Emilie Brunner

Identification of DDB2 Protein as a Transcriptional Regulator of Constitutive SOD2 Gene Expression in Human Breast Cancer Cells

Manganese superoxide dismutase plays a role in breast tumor cell growth, which depends on its constitutive expression. However, the mechanisms responsible for the regulation of constitutive SOD2 gene expression at different malignant phenotype in breast cancers remain to be determined. The present study reports the identification and characterization of a DNA sequence located in the proximal promoter of the SOD2 gene, which forms a complex with a nuclear protein from breast tumor MCF-7 cells. Purification of this complex showed that it contained DDB2 (damaged DNA binding 2), a well known protein involved in nucleotide excision DNA repair and cell cycle regulation. Functional analysis of the proximal promoter of the SOD2 gene or modulation of DDB2 expression allowed us to demonstrate that DDB2 regulates negatively the constitutive expression of the SOD2 gene in breast cancer cells. We demonstrate that the binding of DDB2 was associated with the loss of acetylated H3 histones and the decrease in the binding of Sp1 but not AP-2α transcription factors to the SOD2 proximal promoter. In addition, we show that DDB2 exerts, at least in part, a control of breast cancer cell growth through its negative regulation of constitutive expression of the SOD2 gene. For the first time, these data give supporting evidence that DDB2 is a new transcriptional regulator, and they provide insight into the molecular function of breast cancer cell growth, which will have an important clinical interest.

Regulation of the high basal expression of the manganese superoxide dismutase gene in aggressive breast cancer cells

A high basal expression of manganese superoxide dismutase (MnSOD) has been reported in aggressive breast cancer cells, according to an unknown mechanism, and contributes to their invasive abilities. Here, we report the involvement of Sp1 and nuclear factor-κB (NF-κB) transcription factors in this high basal expression of MnSOD in aggressive breast cancer cells. Suppression or inactivation of Sp1 showed that it plays an essential role in the high MnSOD expression in aggressive breast cancer cells through a unique binding site identified by chromatin immunoprecipitation (ChIP) assay and functional analysis of the MnSOD proximal promoter. Treatment of cells with a specific NF-κB inhibitor peptide decreased significantly high basal MnSOD expression. A ChIP assay showed binding of a constitutive p50/p65 NF-κB complex to the MnSOD intronic enhancer element, associated with hyperacetylation of the H3 histone. Finally, high basal expression of MnSOD resulted in the lack of expression of Damaged DNA binding 2 (DDB2) protein in aggressive breast cancer cells. DDB2 overexpression prevented the binding of Sp1 as well as of NF-κB to their respective elements on the MnSOD gene. These results contribute to a better understanding of MnSOD up-regulation, which may be clinically important in the prediction of breast tumor progression.

Identification of damaged DNA binding 2 protein as a transcriptional regulator of the constitutive sod2 gene expression in human breast cancer cells

Manganese superoxide dismutase plays a role in breast tumor cell growth, which depends on its constitutive expression. However, the mechanisms responsible for the regulation of constitutive SOD2 gene expression at different malignant phenotype in breast cancers remain to be determined. The present study reports the identification and characterization of a DNA sequence located in the proximal promoter of the SOD2 gene, which forms a complex with a nuclear protein from breast tumor MCF-7 cells. Purification of this complex showed that it contained DDB2 (damaged DNA binding 2), a well known protein involved in nucleotide excision DNA repair and cell cycle regulation. Functional analysis of the proximal promoter of the SOD2 gene or modulation of DDB2 expression allowed us to demonstrate that DDB2 regulates negatively the constitutive expression of the SOD2 gene in breast cancer cells. We demonstrate that the binding of DDB2 was associated with the loss of acetylated H3 histones and the decrease in the binding of Sp1 but not AP-2α transcription factors to the SOD2 proximal promoter. In addition, we show that DDB2 exerts, at least in part, a control of breast cancer cell growth through its negative regulation of constitutive expression of the SOD2 gene. For the first time, these data give supporting evidence that DDB2 is a new transcriptional regulator, and they provide insight into the molecular function of breast cancer cell growth, which will have an important clinical interest.

Damaged DNA binding protein 2 plays a role in breast cancer cell growth.

The Damaged DNA binding protein 2 (DDB2), is involved in nucleotide excision repair as well as in other biological processes in normal cells, including transcription and cell cycle regulation. Loss of DDB2 function may be related to tumor susceptibility. However, hypothesis of this study was that DDB2 could play a role in breast cancer cell growth, resulting in its well known interaction with the proliferative marker E2F1 in breast neoplasia. DDB2 gene was overexpressed in estrogen receptor (ER)-positive (MCF-7 and T47D), but not in ER-negative breast cancer (MDA-MB231 and SKBR3) or normal mammary epithelial cell lines. In addition, DDB2 expression was significantly (3.0-fold) higher in ER-positive than in ER-negative tumor samples (P = 0.0208) from 16 patients with breast carcinoma. Knockdown of DDB2 by small interfering RNA in MCF-7 cells caused a decrease in cancer cell growth and colony formation. Inversely, introduction of the DDB2 gene into MDA-MB231 cells stimulated growth and colony formation. Cell cycle distribution and 5 Bromodeoxyuridine incorporation by flow cytometry analysis showed that the growth-inhibiting effect of DDB2 knockdown was the consequence of a delayed G1/S transition and a slowed progression through the S phase of MCF-7 cells. These results were supported by a strong decrease in the expression of S phase markers (Proliferating Cell Nuclear Antigen, cyclin E and dihydrofolate reductase). These findings demonstrate for the first time that DDB2 can play a role as oncogene and may become a promising candidate as a predictive marker in breast cancer.

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.