MicroRNA expression in HTLV-1 infection and pathogenesis

Our laboratory is examining the profiles of microRNA expression in ATLL cells and infected T-cell lines using microarrays and small RNA libraries. Microarray analysis of ATLL samples revealed 6 upregulated and 21 downregulated microRNAs in ATLL cells compared to CD4+ T-cell controls. Potential targets for deregulated microRNAs were identified by integrating microRNA and mRNA expression profiles. Current experiments are aimed at verifying these predicted microRNA-target interactions

Expression Of Mir-34a In T-cells Infected By Human T-lymphotropic Virus 1

Human T-lymphotropic virus 1 (HTLV-1) immortalizes T-cells and is the causative agent of adult T-cell leukemia/lymphoma (ATLL). HTLV-1 replication and transformation are governed by multiple interactions between viral regulatory proteins and host cell factors that remain to be fully elucidated. The present study investigated the impact of HTLV-1 infection on the expression of miR-34a, a microRNA whose expression is downregulated in many types of cancer. Results of RT-PCR assays showed that five out of six HTLV-1-positive cell lines expressed higher levels of miR-34a compared to normal PBMC or purified CD4+ T-cells. ATLL cell line ED, which did not express miR-34a, showed methylation of the miR-34a promoter. Newly infected PBMC and samples from 10 ATLL patients also showed a prominent increase in miR-34a expression compared to PBMC controls. The primary miR-34a transcript expressed in infected cell line C91PL contained binding motifs for NF-kappa B and p53. Pharmacological inhibition of NF-kappa B with Bay 11-7082 indicated that this pathway contributes to sustain miR-34a levels in infected cells. Treatment of infected cell lines with the p53 activator nutlin-3a resulted in a further increase in miR-34a levels, thus confirming it as a transcriptional target of p53. Nutlin-3a-treated cells showed downregulation of known miR-34a targets including the deacetylase SIRT1, which was accompanied by increased acetylation of p53, a substrate of SIRT1. Transfection of C91PL cells with a miR-34a mimic also led to downregulation of mRNA targets including SIRT1 as well as the pro-apoptotic factor BAX. Unlike nutlin-3a, the miR-34a mimic did not cause cell cycle arrest or reduce cell viability. On the other hand, sequestration of miR-34a with a sponge construct resulted in an increase in death of C91PL cells. These findings provide evidence for a functional role for miR-34a in fine-tuning the expression of target genes that influence the turnover of HTLV-1-infected cells

Electromagnetic fields (EMFs) and adenosine receptors modulate prostaglandin E2 and cytokine production in human osteoarthritic synovial fibroblasts

Objective. Synovial fibroblasts (SFs) contribute to the development of osteoarthritis (OA) by the secretion of a wide range of pro-inflammatory mediators, including cytokines and lipid mediators of inflammation (1). Previous studies show that electromagnetic fields (EMFs) may represent a potential therapeutical approach to limit cartilage degradation and to control inflammation associated to OA, and that they may act through the adenosine pathway (2). On this basis the aim of this study was to investigate if EMFs might modulate inflammatory activities of human SFs derived from OA patients (OASFs) and the possible involvement of adenosine receptors (ARs) in mediating EMF effects. Design. SFs obtained from OA patients, undergoing total hip joint replacement surgery, were exposed to EMFs (1.5 mT; 75 Hz) for 24 hours. In control and EMF-exposed cells, ARs were evaluated by western blotting, quantitative real-time RT-PCR and saturation binding experiments and cAMP levels were measured by a specific assay. In the absence and in the presence of interleukin-1β (IL-1β), used as a pro-inflammatory stimulus, prostaglandin E2 (PGE2), cytokine and matrix degrading enzyme production was evaluated in OASFs exposed to EMFs and treated with selective adenosine receptor agonists and antagonists. Results. EMF exposure induced a selective increase in A2A and A3 ARs. These increases were associated to changes in cAMP levels, indicating that ARs were functionally active in EMF-exposed cells. In IL-1β-treated OASFs, functional data obtained in the presence of  A2A and A3 adenosine agonists and antagonists showed that EMFs inhibit the release of (PGE2) and of the proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), whilst stimulate the release of interleukin-10 (IL-10), an antinflammatory cytokine. Further, results show that these effects appear to be mediated by the EMF-induced upregulation of A2A and A3 ARs. No effects of EMFs or ARs have been observed on matrix degrading enzymes production. Conclusions: EMFs display anti-inflammatory effects in human OASFs and these EMF-induced .ffects are in part mediated by the adenosine pathway, specifically by the A2A and A3 ARs activation. Taken together, these results suggest that SFs could represent potential therapeutic targets cells for EMF treatment and open new clinical perspectives to the control of inflammation associated to joint diseases. 1. Martel-Pelletier J et al. Eklem Hastalik Cerrahisi. 2010; 21(1):2-14. 2. De Mattei M et al. Osteoarthritis Cartilage. 2009; 17(2):252-262

