Molecular and Cellular Pathobiology DDB 2 : A Novel Regulator of NFk B 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-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