Heterozygous p53(V172F) mutation in cisplatin-resistant human tumor cells promotes MDM4 recruitment and decreases stability and transactivity of p53.
Heterozygous p53(V172F) mutation in cisplatin-resistant human tumor cells promotes MDM4 recruitment and decreases stability and transactivity of p53.
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DOI：
10.1038/onc.2016.12
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发表时间：
2016-09-08
期刊：
影响因子：
8
通讯作者：
Siddik ZH
中科院分区：
文献类型：
--
作者：
Xie X;Lozano G;Siddik ZH
顺铂是一种重要的抗肿瘤药物，但其临床应用往往受到多因素耐药机制的限制。肿瘤抑制因子p53功能的丧失是一个主要机制，受到DNA结合结构域突变或p53抑制剂MDM 2和MDM 4过表达的失调的影响，MDM 2和MDM 4通过增加p53的蛋白体降解而使p53不稳定。在本研究中，顺铂耐药的2780 CP/Cl-16卵巢肿瘤细胞表达一种杂合的温度敏感性p53 V172 F突变，与亲本A2780细胞中的纯合野生型p53相比，该突变使p53半衰期缩短2- 3倍。尽管2780 CP/Cl-16细胞中p53稳定性降低与MDM 2或MDM 4的中度细胞过表达（<1.5倍）相关，但它们与p53的结合显著增强（5- 8倍）。类似的顺铂耐药2780 CP/Cl-24细胞，表达p53杂合性缺失，保留了p53 V172 F突变和高p53-MDM 4结合，但表现出较低的p53结合MDM 2，这与p53泛素化减少和p53稳定性增强有关。在用野生型（wt）p53或多聚体抑制性p53 L344 P突变体转染的2780 CP/Cl-24细胞中证实了p53作为异聚体p53 wt/p53 V172 F复合物是不稳定的推断，并且在32.5°C的容许温度下生长的两种抗性细胞系中的p53稳定性的标准化进一步支持了这一推断。令人惊讶的是，在2780 CP/Cl-16和2780 CP/Cl-24模型中，顺铂诱导的p53反式活性在37°C下减弱，这与顺铂耐药性相关。然而，在任一耐药细胞系中通过siRNA下调MDM 2或MDM 4在37°C下诱导p53并恢复p21反式激活，顺铂诱导的DNA损伤在32.5°C下也是如此，这与p53-MDM 4结合减少和顺铂耐药性一致。这些结果表明，顺铂介导的p53 V172 F突变调节p53在常温下的稳定性，但它是增加的招聘MDM 4的同源或异聚的突变体-p53 V172 F复合物，抑制p53依赖性的反式激活。这代表了一种新的细胞机制的p53抑制，从而诱导顺铂耐药性。
Cisplatin is an important antitumor agent, but its clinical utility is often limited by multifactorial mechanism of resistance. Loss of tumor suppressor p53 function is a major mechanism, affected by either mutation in the DNA binding domain or dysregulation by overexpression of p53 inhibitors MDM2 and MDM4 that destabilize p53 by increasing its proteosomal degradation. In the present study, cisplatin-resistant 2780CP/Cl-16 ovarian tumor cells expressed a heterozygous, temperature-sensitive p53V172F mutation, which reduced p53 half-life by 2- to 3-fold compared to homozygous wild-type p53 in parental A2780 cells. Although reduced p53 stability in 2780CP/Cl-16 cells was associated with moderate cellular overexpression of MDM2 or MDM4 (<1.5-fold), their binding to p53 was substantially enhanced (5- to 8-fold). The analogous cisplatin-resistant 2780CP/Cl-24 cells, which express loss of p53 heterozygosity, retained the p53V172F mutation and high p53-MDM4 binding, but demonstrated lower p53-bound MDM2 that was associated with reduced p53 ubiquitination and enhanced p53 stability. The inference that p53 was unstable as a hetromeric p53wt/p53V172F complex was confirmed in 2780CP/Cl-24 cells transfected with wild-type (wt) p53 or multimer-inhibiting p53L344P mutant, and further supported by normalization of p53 stability in both resistant cell lines grown at the permissive temperature of 32.5°C. Surprisingly, in 2780CP/Cl-16 and 2780CP/Cl-24 models, cisplatin-induced transactivity of p53 was attenuated at 37°C, and this correlated with cisplatin resistance. However, downregulation of MDM2 or MDM4 by siRNA in either resistant cell line induced p53 and restored p21 transactivation at 37°C, as did cisplatin-induced DNA damage at 32.5°C that coincided with reduced p53-MDM4 binding and cisplatin resistance. These results demonstrate that cisplatin-mediated p53V172F mutation regulates p53 stability at the normothermic temperature, but it is the increased recruitment of MDM4 by the homomeric or heteromeric mutant-p53V172F complex that inhibits p53-dependent transactivation. This represents a novel cellular mechanism of p53 inhibition and, thereby, induction of cisplatin resistance.