A new bioavailable fenretinide formulation with antiproliferative, antimetabolic, and cytotoxic effects on solid tumors.

Fenretinide is a synthetic retinoid characterized by anticancer activity in preclinical models and favorable toxicological profile, but also by a low bioavailability that hindered its clinical efficacy in former clinical trials. We developed a new formulation of fenretinide complexed with 2-hydroxypropyl-beta-cyclodextrin (nanofenretinide) characterized by an increased bioavailability and therapeutic efficacy. Nanofenretinide was active in cell lines derived from multiple solid tumors, in primary spheroid cultures and in xenografts of lung and colorectal cancer, where it inhibited tumor growth independently from the mutational status of tumor cells. A global profiling of pathways activated by nanofenretinide was performed by reverse-phase proteomic arrays and lipid analysis, revealing widespread repression of the mTOR pathway, activation of apoptotic, autophagic and DNA damage signals and massive production of dihydroceramide, a bioactive lipid with pleiotropic effects on several biological processes. In cells that survived nanofenretinide treatment there was a decrease of factors involved in cell cycle progression and an increase in the levels of p16 and phosphorylated p38 MAPK with consequent block in G0 and early G1. The capacity of nanofenretinide to induce cancer cell death and quiescence, together with its elevated bioavailability and broad antitumor activity indicate its potential use in cancer treatment and chemoprevention

Mek inhibition results in marked antitumor activity against metastatic melanoma patient-derived melanospheres and in melanosphere-generated xenografts

One of the key oncogenic pathways involved in melanoma aggressiveness, development and progression is the RAS/BRAF/MEK pathway, whose alterations are found in most patients. These molecular anomalies are promising targets for more effective anti-cancer therapies. Some Mek inhibitors showed promising antitumor activity, although schedules and doses associated with low systemic toxicity need to be defined. In addition, it is now accepted that cancers can arise from and be maintained by the cancer stem cells (CSC) or tumor-initiating cells (TIC), commonly expanded in vitro as tumorspheres from several solid tumors, including melanoma (melanospheres). Here, we investigated the potential targeting of MEK pathway by exploiting highly reliable in vitro and in vivo pre-clinical models of melanomas based on melanospheres, as melanoma initiating cells (MIC) surrogates. MEK inhibition, through PD0325901, provided a successful strategy to affect survival of mutated-BRAF melanospheres and growth of wild type-BRAF melanospheres. A marked citotoxicity was observed in differentated melanoma cells regardless BRAF mutational status. PD0325901 treatment, dramatically inhibited growth of melanosphere-generated xenografts and determined impaired tumor vascularization of both mutated- and wild type-BRAF tumors, in the absence of mice toxicity. These results suggest that MEK inhibition might represent a valid treatment option for patients with both mutated- or wild type-BRAF melanomas, affecting tumor growth through multiple targets. \uc2\ua9 2013 Sette et al.; licensee BioMed Central Ltd

