Given the clinical relevance of the changes in gene expression induced by dysregulation of RRP1B, our current work focuses upon defining the mechanism by which Rrp1b regulates metastasis-associated transcription. A variety of approaches are being employed to define how RRP1B interacts with RNA to regulate transcription. RNA-seq has revealed that RRP1B regulates alternative mRNA splicing, a process which is ubiquitously dysregulated in advanced tumorigenesis. Specifically, we have demonstrated that RRP1B knockdown inducing differential isoform expression in over 600 genes. This activity is mediated through a transcriptionally-dependent interaction with the splicing regulator SRSF1. Additionally, RNA immunoprecipitation sequencing (RIP-seq) has revealed that RRP1B directly interacts with RNA. To our knowledge, this is the first example of a metastasis suppressor with RNA binding activity and our ongoing work will focus on the consequences of this interaction. Specifically, we are in the process of defining how the metastasis-associated 1421C>T (P436L) polymorphism influences the RNA binding activity of RRP1B. These studies will enhance our understanding of the role of RNA binding proteins in tumor progression and metastasis. Over-expression of RRP1B induces a transcriptional profile that accurately predicts patient outcome in breast cancer. However, the mechanism by which RRP1B modulates transcription is unclear. We have defined the chromatin-binding properties of RRP1B were examined to define how it regulates metastasis-associated transcription. To identify genome-wide RRP1B binding sites, high-throughput ChIP-seq was performed in the human breast cancer cell line MDA-MB-231 and HeLa cells using antibodies against endogenous RRP1B. Global changes in repressive marks such as histone H3 lysine 9 trimethylation (H3K9me3) were also examined by ChIP-seq. Analysis of these samples identified 339 binding regions in MDA-MB-231 cells and 689 RRP1B binding regions in HeLa cells. Among these, 136 regions were common to both cell lines. Gene expression analyses of these RRP1B-binding regions revealed that transcriptional repression is the primary result of RRP1B binding to chromatin. ChIP-reChIP assays demonstrated that RRP1B co-occupies loci with decreased gene expression with the heterochromatin-associated proteins, tripartite motif-containing protein 28 (TRIM28/KAP1) and heterochromatin protein 1-alpha (CBX5/HP1alpha). RRP1B occupancy at these loci was also associated with higher H3K9me3 levels, indicative of heterochromatinization mediated by the TRIM28/HP1alpha complex. In addition, RRP1B up-regulation, which is associated with metastasis suppression, induced global changes in histone methylation. Implications: RRP1B, a breast cancer metastasis suppressor, regulates gene expression through heterochromatinization and transcriptional repression, which helps our understanding of mechanisms that drive prognostic gene expression in human breast cancer. Our work with NDN is also focused on how this metastasis modifier regulates transcription. Although the role of NDN in breast cancer is poorly understood, it is known that this gene encodes a transcription factor. We are therefore utilizing ChIP-seq to explore the role of NDN in breast cancer metastasis susceptibility. We are particularly interested in the role of a non-synonymous coding variant of mouse NDN that is present in a number of laboratory mouse strains in transcriptional regulation. Ectopic expression of either allelic variant has divergent effects on the metastatic capacity of several mouse mammary tumor cell lines, with only the wildtype, and not the variant allele, suppressing metastasis. Microarray analysis of these cell lines reveals strong differences in gene expression between the cell lines expressing either allelic variant. Accordingly, ChIP-seq analysis of these same cell lines reveals that the two allelic variants have a distinct set of chromatin interaction peaks. Analysis of these data is ongoing and aims to define the mechanism by which NDN modulates mammary tumor metastasis. These studies will allow us to extend our observations to focus upon the function of human NDN and its putative role in human breast cancer progression and metastasis.
| Lee, Minnkyong; Dworkin, Amy M; Lichtenberg, Jens et al. (2014) Metastasis-associated protein ribosomal RNA processing 1 homolog B (RRP1B) modulates metastasis through regulation of histone methylation. Mol Cancer Res 12:1818-28 |
| Lee, M; Dworkin, A M; Gildea, D et al. (2014) RRP1B is a metastasis modifier that regulates the expression of alternative mRNA isoforms through interactions with SRSF1. Oncogene 33:1818-27 |
| Kanaan, Ziad; Rai, Shesh N; Eichenberger, M Robert et al. (2012) Differential microRNA expression tracks neoplastic progression in inflammatory bowel disease-associated colorectal cancer. Hum Mutat 33:551-60 |
| Alsarraj, Jude; Walker, Renard C; Webster, Joshua D et al. (2011) Deletion of the proline-rich region of the murine metastasis susceptibility gene Brd4 promotes epithelial-to-mesenchymal transition- and stem cell-like conversion. Cancer Res 71:3121-31 |
