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...

Summary In cell lines from advanced lung cancer, breast cancer, and melanoma, endogenous tensin-3 contributes to cell migration, anchorage-independent growth, and tumorigenesis. Although SH2 domains have not been reported previously to be phosphorylated, the tensin-3 SH2 domain is a physiologic substrate for Src. Tyrosines in the SH2 domain contribute to the biological activity of tensin-3, and phosphorylation of these tyrosines can regulate ligand binding. In a mouse breast cancer model, tensin-3 tyrosines are phosphorylated in a Src-associated manner in primary tumors, and experimental metastases induced by tumor-derived cell lines depend on endogenous tensin-3. Thus, tensin-3 is implicated as a proto-oncogene regulated by Src and possessing an SH2 domain with a previously undescribed mechanism for the regulation of ligand binding.

A novel candidate metastasis modifier, ribosomal RNA processing 1 homolog B (Rrp1b), was identified through two independent approaches. First, yeast two-hybrid, immunoprecipitation, and functional assays demonstrated a physical and functional interaction between Rrp1b and the previous identified metastasis modifier Sipa1. In parallel, using mouse and human metastasis gene expression data it was observed that extracellular matrix (ECM) genes are common components of metastasis predictive signatures, suggesting that ECM genes are either important markers or causal factors in metastasis. To investigate the relationship between ECM genes and poor prognosis in breast cancer, expression quantitative trait locus analysis of polyoma middle-T transgene-induced mammary tumor was performed. ECM gene expression was found to be consistently associated with Rrp1b expression. In vitro expression of Rrp1b significantly altered ECM gene expression, tumor growth, and dissemination in metastasis assays. Furthermore, a gene signature induced by ectopic expression of Rrp1b in tumor cells predicted survival in a human breast cancer gene expression dataset. Finally, constitutional polymorphism within RRP1B was found to be significantly associated with tumor progression in two independent breast cancer cohorts. These data suggest that RRP1B may be a novel susceptibility gene for breast cancer progression and metastasis.

The bromodomain-containing chromatin-modifying factor BRD4 is an inherited susceptibility gene for breast cancer progression and metastasis, but its functionality in these settings has yet to be explored. Here we show that deletion of either of the BRD4 bromodomains had modest effects on the metastatic suppression ability of BRD4. In contrast, expression of the natural short isoform of BRD4 that truncates the protein after the SEED domain restored progression and metastatic capacity. Unexpectedly, deletion of the proline-rich region induced mesenchymal-like conversion and acquisition of cancer stem cell-like properties, which are mediated by the carboxy-terminal P-TEFb binding domain. Deletion of this proline-rich region also induced a gene expression signature that predicted poor outcome in human breast cancer data sets and that overlapped G3 grade human breast tumors. Thus our findings suggest that BRD4 may be altering the predisposition of tumors to undergo conversion to a more de-differentiated or primitive state during metastatic progression. Cancer Res; 71(8); 3121-31. Ó2011 AACR.

Pulmonary metastasis remains the leading ca use of death for cancer patients. Opportunities to improve treatment outcomes for patients require new methods to study and view the biology of metastatic progression. Here, we describe an ex vivo pulmonary metastasis assay (PuMA) in which the metastatic progression of GFPexpressing cancer cells, from a single cell to the formation of multicellular colonies, in the mouse lung microenvironment was assessed in real time for up to 21 days. The biological validity of this assay was confirmed by its prediction of the in vivo behavior of a variety of high-and low-metastatic human and mouse cancer cell lines and the discrimination of tumor microenvironments in the lung that were most permissive to metastasis. Using this approach, we provide what we believe to be new insights into the importance of tumor cell interactions with the stromal components of the lung microenvironment. Finally, the translational utility of this assay was demonstrated through its use in the evaluation of therapeutics at discrete time points during metastatic progression. We believe that this assay system is uniquely capable of advancing our understanding of both metastasis biology and therapeutic strategies. IntroductionPulmonary metastasis remains a leading cause of death for cancer patients (1, 2). Opportunities to improve outcomes for these patients require a greater understanding of the biology of metastasis. In addition, there is a need to evaluate novel therapeutics, in a timely manner, that specifically target metastases and metastatic progression. Simple in vitro assay systems are not sufficient to model the complex interaction between cancer cells and the surrounding microenvironment that is necessary for metastasis (3). Accordingly, in vivo models of metastasis, largely in mice, have been necessary. For the most part, these models provide end points of metastatic outcome (i.e., yes or no metastasis) and time to late-stage metastatic events.A "black box" exists during which metastatic progression from single cells to gross metastatic lesions at a secondary site occurs. Recent attempts to shed light on this process have included imaging strategies that allow some of the steps of metastatic progression to be followed in vivo (4). However, these approaches often involve sophisticated and expensive imaging techniques that are time consuming and do not easily allow serial assessment of early metastatic progression at secondary sites, particularly in the lung and at the single-cell level. Challenges associated with studying metastasis have resulted in limited opportunities to include the assessment of novel treatment agents against metastatic end points (5). Therefore, an unmet need in the field of cancer research is a simple assay in which the process of metastatic progression at a secondary site can be reproduced and studied over time.

