Abstract Background Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with a 5-year survival rate of 6%, emphasizing the need for new therapeutic targets and prognostic biomarkers. The damage-specific DNA-binding protein 2 (DDB2) is involved in damaged-DNA repair through the Nucleotide Excision Repair system and has been recently demonstrated as playing a role in mammary, ovarian and colorectal carcinogenesis by stimulating tumour growth, inhibiting migration and invasion and sensitizing tumour cells to chemotherapy. The potent role of DDB2 on pancreatic cancer development has not been explored yet. Given the induction of TGF-β signalling pathway by DDB2 and the TGF-β pathway activation in 47% of PDAC according to the TCGA, we aim to evaluate the role of DDB2 in PDAC. Methods DDB2 expression level was determined in 3 PDAC cell-lines (T3M4, BxPC3, Capan-2) by Western blot and RT-PCR. Knock-out and knockdown DDB2 models were established from DDB2-overexpressing cells (using CRISPR-Cas9 technology and ShRNAs respectively) to evaluate the role of DDB2 protein in cancer cell proliferation, differentiation potential, migration and invasion. Results Overexpression of DDB2 was found in T3M4 and BxPC3 cells and under expression in Capan-2 cells (p  Conclusions These preliminary results show the potent role of DDB2 in PDAC cells proliferation and the regulation of epithelial-mesenchymal transition, cell motility and invasiveness. The next steps of our work will be to explore DDB2 regulation mechanisms to understand whether its expression may be modified using targeted therapies and to elucidate its role as a prognosis marker in fresh frozen samples of patients with PDAC. Legal entity responsible for the study Universite de Lorraine, CNRS UMR 7039 CRAN, Institut de Cancerologie de Lorraine. Funding «Action Incitative du CRAN» funded by the CNRS UMR 7039 CRAN. Disclosure All authors have declared no conflicts of interest.

Introduction Advanced breast cancers do not respond well to therapies and represent a relevant focus for studying molecular mechanisms involved in the tumour progression and drug resistance. The transcription factor NF-κB is often activated constitutively in aggressive breast cancer cells and plays a significant role by inducing many target genes involved in tumour progression and drug resistance. Mechanisms controlling constitutive NF-kB activation are not all clearly understood. Among them, repression of the gene encoding the NF-κB inhibitor, IκBα is not well known. This protein controls NF-κB activation by sequestering it in the cytoplasmic compartment. The present study reports the identification of the hnRNP K/J protein, which is initially known for its role in mRNA splicing and translation, as a repressor of the IκBα gene expression. Material and methods Identification of hnRNP K/J protein on the IκBα promoter was carried out by DNA pull-down coupled with a mass spectrometry analysis, and chromatin immunoprecipitation (ChIP) using a specific polyclonal antibodies. The hnRNP K/J protein was overexpressed in breast cancer cell lines by transient transfection, and consequence on the IκBα expression and the proximal IκBα promoter activity was evaluated by RT-qPCR and gene reporter assay, respectively. The hnRNP K/J protein localization was visualised in cells by Western blotting using nuclear and cytoplasmic extracts. Results and discussions The IκBα gene is expressed higher in nonaggressive compared to aggressive breast cancer cells. We used previous data showing the importance of the proximal promoter at position −495 from the transcription site for DNA pull-down with nuclear protein extract from MCF-7 cells. Mass spectrometry analysis led to identify hnRNP K/J protein, whose the binding to this region of the proximal IκBα promoter was confirmed by ChIP. The IκBα expression at mRNA level and proximal IκBα promoter activity was strongly decreased in hnRNP K/J-overexpressing breast cancer cells, in contrast to the respective parental cells, suggesting the role of hnRNP K/J protein as a gene repressor. Conclusion The identification of hnRNP K/J as a repressor of IκBα gene expression depicts a new molecular mechanism, which may contribute to the high constitutive NF-kB activation in aggressive breast cancer cells and suggests to take it into account in the development of new therapies targeting NF-kB pathway in advanced breast cancers.

