Combining the pan-aurora kinase inhibitor AMG 900 with histone deacetylase inhibitors enhances antitumor activity in prostate cancer

Abstract

Histone deacetylase inhibitors (HDACIs) are being tested in clinical trials for the treatment of solid tumors. While most studies have focused on the reexpression of silenced tumor suppressor genes, a number of genes/pathways are downregulated by HDACIs. This provides opportunities for combination therapy: agents that further disable these pathways through inhibition of residual gene function are speculated to enhance cell death in combination with HDACIs. A previous study from our group indicated that mitotic checkpoint kinases such as PLK1 and Aurora A are downregulated by HDACIs. We used in vitro and in vivo xenograft models of prostate cancer (PCA) to test whether combination of HDACIs with the pan-aurora kinase inhibitor AMG 900 can synergistically or additively kill PCA cells. AMG 900 and HDACIs synergistically decreased cell proliferation activity and clonogenic survival in DU-145, LNCaP, and PC3 PCA cell lines compared to single-agent treatment. Cellular senescence, polyploidy, and apoptosis was significantly increased in all cell lines after combination treatment. In vivo xenograft studies indicated decreased tumor growth and decreased aurora B kinase activity in mice treated with low-dose AMG 900 and vorinostat compared to either agent alone. Pharmacodynamics was assessed by scoring for phosphorylated histone H3 through immunofluorescence. Our results indicate that combination treatment with low doses of AMG 900 and HDACIs could be a promising therapy for future clinical trials against PCA.

Keywords: AMG 900; aurora kinase inhibitor; histone deacetylase inhibitors; prostate cancer; synergy; valproic acid; vorinostat.

Figure 1

AMG 900 effectively targets PCA cells at concentrations above 1 nmol/L. (A) Western blot quantifying the protein levels of phosphorylated aurora A (48 kDa), B (40 kDa) and C (35 kDa) after treatment of PCA cells with AMG 900 (concentrations indicated above each lane) for 48 h. Bands were normalized to total levels of aurora A. (B) Bar graph representing relative clonogenic survival after AMG 900 treatment of PCA cell lines. Colonies were counted in triplicate. PCA, prostate cancer.

Figure 2

Combinations of AMG 900 with HDACIs VPA and vorinostat decrease the proliferation activity and long-term clonogenic survival of PCA cells compared to single-agent use. (A and B) Proliferation activity of PCA cells after treatment with AMG 900 and VPA (A) or vorinostat (B), as measured by MTS assays. (C and D) Quantification of colonies to assess clonogenic survival of PCA cells after treatment with AMG 900 and VPA (C) or vorinostat (D). Ai, AMG 900; +, moderate synergy; ++, synergy; +++, strong synergy; HDACI, histone deacetylase inhibitors; PCA, prostate cancer; SAHA, suberanilohydroxamic acid (vorinostat); VPA, valproic acid.

Figure 3

AMG 900 combined with HDACIs VPA or vorinostat increases cellular senescence in PCA cell lines compared to single-agent use. (A) Western blot for p21, a marker of cellular senescence or a G1/G2 phase cell cycle arrest, after treating DU-145, LNCaP and PC3 cells as indicated above each lane. Bands were normalized to the housekeeper vinculin. Ai, AMG 900. (B) Representative images of PC3 cells after performing an SA β-galactosidase assay. Blue cells are SA β-galactosidase positive cells, indicating cellular senescence. Ai, AMG 900. (C) Quantification of SA β-galactosidase-positive staining after treatment of PCA cells with AMG 900 and/or HDACIs VPA/vorinostat. Ai, AMG 900; * combination treatments with a significantly increased percentage of senescent cells (P ≤ 0.05). HDACI, histone deacetylase inhibitors; PCA, prostate cancer; SAHA, suberanilohydroxamic acid (vorinostat); VPA, valproic acid.

Figure 4

Confocal analysis of DU-145 and PC3 PCA cells treated with AMG 900 (1 or 5 nmol/L) and HDACIs VPA (1 mmol/L) and vorinostat (1 μmol/L) stained for phosphorylated histone H3 (green) and phosphorylated aurora kinase A/B/C (red). AMG 900 causes a decrease in phosphorylated histone H3 and an increase in multipolar spindles in a dose-dependent manner. PC3 cells also demonstrate multipolar spindles when treated with a combination of lower concentrations of AMG 900 and HDACIs. Ai, AMG 900; HDACI, histone deacetylase inhibitors; SAHA, suberanilohydroxamic acid (vorinostat); VPA, valproic acid.

Figure 5

Flow cytometry analysis of PCA cell lines treated with AMG 900 (1 or 5 nmol/L) and HDACIs VPA (1 mmol/L) and vorinostat (1 μmol/L). (A) Scatter plots show a decrease in phosphorylated histone H3 in PCA cells treated with AMG 900 in a dose-dependent manner. Cells treated with a combination of vorinostat and low-dose AMG 900 show a marked decrease in phosphorylated histone H3 as compared to single-agent treatment. (B) Cell cycle analysis indicates that AMG 900 causes an increase in polyploidy in all PCA cell lines in a dose-dependent manner. Low-dose combination of AMG 900 with HDACIs cause an increase in polyploidy as compared to single-agent treatment. Ai, AMG 900. (C) Western blot analysis for cleaved PARP in PCA cells treated with AMG 900 and/or HDACIs (the treatment is indicated above each lane). Bands were normalized to the housekeeper actin. Ai, AMG 900; HDACI, histone deacetylase inhibitors; PCA, prostate cancer; SAHA, suberanilohydroxamic acid (vorinostat).

Figure 6

Low-dose AMG 900 in combination with vorinostat enhances growth suppression of DU-145 xenografts and inhibits histone H3 phosphorylation in vivo, similar to treatment with high-dose AMG 900. (A) Tumor growth curves of NOD/SCID mice bearing established DU-145 tumors treated with vehicle alone, AMG 900 and/or vorinostat for a maximum of 4 weeks. Tumor volumes are represented as mean ± SEM (n = 9). *treatment groups that had a significantly different tumor growth rate compared to the vehicle-treated group (P ≤ 0.05). (B) Representative images of tumor sections stained with phosphorylated histone H3 (red) and DAPI (blue) indicate decreased phosphorylated histone H3-positive cells after AMG 900 treatment and combination treatment. (C) Bar graph quantifying the percentage of phosphorylated histone H3-positive cells per treatment group. Student's t-tests confirm significantly decreased phosphorylated histone H3 staining after high-dose AMG 900 treatment and after combination treatment. *treatment groups that had significantly less phosphorylated histone H3-positive cells compared to the vehicle-treated group (P ≤ 0.05). Ai, AMG 900; NOD, nonobese diabetic; pH3, phosphorylated histone H3; SAHA, suberanilohydroxamic acid (vorinostat); SCID, severe combined immunodeficiency.