Functional Activation of Mutant p53 by Platinum Analogues in Cisplatin-Resistant Cells Is Dependent on Phosphorylation.
Functional Activation of Mutant p53 by Platinum Analogues in Cisplatin-Resistant Cells Is Dependent on Phosphorylation.
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DOI：
10.1158/1541-7786.mcr-16-0257-t
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发表时间：
2017-03
期刊：
影响因子：
--
通讯作者：
Siddik ZH
中科院分区：
文献类型：
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作者：
Xie X;He G;Siddik ZH
肿瘤抑制基因p53功能障碍是癌症耐药的主要原因。最近，我们报道了突变体p53V172F在顺铂耐药的2780CP/Cl-16和2780CP/Cl-24人卵巢肿瘤细胞中通过增加抑制剂MDM4的募集而失活，但其他功能正常。目前的研究表明，与顺铂不同，铂类似物奥沙利铂和DACH-diacetato-dichloro-Pt(IV) （DAP）可以在耐药细胞中稳定和激活p53V172F，这可以通过延长p53半衰期和靶p21 （CDKN1A）和MDM2的反激活来证明。MDM2的增加降低了细胞裂解物中的MDM4水平以及p53免疫复合物，并阻止p53恢复到无活性的p53-MDM2-MDM4结合状态。在敏感的A2780和相应的耐药2780CP/Cl-16和2780CP/Cl-24细胞系中，所有三种药物都证明了p53在Ser15位点的磷酸化。然而，顺铂仅在A2780细胞中诱导Ser20磷酸化，而在耐药细胞中没有；相比之下，DAP和奥沙利铂在所有三种细胞系中都诱导了这种磷酸化。Ser20磷酸化对p53激活更重要的推断得到了异位表达的证实，在p53敲除的A2780细胞中，一个拟磷（S20D）突变p53与野生型p53相比，对MDM2和MDM4的结合都减少了。与此一致，时间研究表明，药物诱导的Ser15磷酸化与p53稳定一致，而Ser20磷酸化与p53反激活一致。顺铂不能激活耐药细胞中磷酸化突变体p53V172F Ser20的通路，但奥沙利铂和DAP可以恢复这种磷酸化，从而重新激活p53功能并绕过顺铂耐药。
Dysfunctionality of the p53 tumor suppressor is a major cause of therapeutic drug resistance in cancer. Recently we reported that mutant, but otherwise functional, p53V172F was inactivated in cisplatin-resistant 2780CP/Cl-16 and 2780CP/Cl-24 human ovarian tumor cells by increased recruitment of the inhibitor MDM4. The current study demonstrates that, unlike cisplatin, platinum analogs oxaliplatin and DACH-diacetato-dichloro-Pt(IV) (DAP), strongly stabilize and activate p53V172F in resistant cells, as indicated by prolonged p53 half-life and transactivation of targets p21 (CDKN1A) and MDM2. This increase in MDM2 reduced MDM4 levels in cell lysates as well as the p53 immunocomplex and prevented reversion of p53 to the inactive p53-MDM2-MDM4 bound state. Phosphorylation of p53 at Ser15 was demonstrated by all three drugs in sensitive A2780 and corresponding resistant 2780CP/Cl-16 and 2780CP/Cl-24 cell lines. However, cisplatin induced Ser20 phosphorylation in A2780 cells only, but not in resistant cells; in contrast, both DAP and oxaliplatin induced this phosphorylation in all three cell lines. The inference that Ser20 phosphorylation is more important for p53 activation was confirmed by ectopic expression of a phosphomimetic (S20D) mutant p53 that displayed reduced binding, relative to wild-type p53, to both MDM2 and MDM4 in p53-knockout A2780 cells. In consonance, temporal studies demonstrated drug-induced Ser15 phosphorylation coincided with p53 stabilization, whereas Ser20 phosphorylation coincided with p53 transactivation. Cisplatin fails to activate the pathway involved in phosphorylating mutant p53V172F at Ser20 in resistant cells, but this phosphorylation is restored by oxaliplatin and DAP that reactivates p53 function and circumvents cisplatin resistance.