The DNA repair protein damaged DNA-binding 2 (DDB2) has been implicated in promoting cell-cycle progression by regulating gene expression. DDB2 is selectively overexpressed in breast tumor cells that are noninvasive, but not in those that are invasive. We found that its overexpression in invasive human breast tumor cells limited their motility and invasiveness in vitro and blocked their ability to colonize lungs in vivo, defining a new function for DDB2 in malignant progression. DDB2 overexpression attenuated the activity of NF-kB and the expression of its target matrix metalloprotease 9 (MMP9). Mechanistic investigations indicated that DDB2 decreased NF-kB activity by upregulating expression of IkBa by binding the proximal promoter of this gene. This effect was causally linked to invasive capacity. Indeed, knockdown of DDB2-induced IkBa gene expression restored NF-kB activity and MMP9 expression, along with the invasive properties of breast tumor cells overexpressing DDB2. Taken together, our findings enlighten understanding of how breast cancer cells progress to an invasive phenotype and underscore potential clinical interest inDDB2 as a prognosticmarker or therapeutic target in this setting. Cancer Res; 73(16); 5040–52. 2013 AACR. Introduction Development of metastatic disease is the primary cause of mortality in patients with breast cancer. This is a multistep event, comprising invasion of mammary carcinoma cells into the adjacent tissues, entry of tumor cells in the systemic circulation, extravasation to distant organs, and finally metastatic colonization, mainly in lungs, liver, bones, and the central nervous system (1). Despite significant advances in diagnosing and treating breast cancer, one of themajor clinical and scientific problems that remain unresolved is the prediction of breast tumor progression toward metastasis. Also, identification of new predictive markers of metastatic development seems relevant. We described recently that the damaged DNA-binding 2 (DDB2) protein, whichwas originally identified as an accessory factor in nucleotide excision repair of UV-induced DNA damage, is involved in breast tumor growth. DDB2 is overexpressed in nonmetastatic breast tumor cells and plays a role in their proliferation by favoring G1–S transition entry and their progression through the S-phase of the cell cycle (2). We reported that DDB2 stimulates the proliferation of nonmetastatic breast tumor cells, at least in part by maintaining a low basal expression of mitochondrial superoxide dismutase (MnSOD) through its binding to a specific and well-characterized DNA sequence in the proximal promoter of the MnSOD gene (3). In addition, in metastatic breast tumor cells, DDB2 is not expressed and a high basal MnSOD level is observed, which is sharp in contrast to nonmetastatic cells. In these cells, the antioxidant enzymewas involved in the invasive ability of these cells through its control of matrix metalloprotease 9 (MMP9) activity (4). On the basis of these results, we hypothesized that the DDB2 protein might be involved in the control of cell migration, invasiveness, and breast tumor progression. To verify this hypothesis, we studied the consequence of DDB2 overexpression in the invasive and metastatic properties of aggressive breast cancer cells. Here, we show in vitro as well as in vivo that DDB2 reduces significantly motility and invasiveness of metastatic breast cancer cells when its gene is overexpressed. We have identified that DDB2 plays this role through its involvement in IkBa gene expression and in consequence in the negative control of constitutive NF-kBactivity. The latter binds Authors' Affiliations: Centre de Recherche en Automatique de Nancy (CRAN), UMR 7039 Centre National de la Recherche Scientifique (CNRS), Universit e de Lorraine, Facult e des Sciences et Technologies; Service D'anatomie et Cytologie Pathologiques, Hôpitaux de Brabois, CHU de Nancy,Universit edeLorraine,Vandoeuvre-l es-Nancy;andCentreR egional de Lutte Contre le Cancer Paul Strauss, Laboratoire de Biologie Tumorale, Strasbourg Cedex, France Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). S. Grandemange and P. Becuwe contributed equally to the cosupervising of this study. Corresponding Authors: Philippe Becuwe, Centre de Recherche en Automatique de Nancy (CRAN), UMR 7039 Centre National de la Recherche Scientifique (CNRS), Universit e de Lorraine, Facult e des Sciences et Technologies, BP 70239, 54506, Vandoeuvre-l es-Nancy Cedex, France. Phone: 33-3-83-68-42-19; Fax: 33-3-83-68-42-19; E-mail: Philippe.Becuwe@univ-lorraine.fr; and St ephanie Grandemange, E-mail: Stephanie.Grandemange@univ-lorraine.fr doi: 10.1158/0008-5472.CAN-12-3655 2013 American Association for Cancer Research. Cancer Research Cancer Res; 73(16) August 15, 2013 5040 on April 6, 2017. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst June 17, 2013; DOI: 10.1158/0008-5472.CAN-12-3655

