Subcellular Targeting of the Euplotes raikovi Kinase Er-MAPK1, as Revealed by Expression in Different Cell Systems.

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.

In vivo and in silico studies to identify mechanisms associated with Nurr1 modulation following early life exposure to permethrin in rats.

The present work was designed to study the mechanisms associated with Nurr1 modulation following early life permethrin (PERM) treatment during rat's life span. Here we demonstrate that PERM exposure in rats, at a dose close to No Observed Adverse Effect Level (NOAEL) for 15days during neonatal brain development leads to its accumulation long after exposure. In striatum from adolescent rats we detected an increase in DNA methyltransferases (DNMTs) such as DNMT1, DNMT3a, Tyrosine hydroxylase, monomeric and aggregated α-synuclein protein levels. Adult rats showed enhanced DNMT3b and α-synuclein aggregation compared to the control group, while with aging a significant decrease in all biomarkers studied was observed. No changes in Nurr1 promoter methylation in adolescent, adult and old rats were found. In silico studies showed clear evidence of a strong binding interaction between PERM and its metabolite 3-phenoxybenzoic acid with the nuclear orphan receptor Nurr1. These findings suggest that an additional interference with the dopaminergic neuron pathway could occur in situ during PERM accumulation in brain. Therefore, Nurr1 modulation in early life PERM-treated rats, depends on age-related adaptive responses in animals.

The water soluble ruthenium(II) organometallic compound [Ru(p-cymene)(bis(3,5 dimethylpyrazol-1-yl)methane)Cl]Cl suppresses triple negative breast cancer growth by inhibiting tumor infiltration of regulatory T cells.

Ruthenium compounds have become promising alternatives to platinum drugs by displaying specific activities against different cancers and favorable toxicity and clearance properties. Here, we show that the ruthenium(II) complex [Ru(p-cymene)(bis(3,5-dimethylpyrazol-1-yl)methane)Cl]Cl (UNICAM-1) exhibits potent in vivo antitumor effects. When administered as four-dose course, by repeating a single dose (52.4mgkg-1) every three days, UNICAM-1 significantly reduces the growth of A17 triple negative breast cancer cells transplanted into FVB syngeneic mice. Pharmacokinetic studies indicate that UNICAM-1 is rapidly eliminated from kidney, liver and bloodstream thanks to its high hydrosolubility, exerting excellent therapeutic activity with minimal side effects. Immunohistological analysis revealed that the efficacy of UNICAM-1, mainly relies on its capacity to reverse tumor-associated immune suppression by significantly reducing the number of tumor-infiltrating regulatory T cells. Therefore, UNICAM-1 appears very promising for the treatment of TNBC.

Identification of relevant conformational epitopes on the HER2 oncoprotein by using Large Fragment Phage Display (LFPD).

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.

Early life permethrin exposure induces long-term brain changes in Nurr1, NF-kB and Nrf-2.

Pesticide exposure during brain development represents an important risk factor for the onset of brain-aging processes. Here, the impact of permethrin administered to rats from 6th to 21st day of life, at a dose near to "no observed adverse effect level" (NOAEL), was studied when animals reached 500 day-old. The permethrin treatment induced a decrease in Nurr1 gene expression in striatum, an increase in hippocampus and cerebellum, while the protein level changed only in striatum where it was increased. NF-kB p65 gene expression was increased in cerebellum, while its protein level augmented in cerebellum and in prefrontal cortex and decreased in hippocampus of treated rats compared to control ones. Nrf-2 gene expression resulted significantly higher only in cerebellum of treated animals. The results suggest that early life permethrin treatment induces long-lasting effects leading to dopaminergic neuronal disorders, monitored by Nurr1 alteration. Moreover the impairment of NF-kB and Nrf-2, important for the balance between pro- and anti-inflammatory systems, confirms that the neonatal permethrin treatment can influence genes involved with the onset of brain-ageing processes.

p130Cas/Cyclooxygenase-2 axis in the control of mesenchymal plasticity of breast cancer cells.

