Copper ions are novel therapeutic agents for uterine leiomyosarcoma

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GYNECOLOGY

Copper ions are novel therapeutic agents for uterine leiomyosarcoma Mamoru Kakuda, MD1; Shinya Matsuzaki, MD, PhD1; Yutaka Ueda, MD, PhD; Mayu Shiomi, MD; Satoko Matsuzaki, MD, PhD; Toshihiro Kimura, MD, PhD; Masami Fujita, MD, PhD; Tomomi Egawa-Takata, MD, PhD; Eiji Kobayashi, MD, PhD; Satoshi Serada, PhD; Kiyoshi Yoshino, MD, PhD; Tetsuji Naka, MD, PhD; Tadashi Kimura, MD, PhD

BACKGROUND: Multidrug resistance is a major concern in uterine

leiomyosarcoma treatment. Development of effective chemotherapies and management of drug resistance in patients is necessary. The copper efflux transporter adenosine triphosphatase copper transporting beta is a member of the P-type adenosine triphosphatase family and is also known as a strong platinum efflux transporter. Various reports have shown the association between adenosine triphosphatase copper transporting beta and platinum resistance; however, suitable inhibitors or methods for inhibiting platinum efflux via adenosine triphosphatase copper transporting beta are not developed. OBJECTIVE: Our study focused on platinum resistance in uterine leiomyosarcoma. The role of adenosine triphosphatase copper transporting beta in uterine leiomyosarcoma resistance to platinum drugs was investigated both in vitro and in vivo. STUDY DESIGN: Adenosine triphosphatase copper transporting beta expression was investigated by Western blotting and the efficacy of copper sulfate pretreatment and cisplatin administration in adenosine triphosphatase copper transporting betaeexpressing cells was investigated both in vitro and in vivo. RESULTS: Western blot analysis of SK-LMS-1 cells (uterine leiomyosarcoma cell line) revealed strong adenosine triphosphatase copper transporting beta expression. A permanent SK-LMS-ATPase copper transporting betaesuppressed cell line (SK-LMS-7B cells) was generated,

U

terine leiomyosarcoma (LMS) arises from uterine smooth muscles and is a relatively rare tumor, constituting only 2e3% of all uterine malignancies.1 Compared with other uterine cancers, LMS is aggressive and is associated with a high risk of recurrence and death, regardless of the stage at presentation.1,2 Five year survival estimates as per the International Federation of Gynecology and Obstetrics stage are 76%, 60%, 45%, and 29% for stages IeIV, respectively.3

Cite this article as: Kakuda M, Matsuzaki, S, Ueda Y, et al. Copper ions are novel therapeutic agents for uterine leiomyosarcoma. Am J Obstet Gynecol 2019;xxx;xx-xx 0002-9378/$36.00 ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.ajog.2019.07.030

and cisplatin exhibited a significant antitumor effect in SK-LMS-7B cells, both in vitro (SK-LMS-1 cells, half-maximal inhibitory concentration, 17.2 mM; SK-LMS-7B cells, half-maximal inhibitory concentration, 4.2 mM, P < .01) and in xenografts compared with that in SK-LMS-1 cells (5.8% vs 62.8%, P < .01). Copper sulfate was identified as a preferential inhibitor of platinum efflux via adenosine triphosphatase copper transporting beta. In SK-LMS-1 cells pretreated with 15 mM copper sulfate for 3 hours, the cisplatin half-maximal inhibitory concentration decreased significantly compared with that in untreated cells and resulted in significantly increased intracellular platinum accumulation (1.9 pg/cell vs 8.6 pg/cell, P < .01). The combination of copper sulfate pretreatment with cisplatin administration was also effective in vivo and caused cisplatin to exhibit significantly increased antitumor effects in mice with SK-LMS-1 xenografts (3.1% vs 62.7%, P < .01). CONCLUSION: Our study demonstrates that adenosine triphosphatase copper transporting beta is overexpressed in uterine leiomyosarcoma cells and that copper sulfate, which acts as an inhibitor of platinum efflux via adenosine triphosphatase copper transporting beta, may be a therapeutic agent in the treatment of uterine leiomyosarcoma. Key words: adenosine triphosphatase copper transporting beta, copper

