Nutlin-3a suppresses poly (ADP-ribose) polymerase 1 by mechanisms different from conventional PARP1 suppressors in a human breast cancer cell line
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Masaki Kobayashi1,2,*, Yuka Ishizaki1,*, Mika Owaki1,*, Yoko Matsumoto1, Yuri Kakiyama1, Shunsuke Hoshino1,2, Ryoma Tagawa1, Yuka Sudo2, Naoyuki Okita3, Kazunori Akimoto2,4 and Yoshikazu Higami1,2
1 Laboratory of Molecular Pathology & Metabolic Disease, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba 278-8510, Japan
2 Translational Research Center, Research Institute of Science and Technology, Tokyo University of Science, Noda, Chiba 278-8510, Japan
3 Division of Pathological Biochemistry, Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University, Sanyo-onoda, Yamaguchi 756-0884, Japan
4 Laboratory of Medicinal and Life Science, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba 278-8510, Japan
* Co-first authors
|Masaki Kobayashi,||email:||[email protected]|
|Yoshikazu Higami,||email:||[email protected]|
Keywords: nutlin-3a; PARP1; breast cancer; proteasomal degradation; autoPARylation
Received: December 21, 2019 Accepted: April 14, 2020 Published: May 05, 2020
Poly (ADP-ribose) polymerase 1 (PARP1) plays important roles in single strand DNA repair. PARP1 inhibitors enhance the effects of DNA damaging drugs in homologous recombination-deficient tumors including tumors with breast cancer susceptibility gene (BRCA1) mutation. Nutlin-3a, an analog of cis-imidazoline, inhibits degradation of murine double minute 2 (MDM2) and stabilizes p53. We previously reported that nutlin-3a induces PARP1 degradation in p53-dependent manner in mouse fibroblasts, suggesting nutlin-3a may be a PARP1 suppressor. Here, we investigated the effects of nutlin-3a on PARP1 in MCF-7, a human breast cancer cell line. Consistent with our previous results, nutlin-3a reduced PARP1 levels in dose- and time-dependent manners in MCF-7 cells, but this reduction was suppressed in p53 knockdown cells. RITA, a p53 stabilizer that binds to p53 itself, failed to reduce PARP1 protein levels. Moreover, transient MDM2 knockdown repressed nutlin-3a-mediated PARP1 reduction. The MG132 proteasome inhibitor, and knockdown of checkpoint with forkhead and ring finger domains (CHFR) and ring finger protein 146 (RNF146), E3 ubiquitin ligases targeting PARP1, suppressed nutlin-3a-induced PARP1 reduction. Short-term nutlin-3a treatment elevated the levels of PARylated PARP1, suggesting nutlin-3a promoted PARylation of PARP1, thereby inducing its proteasomal degradation. Furthermore, nutlin-3a-induced PARP1 degradation enhanced DNA-damaging effects of cisplatin in BRCA1 knockdown cells. Our study revealed that nutlin-3a is a PARP1 suppressor that induces PARP1 proteasomal degradation by binding to MDM2 and promoting autoPARylation of PARP1. Further analysis of the mechanisms in nutlin-3a-induced PARP1 degradation may lead to the development of novel PARP1 suppressors applicable for cancers with BRCA1 mutation.
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