HDM2 regulation by AURKA promotes cell survival in gastric cancer

Abstract

Purpose: Suppression of P53 (tumor protein 53) transcriptional function mediates poor therapeutic response in patients with cancer. Aurora kinase A (AURKA) and human double minute 2 (HDM2) are negative regulators of P53. Herein, we examined the role of AURKA in regulating HDM2 and its subsequent effects on P53 apoptotic function in gastric cancer.

Experimental design: Primary tumors and in vitro gastric cancer cell models with overexpression or knockdown of AURKA were used. The role of AURKA in regulating HDM2 and cell survival coupled with P53 expression and activity were investigated.

Results: Overexpression of AURKA enhanced the HDM2 protein level; conversely, knockdown of endogenous AURKA decreased expression of HDM2 in AGS and SNU-1 cells. Dual co-immunoprecipitation assay data indicated that AURKA was associated with HDM2 in a protein complex. The in vitro kinase assay using recombinant AURKA and HDM2 proteins followed by co-immunoprecipitation revealed that AURKA directly interacts and phosphorylates HDM2 protein in vitro. The activation of HDM2 by AURKA led to induction of P53 ubiquitination and attenuation of cisplatin-induced activation of P53 in gastric cancer cells. Inhibition of AURKA using an investigational small-molecule specific inhibitor, alisertib, decreased the HDM2 protein level and induced P53 transcriptional activity. These effects markedly decreased cell survival in vitro and xenograft tumor growth in vivo. Notably, analysis of immunohistochemistry on tissue microarrays revealed significant overexpression of AURKA and HDM2 in human gastric cancer samples (P < 0.05).

Conclusion: Collectively, our novel findings indicate that AURKA promotes tumor growth and cell survival through regulation of HDM2-induced ubiquitination and inhibition of P53. Clin Cancer Res; 20(1); 76-86. ©2013 AACR.

Figure 1. AURKA promotes cell survival through regulation of HDM2 and P53 expression in GC cell lines

A, Protein extracts from a panel of GC cell lines were subjected to Western blot analysis of AURKA, P53, and HDM2 proteins. B, Western blot analysis of HDM2, P53, P21, BAX, and AURKA proteins following adenovirus-mediated transient overexpression of AURKA, control adenovirus, siRNA- mediated knockdown of AURKA or control siRNA in AGS and SNU-1 cells is shown. Overexpression of AURKA led to an increase in the protein level of HDM2 and a decrease in P53. Conversely, inhibition of AURKA reduced the level of HDM2 with an increase in P53. C, AGS cells were transiently transfected with pcDNA3.1 empty vector, AURKA, or kinase-dead mutant AURKA D274A. Western blot analysis indicated that AURKA-mediated increase in the HDM2 protein level is dependent on AURKA kinase activity. D, AGS and SNU-1 cells were treated with alisertib (0.5 μmol/L) for 48 hours, and cell lysates were subjected to Western blot analysis. The data showed that treatment with alisertib reduced the level HDM2 and increased P53. E, Clonogenic cell survival analysis for AGS and SNU-1 cells was performed following treatment with alisertib (0.25–5.0 μmol/L). The data indicated a dose-dependent decrease in cell survival. Ctrl AD, Control Adenovirus; AURKA AD, Aurora Kinase A Adenovirus; AURKA Mut, Aurora Kinase A D274A Mutant; ** P<0.01.

Figure 2. AURKA expression increases the HDM2 protein level in GC cell lines

A & B, Dual immunofluorescence (IF) analysis for AURKA (Green) and HDM2 (Red) was performed on AGS and SNU-1 cells transiently transfected with pcDNA3.1 or AURKA. The nucleus was stained with DAPI (Blue). After merging AURKA, HDM2, and DAPI images together (MERGE), the data showed that overexpression of AURKA leads to a significant increase in HDM2 protein level. The percentage of cells that presented expression of HDM2 was averaged from six different microscopic fields for more than 100 cells. The IF data are represented as the mean of three different experiments.

Figure 3. AURKA-mediated regulation of HDM2 promotes P53 ubiquitination and attenuates CDDP-induced P53 expression in GC cells

A, AGS cells transiently expressing AURKA or pcDNA3.1 were treated with CDDP (5.0–10.0 μmol/L) for 12 hours and analyzed by immunoblotting. The data indicated that CDDP treatment induced P53 and P21 expression in control cells. In contrast, AURKA expression abrogated these effects in response to CDDP. B, Immunoprecipitation of endogenous P53 was done in AGS cells transiently expressing pcDNA3.1, AURKA, or HDM2. Western blot analysis showed that overexpression of AURKA significantly induced P53 ubiquitination. C, AGS cells transiently expressing pcDNA3.1, AURKA, or HDM2 were treated with Nutlin3A (4.0 μmol/L) for 12 hours. Western blot data suggested that AURKA-induced suppression of P53 and P21 expression is mediated by HDM2. IgG, immunoglobin G; IB, immunoblotting.

Figure 4. AURKA directly associates and phosphorylates HDM2 protein

A, In vitro kinase assay followed by a subsequent IP with AURKA antibody was done on recombinant AURKA and HDM2 proteins. Western blot analysis of the immunoprecipitates showed that AURKA-mediated interaction of HDM2 is dependent on AURKA kinase activity. B, Western blot analysis of in vitro kinase assay with recombinant AURKA, HDM2, AKT and GPX7 proteins indicated that AURKA directly phosphorylates HDM2 in a concentration-dependent manner. AKT and GPX7 recombinant proteins were used as positive and negative controls, respectively. GPX7, glutathione peroxidase enzyme 7.

Figure 5. Alisertib exhibits anti-tumor activity, suppresses HDM2, and enhances P53 function in vivo

AGS xenograft tumors were treated with alisertib (30mg/kg) for 21 days and tumor size was measured every four days. A, The data indicated that alisertib has significant anti-tumor activity against AGS xenografts. B & C, IHC of AGS xenograft tumors showed that inhibition of AURKA suppressed HDM2 expression and induced P53 in vivo. D, qRT-PCR analysis of AGS xenograft tumors revealed that blocking AURKA with alisertib enhanced P53 transcriptional activity as indicated by elevated mRNA levels of P21, BAX, NOXA and PUMA downstream target genes. ** P<0.01.

Figure 6. Frequent overexpression and direct correlation between AURKA and HDM2 in human gastric cancer tissues

A & B, Representative images of AURKA and HDM2 IHC staining in normal gastric (NT) and gastric cancer (GC) tissue samples are shown at x40 magnification. The data showed that AURKA and HDM2 protein expression levels are significantly higher in GC than NT. C & D, IHC analysis of human gastric tissue array exhibited significantly high expression scores for AURKA and HDM2 proteins in GC than NT (P<0.05). E, AURKA and HDM2 protein expression correlation analysis in human gastric tissue array indicated strong direct correlation between AURKA and HDM2 expression (P=0.04, r=0.41, n=24) in GC.