transporter, platinum resistance, platinum transporter, uterine leiomyosarcoma

The first treatment should be surgical management. However, the recurrence rate of LMS has been reported as approximately 60e70%, with poor prognosis despite complete surgical resection.4e6 The National Cancer Database of the United States concluded that chemotherapy adds only 8.5 months benefit compared with untreated patients with metastatic LMS (19.4 vs 10.9 months) in an observational cohort study of patients diagnosed between 1998 and 2013.7 Doxorubicin is still the first-line treatment in uterine sarcomas. In a recently published phase III clinical trial, the median progression-free survival was reported as 23.3 weeks, the median overall survival was 76.3 weeks, and the response rate was 20%.8 To improve the survival rate of LMS, addition of trabectedin to doxorubicin,9 a phase III trabectedin trial,10 and a phase II

pazopanib trial have been reported.11 These studies showed that various agents have some activity, but effective chemotherapeutics for patients with LMS are limited because of poor responses, and standard chemotherapeutic regimens for advanced and recurrent forms of LMS have not been established yet.3 Platinum drugs are effective against a wide spectrum of solid neoplasms including ovarian, testicular, bladder, colorectal, lung, and head and neck cancers. They are considered important in treating gynecological cancers because chemotherapy regimens for treating ovarian serous adenocarcinoma (the most common subtype of ovarian carcinoma), which include platinum drugs, have demonstrated an efficacy of 81%.12e14 However, their efficacy is only 3% in LMS, which is frequently resistant

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AJOG at a Glance Why was the study conducted? Adenosine triphosphatase copper transporting beta (ATP7B) is a copper and platinum efflux transporter that plays an important role in platinum resistance. It belongs to the P-type adenosine triphosphatase family of transporters and is involved in copper homeostasis. ATP7B is expressed in normal liver, kidney, and placenta tissues and is located in the trans-Golgi network of these cells. A strong association between platinum resistance and ATP7B has been reported, and blocking ATP7B function is considered difficult. What are the key findings? The method of blocking the platinum efflux of ATP7B was investigated in uterine leiomyosarcoma, one of the strongest platinum-resistant gynecological tumors. We successfully showed that ATP7B suppression can improve the platinum resistance of uterine leiomyosarcoma cells in vitro and in vivo. Next, we showed that copper sulfate (CuSO4) pretreatment before cisplatin administration significantly improves the platinum resistance of uterine leiomyosarcoma cells compared with that in leiomyosarcoma cells without treatment, by increasing intracellular platinum accumulation. Improvement of platinum resistance was also confirmed in vivo by CuSO4 pretreatment before cisplatin administration. What does this study add to what is already known? We demonstrated that CuSO4 pretreatment before cisplatin administration improves platinum sensitivity in uterine leiomyosarcoma cells by inhibiting platinum efflux via ATP7B. Although our analysis was performed in uterine leiomyosarcoma cells, similar results are expected in other ATP7B-expressing cancers such as platinum-resistant ovarian and endometrial cancer and colorectal, lung, and gastric cancers.

to platinum drugs, leading to poor prognosis.3,15 Therefore, investigation of platinum resistance to increase platinum sensitivity is warranted to improve the survival rates of patients with LMS. Platinum resistance is a major concern in cancer treatment, and its underlying mechanisms have been investigated thoroughly. We previously reported Annexin A4 induced platinum resistance in ovarian and endometrial carcinoma and showed that Annexin A4 promotes platinum efflux via adenosine triphosphatase copper transporting alpha (ATP7A), thereby decreasing intracellular platinum accumulation.16,17 These studies demonstrated that Annexin A4 and ATP7A are therapeutic targets in platinum-resistant cancers, as are various other platinum transporters.18 Platinum transporters such as multiple drug resistance 1, multidrug resistance-associated protein 1, multidrug resistance-associated protein 2, ATP7A, adenosine triphosphatase

copper transporting beta (ATP7B), copper transporter receptor 1 (CTR1), and copper transporter receptor 2 are associated with platinum resistance. Previous studies have reported that ATP7A, ATP7B, and CTR1 are major regulators of human copper homeostasis and copper ions, and platinum drugs may share the same transport system in the cell. Therefore, they also play important roles in the transport of platinum drugs.13,18e22 In particular, previous reports have shown that ATP7B and CTR1 are strongly associated with platinum resistance.23e27 Although these transporters are thought to be important in the development of platinum drug resistance, only a small number of clinical trials have been performed to investigate their role.28,29 Previous studies have reported that CTR1 is a high-affinity copper (Cu)uptake transporter and a major uptake transporter of platinum drugs.18,25 Cu

