Rrp1b, a New Candidate Susceptibility Gene for Breast Cancer Progression and Metastasis

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

Allelic Variation and Differential Expression of the mSIN3A Histone Deacetylase Complex Gene Arid4b Promote Mammary Tumor Growth and Metastasis

Accumulating evidence suggests that breast cancer metastatic progression is modified by germline polymorphism, although specific modifier genes have remained largely undefined. In the current study, we employ the MMTV-PyMT transgenic mouse model and the AKXD panel of recombinant inbred mice to identify AT–rich interactive domain 4B (Arid4b; NM_194262) as a breast cancer progression modifier gene. Ectopic expression of Arid4b promoted primary tumor growth in vivo as well as increased migration and invasion in vitro, and the phenotype was associated with polymorphisms identified between the AKR/J and DBA/2J alleles as predicted by our genetic analyses. Stable shRNA–mediated knockdown of Arid4b caused a significant reduction in pulmonary metastases, validating a role for Arid4b as a metastasis modifier gene. ARID4B physically interacts with the breast cancer metastasis suppressor BRMS1, and we detected differential binding of the Arid4b alleles to histone deacetylase complex members mSIN3A and mSDS3, suggesting that the mechanism of Arid4b action likely involves interactions with chromatin modifying complexes. Downregulation of the conserved Tpx2 gene network, which is comprised of many factors regulating cell cycle and mitotic spindle biology, was observed concomitant with loss of metastatic efficiency in Arid4b knockdown cells. Consistent with our genetic analysis and in vivo experiments in our mouse model system, ARID4B expression was also an independent predictor of distant metastasis-free survival in breast cancer patients with ER+ tumors. These studies support a causative role of ARID4B in metastatic progression of breast cancer

Caffeine Suppresses Metastasis in a Transgenic Mouse Model: A Prototype Molecule for Prophylaxis of Metastasis

A significant fraction of cancer patients have occult disseminated tumors at the time of primary diagnosis, which usually progress to become clinically relevant lesions. Since the majority of cancer mortality is associated with metastatic disease, the ability to inhibit the growth of the secondary tumors would significantly reduce cancer-related morbidity and mortality. We have investigated whether caffeine, which has been shown to suppress tumor cell invasiveness and experimental metastasis, can suppress metastasis in a spontaneous transgene-induced mammary tumor model. Chronic exposure to caffeine prior to the appearance of palpable mammary tumors significantly reduced both tumor burden and metastatic colonization. However, when caffeine exposure began after the appearance of frank tumors, caffeine suppressed metastasis without changing primary tumor burden. The means by which caffeine suppressed metastatic activity may be associated with inhibition of malignant transformation of mammary epithelial cells, inhibition of conversion of dormant tumor cells to micrometastases, micrometastases to macrometastases, or inhibition of tumor cell adhesion and motility. Gene and protein expression patterns resulting from caffeine treatment showed that metastasis suppression may be associated with up-regulation the mRNA expression of multiple extracellular matrix genes, including Fbln1, Bgn, Sparc, Fbn1, Loxl1, Colla1, Col3a1, Col5a1, ColS5a2, ColSa3, Col6a1, Col6a2, and Col6a3. These data suggested that caffeine or other methyl xanthine derivatives may improve the clinical outcome in patients prior to and following the diagnosis of metastatic disease, and could potentially reduce the morbidity and mortality associated with disseminated tumors

Cellular processes regulated by <i>Arid4b</i> knockdown.

<p>Biological processes are listed in order of statistical significance after applying a cutoff based on z-scores with an absolute value greater than 2. Negative z-scores indicate downregulation; positive z-scores indicate upregulation.</p

High expression of <i>ARID4B</i> is associated with poor clinical outcome.

<p>In patients with ER-positive tumors who were node negative at diagnosis (A) distant metastasis-free survival (DMFS) was significantly lower (p = .009) in patients with high expression (blue) compared to middle (red) or low (gray) expression of <i>ARID4B</i>, and multivariate analysis of 440 cases (B) was performed to determine metastatic progression hazard ratios of 0.54 and 0.42 for median and low <i>ARID4B</i> terciles, respectively, compared to the high <i>ARID4B</i> tercile. The association of high <i>ARID4B</i> with poor DMFS was also highly significant (p = 3.05×10∧−4) among ER-positive patients in the absence of adjuvant therapy (C) with similar hazard ratios (D) of 0.53 and 0.49 for middle and low <i>ARID4B</i> groups compared to high <i>ARID4B</i>.</p

<i>Arid4b</i> germline amino acid sequence variants.

<p>Substitutions in the AKR allele are shown relative to a consensus sequence that is identical between DBA, FVB, and C57Bl/6. The+symbol indicates conserved substitutions.</p

ARID4B binds BRMS1.

<p>HA-BRMS1 was coprecipitated with FLAG-ARID4B (A) and FLAG-ARID4B was coprecipitated with myc-BRMS1 (B). No difference in binding to BRMS1 was observed between the AKR and DBA alleles of ARID4B.</p

<i>Arid4b</i> expression is correlated with tumor growth and metastasis.

<p>Gene expression microarray data from 18 of the AKXD recombinant inbred strains shows expression of <i>Arid4b</i> is positively correlated with tumor burden (A) and metastatic density (B). The formula used to calculate metastatic density was ln[(metastases/um²)×10⁸].</p

Stable shRNA–mediated knockdown of <i>Arid4b</i> inhibits lung metastasis.

<p>ARID4B protein levels were analyzed by western blot to determine level of knockdown in cell lines stably expressing different <i>Arid4b</i> shRNAs. Experiment was performed in triplicate and a representative blot is shown in panel A. Lysate from 293 cells transiently transfected with <i>Arid4b</i> was used as a positive control (+C). Untransduced 6DT1 and scrambled control lines are designated “untr” and “scr” respectively. The lower non-specific (n.s.) band most likely represents ARID4A, based on a nearly identical epitope sequence and consistent with the observed difference in molecular weight. Expression of the non-specific band remained relatively unchanged across the panel of stable lines. Densitometry was performed to quantitate knockdown at the protein level in the <i>Arid4b</i> shRNA lines relative to the scrambled control line (B) Data are shown as the mean ± standard error of three independent experiments. Kruskal-Wallis followed by Conover-Inman post hoc tests were used to determine statistical significance versus the scrambled control cell line. *, p<.05; **, p<.01; ***, p<.001. Primary tumors were weighed (C) and lung surface metastases were counted (D) at 3.5 weeks after orthotopic implantation of 10∧5 cells from the scrambled control, H3, and H4 lines into the mammary fat pad of FVB mice (n = 10 per cohort). Horizontal bars represent median values and statistical significance was determined using Kruskal-Wallis tests followed by Conover-Inman correction for multiple comparisons.</p

Differential binding of the AKR and DBA variants of ARID4B to the mSIN3A complex.

<p>Western blots (A) show equivalent input of V5-ARID4B, mSIN3A, mSDS3, and equivalent pull-down of the two variants using an anti-V5 antibody. Decreased binding of the DBA variant of ARID4B to mSIN3A and mSDS3 was observed relative to the AKR variant. Experiment was performed in triplicate and densitometry analysis (B) revealed a 51% reduction in mSIN3A binding (p = .037) and a 37% reduction in mSDS3 binding (p = .026) for the DBA variant relative to AKR. Statistical significance was determined using paired t-tests. *, p<.05.</p