Lara Paracchini

Blockade of the IL-1R1/TLR4 pathway mediates disease-modification therapeutic effects in a model of acquired epilepsy

We recently discovered that forebrain activation of the IL-1 receptor/Toll-like receptor (IL-1R1/TLR4) innate immunity signal plays a pivotal role in neuronal hyperexcitability underlying seizures in rodents. Since this pathway is activated in neurons and glia in human epileptogenic foci, it represents a potential target for developing drugs interfering with the mechanisms of epileptogenesis that lead to spontaneous seizures. The lack of such drugs represents a major unmet clinical need. We tested therefore novel therapies inhibiting the IL-1R1/TLR4 signaling in an established murine model of acquired epilepsy. We used an epigenetic approach by injecting a synthetic mimic of micro(mi)RNA-146a that impairs IL1R1/TLR4 signal transduction, or we blocked receptor activation with antiinflammatory drugs. Both interventions when transiently applied to mice after epilepsy onset, prevented disease progression and dramatically reduced chronic seizure recurrence, while the anticonvulsant drug carbamazepine was ineffective. We conclude that IL-1R1/TLR4 is a novel potential therapeutic target for attaining disease-modifications in patients with diagnosed epilepsy.

miRNA Landscape in Stage I Epithelial Ovarian Cancer Defines the Histotype Specificities

Purpose: Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic diseases, with survival rate virtually unchanged for the past 30 years. EOC comprises different histotypes with molecular and clinical heterogeneity, but up till now the present gold standard platinum-based treatment has been conducted without any patient stratification. The aim of the present study is to generate microRNA (miRNA) profiles characteristic of each stage I EOC histotype, to identify subtype-specific biomarkers to improve our understanding underlying the tumor mechanisms. Experimental Design: A collection of 257 snap-frozen stage I EOC tumor biopsies was gathered together from three tumor tissue collections and stratified into independent training (n = 183) and validation sets (n = 74). Microarray and quantitative real-time PCR (qRT-PCR) were used to generate and validate the histotype-specific markers. A novel dedicated resampling inferential strategy was developed and applied to identify the highest reproducible results. mRNA and miRNA profiles were integrated to identify novel regulatory circuits. Results: Robust miRNA markers for clear cell and mucinous histotypes were found. Specifically, the clear cell histotype is characterized by a five-fold (log scale) higher expression of miR-30a and miR-30a*, whereas mucinous histotype has five-fold (log scale) higher levels of miR-192/194. Furthermore, a mucinous-specific regulatory loop involving miR-192/194 cluster and a differential regulation of E2F3 in clear cell histotype were identified. Conclusions: Our findings showed that stage I EOC histotypes have their own characteristic miRNA expression and specific regulatory circuits. Clin Cancer Res; 19(15); 4114–23. ©2013 AACR.

Identification of high-grade serous ovarian cancer miRNA species associated with survival and drug response in patients receiving neoadjuvant chemotherapy: a retrospective longitudinal analysis using matched tumor biopsies

BACKGROUND Neoadjuvant chemotherapy (NACT) has been recognized as a reliable therapeutic strategy in patients with unresectable advanced epithelial ovarian cancer (EOC). The molecular events leading to platinum (Pt) response in NACT settings have hitherto not been explored. In the present work, longitudinal changes of miRNA expression profile were investigated to identify miRNA families with prognostic role in high-grade serous EOC patients who received the NACT regimen. PATIENTS AND METHODS One hundred sixty-four matched tumor biopsies taken at initial laparoscopic evaluation and at interval-debulking surgery (IDS) after four courses of Pt-based therapy were selected from 82 stage IIIC-IV high-grade serous-EOC patients that were judged unsuitable for complete primary debulking and subjected the NACT protocol. miRNA profiling by microarray, real-time PCR and immuno-histochemical staining for Smad2 phosphorylation (P-Smad2) were used for data analysis. RESULTS Analysis revealed that 369 miRNAs were differentially expressed in matched biopsies (referred to as DEMs). DEMs were not scattered across the genome, but clustered into families: miR-199, let-7, miR-30, miR-181 and miR-29. Multivariate analysis showed that miR-199a-3p, miR-199a-5p, miR-181a-5p and let-7g-5p associated with overall and progression-free survival (P < 0.05); miR-199a-3p, miR-199a-5p and miR-181a-5p associated with residual tumor volume and Pt-free interval (P < 0.05). Immuno-histochemical staining confirmed an enrichment of P-Smad2, a marker of transforming growth factor-β activation, in tumors from patients with shorter PFS and OS, and with high levels of expression of miR-181a-5p (P < 0.05). Kaplan-Meier curves plotting concomitant expression of P-Smad2 and miR-181a-5p show significant differences in PFS and OS compared with those depicting the expression of each biomarker alone (P < 0.001). CONCLUSIONS This study describes several miRNA families with a prognostic role in the NACT setting. It also confirms that concomitant analysis of P-Smad2 and miR-181a-5p in surgical samples may be capable of identifying those ovarian cancer patients with poor outcome and little chance of response to Pt-based NACT.

