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Overexpression of CCDC69 Activates P14arf/MDM2/P53 Pathway and Confers Cisplatin Sensitivity Long Cui1,4* , Fang Zhou2, Cui Chen3 and Chi Chiu Wang4,5,6

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Overexpression of CCDC69 Activates P14arf/MDM2/P53 Pathway and Confers Cisplatin Sensitivity Long Cui1,4* , Fang Zhou2, Cui Chen3 and Chi Chiu Wang4,5,6 Cui et al. Journal of Ovarian Research (2019) 12:4 https://doi.org/10.1186/s13048-019-0479-3 RESEARCH Open Access Overexpression of CCDC69 activates p14ARF/MDM2/p53 pathway and confers cisplatin sensitivity Long Cui1,4* , Fang Zhou2, Cui Chen3 and Chi Chiu Wang4,5,6 Abstract Objectives: The aim of the study is to explore the relationship between CCDC69 expression and resistance of ovarian cancer cells to cisplatin and reveal the underlying mechanism. Methods: One hundred thirty five ovarian cancer patients with intact chemo-response information from The Cancer Genome Atlas (TCGA) database were included and analyzed. Stable CCDC69 overexpressing 293 and ovarian cancer A2780 cell lines were established and subjected to examine cell apoptosis and cell cycle distribution using CCK-8 assay and flow cytometry. Cell cycle and apoptosis pathway were evaluated by immunoblots. Stability of p14ARF/MDM2/p53 pathway related proteins were determined by half-life analysis and ubiquitination experiments. Results: We found that CCDC69 expression was significantly higher in chemo-sensitive groups compared with chemo- resistant groups from TCGA database. High CCDC69 expression was associated longer survival. CCDC69 overexpressing 293 and A2780 cells with wildtype p53 and contributes to cisplatin sensitivity following treatment with cisplatin. We further found over-expression of CCDC69 activated p14ARF/MDM2/p53 pathway. Importantly, we also demonstrated that CCDC69 expression extended p53 and p14ARF protein half-life and shortened MDM2 protein half-life. Ubiquitination assay revealing a decrease in p14 ubiquitination in CCDC69 over-expression cells comparing to cells expressing empty vector. Conclusions: It is tempting to conclude that targeting CCDC69 may play a role in cisplatin resistance. Keywords: Cell cycle, Chemoresistance,ovarian cancer, Apoptosis, p14ARF/MDM2/p53 Introduction chemo-resistance in ovarian cancer may improve the Epithelial ovarian cancer (EOC) is the second most com- therapeutic outcomes. mon and accounts for greatest number of death from Cisplatin has been the effective and widely used drugs gynecologic malignancies in the world wide [1, 2]. against various human cancers, including ovarian cancer [5]. High-grade serous ovarian cancer (HGSC) is an aggres- In response to cisplatin, p53, a key regulator of cell cycle sive subtypes and often associated with relatively un- checkpoints, activates a number of genes regulating cells favorable clinical outcomes [3]. Majority of advanced cycle and/or apoptosis [6]. As transcriptional target of p53, ovarian cancer patients initially responsive to chemo- p21 plays an important role in the maintenance of and G1 therapy; however, became increasingly ineffective and and G2 checkpoints following DNA damage such as cis- eventually results in treatment failure [4]. Thus, explor- platin exposure [6–8]. In addition, p21 can regulate cell cycle ing and understanding the mechanisms responsible for progression dependent or independent of p53 following treatment with cisplatin [9, 10]. Previous reports have shown that abrogation of G2/M cell cycle checkpoint is related to * Correspondence: [email protected] enhancement of cisplatin-induced cytotoxicity [11, 12]. Ex- 1Department of Obstetrics and Gynaecology, Guangzhou Women and pression of the p53 protein is posttranscriptionally regulated Children Hospital, Guangzhou 511400, Guangdong, China 4Department of Obstetrics and Gynecology, Prince of Wales Hospital, The by MDM2. The MDM2 binds to p53 and blocking its activa- Chinese University of Hong Kong, Hong Kong, SAR, China tion domain [13], thus promoting degradation of p53 via Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cui et al. Journal of Ovarian Research (2019) 12:4 Page 2 of 8 ubiquitin-proteasome pathway [14, 15]. Under the DNA 10% (v/v) fetal bovine serum (Invitrigen) and 1% damage pressure, the p53 phosphorylation attenuates antibiotic-antimycotic. The human embryonic kidney MDM2 binding ability and then leads to p53 accumulation cells 293 was purchased from ATCC (Bethesda, MD, [16]. Until recent years, investigators have identified another USA). 