (RMS) by the anti-MYCN-PNA in RMS cell lines (four ARMS and four ERMS) and in a xenograft RMS mouse model. Rhabdomyosarcoma is the most common pediatric soft-tissue sarcoma, comprising two main subgroups [Alveolar (ARMS) and Embryonal (ERMS)]. ARMS is associated with a poorer prognosis. MYCN amplification is a feature of both the ERMS and ARMS, but the MYCN amplification and expression levels shows a significant correlation and are greater in ARMS, in which they are associated with adverse outcome. We found that MYCN mRNA and protein levels were higher in the four ARMS (RH30, RH4, RH28 and RMZ-RC2) than in the four ERMS (RH36, SMS-CTR, CCA and RD) cell lines. The potent inhibition of MYCN transcription was highly specific, it did not affect the MYC expression, it was followed by cell-growth inhibition in the RMS cell lines which correlated with the MYCN expression rate, and it led to complete cell-growth inhibition in ARMS cells. We used a mutated- PNA as control. MYCN silencing induced apoptosis. Global gene expression analysis (Affymetrix microarrays) in ARMS cells treated with the anti-MYCN-PNA revealed genes specifically induced or repressed, with both genes previously described as targets of N-myc or Myc, and new genes undescribed as targets of N-myc or Myc (mainly involved in cell cycle, apoptosis, cell motility, metastasis, angiogenesis and muscle development). The changes in the expression of the most relevant genes were confirmed by Real-Time PCR and western blot, and their expression after the MYCN silencing was evaluated in the other RMS cell lines. The in vivo study, using an ARMS xenograft murine model evaluated by micro-PET, showed a complete elimination of the metabolic tumor signal in most of the cases (70%) after anti-MYCN-PNA treatment (without toxicity), whereas treatment with the mutated-PNA had no effect. Our results strongly support the development of MYCN anti-gene therapy for the treatment of RMS, particularly for poor prognosis ARMS, and of other MYCN-expressing tumors.

Ruolo dell'Enantiomero (R)-9-HSA nel controllo della proliferazione in una linea di Adenocarcinoma del Colon umano

9-hydroxystearic acid (9-HSA) is an endogenous lipoperoxidation product and its administration to HT29, a colon adenocarcinoma cell line, induced a proliferative arrest in G0/G1 phase mediated by a direct activation of the p21WAF1 gene, bypassing p53. We have previously shown that 9-HSA controls cell growth and differentiation by inhibiting histone deacetylase 1 (HDAC1) activity, showing interesting features as a new anticancer drug. The interaction of 9-HSA with the catalytic site of the 3D model has been tested with a docking procedure: noticeably, when interacting with the site, the (R)-9-enantiomer is more stable than the (S) one. Thus, in this study, (R)- and (S)-9-HSA were synthesized and their biological activity tested in HT29 cells. At the concentration of 50 M (R)-9-HSA showed a stronger antiproliferative effect than the (S) isomer, as indicated by the growth arrest in G0/G1. The inhibitory effect of (S)-9-HSA on HDAC1, HDAC2 and HDAC3 activity was less effective than that of the (R)-9-HSA in vitro, and the inhibitory activity of both the (R)- and the (S)-9-HSA isomer, was higher on HDAC1 compared to HDAC2 and HDAC3, thus demonstrating the stereospecific and selective interaction of 9-HSA with HDAC1. In addition, histone hyperacetylation caused by 9-HSA treatment was examined by an innovative HPLC/ESI/MS method. Analysis on histones isolated from control and treated HT29 confirmed the higher potency of (R)-9-HSA compared to (S)-9-HSA, severely affecting H2A-2 and H4 acetylation. On the other side, it seemed of interest to determine whether the G0/G1 arrest of HT29 cell proliferation could be bypassed by the stimulation with the growth factor EGF. Our results showed that 9-HSA-treated cells were not only prevented from proliferating, but also showed a decreased [3H]thymidine incorporation after EGF stimulation. In this condition, HT29 cells expressed very low levels of cyclin D1, that didn’t colocalize with HDAC1. These results suggested that the cyclin D1/HDAC1 complex is required for proliferation. Furthermore, in the effort of understanding the possible mechanisms of this effect, we have analyzed the degree of internalization of the EGF/EGFR complex and its interactions with HDAC1. EGF/EGFR/HDAC1 complex quantitatively increases in 9-HSA-treated cells but not in serum starved cells after EGF stimulation. Our data suggested that 9-HSA interaction with the catalytic site of the HDAC1 disrupts the HDAC1/cyclin D1 complex and favors EGF/EGFR recruitment by HDAC1, thus enhancing 9-HSA antiproliferative effects. In conclusion 9-HSA is a promising HDAC inhibitor with high selectivity and specificity, capable of inducing cell cycle arrest and histone hyperacetylation, but also able to modulate HDAC1 protein interaction. All these aspects may contribute to the potency of this new antitumor agent.

