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Abstract The histone deacetylase sirtuin 6 (SIRT6) has been endowed with anti-cancer capabilities in many tumor types. Here, we investigate the impact of SIRT6-overexpression (SIRT6-OE) in Delta16HER2 mice, which are a bona fide model of HER2-positive breast cancer. After an initial delay in the tumor onset, SIRT6-OE induces a more aggressive phenotype of Delta16HER2 tumors promoting the formation of higher number of tumor foci and metastases than controls. This phenotype of SIRT6-OE tumors is associated with cancer stem cell (CSC)-like features and tumor dormancy, and low senescence and oxidative DNA damage. Accordingly, a sub-set of HER2-positive breast cancer patients with concurrent SIRT6-OE has a significant poorer relapse-free survival (RFS) probability than patients with low expression of SIRT6. ChIP-seq, RNA-seq and RT-PCR experiments indicate that SIRT6-OE represses the expression of the T-box transcription factor 3 (Tbx3) by deacetylation of H3K9ac. Accordingly, loss-of-function mutations of TBX3 or low TBX3 expression levels are predictive of poor prognosis in HER2-positive breast cancer patients. Our work indicates that high levels of SIRT6 are indicative of poor prognosis and high risk of metastasis in HER2-positive breast cancer and suggests further investigation of TBX3 as a downstream target of SIRT6 and co-marker of poor-prognosis. Our results point to a breast cancer subtype-specific effect of SIRT6 and warrant future studies dissecting the mechanisms of SIRT6 regulation in different breast cancer subtypes.

DNA vaccination has been extensively studied as a promising strategy for tumor treatment. Despite the efforts, the therapeutic efficacy of DNA vaccines has been limited by their intrinsic poor cellular internalization. Electroporation, which is based on the application of a controlled electric field to enhance DNA penetration into cells, has been the method of choice to produce acceptable levels of gene transfer in vivo. However, this method may cause cell damage or rupture, non-specific targeting, and even degradation of pDNA. Skin irritation, muscle contractions, pain, alterations in skin structure, and irreversible cell damage have been frequently reported. To overcome these limitations, in this work, we use a microfluidic platform to generate DNA-loaded lipid nanoparticles (LNPs) which are then characterized by a combination of dynamic light scattering (DLS), synchrotron small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). Despite the clinical successes obtained by LNPs for mRNA and siRNA delivery, little is known about LNPs encapsulating bulkier DNA molecules, the clinical application of which remains challenging. For in vitro screening, LNPs were administered to human embryonic kidney 293 (HEK-293) and Chinese hamster ovary (CHO) cell lines and ranked for their transfection efficiency (TE) and cytotoxicity. The LNP formulation exhibiting the highest TE and the lowest cytotoxicity was then tested for the delivery of the DNA vaccine pVAX-hECTM targeting the human neoantigen HER2, an oncoprotein overexpressed in several cancer types. Using fluorescence-activated cell sorting (FACS), immunofluorescence assays and fluorescence confocal microscopy (FCS), we proved that pVAX-hECTM-loaded LNPs produce massive expression of the HER2 antigen on the cell membrane of HEK-293 cells. Our results provide new insights into the structure–activity relationship of DNA-loaded LNPs and pave the way for the access of this gene delivery technology to preclinical studies.