The protein Damaged DNA Binding-2 (DDB2) is well known for its role in DNA repair by nucleotide excision repair. Interestingly, DDB2 is differentially expressed in breast cancer expressing the estrogen receptor alpha or not. Recent works performed in our laboratory showed a new role of DDB2 in the control of proliferation and invasive abilities in different breast tumor cells through its involvement in the transcriptional regulation of target genes. Two genes involved in tumorigenic processes, MnSOD (manganese superoxide dismutase) and IκBα (inhibitor alpha of Nuclear Factor-kappa B), have been found to be regulated by DDB2. In addition, transcriptomic analyses on cells that differentially express DDB2 showed that several genes involved in the regulation of cellular metabolism are modulated. Our aim is now to focus on the effects of DDB2 expression on cellular metabolism and glycolysis. The results indicated that the overexpression of DDB2 leads to a respiratory chain dysfunction and an increase of the glycolytic pathway. Moreover, we observed an increased production of reactive oxygen species in these cells, compared to parental cells. As mitochondria are involved in cell death, we performed different experiments to evaluate the impact of DDB2 in the response to anticancer agents (Doxorubicin, and 5-fluorouracile (5-FU)) commonly used in the treatment of breast cancer. Interestingly, the cells exhibit a greater sensitivity to anticancer drugs when DDB2 is overexpressed. As these two agents are related to DNA damaged, we have also used other molecules, the apoptotic inducer, TNFα (Tumor Necrosis Factor alpha), and the Paclitaxel (an antimicrotubule agent) to precise the role of DDB2 on cell death. Similar results were obtained, thus demonstrating the influence of DDB2 overexpression in the response to cell death. The identification of molecular mechanisms responsible for these cellular modifications could place DDB2 and these target genes as predictive markers of sensitivity to anticancer drugs in breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-01-04.

The main objective of this study was to prepare two types of nanoparticles with poly(d,l-lactide-co-glycolide) (PLGA) and polyethylenimine (PEI) polymers. Plasmid DNA (pDNA) was adsorbed either on PLGA/PEI nanoparticles, or as PEI/DNA complex onto the surface of PLGA nanoparticles. Both types of nanoparticles were prepared by the double emulsion method. The nanoparticles were characterized by their size, zeta potential and pDNA or PEI/DNA complex adsorption. The PEI/DNA complex adsorption was confirmed with ethidium bromide assay. pDNA adsorption onto PLGA/PEI nanoparticles (PLGA/PEI–DNA) was studied by electrophoresis on agarose gel. Cytotoxicity and transfection efficiency of both types of nanoparticle and PEI/DNA complexes formulations were studied in head and neck squamous carcinoma cell line (FaDu). To improve endosomal release, photochemical internalization (PCI) was used. The zeta potential increased when the PEI/DNA complex adsorbed onto PLGA nanoparticles (PLGA–PEI/DNA). Optimal pDNA adsorption efficiency was achieved for nitrogen/phosphorous ratio≥20/1. In vitro transfection and cells viability on FaDu cells with or without PCI were found to be variable depending on the type and concentration of nanoparticles. The results showed that transfection efficiency for PLGA/PEI–DNA or PLGA–PEI/DNA nanoparticles ranged between 2 and 80%, respectively. PCI was found to slightly improve the transfection efficiency for all formulations.

Cetuximab (Erbitux) is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody whose activity is related to the inhibition of EGFR downstream signaling pathways. P53 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) have been reported to control the functionality of PI3K/AKT signaling. In this study we evaluated whether reintroducing P53 using non-viral gene transfer enhances PTEN-mediated inhibition of PI3K/AKT signaling by cetuximab in PC3 prostate adenocarcinoma cell line bearing p53 and pten mutations. Signaling phosphoproteins expression was analyzed using Bio-Plex phosphoprotein array and western blot. Apoptosis induction was evaluated from BAX expression, caspase-3 activation and DNA fragmentation analyses. The results presented show that p53 and pten gene transfer additionally mediated cell growth inhibition and apoptosis induction by restoral of signaling functionality, which enabled the control of PI3K/AKT and MAPKinase signaling pathways by cetuximab in PC3 cells. These results highlight the interest of the analysis of signaling phosphoproteins expression as molecular predictive markers for response to cetuximab and show that p53 and pten mutations could be key determinants of cell response to cetuximab through the functional impact of these mutations on cell signaling.

