Teresa Bellissimo

Epigenetic silencing of miR-145-5p contributes to brain metastasis.

Brain metastasis is a major cause of morbidity and mortality of lung cancer patients. We assessed whether aberrant expression of specific microRNAs could contribute to brain metastasis. Comparison of primary lung tumors and their matched metastatic brain disseminations identified shared patterns of several microRNAs, including common down-regulation of miR-145-5p. Down-regulation was attributed to methylation of miR-145s promoter and affiliated elevation of several protein targets, such as EGFR, OCT-4, MUC-1, c-MYC and, interestingly, tumor protein D52 (TPD52). In line with these observations, restored expression of miR-145-5p and selective depletion of individual targets markedly reduced in vitro and in vivo cancer cell migration. In aggregate, our results attribute to miR-145-5p and its direct targets pivotal roles in malignancy progression and in metastasis.

Oncogenic microRNAs characterization in clear cell renal cell carcinoma

A key challenge for the improvement of clear cell renal cell carcinoma (ccRCC) management could derive from a deeper characterization of the biology of these neoplasms that could greatly improve the diagnosis, prognosis and treatment choice. The aim of this study was to identify specific miRNAs that are deregulated in tumor vs. normal kidney tissues and that could impact on the biology of ccRCC. To this end we selected four miRNAs (miR-21-5p, miR-210-3p, miR-185-5p and miR-221-3p) and their expression has been evaluated in a retrospective cohort of formalin-fixed paraffin-embedded (FFPE) tissues from 20 ccRCC patients who underwent surgical nephrectomy resection. miR-21-5p and miR-210-3p resulted the most significantly up-regulated miRNAs in this patient cohort, highlighting these onco-miRNAs as possible relevant players involved in ccRCC tumorigenesis. Thus, this study reports the identification of specific oncogenic miRNAs that are altered in ccRCC tissues and suggests that they might be useful biomarkers in ccRCC management.

MicroRNAs: short non-coding players in cancer chemoresistance

Chemoresistance is one of the main problems in the therapy of cancer. There are a number of different molecular mechanisms through which a cancer cell acquires resistance to a specific treatment, such as alterations in drug uptake, drug metabolism and drug targets. There are several lines of evidence showing that miRNAs are involved in drug sensitivity of cancer cells in different tumor types and by different treatments. In this review, we provide an overview of the more recent and significant findings on the role of miRNAs in cancer cell drug resistance. In particular, we focus on specific miRNA mechanisms of action that in various steps lead from drug cell sensitivity to drug cell resistance. We also provide evidence on how miRNA profiling may unveil relevant predictive biomarkers for therapy outcomes.

Circulating miR-21-5p and miR-148a-3p as emerging non-invasive biomarkers in thymic epithelial tumors

ABSTRACT Thymic epithelial cells give rise to both thymoma and thymic carcinoma. A crucial advance in thymic epithelial tumors (TET) management may derive from the identification of novel molecular biomarkers able to improve diagnosis, prognosis and treatment planning.In a previous study, we identified microRNAs that were differentially expressed in tumor vs normal thymic tissues. Among the microRNAs resulted up-regulated in TET tissues, we evaluated miR-21-5p, miR-148a-3p, miR-141-3p, miR-34b-5p, miR-34c-5p, miR-455-5p as blood plasma circulating non-invasive biomarkers for TET management.We firstly report that the expression levels of specific onco-miRNAs, that we found upregulated in the blood plasma collected from TET patients at surgery, resulted significantly reduced in follow-up samples.This pilot study suggests that circulating miR-21-5p and miR-148a-3p could represent novel non-invasive biomarkers to evaluate the efficacy of therapy and the prognosis of TET.

Identification of post-transcriptional regulatory networks during myeloblast-to-monocyte differentiation transition

Treatment of leukemia cells with 1,25-dihydroxyvitamin D3 may overcome their differentiation block and lead to the transition from myeloblasts to monocytes. To identify microRNA-mRNA networks relevant for myeloid differentiation, we profiled the expression of mRNAs and microRNAs associated to the low- and high-density ribosomal fractions in leukemic cells and in their differentiated monocytic counterpart. Intersection between mRNAs shifted across the fractions after treatment with putative target genes of modulated microRNAs showed a series of molecular networks relevant for the monocyte cell fate determination, as for example the post-transcriptional regulation of the Polo-like kinase 1 (PLK1) by miR-22–3p and let-7e-5p.

