Functional Activation of Mutant p53 by Platinum Analogues in Cisplatin-Resistant Cells Is Dependent on Phosphorylation

Abstract

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. Implications 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.

Coexisting Molecular Determinants of Acquired Oxaliplatin Resistance in Human Colorectal and Ovarian Cancer Cell Lines

“…Similarly, increased expression of the inhibitor of apoptosis BIRC4 (XIAP) could also contribute to oxaliplatin resistance and counteract the increased expression of the cell death execution caspases 6 and 7 in the platinum-resistant ovarian cancer cell lines. These data are in line with the decrease in annexin A3 in l-OHP resistant colon cancer cells [43], as well as an activation of mutated p53 in l-OHP resistant ovarian cancer cells [44] and a change in multiple apoptosis regulating genes in resistant colon cancer cells [45].…”

Dysregulated Phosphorylation of p53, Autophagy and Stemness Attributes the Mutant p53 Harboring Colon Cancer Cells Impaired Sensitivity to Oxaliplatin

“…Phosphorylation, a key posttranslational modification is known for its potent role in rendering wild-type p53 protein’s functionality as a tumor suppressor ( 27 ). In particular, the stabilization and activation of p53 in response to genotoxic stress induced by platinum-based therapeutics are reported to be facilitated via phosphorylation of the serine residues ( 28 , 29 ). Based on these reports, we intended to determine the phosphorylation of N-terminal and C-terminal serine residues of p53 protein in wild-type p53 bearing HCT 116 and mutant p53 bearing HT 29 colon cancer cells in response to OX treatment.…”

“…Among them phosphorylation forms the key modification that stabilizes the p53 protein and thereby activates its functional role as a tumor suppressor in response to cellular stress or DNA damage ( 55 ). The stabilization and activation of p53 in response to platinum-based drug treatments (DNA-damaging) are reported to be facilitated through the phosphorylation of N-terminus serine residues especially Ser15, Ser20, and Ser37 by reducing p53 affinity to its negative regulator Hdm2 ( 29 , 56 ).…”

Non-coding RNA transcripts, incredible modulators of cisplatin chemo-resistance in bladder cancer through operating a broad spectrum of cellular processes and signaling mechanism

“…Although it is not clearly elucidated what modulates NEAT1 transcription, c-MYC, OCT4 (octamer-binding transcription factor 4), C/EBPβ, and p53 are determined to bind at the NEAT1's promoter to affect its transcription activity. These transcription factors were also found to be interestingly correlated with cisplatin resistance [ 158 , [190] , [191] , [192] ]. Using the ChIP-qPCR assay, the free proteins of c-MYC, OCT4, and p53 are overexpressed in cisplatin-resistant BC cells, and also c-MYC, OCT4, and p53 are greatly concentrated on NEAT1 promoter when bladder tumor cells are affected by cisplatin.…”