New circulating microRNAs in serum samples from malignant pleura mesothelioma affected patients as new biomarkers of this neoplasm

Background: Il Mesotelioma maligno della pleura (MPM) è una neoplasia mortale con un'incidenza crescente in tutto il mondo. Il MPM è resistente alle terapie convenzionali e la sua diagnosi avviene in fase tardiva con una sopravvivenza media di 12 mesi. L'esposizione all'amianto è il principale fattore di rischio per l’insorgenza del MPM, che colpisce i lavoratori, anche decenni dopo l'esposizione a questo minerale cancerogeno. L'identificazione di nuovi e specifici marcatori è di fondamentale importanza per una diagnosi precoce, il trattamento e per indagare l’evoluzione di questa neoplasia. Negli ultimi anni i microRNA (miRNA), da cellule o sieri, sono stati proposti come nuovi biomarcatori. Recenti studi hanno dimostrato l'espressione differenziale dei miRNA maturi in molti tumori umani, suggerendo il loro ruolo potenziale come oncogeni o geni soppressori tumorali. Lo scopo di questa indagine è stato quello di identificare miRNA circolanti come biomarcatori putativi per il MPM e potenziali bersagli per terapie innovative. Metodi: In questo studio, ho valutato l’espressione dei miRNA tramite analisi comparativa in campioni di siero di pazienti affetti da mesothelioma maligno della pleura (MPM), lavoratori ex-esposti alle fibre di amianto (WEA) e soggetti sani (HS), con la tecnica del microarray. I risultati sono stati confermati tramite RT-qPCR. Resultati: L'analisi comparativa dei miRNA è stata eseguita per valutarne i profili di espressione nel siero delle tre coorti in studio. In generale, ho rilevato più miRNA nel siero di HS rispetto a MPM e WEA, inoltre nel gruppo WEA si evidenzia un ridotto numero di miRNA rispetto a quelli rivelato in MPM e HS. L’analisi al microarray ha rilevato diversi profili di espressione dei miRNA in campioni di siero MPM, WEA e HS; in particolare i miR-197-3p, miR-1281 e miR-32-3p sono stati rilevati come i principali miRNA disregolati. Al fine di convalidare i dati del microarray, è stata eseguita un’analisi RT-qPCR di tre miRNA differenzialmente espressi nelle coorti MM, WEA e HS. I mir-197-3p, miR-1281 e miR-32-3p sono stati trovati up-regolati nel confronto MPM vs HS. È possibile che le fibre di amianto possano regolare negativamente l'espressione dei miRNA, in un modo simile a quello rilevato per il sistema immunitario, che appare down-regolato nel gruppo WEA. L’espressioni dei mir-197-3p e miR-32-3p sono simili nei gruppi WEA e HS, mentre sono up-regulate nel gruppo MPM. L’espressione del mir-1281 è risultata simile nei gruppi MPM e WEA, e risulta up-regolata rispetto al gruppo HS. Conclusioni: In questo studio, tre miRNA circolanti, miR-197-3p, miR-1281 e miR-32-3p, sono stati trovati disregolati. È noto che i miRNA regolano l'espressione genica legandosi al 3'UTR dell’RNA mesaggero di geni bersaglio. Ulteriori studi sono necessari per valutare la relazione tra questi miRNA e i loro mRNA target, nell’ipotesi che questi geni target siano coinvolti nella progressione del ciclo cellulare e nella mobilità delle cellule, promuovendo così la trasformazione cellulare e l’insorgenza/progressione del MPM, come evidenziato in altri tumori umani.Background: Malignant pleural mesothelioma (MPM) is a fatal cancer, with an increasing incidence world-wide. MPM, which is resistant to conventional therapies, is diagnosed in a late stage with a median survival of 12 months. Exposure to asbestos is the main risk factor for the MPM, which may occur in workers even decades after exposure to this tumorigenic mineral. The identification of new and specific markers is of a paramount importance for an early detection, diagnosis, treatment and to understand the evolution of this malignancy. In recent years, microRNAs (miRNAs), from cells or sera, have been proposed as new biomarkers. Recent studies have shown the differential expression of mature miRNAs in several human cancers, suggesting their potential role as oncogenes or tumor suppressor genes. The aim of this investigation was to identify extracellular miRNAs as putative biomarkers for MPM and potential targets for innovative therapies. Methods: In this study, I investigated miRNA expression on the comparative analysis in serum samples from MPM affected patients, workers ex-exposed to asbestos fibers (WEA) and healthy subjects (HS), with microarray approach. These results were confirmed by real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). Results: A comparative analysis of miRNAs was performed to evaluate their expression profiles in the sera in the three cohorts considered. In general, I detected more miRNAs in sera of HS compared to MPM and WEA, whereas in WEA a reduced number of miRNA was shown compared to those revealed in MPM and HS. Microarray analysis showed different miRNA expression profiles in serum samples from MPM, WEA and HS, being miR-197-3p, miR-1281 and miR-32-3p the most relevant miRNA found dysregualted. In order to validate the microarray data, RT-qPCR analyses were carried out on three miRNAs resulted differentially expressed in MPM, WEA and HS cohorts. MiR-197-3p, miR-1281 and miR-32-3p were found up-regulated in MPM vs HS. It is possible that the asbestos fibers may regulated negatively the miRNA expression, in a way similar to that detected for the immune system, which appeared down-regulated in WEA. Mir-197-3p and mir-32-3p expression in WEA and HS groups were similar, whereas it was up-regulated in MPM group. MiR-1281 expression was similar in MPM and WEA groups, while it was up-regulated compared to HS group. Conclusions: In this study, three circulating miRNAs, namely miR-197-3p, miR-1281 and miR-32 3p, were found dysregulated. It is known that miRNAs regulate the gene expression by binding to mRNA 3'UTR of target genes. Further studies are needed to evaluate the relationship between these miRNAs and their mRNA targets. It would be possible that these targets/genes are involved in the cell cycle progression and cell mobility, thus promoting cell transformation and the MPM onset/progression, as shown in other human cancers