The development of new vectors to deliver DNA into cells for therapy of cancers or genetic diseases has been a major area of research for many years. However, the clinical application of this technology requires the development of efficient, reliable and sterile vectors enabling the transfer of genes in vivo. Non viral, polymer or lipid-based vectors offer a new impetus to gene therapy because they are less toxic than viral vectors (no endogenous recombination, fewer immunological reactions, easy production and delivery of large-sized plasmid). The aim of this study is to develop a new tool for DNA delivery composed of methacrylic polymeric (Eudragit® RS and RL) nanoparticles. These nanoparticles were prepared by two methods: nanoprecipitation and double emulsion. The nanoparticles were characterized by their size, zeta potential and amount of DNA adsorption. Cytotoxicity tests based on mitochondrial activity (MTT test) revealed that the nanoparticles had limited cytotoxicity and that this depended on both the cell type and the nanoparticle concentration. Transgene expression was observed using the Green Fluorescence Protein gene as reporter gene, and was evaluated by flow cytometry in FaDu, MDA-MB 231 and MCF-7 cell lines. The results showed that transfection rates ranging between 4 and 7% were achieved in FaDu and MDA-MB 231 cells with nanoparticles prepared by the nanoprecipitation method. In MCF-7 cells transfected with nanoparticles prepared by either the double emulsion or the nanoprecipitation method, the transfection efficiency was between 2 and 4%. Nanoparticles prepared by nanoprecipitation were slightly more efficient than nanoparticles prepared from a double emulsion. Particle size was not an important factor for transfection, since no significant difference was observed with size between 50 and 350 nm. We showed that Eudragit® RS and RL nanoparticles could introduce the transgene into different types of cells, but were generally less effective than the lipofectamine control.

This study tests the hypothesis that the activators of peroxisome proliferator-activated receptors (PPARs) and 9-cis-retinoic acid receptor (RXR) regulate human semaphorin 6B (Sema6B) gene expression. The human MCF-7 breast adenocarcinoma cell line was chosen because it expresses Sema6B at a high level. The Sema6B mRNA level was analyzed by RT-PCR and the semaphorin 6B protein content was determined using a polyclonal antibody that we have produced and characterized. Treatments with fenofibrate (a PPARalpha activator) and troglitazone (a PPARgamma ligand) strongly decreased the Sema6B mRNA. The drop in Sema6B mRNA level and in protein content was more important when the treatment combined the action of fenofibrate or troglitazone and 9-cis-retinoic acid. On the other hand, no significant change was observed in the Sema6B mRNA and protein levels when the cells were exposed to the combined action of GW610742 (a PPARbeta activator) and 9-cis-retinoic acid. These data suggest that PPARalpha/RXR and PPARgamma/RXR heterodimers are involved in the regulation of Sema6B gene expression and open new perspectives concerning the participation of these nuclear receptors in cell recognition and migration.

Mitochondrial dysfunctions are frequently reported in cancer cells, but their direct involvement in tumorigenesis remains unclear. To understand this relation, we stimulated mitochondrial activity by overexpression of the mitochondrial triiodothyronine receptor (p43) in human dermal fibroblasts. In all clones, this stimulation induced morphologic changes and cell fusion in myotube-like structures associated with the expression of several muscle-specific genes (Myf5, desmin, connectin, myosin, AchRalpha). In addition, these clones displayed all the in vivo and in vitro features of cell transformation. This phenotype was related to an increase in c-Jun and c-Fos expression and extinction of tumor suppressor gene expression (p53, p21WAF1, Rb3). Lastly, reactive oxygen species (ROS) production was increased in positive correlation to the stimulation of mitochondrial activity. The direct involvement of mitochondrial activity in this cell behavior was studied by adding chloramphenicol, an inhibitor of mitochondrial protein synthesis, to the culture medium. This inhibition resulted in partial restoration of the normal phenotype, with the loss of the ability to fuse, a strong decrease in muscle-specific gene expression, and potent inhibition of the transformed phenotype. However, expression of tumor suppressor genes was not restored. Similar results were obtained by using N-acetylcysteine, an inhibitor of ROS production. These data indicate that stimulation of mitochondrial activity in human dermal fibroblasts induces cell transformation through events involving ROS production.

