Abstract 3420: A survival-associated polymorphism within metastasis suppressor RRP1B directs RRP1B-chromatin interactions and self-regulation of gene expression
Authors: Dworkin Amy M. , Lee Minnkyong , Lichtenberg Jens , Gildea Derek , Trivedi Niraj , Wolfsberg Tyra , Crawford Nigel P. 
Published: August 2012

Previous studies demonstrate that activation of Ribosomal RNA Processing 1 Homolog B (Rrp1b) suppresses the metastatic capacity of the aggressive Mvt-1 mouse mammary tumor cell line. Over-expression of Rrp1b induces a gene expression signature that can be used to accurately predict survival in human breast cancer. Also, human RRP1B contains a non-synonymous coding SNP (rs9306160) associated with survival in multiple breast cancer cohorts. However, the mechanism by which RRP1B modulates metastasis is unclear. Co-IF, tandem affinity purification and co-IP reveal that RRP1B co-localizes with double stranded DNA in the nucleus. Significantly, these analyses show that RRP1B physically interacts with numerous DNA binding proteins, many of which are prominent regulators of gene expression. We hypothesize that RRP1B regulates gene expression in tumors prone to metastasis through either direct or indirect interaction with target regions. We performed ChIP-seq analysis of the human breast cancer cell lines MDA-MB231 and MCF7 to define RRP1B bound genomic loci. ChIP-seq shows that RRP1B reproducibly interacts with 180 chromatin regions including known metastasis genes, oncogenes and tumor suppressors. Interestingly, RRP1B interacts with its own genomic locus at exon 13, which contains the metastasis-associated SNP (rs9306160; G1421A). To further characterize this interaction, MDA-MB231 cell lines stably over-expressing either the RRP1B 1421G (WT) or the RRP1B 1421A (Variant) allele were created. Growth curves and soft agar assays were both significantly different between the WT and Variant allele cell lines. Multiple clonal isolates were then transfected with a luciferase reporter construct containing the 1.5 kb RRP1B exon 13 peak binding region to examine the transcriptional activity. These studies indicated that binding of RRP1B to its own exon 13 negatively regulates its expression. Significantly, the magnitude of this decrease in RRP1B transcription was greater in cell lines over-expressing the WT allele, which is associated with the poorer overall survival in breast cancer. Global analysis of gene expression in the cell lines over-expressing the two allelic variants was performed using microarrays. A distinct gene expression signature was found to delineate the cells over-expressing the two allelic variants. A number of these genes overlap with RRP1B ChIP-seq peaks, indicating that RRP1B may be differentially binding to the regulatory regions of the genes identified on microarray analysis. In conclusion, we have demonstrated that RRP1B regulates its own expression through binding to its own locus, and that this ability to self-regulate is modulated by a germline polymorphism within the region of chromatin bound by RRP1B. These observations give us further insight into the mechanism of action of this germline-encoded metastasis suppressor.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3420. doi:1538-7445.AM2012-3420