ZZW-115–dependent inhibition of NUPR1 nuclear translocation sensitizes cancer cells to genotoxic agents
Wenjun Lan, Patricia Santofimia-Castaño, Mirna Swayden, Yi Xia, Zhengwei Zhou, Stephane Audebert, Luc Camoin, Can Huang, Ling Peng, Ana Jiménez-Alesanco, Adrián Velázquez-Campoy, Olga Abián, Gwen Lomberk, Raul Urrutia, Bruno Rizzuti, Vincent Geli, Philippe Soubeyran, José L. Neira, Juan Iovanna
Wenjun Lan, Patricia Santofimia-Castaño, Mirna Swayden, Yi Xia, Zhengwei Zhou, Stephane Audebert, Luc Camoin, Can Huang, Ling Peng, Ana Jiménez-Alesanco, Adrián Velázquez-Campoy, Olga Abián, Gwen Lomberk, Raul Urrutia, Bruno Rizzuti, Vincent Geli, Philippe Soubeyran, José L. Neira, Juan Iovanna
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Research Article Cell biology Oncology

ZZW-115–dependent inhibition of NUPR1 nuclear translocation sensitizes cancer cells to genotoxic agents

Abstract

Establishing the interactome of the cancer-associated stress protein Nuclear Protein 1 (NUPR1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair, and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1, thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further report that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system, indicating that NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation-dependent functions of key proteins involved in the DDR.

Authors

Wenjun Lan, Patricia Santofimia-Castaño, Mirna Swayden, Yi Xia, Zhengwei Zhou, Stephane Audebert, Luc Camoin, Can Huang, Ling Peng, Ana Jiménez-Alesanco, Adrián Velázquez-Campoy, Olga Abián, Gwen Lomberk, Raul Urrutia, Bruno Rizzuti, Vincent Geli, Philippe Soubeyran, José L. Neira, Juan Iovanna

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Figure 5

NUPR1 interacts with DNA repair proteins at DNA lesions, and its inhibition induces alterations of protein posttranslational modifications (PTM) profiles.

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NUPR1 interacts with DNA repair proteins at DNA lesions, and its inhibit...
(A) PLA was performed in MiaPaCa-2 cells, treated or not with ZZW-115 (5 μM) for 6 hours, transfected with a plasmid expressing NUPR1-Flag, using rabbit antibodies against MRE11 or TP53 and mouse anti-Flag. A representative experiment is shown (n = 3). ImageJ was used to count the number of red dots. Data represent mean ± SEM of 6 fields. Student’s 2-tailed unpaired t test was used. **P < 0.01; ***P < 0.001. (B) PLA was performed in MiaPaCa-2 cells (treated or no with ZZW-115 at 1.5 μM, 5-FU at 10 μM, or the combination of both for 24 hours) using rabbit anti-NUPR1 and mouse anti-γH2AX antibodies. Nontreated or treated with ZZW-11A representative experiment is shown (n = 3). ImageJ was used to count the number of red dots. Data represent mean ± SEM of 6 fields. One-way ANOVA with Tukey’s post hoc test was used. **P < 0.01. (C) Variations of SUMOylation of 3 identified DNA repair proteins and variations following the different treatments were studied by Western blot after purification of SUMOylated proteins in denaturing condition by Ni2+ pull-down. The upper parts of nitrocellulose filters were immunoblotted using anti-MRE11–, -TP53–, and -KDM1A–specific antibodies. Lower parts of filters were blotted using anti-Flag antibody in order to control the equal amount of precipitated material. Signals were quantified by densitometry, and values of the ratio of SUMOylated bands over precipitated SUMO1 are shown. Asterisk represents nonspecific bands (n = 1). (D) PLA was performed on MiaPaCa-2–6HF–SUMO1 cells transfected with siRNA against NUPR1 and treated with 5-FU and/or ZZW-115 (same conditions as mentioned above). SUMOylation of TP53 was quantified by mouse anti-Flag and rabbit anti-TP53 antibodies. A representative experiment is shown (n = 3). ImageJ was used to count the number of red dots. Data represent mean ± SEM of 6 fields. One-way ANOVA with Tukey’s post hoc test was used. ***P < 0.001.