Abstract 3029: Targeting Annexin A4 with antisense oligonucleotides improves platinum resistance of ovarian cancer
Authors: Nakagawa Satoshi , Kakubari Reisa , Matsuzaki Shinya , Ueda Yutaka , Hiramatsu Kosuke , Serada Satoshi , Fujimoto Minoru , Kimura Tadashi , Naka Tetsuji 
Published: August 2019

Abstract
[Introduction] Ovarian cancer (OvCa) is one of the main causes of gynecologic cancer death in many countries and the number of patients is increasing. The standard treatment for advanced OvCa is cytoreductive surgery and adjuvant chemotherapy. The key drug for OvCa chemotherapy is platinum. However more than 70% of advanced stage OvCa relapse within 5 years, and the major issue to treat them is platinum resistance. We previously reported that Annexin (Anx) A4 protein was associating with chemoresistance to platinum-based cancer drugs. Nucleic acid therapeutics is promising therapy to target molecule existing intra cell membrane. The aim of this study is to evaluate the usefulness of antisense oligonucleotides (ASOs) and toovercome the platinum chemoresistance with ASOs by suppressing the expression of Anx A4 in cancer cells.
[Methods] Cell line: To assess AnxA4 ASOs, two human ovarian cancer cell lines, RMG-1 and OVISE, which have platinum resistance and strongly express Anx A4, were used in each experiment. Anx A4 ASOs: We selected targeting Annexin A4 mRNA sequence in silico and made 16 ASOs. To enhance the stability, 2’, 4’-bridged nucleic acid were inserted in both 3’and 5’ends. And we analyzed suppression of Annexin A4 in ovarian clear cell cancer cell lines in vitro using real time PCR and western blotting. Platinum resistance verification: In vitro, cells were transfected with ASOs using lipofectamine 2000 and were exposed to various concentrations of cisplatin (0 - 100 μM) for 72 hr. Then, drug concentrations resulting in a 50% inhibition of cell growth (IC50values) were calculated. In vivo, we used ICRnu/nu mice xenografted subcutaneously with RMG-I and OVISE cells. Intraperitoneal injection of cisplatin 3mg/kg after intratumoral administration of ASO 1mg/kg each twice a week were given to xenograft mice.
[Result] Of the 16 types of antisense oligonucleotides, we focused on 2 type of ASO, #7, #9 which had stronger knockdown effect for Anx A4 expression. In RMG-I cell, IC50value of Anx A4 ASO transfected cells were 6.42 µM in compared with 11.12 µM in control ASO transfected cells. (p=0.012) In OVISE cells, IC50value was also significantly higher in transfected Anx A4 ASO cells. (10.30 µM, 4.66 µM, p&lt;0.0001) Significantly more amount of platinum had accumulated in Anx A4 ASO transfected cells in compared with control ASO transfected cells and no treatment cells.In vivo, tumor growth of mice treated with cisplatin+Anx A4 ASOs were significantly inhibited compared to cisplatin + control ASOs in both the RMG-1 and OVISE cell lines xenograft mice.
[Conclusion] By transfection of ANXA4 ASO, platinum resistance has been improved both in vitro and in vivo. Targeting ANX A4 with nucleic therapeutics could be an option for platinum resistant ovarian cancer.
Citation Format: Satoshi Nakagawa, Reisa Kakubari, Shinya Matsuzaki, Yutaka Ueda, Kosuke Hiramatsu, Satoshi Serada, Minoru Fujimoto, Tadashi Kimura, Tetsuji Naka. Targeting Annexin A4 with antisense oligonucleotides improves platinum resistance of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3029.