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. We are continuing to analyze RIP-seq data to enhance our understanding of the role of RNA binding proteins in tumor progression and metastasis. Our earlier work also demonstrates that RRP1B is a component of protein complexes with chromatin binding activity. We previously utilized ChIP-seq to demonstrate that RRP1B interacts with multiple genomic loci to regulate gene expression. Our analyses revealed that RRP1B is regulating gene expression through a variety of mechanisms. One particularly relevant mechanism of regulation is suppression of gene expression by RRP1B-mediated binding of the TRIM28/HP1-alpha heterochromatin complex. This is associated with an increase in histone H3 tri-methyl lysine 9 levels at RRP1B bound genomic loci, implying that RRP1B binding induces changes in epigenetic markers. Our recent analyses have revealed that changes in RRP1B levels have a strong effect on global histone methylation, which demonstrates that a prominent mechanism by which RRP1B is modulating metastasis-associated gene expression is through epigenetic reprogramming. 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, 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|
|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|