Precision digital mapping of endogenous and induced genomic DNA breaks by INDUCE-seq.

Understanding how breaks form and are repaired in the genome depends on the accurate measurement of the frequency and position of DNA double strand breaks (DSBs). This is crucial for identification of a chemical's DNA damage potential and for safe development of therapies, including genome editing technologies. Current DSB sequencing methods suffer from high background levels, the inability to accurately measure low frequency endogenous breaks and high sequencing costs. Here we describe INDUCE-seq, which overcomes these problems, detecting simultaneously the presence of low-level endogenous DSBs caused by physiological processes, and higher-level recurrent breaks induced by restriction enzymes or CRISPR-Cas nucleases. INDUCE-seq exploits an innovative NGS flow cell enrichment method, permitting the digital detection of breaks. It can therefore be used to determine the mechanism of DSB repair and to facilitate safe development of therapeutic genome editing. We further discuss how the method can be adapted to detect other genomic features.

Pandemic knowledge and regulation effectiveness: Evidence from COVID-19.

The spread of COVID-19 led countries around the world to adopt lockdown measures of varying stringency, with the purpose of restricting the movement of people. However, the effectiveness of these measures on mobility has been markedly different. Employing a difference-in-differences design, we analyse the effectiveness of movement restrictions across different countries. We disentangle the role of regulation (stringency measures) from the role of people's knowledge about the spread of COVID-19. We proxy COVID-19 knowledge by using Google Trends data on the term "Covid". We find that lockdown measures have a higher impact on mobility the more people learn about COVID-19. This finding is driven by countries with low levels of trust in institutions and low levels of education.

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.

In Vivo Evaluation of a New Recombinant Hyaluronidase to Improve Gene Electro-Transfer Protocols for DNA-Based Drug Delivery against Cancer.

Cancer vaccines based on plasmid DNA represent a good therapeutic perspective, despite their low potency. Animal-derived hyaluronidases (Hyals) are employed in oncological clinical practice. Hyal has been also demonstrated to be a good enhancer of intramuscular Gene Electro-Transfer (GET) efficiency in anti-cancer preclinical protocols, with increased transfected cells and higher expression of the encoded genes. Nevertheless, the use of animal-derived Hyals results limited respect to their potentialities, since such preparations could be affected by low purity, variable potency and uncertain safety. To improve the delivery of intramuscular GET-based protocols in mouse, we investigated a new recombinant Hyal, the rHyal-sk, to assess in vivo safety and activity of this treatment at cellular and biochemical levels. We evaluated the cellular events and the inflammation chemical mediators involved at different time points after rHyal-sk administration plus GET. Our results demonstrated the in vivo safety and efficacy of rHyal-sk when injected once intramuscularly in association with GET, with no toxicity, good plasmid in-take ability, useful inflammatory response activation, and low immunogenicity. Following these findings, we would recommend the use of the new rHyal-sk for the delivery of DNA-based vaccines and immunotherapy, as well as into clinical practice, for tumor disease treatments.

Gene Electrotransfer of Plasmid-Encoding IL-12 Recruits the M1 Macrophages and Antigen-Presenting Cells Inducing the Eradication of Aggressive B16F10 Murine Melanoma.

Cancer immunotherapy is currently one of the leading approaches in cancer treatment. Gene electrotransfer of plasmids encoding interleukin 12 (IL-12) into the cells leads to the production of IL-12, which drives immune cell polarization to an antitumoral response. One of the cell types that shows great promise in targeting tumor cells under the influence of IL-12 cytokine milieu is that of macrophages. Therefore, the aim of this study was to evaluate gene electrotransfer of antibiotic resistance-free plasmid DNA-encoding murine IL-12 (mIL-12) in mice bearing aggressive B16F10 murine melanoma. IL-12 electrotransfer resulted in the complete long-term eradication of the tumors. Serum mIL-12 and murine interferon γ (mIFNγ) were increased after IL-12 gene electrotransfer. Further on, hematoxylin and eosin (HE) staining showed increased infiltration of immune cells that lasted from day 4 until day 14. Immunohistochemistry (IHC) staining of F4/80, MHCII, and CD11c showed higher positive staining in the IL-12 gene electrotransfer group than in the control groups. Immune cell infiltration into the tumors and the high density of MHCII- and CD11c-positive cells suggest an antitumor polarization of macrophages and the presence of antigen-presenting cells that contributes to the important antitumor effectiveness of IL-12.

Dysregulation of EGFR Pathway in EphA2 Cell Subpopulation Significantly Associates with Poor Prognosis in Colorectal Cancer.

PURPOSE: EphA2 receptor is involved in multiple cross-talks with other cellular networks, including EGFR, FAK, and VEGF pathways, with which it collaborates to stimulate cell migration, invasion, and metastasis. Colorectal cancer (CRC) EphA2 overexpression has also been correlated to stem-like properties of cells and tumor malignancy. We investigated the molecular cross-talk and miRNAs modulation of the EphA2 and EGFR pathways. We also explored the role of EphA2/EGFR pathway mediators as prognostic factors or predictors of cetuximab benefit in patients with CRC. EXPERIMENTAL DESIGN: Gene expression analysis was performed in EphA2high cells isolated from CRC of the AOM/DSS murine model by FACS-assisted procedures. Six independent cohorts of patients were stratified by EphA2 expression to determine the potential prognostic role of a EphA2/EGFR signature and its effect on cetuximab treatment response. RESULTS: We identified a gene expression pattern (EphA2, Efna1, Egfr, Ptpn12, and Atf2) reflecting the activation of EphA2 and EGFR pathways and a coherent dysregulation of mir-26b and mir-200a. Such a pattern showed prognostic significance in patients with stage I-III CRC, in both univariate and multivariate analysis. In patients with stage IV and WT KRAS, EphA2/Efna1/Egfr gene expression status was significantly associated with poor response to cetuximab treatment. Furthermore, EphA2 and EGFR overexpression showed a combined effect relative to cetuximab resistance, independently from KRAS mutation status. CONCLUSIONS: These results suggest that EphA2/Efna1/Egfr genes, linked to a possible control by miR-200a and miR-26b, could be proposed as novel CRC prognostic biomarkers. Moreover, EphA2 could be linked to a mechanism of resistance to cetuximab alternative to KRAS mutations. Clin Cancer Res; 23(1); 159-70. ©2016 AACR.

Novel insights into Notum and glypicans regulation in colorectal cancer.

The connection between colorectal cancer (CRC) and Wnt signaling pathway activation is well known, but full elucidation of the underlying regulation of the Wnt/ß-catenin pathway and its biological functions in CRC pathogenesis is still needed. Here, the azoxymethane/dextran sulfate sodium salt (AOM/DSS) murine model has been used as an experimental platform able to mimic human sporadic CRC development with predictable timing. We performed genome-wide expression profiling of AOM/DSS-induced tumors and normal colon mucosa to identify potential novel CRC biomarkers. Remarkably, the enhanced expression of Notum, a conserved feedback antagonist of Wnt, was observed in tumors along with alterations in Glypican-1 and Glypican-3 levels. These findings were confirmed in a set of human CRC samples. Here, we provide the first demonstration of significant changes in Notum and glypicans gene expression during CRC development and present evidence to suggest them as potential new biomarkers of CRC pathogenesis.