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ajog.org chelation has been reported to lower the levels and decrease Cu intake, causing induction of CTR1 expression and an elevated intracellular accumulation of platinum drugs. Cu-lowering agents such as trientine may have the same effect. Fu et al29 treated 5 patients showing platinum-resistant tumors, with Culowering agents, and provided the first preliminary data in human subjects, demonstrating partial overcoming of platinum resistance in some patients. This resulted in a follow-up study on 55 patients with various cancers (the most common cancers were head and neck [n ¼ 13], nonesmall-cell lung [n ¼ 10], and epithelial ovarian [n ¼ 8]) cancer that had failed to respond to standard treatments (45 had failed to respond to platinum drugs).28 Trientine was administered to all patients prior to carboplatin, and 1 patient exhibited a partial response, whereas the disease was stabilized in 8 patients with tolerable side effects. Although these effects were not fully confirmed, these studies demonstrate that Cu transporters are potential therapeutics for some platinum-resistant cancers. ATP7B may also serve as a therapeutic target in these cancers but has not yet been studied in clinical settings. As mentioned in the previous text, CTR1 targeting therapy is already used in the clinical setting; however, the results of the clinical study were anything but satisfactory. Therefore, we focused on ATP7A and ATP7B. Although ATP7A and ATP7B are considered strong platinum and Cu efflux transporters in cancer cells, they are not tested clinically because of the lack of inhibitors or an inhibitor against platinum efflux. In this study, we investigated ATP7A and ATP7B expression in LMS cells and established a method for inhibition of platinum efflux using them. We focused on Cu2þ because Cu2þ is considered an outlier in the Irving-Williams series, suggesting that it has a strong competitive edge over cisplatin for the same binding site30; thus, copper sulfate (CuSO4) may favor intracellular platinum accumulation.

ajog.org Materials and Methods Cell lines The human leiomyosarcoma cell lines, SK-LMS-1 and SK-UT1, were obtained from the American Type Culture Collection (Manassas, VA). The human leiomyosarcoma cell lines SKN; the human ovarian serous carcinoma cell line OVSAHO, SKOV3; and the ovarian clear cell carcinoma cell lines OVTOKO, OVISE, and RMG-1 were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). The human ovarian serous carcinoma cell line A2780 was obtained from the European Collection of Animal Cell Culture (Salisbury, Scotland). SK-LMS-1 and SK-UT1 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT) and 100 U/mL penicillin and 100 mg/mL streptomycin (Nacalai Tesque, Kyoto, Japan) at 37 C under a humidified atmosphere of 5% CO2. SKN cells were maintained in Ham’s F12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. OVSAHO, A2780, OVISE, and OVTOKO cells were maintained in Roswell Park Memorial Institute 1640 medium (Wako Pure Chemical Industries) supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin.

Western blotting Cells were prepared, as described previously.17 Proteins were transferred to polyvinylidene difluoride membranes treated with chicken polyclonal antiATP7A antibody (ab13995; Abcam, Cambridge, United Kingdom) or rabbit monoclonal anti-ATP7B antibody (SAB1403592; Sigma-Aldrich, St Louis, MO). Additional information can be found in the Supporting Information on Material and Methods.

Immunohistochemistry Informed consent was obtained from all donors, and all studies involving human subjects were approved by the Institutional Review Boards No. 17217

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of Osaka University Hospital. Surgically resected LMS tissues were obtained from patients with uterine leiomyosarcoma. The sections were then analyzed for the expression of ATP7B, as described previously with some technical modifications.31,32 Immunohistochemical staining for ATP7B was performed using the avidinbiotin-peroxidase complex method with a rabbit monoclonal anti-ATP7B antibody (SAB1403592; Sigma-Aldrich) and the Vectastain avidin-biotin-peroxidase complex kit (PK-4001; Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocol. Additional information can be found in the Supporting Information on Material and Methods.

Small interfering RNA transfection Two commercial small interfering RNAs (siRNAs) against ATP7A and ATP7B and a nonspecific control siRNA were obtained from Qiagen (Venlo, The Netherlands) and designated SK-LMSC, SK-LMS-si7A2, SK-LMS-si7A3, SK-LMS-si7B1, and SK-LMS-si7B2, respectively. Cells were transfected with siRNA using Lipofectamine 3000 Reagent (Invitrogen) according to the manufacturer’s instructions. Selective silencing of ATP7A and ATP7B was confirmed by Western blotting.

