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(RRP1B) Modulates Metastasis Through Regulation of Histone Methylation

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(RRP1B) Modulates Metastasis Through Regulation of Histone Methylation Published OnlineFirst August 4, 2014; DOI: 10.1158/1541-7786.MCR-14-0167 Molecular Cancer Oncogenes and Tumor Suppressors Research Metastasis-Associated Protein Ribosomal RNA Processing 1 Homolog B (RRP1B) Modulates Metastasis through Regulation of Histone Methylation Minnkyong Lee1, Amy M. Dworkin1, Jens Lichtenberg1, Shashank J. Patel1, Niraj S. Trivedi2, Derek Gildea2, David M. Bodine1, and Nigel P.S. Crawford1 Abstract Overexpression of ribosomal RNA processing 1 homolog B (RRP1B) induces a transcriptional profile that accurately predicts patient outcome in breast cancer. However, the mechanism by which RRP1B modulates transcription is unclear. Here, 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-a (CBX5/HP1a). RRP1B occupancy at these loci was also associated with higher H3K9me3 levels, indicative of heterochromatinization mediated by the TRIM28/HP1a complex. In addition, RRP1B upregulation, which is associated with metastasis suppression, induced global changes in histone methylation. Implications: RRP1B, a breast cancer metastasis suppressor, regulates gene expression through heterochroma- tinization and transcriptional repression, which helps our understanding of mechanisms that drive prognostic gene expression in human breast cancer. Mol Cancer Res; 12(12); 1818–28. Ó2014 AACR. Introduction tions (3). The sequential acquisition of random somatic Breast carcinoma is the most commonly diagnosed malig- mutations within the primary tumor is the most widely nancy in women in the United States, with over 235,000 accepted model of metastasis at the molecular level. How- new cases and over 40,000 deaths in 2012 (1). Metastasis is ever, it is becoming increasingly clear that although somatic mutation is the primary driver of metastasis, the propensity the main cause of death in breast cancer and metastatic fl disease is considered incurable (2). Tumorigenesis and of a primary tumor to metastasize is strongly in uenced by metastasis form a spectrum of progression that are induced interindividual germline variation (reviewed in ref. 4). by genetic alterations and disruption of epigenetic modifica- The consideration of metastasis susceptibility as a her- itable trait with a complex inheritance pattern has led to the identification of several germline-encoded metas- tasis modifier genes, including ribosomal RNA processing 1Genetics and Molecular Biology Branch, National Human Genome fi Research Institute, NIH, Bethesda, Maryland. 2Genome Technology 1 homolog B (RRP1B). RRP1B was identi ed as a germline Branch, National Human Genome Research Institute, NIH, Bethesda, susceptibility gene for breast cancer metastasis using Maryland. expression quantitative trait locus mapping in the FVB/ Note: Supplementary data for this article are available at Molecular Cancer N-Tg(MMTV-PyVT)634Mul/J mouse mammary tumor- Research Online (http://mcr.aacrjournals.org/). igenesis model (5). Specifically, two concurrent and inde- M. Lee and A.M. Dworkin contributed equally to this article. pendent experimental results identified Rrp1b as a novel fi fi Corresponding Author: Nigel P.S. Crawford, National Human Genome germline modi er of metastasis: rst, RRP1B is a binding Research Institute, NIH, 50 South Dr. Rm 5154, Bethesda, MD 20892. partner of the metastasis modifier SIPA1; second, Phone: 301-402-6078; Fax: 301-480-8711; E-mail: [email protected] RRP1B is a germline regulator of extracellular matrix gene doi: 10.1158/1541-7786.MCR-14-0167 expression, which is a class of genes frequently dysregu- Ó2014 American Association for Cancer Research. lated in tumors prone to metastasizing (5). Following its 1818 Mol Cancer Res; 12(12) December 2014 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst August 4, 2014; DOI: 10.1158/1541-7786.MCR-14-0167 RRP1B Regulates Histone H3K9 Trimethylation identification using modifier locus mapping, the proper- After viral infection, cells were selected in normal growth ties of Rrp1b were investigated by ectopic expression in the medium containing 10 mg/mL puromycin (Sigma-Aldrich), highly metastatic Mvt-1 mouse mammary tumor cell line. transferred to 96-well plates, and individual clones were These experiments