Myc-mediated control of gene transcription in cancer cells

The Myc oncoproteins belong to a family of transcription factors composed by Myc, N-Myc and L-Myc. The most studied components of this family are Myc and N-Myc because their expressions are frequently deregulated in a wide range of cancers. These oncoproteins can act both as activators or repressors of gene transcription. As activators, they heterodimerize with Max (Myc associated X-factor) and the heterodimer recognizes and binds a specific sequence elements (E-Box) onto gene promoters recruiting histone acetylase and inducing transcriptional activation. Myc-mediated transcriptional repression is a quite debated issue. One of the first mechanisms defined for the Myc-mediated transcriptional repression consisted in the interaction of Myc-Max complex Sp1 and/or Miz1 transcription factors already bound to gene promoters. This interaction may interfere with their activation functions by recruiting co-repressors such as Dnmt3 or HDACs. Moreover, in the absence of , Myc may interfere with the Sp1 activation function by direct interaction and subsequent recruitment of HDACs. More recently the Myc/Max complex was also shown to mediate transcriptional repression by direct binding to peculiar E-box. In this study we analyzed the role of Myc overexpression in Osteosarcoma and Neuroblastoma oncogenesis and the mechanisms underling to Myc function. Myc overexpression is known to correlate with chemoresistance in Osteosarcoma cells. We extended this study by demonstrating that c-Myc induces transcription of a panel of ABC drug transporter genes. ABCs are a large family trans-membrane transporter deeply involved in multi drug resistance. Furthermore expression levels of Myc, ABCC1, ABCC4 and ABCF1 were proved to be important prognostic tool to predict conventional therapy failure. N-Myc amplification/overexpression is the most important prognostic factor for Neuroblastoma. Cyclin G2 and Clusterin are two genes often down regulated in neuroblastoma cells. Cyclin G2 is an atypical member of Cyclin family and its expression is associated with terminal differentiation and apoptosis. Moreover it blocks cell cycle progression and induces cell growth arrest. Instead, CLU is a multifunctional protein involved in many physiological and pathological processes. Several lines of evidences support the view that CLU may act as a tumour suppressor in Neuroblastoma. In this thesis I showed that N-Myc represses CCNG2 and CLU transcription by different mechanisms. • N-Myc represses CCNG2 transcription by directly interacting with Sp1 bound in CCNG2 promoter and recruiting HDAC2. Importantly, reactivation of CCNG2 expression through epigenetic drugs partially reduces N-Myc and HDAC2 mediated cell proliferation. • N-Myc/Max complex represses CLU expression by direct binding to a peculiar E-box element on CLU promoter and by recruitment of HDACs and Polycomb Complexes, to the CLU promoter. Overall our findings strongly support the model in which Myc overexpression/amplification may contribute to some aspects of oncogenesis by a dual action: i) transcription activation of genes that confer a multidrug resistant phenotype to cancer cells; ii), transcription repression of genes involved in cell cycle inhibition and cellular differentiation.