Protection of Brain Injury by Amniotic Mesenchymal Stromal Cell-Secreted Metabolites

Objectives:To define the features of human amniotic mesenchymal stromal cell secretome and its protective properties in experimental models of acute brain injury. Design:Prospective experimental study. Setting:Laboratory research. Subjects:C57Bl/6 mice. Interventions:Mice subjected to sham or traumatic brain injury by controlled cortical impact received human amniotic mesenchymal stromal cells or phosphate-buffered saline infused intracerebroventricularly or intravenously 24 hours after injury. Organotypic cortical brain slices exposed to ischemic injury by oxygen-glucose deprivation were treated with human amniotic mesenchymal stromal cells or with their secretome (conditioned medium) in a transwell system. Measurements and Main Results:Traumatic brain injured mice receiving human amniotic mesenchymal stromal cells intravenously or intracerebroventricularly showed early and lasting functional and anatomical brain protection. cortical slices injured by oxigen-glucose deprivation and treated with human amniotic mesenchymal stromal cells or conditioned medium showed comparable protective effects (neuronal rescue, promotion of M2 microglia polarization, induction of trophic factors) indicating that the exposure of human amniotic mesenchymal stromal cells to the injured tissue is not necessary for the release of bioactive factors. Using sequential size-exclusion and gel-filtration chromatography, we identified a conditioned medium subfraction, which specifically displays these highly protective properties and we found that this fraction was rich in bioactive molecules with molecular weight smaller than 700 Da. Quantitative RNA analysis and mass spectrometry-based peptidomics showed that the active factors are not proteins or RNAs. The metabolomic profiling of six metabolic classes identified a list of molecules whose abundance was selectively elevated in the active conditioned medium fraction. Conclusions:Human amniotic mesenchymal stromal cell-secreted factors protect the brain after acute injury. Importantly, a fraction rich in metabolites, and containing neither proteic nor ribonucleic molecules was protective. This study indicates the profiling of protective factors that could be useful in cell-free therapeutic approaches for acute brain injury.

Circulating miRNA landscape identifies miR-1246 as promising diagnostic biomarker in high-grade serous ovarian carcinoma: a validation across two independent cohorts.

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecologic neoplasm, with five-year survival rate below 30%. Early disease detection is of utmost importance to improve HGSOC cure rate. Sera from 168 HGSOC patients and 65 healthy controls were gathered together from two independent collections and stratified into a training set, for miRNA marker identification, and a validation set, for data validation. An innovative statistical approach for microarray data normalization was developed to identify differentially expressed miRNAs. Signature validation in both the training and validation sets was performed by quantitative Real Time PCR (RT-qPCR). In both the training and validation sets, miR-1246, miR-595 and miR-2278 emerged significantly over expressed in the sera of HGSOC patients compared to healthy controls. Receiver Operating Characteristic curve analysis revealed miR-1246 as the best diagnostic biomarker, with a sensitivity of 87%, a specificity of 77% and an accuracy of 84%. This study is the first step in the identification of circulating miRNAs with diagnostic relevance for HGSOC. According to its specificity and sensitivity, circulating miR-1246 levels are worthy to be further investigated as potential diagnostic biomarker for HGSOC.

Lurbinectedin reduces tumour-associated macrophages and the inflammatory tumour microenvironment in preclinical models

Background:Lurbinectedin is a novel anticancer agent currently undergoing late-stage (Phase II /III) clinical evaluation in platinum-resistant ovarian, BRCA1/2-mutated breast and small-cell lung cancer. Lurbinectedin is structurally related to trabectedin and it inhibits active transcription and the DNA repair machinery in tumour cells.Methods:In this study we investigated whether lurbinectedin has the ability to modulate the inflammatory microenvironment and the viability of myeloid cells in tumour-bearing mice.Results:Administration of lurbinectedin significantly and selectively decreased the number of circulating monocytes and, in tumour tissues, that of macrophages and vessels. Similar findings were observed when a lurbinectedin-resistant tumour variant was used, indicating a direct effect of lurbinectedin on the tumour microenviroment. In vitro, lurbinectedin induced caspase-8-dependent apoptosis of human purified monocytes, whereas at low doses it significantly inhibited the production of inflammatory/growth factors (CCL2, CXCL8 and VEGF) and dramatically impaired monocyte adhesion and migration ability. These findings were supported by the strong inhibition of genes of the Rho-GTPase family in lurbinectedin-treated monocytes.Conclusions:The results illustrate that lurbinectedin affects at multiple levels the inflammatory microenvironment by acting on the viability and functional activity of mononuclear phagocytes. These peculiar effects, combined with its intrinsic activity against cancer cells, make lurbinectedin a compound of particular interest in oncology.

