Stéphanie Grandemange

Manganese superoxide dismutase in breast cancer: From molecular mechanisms of gene regulation to biological and clinical significance

Breast cancer is one of the most common malignancies of all cancers in women worldwide. Many difficulties reside in the prediction of tumor metastatic progression because of the lack of sufficiently reliable predictive biological markers, and this is a permanent preoccupation for clinicians. Manganese superoxide dismutase (MnSOD) may represent a rational candidate as a predictive biomarker of breast tumor metastatic progression, because its gene expression is profoundly altered between early and advanced breast cancer, in contrast to expression in the normal mammary gland. In this review, we report the characterization of some gene polymorphisms and molecular mechanisms of SOD2 gene regulation, which allows a better understanding of how MnSOD is decreased in early breast cancer and increased in advanced breast cancer. Several studies display the biological significance of MnSOD level in proliferation as well as in invasive and angiogenic abilities of breast tumor cells by controlling superoxide anion radical (O2(•-)) and hydrogen peroxide (H2O2). Particularly, they report how these reactive oxygen species may activate some signaling pathways involved in breast tumor growth. Emerging understanding of these findings provides an interesting framework for guiding translational research and suggests a way to define precisely the clinical interest of MnSOD as a prognostic and/or predicting marker in breast cancer, by associating with some regulators involved in SOD2 gene regulation and other well-known biomarkers, in addition to the typical clinical parameters.

Stimulation of Mitochondrial Activity by p43 Overexpression Induces Human Dermal Fibroblast Transformation

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.

DDB2: A Novel Regulator of NF-κB and Breast Tumor Invasion

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-κB and the expression of its target matrix metalloprotease 9 (MMP9). Mechanistic investigations indicated that DDB2 decreased NF-κB activity by upregulating expression of IκBα by binding the proximal promoter of this gene. This effect was causally linked to invasive capacity. Indeed, knockdown of DDB2-induced IκBα gene expression restored NF-κB 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 in DDB2 as a prognostic marker or therapeutic target in this setting.

From non-covalent binding to irreversible DNA lesions: nile blue and nile red as photosensitizing agents

We report a molecular modeling study, coupled with spectroscopy experiments, on the behavior of two well known organic dyes, nile blue and nile red, when interacting with B-DNA. In particular, we evidence the presence of two competitive binding modes, for both drugs. However their subsequent photophysical behavior is different and only nile blue is able to induce DNA photosensitization via an electron transfer mechanism. Most notably, even in the case of nile blue, its sensitization capabilities strongly depend on the environment resulting in a single active binding mode: the minor groove. Fluorescence spectroscopy confirms the presence of competitive interaction modes for both sensitizers, while the sensitization via electron transfer, is possible only in the case of nile blue.

Δ2-Troglitazone promotes cytostatic rather than pro-apoptotic effects in breast cancer cells cultured in high serum conditions

ABSTRACT We have previously shown that Δ2-Troglitazone (Δ2-TGZ) displayed anticancer effects on breast cancer cell lines grown in low serum conditions (1% fetal calf serum (FCS)). The present study was performed in order to characterize the effects of Δ2-TGZ in high serum containing medium and to determine if starvation could influence the response of breast cancer cells to this compound, keeping in mind the potential interest for breast cancer therapy. We observed that in high serum conditions (10% FCS), a 48 h treatment with Δ2-TGZ induced a decrease in cell numbers in MDA-MB-231 and MCF-7 breast cancer cell lines. The IC50 values were higher than in low serum conditions. Furthermore, in contrast to our previous results obtained in 1% FCS conditions, we observed that in 10% FCS-containing medium, MCF-7 cells were more sensitive to Δ2-TGZ than MDA-MB-231 cells. Δ2-TGZ also induced endoplasmic reticulum (ER) stress mainly in MDA-MB-231 cells. Besides, in high serum conditions, Δ2-TGZ induced a G0/G1 cell cycle arrest, an inhibition of BrdU incorporation and a reduced level of cyclin D1. We observed a limited cleavage of PARP and a limited proportion of cells in sub-G1 phase. Thus, in high serum conditions, Δ2-TGZ displayed cytostatic effects rather than apoptosis as previously reported in 1% FCS-containing medium. Our results are in accordance with studies suggesting that serum starvation could potentiate the action of diverse anti-cancer agents.