Cellular and molecular effects of cross reacting material 197 (CRM197) on HT-29 colon cancer cell line

Cross Reacting Material 197(CRM197) is a Diphteria toxin non toxic mutant that had shown anti-tumor activity in mice and humans. CRM197 is utilized as a specific inhibitor of heparin-binding epidermal growth factor (HB-EGF), that competes for the epidermal growth factor receptor (EGFR), overexpressed in colorectal cancer and implicated in its progression. We evaluated the effects of CRM197 on HT-29 human colon cancer cell line behaviour and, for CRM197 recognized ability to inhibit HB-EGF, its possible effects on EGFR activation. In particular, while HT-29 does not show any reduction of viability after CRM197 treatment, or changes in cell cycle distribution, in EGFR localization or activation, they show a change in gene expression profile analyzed by microarray. This is the first study where the CRM197 treatment on HT-29 show the alteration of a specific and selected number of genes.

Drugs down-regulating E2F-1 expression hinders cell proliferation through a p53-indipendent mechanism

E2F-1 is a transcription factor that plays a key role in cell-cycle control at G1/S check-point level by regulating the timely expression of many target genes whose products are required for S phase entry and progression. In mammalian cells, E2F-1 is negatively regulated by hypo-phosphorylated Retinoblastoma protein (pRb) whereas it is protected against degradation by its binding to Mouse Double Minute 2 protein (MDM2). In this study we experimented a drug combination in order to obtain a strong down-regulation of E2F-1 by acting on two different mechanisms of E2F-1 regulation mentioned above. This was achieved by combining drugs inhibiting the phosphorylation of pRb with drugs inactivating the MDM2 binding capability. The mechanism of action of these drugs in down-regulating E2F-1 level and activity is p53 independent. As expected, when combined, these drugs strongly inhibits E2F-1 and hinder cell proliferation in p53-/- and p53-mutated cells by blocking them in G1 phase of cell cycle, suggesting that E2F-1 down-regulation may represent a valid chemotherapeutic approach to inhibit proliferation in tumors independently of p53 status.

Epigenetic role of N-Myc in Neuroblastoma

Childhood neuroblastoma is the most common solid tumour of infancy and highly refractory to therapy. One of the most powerful prognostic indicators for this disease is the N-Myc gene amplification, which occurs in approximately 25% of all neuroblastomas. N-Myc is a member of transcription factors belonging to a subclass of the larger group of proteins sharing Basic-Region/Helix–Loop–Helix/Leucin-Zipper (BR/HLH/LZ) motif. N-Myc oncoproteins may determine activation or repression of several genes thanks to different protein-protein interactions that may modulate its transcriptional regulatory ability and therefore its potential for oncogenicity. Chromatin modifications, including histone methylation, have a crucial role in transcription de-regulation of many cancer-related genes. Here, it was investigated whether N-Myc can functionally and/or physically interact with two different factors involved in methyl histone modification: WDR5 (core member of the MLL/Set1 methyltransferase complex) and the de- methylase LSD1. Co-IP assays have demonstrated the presence of both N-Myc-WDR5 and N-Myc-LSD1 complexes in two neuroblastoma cell lines. Human N-Myc amplified cell lines were used as a model system to investigate on transcription activation and/or repression mechanisms carried out by N-Myc-LSD1 and N-Myc-WDR5 protein complexes. qRT-PCR and immunoblot assays underlined the ability of both complexes to positively (N-Myc-WDR5) and negatively (N-Myc-LSD1) influence transcriptional regulation of crititical neuroblastoma N-Myc-related genes, MDM2, p21 and Clusterin. Ch-IP experiments have revealed the binding of the N-Myc complexes above mentioned to the gene promoters analysed. Finally, pharmacological treatment pointed to abolish N-Myc and LSD1 activity were performed to test cellular alterations, such as cell viability and cell cycle progression. Overall, the results presented in this work suggest that N-Myc can interact with two distinct histone methyl modifiers to positively and negatively affect gene transcription in neuroblastoma.