Background Tumor endothelial marker 1 (TEM1) is a protein expressed in the tumor-associated endothelium and/or stroma of various types of cancer. We previously demonstrated that immunization with a plasmid-DNA vaccine targeting TEM1 reduced tumor progression in three murine cancer models. Radiation therapy (RT) is an established cancer modality used in more than 50% of patients with solid tumors. RT can induce tumor-associated vasculature injury, triggering immunogenic cell death and inhibition of the irradiated tumor and distant non-irradiated tumor growth (abscopal effect). Combination treatment of RT with TEM1 immunotherapy may complement and augment established immune checkpoint blockade.Methods Mice bearing bilateral subcutaneous CT26 colorectal or TC1 lung tumors were treated with a novel heterologous TEM1-based vaccine, in combination with RT, and anti-programmed death-ligand 1 (PD-L1) antibody or combinations of these therapies, tumor growth of irradiated and abscopal tumors was subsequently assessed. Analysis of tumor blood perfusion was evaluated by CD31 staining and Doppler ultrasound imaging. Immunophenotyping of peripheral and tumor-infiltrating immune cells as well as functional analysis was analyzed by flow cytometry, ELISpot assay and adoptive cell transfer (ACT) experiments.Results We demonstrate that addition of RT to heterologous TEM1 vaccination reduces progression of CT26 and TC1 irradiated and abscopal distant tumors as compared with either single treatment. Mechanistically, RT increased major histocompatibility complex class I molecule (MHCI) expression on endothelial cells and improved immune recognition of the endothelium by anti-TEM1 T cells with subsequent severe vascular damage as measured by reduced microvascular density and tumor blood perfusion. Heterologous TEM1 vaccine and RT combination therapy boosted tumor-associated antigen (TAA) cross-priming (ie, anti-gp70) and augmented programmed cell death protein 1 (PD-1)/PD-L1 signaling within CT26 tumor. Blocking the PD-1/PD-L1 axis in combination with dual therapy further increased the antitumor effect and gp70-specific immune responses. ACT experiments show that anti-gp70 T cells are required for the antitumor effects of the combination therapy.Conclusion Our findings describe novel cooperative mechanisms between heterologous TEM1 vaccination and RT, highlighting the pivotal role that TAA cross-priming plays for an effective antitumor strategy. Furthermore, we provide rationale for using heterologous TEM1 vaccination and RT as an add-on to immune checkpoint blockade as triple combination therapy into early-phase clinical trials.

A class of phosphane gold(I) compounds, made of azoles and phosphane ligands, was evaluated for a screening on the regards of Breast Cancer cell panels (BC). The compounds possess N-Au-P or Cl-Au-P bonds around the central metal, and they differ for the presence of aprotic or protic polar groups in the azoles and/or the phosphane moieties to tune their hydrophilicity. Among the six candidates, only the compounds having the P-Au-N environment and not displaying neither the hydroxyl nor carboxyl groups in the ligands were found active. The compounds were screened by MTT tests in SKBR3, A17, and MDA-MB231 cancer cells, and two compounds (namely the 4,5-dicyano-imidazolate-1yl-gold(I)-(triphenylphosphane, 5, and 4,5-dichloro-imidazolate-1yl-gold(I)-triphenylphosphane, 6) were found very cytotoxic, with the most active with an IC50 value of 3.46 μM in MDA-MB231 cells. By performing enzymatic assays in the treated cells lysates, the residual enzymatic activity of dihydrofolate reductase (DHFR) has been measured after cell treatment for 4 or 12 h in comparison with control cells. Upon 12 h of treatment, the activity of DHFR was significantly reduced in both SKBR3 and A17 cells by compounds 5 and 6, but not in human MDA-MB231 cells; interestingly, it was found remarkably high after 4 h of treatment, revealing a time dependence for the DHFR enzymatic assays. The DHFR inhibition data have been compared to those for the thioredoxin reductase (TrxR), the most recognized molecular target for gold compounds. For this latter, similar residual activities (i.e., 37 and 49% for the match of SKBR3 cells and compound 5 or 6, respectively) were found. Binding studies on the regards of ct-DNA (calf-thymus-DNA) and of plasma transporters proteins, such as BSA (bovine serum albumin) and ATF (apo transferrin), were performed. As expected for gold compounds, the data support strong binding to proteins (Ksv values range: 1.51 ÷ 2.46 × 104 M−1) and a weaker interaction with ct-DNA's minor groove (Ksv values range: 1.55 ÷ 6.12 × 103 M−1).

Breast cancers (BCs) may present dramatic diagnoses, both for ineffective therapies and for the limited outcomes in terms of lifespan. For these types of tumors, the search for new drugs is a primary necessity. It is widely recognized that gold compounds are highly active and extremely potent as anticancer agents against many cancer cell lines. The presence of the metal plays an essential role in the activation of the cytotoxicity of these coordination compounds, whose activity, if restricted to the ligands alone, would be non-existent. On the other hand, gold exhibits a complex biochemistry, substantially variable depending on the chemical environments around the central metal. In this review, the scientific findings of the last 6–7 years on two classes of gold(I) compounds, containing phosphane or carbene ligands, are reviewed. In addition to this class of Au(I) compounds, the recent developments in the application of Auranofin in regards to BCs are reported. Auranofin is a triethylphosphine-thiosugar compound that, being a drug approved by the FDA—therefore extensively studied—is an interesting lead gold compound and a good comparison to understand the activities of structurally related Au(I) compounds.

