Xie X, Lozano G, Siddik Z H
Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Oncogene. 2016 Sep 8;35(36):4798-806. doi: 10.1038/onc.2016.12. Epub 2016 Feb 15.
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 that is affected by either mutation in the DNA-binding domain or dysregulation by overexpression of p53 inhibitors MDM2 and MDM4, which destabilize p53 by increasing its proteosomal degradation. In the present study, cisplatin-resistant 2780CP/Cl-16 ovarian tumor cells expressed a heterozygous, temperature-sensitive p53(V172F) mutation, which reduced p53 half-life by two- to threefold compared with homozygous wild-type (wt) 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 (five- to eightfold). The analogous cisplatin-resistant 2780CP/Cl-24 cells, which express loss of p53 heterozygosity, retained the p53(V172F) 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 heteromeric p53(wt)/p53(V172F) complex was confirmed in 2780CP/Cl-24 cells transfected with wt p53 or multimer-inhibiting p53(L344P) 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 small interfering RNA 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 p53(V172F) mutation regulates p53 stability at the normothermic temperature, but it is the increased recruitment of MDM4 by the homomeric or heteromeric mutant p53(V172F) complex that inhibits p53-dependent transactivation. This represents a novel cellular mechanism of p53 inhibition, and, thereby, induction of cisplatin resistance.
顺铂是一种重要的抗肿瘤药物，但其临床应用常常受到多因素耐药机制的限制。肿瘤抑制因子p53功能的丧失是一种主要机制，这一机制要么是由DNA结合域的突变引起，要么是由p53抑制剂MDM2和MDM4的过表达导致失调所影响，MDM2和MDM4通过增加p53的蛋白酶体降解使其不稳定。在本研究中，顺铂耐药的2780CP/Cl-16卵巢肿瘤细胞表达一种杂合的、温度敏感的p53(V172F)突变，与亲本A2780细胞中的纯合野生型（wt）p53相比，该突变使p53半衰期缩短了两到三倍。尽管2780CP/Cl-16细胞中p53稳定性的降低与MDM2或MDM4的适度细胞过表达（<1.5倍）相关，但其与p53的结合显著增强（五到八倍）。类似的顺铂耐药2780CP/Cl-24细胞，其p53杂合性缺失，保留了p53(V172F)突变和高p53-MDM4结合，但显示出与p53结合的MDM2较低，这与p53泛素化减少和p53稳定性增强相关。p53作为异源p53(wt)/p53(V172F)复合物不稳定的推断在转染wt p53或多聚体抑制性p53(L344P)突变体的2780CP/Cl-24细胞中得到证实，并在32.5 °C的允许温度下生长的两种耐药细胞系中p53稳定性的正常化进一步得到支持。令人惊讶的是，在2780CP/Cl-16和2780CP/Cl-24模型中，顺铂诱导的p53转录活性在37 °C时减弱，这与顺铂耐药相关。然而，在任一耐药细胞系中通过小干扰RNA下调MDM2或MDM4可诱导p53并在37 °C时恢复p21转录激活，在32.5 °C时顺铂诱导的DNA损伤也可如此，这与p53-MDM4结合减少和顺铂耐药相一致。这些结果表明，顺铂介导的p53(V172F)突变在常温下调节p53稳定性，但正是同源或异源突变体p53(V172F)复合物对MDM4的募集增加抑制了p53依赖的转录激活。这代表了一种新 的p53抑制细胞机制，并由此诱导了顺铂耐药。
