Rrp1b, a new candidate susceptibility gene for breast cancer progression and metastasis

Abstract

A novel candidate metastasis modifier, ribosomal RNA processing 1 homolog B (Rrp1b), was identified through two independent approaches. First, yeast two-hybrid, immunoprecipitation, and functional assays demonstrated a physical and functional interaction between Rrp1b and the previous identified metastasis modifier Sipa1. In parallel, using mouse and human metastasis gene expression data it was observed that extracellular matrix (ECM) genes are common components of metastasis predictive signatures, suggesting that ECM genes are either important markers or causal factors in metastasis. To investigate the relationship between ECM genes and poor prognosis in breast cancer, expression quantitative trait locus analysis of polyoma middle-T transgene-induced mammary tumor was performed. ECM gene expression was found to be consistently associated with Rrp1b expression. In vitro expression of Rrp1b significantly altered ECM gene expression, tumor growth, and dissemination in metastasis assays. Furthermore, a gene signature induced by ectopic expression of Rrp1b in tumor cells predicted survival in a human breast cancer gene expression dataset. Finally, constitutional polymorphism within RRP1B was found to be significantly associated with tumor progression in two independent breast cancer cohorts. These data suggest that RRP1B may be a novel susceptibility gene for breast cancer progression and metastasis.

Figure 1. Rrp1b Forms a Complex with Sipa-1 and Inhibits its RapGAP Activity

(A) and (B) Complex formation between Rrp1b and Sipa1, and between AQP2 and Sipa1. AQP2 or Rrp1b-V5 (A) or Rrp1b-HA (B) was expressed singly or coexpressed with Sipa1-V5 in 293 cells as indicated. In (A), anti-Sipa1 immunocomplexes were immunoblotted with anti-V5 antibodies (top western blot (WB) panel) or anti-AQP2 antibodies (next WB panel). In (B), anti-Rrp1b-HA immunocomplexes were immunoblotted with anti-Sipa1-V5 antibody (WB panel). In the two lower panels of A and B, the immunoblots show the expression in cell extracts of exogenous Rrp1b-V5, Sipa1-V5, AQP2, and Rrp1b-HA. (C) Inhibition of Sipa1 RapGAP Activity by AQP2 and Rrp1b. In cells expressing Epac, Sipa1-V5 was expressed with AQP2 or Rrp1b-V5 as indicated. The in vivo Rap1GTP levels were assayed by RalGDS-RBD pull-down (top panel). In the lower panels, the immunoblots show the expression of endogenous Rap1, and the exogenous proteins: Sipa1-V5, Rrp1b-V5, AQP2, and Epac-HA. The Rap1 and EpacHA immunoblots also serve as loading controls.

Figure 2. eQTL Analysis in the AKXD RI Mouse Panel

Analysis of metastasis class-predictive ECM gene component expression patterns in PyMT transgene-induced tumors in AKXD recombinant inbred mice reveals the presence of three relatively consistent eQTLs on chromosomes 7, 17, and 18. The diagram on the left shows an eQTL cluster map generated with WebQTL. The warm hues reflect an LRS for increased transcription in mice with the DBA/2J genotype at any given locus, and the cool hues represent LRS for elevated transcription in mice with the AKR/J allele. The strongest degree of linkage is observed with the chromosome 17 eQTL, as illustrated in the three QTL scans on the right. In total, nine of the 16 metastasis-predictive probes displayed a LRS that was at least suggestive of linkage (denoted by the warm hues on the heat map to the left) suggesting that their expression is controlled in some respect by the chromosome 17 locus. These probes are located within the metastasis-predictive ECM genes Serping1, Nid1, Col1a1, Col1a2, Col3a1, Fbn1, and Mmp2. Rrp1b lies in very close physical proximity to the peak LRS within the chromosome 17 locus and displays a high degree of correlation of expression with a number of class-predictive ECM genes.

Figure 3. Ectopic Expression of Rrp1b Reduces (A) Tumor Growth and (B) Metastasis Burden in the Highly Metastatic Mvt-1 Cell Line

Mvt-1 cell lines were stably transfected with a mammalian expression vector encoding either Rrp1b or β-galactosidase. Following isolation of individual clones by serial dilution and confirmation of ectopic gene expression, clonal isolates were subcutaneously implanted in FVB/NJ mice. Those mice implanted with Mvt-1 clones ectopically expressing Rrp1b developed smaller primary tumors and fewer pulmonary metastases compared to mice implanted with a clonal isolate ectopically expressing β-galactosidase.

Figure 4. The RRP1B Microarray Expression Signature Predicts Survival into Good and Poor Outcome Groups Based on Tumor Gene Expression

(A) The RRP1B signature was highly predictive of overall survival in the Dutch Rosetta dataset, with the cumulative proportion survival being estimated to be 72% versus 34% for the good and poor prognosis RRP1B signatures, respectively (RRP1B signature HR = 3.28, 95% CI = 2.11–5.11). (B) Indeed, it appears that the RRP1B signature possesses a similar ability to predict survival in this dataset than the 70-gene signature described by van't Veer et al. [10]. Specifically, the survival for the good and poor prognosis 70-gene signatures were estimated to be 73% versus 47%, respectively (70 gene signature HR = 4.49, 95% CI = 2.65–7.61). (C) To stratify patients more adequately based upon their disease characteristics, RRP1B signature gene expression was examined with respect to tumor LN and ER status. Overall survival in the Dutch Rosetta patients with negative LNs was 79% versus 30% for the good and poor prognosis RRP1B signatures, respectively (HR = 4.34, 95% CI = 2.36–7.99). (D) Similarly, the RRP1B signature could sub-stratify patients with positive LNs into good and poor prognosis groups, with survivals being 65% versus 37%, respectively (HR = 2.38, 95% CI = 1.22–4.62). (E) A similar stratification effect by tumor RRP1B signature gene expression was observed in patients with ER positive tumors, with an overall survival in patients with ER positive tumors being 72% versus 50% for the good and poor prognosis RRP1B signatures, respectively (HR = 2.36, 95% CI = 1.17–4.77). (F) However, it did not prove possible to stratify patients with ER negative tumors based upon their RRP1B expression signature. This likely reflects the lack of individuals with this disease subtype in this cohort.

Figure 5. RRP1B 1421G→A SNP and Breast Cancer Survival

Kaplan-Meier survival analysis. The survival of patients with the variant allele (A/G or A/A; n = 92) is significantly better than the survival of patients who are homozygous carriers of the common allele (G/G; n = 150). Two-sided log-rank test: p = 0.025.

Figure 6. Haplotype Structure Surrounding rs9306160 in the CEPH Families

The LD structure of rs9306160. Pairwise r² values are shown with gray scale; the higher the r² value the darker the color. The red line and arrow mark the location of rs9306160. The large block in the middle represents the 210 kb LD block where 104 SNPs are in high LD with rs9306160. The figure was generated by the program HaploView [42].