In recent years, lipid nanoparticles (LNPs) have gained considerable attention in numerous research fields ranging from gene therapy to cancer immunotherapy and DNA vaccination. While some RNA-encapsulating LNP formulations passed clinical trials, DNA-loaded LNPs have been only marginally explored so far. To fulfil this gap, herein we investigated the effect of several factors influencing the microfluidic formulation and transfection behavior of DNA-loaded LNPs such as PEGylation, total flow rate (TFR), concentration and particle density at the cell surface. We show that PEGylation and post-synthesis sample concentration facilitated formulation of homogeneous and small size LNPs with high transfection efficiency and minor, if any, cytotoxicity on human Embryonic Kidney293 (HEK-293), spontaneously immortalized human keratinocytes (HaCaT), immortalized keratinocytes (N/TERT) generated from the transduction of human primary keratinocytes, and epidermoid cervical cancer (CaSki) cell lines. On the other side, increasing TFR had a detrimental effect both on the physicochemical properties and transfection properties of LNPs. Lastly, the effect of particle concentration at the cell surface on the transfection efficiency (TE) and cell viability was largely dependent on the cell line, suggesting that its case-by-case optimization would be necessary. Overall, we demonstrate that fine tuning formulation and microfluidic parameters is a vital step for the generation of highly efficient DNA-loaded LNPs.

p140Cap adaptor proteins interfere with adhesion and growth factor-dependent signalling in cancer cells but the mechanisms are unclear. Here the authors show that p140Cap interferes with ERBB2-dependent activation of Rac GTPase-controlled circuitries reducing metastasis and cancer progression.

In the ciliate Euplotes raikovi, a 631-amino acid Er-MAPK1 protein kinase was found to localize in nucleoli of the transcriptionally active nucleus (macronucleus) and act as a key component of an autocrine, cell-growth promoting self-signaling mechanism. While its 283-amino acid N-terminal domain includes all the structural specificities of the mitogen-activated protein kinases required for a catalytic function, the 348-amino acid C-terminal domain is structurally unique with undetermined functions. By expressing the two Er-MAPK1 domains tagged with the green fluorescent protein in mammalian fibroblasts, the yeast Schizosaccharomyces pombe and the ciliate Tetrahymena thermophila, evidence was obtained that the C-terminal domain contains all the sequence information responsible for the Er-MAPK1 subcellular localization. However, in fibroblasts and S. pombe this information determined a nucleolar localization of the GFP-tagged C-terminal domain, and a ciliary localization in T. thermophila. In the light of these findings, the Er-MAPK1 localization in E. raikovi was re-examined via immunoreactions and shown to be ciliary besides that nuclear, as is the case for the mammalian intestinal cell kinase with which the Er-MAPK1 N-terminal domain shares a strong sequence identity and a catalytic function.

Mosquitoes can transmit many infectious diseases, such as malaria, dengue, Zika, yellow fever, and lymphatic filariasis. Current mosquito control strategies are failing to reduce the severity of outbreaks that still cause high human morbidity and mortality worldwide. Great expectations have been placed on genetic control methods. Among other methods, genetic modification of the bacteria colonizing different mosquito species and expressing anti-pathogen molecules may represent an innovative tool to combat mosquito-borne diseases. Nevertheless, this emerging approach, known as paratransgenesis, requires a detailed understanding of the mosquito microbiota and an accurate characterization of selected bacteria candidates. The acetic acid bacteria Asaia is a promising candidate for paratransgenic approaches. We have previously reported that Asaia symbionts play a beneficial role in the normal development of Anopheles mosquito larvae, but no study has yet investigated the role(s) of Asaia in adult mosquito biology. Here we report evidence on how treatment with a highly specific anti-Asaia monoclonal antibody impacts the survival and physiology of adult Anopheles stephensi mosquitoes. Our findings offer useful insight on the role of Asaia in several physiological systems of adult mosquitoes, where the influence differs between males and females.

