Vladia Monsurrò

MicroRNAs linking inflamm-aging, cellular senescence and cancer

Epidemiological and experimental data demonstrate a strong correlation between age-related chronic inflammation (inflamm-aging) and cancer development. However, a comprehensive approach is needed to clarify the underlying molecular mechanisms. Chronic inflammation has mainly been attributed to continuous immune cells activation, but the cellular senescence process, which may involve acquisition of a senescence-associated secretory phenotype (SASP), can be another important contributor, especially in the elderly. MicroRNAs (miRs), a class of molecules involved in gene expression regulation, are emerging as modulators of some pathways, including NF-κB, mTOR, sirtuins, TGF-β and Wnt, that may be related to inflammation, cellular senescence and age-related diseases, cancer included. Interestingly, cancer development is largely avoided or delayed in centenarians, where changes in some miRs are found in plasma and leukocytes. We identified miRs that can be considered as senescence-associated (SA-miRs), inflammation-associated (inflamma-miRs) and cancer-associated (onco-miRs). Here we review recent findings concerning three of them, miR-21, -126 and -146a, which target mRNAs belonging to the NF-κB pathway; we discuss their ability to link cellular senescence, inflamm-aging and cancer and their changes in centenarians, and provide an update on the possibility of using miRs to block accumulation of senescent cells to prevent formation of a microenvironment favoring cancer development and progression.

An integrated humoral and cellular response is elicited in pancreatic cancer by α-enolase, a novel pancreatic ductal adenocarcinoma-associated antigen†

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with a very poor 5‐year survival rate. α‐Enolase is a glycolytic enzyme that also acts as a surface plasminogen receptor. We find that it is overexpressed in PDAC and present on the cell surface of PDAC cell lines. The clinical correlation of its expression with tumor status has been reported for lung and hepatocellular carcinoma. We have previously demonstrated that sera from PDAC patients contain IgG autoantibodies to α‐enolase. The present work was intended to assess the ability of α‐enolase to induce antigen‐specific T cell responses. We show that α‐enolase‐pulsed dendritic cells (DC) specifically stimulate healthy autologous T cells to proliferate, secrete IFN‐γ and lyse PDAC cells but not normal cells. In vivo, α‐enolase‐specific T cells inhibited the growth of PDAC cells in immunodeficient mice. In 8 out of 12 PDAC patients with circulating IgG to α‐enolase, the existence of α‐enolase‐specific T cells was also demonstrated. Taken as a whole, these results indicate that α‐enolase elicits a PDAC‐specific, integrated humoral and cellular response. It is thus a promising and clinically relevant molecular target candidate for immunotherapeutic approaches as new adjuvants to conventional treatments in pancreatic cancer.

Induction of interferon pathways mediates in vivo resistance to oncolytic adenovirus.

Oncolytic adenoviruses are an emerging experimental approach for treatment of tumors refractory to available modalities. Although preclinical results have been promising, and clinical safety has been excellent, it is also apparent that tumors can become virus resistant. The resistance mechanisms acquired by advanced tumors against conventional therapies are increasingly well understood, which has allowed development of countermeasures. To study this in the context of oncolytic adenovirus, we developed two in vivo models of acquired resistance, where initially sensitive tumors eventually gain resistance and relapse. These models were used to investigate the phenomenon on RNA and protein levels using two types of analysis of microarray data, quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. Interferon (IFN) signaling pathways were found upregulated and Myxovirus resistance protein A (MxA) expression was identified as a marker correlating with resistance, while transplantation experiments suggested a role for tumor stroma in maintaining resistance. Furthermore, pathway analysis suggested potential therapeutic targets in oncolytic adenovirus-resistant cells. Improved understanding of the antiviral phenotype causing tumor recurrence is of key importance in order to improve treatment of advanced tumors with oncolytic adenoviruses. Given the similarities between mechanisms of action, this finding might be relevant for other oncolytic viruses as well.

