Abstract
There is accumulating evidence for a role of germ line variation in breast cancer metastasis. We have recently identified a novel metastasis susceptibility gene, Rrp1b (ribosomal RNA processing 1 homolog B). Overexpression of Rrp1b in a mouse mammary tumor cell line induces a gene expression signature that predicts survival in breast cancer. Here we extend the analysis of RRP1B function by demonstrating that the Rrp1b activation gene expression signature accurately predicted the outcome in three of four publicly available breast carcinoma gene expression data sets. In addition, we provide insights into the mechanism of RRP1B. Tandem affinity purification demonstrated that RRP1B physically interacts with many nucleosome binding factors, including histone H1X, poly(ADP-ribose) polymerase 1, TRIM28 (tripartite motif-containing 28), and CSDA (cold shock domain protein A). Co-immunofluorescence and co-immunoprecipitation confirmed these interactions and also interactions with heterochromatin protein-1alpha and acetyl-histone H4 lysine 5. Finally, we investigated the effects of ectopic expression of an RRP1B allelic variant previously associated with improved survival in breast cancer. Gene expression analyses demonstrate that, compared with ectopic expression of wild type RRP1B in HeLa cells, the variant RRP1B differentially modulates various transcription factors controlled by TRIM28 and CSDA. These data suggest that RRP1B, a tumor progression and metastasis susceptibility candidate gene, is potentially a dynamic modulator of transcription and chromatin structure.
Highlights
The concept of germ line polymorphism as a significant variable in susceptibility to metastasis in breast cancer is gaining wider acceptance in the scientific community [1]
Subsequent studies were based on two observations: that 1) knockdown of Sipa1 dysregulates the expression of extracellular matrix (ECM)3 genes,4 and 2) ECM genes are frequent components of prognostic microarray gene expression profiles observed in both human breast cancer (e.g. Refs. 7–9) and mouse models of mammary tumorigenesis (10 –12)
Characterization of Rrp1b signature genes associated with survival in each of the breast cancer data sets revealed overlapping but not identical gene expression signatures
Summary
Survival Analysis Using the Mvt-1/Rrp1b Microarray Gene Expression Signature—Generation of the Mvt-1/Rrp1b gene expression signature has been described elsewhere [14]. HEK293 cells were transfected with either RRP1B or eGFP C-TAP pDest-472 vectors using Fugene (Roche Applied Science). HeLa cells were seeded in 2-well chamber slides and transfected with the HA-RRP1B pDest-530 vector as described above. Cells were washed with PBS at 4 °C and incubated for 10 min in ice-cold cytoskeleton buffer (10 mM NaCl, 300 mM sucrose, 10 mM PIPES, pH 6.8, 3 mM MgCl2, 0.5% Triton X-100, 1ϫ Halt protease inhibitor mixture (Pierce), 20 units/ml recombinant RNasin (Promega) ribonuclease inhibitor). Quantitative Real Time PCR Gene Expression Analysis— cDNA was synthesized from RNA isolated from transfected cell lines using the iScript reverse transcription-PCR System (BioRad) by following the manufacturer’s protocol. Significance levels for comparisons were performed using the Mann-Whitney U test, with relative quantities of RRP1B-transfected and lacZtransfected HeLa cells used for analyses
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