EphB2 stem-related and EphA2 progression-related miRNA-based networks in progressive stages of CRC evolution: clinical significance and potential miRNA drivers.

EphB2 and EphA2 control stemness and differentiation in the intestinal mucosa, but the way they cooperate with the complex mechanisms underlying tumor heterogeneity and how they affect the therapeutic outcome in colorectal cancer (CRC) patients, remain unclear. MicroRNA (miRNA) expression profiling along with pathway analysis provide comprehensive information on the dysregulation of multiple crucial pathways in CRC.Through a network-based approach founded on the characterization of progressive miRNAomes centered on EphA2/EphB2 signaling during tumor development in the AOM/DSS murine model, we found a miRNA-dependent orchestration of EphB2-specific stem-like properties in earlier phases of colorectal tumorigenesis and the EphA2-specific control of tumor progression in the latest CRC phases. Furthermore, two transcriptional signatures that are specifically dependent on the EphA2/EphB2 signaling pathways were identified, namely EphA2, miR-423-5p, CREB1, ADAMTS14, and EphB2, miR-31-5p, mir-31-3p, CRK, CXCL12, ARPC5, SRC.EphA2- and EphB2-related signatures were validated for their expression and clinical value in 1663 CRC patients. In multivariate analysis, both signatures were predictive of survival and tumor progression.The early dysregulation of miRs-31, as observed in the murine samples, was also confirmed on 49 human tissue samples including preneoplastic lesions and tumors. In light of these findings, miRs-31 emerged as novel potential drivers of CRC initiation.Our study evidenced a miRNA-dependent orchestration of EphB2 stem-related networks at the onset and EphA2-related cancer-progression networks in advanced stages of CRC evolution, suggesting new predictive biomarkers and potential therapeutic targets.

Aberrant promoter methylation of beta-1,4 galactosyltransferase 1 as potential cancer-specific biomarker of colorectal tumors.

Epigenetic alterations, such as CpG islands methylation and histone modifications, are recognized key characteristics of cancer. Glycogenes are a group of genes which epigenetic status was found to be changed in several tumors. In this study, we determined promoter methylation status of the glycogene beta-1,4-galactosyltransferase 1 (B4GALT1) in colorectal cancer patients. Methylation status of B4GALT1 was assessed in 130 colorectal adenocarcinomas, 13 adenomas, and in paired normal tissue using quantitative methylation specific PCR (QMSP). B4GALT1 mRNA expression was evaluated in methylated/unmethylated tumor and normal specimens. We also investigated microsatellite stability and microsatellite instability status and KRAS/BRAF mutations. Discriminatory power of QMSP was assessed by receiving operating curve (ROC) analysis on a training set of 24 colorectal cancers and paired mucosa. The area under the ROC curve (AUC) was 0.737 (95% confidence interval [CI]:0.591-0.881, P = 0.005) with an optimal cutoff value of 2.07 yielding a 54% sensitivity (95% CI: 35.1%-72.1%) and a specificity of 91.7% (95% CI: 74.1%-97.7%). These results were confirmed in an independent validation set where B4GALT1 methylation was detected in 52/106 patients. An inverse correlation was observed between methylation and B4GALT1 mRNA expression levels (r = -0.482, P = 0.037). Significant differences in methylation levels and frequencies was demonstrated in invasive lesions as compared with normal mucosa (P = 0.0001) and in carcinoma samples as compared with adenoma (P = 0.009). B4GALT1 methylation is a frequent and specific event in colorectal cancer and correlates with downregulation of mRNA expression. These results suggest that the glycogene B4GALT1 represent a valuable candidate biomarker of invasive phenotype of colorectal cancer.

The AOM/DSS murine model for the study of colon carcinogenesis: From pathways to diagnosis and therapy studies.

Colorectal cancer (CRC) is a major health problem in industrialized countries. Although inflammation-linked carcinogenesis is a well accepted concept and is often observed within the gastrointestinal tract, the underlying mechanisms remain to be elucidated. Inflammation can indeed provide initiating and promoting stimuli and mediators, generating a tumour-prone microenvironment. Many murine models of sporadic and inflammation-related colon carcinogenesis have been developed in the last decade, including chemically induced CRC models, genetically engineered mouse models, and xenoplants. Among the chemically induced CRC models, the combination of a single hit of azoxymethane (AOM) with 1 week exposure to the inflammatory agent dextran sodium sulphate (DSS) in rodents has proven to dramatically shorten the latency time for induction of CRC and to rapidly recapitulate the aberrant crypt foci-adenoma-carcinoma sequence that occurs in human CRC. Because of its high reproducibility and potency, as well as the simple and affordable mode of application, the AOM/DSS has become an outstanding model for studying colon carcinogenesis and a powerful platform for chemopreventive intervention studies. In this article we highlight the histopathological and molecular features and describe the principal genetic and epigenetic alterations and inflammatory pathways involved in carcinogenesis in AOM/DSS-treated mice; we also present a general overview of recent experimental applications and preclinical testing of novel therapeutics in the AOM/DSS model.

DNA vaccination strategies for anti-tumour effective gene therapy protocols.