Objective: Available treatment for obesity and type 2 diabetes mellitus (T2DM) is suboptimal. Thus, identifying novel molecular target(s) exerting protective effects against these metabolic imbalances is of enormous medical significance. Sirt6 loss- and gain-of-function studies have generated confounding data regarding the role of this sirtuin on energy and glucose homeostasis, leaving unclear whether activation or inhibition of SIRT6 may be beneficial for the treatment of obesity and/or T2DM. Methods: To address these issues, we developed and studied a novel mouse model designed to produce eutopic and physiological overexpression of SIRT6 (Sirt6BAC mice). These mutants and their controls underwent several metabolic analyses. These include whole-blood reverse phase high-performance liquid chromatography assay, glucose and pyruvate tolerance tests, hyperinsulinemic-euglycemic clamp assays, and assessment of basal and insulin-induced level of phosphorylated AKT (p-AKT)/AKT in gastrocnemius muscle. Results: Sirt6BAC mice physiologically overexpress functionally competent SIRT6 protein. While Sirt6BAC mice have normal body weight and adiposity, they are protected from developing high-caloric-diet (HCD)-induced hyperglycemia and glucose intolerance. Also, Sirt6BAC mice display increased circulating level of the polyamine spermidine. The ability of insulin to suppress endogenous glucose production was significantly enhanced in Sirt6BAC mice compared to wild-type controls. Insulin-stimulated glucose uptake was increased in Sirt6BAC mice in both gastrocnemius and soleus muscle, but not in brain, interscapular brown adipose, or epididymal adipose tissue. Insulin-induced p-AKT/AKT ratio was increased in gastrocnemius muscle of Sirt6BAC mice compared to wild-type controls. Conclusions: Our data indicate that moderate, physiological overexpression of SIRT6 enhances insulin sensitivity in skeletal muscle and liver, engendering protective actions against diet-induced T2DM. Hence, the present study provides support for the anti-T2DM effect of SIRT6 and suggests SIRT6 as a putative molecular target for anti-T2DM treatment.

In veterinary medicine, there have been different experiences with the plasmid DNA vaccination. In this area and with the hypothesis to demonstrate the effectiveness of different plasmids encoding porcine respiratory and reproductive syndrome (PRRS), five DNA vaccines against PRRS were evaluated for their innocuity and efficacy in pigs. Eighteen animals were divided into five groups which were injected with five (A, B, C, D, E) different DNA vaccines. Albeit, none of the proposed vaccines were able to protect the animals against PRRS virus. Only vaccines A and B were able to reduce the clinical signs of the infection. ELISA IgM were detected 30 days after the first vaccination in the pigs injected by Vaccine A or B. ELISA IgG were detected 90 days after the first vaccination in the pigs injected by Vaccine B or C. Neutralizing antibody were detected Post Challenge Days 61 (PCD) in all groups. In the pigs inoculated with Vaccine C, IFN-g were detected 90 days after first vaccination, and after challenge exposure they increased. In the other groups, the IFN-g were detected after challenge infection. Pigs injected with each of the vaccines A, B, C, D and E showed a significantly higher level of CD4−CD8+ lymphocytes (p < 0.001) after infection in comparison with their controls.

Nature Communications 8: Article number: 14797 (2017); Published online 16 March 2017; Updated 30 March 2018 In the original version of this Article, the affiliation details for Anna Sapino were incorrectly given as Department of Medical Sciences, University of Torino, 10126 Torino, Italy instead ofCandiolo Cancer Institute-FPO, IRCCS, Str.

Skin represents an attractive target for DNA vaccine delivery because of its natural richness in APCs, whose targeting may potentiate the effect of vaccination. Nevertheless, intramuscular electroporation is the most common delivery method for ECTM vaccination. In this study we assessed whether intradermal administration could deliver the vaccine into different cell types and we analyzed the evolution of tissue infiltrate elicited by the vaccination protocol. Intradermal electroporation (EP) vaccination resulted in transfection of different skin layers, as well as mononuclear cells. Additionally, we observed a marked recruitment of reactive infiltrates mainly 6–24 hours after treatment and inflammatory cells included CD11c+. Moreover, we tested the efficacy of intradermal vaccination against Her2/neu antigen in cellular and humoral response induction and consequent protection from a Her2/neu tumor challenge in Her2/neu nontolerant and tolerant mice. A significant delay in transplantable tumor onset was observed in both BALB/c (p≤0,0003) and BALB-neuT mice (p=0,003). Moreover, BALB-neuT mice displayed slow tumor growth as compared to control group (p