A systems biology approach to investigate the mechanism of action of trabectedin in a model of myelomonocytic leukemia

This study was designed to investigate the mode of action of trabectedin in myelomonocytic leukemia cells by applying systems biology approaches to mine gene expression profiling data and pharmacological assessment of the cellular effects. Significant enrichment was found in regulons of target genes inferred for specific transcription factors, among which MAFB was the most upregulated after treatment and was central in the transcriptional network likely to be relevant for the specific therapeutic effects of trabectedin against myelomonocytic cells. Using the Connectivity Map, similarity among transcriptional signatures elicited by treatment with different compounds was investigated, showing a high degree of similarity between transcriptional signatures of trabectedin and those of the topoisomerase I inhibitor, irinotecan, and an anti-dopaminergic antagonist, thioridazine. The study highlights the potential importance of systems biology approaches to generate new hypotheses that are experimentally testable to define the specificity of the mechanism of action of drugs.

Exome sequencing in an Italian family with Alzheimer’s disease points to a role for seizure-related gene 6 (SEZ6) rare variant R615H

BackgroundThe typical familial form of Alzheimer’s disease (FAD) accounts for about 5% of total Alzheimer’s disease (AD) cases. Presenilins (PSEN1 and PSEN2) and amyloid-β (A4) precursor protein (APP) genes carry all reported FAD-linked mutations. However, other genetic loci may be involved in AD. For instance, seizure-related gene 6 (SEZ6) has been reported in brain development and psychiatric disorders and is differentially expressed in the cerebrospinal fluid of AD cases.MethodsWe describe a targeted exome sequencing analysis of a large Italian kindred with AD, negative for PSEN and APP variants, that indicated the SEZ6 heterozygous mutation R615H is associated with the pathology.ResultsWe overexpressed R615H mutation in H4-SW cells, finding a reduction of amyloid peptide Aβ(1–42). Sez6 expression decreased with age in a mouse model of AD (3xTG-AD), but independently from transgene expression.ConclusionsThese results support a role of exome sequencing for disease-associated variant discovery and reinforce available data on SEZ6 in AD models.

Abstract B18: miRNA landscape analysis of stage I EOC, identifies miR-199a-5p associated to poor prognosis in grade 3 subgroup

Introduction. Within stage I epithelial ovarian cancer (EOC), the current clinic-pathological parameters, like tumor grade, fail to accurately stratify patient prognosis and it is therefore crucial for optimal treatment that the biological properties of stage I EOCs are further elucidated. We have previously demonstrated miR-200c as a predictor of survival, and a biomarker of relapse (Marchini et al. Lancet Oncology, 2011), suggesting that miRNA profile could be a useful tool to dissect molecular networks in stage I EOC. The aim of the current study is to identify a miRNA signature for each tumor grade, that integrated with clinical variables would be used to improve stage I patients stratification. Experimental procedures. A cohort of 219 snap frozen tumor biopsies, with median follow up of seven years, was gathered together from three independent Italian tumor tissue collections. miRNA landscape was generated with commercially available arrays (Agilent, Palo Alto CA) and analysis performed as recently published (Calura et al., CCR 2013). Signature validation was performed by qRT-PCR using commercially available primers and reagents (Qiagen, Milano, Italy). Results. The entire cohort of patients was stratified by sub-stage, grade and relapse into a training set (n= 151), used for miRNA landscape generation, and a validation set (n= 68) used for qRT-PCR validation. “Resampling score” (RS) strategy (Calura et al., CCR 2013) reported that the largest number of miRNAs found differentially expressed is between grade three and grade one (n= 72), while the comparison between grade one versus borderline tumors showed the lowest number (n= 14). Signature validation in both training and validation set by qRT-PCR of the top seven selected miRNAs with highest RS, confirmed hsa-miR-376c, hsa-miR-377 and hsa-miR-214 as down-regulated in grade three compared to the other grades; hsa-miR-96 expression increases directly from grade one to grade three, while hsa-miR-199a-5p was down-regulated in grade three compared to borderline tumors. No differences were observed for hsa-miR-183 and hsa-miR-29c. miRNA expression profile was correlated to clinical variables in both univariate and multivariate model and only miR-199a-5p, resulted associated to PFS in multivariate Cox proportional hazard model. Conclusions. In the present study we observed that, regardless of tumor histological subtype (Calura et al. CCR 2013), known morphological differences across tumor grades mirror molecular differences in term of miRNA expression profile. To optimize patients stratification and thus improving clinical management of stage I EOC, we are now drawing new miRNA-based networks (i.e. miRNA-gene expression integration) for each tumor grade that will be correlated with known clinical parameters. Citation Format: Enrica Calura, Robert Fruscio, Lara Paracchini, Eliana Bignotti, Paolo Martini, Antonella Ravaggi, Mariacristina Di Marino, Gabriele Sales, Luca Beltrame, Federica Dell9Orto, Romina Baldo, Sergio Pecorelli, Enrico Sartori, laura Zanotti, Dionyssios Katsaros, Germana Tognon, Maurizio D9Incalci, Chiara Romualdi, Sergio Marchini. miRNA landscape analysis of stage I EOC, identifies miR-199a-5p associated to poor prognosis in grade 3 subgroup. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B18.