INTRODUCTION: Intrinsic plasticity of breast carcinoma cells allows them to undergo a transient and reversible conversion into mesenchymal cells to disseminate into distant organs, where they can re-differentiate to an epithelial-like status to form a cohesive secondary mass. The p130Cas scaffold protein is overexpressed in human ER+ and HER2+ breast cancer where it contributes to cancer progression, invasion and resistance to therapy. However, its role in regulating mesenchymal aggressive breast cancer cells remains to be determined. The aim of this study was to investigate the molecular and functional involvement of this adaptor protein in breast cancer cell plasticity. METHODS: We used silencing strategies and rescue experiments to evaluate phenotypic and biochemical changes from mesenchymal to epithelial traits in breast tumor cell lines. In the mouse A17 cell model previously related to mesenchymal cancer stem cells and basal-like breast cancer, we biochemically dissected the signaling pathways involved and performed functional in vivo tumor growth ability assays. The significance of the signaling platform was assessed in a human setting through the use of specific inhibitors in aggressive MDA-MB-231 subpopulation LM2-4175 cells. To evaluate the clinical relevance of the results, we analyzed publicly available microarray data from the Netherlands Cancer Institute and from the Koo Foundation Sun Yat-Sen Cancer Center. RESULTS: We show that p130Cas silencing induces loss of mesenchymal features, by downregulating Vimentin, Snail, Slug and Twist transcriptional factors, resulting in the acquirement of epithelial-like traits. Mechanistically, p130Cas controls Cyclooxygenase-2 transcriptional expression, which in turn contributes to p130Cas-dependent maintenance of mesenchymal phenotype. This cascade of events also compromises in vivo tumor growth through inhibition of cell signaling controlling cell cycle progression. c-Src and JNK kinases are sequential players in p130Cas/ Cyclooxygenase-2 axis and their pharmacological inhibition is sufficient to downregulate Cyclooxygenase-2 leading to an epithelial phenotype. Finally, in silico microarray data analysis indicates that p130Cas and Cyclooxygenase-2 concomitant overexpression predicts poor survival and high probability of breast tumor recurrence. CONCLUSIONS: Overall, these data identify a new p130Cas/Cyclooxygenase-2 axis as a crucial element in the control of breast tumor plasticity, opening new therapeutic strategies leading to inhibition of these pathways in aggressive breast carcinoma.

Leukocyte Nurr1 as peripheral biomarker of early-life environmental exposure to permethrin insecticide.

The effect of a low dose of the insecticide permethrin administered during early-life was evaluated on leukocytes inflammation mediators on 300- and 500-day-old rats. Nurr1, NF-κB-p65, Nrf2, lipid peroxidation and GSH levels increased with age but compared to the control group, treatment with permethrin induced a significant increase only of Nurr1 and lipid peroxidation in oldest rats. TNF-α and Rantes increased, while IL-1ß, IL-2, IL-13 decreased in oldest treated rats. The results propose Nurr1, TNF-α, Rantes, GSH and plasma lipid peroxidation as peripheral biomarkers for monitoring the impact of early-life environmental exposure to xenobiotics in old age.

The impact of early life permethrin exposure on development of neurodegeneration in adulthood.

Early life environmental exposure to pesticides could play a critical role in the onset of age-related diseases. The present study aims to evaluate in brain, plasma and leukocytes of 300 day-old rats, the effect of a low dose of the insecticide permethrin administered during early life (1/50 LD(50), from 6th to 21st day of life). The outcomes show that Nurr1, mRNA and protein expression, as well as calcium and NO levels are decreased in striatum. Moreover, the pesticide induces an imbalance in glutamate, calcium and NO in hippocampus. Low calcium concentrations in leukocytes and in plasma were observed, while increased NO and decreased SOD plasma levels were measured. The results suggest that permethrin intake at a dose close to the NOAEL (25 mg/kg) during the perinatal period can interact with Nurr1 by reducing its expression on striatum nucleus. Consequently, the maintenance of dopaminergic neurons as well as Nurr1 inhibitory effect on the production of proinflammatory mediators fails. The changes in biological markers found in our animal model could represent the basis to study neurodegenerative diseases whose development depends on individual gene signature and life style.

Factors determining the superior performance of lipid/DNA/protammine nanoparticles over lipoplexes.

The utility of using a protammine/DNA complex coated with a lipid envelope made of cationic 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) for transfecting CHO (Chinese hamster ovary cells), HEK293 (human embryonic kidney cells), NIH 3T3 (mouse embryonal cells), and A17 (murine cancer cells) cells was examined. The widely used DOTAP/DNA lipoplex was employed as a reference. In all the tested cell lines lipid/protamine/DNA (LPD) nanoparticles were more efficient in transfecting cells than lipoplexes even though the lipid composition of the lipid envelope was the same in both devices. Physical-chemical properties were found to control the ability of nanocarriers to release DNA upon interaction with cellular membranes. LPD complexes easily release their DNA payload, while lipoplexes remain largely intact and accumulate at the cell nucleus. Collectively, these data explain why LPD nanoparticles often exhibit superior performances compared to lipoplexes in trasfecting cells and represent a promising class of nanocarriers for gene delivery.

The human splice variant Δ16HER2 induces rapid tumor onset in a reporter transgenic mouse.

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.

Mesenchymal/stromal gene expression signature relates to basal-like breast cancers, identifies bone metastasis and predicts resistance to therapies.

