Dar Altaf A, Belkhiri Abbes, Ecsedy Jeffrey, Zaika Alexander, El-Rifai Wael
Departments of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Cancer Res. 2008 Nov 1;68(21):8998-9004. doi: 10.1158/0008-5472.CAN-08-2658.
We investigated the role of Aurora kinase A (AURKA) in regulating p73-dependent apoptosis using the p53-deficient cancer cell lines H1299, TE7, and HCT116p53(-/-). Overexpression of AURKA led to down-regulation of the TAp73-induced activation of the p53/p73-dependent luciferase reporter plasmid (pG13-luc). The reduction in the TAp73 transcription activity was confirmed by measuring the activity of luciferase reporters for p21/WAF1, and PUMA. The siRNA knockdown of endogenous AURKA reversed these effects and Western blot analysis showed a significant increase in the protein level of TAp73 and its downstream transcription targets, PUMA, NOXA, and p21/WAF1. The coexpression of AURKA together with TAp73 inhibited the activation of the pG13-luc, PUMA-luc, and p21/WAF1-luc reporter plasmids with reduction in the protein levels of TAp73 transcription targets. Treatment with AURKA-selective small molecule inhibitor MLN8054 led to a significant increase in the activities of pG13-luc, PUMA-luc, and p21/WAF1-luc reporter plasmids. This effect was accompanied by a significant increase in the mRNA and protein levels of several TAp73 transcription targets: p21/WAF1, PUMA, and NOXA. Flow cytometry cell cycle analysis, after MLN8054 treatment, showed more than a 2-fold increase in cell death. The apoptotic outcome was corroborated by showing an increase in cleaved caspase-3 protein levels by Western blot. Using terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling assay, we showed that the expression of dominant-negative mutant TAp73 expression plasmid (p73DD) counteracted the MLN8054-induced cell death. Taken together, our results indicate that AURKA regulates TAp73-dependent apoptosis and highlight the potential of the AURKA inhibitor MLN8054 in treating cancers that are defective in p53 signaling.
我们使用p53缺陷型癌细胞系H1299、TE7和HCT116p53(-/-)，研究了极光激酶A（AURKA）在调节p73依赖性细胞凋亡中的作用。AURKA的过表达导致TAp73诱导的p53/p73依赖性荧光素酶报告质粒（pG13-luc）激活下调。通过测量p21/WAF1和PUMA的荧光素酶报告基因活性，证实了TAp73转录活性的降低。内源性AURKA的siRNA敲低逆转了这些效应，蛋白质印迹分析显示TAp73及其下游转录靶点PUMA、NOXA和p21/WAF1的蛋白质水平显著增加。AURKA与TAp73共表达抑制了pG13-luc、PUMA-luc和p21/WAF1-luc报告质粒的激活，同时TAp73转录靶点的蛋白质水平降低。用AURKA选择性小分子抑制剂MLN8054处理导致pG13-luc、PUMA-luc和p21/WAF1-luc报告质粒的活性显著增加。这种效应伴随着几个TAp73转录靶点的mRNA和蛋白质水平显著增加：p21/WAF1、PUMA和NOXA。MLN8054处理后的流式细胞术细胞周期分析显示细胞死亡增加了2倍以上。蛋白质印迹显示裂解的caspase-3蛋白水平增加，证实了凋亡结果。使用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记试验，我们显示显性负性突变体TAp73表达质粒（p73DD）的表达抵消了MLN8054诱导的细胞死亡。综上所述，我们的结果表明AURKA调节TAp73依赖性细胞凋亡，并突出了AURKA抑制剂MLN8054在治疗p53信号缺陷癌症中的潜力。
