Role of the Damaged DNA-Binding 2 protein in the tumor progression through its control on the Nuclear Factor-kappa B activation in breast tumor cells.
Résumé
Body: The Damaged DNA-Binding 2 protein (DDB2) is known to play a role in nucleotide excision repair in normal cells exposed to ultraviolet light. However, we have shown that DDB2 is overexpressed in nonmetastatic breast tumor cells and plays a role in their proliferation by favouring G1/S transition entry and progression through S phase of cell cycle, by being involved in the transcriptional regulation of genes, such as the mitochondrial superoxide dismutase (Kattan et al. PLoS ONE 3: e2002, 2008; Minig et al. J. Biol. Chem. 284: 14165-76, 2009). To define more precisely the importance of DDB2 in the breast cancer cell growth, a comparative microarray between breast tumor cells expressing or not DDB2 was done to identify a new DDB2 target genes. Among the 664 potential DDB2 target genes, we have focused our attention to the gene encoding the cytoplasmic inhibitor of Nuclear Factor-kappa B (NF-κB) transcription factor, I kappa B alpha (IκBα). Our results show that expression of IκBα is strictly correlated with that of DDB2, as confirmed by RT-PCR and Western blot analyses. A computer analysis followed by a chromatin immunoprecipitation assay has identified a binding site of DDB2 in the promoter region of the IκBα gene, strongly suggesting a direct involvement of this protein in the regulation of the gene expression. Related to these results, we have observed by gel shift and supershift assays using nuclear extracts, as well as by measuring the NF-κB activity with a NF-κB driven luciferase gene reporter and by immunocytochemistry showing the nuclear translocation of p50 and p65 proteins, that the overexpression of DDB2 leads to a strong decrease in the constitutive activity of the NF-κB complex in metastatic breast tumor cells transfected with the DDB2 gene. When DDB2 is overexpressed, its control on the constitutive NF-κB activity leads to a significant biological consequences on metastatic breast tumor cells, such as a decrease in their adherence on diverse matrix proteins (fibronectin, collagens I and IV and laminin) as well as their migratory abilities (wound healing assay and Boyden chambers experiments). In conclusion, these results suggest that DDB2 has a real clinical interest as a new potential molecular marker of breast tumor progression toward metastasis, through its potential involvement in the regulation of the IκBα gene and thus in the control of NF-κB activation. This work was supported by the French "Ligue contre le Cancer".