Specific IgG antibodies react to mimotopes of BK Polyomavirus, a small DNA tumor virus, in healthy adult sera

BK polyomavirus (BKPyV) was isolated in 1971 from the urine of a kidney transplant patient. Soon 25 after its identification, BKPyV was characterised as a kidney-tropic virus, which is responsible of a 26 significant fraction of the rejection of transplant kidney in the host. Moreover, in experimental 27 conditions BKPyV is able to transform different types of animal and human cells and to induce 28 tumours of different histotypes in experimental animals. BKPyV DNA sequences have been 29 detected in healthy individuals and cancer patients using polymerase chain reaction/Shouthern blot 30 hybridisation methods. Serum antibodies against this polyomavirus were revealed using 31 immunological techniques, which however cross-react with other polyomaviruses, such as JC 32 (JCPyV) and Simian Virus 40 (SV40). These non-specific data indicate the need of novel 33 immunological methods and new investigations to check in a specific manner BKPyV spread in 34 humans. To this aim, mimotopes from BKPyV structural capsid protein 1 (VP1) were employed for 35 specific immunological reactions to IgG antibodies of human serum samples. An indirect enzyme36 linked immunosorbent assay (ELISA) with synthetic peptides mimicking immunogenic epitopes of 37 BKPyV VP1 was set up and employed to test sera of healthy adult subjects. Data from this 38 innovative immunological assay indicates that serum antibodies against BKPyV VP1 mimotopes 39 are detectable in healthy subjects ranging from 18-90 year old. The overall prevalence of serum 40 samples that reacted to BKPyV VP1 mimotopes was 72%. The strong points from this investigation 41 are the novelty of the immunological method, its simplicity of the approach and the specificity of 42 BKPyV antibody reaction to VP1 mimotopes

