Abstract
p53 mutants can form amyloid-like structures that accumulate in cells. p53 reactivation with induction of massive apoptosis-1 (PRIMA-1) and its primary active metabolite, 2-methylene-3-quinuclidinone (MQ), can restore unfolded p53 mutants to a native conformation that induces apoptosis and activates several p53 target genes. However, whether PRIMA-1 can clear p53 aggregates is unclear. In this study, we investigated whether PRIMA-1 can restore aggregated mutant p53 to a native form. We observed that the p53 mutant protein is more sensitive to both PRIMA-1 and MQ aggregation inhibition than WT p53. The results of anti-amyloid oligomer antibody assays revealed that PRIMA-1 reverses mutant p53 aggregate accumulation in cancer cells. Size-exclusion chromatography of the lysates from mutant p53-containing breast cancer and ovarian cell lines confirmed that PRIMA-1 substantially decreases p53 aggregates. We also show that MDA-MB-231 cell lysates can "seed" aggregation of the central core domain of recombinant WT p53, corroborating the prion-like behavior of mutant p53. We also noted that this aggregation effect was inhibited by MQ and PRIMA-1. This study provides the first demonstration that PRIMA-1 can rescue amyloid-state p53 mutants, a strategy that could be further explored as a cancer treatment.
Highlights
P53 mutants can form amyloid-like structures that accumulate in cells. p53 reactivation with induction of massive apoptosis-1 (PRIMA-1) and its primary active metabolite, 2-methylene-3-quinuclidinone (MQ), can restore unfolded p53 mutants to a native conformation that induces apoptosis and activates several p53 target genes
Through immunoprecipitation assays using the anti-amyloid oligomer antibody A11, we show that PRIMA-1 mobilizes amyloid-state p53, promoting its partial reactivation and de-aggregation, leading to apoptosis
We provide the first demonstration of the molecular mechanism through which PRIMA-1 rescues amyloid mutant p53 and thereby decreases dominantnegative and GoF effects
Summary
GoF, gain-of-function; MQ, 2-methylene-3-quinuclidinone; SEC, size-exclusion chromatography; ThT, thioflavin T; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DAPI, 4Ј,6-diamidino-2-phenylindole. The importance of a drug targeting mutant p53 is undeniable because the number of candidate cases for possible treatment is very large These off-target effects appear to be positive because they seem to cooperate in the reactivation of mutant p53 and boost the anticancer effect of PRIMA-1 and its analogs [26]. Size-exclusion chromatography (SEC) of the lysates from the cancer cell lines containing mutant p53 corroborated that PRIMA-1 led to a substantial decrease in p53 aggregates. Our findings reinforce the notion that mutant p53 aggregation is an excellent target for the development of new antineoplastic drugs
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