BACKGROUND: Mounting 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 FINDINGS: By 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/SIGNIFICANCE: Our results demonstrate that mesenchymalization is an intrinsic property of the most aggressive tumors and it relates to therapy resistance as well as bone metastasis.

Tailoring lipoplex composition to the lipid composition of plasma membrane: a Trojan horse for cell entry?

The first interaction between lipoplexes and cells is charge-mediated and not specific. Endocytosis is considered to be the main pathway for lipoplex entry. Upon interaction between lipoplexes and the plasma membrane, intermixing between lipoplex and membrane lipids is necessary for efficient endocytosis. Here we study the mechanism of the different endocytic pathways in lipid-mediated gene delivery. We show that DC-Chol-DOPE/DNA lipoplexes preferentially use a raft-mediated endocytosis, while DOTAP-DOPC/DNA systems are mainly internalized by not specific fluid phase macropinocitosys. On the other hand, most efficient multicomponent lipoplexes, incorporating different lipid species in their lipid bilayer, can use multiple endocytic pathways to enter cells. Our data demonstrate that efficiency of endocytosis is regulated by shape coupling between lipoplex and membrane lipids. We suggest that such a shape-dependent coupling regulates efficient formation of endocytic vesicles thus determining the success of internalization. Our results suggest that tailoring the lipoplex lipid composition to the patchwork-like plasma membrane profile could be a successful machinery of coordinating the endocytic pathway activities and the subsequent intracellular processing.

p130Cas is an essential transducer element in ErbB2 transformation.

The ErbB2 oncogene is often overexpressed in breast tumors and associated with poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration, and proliferation in normal and pathological cells. The functional role of p130Cas in ErbB2-dependent breast tumorigenesis was assessed by its silencing in breast cancer cells derived from mouse mammary tumors overexpressing ErbB2 (N202-1A cells), and by its reexpression in ErbB2-transformed p130Cas-null mouse embryonic fibroblasts. We demonstrate that p130Cas is necessary for ErbB2-dependent foci formation, anchorage-independent growth, and in vivo growth of orthotopic N202-1A tumors. Moreover, intranipple injection of p130Cas-stabilized siRNAs in the mammary gland of Balbc-NeuT mice decreases the growth of spontaneous tumors. In ErbB2-transformed cells, p130Cas is a crucial component of a functional molecular complex consisting of ErbB2, c-Src, and Fak. In human mammary cells, MCF10A.B2, the concomitant activation of ErbB2, and p130Cas overexpression sustain and strengthen signaling, leading to Rac1 activation and MMP9 secretion, thus providing invasive properties. Consistently, p130Cas drives N202-1A cell in vivo lung metastases colonization. These results demonstrate that p130Cas is an essential transducer in ErbB2 transformation and highlight its potential use as a novel therapeutic target in ErbB2 positive human breast cancers.

A better immune reaction to Erbb-2 tumors is elicited in mice by DNA vaccines encoding rat/human chimeric proteins.

The Erbb-2 (neu in rat and Her-2 in humans) tyrosine kinase receptor is an oncoantigen (i.e., a tumor-associated molecule directly involved in cancer progression). Because oncoantigens are self-tolerated molecules, to trigger a response circumventing tolerance, we generated two plasmids (RHuT and HuRT) coding for chimeric neu-Her-2 extracellular and transmembrane proteins that are expressed on the cell membrane of the transfected cells and recognized by monoclonal antibodies reacting against neu and Her-2. RHuT encodes a protein in which the 410 NH(2)-terminal residues are from the neu extracellular domain and the remaining residues from Her-2. Almost symmetrically, HuRT encodes for a protein in which the 390 NH(2)-terminal residues are from Her-2 and the remainder from neu. The ability of RHuT and HuRT to elicit a protective response to neu and Her-2 in wild-type mice and in transgenic mice tolerant to neu and Her-2 proteins was compared with that of plasmids coding for the fully rat or fully human extracellular and transmembrane domains of the Erbb-2 receptor. In most cases, RHuT and HuRT elicited a stronger response, although this chimeric benefit is markedly modulated by the location of the heterologous moiety in the protein coded by the plasmid, the immune tolerance of the responding mouse, and the kind of Erbb-2 orthologue on the targeted tumor.

Structural stability and increase in size rationalize the efficiency of lipoplexes in serum.

We have investigated the effect of serum on nanometric structure, size, surface potential, DNA-binding capacity, and transfection efficiency of DDAB-DOPE/DNA and DC-Chol-DOPE/DNA lipoplexes as a function of membrane charge density and cationic lipid/DNA charge ratio. In the absence of serum, the nanometric structure and DNA binding capacity of lipoplexes determined the transfection efficiency. When serum was added, the transfection efficiency of all lipoplex formulations was found to increase. We identified structural stability and an increase in size in serum as major parameters regulating the efficiency of lipofection. By extrapolation, we propose that serum, regulating the size of resistant lipid-DNA complexes, can control the mechanism of internalization of lipoplexes and, in turn, their efficiency.