Metastasis is a complex process utilizing both tumor-cell-autonomous properties and host-derived factors, including cellular immunity. We have previously shown that germline polymorphisms can modify tumor cell metastatic capabilities through cell-autonomous mechanisms. However, how metastasis susceptibility genes interact with the tumor stroma is incompletely understood. Here, we employ a complex genetic screen to identify Cadm1 as a novel modifier of metastasis. We demonstrate that Cadm1 can specifically suppress metastasis without affecting primary tumor growth. Unexpectedly, Cadm1 did not alter tumor-cell-autonomous properties such as proliferation or invasion, but required the host's adaptive immune system to affect metastasis. The metastasis-suppressing effect of Cadm1 was lost in mice lacking T cell–mediated immunity, which was partially phenocopied by depleting CD8+ T cells in immune-competent mice. Our data show a novel function for Cadm1 in suppressing metastasis by sensitizing tumor cells to immune surveillance mechanisms, and this is the first report of a heritable metastasis susceptibility gene engaging tumor non-autonomous factors.

Microarray expression signature analyses have suggested that extracellular matrix (ECM) gene dysregulation is predictive of metastasis in both mouse mammary tumorigenesis and human breast cancer. We have previously demonstrated that such ECM dysregulation is influenced by hereditary germline-encoded variation. To identify novel metastasis efficiency modifiers, we performed expression QTL (eQTL) mapping in recombinant inbred mice by characterizing genetic loci modulating metastasis-predictive ECM gene expression. Three reproducible eQTLs were observed on chromosomes 7, 17 and 18. Candidate genes were identified by correlation analyses and known associations with metastasis. Seven candidates were identified (Ndn, Pi16, Luc7l, Rrp1b, Brd4, Centd3 and Csf1r). Stable transfection of the highly metastatic Mvt-1 mouse mammary tumor cell line with expression vectors encoding each candidate modulated metastasis-predictive ECM gene expression. Implantation of these cells into mice demonstrated that candidate gene ectopic expression impacts tumor progression. Gene expression analyses facilitated the construction of a transcriptional network that we have termed the 'Diasporin Pathway'. This pathway contains the seven candidates, as well as metastasis-predictive ECM genes and metastasis suppressors. Brd4 and Rrp1b appear to form a central node within this network, which likely is a consequence of their physical interaction with the metastasis efficiency modifier Sipa1. Furthermore, we demonstrate that the microarray gene expression signatures induced by activation of ECM eQTL genes in the Mvt-1 cell line can be used to accurately predict survival in a human breast cancer cohort. These data imply that the Diasporin Pathway may be an important nexus in tumor progression in both mice and humans.

Recent studies suggest that BET inhibitors are effective anti-cancer therapeutics. Here we show that BET inhibitors are effective against murine primary mammary tumors, but not pulmonary metastases. BRD4, a target of BET inhibitors, encodes two isoforms with opposite effects on tumor progression. To gain insights into why BET inhibition was ineffective against metastases the pro-metastatic short isoform of BRD4 was characterized using mass spectrometry and cellular fractionation. Our data show that the pro-metastatic short isoform interacts with the LINC complex and the metastasis-associated proteins RRP1B and SIPA1 at the inner face of the nuclear membrane. Furthermore, histone binding arrays revealed that the short isoform has a broader acetylated histone binding pattern relative to the long isoform. These differential biochemical and nuclear localization properties revealed in our study provide novel insights into the opposing roles of BRD4 isoforms in metastatic breast cancer progression.