Massimo Mallardo

Epstein–Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-κB pathway

The Epstein–Barr virus (EBV)-encoded latent membrane protein-1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, substantially contributes to EBVs oncogenic potential by activating nuclear factor-κB (NF-κB). miR-155 is an oncogenic miRNA critical for B-cell maturation and immunoglobulin production in response to antigen. We report that miR-155 expression is much higher in EBV-immortalized B cells than in EBV-negative B cells. LMP1, but not LMP2, up-regulated the expression of miR-155, when transfected in EBV-negative B cells. We analyzed two putative NF-κB binding sites in the miR-155 promoter; both sites recruited NF-κB complex, in nuclear extract from EBV-immortalized cells. The exogenous expression of LMP1, in EBV-negative background, is temporally correlated to induction of p65 with binding on both NF-κB sites and with miR-155 overexpression. The induction of p65 binding together with increased RNA polymerase II binding, confirms that LMP1-mediated activation of miR-155 occurs transcriptionally. In reporter assays, miR-155 promoter lacking NF-κB binding sites was no longer activated by LMP1 expression and an intact AP1 site is needed to attain maximum activation. Finally, we demonstrate that LMP1-mediated activation of miR-155 in an EBV-negative background correlates with reduction of protein PU.1, which is a possible miR target.

An NF-kappaB site in the 5'-untranslated leader region of the human immunodeficiency virus type 1 enhances the viral expression in response to NF-kappaB-activating stimuli.

The 5′-untranslated leader region of human immunodeficiency virus, type 1 (HIV-1), includes a complex array of putative regulatory elements whose role in the viral expression is not completely understood. Here we demonstrate the presence of an NF-κB-responsive element in the trans-activation response (TAR) region of HIV-1 that confers the full induction of HIV-1 long terminal repeat (LTR) in response to NF-κB-activating stimuli, such as DNA alkylating agents, phorbol 12-myristate 13-acetate, and tumor necrosis factor-α. The TAR NF-κB site GGGAGCTCTC spans from positions +31 to +40 and cooperates with the NF-κB enhancer upstream of the TATA box in the NF-κB-mediated induction of HIV-1 LTR. The conclusion stems from the following observations: (i) deletion of the two NF-κB sites upstream of the TATA box reduces, but does not abolish, the HIV-1 LTR activation by NF-κB inducers; (ii) deletion or base pair substitutions of the TAR NF-κB site significantly reduce the HIV-1 LTR activation by NF-κB inducers; (iii) deletions of both the NF-κB sites upstream of the TATA box and the TAR NF-κB site abolish the activation of HIV-1 LTR in response to NF-κB inducers. Moreover, the p50·p65 NF-κB complex binds to the TAR NF-κB sequence and trans-activates the TAR NF-κB-directed expression. The identification of an additional NF-κB site in the HIV-1 LTR points to the relevance of NF-κB factors in the HIV-1 life cycle.

miR-155 is up-regulated in primary and secondary glioblastoma and promotes tumour growth by inhibiting GABA receptors.

An altered expression of microRNAs (miRNAs) contributes both to the development of cancer and to the progression of the disease. Malignant tumours and tumour cell lines have widespread deregulated expressions of miRNAs compared to normal tissues. In this study, we investigated the expression profiles of 340 mammalian miRNAs in 93 cases of multiform glioblastoma (primary and secondary glioblastoma tumours), by means of DNA microarrays. We show that the expression profiles of 10 miRNAs can distinguish primary from secondary glioblastoma types. Moreover, we found elevated miR-155 levels in primary and secondary glioblastoma tissues as well as in glioblastoma primary cultures. We hypothesised that γ-aminobutyric acid A receptor 1 (GABRA1) is a miR-155 target, and studied the correlation between miR-155 up-regulation and the GABRA1 protein in cultured glioblastoma cells by miRNA silencing. We show that a decrease in miR-155 expression to normal levels restores the expression of GABRA1, making glioblastoma cells sensitive to signals that inhibit cell proliferation mediated by GABRA1. In conclusion, the expression patterns of different miRNAs characterise primary and secondary glioblastomas. The aberrant overexpression of miR-155 contributes to the malignant phenotype of glioblastoma cells removing growth inhibition.

Non-protein coding RNA biomarkers and differential expression in cancers: a review.

BackgroundIn these years a huge number of human transcripts has been found that do not code for proteins, named non-protein coding RNAs. In most cases, small (miRNAs, snoRNAs) and long RNAs (antisense RNA, dsRNA, and long RNA species) have many roles, functioning as regulators of other mRNAs, at transcriptional and post-transcriptional level, and controlling protein ubiquitination and degradation. Various species of npcRNAs have been found differentially expressed in different types of cancer. This review discusses the published data and new results on the expression of a subset of npcRNAs.ConclusionThese results underscore the complexity of the RNA world and provide further evidence on the involvement of functional RNAs in cancer cell growth control.

FKBP51 and the NF-κB regulatory pathway in cancer

Constitutive activation of NF-κB occurs in a significant percentage of human cancers. Genetic abnormalities of tumors often enhance normal NF-κB signaling. Chronic inflammation is also associated with constitutive NF-κB activation and increases the risk of cancer. Aberrant NF-κB activation favors cellular transformation, sustains cancer survival, and contributes to tumor invasion. Strategies to inhibit NF-κB represent a promising therapeutic option against cancer. In the last decade, several studies point to the large immunophilin FKBP51 as an important element for the control of NF-κB activation in human neoplasia. This article is an overview of the causes of aberrant NF-κB regulation in cancer and highlights recent papers that implicate FKBP51 in such deregulation.