293 cells were cultured in high-glucose DMEM important regulator in this p53/MDM2 pathway, the (Invitrogen) containing 10% fetal bovine serum (FBS) cyclin-dependent kinase inhibitor p14ARF (termed p19ARF in (Invitrogen) and 100 U/ml of penicillin G/streptomycin. the mouse) [17]. The p14ARF protein is an alternative reading These mammalian cells were cultured in an incubator frame protein product of the CDKN2A/INK4A locus [18]. with an atmosphere of 37 °C in 5% CO2. Cell culture Theotheroneisp16INK4A protein [17]. It has been shown medium was refreshed every two days. that the p14ARF binds to the p53/MDM2 complex and in- hibits MDM2-mediated degradation of p53 [19]. p14ARF can Mutation analysis of p53 inhibit cell cycle progression in both G1 and G2/M phases Polymerase chain reaction (PCR) amplification and dir- and/or cell apoptosis in a p53-dependent and independent ect sequencing were used to screen for DNA variations manner [20, 21]. Furthermore, apoptosis stimulated by in the coding and the 5′ and 3′ flanking regions of p53. p14ARF apoptosis is enhanced by loss of p21 mediated cell Primers for exons 5–8 are modified from Martin et al. cycle checkpoint control [22]. [29]. Primers used for sequencing not shown Add- Coiled-coil domain-containing (CCDC) proteins have itional file 1: Table S1. All purified PCR products were variety of functions in regulating cell cycle progression sequenced for variations using an ABI PRISM® Big Dye™ and mediating apoptosis following DNA damage in Terminator version 3.1 Cycle sequencing kit (Applied eukaryotic cells [23, 24]. In recent years, several CCDC Biosystems, Foster City, CA, USA) and an ABI 3100 au- proteins have been widely studied as tumor suppressors tomated sequencer (Applied Biosystems). in several types of cancer, such as breast and prostate cancers non-small cell lung cancer [25–27]. However, Viral packaging and lentiviral transduction the studies on the role of coiled-coil domain-containing For the production of lentiviral particles, 293 (1 × 106) 69 (CCDC69) in ovarian cancer were limited. As one of were transfected with CCDC69 lentiviral construct these family members, CCDC69 locates on 5q33.1 and is (pLenti-CCDC69-GFP) or control plasmid (pLen- responsible for mitotic spindle and DNA replication ti-C-GFP, Origene, Bothell, WA, USA) together with [28]. In the present study, we found that CCDC69 ex- pRSV-Rev and psPAX2 packaging vectors using Lipofec- pression is significantly higher in chemo-sensitive groups tamine 2000 transfection reagent (Invitrogen). To gener- compared with chemo-resistant groups from The Cancer ation stable cell lines, viral supernatant were harvested Genome Atlas (TCGA) database. We also found women and filter through a 0.45 μm filter to remove cellular with high CCDC69 expression was related to longer sur- debris. The viral supernatant and 4.5 mL of fresh growth vival. To further elucidate the role of CCDC69, we then sta- medium were added to plates and incubate the cells at bly expressed CCDC69 in 293 cells and human ovarian 37 °C with 5% CO2 for 4 h. And then removed the trans- cancer cell lines A2780 with functional p53. Our data duction medium and added 10 mL of fresh growth showed that expression of CCDC69 abrogates G2/M arrest medium. Incubated the cells for three more days in the followed by apoptosis in these p53 wildtype cells. Import- presence of puromycin, and resistant colonies were se- antly, we also demonstrated that CCDC69 expression ex- lected for subsequent experiments. tended p53 and p14ARF protein half-life and shortened MDM2 protein half-life due to deubiquitination of p14ARF. Cell viability assay Cell viability was measured by MTT assay using the Cell Materials and methods Counting Kit-8 (CCK-8, Sigma-Aldrich) following the Chemo-response and survival analysis using public manufacturer’s instructions. The absorbance at 450 nm datasets was measured in the microplate reader. The percentage TCGA clinical and CCDC69 expression mRNA data of cell survival at each dose of cisplatin = Mean of A450 were retrieved from published The Cancer Genome (drug-treated cells)/Mean of A450 (untreated cells). Atlas (TCGA) through the Computational Biology Cen- IC50 values were calculated using GraphPad Prism Soft- ter Portal (cBio): http://www.cbioportal.org/.The cgdsr ware Version 5 (GraphPad Software Inc., CA, USA) and extension package was used to execute the retrieval. plotted in dose response curves. Cell lines Flow cytometry analysis of cell apoptosis using Annexin Human ovarian cancer cell line A2780 was purchased V-FITC/PI staining from Sigma-Aldrich and routinely maintained in RPMI The experiments was performed according to the man- 1640 (Invitrogen) supplemented with heat-inactivated ual of Alexa Fluor® 488 Annexin V/Dead
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