Anti-viral state segregates two molecular phenotypes of pancreatic adenocarcinoma: potential relevance for adenoviral gene therapy

BackgroundPancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer mortality for which novel gene therapy approaches relying on tumor-tropic adenoviruses are being tested.MethodsWe obtained the global transcriptional profiling of primary PDAC using RNA from eight xenografted primary PDAC, three primary PDAC bulk tissues, three chronic pancreatitis and three normal pancreatic tissues. The Affymetrix GeneChip HG-U133A was used. The results of the expression profiles were validated applying immunohistochemical and western blot analysis on a set of 34 primary PDAC and 10 established PDAC cell lines. Permissivity to viral vectors used for gene therapy, Adenovirus 5 and Adeno-Associated Viruses 5 and 6, was assessed on PDAC cell lines.ResultsThe analysis of the expression profiles allowed the identification of two clearly distinguishable phenotypes according to the expression of interferon-stimulated genes. The two phenotypes could be readily recognized by immunohistochemical detection of the Myxovirus-resistance A protein, whose expression reflects the activation of interferon dependent pathways. The two molecular phenotypes discovered in primary carcinomas were also observed among established pancreatic adenocarcinoma cell lines, suggesting that these phenotypes are an intrinsic characteristic of cancer cells independent of their interaction with the hosts microenvironment. The two pancreatic cancer phenotypes are characterized by different permissivity to viral vectors used for gene therapy, as cell lines expressing interferon stimulated genes resisted to Adenovirus 5 mediated lysis in vitro. Similar results were observed when cells were transduced with Adeno-Associated Viruses 5 and 6.ConclusionOur study identified two molecular phenotypes of pancreatic cancer, characterized by a differential expression of interferon-stimulated genes and easily recognized by the expression of the Myxovirus-resistance A protein. We suggest that the detection of these two phenotypes might help the selection of patients enrolled in virally-mediated gene therapy trials.

Next generation sequencing: new tools in immunology and hematology

One of the hallmarks of the adaptive immune system is the specificity of B and T cell receptors. Thanks to somatic recombination, a large repertoire of receptors can be generated within an individual that guarantee the recognition of a vast number of antigens. Monoclonal antibodies have limited applicability, given the high degree of diversity among these receptors, in BCR and TCR monitoring. Furthermore, with regard to cancer, better characterization of complex genomes and the ability to monitor tumor-specific cryptic mutations or translocations are needed to develop better tailored therapies. Novel technologies, by enhancing the ability of BCR and TCR monitoring, can help in the search for minimal residual disease during hematological malignancy diagnosis and follow-up, and can aid in improving bone marrow transplantation techniques. Recently, a novel technology known as next generation sequencing has been developed; this allows the recognition of unique sequences and provides depth of coverage, heterogeneity, and accuracy of sequencing. This provides a powerful tool that, along with microarray analysis for gene expression, may become integral in resolving the remaining key problems in hematology. This review describes the state of the art of this novel technology, its application in the immunological and hematological fields, and the possible benefits it will provide for the hematology and immunology community.

Administration of Reconstituted Polyphenol Oil Bodies Efficiently Suppresses Dendritic Cell Inflammatory Pathways and Acute Intestinal Inflammation

Polyphenols are natural compounds capable of interfering with the inflammatory pathways of several in vitro model systems. In this study, we developed a stable and effective strategy to administer polyphenols to treat in vivo models of acute intestinal inflammation. The in vitro suppressive properties of several polyphenols were first tested and compared for dendritic cells (DCs) production of inflammatory cytokines. A combination of the polyphenols, quercetin and piperine, were then encapsulated into reconstituted oil bodies (OBs) in order to increase their stability. Our results showed that administration of low dose reconstituted polyphenol OBs inhibited LPS-mediated inflammatory cytokine secretion, including IL-6, IL-23, and IL-12, while increasing IL-10 and IL-1Rα production. Mice treated with the polyphenol-containing reconstituted OBs (ROBs) were partially protected from dextran sodium sulfate (DSS)-induced colitis and associated weight loss, while mortality and inflammatory scores revealed an overall anti-inflammatory effect that was likely mediated by impaired DC immune responses. Our study indicates that the administration of reconstituted quercetin and piperine-containing OBs may represent an effective and potent anti-inflammatory strategy to treat acute intestinal inflammation.