Toward the rational design of lipid gene vectors: shape coupling between lipoplex and anionic cellular lipids controls the phase evolution of lipoplexes and the efficiency of DNA release.

A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interaction with anionic cellular lipids, resulting in DNA release. At the early stages of interaction, we found a universal behavior of lipoplex/anionic lipid (AL) mixtures: the lipoplex structure is slightly perturbed, while the one-dimensional DNA lattice between cationic membranes is largely diluted by ALs. This finding is in excellent agreement with previous suggestions on the mechanism of DNA unbinding from lipoplexes by ALs. Upon further interaction, the propensity of a given lipoplex structure to be solubilized by anionic cellular lipids strongly depends on the shape coupling between lipoplex and ALs. Furthermore, we investigated the effect of the membrane charge density and a general correlation resulted: the higher the membrane charge density of anionic membranes, the higher their ability to solubilize the structure of lipoplexes and to promote DNA release. Lastly, the formation of nonlamellar phases in lipoplex/AL mixtures is regulated by the propensity of anionic cellular lipids to adopt nonlamellar phases. Remarkably, also phase transition rates and DNA release were found to be strongly affected by the shape coupling between lipoplex and ALs. It thus seems likely that the structural and phase evolution of lipoplexes may only be meaningful in the context of specific anionic cellular membranes. These results highlight the phase properties of the carrier lipid/cellular lipid mixtures as a decisive factor for optimal DNA release and suggest a potential strategy for the rational design of efficient cationic lipid carriers.

Enhanced transfection efficiency of multicomponent lipoplexes in the regime of optimal membrane charge density.

Recently, membrane charge density of lipid membranes, sigma M, has been recognized as a universal parameter that controls the transfection efficiency of complexes made of binary cationic liposomes and DNA (binary lipoplexes). Three distinct regimes, most likely related to interactions between complexes and cells, have also been identified. The purpose of this work was to investigate the transfection efficiency behavior of multicomponent lipoplexes in the regime of optimal membrane charge density (1< sigma M < 2 x 10 (-2) e/A (2)) and compare their performance with that of binary lipoplexes usually employed for gene delivery purposes. We found remarkable differences in transfection efficiency due to lipid composition, with maximum in efficiency being obtained when multicomponent lipoplexes were used to transfect NIH 3T3 cells, while binary lipoplexes were definitely less efficient. These findings suggested that multicomponent systems are especially promising lipoplex candidates. With the aim of providing new insights into the mechanism of transfection, we investigated the structural evolution of lipoplexes when interacting with anionic (cellular) lipids by means of synchrotron small-angle X-ray diffraction (SAXD), while the extent of DNA release upon interaction with anionic lipids was measured by electrophoresis on agarose gels. Interestingly, a clear trend was found that the transfection activity increased with the number of lipid components. These results highlight the compositional properties of carrier lipid/cellular lipid mixtures as decisive factors for transfection and suggest a strategy for the rational design of superior cationic lipid carriers.

Protective immunity against neu-positive carcinomas elicited by electroporation of plasmids encoding decreasing fragments of rat neu extracellular domain.

We have shown that electroporation of plasmid carrying extracellular and transmembrane domains (EC-TM plasmid) encoded by the rat neu oncogene triggers a protective immune response toward rat p185(neu)-positive tumors in both wild-type BALB/c mice and cancer-prone rat neu-transgenic BALB-neuT mice. To identify the critical fragments that confer this protective immunity, mice were electroporated with plasmids encoding the TM domain associated with decreasing fragments of the EC domain and the antitumor protection afforded, the titer of antibody, and cytotoxic T lymphocyte (CTL) activity elicited to Neu protein were evaluated. Plasmids encoding EC fragments shortened by 70 (EC1-TM plasmid), 150 (EC2-TM), 230 (EC3-TM), 310 (EC4-TM), and 390 (EC5-TM) NH(2)-terminal residues afforded effective protection. Plasmids encoding shorter truncated proteins were ineffective. When the immunogenic protein was retained in the cytoplasm (EC1-TM, EC2-TM, and EC5-TM), only a CTL response was elicited, whereas when it was also expressed on the membrane (EC4-TM) both CTLs and antibodies were induced. EC4-TM encoding a truncated protein with an EC portion of only 344 amino acids conferred protection on both BALB/c and BALB-neuT mice comparable to that of EC-TM.