Thymic Epithelial Tumors phenotype relies on miR-145-5p epigenetic regulation

BackgroundThymoma and thymic carcinoma are the most frequent subtypes of thymic epithelial tumors (TETs). A relevant advance in TET management could derive from a deeper molecular characterization of these neoplasms. We previously identified a set of microRNA (miRNAs) differentially expressed in TETs and normal thymic tissues and among the most significantly deregulated we described the down-regulation of miR-145-5p in TET. Here we describe the mRNAs diversely regulated in TETs and analyze the correlation between these and the miRNAs previously identified, focusing in particular on miR-145-5p. Then, we examine the functional role of miR-145-5p in TETs and its epigenetic transcriptional regulation.MethodsmRNAs expression profiling of a cohort of fresh frozen TETs and normal tissues was performed by microarray analysis. MiR-145-5p role in TETs was evaluated in vitro, modulating its expression in a Thymic Carcinoma (TC1889) cell line. Epigenetic transcriptional regulation of miR-145-5p was examined by treating the TC1889 cell line with the HDAC inhibitor Valproic Acid (VPA).ResultsStarting from the identification of a 69-gene signature of miR-145-5p putative target mRNAs, whose expression was inversely correlated to that of miR-145-5p, we followed the expression of some of them in vitro upon overexpression of miR-145-5p; we observed that this resulted in the down-regulation of the target genes, impacting on TETs cancerous phenotype. We also found that VPA treatment of TC1889 cells led to miR-145-5p up-regulation and concomitant down-regulation of miR-145-5p target genes and exhibited antitumor effects, as indicated by the induction of cell cycle arrest and by the reduction of cell viability, colony forming ability and migration capability. The importance of miR-145-5p up-regulation mediated by VPA is evidenced by the fact that hampering miR-145-5p activity by a LNA inhibitor reduced the impact of VPA treatment on cell viability and colony forming ability of TET cells. Finally, we observed that VPA was also able to enhance the response of TET cells to cisplatin and erlotinib.ConclusionsAltogether our results suggest that the epigenetic regulation of miR-145-5p expression, as well as the modulation of its functional targets, could be relevant players in tumor progression and treatment response in TETs.

Surface Plasmon Resonance: A Useful Strategy for the Identification of Small Molecule Argonaute 2 Protein Binders

Surface plasmon resonance (SPR) is one of the most important techniques for the detection and the characterization of molecular interactions. SPR technology is a label-free approach for monitoring biomolecular interactions in real time. The binding of analytes to molecules immobilized on a thin metal film (ligand) determines a change in the refractive index and, therefore in the angle of extinction of light, is reflected when polarized light hits the film, monitored in real time as a change in the position of the dip in reflected intensity. Since SPR detects mass, the technique is label-free.Here, we describe the use of SPR techniques to study the interaction between Argonaute 2 and small molecular compounds selected by means of high-throughput docking screening.

Small Molecules Targeting the miRNA-Binding Domain of Argonaute 2: From Computer-Aided Molecular Design to RNA Immunoprecipitation

The development of small-molecule-based target therapy design for human disease and cancer is object of growing attention. Recently, specific microRNA (miRNA) mimicking compounds able to bind the miRNA-binding domain of Argonaute 2 protein (AGO2) to inhibit miRNA loading and its functional activity were described. Computer-aided molecular design techniques and RNA immunoprecipitation represent suitable approaches to identify and experimentally determine if a compound is able to impair the loading of miRNAs on AGO2 protein. Here, we describe these two methodologies that we recently used to select a specific compound able to interfere with the AGO2 functional activity and able to improve the retinoic acid-dependent myeloid differentiation of leukemic cells.

Contribution of miR-145-5p/Ago2 complex to the regulation of epithelial-mesenchymal transition

The epithelial-mesenchymal transition (EMT) is essential for cell fate determination during development but it is involved in pathological processes like cancer as well, being one of the first steps in the mechanisms leading to metastasis. miR-145-5p is one of the most widely recognized tumor-suppressor miRNAs, able to regulate cell migration and EMT through the contribution of the RISC complex in which Argonaute (Ago) proteins are required for target recognition and gene silencing [1]. Ago2 is an important member of the Ago family and its overexpression correlates with a transformed phenotype in breast cancer cells [2]. With the aim to unravel miR-145-5p/Ago2 contribution to the suppression of cancer progression in epithelial tumors, here we show that: i) miR-145-5p and Ago2 are down-regulated in breast tumor vs normal tissues; ii) the restored expression of miR-145-5p in breast cancer cell lines results in the reduction of tumor phenotype; iii) Ago2 expression is positively and specifically regulated by miR-145-5p; iv) miR-145-5p-dependent Ago2 induction is necessary for the inhibition of cell migration; v) when Ago2 is depleted, the formation of an alternative miR-145-5p/Ago1 active complex redirects miR-145-5p tumor suppressor function and correlates with a more invasive phenotype in breast cancer cells. These results open to the identification of miR-145-5p/Ago2-dependent molecular networks involved in the maintenance and progression of cancer phenotype.

The Methylated DNA Immunoprecipitation [MeDIP] to Investigate the Epigenetic Remodeling in Cell Fate Determination and Cancer Development

Epigenetic mechanisms such as DNA methylation, posttranslational modifications of histone proteins, remodeling of nucleosomes, and the expression of noncoding RNAs contribute to the regulation of gene expression for the cell fate determination and tissue development. The disruption of these epigenetic mechanisms, in conjunction with genetic alterations, is a decisive element for cancer development and progression. The cancer phenotype is characterized by global DNA hypomethylation and gene-specific hypermethylation. The methylated DNA immunoprecipitation [MeDIP] is a useful approach currently used to clarify the functional consequences of DNA methylation on cell fate determination and cancer development.