Abstract 3172: Inhibition of coiled coil domain containing protein 69 (CCDC69) enhance platinum-mediated apoptosis in ovarian cancer cells

Abstract

Abstract Background and Objective:Molecular mechanisms of chemo-resistance in ovarian cancer are poorly understood. To identify gene involved in ovarian cancer chemo-resistance, 135 ovarian cancer patients with intact chemo-response information from The Cancer Genome Atlas (TCGA) database were included. Our analysis revealed that the level of CCDC69 mRNA is differentially expressed between chemo-sensitive group and chemo-resistant group. Moreover, there was a significant negatively correlation between CCDC69 promoter methylation and mRNA expression. The aim of the study is to examine the role of CCDC69 in the underlying mechanism of chemo-resistance. Methods: The expression levels of CCDC69 were detected in chemo-sensitive ovarian cancer A2780, chemo-resistant A2780cis and SKOV3 cell lines using quantitative RT-PCR and immunoblots. Promoter methylation status of CCDC69 were investigated by bisulfite sequencing. Silencing CCDC69 in A2780cis and SKOV3 cells were performed by Small-interfering RNAs (siRNAs) and CRISPR-Cas9. Cell viabilities after cisplatin treatment were evaluated by MTT and colony formation assays. Apoptosis was assessed by Annexin V/PI staining and caspases 3/8 activity. Cell cycle distributions and mitochondrial membrane potential (ΔΨm) were measured by flow cytometry. Relevant pathway proteins were determined by immunoblotting assays. Results: Heavy CpG methylation (73.1% and 74.3%) was found in A2780 and A2780cis cells. Restoration in the expression of CCDC69 were found in A2780 and A2780cis cells after 5-Aza-dC treatment. In fact, the expression levels of CCDC69 were about 3-4 fold higher in chemo-resistant A2780cis cells than its parental chemo-sensitive A2780 cells. Inhibition of CCDC69 in chemo-resistant A2780cis cells by si-RNA significantly increased sensitive to cisplatin treatment (p<0.05). When knockout CCDC69 in chemo-resistant A2780cis and SKOV3 cells by CRISPR-Cas9, the CCDC69KO chemo-resistant A2780cis and CCDC69KO SKOV3 cells were also shown increased sensitive to cisplatin treatment (p<0.001). Moreover, treating CCDC69KO A2780cis cells with cisplatin, abolished G1 and G2/M arrest, more cleaved caspase 3&8 activities, greater ΔΨm loss and higher levels of Bax were observed. When restoring CCDC69 expression in CCDC69KO A2780cis cells by transient transfection, it attenuated sensitivity to cisplatin. By immunoblotting, disruption of p14ARF-Mdm2-p53 cell cycle and/or apoptosis pathway, enhancement of MDM2 expression, and increase of p53 nuclear export were found in CCDC69KO A2780cis cells. Additionally, inhibition of c-Myc enhance cisplatin sensitivities in CCDC69KO A2780cis cells, overexpression of c-Myc reduce apoptosis in CCDC69KO SKOV3 cells. Summary: Our results show that silenced CCDC69 expression induced platinum-mediated apoptosis in ovarian cancer cells by abrogating c-Myc mediated apoptosis and cell cycle control networks. Citation Format: Long Cui, Liang Bo, Yihua Yang, Minhui Zhu, Joseph Kwong, Hongliang Zheng, Chi Chiu Wang. Inhibition of coiled coil domain containing protein 69 (CCDC69) enhance platinum-mediated apoptosis in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3172. doi:10.1158/1538-7445.AM2017-3172