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.gene expression signatures ͉ metastasis ͉ mouse models T he majority of deaths attributable to solid cancers result from the pathophysiological impact of metastasis. This is starkly illustrated when one considers that the median survival of patients with metastatic breast cancer is Ϸ2-4 years (1), compared with an Ϸ80% survival rate for women whose disease remains nonmetastatic. Advanced disseminated breast cancer thus remains an incurable condition regardless of new treatments (2). It is therefore important to develop a comprehensive understanding of the metastasis biology to identify patients at higher risk of tumor dissemination. This in turn may permit development of therapies and initiation of more aggressive treatment in women with poorer prognoses to reduce the incidence and extent of metastatic disease. Conversely, it may also prove possible to identify women at low risk of metastasis, thus sparing them needless adjuvant therapy.Our laboratory has demonstrated that germ-line genetic variation influences tumor progression. Specifically, in a model system, the F 1 progeny of the highly metastatic polyoma middle-T (PyMT) transgenic mouse and different inbred laboratory mouse strains display wide variations in metastatic efficiency after mammary tumor development (3). The most likely explanation for this observation is that germ-line variation modulates tumor progression. Subsequent identification of heritable loci modulating metastatic efficiency support this hypothesis (4, 5). Positional cloning subsequently identified Sipa1, a GTPase activating protein (GAP) that negatively regulates Rap-GTPases, as the first polymorphic metastasis efficiency gene in mice (6). Studies of human cancer have suggeste...

Integrated cross-species transcriptional network analysis of metastatic susceptibility

“…Consistent with this possibility, examination of the gene list for the TPX2 network reveals the presence of two genes previously identified in our mouse mammary tumor genetic screens [CDC25A (24), RRP1B (25)]. Additionally, four TPX2 network genes [RFC3, RFC4 (26), JMJD6 (27), CNOT1 (28)] are known to physically interact with the metastasis susceptibility protein BRD4 (19). Further investigation into the role of these critical node genes may reveal interesting and important insights into the etiology of metastatic breast cancer.…”

“…To test this hypothesis, transcriptional analysis of the metastasis susceptibility gene Bromodomain 4 (Brd4) (19) was performed. Brd4 is transcribed as two isoforms, the longer of which is antimetastatic (19) and induces a gene signature associated with longer DMFS, but the short isoform is prometastatic and induces a gene signature indicative of poorer DMFS (20). The expression of the TPX2 network was therefore examined in the highly metastatic Mvt1 mouse mammary tumor cell line (17) stably expressing either the long or short Brd4 isoforms.…”

“…The Brd4-expressing cell line has been previously described (19,20). Briefly, the highly metastatic Mvt1 cell line (17) was transfected with long isoform Brd4 expression vectors (19) or transduced with short isoform-expressing lentiviruses (20).…”

“…Individual clones were generated, total RNA isolated, and arrayed on Affymetrix MOE430 v2 chips by the K.W.H. laboratory, as previously described (19) or by the Microarray Core in the NCI Laboratory of Molecular Technology (20). Expression data were normalized using the Partek Genomics Suite then loaded into BRB ArrayTools version 4.2.0-Beta_2 (42).…”

BRD4 is an atypical kinase that phosphorylates Serine2 of the RNA Polymerase II carboxy-terminal domain

“…More recently, it has been characterized as a key determinant in acute myeloid leukemia, multiple myeloma, Burkitt's lymphoma, NUT midline carcinoma, colon cancer, and inflammatory disease (2)(3)(4)(5)(6)(7). It suppresses tumor metastasis in mice, and its expression is a prognostic signature of breast cancer survival (8). BRD4 has been proposed to be a structural scaffold that regulates transcription indirectly by recruiting the elongation factor, PTEFb, to the transcription preinitiation complex (9).…”