Localization of decorin gene expression in normal human breast tissue and in benign and malignant tumors of the human breast

The small extracellular matrix proteoglycan decorin which possesses a potent antitumor activity has been shown to be present in various amounts in the stroma of several tumors including those of the breast. Regarding decorin in breast malignancies the published data are conflicting, i.e., whether breast cancer cells express it or not. Here, we first compared decorin gene expression levels between healthy human breast tissue and selected types of human breast cancer using GeneSapiens databank. Next, we localized decorin mRNA in tissue specimen of normal human breast, intraductal breast papillomas and various histologic types of human breast cancer using in situ hybridization (ISH) with digoxigenin-labeled RNA probes for decorin. We also examined the effect of decorin transduction on the behavior of cultured human breast cancer MCF7 cells. Analysis of GeneSapiens databank revealed that in various human breast cancers decorin expression is significant. However, ISH results clearly demonstrated that human breast cancer cells independently of the type of the cancer do not express decorin mRNA. This was also true for papilloma-forming cells of the human breast. Indeed, decorin gene expression in healthy human breast tissue as well as in benign and malignant tumors of human breast was shown to take place solely in cells of the original stroma. Decorin transduction using decorin adenoviral vector in decorin-negative MCF7 cells resulted in a significant decrease in the proliferation of these cells and changed cell cohesion. Decorin-transduced MCF7 cells also exhibited increased apoptosis. In conclusion, our study shows that in human breast tissue only cells of the original stroma are capable of decorin gene expression. Our study also shows that transduction of decorin in decorin-negative human breast cancer cells markedly modulates the growth pattern of these cells.     

Regulation of BRCA2 gene expression by the SLUG repressor protein in human breast cells

The expression of the breast cancer susceptibility protein BRCA2 is highly regulated in human breast, ovary, and pancreatic cells. BRCA2 is not expressed in the non-dividing cells, and expression is cell cycle stage-dependent and is elevated in the sporadic cancer cells. Mutational analysis of the upstream sequence of the human BRCA2 gene revealed an E2-box-containing silencer at the -701 to -921 position. The E2-box is essential for the cell-cycle stage-dependent activity of the silencer. We affinity-purified a 29-kDa silencer-binding protein (SBP) from the nuclear extracts of human breast cells BT-549 and MDA-MB-231. We explored whether the E2-box-binding repressor protein SLUG, which is of similar molecular size, is involved in the silencing process. Supershift assay with the purified SBP and anti-SLUG antibody revealed the identity of the SBP as SLUG. We found that silencer is inactive in the human breast cancer cells such as MDA-MB-468 and MCF-7 that do not express SLUG, further suggesting the involvement of SLUG in the BRCA2 gene silencing. Inducible expression of human SLUG in the dividing MDA-MB-468 cells reduced BRCA2 RNA levels with the activation of the silencer. Furthermore, small interfering RNA-mediated knockdown of SLUG mRNA in the BT-549 cells caused inhibition of the silencer function. Chromatin immunoprecipitation assays suggested that SLUG mediates its action by recruiting C-terminal-binding protein-1 (CtBP-1) and histone deacetylase-1 (HDAC-1) at the silencer E2-box. The general HDAC inhibitor, trichostatin A, inhibited the SLUG-mediated regulation of the silencer function. It thus appears that SLUG is a negative regulator for BRCA2 gene expression.