BRCA1 Suppresses Osteopontin-mediated Breast Cancer

Abstract

BRCA1 is a well described breast cancer susceptibility gene thought to be involved primarily in DNA repair. However, mutation within the BRCA1 transcriptional domain is also implicated in neoplastic transformation of mammary epithelium, but responsible mechanisms are unclear. Here we show in a rat mammary model system that wild type (WT) BRCA1 specifically represses the expression of osteopontin (OPN), a multifunctional estrogen-responsive gene implicated in oncogenic transformation, particularly that of the breast. WT.BRCA1 selectively binds OPN-activating transcription factors estrogen receptor ␣, AP-1, and PEA3, inhibits OPN promoter transactivation, and suppresses OPN mRNA and protein both from an endogenous gene and a relevant model inducible gene. WT.BRCA1 also inhibits OPN-mediated neoplastic transformation characterized by morphology change, anchorage-independent growth, adhesion to fibronectin, and invasion through Matrigel. A mutant BRCA1 allele (Mut.BRCA1) associated with familial breast cancer lacks OPN suppressor effects, binds to WT.BRCA1, and impedes WT.BRCA1 suppression of OPN. Stable transfection of rat breast tumor cell lines with Mut.BRCA1 dramatically up-regulates OPN protein and induces anchorageindependent growth. In human primary breast cancer, BRCA1 mutation is significantly associated with OPN overexpression. Taken together, these data suggest that BRCA1 mutation may confer increased tissue-specific cancer risk, in part by disruption of BRCA1 suppression of OPN gene transcription.

Agelastatin A: a novel inhibitor of osteopontin-mediated adhesion, invasion, and colony formation

“…At time points of 3,6,12,24,48,72, and 96 h, cells were removed by trypsination and counted using a hemocytometer. All assays were carried out in triplicate as described previously (19).…”

“…Densitometry data were obtained using a digital imaging system (Syngene, Genetool). All Western blot experiments were repeated at least three times to ensure reproducibility as described previously (19,20).…”

“…Cell Adhesion Assay The adhesive ability of cells was determined as described previously (19,21). Briefly, cells were plated out at a known density (2 Â 10 5 per dish) in a 24-well plate in conditioned medium and allowed to adhere to laminin-coated plates for 15 min at 37jC, 5% CO 2 .…”

“…Soft Agar Assays Anchorage-independent growth was determined as described previously (19). Briefly, 5 mL of 1.6% (w/v) agarose were plated in a 100-mm diameter tissue culture dish and allowed to harden.…”

“…Matrigel Invasion Assays Biocoat 250 Ag/mL Matrigel invasion chambers (diameter, 6.4 mm; Falcon) were used to assess the invasiveness of cells as described previously (19). Briefly, 1 Â 10 6 cells were resuspended in 100 AL serum-free DMEM [containing (-)-agelastatin A for treated samples] and added to the cell culture inserts of the upper invasion chambers.…”