Bromodomain 4 activation predicts breast cancer survival

Abstract

Previous work identified the Rap1 GTPase-activating protein Sipa1 as a germ-line-encoded metastasis modifier. The bromodomain protein Brd4 physically interacts with and modulates the enzymatic activity of Sipa1. In vitro analysis of a highly metastatic mouse mammary tumor cell line ectopically expressing Brd4 demonstrates significant reduction of invasiveness without altering intrinsic growth rate. However, a dramatic reduction of tumor growth and pulmonary metastasis was observed after s.c. implantation into mice, implying that activation of Brd4 may somehow be manipulating response to tumor microenvironment in the in vivo setting. Further in vitro analysis shows that Brd4 modulates extracellular matrix gene expression, a class of genes frequently present in metastasis-predictive gene signatures. Microarray analysis of the mammary tumor cell lines identified a Brd4 activation signature that robustly predicted progression and/or survival in multiple human breast cancer datasets analyzed on different microarray platforms. Intriguingly, the Brd4 signature also almost perfectly matches a molecular classifier of low-grade tumors. Taken together, these data suggest that dysregulation of Brd4-associated pathways may play an important role in breast cancer progression and underlies multiple common prognostic signatures.

Fig. 1.

Ectopic expression of Brd4 positively regulates cell invasion and migration. (A) Four separate clonal isolates of Mvt-1/Brd4 and Mvt-1/β-Gal cells were grown on the top of a solidified basement membrane extract. Both the invasive properties and the ability to form cell extensions were significantly reduced in the Brd4 cells compared with the control cells. (B and C) Matrigel invasion and transwell migration assays of the four clonal isolates of Mvt-1/Brd4 and Mvt-1/β-Gal cells. Both the invasion and the migration properties of the Mvt-1 cells were reduced by ectopic expression of Brd4.

Fig. 2.

Ectopic expression of Brd4 in the highly metastatic mouse mammary tumor cell line Mvt-1 reduces tumor growth and pulmonary surface metastasis after s.c. implantation of cells into FVB/NJ mice. (A) A significant reduction in tumor growth was observed in the Mvt-1/Brd4 cells with the average tumor weight for the Mvt-1/Brd4 clones being 91 mg ± 42 mg compared with 595 mg ± 308 mg for the two Mvt-1/β-Gal clones (P < 0.001). (B) A similar reduction in pulmonary surface metastasis was observed with average lung surface metastasis count being 1.4 ± 2.5 for the Mvt-1/Brd4 clones compared with 11.1 ± 5.8 for the Mvt-1/β-Gal clones (P < 0.001).

Fig. 3.

The Mvt-1/Brd4 microarray expression signature predicts survival and/or relapse in multiple breast cancer microarray datasets. (A) The Brd4 signature consistently and robustly predicts survival and/or relapse in four separate breast cancer microarray datasets performed on Affymetrix GeneChips. A significant difference in the overall likelihood of survival was observed in the GSE1456 dataset with 8-year survival being 95.9% vs. 65.5% for the good and poor prognosis Brd4 signatures, respectively (Upper Left). A similar effect was observed in the GSE3494 dataset with 12-year survival being 80.6% vs. 57.5% for the good and poor prognosis Brd4 signatures, respectively (Upper Right). The end point for the GSE2034 and GSE4922 differ in that disease-free survival was measured. A similar effect was seen in both cohorts with 10-year disease-free survival being 68.9% vs. 54.2% in the GSE2034 dataset (Lower Left) and 71.3% vs. 47.6% in the GSE4922 dataset (Lower Right) for the good and poor prognosis Brd4 signatures, respectively. (B) The Brd4 signature was also highly predictive of overall survival in the Dutch Rosetta dataset, with the overall survival being estimated to be 78.5% vs. 45.1% for the good and poor prognosis Brd4 signatures, respectively (Brd4 signature hazard ratio = 5.50, 95% C.I. = 3.12–9.69) (Left) compared with 72.6% vs. 47.0%, respectively (70 gene signature hazard ratio = 4.49, 95% C.I. = 2.65–7.61) for the same analysis, using the Rosetta 70 gene signature.