Polyphenol administration impairs T-cell proliferation by imprinting a distinct dendritic cell maturational profile

Currently little is known as to how nutritionally derived compounds may affect dendritic cell (DC) maturation and potentially prevent inappropriate inflammatory responses that are characteristic of chronic inflammatory syndromes. Previous observations have demonstrated that two polyphenols quercetin and piperine delivered through reconstituted oil bodies (ROBs‐QP) can influence DC maturation in response to LPS leading to a modulated inflammatory response. In the present study, we examined the molecular effects of ROBs‐QP exposure on DC differentiation in mice and identified a unique molecular signature in response to LPS administration that potentially modulates DC maturation and activity in inflammatory conditions. Following LPS administration, ROBs‐QP‐exposed DCs expressed an altered molecular profile as compared with control DCs, including cytokine and chemokine production, chemokine receptor repertoire, and antigen presentation ability. In vivo ROBs‐QP administration suppresses antigen‐specific T‐cell division in the draining lymph nodes resulting from a reduced ability to create stable immunological synapse. Our data demonstrate that polyphenols exposure can drive DCs toward a new anti‐inflammatory molecular profile capable of dampening the inflammatory response, highlighting their potential as complementary nutritional approaches in the treatment of chronic inflammatory syndromes.

From Oxidative Stress Damage to Pathways, Networks, and Autophagy via MicroRNAs

Oxidative stress can alter the expression level of many microRNAs (miRNAs), but how these changes are integrated and related to oxidative stress responses is poorly understood. In this article, we addressed this question by using in silico tools. We reviewed the literature for miRNAs whose expression is altered upon oxidative stress damage and used them in combination with various databases and software to predict common gene targets of oxidative stress-modulated miRNAs and affected pathways. Furthermore, we identified miRNAs that simultaneously target the predicted oxidative stress-modulated miRNA gene targets. This generated a list of novel candidate miRNAs potentially involved in oxidative stress responses. By literature search and grouping of pathways and cellular responses, we could classify these candidate miRNAs and their targets into a larger scheme related to oxidative stress responses. To further exemplify the potential of our approach in free radical research, we used our explorative tools in combination with ingenuity pathway analysis to successfully identify new candidate miRNAs involved in the ubiquitination process, a master regulator of cellular responses to oxidative stress and proteostasis. Lastly, we demonstrate that our approach may also be useful to identify novel candidate connections between oxidative stress-related miRNAs and autophagy. In summary, our results indicate novel and important aspects with regard to the integrated biological roles of oxidative stress-modulated miRNAs and demonstrate how this type of in silico approach can be useful as a starting point to generate hypotheses and guide further research on the interrelation between miRNA-based gene regulation, oxidative stress signaling pathways, and autophagy.

Seventh annual meeting of the Italian Network for Tumor Biotherapy (NIBIT), Siena, 1–3 October 2009

Abbreviations ACT Adoptive cell therapy APC Antigen-presenting cells AS Adjuvant system CIK Cytokine-induced killer COA Colon antigen CRC Colorectal cancer CSC Cancer stem cells CTC Circulating tumor cells CTL Cytotoxic T lymphocytes CTLA-4 Cytotoxic T lymphocyte-associated antigen-4 CTX Cyclophosphamide DC Dendritic cells DXR Doxorubicin HDI High-dose interferon-alpha-2b IDO Indoleamine 2,3-dioxygenase IFN Interferon IL Interleukin mAb Monoclonal antibody MM Metastatic melanoma NB Neuroblastoma NGR Asn-Gly-Arg NHL Non-Hodgkin lymphoma NK Natural killer PC Pancreatic cancer