Manuela Ferracin

A MicroRNA Signature of Hypoxia

ABSTRACT Recent research has identified critical roles for microRNAs in a large number of cellular processes, including tumorigenic transformation. While significant progress has been made towards understanding the mechanisms of gene regulation by microRNAs, much less is known about factors affecting the expression of these noncoding transcripts. Here, we demonstrate for the first time a functional link between hypoxia, a well-documented tumor microenvironment factor, and microRNA expression. Microarray-based expression profiles revealed that a specific spectrum of microRNAs (including miR-23, -24, -26, -27, -103, -107, -181, -210, and -213) is induced in response to low oxygen, at least some via a hypoxia-inducible-factor-dependent mechanism. Select members of this group (miR-26, -107, and -210) decrease proapoptotic signaling in a hypoxic environment, suggesting an impact of these transcripts on tumor formation. Interestingly, the vast majority of hypoxia-induced microRNAs are also overexpressed in a variety of human tumors.

miR‐34a predicts survival of Ewing's sarcoma patients and directly influences cell chemo‐sensitivity and malignancy

Identification of factors to detect chemotherapy‐resistant tumours at diagnosis is a first priority for risk‐adapted therapy in the oncology of children and young adults, where more individualized, effective, and less toxic treatments are highly desirable. In this study, we analysed the miRNAs discriminating Ewings sarcoma (EWS) patients with different clinical outcomes in order to identify new indicators of prognosis. miRNA expression was investigated in 49 primary EWSs by using the Agilent human miRNA microarray v.2 and/or qRT‐PCR. Statistical power of the samples studied for miRNA expression was verified, indicating adequate sample size. Microarray analysis defined a signature of five miRNAs (miR‐34a, miR‐23a, miR‐92a, miR‐490‐3p, and miR‐130b) as an independent predictor of risk for disease progression and survival. Validation analysis in the extended sample set indicated that both miR‐34a and miR‐490‐3p achieved sufficient statistical power to predict prognosis. Results were particularly robust for miR‐34a, which appeared associated with either event‐free or overall survival and emerged as a significant predictor also after multivariate analysis. Patients with the highest expression of miR‐34a did not experience adverse events in 5 years; in contrast, patients with the lowest expression recurred within 2 years. High expression of miR34a can be detected also in paraffin‐embedded tissues by in situ hybridization, thus contributing to an easy routine evaluation of this miRNA. Functional analysis of miR‐34a in EWS cell lines indicated that when miR‐34a expression was enforced, cells were less proliferative, less malignant, and sensitized to doxorubicin and vincristine. Expression of miR‐34a could be increased in p53wt cells by treatment with nutlin‐3a. Accordingly, nutlin‐3a synergizes with doxorubicin. Overall, our data indicate that miR‐34a expression is a strong predictor of outcome in EWS. Restoration of miR‐34a activity may be useful to decrease malignancy and increase tumour sensitivity to current drugs, so sparing excessive long‐term toxicity to EWS patients. Copyright

MicroRNAs dysregulation in human malignant pleural mesothelioma.

Background:Malignant pleural mesothelioma (MPM) is a rare but aggressive asbestos-related cancer that develops by mesothelial cell transformation. At present, there are no effective therapies for MPM. Great efforts have been made in finding specific markers/mechanisms for MPM onset, including studies into microRNAs (miRNAs). Recent studies have shown the differential expression of mature miRNAs in several human cancers, suggesting their potential role as oncogenes or tumor suppressor genes. Methods:In this study, we investigated miRNAs profile in five human normal pleural mesothelial short-term cell cultures (HMCs) and five MPMs, with microarray approach. These results were confirmed by real-time quantitative reverse-transcriptase polymerase chain reaction and Western blotting. Results:A comparative analysis of miRNA expression in MPM and HMCs was carried out. Microarray profiling showed different miRNA expression between MPM and HMCs. Specifically, members of the oncomiRNA miR 17-92 cluster and its paralogs, namely miR 17-5p, 18a, 19b, 20a, 20b, 25, 92, 106a, 106b, were markedly upregulated. Besides, in our investigation, additional miRNAs, such as miR-7, miR-182, miR-214, and miR-497 were found to be dysregulated in MPM. Conclusions:These data are in agreement with results that have previously been reported on dysregulated miRNAs for other solid human tumors. Moreover, in our investigation, additional miRNAs were found to be dysregulated in MPM. Interestingly, gene products that regulate the cell cycle are targets and predicted targets for these miRNAs. Our data suggest that specific miRNAs could be key players in MPM development/progression. In addition, some of these miRNAs may represent MPM markers and potential targets for new therapeutic approaches.

Anti-CD38 antibody therapy: windows of opportunity yielded by the functional characteristics of the target molecule.