Hypolipidemic drugs (HP drugs) are xenobiotics belonging to the peroxisome proliferator family which are used as pharmaceuticals in the treatment of human hyperlipidemia and hypercholesterolemia. They cause hepatocarcinogenesis in rodents by increasing cell proliferation. One hypothesis is that this hepatocarcinogenic effect is caused by induced oxidative stress resulting from the overproduction of reactive oxygen species (ROS) and from a decreasing antioxidant defense. In addition, ROS play a role in hepatocellular proliferation by activation of NF-kappa B and AP-1, leading to an increase in mitogenic cytokines such as tumor necrosis factor-alpha. No liver cancer incidence has been noted in individuals treated with HP drugs for brief periods of time, However, the observation that old rats and mice are more susceptible than young individuals to the hepatocarcinogenic effect caused by long term exposure to HP drugs raises the question of a potential health risk for the human population. In vitro, HP drugs cause an apoptogenic effect in human hepatocytes. This effect is related to a moderate antioxidant response, dysfunction of mitochondria caused by an overproduction of ROS and release of apoptogenic factors, Finally, the apoptogenic effect of HP drugs is observed in human hepatomas, suggesting a clinical interest of these agents in antitumoral activity.

We showed that the metabolism of arachidonic acid (AA) in HepG2 cells generates reactive oxygen species (ROS), which activate the p38 mitogen-activated protein kinase (MAPK) pathway and the redox-sensitive transcription factors AP-1 and NF-kappaB, leading to the induction of the antioxidant manganese superoxide dismutase gene. The present study reports that AA decreases the HepG2 cell growth by 40% and 55% after a treatment for 24 and 48 h, respectively. This effect was blocked by an inhibitor of lipoxygenase/cytochrome P450 monooxygenase pathways and by the antioxidants. In addition, AA induced an oxidative stress, as an accumulation of malondialdehyde (MDA)-modified proteins, resulting to a generation of MDA and H2O2 was observed after 24 h. This AA-induced oxidative stress was associated with the lack of an increase in the H2O2-degrading enzyme level. In contrast, 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable analog of AA, had not effect. The peroxisome proliferator-activated receptor gamma (PPARgamma) with AA metabolites as ligands was upregulated by the fatty acid but was not involved in the AA effect because its transcriptional activity estimated by reporter gene assays was negatively controlled by p38 MAPK pathway. These findings suggest that the effect of AA on human hepatoma cell growth by inducing an oxidative stress may present a clinical interest in the treatment of the liver cancer. (C) 2004 Elsevier SAS. All rights reserved.

(2003). Superoxide dismutase in Plasmodium: a current survey. Redox Report: Vol. 8, No. 5, pp. 265-267.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the nuclear hormone receptor superfamily that can be activated by natural fatty acids and various xenobiotics, including clofibrate. This transcription factor primarily regulates genes involved in lipid metabolism and homeostasis. We present the expression pattern of the PPARalpha subtype in the adult jerboa Jaculus orientalis, determined by RT-PCR and Western blotting using specific probes and a polyclonal antibody for PPARalpha, respectively. PPARalpha is highly expressed in liver and kidney, and to a lesser extent in duodenum and colon. PPARalpha expression is increased at the mRNA and protein levels in liver and duodenum of jerboa treated for 2 weeks with the peroxisome proliferator (PP) clofibrate. The induction is tissue-specific as no significant changes are observed in kidney and colon. The present data indicate that the PP-induced PPARalpha gene expression is not dependent on the PPARalpha content in target cells.

This work demonstrated the constitutive expression of peroxisome proliferator-activated receptor (PPAR)-gamma and PPAR-alpha in rat synovial fibroblasts at both mRNA and protein levels. A decrease in PPAR-gamma expression induced by 10 microg/ml lipopolysaccharide (LPS) was observed, whereas PPAR-alpha mRNA expression was not modified. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) dose-dependently decreased LPS-induced cyclooxygenase (COX)-2 (-80%) and inducible nitric oxide synthase (iNOS) mRNA expression (-80%), whereas troglitazone (10 microM) only inhibited iNOS mRNA expression (-50%). 15d-PGJ(2) decreased LPS-induced interleukin (IL)-1 beta (-25%) and tumor necrosis factor (TNF)-alpha (-40%) expression. Interestingly, troglitazone strongly decreased TNF-alpha expression (-50%) but had no significant effect on IL-1 beta expression. 15d-PGJ(2) was able to inhibit DNA-binding activity of both nuclear factor (NF)-kappa B and AP-1. Troglitazone had no effect on NF-kappa B activation and was shown to increase LPS-induced AP-1 activation. 15d-PGJ(2) and troglitazone modulated the expression of LPS-induced iNOS, COX-2, and proinflammatory cytokines differently. Indeed, troglitazone seems to specifically target TNF-alpha and iNOS pathways. These results offer new insights in regard to the anti-inflammatory potential of the PPAR-gamma ligands and underline different mechanisms of action of 15d-PGJ(2) and troglitazone in synovial fibroblasts.