demonstrated that Rrp1b dysregulation selected by limiting dilution. Ectopic expression of RRP1B in induced a gene expression signature that predicts survival single clonal isolate colonies was confirmed by qPCR as in multiple human breast cancer datasets with a high previously described (6). degree of reproducibility (5, 6). The relevance of RRP1B was further highlighted by the finding that a coding siRNA transfection germline polymorphism within human RRP1B is consis- Two different sequences of RRP1B Silencer Select siRNA tently associated with clinical outcome in multiple breast (Life Technologies, ID s22978 and s225927) were trans- cancer cohorts representing over 2,000 patients with fected in MDA-MB-231 cells using Lipofectamine RNAi- breast cancer (5, 7). MAX Transfection Reagent (Life Technologies) according Subsequent protein–protein interaction analyses revealed to the manufacturer's reverse transfection protocol. For several probable mechanisms by which Rrp1b dysregulation RNA isolation, cells were plated at 1 Â 105 per 24-well has such profound and clinically relevant effects on global gene and collected 48 hours after transfection. For protein analysis expression (6). Most notably, these initial studies suggested and ChIP assays, cells were plated in 6-well and 150 mm that RRP1B is most likely a facultative heterochromatin plates at 2 Â 105 per mL. protein because it colocalizes with several heterochromatin- associated histone marks. Concomitantly, RRP1B was shown Cell growth and invasion assays to physically interact with a number of heterochromatin- MDA-MB-231 clonal isolates infected with either HA- associated proteins, including tripartite motif-containing pro- RRP1B overexpression or control lentiviral particles were tein 28 (TRIM28; KAP1) and heterochromatin protein 1-a seeded in triplicate on 24 mm plates at a density of 105 cells a – (HP1 ;refs.811), which are potent inducers of gene per plate and incubated in 5% CO2 at 37 C for the indicated silencing. TRIM28 interacts with and recruits SETDB1 (SET periods of time. After incubation, the cells were harvested domain, bifurcated 1; ref. 9), a histone methyltransferase and the number of cells was counted. For soft agar growth which has been shown to colocalize with and mediate tri- assays, cells were plated in triplicate in 0.3% soft agar and methylation of histone H3 at lysine 9 (H3K9me3; ref. 12). allowed to grow for 7 days. Colonies were quantified after This creates high-affinity binding sites for the TRIM28/ staining with 0.005% crystal violet. Cell invasion assays were HP1a complex (13). H3K9me3 is a well-known marker of performed as previously described (18) using the MDA-MB- heterochromatin (14), and a strong association between 231 clonal isolate cell lines described above. TRIM28 and H3K9me3 has been reported (15, 16). In our current study, we aim to elucidate the mechanism Lung colonization assays by which RRP1B regulates metastasis-associated gene One million MDA-MB-231 RRP1B or control cells were expression. We utilized a variety of approaches to define intravenously injected via the tail vein of NU/J female mice the mechanism by which RRP1B suppresses gene expres- (Jackson Laboratory). Twenty-six mice were injected with sion. We have used chromatin immunoprecipitation (ChIP) MDA-MB-231 RRP1B cells and 11 mice were injected with assays to identify chromatin regions bound by RRP1B, and control cells. Lungs were harvested 90 days postinjection and demonstrated that these interactions are often associated pulmonary surface metastases were counted. All animal with binding of the transcriptional repressors HP1a and experiments were performed in compliance with the Nation- TRIM28. Furthermore, we demonstrate that an association al Human Genome Research Institute (Bethesda, MD) of these proteins at discrete genomic loci is concurrent with Animal Care and Use Committee's guidelines. H3K9me3 and downregulation of gene expression. Taken together, these data demonstrate that the clinically relevant Total RNA isolation changes in gene expression induced by RRP1B dysregulation Total RNA was isolated from cells using RNeasy Mini Kit and subsequent effects upon metastasis in breast cancer are, (QIAGEN). All samples were subjected to on-column at least in part, due to regulation of epigenetic mechanisms. DNase I digestion, and RNA quality and quantity were determined
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