Measurement of half-maximal inhibitory concentration (IC50) values after treatment with cisplatin Cells were suspended in DMEM supplemented with 10% FBS and were seeded in 96 well plates (1000 cells per well; Costar, Corning, Corning, NY) for 24 hours. They were then exposed to various concentrations of cisplatin (0e50 mM) for 72 hours. Cell proliferation was evaluated using the WST-8 assay (Cell Counting Kit-SF; Nacalai Tesque) after treatment at the time points indicated by the manufacturer. The absorption of WST-8 was measured at a wavelength of 450 nm (reference wavelength, 630 nm) using a Model 680 Microplate Reader (Bio-Rad Laboratories, Hercules, CA). Absorbance values for treated cells indicative of

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proliferation rates were expressed as percentages relative to values for untreated controls, and the drug concentrations resulting in a 50% inhibition of cell growth (IC50 values) were calculated.

Quantification of intracellular platinum accumulation Cisplatin accumulation in cells was analyzed according to a previously established method,16,17 with minor modifications. In brief, 4  106 cells (SKLMS-1, SK-LMS-C, SK-LMS-si7B1, and SK-LMS-si7B2 cells) were seeded into two 150-mm tissue culture dishes and incubated for 24 hours. The cells were exposed to 100 mM cisplatin for 60 minutes at 37 C and then washed twice with phosphate-buffered saline (PBS). After 3 hours of incubation in cisplatin-free DMEM (supplemented with 10% FBS), whole extracts were prepared, and the concentration of intracellular platinum was determined using an Agilent 7500ce inductively coupled plasma mass spectrometer (Agilent, Santa Clara, CA). The absolute concentration of platinum in each sample was determined from a calibration curve prepared with a platinum standard solution.

Generation of ATP7B short hairpin RNA stably transfected cell lines To generate cell lines with stably suppressed ATP7B, the pRS-ATP7B suppression plasmid was obtained from OriGene Technologies (TF314561; Rockville, MD). SK-LMS-1 cells were transfected with the pRS-ATP7B suppression plasmid, as described previously).16 Transfected cells were selected with 0.5 mg/mL puromycin (Invitrogen). Clones were maintained in 0.3 mg/mL puromycin to obtain stable suppression. Two stable ATP7B-suppressing cell lines were established and designated SK-LMS-ATP7B-7B-21, and SK-LMSATP7B-33. A control cell line, SK-LMS1, was also established and stably transfected with an empty vector. This cell line was designated SK-LMS-CV.

In vivo model of cisplatin resistance improvement Four week old female Institute of Cancer Research (ICR) nu/nu mice were

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FIGURE 1

Silencing ATP7B expression in SK-LMS-1 improves platinum resistance in vitro

A, Western blot analysis for ATP7A and ATP7B in 8 gynecological cancer cell lines. Moderate to strong ATP7A expression was observed in SK-LMS-1 cells, and ATP7B was strongly expressed in SK-LMS-1 cells. B, Knockdown of ATP7A and ATP7B expression by siRNA in SK-LMS-1 cells, confirmed by Western blotting. C, Sensitivity to cisplatin, as determined by IC50, was investigated in SK-LMS-1, SK-LMS-C, SK-LMS-si7A-2, SK-LMS-si7A-3, SKLMS-si7B-1, and SK-LMS-si7B-2 cells. Asterisk indicates P < .05; double asterisk indicates P < .01. D, Intracellular platinum accumulation was investigated after treatment with 100 mM cisplatin for 60 minutes and further incubation with cisplatin-free medium for 180 minutes and was determined by ICP-MS (Agilent) analyses in SK-LMS-1, SK-LMS -C, SK-LMS-si7B-1, and SK-LMS-si7B-2 cells. Asterisk indicates P < .05; double asterisk indicates P < .01. ATP7A, adenosine triphosphatase copper transporting alpha; ATP7B, adenosine triphosphatase copper transporting beta; IC50, half-maximal inhibitory concentration; ICP-MS, inductively coupled plasma mass spectrometer; N.S., not significant; siRNA, silent interfering RNA. Kakuda et al. Novel CuSO4 therapy for leiomyosarcoma. Am J Obstet Gynecol 2019.