After more than 15 years of experimentation, DNA vaccines have become a promising perspective for tumour diseases, and animal models are widely used to study the biological features of human cancer progression and to test the efficacy of vaccination protocols. In recent years, immunisation with naked plasmid DNA encoding tumour-associated antigens or tumour-specific antigens has revealed a number of advantages: antigen-specific DNA vaccination stimulates both cellular and humoral immune responses; multiple or multi-gene vectors encoding several antigens/determinants and immune-modulatory molecules can be delivered as single administration; DNA vaccination does not induce autoimmune disease in normal animals; DNA vaccines based on plasmid vectors can be produced and tested rapidly and economically. However, DNA vaccines have shown low immunogenicity when tested in human clinical trials, and compared with traditional vaccines, they induce weak immune responses. Therefore, the improvement of vaccine efficacy has become a critical goal in the development of effective DNA vaccination protocols for anti-tumour therapy. Several strategies are taken into account for improving the DNA vaccination efficacy, such as antigen optimisation, use of adjuvants and delivery systems like electroporation, co-expression of cytokines and co-stimulatory molecules in the same vector, different vaccination protocols. In this review we discuss how the combination of these approaches may contribute to the development of more effective DNA vaccination protocols for the therapy of lymphoma in a mouse model.

Recent advances in epitope design for immunotherapy of cancer.

Eradication of cancer cells is imperative for a successful treatment of tumours. In addition to the existent chemotherapy or radiation therapy, other novel immunotherapeutic strategies to fight tumours are currently under investigation. One of these is cancer vaccination, an approach aimed at inducing effective immune responses in the host against defined tumour antigens. Among several classes of cancer vaccines, the subunit vaccines based on the single and multi epitope-approach are worthy of note as they offer an exquisite specificity in targeting only tumour cells. In this review we will focus on the significant advances made in the development and use of epitope-based cancer vaccines, reporting a selection of important and recent patents on tumour antigen discovery and epitope design for immunotherapy of cancer.

Electroporation of skeletal muscle induces danger signal release and antigen-presenting cell recruitment independently of DNA vaccine administration.

BACKGROUND: Plasmid DNA vaccination combined with electroporation (EP) provides a promising approach for the prevention of infectious diseases and for cancer immunotherapy. This technology has been described as being effective in activating humoral and cellular immune response in the host as well as in enhancing expression of the encoded antigen. Several reports showed EP has adjuvant-like properties when combined with plasmid DNA injection although the effect in the absence of DNA has not been investigated. OBJECTIVE: The aim of this study is to clarify whether the application of EP alone to the skeletal muscle is able to recruit and trigger cells involved in antigen presentation and immune response. METHODS: Mouse skeletal muscle treated by EP were observed and processed for clinical, histological and immunohistochemistry analysis at different time points. RESULTS: We demonstrate that EP induces transient morphological changes in the muscle with early production of endogenous cytokines responsible for signalling danger at the local level. Moreover, it causes the recruitment of inflammatory cells independently of the DNA injection and the activation of a danger pro-inflammatory pathway, resulting in T-lymphocyte migration. CONCLUSIONS: Our data indicate EP by itself is able to recruit and trigger cells involved in antigen presentation and immune response; hence, the idea that EP has adjuvant-like properties owing to a moderate tissue injury and generation of a pro-inflammatory context with cytokine release that enhances the immune response. We suggest EP may be of practical use in clinical protocols, contributing to the development of DNA vaccination strategies.

Strategies for effective naked-DNA vaccination against infectious diseases.

To date, vaccination is an active area of investigation for its application to a great variety of human diseases including infections and cancer. In particular, naked-DNA vaccination has arisen as effective strategy in the preventive medicine field with promising future prospects. The ability of plasmid DNA to activate the humoural and the cellular arms of the immune system against the encoded antigen have resulted in intensive study of new strategies aimed at increasing the DNA vaccine immunogenicity. Nevertheless, plasmid-based vaccines emerged as a safer and advantageous alternative with respect to viral vector vaccines. Recent advances in both the immunological and biotechnological research field made it possible to enhance significantly the DNA vaccine potency. Most of these approaches are based on both the discovery of novel delivery systems and the implementation of plasmid constructs, achieved through genetic engineering. In this review, we will describe some of the most relevant patents issued in the last ten years, supporting the progress made in naked-DNA vaccination against infectious diseases.

Adjuvants in vaccines and for immunisation: current trends.

Vaccines represent one of the most successful strategies in medical science. From the mechanistic perspective, vaccination works by manipulating the immune response through selecting, activating and expanding the memory of B and T cells. To determine the magnitude and quality of immune response, suitable vaccine adjuvants are required; therefore, much effort is going into finding new, effective and non-toxic adjuvant formulations focussed on the activation of key immune targets for inducing a long-term, potent and safe immune response. Significant research is being done in this area, to develop new classes of vaccines for use not only against infectious diseases, but also in the treatment of autoimmune disorders, allergies, chronic inflammatory diseases and cancer. Here the authors review and classify some of the main vaccine adjuvants on the basis of their immunomodulating properties on the immune system.

A combined analytical approach reveals novel EXT1/2 gene mutations in a large cohort of Italian multiple osteochondromas patients.

Multiple osteochondromas (MO), also known as hereditary multiple exostoses (HME), is one of the most common hereditary musculoskeletal diseases in Caucasians (1/50,000) with wide clinical variability and genetic heterogeneity. Two genes have thus far been identified as causing the disease, namely EXT1 and EXT2. Various methods to detect mutations in the EXT genes have been used. Here a cohort of 100 MO patients belonging to unrelated Italian families have been analyzed by single-strand conformation polymorphism (SSCP) analysis or by denaturing high performance liquid chromatography (DHPLC). However, neither of these techniques can detect deletions or duplications of entire exons. Families that were negative at SSCP/DHPLC analysis underwent two-color multiple ligation-dependent probe amplification (MLPA) analysis. By these complementary techniques mutation detection was significantly improved and 26 novel mutations have been revealed as well as 18 previously described mutations to give a total of 44 different mutations. Thus we can conclude that combining MLPA with DHPLC in point-mutations negative MO families, the detection of mutations in EXT genes can significantly improve the identification of both point-mutations and mid-size rearrangements. More important, we were able to characterize all those patients who were negative at the first PCR-based method screening.