Allelic variation and differential expression of the mSIN3A histone deacetylase complex gene Arid4b promote mammary tumor growth and metastasis

Abstract

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.

Figure 1. Arid4b expression is correlated with tumor growth and metastasis.

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

Figure 2. Arid4b germline amino acid sequence variants.

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.

Figure 3. Ectopic expression of Arid4b increases orthotopic tumor growth, tumor cell migration, and invasion.

Scatter plot bars represent median tumor mass (A) four weeks after orthotopic implantation of 10∧5 cells stably expressing lacZ (n = 20), Arid4b AKR allele (n = 17) or Arid4b DBA allele (n = 20). Migrating and invading cells (B) were counted in five 400× fields per experiment and data are presented as the mean ± standard error of three independent experiments. Statistical significance was determined using Kruskal-Wallis tests followed by Conover-Inman post hoc tests. *, p<0.05; ***, p<0.001.

Figure 4. Stable shRNA–mediated knockdown of Arid4b inhibits lung metastasis.

ARID4B protein levels were analyzed by western blot to determine level of knockdown in cell lines stably expressing different Arid4b shRNAs. Experiment was performed in triplicate and a representative blot is shown in panel A. Lysate from 293 cells transiently transfected with Arid4b 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 Arid4b 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.

Figure 5. Differential binding of the AKR and DBA variants of ARID4B to the mSIN3A complex.

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.

Figure 6. ARID4B binds BRMS1.

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.

Figure 7. Expression of Tpx2 network genes in Arid4b knockdown cell lines.

The nine labeled hub genes were conserved across mouse and human breast cancer data sets. Red indicates statistically significant (p<.05) downregulation; green indicates upregulation.

Figure 8. High expression of ARID4B is associated with poor clinical outcome.

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 ARID4B, 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 ARID4B terciles, respectively, compared to the high ARID4B tercile. The association of high ARID4B 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 ARID4B groups compared to high ARID4B.