Contrast-enhanced ultrasound (CEUS) is an advanced approach to in vivo assessment of tumor vascularity and is being increasingly adopted in clinical oncology. It is based on 1- to 10 μm-sized gas microbubbles, which can cross the capillary beds of the lungs and are effective echo enhancers. It is known that high cell density, high transendothelial fluid exchange, and poorly functioning lymphatic circulation all provoke solid stress, which compresses vessels and drastically reduces tumor blood flow. Given their size, we supposed that the perfusion of microbubbles is affected by anatomic features of tumor vessels more than are contrast agents traditionally used in dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Here, we compared dynamic information obtained from CEUS and DCE-MRI on two experimental tumor models exhibiting notable differences in vessel anatomy. We found that tumors with small, flattened vessels show a much higher resistance to microbubble perfusion than to MRI contrast agents, and appear scarcely vascularized at CEUS examination, despite vessel volume adequate for normal function. Thus, whereas CEUS alone could induce incorrect diagnosis when tumors have small or collapsed vessels, integrated analysis using CEUS and DCE-MRI allows in vivo identification of tumors with a vascular profile frequently associated with malignant phenotypes.

We developed a new phage-display based approach, the Large Fragment Phage Display (LFPD), that can be used for mapping conformational epitopes on target molecules of immunological interest. LFPD uses a simplified and more effective phage-display approach in which only a limited set of larger fragments (about 100 aa in length) are expressed on the phage surface. Using the human HER2 oncoprotein as a target, we identified novel B-cell conformational epitopes. The same homologous epitopes were also detected in rat HER2 and all corresponded to the epitopes predicted by computational analysis (PEPITO software), showing that LFPD gives reproducible and accurate results. Interestingly, these newly identified HER2 epitopes seem to be crucial for an effective immune response against HER2-overexpressing breast cancers and might help discriminating between metastatic breast cancer and early breast cancer patients. Overall, the results obtained in this study demonstrated the utility of LFPD and its potential application to the detection of conformational epitopes on many other molecules of interest, as well as, the development of new and potentially more effective B-cell conformational epitopes based vaccines.

Several transgenic mice models solidly support the hypothesis that HER2 (ERBB2) overexpression or mutation promotes tumorigenesis. Recently, a HER2 splice variant lacking exon-16 (Δ16HER2) has been detected in human breast carcinomas. This alternative protein, a normal byproduct of HER2, has an increased transforming potency compared to wild-type (wt) HER2 receptors. To examine the ability of Δ16HER2 to transform mammary epithelium in vivo and to monitor Δ16HER2-driven tumorigenesis in live mice, we generated and characterized a mouse line that transgenically expresses both human Δ16HER2 and firefly luciferase under the transcriptional control of the MMTV promoter. All the transgenic females developed multifocal mammary tumors with a rapid onset and an average latency of 15.11 weeks. Immunohistochemical analysis revealed the concurrent expression of luciferase and the human Δ16HER2 oncogene only in the mammary gland and in strict correlation with tumor development. Transgenic Δ16HER2 expressed on the tumor cell plasma membrane from spontaneous mammary adenocarcinomas formed constitutively active homodimers able to activate the oncogenic signal transduction pathway mediated through Src kinase. These new transgenic animals demonstrate the ability of the human Δ16HER2 isoform to transform "per se" mammary epithelium in vivo. The high tumor incidence as well as the short latency strongly suggests that the Δ16HER2 splice variant represents the transforming form of the HER2 oncoprotein.

BackgroundMounting clinical and experimental evidence suggests that the shift of carcinomas towards a mesenchymal phenotype is a common paradigm for both resistance to therapy and tumor recurrence. However, the mesenchymalization of carcinomas has not yet entered clinical practice as a crucial diagnostic paradigm.Methodology/principal findingsBy integrating in silico and in vitro studies with our epithelial and mesenchymal tumor models, we compare herein crucial molecular pathways of previously described carcinoma-derived mesenchymal tumor cells (A17) with that of both carcinomas and other mesenchymal phenotypes, such as mesenchymal stem cells (MSCs), breast stroma, and various types of sarcomas. We identified three mesenchymal/stromal-signatures which A17 cells shares with MSCs and breast stroma. By using a recently developed computational approach with publicly available microarray data, we show that these signatures: 1) significantly relates to basal-like breast cancer subtypes; 2) significantly relates to bone metastasis; 3) are up-regulated after hormonal treatment; 4) predict resistance to neoadjuvant therapies.Conclusions/significanceOur results demonstrate that mesenchymalization is an intrinsic property of the most aggressive tumors and it relates to therapy resistance as well as bone metastasis.