P2X7 targeting inhibits growth of human mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive tumor refractory to anti-blastic therapy. MPM cells show several genetic and biochemical defects, e.g. overexpression of oncogenes, downregulation of onco-suppressor genes, dysregulation of microRNA, or alteration of intracellular Ca2+ homeostasis and of apoptosis. No information is as yet available on purinergic signalling in this tumor. Signalling via the P2X7 (P2RX7 or P2X7R) purinergic receptor is attracting increasing attention as a pathway involved in cancer cell death or proliferation. In this report we show that the P2X7R is expressed by three MPM cell lines established from MPM patients but not by mesothelial cells from healthy subjects (healthy mesothelial cells, HMCs). MPM cell proliferation was inhibited by in vitro incubation in the presence of selective P2X7R antagonists, as well as by stimulation with the P2X7R agonist BzATP. Systemic administration of the selective P2X7R blocker AZ10606120 inhibited in vivo growth of MPM tumors whether implanted subcutaneously (s.c.) or intraperitoneally (i.p.). Our findings suggest that the P2X7R might be a novel target for the therapy of mesothelioma

Merkel cell carcinomas arising in autoimmune disease affected patients treated with biologic drugs including anti-TNF

open12siThis study has been supported in parts by Associazione Italiana per la Ricerca sul Cancro (AIRC), grant IG16046; Associazione Sammarinese per la Lotta contro le Leucemie e le Emopatie Maligne (ASLEM), grant CFR/2015; LIONS Club International, District 108 TB, Italy, grant UNIFE 2015; and Fondazione Cassa di Risparmio di Cento, grant 2014 (all to M. Tognon).Purpose: The purpose of this investigation was to characterize Merkel cell carcinomas (MCC) arisen in patients affected by autoimmune diseases and treated with biologic drugs. Experimental Design: Serum samples from patients with MCC were analyzed for the presence and titer of antibodies against antigens of the oncogenic Merkel cell polyomavirus (MCPyV). IgG antibodies against the viral oncoproteins large T (LT) and small T (ST) antigens and the viral capsid protein-1 were analyzed by indirect ELISA. Viral antigens were recombinant LT/ST and virus-like particles (VLP), respectively. MCPyV DNA sequences were studied using PCR methods in MCC tissues and in peripheral blood mononuclear cells (PBMC). Immunohistochemical (IHC) analyses were carried out in MCC tissues to reveal MCPyV LT oncoprotein. Results: MCPyV DNA sequences identified in MCC tissues showed 100% homology with the European MKL-1 strain. PBMCs from patients tested MCPyV-negative. Viral DNA loads in the three MCC tissues were in the 0.1 to 30 copy/cell range. IgG antibodies against LT/ST were detected in patients 1 and 3, whereas patient 2 did not react to the MCPyV LT/ST antigen. Sera from the three patients with MCC contained IgG antibodies against MCPyV VP1. MCC tissues tested MCPyV LT-antigen–positive in IHC assays, with strong LT expression with diffuse nuclear localization. Normal tissues tested MCPyV LT–negative when employed as control. Conclusions: We investigated three new MCCs in patients affected by rheumatologic diseases treated with biologic drugs, including TNF. A possible cause–effect relationship between pharmacologic immunosuppressive treatment and MCC onset is suggested. Indeed, MCC is associated with MCPyV LT oncoprotein activity.openRotondo, John Charles; Bononi, Ilaria; Puozzo, Andrea; Govoni, Marcello; Foschi, Valentina; Lanza, Giovanni; Gafa, Roberta; Gaboriaud, Pauline; Touzã©, Françoise Antoine; Selvatici, Rita; Martini, Fernanda; Tognon, MauroRotondo, John Charles; Bononi, Ilaria; Puozzo, Andrea; Govoni, Marcello; Foschi, Valentina; Lanza, Giovanni; Gafa, Roberta; Gaboriaud, Pauline; Touzã©, Françoise Antoine; Selvatici, Rita; Martini, Fernanda; Tognon, Maur