In vivo use of monoclonal antibodies (mAbs) has become a mainstay of routine clinical practice in the treatment of various human diseases. A number of molecules can serve as targets, according to the condition being treated. Now entering human clinical trials, CD38 molecule is a particularly attractive target because of its peculiar pattern of expression and its twin role as receptor and ectoenzyme. This review provides a range of analytical perspectives on the current progress in and challenges to anti-CD38 mAb therapy. We present a synopsis of the evidence available on CD38, particularly in myeloma and chronic lymphocytic leukemia (CLL). Our aim is to make the data from basic science helpful and accessible to a diverse clinical audience and, at the same time, to improve its potential for in vivo use. The topics covered include tissue distribution and signal implementation by mAb ligation and the possibility of increasing cell density on target cells by exploiting information about the molecule’s regulation in combination with drugs approved for in vivo use. Also analyzed is the behavior of CD38 as an enzyme: CD38 is a component of a pathway leading to the production of adenosine in the tumor microenvironment, thus inducing local anergy. Consequently, not only might CD38 be a prime target for mAb-mediated therapy, but its functional block may contribute to general improvement in cancer immunotherapy and outcomes.

Quantification of Circulating miRNAs by Droplet Digital PCR: Comparison of EvaGreen- and TaqMan-Based Chemistries

Droplet digital PCR (ddPCR) has been successfully used with TaqMan assays to assess gene expression through the quantification of mRNA and miRNA. Recently, a new ddPCR system that can also run DNA-binding dye-based assays has been developed but it has not yet been tested for miRNA. We tested and compared the feasibility of quantifying miRNA with the new QX200 Droplet Digital PCR system when used with EvaGreen dye– and TaqMan probe–based assays. RNA from plasma and serum of 28 patients with cancer and healthy persons was reverse-transcribed and quantified for two circulating miRNAs and one added exogenous miRNA, with both EvaGreen dye–based miRCURY LNA miRNA assays and TaqMan assays. Amplification and detection of target miRNAs were performed on the QX200 ddPCR system. Conditions required to run miRCURY LNA miRNA assays were optimized. The EvaGreen-based assay was precise, reproducible over a range of concentrations of four orders of magnitude, and sensitive, detecting a target miRNA at levels down to 1 copy/μL. When this assay was compared with TaqMan assays, high concordance was obtained for two endogenous miRNAs in serum and plasma (Pearson r > 0.90). EvaGreen dye–based and TaqMan probe–based assays can be equally used with the ddPCR system to quantify circulating miRNAs in human plasma and serum. This study establishes the basis for using EvaGreen dye–based assays on a ddPCR system for quantifying circulating miRNA biomarkers and potentially other low-abundance RNA biomarkers in human biofluids. See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.” Cancer Epidemiol Biomarkers Prev; 23(12); 2638–42. ©2014 AACR.

Mutated β-catenin evades a microRNA-dependent regulatory loop

hsa-mir-483 is located within intron 2 of the IGF2 gene. We have previously shown oncogenic features of miR-483-3p through cooperation with IGF2 or by independently targeting the proapoptotic gene BBC3/PUMA. Here we demonstrate that expression of miR-483 can be induced independently of IGF2 by the oncoprotein β-catenin through an interaction with the basic helix–loop–helix protein upstream stimulatory transcription factor 1. We also show that β-catenin itself is a target of miR-483-3p, triggering a negative regulatory loop that becomes ineffective in cells harboring an activating mutation of β-catenin. These results provide insights into the complex regulation of the IGF2/miR-483 locus, revealing players in the β-catenin pathway.

The P2X7 receptor is a key modulator of the PI3K/GSK3β/VEGF signaling network: evidence in experimental neuroblastoma

Neuroblastoma (NB) is an aggressive pediatric tumor, responsible for 15% of cancer-related deaths in childhood, lacking an effective treatment in its advanced stages. The P2X7 receptor for extracellular ATP was associated to NB cell proliferation and recently emerged as a promoter of tumor engraftment, growth and vascularization. In an effort to identify new therapeutic options for neuroblastoma, we studied the role of P2X7 receptor in NB biology. We first analyzed the effect of P2X7 activation or down-modulation of the main biochemical ways involved in NB progression: the PI3K/Akt/GSK3β/MYCN and the HIF1α/VEGF pathways. In ACN human NB cells, P2X7 stimulation enhanced PI3K/Akt, while decreasing GSK3β activity. In the same model, P2X7 silencing or antagonist administration reduced the activity of PI3K/Akt and increased that of GSK3β, leading to a decrease in cellular glycogen stores. Similarly, P2X7 downmodulation caused a reduction in HIF1α levels and vascular endothelial growth factor (VEGF) secretion. Systemic administration of two different P2X7 antagonists (AZ10606120 or A740003) in nude/nude mice reduced ACN-derived tumor growth. An even stronger effect of P2X7 blockade was obtained in a syngeneic immune-competent neuroblastoma model: Neuro2A cells injected in AlbinoJ mice. Together with tumor regression, treatment with P2X7 antagonists caused downmodulation of the Akt/HIF1α axis, leading to reduced VEGF content and decreased vessel formation. Interestingly, in both experimental models, P2X7 antagonists strongly reduced the expression of the probably best-accepted oncogene in NB: MYCN. Finally, we associated P2X7 overexpression with poor prognosis in advanced-stage NB patients. Taken together, our data suggest that P2X7 receptor is an upstream regulator of the main signaling pathways involved in NB growth, metabolic activity and angiogenesis, and a promising therapeutic target for neuroblastoma treatment.

