Abstract 1200: Novel strategy to overcome platinum resistance in uterine leiomyosarcoma; blocking ATP7B by copper ion
Authors: Kakuda Mamoru , Matsuzaki Shinya , Nakae Ruriko , Tanaka Yusuke , Kobayashi Eiji , Ueda Yutaka , Yoshino Kiyoshi , Kimura Tadashi 
Published: July 2017

Abstract
Objective: Resistance to platinum drugs remains a significant problem. Our objective is to elucidate the role of ATP7B, a copper transporter which excretes intracellular platinum giving drug resistance, in gynecologic malignancies and to develop a method which can overcome the platinum resistance.
Methods: The expression of ATP7B was evaluated in 3 uterine leiomyosarcoma (LMS) cell lines (SK-LMS, SK-UT1 and SKN) and 26 clinical samples of LMS by immunohistochemical (IHC) staining, respectively. The IC50 values of cisplatin were measured by modified MTT assay. Intracellular platinum accumulation was determined by ICP-MS analysis in SK-LMS cells and ATP7B-stably suppressed cells (SK-LMS-7B cells), which transfected the pRS ATP7B shRNA vector. To analyze in vivo platinum sensitivity, SK-LMS and SK-LMS-7B cells were subcutaneously injected into nude mice. Cisplatin (3 mg/kg) or PBS was administered i.p. to xenografted mice twice weekly for 28 days. Further, we attempted to show the improvement of platinum sensitivity by using premedication for CuSO4 before cisplatin treatment in vitro and in vivo model.
Results: Expression of ATP7B was observed in all 3 LMS cell lines. In clinical samples, ATP7B was over expressed in 14 of 26 (53.8%) cases. In drug resistance analysis, significantly lower IC50 values of cisplatin were observed in SK-LMS-7B cells (4.3μM, p &lt; 0.01) compared with SK-LMS cells (17μM) indicating knockdown of ATP7B improved platinum sensitivity. Also, significantly higher platinum accumulation was detected in SK-LMS-7B cells (7.2ng/dish, p &lt; 0.01) compared with SK-LMS cells (52ng/dish). In in vivo analyses, a significantly higher anti-tumor effect of cisplatin was observed in SK-LMS-7B-xenografted mice compared with SK-LMS-xenografted mice (56±27 μg vs 214±68 μg, p&lt;0.05). Pretreatment with CuSO4 (15μM) before cisplatin treatment for SK-LMS cells significantly reduced IC50-values (17 μM to 3.9 μM, p&lt;0.05). In addition, an elevated intracellular platinum accumulation was observed in cells from pretreatment group (15.0 to 68.4 ng/dish, p&lt;0.05). A significant improvement of platinum sensitivity was observed in the premedication group (CuSO4 premedication group; 65±40 μg vs no pretreatment group; 230±60 μg, p&lt;0.05).
Conclusion: ATP7B contributes platinum resistance in LMS. Presumably, ATB7B preferentially excrete copper ion thereby platinum can be accumulate in cells. Thus, pretreatment with CuO4 effectively improved platinum sensitivity in vitro and in vivo by blocking ATP7B.
Citation Format: Mamoru Kakuda, Shinya Matsuzaki, Ruriko Nakae, Yusuke Tanaka, Eiji Kobayashi, Yutaka Ueda, Kiyoshi Yoshino, Tadashi Kimura. Novel strategy to overcome platinum resistance in uterine leiomyosarcoma; blocking ATP7B by copper ion [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 1200. doi:10.1158/1538-7445.AM2017-1200