Renard C. Walker

Bromodomain 4 activation predicts breast cancer survival

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.

The Diasporin Pathway: a tumor progression-related transcriptional network that predicts breast cancer survival

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.

Modeling metastasis biology and therapy in real time in the mouse lung

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 GFP-expressing 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.

The Tensin-3 Protein, Including its SH2 Domain, Is Phosphorylated by Src and Contributes to Tumorigenesis and Metastasis

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 an oncoprotein regulated by Src and possessing an SH2 domain with a previously undescribed mechanism for the regulation of ligand binding.

Cadm1 Is a Metastasis Susceptibility Gene That Suppresses Metastasis by Modifying Tumor Interaction with the Cell-Mediated Immunity

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 hosts 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.

Deletion of the Proline-Rich Region of the Murine Metastasis Susceptibility Gene Brd4 Promotes Epithelial-to-Mesenchymal Transition- and Stem Cell-Like Conversion

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.

The Origins of Breast Cancer Prognostic Gene Expression Profiles

Recent high profile clinical trials show that microarray-based gene expression profiling has the potential to become an important tool for predicting prognosis in breast cancer. Earlier work in our laboratory using mouse models and human breast cancer populations has enabled us to show that metastasis susceptibility is an inherited trait. This same combined approach facilitated the identification of a number of candidate genes that, when dysregulated, have the potential to induce prognostic gene expression profiles in human data sets. To investigate if these gene expression signatures were of somatic or germline origin and to assess the contribution of different cell types to the induction of these signatures, we have performed a series of expression profiling experiments in a mouse model of metastatic breast cancer. These results show that both the tumor epithelium and invading stromal tissues contribute to the development of prognostic gene signatures. Furthermore, analysis of normal tissues and tumor transplants suggests that prognostic signatures result from both somatic and inherited components, with the inherited components being more consistently predictive.

BRD4 short isoform interacts with RRP1B, SIPA1 and components of the LINC complex at the inner face of the nuclear membrane.

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.

Allelic variation and differential expression of the mSIN3A histone deacetylase complex gene Arid4b promote mammary tumor growth and metastasis.

Accumulating evidence suggests that breast cancer metastatic progression is modified by germline polymorphism, although specific modifier genes have remained largely undefined. In the current study, we employ the MMTV-PyMT transgenic mouse model and the AKXD panel of recombinant inbred mice to identify AT–rich interactive domain 4B (Arid4b; NM_194262) as a breast cancer progression modifier gene. Ectopic expression of Arid4b promoted primary tumor growth in vivo as well as increased migration and invasion in vitro, and the phenotype was associated with polymorphisms identified between the AKR/J and DBA/2J alleles as predicted by our genetic analyses. Stable shRNA–mediated knockdown of Arid4b caused a significant reduction in pulmonary metastases, validating a role for Arid4b as a metastasis modifier gene. ARID4B physically interacts with the breast cancer metastasis suppressor BRMS1, and we detected differential binding of the Arid4b alleles to histone deacetylase complex members mSIN3A and mSDS3, suggesting that the mechanism of Arid4b action likely involves interactions with chromatin modifying complexes. Downregulation of the conserved Tpx2 gene network, which is comprised of many factors regulating cell cycle and mitotic spindle biology, was observed concomitant with loss of metastatic efficiency in Arid4b knockdown cells. Consistent with our genetic analysis and in vivo experiments in our mouse model system, ARID4B expression was also an independent predictor of distant metastasis-free survival in breast cancer patients with ER+ tumors. These studies support a causative role of ARID4B in metastatic progression of breast cancer.

Inherited Variation in miR-290 Expression Suppresses Breast Cancer Progression by Targeting the Metastasis Susceptibility Gene Arid4b

The metastatic cascade is a complex and extremely inefficient process with many potential barriers. Understanding this process is of critical importance because the majority of cancer mortality is associated with metastatic disease. Recently, it has become increasingly clear that microRNAs (miRNA) play important roles in tumorigenesis and metastasis, yet few studies have examined how germline variations may dysregulate miRNAs, in turn affecting metastatic potential. To explore this possibility, the highly metastatic MMTV-PyMT mice were crossed with 25 AKXD (AKR/J × DBA/2J) recombinant inbred strains to produce F1 progeny with varying metastatic indices. When mammary tumors from the F1 progeny were analyzed by miRNA microarray, miR-290 (containing miR-290-3p and miR-290-5p) was identified as a top candidate progression-associated miRNA. The microarray results were validated in vivo when miR-290 upregulation in two independent breast cancer cell lines suppressed both primary tumor and metastatic growth. Computational analysis identified breast cancer progression gene Arid4b as a top target of miR-290-3p, which was confirmed by luciferase reporter assay. Surprisingly, pathway analysis identified estrogen receptor (ER) signaling as the top canonical pathway affected by miR-290 upregulation. Further analysis showed that ER levels were elevated in miR-290-expressing tumors and positively correlated with apoptosis. Taken together, our results suggest miR-290 targets Arid4b while simultaneously enhancing ER signaling and increasing apoptosis, thereby suppressing breast cancer progression. This, to the best of our knowledge, is the first example of inherited differences in miRNA expression playing a role in breast cancer progression.