6-(Methylsulfonyl) hexyl isothiocyanate as potential chemopreventive agent: molecular and cellular profile in leukaemia cell lines

Despite numerous therapeutic interventions cancer is still today the second leading cause of death. A growing interest has been addressed to isothiocytanates and more recently, the 6- (methylsulfonyl) hexyl isothiocyanate (6-MITC), the main constituent of the rhizome of Wasabia Japonica, has stimulated the interest of researchers. Aim of the research was to study if 6-MITC is able to modulate the main mechanisms underlying chemopreventive process in leukemic cells lines, verify the selectivity of action and the safety of use in terms of mutagenicity. The study was conducted on different cell types. In particular, Jurkat and HL-60 cells were treated with increasing concentrations of 6-MITC and cell viability, induction of apoptosis, cell cycle analysis, autophagy modulation and stimulation of differentiation were evaluated by flow cytometry. PBL, the non-transformed counterparty of leukemia cells, was used to analyse the selectivity of action by studying the same mechanisms previously indicated. Finally, safety of use and antimutagenicity were studied in TK6 cells adopting an automated protocol in flow cytometry. The achieved results have demonstrated that isothiocyanate modulates many signaling pathways involved in chemopreventive mechanism. In fact, 6-MITC induces apoptosis of both transformed cells, limits tumor growth by slowing down the cell cycle of Jurkat cells and blocks HL-60 cell cycle, increases the autophagic flux and induces cytodifferentiation of promyelocytic HL-60 into macrophage and granulocytic phenotypes. Furthermore, the results obtained with 6-MITC on PBL from healthy donors suggest that the isothiocyante is a good selective cytotoxic agent. Essential feature of a good chemopreventive agent is selectivity toward cancer cells and low toxicity towards non-transformed cells. Finally, the analysis of the micronuclei revealed that 6-MITC is not mutagenic, ensuring safe use, and that instead, it is able to counteract the mutagenic activity of the aneuploidogen Vinblastine, demonstrating another important and interesting chemopreventive activity.

Harnessing the cellular and molecular complexity of high-risk neuroblastoma to investigate novel potential treatment approaches

Neuroblastoma (NB) is the most common type of tumor in infants and the third most common cancer in children. Current clinical practices employ a variety of strategies for NB treatment, ranging from standard chemotherapy to immunotherapy. Due to a lack of knowledge about the molecular mechanisms underlying the disease's onset, aggressive phenotype, and therapeutic resistance, these approaches are ineffective in the majority of instances. MYCN amplification is one of the most well-known genetic alterations associated with high risk in NB. The following work is divided into three sections and aims to provide new insights into the biology of NB and hypothetical new treatment strategies. First, we identified RUNX1T1 as a key gene involved in MYCN-driven NB onset in a transgenic mouse model. Our results suggested that that RUNX1T1 may recruit the Co-REST complex on target genes that regulate the differentiation of NB cells and that the interaction with RCOR3 is essential. Second, we provided insights into the role of MYCN in dysregulating the CDK/RB/E2F pathway controlling the G1/S transition of the cell cycle. We found that RB is dispensable in regulating MYCN amplified NB's cell cycle, providing the rationale for using cyclin/CDK complexes inhibitors in NBs carrying MYCN amplification and relatively high levels of RB1 expression. Third, we generated an M13 bacteriophage platform to target GD2-expressing cells in NB. Here, we generated a recombinant M13 phage capable of binding GD2-expressing cells selectively (M13GD2). Our results showed that M13GD2 chemically conjugated with the photosensitizer ECB04 preserves the retargeting capability, inducing cell death even at picomolar concentrations upon light irradiation. These results provided proof of concept for M13 phage employment in targeted photodynamic therapy for NB, an exciting strategy to overcome resistance to classical immunotherapy.