SLUG‐mediated repression of BRCA2 gene expression in invasive breast tumor cells

Abstract

The expression of the breast cancer susceptibility protein BRCA2 is responsible for suppression of growth of breast tumor cells. Its expression is often elevated in the non-invasive breast tumor cells. Mutational analysis of the upstream sequence of 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 invasive 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. Super-shift 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 non-invasive human breast cancer cells like MBA-MD-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 MDA-MB-468 cells reduced BRCA2 RNA levels with the activation of the silencer. Furthermore, siRNA–mediated knock down 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 CtBP-1 and 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 in the invasive breast tumor cells. Supported by the MMC/VICC cancer partnership grant #1U54CA091408-010003 from NCI and the DOD grant # DAMD17-00-1-0341 to GC