Endoplasmic reticulum stress reduces the export from the ER and alters the architecture of post-ER compartments

In eukaryotic cells several physiologic and pathologic conditions generate the accumulation of unfolded proteins in the endoplasmic reticulum (ER), leading to ER stress. To restore normal function, some ER transmembrane proteins sense the ER stress and activate coordinated signalling pathways collectively called the Unfolded Protein Response (UPR). Little is known on how the UPR relates to post-ER compartments and to the export from the ER of newly synthesized proteins. Here, we report that the ER stress response induced by either thapsigargin or nitric oxide modifies the dynamics of the intracellular distribution of ERGIC-53 and GM130, two markers of the ER Golgi Intermediate Compartment and of the cis-Golgi, respectively. In addition, induction of ER stress alters the morphology of the ERGIC and the Golgi complex and interferes with the reformation of both compartments. Moreover, ER stress rapidly reduces the transport to the Golgi complex of the temperature sensitive mutant of the Vesicular Stomatitis Virus G Glycoprotein (VSV-G) fused with the Green Fluorescent Protein (ts045G), without apparently decreasing the amount of the protein competent for export. Interestingly, a parallel rapid reduction of the number of Sec31 labelled fluorescent puncta on the ER membranes does occur, thus suggesting that the ER stress alters the ER export and the dynamic of post-ER compartments by rapidly targeting the formation of COPII-coated transport intermediates.

Non-coding RNAs change their expression profile after Retinoid induced differentiation of the promyelocytic cell line NB4

BackgroundThe importance of non-coding RNAs (ncRNAs) as fine regulators of eukaryotic gene expression has emerged by several studies focusing on microRNAs (miRNAs). miRNAs represent a newly discovered family of non coding-RNAs. They are thought to be crucial players of human hematopoiesis and related tumorigenesis and to represent a potential tool to detect the early stages of cancer. More recently, the expression regulation of numerous long ncRNAs has been linked to cell growth, differentiation and cancer although the molecular mechanism of their function is still unknown.NB4 cells are promyelocytic cells that can be induced to differentiation upon retinoic acid (ATRA) treatment and represent a feasible model to study changes of non coding RNAs expression between cancer cells and their terminally differentiated counterpart.Findingswe screened, by microarray analysis, the expression of 243 miRNAs and 492 human genes transcribing for putative long ncRNAs different from miRNAs in NB4 cells before and after ATRA induced differentiation. Our data show that 8 miRNAs, and 58 long ncRNAs were deregulated by ATRA induced NB4 differentiation.Conclusionour data suggest that ATRA-induced differentiation lead to deregulation of a large number of the ncRNAs that can play regulatory roles in both tumorigenesis and differentiation.

Pharmacological folding chaperones act as allosteric ligands of Frizzled4

Upon binding, ligands can chaperone their protein targets by preventing them from misfolding and aggregating. Thus, an organic molecule that works as folding chaperone for a protein might be its specific ligand, and, similarly, the chaperone potential could represent an alternative readout in a molecular screening campaign toward the identification of new hits. Here we show that small molecules selected for acting as pharmacological chaperones on a misfolded mutant of the Frizzled4 (Fz4) receptor bind and modulate wild-type Fz4, representing what are to our knowledge the first organic ligands of this until-now-undruggable GPCR. The novelty and the advantages of the screening platform, the allosteric binding site addressed by these new ligands and the mechanism they use to modulate Fz4 suggest new avenues for development of inhibitors of the Wnt-β-catenin pathway and for drug discovery.

Evaluation of low doses BPA-induced perturbation of glycemia by toxicogenomics points to a primary role of pancreatic islets and to the mechanism of toxicity.

Epidemiologic and experimental studies have associated changes of blood glucose homeostasis to Bisphenol A (BPA) exposure. We took a toxicogenomic approach to investigate the mechanisms of low-dose (1 × 10−9 M) BPA toxicity in ex vivo cultures of primary murine pancreatic islets and hepatocytes. Twenty-nine inhibited genes were identified in islets and none in exposed hepatocytes. Although their expression was slightly altered, their impaired cellular level, as a whole, resulted in specific phenotypic changes. Damage of mitochondrial function and metabolism, as predicted by bioinformatics analyses, was observed: BPA exposure led to a time-dependent decrease in mitochondrial membrane potential, to an increase of ROS cellular levels and, finally, to an induction of apoptosis, attributable to the bigger Bax/Bcl-2 ratio owing to activation of NF-κB pathway. Our data suggest a multifactorial mechanism for BPA toxicity in pancreatic islets with emphasis to mitochondria dysfunction and NF-κB activation. Finally, we assessed in vitro the viability of BPA-treated islets in stressing condition, as exposure to high glucose, evidencing a reduced ability of the exposed islets to respond to further damages. The result was confirmed in vivo evaluating the reduction of glycemia in hyperglycemic mice transplanted with control and BPA-treated pancreatic islets. The reported findings identify the pancreatic islet as the main target of BPA toxicity in impairing the glycemia. They suggest that the BPA exposure can weaken the response of the pancreatic islets to damages. The last observation could represent a broader concept whose consideration should lead to the development of experimental plans better reproducing the multiple exposure conditions.

Cancer-Associated CD43 Glycoforms as Target of Immunotherapy

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer–mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy. Mol Cancer Ther; 13(3); 752–62. ©2013 AACR.