EGFR Inhibition Abrogates Leiomyosarcoma Cell Chemoresistance through Inactivation of Survival Pathways and Impairment of CSC Potential

Background: Tumor cells with stem-like phenotype and properties, known as cancer stem cells (CSC), have been identified in most solid tumors and are presumed to be responsible for driving tumor initiation, chemoresistance, relapse, or metastasis. A subpopulation of cells with increased stem-like potential has also been identified within sarcomas. These cells are endowed with increased tumorigenic potential, chemoresistance, expression of embryonic markers, and side population(SP) phenotype. Leiomyosarcomas (LMS) are soft tissue sarcomas presumably arising from undifferentiated cells of mesenchymal origin, the Mesenchymal Stem Cells (MSC). Frequent recurrence of LMS and chemoresistance of relapsed patients may likely result from the failure to target CSC. Therefore, therapeutic cues coming from the cancer stem cell (CSC) field may drastically improve patient outcome. Methodology/Principal Findings: We expanded LMS stem-like cells from patient samples in vitro and examined the possibility to counteract LMS malignancy through a stem-like cell effective approach. LMS stem-like cells were in vitro expanded both as "tumor spheres" and as "monolayers" in Mesenchymal Stem Cell (MSC) conditions. LMS stem-like cells displayed MSC phenotype, higher SP fraction, and increased drug-extrusion, extended proliferation potential, self-renewal, and multiple differentiation ability. They were chemoresistant, highly tumorigenic, and faithfully reproduced the patient tumor in mice. Such cells displayed activation of EGFR/AKT/MAPK pathways, suggesting a possibility in overcoming their chemoresistance through EGFR blockade. IRESSA plus Vincristine treatment determined pathway inactivation, impairment of SP phenotype, high cytotoxicity in vitro and strong antitumor activity in stem-like cell-generated patient-like xenografts, targeting both stem-like and differentiated cells. Conclusions/Significance: EGFR blockade combined with vincristine determines stem-like cell effective antitumor activity in vitro and in vivo against LMS, thus providing a potential therapy for LMS patients. \uc2\ua9 2012 Sette et al

A retrospective multicentric observational study of trastuzumab emtansine in HER2 positive metastatic breast cancer: A real-world experience

We addressed trastuzumab emtansine (T-DM1) efficacy in HER2+ metastatic breast cancer patients treated in real-world practice, and its activity in pertuzumab-pretreated patients. We conducted a retrospective, observational study involving 23 cancer centres, and 250 patients. Survival data were analyzed by Kaplan Meier curves and log rank test. Factors testing significant in univariate analysis were tested in multivariate models. Median follow-up was 15 months and median T-DM1 treatment-length 4 months. Response rate was 41.6%, clinical benefit 60.9%. Median progression-free and median overall survival were 6 and 20 months, respectively. Overall, no differences emerged by pertuzumab pretreatment, with median progression-free and median overall survival of 4 and 17 months in pertuzumab-pretreated (p=0.13), and 6 and 22 months in pertuzumab-na\uc3\uafve patients (p=0.27). Patients who received second-line T-DM1 had median progression-free and median overall survival of 3 and 12 months (p=0.0001) if pertuzumab-pretreated, and 8 and 26 months if pertuzumab-na\uc3\uafve (p=0.06). In contrast, in third-line and beyond, median progression-free and median overall survival were 16 and 18 months in pertuzumab-pretreated (p=0.05) and 6 and 17 months in pertuzumab-na\uc3\uafve patients (p=0.30). In multivariate analysis, lower ECOG performance status was associated with progression-free survival benefit (p < 0.0001), while overall survival was positively affected by lower ECOG PS (p < 0.0001), absence of brain metastases (p 0.05), and clinical benefit (p < 0.0001). Our results are comparable with those from randomized trials. Further studies are warranted to confirm and interpret our data on apparently lower T-DM1 efficacy when given as second-line treatment after pertuzumab, and on the optimal sequence order