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ajog.org obtained from Charles River Japan (Yokohama, Japan). For subcutaneous xenograft experiments, 2.5  106 SKLMS-1, SK-LMS-CV, SK-LMS-7B21, and SK-LMS-7B33 cells were suspended in 100 mL of PBS and injected subcutaneously into the backs of the ICR nu/nu mice (n ¼ 5 per group). Ten days after xenograft establishment, tumors measured 100 mm3. Mice were then randomly divided into 2 groups and administered cisplatin (3 mg/kg) or PBS intraperitoneally (i.p.) twice weekly for 4 weeks. Tumor volumes were determined by measuring the length and width and calculating the volume as (width 2  length)/2. Fortynine days after tumor implantation, mice were killed and tumors were removed and weighed.

Effect of pretreatment with CuSO4 on platinum sensitivity in vitro Cells were suspended in DMEM supplemented with 10% FBS and were seeded in 96-well plates (1000 cells per well; Costar, Corning) for 24 hours. Cells were pretreated with CuSO4 before the administration of cisplatin (451657; Sigma-Aldrich). In particular, 15 mM CuSO4 was administrated and cisplatin was added at various concentrations after 3 hours of CuSO4 treatment. Cells were then exposed to various concentrations of cisplatin (0e200 mM) for 72 hours, and the IC50 value was determined as described above. Additional information can be found in the Supporting Information on Material and Methods.

Quantification of intracellular platinum accumulation with pretreated CuSO4 To investigate the effect of pretreatment with CuSO4, 6 treatment groups were established: SK-LMS-1, SK-LMS-1 with CuSO4 before treatment, SK-LMS-CV, SK-LMS-CV with CuSO4 before treatment, SK-LMS-ATP7B-7B-21, and SKLMS-ATP7B-7B-33. Cells (4  106) from each group were investigated for the intracellular platinum accumulation. Additional information can be found in the Supporting Information on Material and Methods.

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TABLE 1

Demographic characteristics of leiomyosarcoma patients Variables

Leiomyosarcoma

Number of cases

14

Age, median (range)

51 (38e60)

FIGO stage I

7

II

2

III

5

IV

0

FIGO, International Federation of Gynecology and Obstetrics. Kakuda et al. Novel CuSO4 therapy for leiomyosarcoma. Am J Obstet Gynecol 2019.

Immunofluorescence for ATP7B Immunofluorescence staining was performed 2 days after cells were seeded on coverslips (3000 cells/well). Before staining, cells in the treatment groups were pretreated with 10 mM cisplatin or 50 mM CuSO4 for 3 hours. Additional information can be found in the Supporting Information on Material and Methods.

In vivo model of cisplatin resistance after pretreatment with CuSO4 For subcutaneous xenograft experiments, 2.5  106 SK-LMS-1 cells were suspended in 100 mL of PBS and injected subcutaneously into the backs of the ICR nu/nu mice (n ¼ 5 per group). At 10 days after xenograft establishment, the mice were randomly divided into 6 groups (groups 1e6). In group 1, xenograft mice were administered cisplatin (3 mg/kg) i.p. twice weekly for 4 weeks. In group 2, xenograft mice were administered PBS i.p. twice weekly for 4 weeks. In group 3, xenograft mice were administered cisplatin (3 mg/kg) i.p. 3 hours after the administration of CuSO4 (0.25 mg/kg) i.p. twice weekly for 4 weeks. In group 4, xenograft mice were administered PBS i.p. 3 hours after the administration of CuSO4 (0.25 mg/kg) i.p. twice weekly for 4 weeks. In group 5, xenograft mice were administered cisplatin (3 mg/kg) i.p. 3 hours after the administration of CuSO4 (1 mg/kg) i.p. twice weekly for 4 weeks. In group 6, xenograft mice were

administered PBS i.p. 3 hours after the administration of CuSO4 (1 mg/kg) i.p. twice weekly for 4 weeks. Groups 1 and 2 were labeled no premedication groups, group 3 and 4 were labeled low-dose groups, and groups 5 and 6 were labeled high-dose groups. Additional information can be found in the Supporting Information on Material and Methods.

Statistical analysis Statistical analyses were performed using 1-way analysis of variance followed by Dunnett’s analysis to evaluate the significance of differences. For all analyses, P < .05 was considered statistically significant.