STAT3-mediated activation of microRNA cluster 17~92 promotes proliferation and survival of ALK-positive anaplastic large cell lymphoma

Systemic anaplastic large cell lymphoma is a category of T-cell non-Hodgkin’s lymphoma which can be further subdivided into two distinct entities (ALK+ and ALK−) based on the presence or absence of ALK gene rearrangements. Among several pathways triggered by ALK signaling, constitutive activation of STAT3 is strictly required for ALK-mediated transformation and survival. Here we performed genome-wide microRNA profiling and identified 48 microRNA concordantly modulated by the inducible knock-down of ALK and STAT3. To evaluate the functional role of differentially expressed miRNA, we forced their expression in ALK+ anaplastic large cell lymphoma cells, and monitored their influence after STAT3 depletion. We found that the expression of the microRNA-17~92 cluster partially rescues STAT3 knock-down by sustaining proliferation and survival of ALK+ cells. Experiments in a xenograft mouse model indicated that forced expression of microRNA-17~92 interferes with STAT3 knock-down in vivo. High expression levels of the microRNA-17~92 cluster resulted in down-regulation of BIM and TGFβRII proteins, suggesting that their targeting might mediate resistance to STAT3 knock-down in anaplastic large cell lymphoma cells. We speculate that the microRNA-17~92 cluster is involved in lymphomagenesis of STAT3+ ALCL and that its inhibition might represent an alternative avenue to interfere with ALK signaling in anaplastic large cell lymphomas.

microRNAome Expression in Chronic Lymphocytic Leukemia: Comparison with Normal B-cell Subsets and Correlations with Prognostic and Clinical Parameters

Purpose: Despite its indolent nature, chronic lymphocytic leukemia (CLL) remains an incurable disease. To establish the potential pathogenic role of miRNAs, the identification of deregulated miRNAs in CLL is crucial. Experimental Design: We analyzed the expression of 723 mature miRNAs in 217 early-stage CLL cases and in various different normal B-cell subpopulations from tonsils and peripheral blood. Results: Our analyses indicated that CLL cells exhibited a miRNA expression pattern that was most similar to the subsets of antigen-experienced and marginal zone–like B cells. These normal subpopulations were used as reference to identify differentially expressed miRNAs in comparison with CLL. Differences related to the expression of 25 miRNAs were found to be independent from IGHV mutation status or cytogenetic aberrations. These differences, confirmed in an independent validation set, led to a novel comprehensive description of miRNAs potentially involved in CLL. We also identified miRNAs whose expression was distinctive of cases with mutated versus unmutated IGHV genes or cases with 13q, 11q, and 17p deletions and trisomy 12. Finally, analysis of clinical data in relation to miRNA expression revealed that miR26a, miR532-3p, miR146-5p, and miR29c* were strongly associated with progression-free survival. Conclusion: This study provides novel information on miRNAs expressed by CLL and normal B-cell subtypes, with implication on the cell of origin of CLL. In addition, our findings indicate a number of deregulated miRNAs in CLL, which may play a pathogenic role and promote disease progression. Collectively, this information can be used for developing miRNA-based therapeutic strategies in CLL. Clin Cancer Res; 20(15); 4141–53. ©2014 AACR.

Identification of miRNAs differentially expressed in human epilepsy with or without granule cell pathology.

The microRNAs (miRNAs) are small size non-coding RNAs that regulate expression of target mRNAs at post-transcriptional level. miRNAs differentially expressed under pathological conditions may help identifying mechanisms underlying the disease and may represent biomarkers with prognostic value. However, this kind of studies are difficult in the brain because of the cellular heterogeneity of the tissue and of the limited access to fresh tissue. Here, we focused on a pathology affecting specific cells in a subpopulation of epileptic brains (hippocampal granule cells), an approach that bypasses the above problems. All patients underwent surgery for intractable temporal lobe epilepsy and had hippocampal sclerosis associated with no granule cell pathology in half of the cases and with type-2 granule cell pathology (granule cell layer dispersion or bilamination) in the other half. The expression of more than 1000 miRNAs was examined in the laser-microdissected dentate granule cell layer. Twelve miRNAs were differentially expressed in the two groups. One of these, miR487a, was confirmed to be expressed at highly differential levels in an extended cohort of patients, using RT-qPCR. Bioinformatics searches and RT-qPCR verification identified ANTXR1 as a possible target of miR487a. ANTXR1 may be directly implicated in granule cell dispersion because it is an adhesion molecule that favors cell spreading. Thus, miR487a could be the first identified element of a miRNA signature that may be useful for prognostic evaluation of post-surgical epilepsy and may drive mechanistic studies leading to the identification of therapeutic targets.