Role of microRNAs in T-cell activation and transformation by human T-cell Leukemia virus type 1

Human T-Lymphotropic virus type 1 (HTLV-1) is the causative agent of two distinct pathologies, adult T-cell leukemia/lymphoma (ATLL), an aggressive malignancy of mature CD4+ T-cells, and tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM), a demyelinating neurodegenerative disease. Despite intense study, many aspects of HTLV-1 replication, persistence and pathogenesis remain to be understood. The work described in the present thesis was aimed at defining the role of microRNAs (miRNAs) in HTLV-1 infection and ATLL pathogenesis. We generated small RNA libraries from normal CD4+ cells (resting and stimulated) and two T-cell lines chronically infected with HTLV-1 (MT-2 and C91PL). Libraries were analyzed by 454 mass sequencing and data were processed through a series of computational steps to identify known and candidate new miRNAs for each library. Comparison of frequencies of known miRNAs in the different libraries led to the identification of 14 downregulated miRNAs and 4 upregulated species in infected cell lines vs. resting CD4+ cells, while 21 miRNAs were differentially expressed (16 downregulated, 5 upregulated) in stimulated compared to resting CD4+ cells. We validated the expression of some new miRNA candidates identified by bioinformatic analysis of the libraries through end point and quantitative RT-PCR. Two sequences mapped to the HTLV-1 genome, suggesting that the virus may produce its own miRNAs under certain conditions. We examined the profiles of known miRNA expression in ATLL cells and normal resting and activated T CD4+ lymphocytes using microarrays. On the basis of miRNA expression, cluster analysis of ATLL samples and CD4+ controls showed that the resting controls were highly related to each other, while the tumor samples exhibited some heterogeneity. Statistical analysis revealed 6 upregulated and 21 downregulated miRNAs in ATLL cells compared to CD4+ T-cell controls. Several of the differentially expressed miRNAs identified in the libraries and by microarray analysis were validated by real time RT-PCR. Since miRNA-mRNA interactions often result in degradation of the target mRNA, integration of results from target prediction programs with expression profiles for miRNAs and mRNAs can aid in identifying genuine mRNA targets. This approach was applied to miRNA and mRNA microarray data obtained for our ATLL and resting CD4+ samples. Potential targets for 12 miRNAs differentially expressed in ATLL cells were identified by integrating miRNA and mRNA expression profiles. Functional enrichment analysis of predicted targets revealed the presence of several genes belonging to the cAMP signalling pathway, which is known to be activated upon HTLV-1 transformation. We also investigated the role of miR-34a, consistently upregulated in ATLL samples and HTLV-1 infected cells lines. Knockdown of miR-34a in infected cell lines determined an increased in cell death, suggesting that miR-34a could play an important role in the expansion of HTLV-1 infected cells and thereby in ATLL development.Il virus T-linfotropico umano di tipo 1 (HTLV-1) è l’agente eziologico della leucemia/linfoma a cellule T dell’adulto (ATLL, adult T-cell leukemia/lymphoma) e della paraparesi spastica tropicale/mielopatia associata ad HTLV (TSP/HAM, Tropical spastic paraparesis/HTLV-associated myelopathy), una patologia degenerativa del sistema nervoso centrale. Recenti evidenze suggeriscono che i microRNA (miRNA) contribuiscano a questo processo di trasformazione mediata da HTLV-1. Le ricerche condotte nel corso del mio dottorato sono state mirate ad approfondire il ruolo dei microRNA (miRNA) nell’infezione di cellule T da parte di HTLV-1 e nella patogenesi dell’ATLL. Sono state realizzate librerie di cDNA di piccoli RNA, a partire da linfociti T CD4+ normali (resting e attivati) e da due linee cellulari cronicamente infettate con HTLV-1 (C91PL e MT-2). Le librerie sono state analizzate attraverso il sequenziamento di massa 454 e l’analisi bioinformatica delle sequenze ottenute ha permesso l’identificazione dei miRNA noti e nuovi miRNA candidati presenti in ciascuna libreria. Il confronto delle frequenze dei miRNA noti nelle diverse librerie ha evidenziato la presenza di 14 e 4 miRNA rispettivamente downregolati e upregolati nelle linee cellulari infettare rispetto ai linofociti T CD4+ resting, mentre 21 miRNA sono risultati differenzialmente espressi in linfociti T CD4+ stimolati in confronto ai linfociti T CD4+ resting (16 downregolati, 5 upregolati). L’espressione di diversi nuovi miRNA, individuati dall’analisi bioinformatica delle librerie, è stata validata attraverso RT-PCR end-point o RT-PCR quantitativa. Inoltre la nostra analisi ha rivelato nelle librerie da cellule infettate 2 sequenze che mappano in regioni trascritte del genoma di HTLV-1 e che potrebbero rappresentare dei miRNA virali. Attraverso l’impiego di microarray il profilo di espressione dei miRNA noti è stato analizzato in pazienti ATLL e in linfociti T CD4+ resting e stimolati. In base ai profili di espressione di miRNA ottenuti i campioni sono stati raggruppati in cluster che indicano una forte similitudine all’interno dei campioni di linfocititi T CD4+ resting, mentre i campioni di ATLL hanno profili di espressione di miRNA più eterogenei. L’analisi statistica ha evidenziato 21 miRNA downregolati e 6 upregolati nei pazienti ATLL vs linfociti T CD4+ resting. Diversi miRNA differenzialmente espressi identificati attraverso l’analisi delle librerie e dei microarray sono stati validati tramite RT-PCR quantitativa. Dal momento che l’interazione miRNA-mRNA spesso comporta la degradazione del messaggero bersaglio, l’analisi integrata dei risultati dei programmi di predizione di bersagli con i profili di espressione di miRNA e geni può aiutare nell’identificazione di target. Abbiamo applicato questo approccio ai dati di espressione di miRNA e geni ottenuti per i nostri campioni di ATLL e linfociti T CD4+ resting. Dall’integrazione dei profili di espressione di miRNA e mRNA sono stati identificati i target putativi per 12 miRNA differenzialmente espressi nei pazienti ATLL. L’arricchimento funzionale dei geni bersaglio predetti ha evidenziato la presenza di diversi geni coinvolti nella via di segnale di cAMP, noto per essere presente ad alti livelli in cellule trasformate da HTLV-1. Infine abbiamo indagato il significato funzionale di miR-34a, che risulta essere consistentemente upregolato in pazienti ATLL e linee cellulari infettate. Il silenziamento di miR-34a in linee cellulari infettate determina un aumento della morte cellulare, suggerendo che la deregolazione di questo miRNA possa svolgere un ruolo importante nell’espansione della popolazione di cellule infettate da HTLV-1 e quindi nello sviluppo dell’ATLL

Mathematically defined tissue engineering scaffold architectures prepared by stereolithography

The technologies employed for the preparation of conventional tissue engineering scaffolds restrict the materials choice and the extent to which the architecture can be designed. Here we show the versatility of stereolithography with respect to materials and freedom of design. Porous scaffolds are designed with computer software and built with either a poly(d,l-lactide)-based resin or a poly(d,l-lactide-co-ε-caprolactone)-based resin. Characterisation of the scaffolds by micro-computed tomography shows excellent reproduction of the designs. The mechanical properties are evaluated in compression, and show good agreement with finite element predictions. The mechanical properties of scaffolds can be controlled by the combination of material and scaffold pore architecture. The presented technology and materials enable an accurate preparation of tissue engineering scaffolds with a large freedom of design, and properties ranging from rigid and strong to highly flexible and elastic