Results ATP7A and ATP7B are expressed in uterine LMS cells ATP7A and ATP7B expression in 3 LMS (SK-LMS-1, SKN, and SK-UT1) and 5 ovarian cancer cell lines (A2780, SKOV3, OVTOKO, OVISE, and RMGI) was investigated by Western blotting. Strong ATP7A expression was observed in SKN cells, whereas moderate to strong expression was observed in SK-LMS-1 cells, and weak expression was observed in SK-UT1, A2780, SKOV3, OVTOKO, OVISE, and RMG-I cells. Strong ATP7B expression was observed in SK-LMS-1 cells, and weak expression was observed in SKN, SKUT1, A2780, SKOV3, and OVISE cells (Figure 1A).

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FIGURE 2

Immunohistochemical LMS analysis and generation of SK-LMS-1 with ATP7B suppression

ajog.org IC50 ¼ 4.3 mM, P < .01; SK-LMS-si7B2, IC50 ¼ 4.4 mM, P < .01) were significantly lower than the controls (IC50 ¼ 16.8 mM; Figure 1C). This suggests that ATP7B, but not ATP7A, is associated with SK-LMS-1 cell platinum resistance. To determine the mechanism underlying improved platinum drug sensitivity in SK-LMSATP7B-silenced cells, platinum accumulation following cisplatin exposure was investigated in SK-LMC-C and ATP7B-silenced cells. Significantly higher platinum accumulation was observed in SK-LMS-si7B1 (6.9 pg/cell, P <.01) and SK-LMS-si7B2 cells (6.3 pg/ cell, P <.01) than in SK-LMS-C cells (0.9 pg/cell) and untransfected controls (0.9 pg/cell; Figure 1D). This suggests that ATP7B silencing induces increased intracellular platinum accumulation following cisplatin exposure, resulting in amelioration of platinum resistance.

ATP7B is expressed in clinical LMS samples

A, Representative positive and negative images of IHC staining in LMS specimens. The left panel shows 40 magnification and the right panel shows 200 magnification. B, Establishment of SKLMS-1 cells with stable ATP7B suppression by ATP7B shRNA transfection into SK-LMS-1 cells with high ATP7B expression levels. Suppressed expression of ATP7B was confirmed by Western blotting. Asterisk indicates P < .05; double asterisk indicates P < .01. C, Sensitivity to cisplatin, as determined by IC50, was investigated in SK-LMS-1, SK-LMS-CV, SK-LMS-7B-21, and SK-LMS 7B33 cells. ATP7B, adenosine triphosphatase copper transporting beta; IC50, half-maximal inhibitory concentration; IHC, Immunohistochemistry; LMS, leiomyosarcoma; N.S., not significant; shRNA, short hairpin RNA. Kakuda et al. Novel CuSO4 therapy for leiomyosarcoma. Am J Obstet Gynecol 2019.

ATP7B silencing improves platinum resistance of SK-LMS-1 cells Cells were transfected with two siRNAs to suppress either ATP7A or ATP7B expression, and suppression was confirmed by Western blotting (Figure 1B). Cisplatin IC50 values were also determined for both control and knockdown cell lines. The

cisplatin IC50 values in 2 types of ATP7Asilenced SK-LMS-1 cells (SK-LMS-si7A2, IC50 ¼ 11.3 mM, P ¼ .35; SK-LMS-si7A3, IC50 ¼ 11.7 mM, P ¼ 0.30) were not significantly different compared with control cells (IC50 ¼ 16.0 mM). However, cisplatin IC50 values in 2 types of ATP7Bsilenced SK-LMS-1 cells (SK-LMS-si7B1,

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Immunohistochemical staining was performed for 14 LMS specimens. Patients’ clinical and demographic data are summarized in Table 1, and representative images indicating the presence of ATP7B expression are presented in Figure 2A. Among the 14 specimens, 8 cases (57.2%) scored more than 4 points (high group), 3 cases (21.4%) scored 1e3 points (low group), and 3 cases (21.4 %) scored 0-1 points (negative group).

Platinum drug sensitivity improves upon stable knockdown of ATP7B in SK-LMS-1 cells SK-LMS-1 cells with elevated ATP7B expression were used to generate stable ATP7B-supressed cell lines (SKLMS-7B-21 and SK-LMS-7B-33 cells) and 1 empty vector-transfected cell line (SK-LMS-CV cells). Abbreviations and definitions of cell lines are shown in Table 2. The suppression of ATP7B was confirmed by Western blotting (Figure 2B). Cisplatin IC50 values were significantly lower in SK-LMS-7B-21 (4.2 mM, P < .01) and SK-LMS-7B-33 cells (3.4 mM, P < .01) than in SKLMS-1 (17.2 mM) and SK-LMS-CV cells (15.8 mM; Figure 2C).