PO-164 Identification of the heterogeneous nuclear ribonucleoprotein k/j (hnrnp k/j) as a repressor of the inhibitor kappa b alpha (iκbα) gene in aggressive breast cancer cells

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.

Photophysics of the Singlet Oxygen Sensor Green Chromophore: Self-Production of 1O2 Explained by Molecular Modeling

We report a combined computational and experimental study to rationalize the behavior of a well-known singlet oxygen (1O2) probe, that is, the chromophore of the Singlet Oxygen Sensor Green: a fluoresceine-based sensor. In particular, we evidence that the presence of an intramoleculer charge transfer state that is no more present upon reaction with 1O2 explains the fluorescence enhancement observed in the presence of reactive oxygen species. Furthermore, we also unequivocally show the photophysical pathways leading to the fluorescence enhancement of fluoresceine upon irradiation with UVA lights and also in the absence of any oxygen activator. More specifically, we evidence that the presence of a possible intersystem crossing upon population of higher energy singlet electronic excited states will lead to the population of the fluoresceine triplet manifold and hence to the self-production of 1O2.

The Damaged DNA Binding 2 protein: a new modulator of TGFβ1 signaling pathway and membrane nanomechanics in breast cancer cells

Posttranslational modification (PTM) of proteins is a versatile cellular process to regulate the activities of proteins. The high regioselectivity and catalysis rate of posttranslationally modifying ...Dynamic modeling showed that the topology of fatty-acid betaoxidation makes this pathway intrinsically vulnerable to substrate overload: at a high influx of palmitoyl-CoA into the pathway the flux dropped and intermediate CoA-esters accumulated extremely(Van Eunen et al., 2013 PLoS Comput Biol). We show here that inborn errors in fatty-acid metabolism aggravate the risk of amitochondrial catastrophe.We applied the previously constructed dynamic model to study the impact of multiple acyl-CoA dehydrogenase deficiency(MADD) and medium-chain acyl-CoA dehydrogenase deficiency(MCADD) on the kinetics of fatty acid oxidation. We explored the relation between the deficiencies and metabolite profiles and calculated which profiles might enhance the risk of pathway overload. MADD patients show accumulation of acylcarnitines acrossall chain lengths. In contrast, MCADD patients accumulate the medium-chain acylcarnitines. A linear non-competition model could not explain this, as it predicted exclusive accumulation of longer chain-length metabolites in MADD. This provides the first experimental evidence that molecular competition at the enzyme level is physiologically relevant for fatty-acid oxidation. Subsequently,this more realistic competition model was fitted to either mouse liver data or to disease-specific patient plasma data. When the substrate concentration was varied, both MADD and MCADD enhanced the accumulation of intermediate metabolite sand the flux declined already at lower substrate concentrations compared to the model without enzyme deficiencies.We hypothesize that the pathway structure of the beta-oxidationin which substrates compete for enzymes, is at the basis of the disease phenotypes associated with enzyme deficiencies.

A non-negative multilinear block tensor decomposition approach to flow cytometry data analysis

The paper presents a novel approach to the processing of flow cytometry data sequences. It consists in decomposing a sequence of multidimensional probability density functions by using multilinear block tensor decomposition approach [1]. The identifiability of the model is also addressed as well as the data processing. To illustrate the effectiveness of the approach, a study of the T47D cell line mitochondrial membrane potential as a function of the CCCP1 decoupling agent concentration is performed. The cell sorting capacity of the method is significantly improved as compared to classical clustering methods.

DDB2, a new regulator of metabolism and cell death in human breast tumor cells

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.