Jude Alsarraj

Mechanisms of metastasis

Metastasis is an enormously complex process that remains to be a major problem in the management of cancer. The fact that cancer patients might develop metastasis after years or even decades from diagnosis of the primary tumor makes the metastatic process even more complex. Over the years many hypotheses were developed to try to explain the inefficiency of the metastatic process, but none of these theories completely explains the current biological and clinical observations. In this review we summarize some of the proposed models that were developed in attempt to understand the mechanisms of tumor dissemination and colonization as well as metastatic progression.

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.

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.

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.

Gene expression profiles and breast cancer metastasis: a genetic perspective

The majority of cancer mortality is attributed to metastasis, which is the spread of tumor cells to a secondary site. Several studies have demonstrated that the genetic background on which a tumor arises has a major effect on both metastatic efficiency and on predictive gene expression profiles. These observations suggest that there is variability in metastasis frequency between individuals and that some individuals could be more prone to secondary tumor formation and development than others. Thus, genetic background might have important clinical implications in metastasis detection, management and prevention.

Bromodomain-Containing Protein 4: A Dynamic Regulator of Breast Cancer Metastasis through Modulation of the Extracellular Matrix.

Metastasis is an extremely complex process that accounts for most cancer-related deaths. Malignant primary tumors can be removed surgically, but the cells that migrate, invade, and proliferate at distant organs are often the cells that prove most difficult to target therapeutically. There is growing evidence that host factors outside of the primary tumors are of major importance in the development of metastasis. Recently, we have shown that the bromodomain-containing protein 4 or bromodomain 4 (Brd4) functions as an inherited susceptibility gene for breast cancer progression and metastasis. In this paper, we will discuss that host genetic background on which a tumor arises can significantly alter the biology of the subsequent metastatic disease, and we will focus on the role of Brd4 in regulating metastasis susceptibility.

Abstract 5269: Mechanistic insights into regulation of metastasis by Sipa1

Metastasis is the major cause for morbidity and mortality of cancer patients. Still, the molecular mechanisms underlying metastasis are incompletely understood. Conceivably, a better knowledge of the metastatic process will enable the design of better treatments for cancer patients in the future. Previously, we have shown that genetic background has a strong influence on metastasis susceptibility in breast cancer mouse models. Single nucleotide polymorphisms (SNP) in the Sipa1 gene are associated with metastasis in mice and human breast cancer patients. Consistent with this observation, we have shown that Sipa1 regulates metastasis in breast cancer cells; however, the molecular mechanisms remain largely unknown. We identified several potential binding partners of SIPA1 in a yeast-two-hybrid screen, and confirmed interactions of SIPA1 with BRD4, RRP1B and SUN2 in subsequent experiments. Our analysis suggests that several complexes of SIPA1 exist in different compartments of the cell. A structure-function analysis that we have begun to carry out indicates that Sipa1 regulates metastasis in several ways, depending on its interaction partners and subcellular localization. Furthermore, the enzymatic GTPase-activating function of Sipa1 appears to play a critical role in regulating metastasis. These results could facilitate developing novel therapeutic approaches for the treatment of metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5269. doi:10.1158/1538-7445.AM2011-5269

Abstract 5761: An alternative isoform of the metastasis susceptibility gene Brd4 promotes EMT and metastatic progression

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Recent work from our laboratory has identified the bromodomain 4 (Brd4) as a suppressor of tumor progression and metastasis. Brd4 however has an alternatively spliced isoform that truncates the protein and substitutes a different 3′-UTR. Unlike the longer isoform, expression of the short isoform (ΔC) does not impair primary tumor growth of a highly metastatic mammary tumor cell line orthotopically implanted into the mammary fat pad, although pulmonary metastasis is significantly reduced compared to control tumors. Unexpectedly, a dramatic enhancement of metastatic colonization is observed following injection of the cells directly into the circulation of mice. In vivo invasion of the cell line was not impaired by expression of the short isoform suggesting that the inability of the orthotopic tumors to metastasize was not due to the inability of the tumor cells to escape from the primary tumor. Consistent with the tail vein experiments, in vitro analyses demonstrate that ΔC-expressing cells exhibit a more malignant phenotype with an increased ability to grow in a three dimensional culture compared to control cells. Furthermore, ΔC-expressing cells are able to form tumorspheres in serum-free and non adherent conditions, suggesting a reversion to a more stem-like state. Microarray gene expression analysis demonstrates that ectopic expression of ΔC modulates the expression of a subset of EMT markers and stem cell markers. Microarray analysis further shows that ectopic expression of ΔC induces a gene expression signature that predicts poor survival in multiple human breast cancer datasets. This expression signature nearly perfectly matches a molecular classifier of high-grade tumors. Taken together, these data suggest that the short isoform may be a metastasis-promoting molecule and that metastasis susceptibility mediated by Brd4 may be the result of a complex equilibrium between the metastasis-suppressing long isoform and the metastasis-promoting shorter isoform. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5761.