Our studies of RRP1B and NDN have been completed in the last year and no further work on either of these projects will be carried forward to the next fiscal year. Given the clinical relevance of the changes in gene expression induced by dysregulation of RRP1B, work in the last year has focused upon defining the mechanism by which Rrp1b regulates metastasis-associated transcription. A variety of approaches have been 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, we have defined the chromatin-binding properties of RRP1B to further 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 has also focused on how this metastasis modifier regulates transcription. Although the role of NDN in breast cancer has been poorly understood, it is known that this gene encodes a transcription factor. Differential expression of Ndn induces a gene-expression signature that predicts prognosis in human breast cancer. Additionally, a non-synonymous germline single nucleotide polymorphism (T50C; V17A) in Ndn distinguishes mouse strains with differing metastatic capacities. To better understand how hereditary factors influence metastasis in breast cancer, we characterized NDN-mediated transcription. Haplotype analysis in the well-characterized The Cancer Genome Atlas breast cancer cohort revealed that NDN germline variation is associated with both NDN expression levels and patient outcome. To examine the role of NDN in mammary tumor metastasis and transcriptional regulation, mouse mammary tumor cell lines stably over-expressing either the wildtype 50T or variant 50C Ndn allele were generated. Cells over-expressing Ndn 50T, but not Ndn 50C, exhibited significant decrease in cell invasiveness and pulmonary metastases compared to control cells. Transcriptome analyses identified a 71-gene expression signature that distinguishes cells over-expressing the two Ndn allelic variants. Furthermore, ChIP assays revealed c-Myc, a target gene of NDN, to be differentially regulated by the allelic variants. These data demonstrate that NDN and the T50C allele regulate gene expression and metastasis efficiency.
| 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 |