ajog.org ATP7B knockdown improves cisplatin sensitivity in xenograft models SK-LMS-1, SK-LMS-CV, SK-LMS-7B21, and SK-LMS-7B-33 cells were subcutaneously injected into nude mice. After tumors were established, either cisplatin or a PBS control was administered twice a week for 4 weeks. On day 39 following tumor establishment, average tumor volumes were 635.6  63.4 mm3 in PBS-treated SKLMS-CV control mice and 659.1  74.6 mm3 in cisplatin-treated SK-LMSCV mice, which were not significantly different (P ¼ .82). The parent SKLMS-1 and SK-LMS-CV xenografts exhibited similar resistance to cisplatin (Figure 3A, upper left and lower left panels) (P ¼ .64). In SK-LMS-7B-21-xenografted mice, the average day 39 tumor volume upon cisplatin treatment (254.8  16.6 mm3) was significantly lower than that with PBS treatment (685.8  46.5 mm3; P < .001; Figure 3A, upper right panel). A similar cisplatin response was observed in SK-LMS-7B-33xenografted mice (138.0  95.0 v s533.0  81.3 mm3; P < .001; Figure 3B, lower right panel). In SKLMS-7B-21-xenografted mice, the weights of the PBS-treated (361.0  28.8 mg) and cisplatin-treated tumors (179.5  14.6 mg) were significantly different (P ¼ .002; Figure 3B) as were those from SK-LMS-7B-33 xenografts (348.4  22.1 vs 138.0  38.4 mg; P ¼ .005). Thus, ATP7B overexpression in SK-LMS-1 cells confers a significant platinum resistance with respect to in vivo tumor growth (Figure 3B).

Pretreatment with CuSO4 increases cisplatin sensitivity of SK-LMS cells in vitro It is unclear whether CuSO4 or cisplatin is the preferred substrate of ATP7B. SK-LMS-1 cells were pretreated with CuSO4 prior to cisplatin to determine the noncytotoxic CuSO4 concentration; the CuSO4 IC50 value in SK-LMS-1 cells was 60 mM (data not shown). There was no significant decrease in cell viability between

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TABLE 2

Abbreviations and definitions of cell lines List of cell lines

Definitions

Parental cells SK-LMS-1

Parental leiomyosarcoma cell lines

SK-UT1 SKN siRNA-transfected cells SK-LMS-C

Control siRNA transfected SK-LMS-1 cells

SK-LMS-si7A2

ATP7A silencing SK-LMS-1 cells

SK-LMS-si7A3 SK-LMS-si7B1

ATP7B silencing SK-LMS-1 cells

SK-LMS-si7B2 shRNA stably transfected cell lines SK-LMS-CV

Control vector transfected SK-LMS-1 cells

SK-LMS-7B21

Permanent ATP7B-suppressed SK-LMS-1 cells

SK-LMS-7B33 ATP7B, adenosine triphosphatase copper transporting beta; ATP7B, adenosine triphosphatase copper transporting beta; shRNA, short hairpin RNA; siRNA, silent interfering RNA. Kakuda et al. Novel CuSO4 therapy for leiomyosarcoma. Am J Obstet Gynecol 2019.

treatment with 15 mM CuSO4 for 75 hours or 2.5 mM cisplatin for 72 hours compared with untreated SK-LMS-1 cells (93.1% and 94.4% vs 100%; P ¼ 0.684 and P ¼ .772, respectively; Figure 4A). Therefore, 15 mM CuSO4 was determined to be a suitable pretreatment prior to cisplatin. After 3 hours of continuous pretreatment with 15 mM CuSO4 followed by a further addition of 2.5 mM cisplatin treatment for 72 hours, a significant decrease in cell viability was observed (55.0%, P < .01). The cisplatin IC50 in pretreated SK-LMS-1 cells (3.7 mM, P < .01) was significantly lower than that in untreated SK-LMS-1 cells (17.5 mM) (Figure 4B), whereas the cisplatin IC50 in pretreated SK-LMS-1 cells was similar to those in SK-LMS-7B-21 and SK-LMS7B-33 cells (4.4 mM and 7.1 mM; P > .05). Additionally, platinum accumulation in pretreated SK-LMS-1 cells (8.6 pg/ cell, P < .01) was significantly higher than that in untreated SK-LMS-1 (1.9 pg/cell) and SK-LMS-CV cells (1.2 pg/ cell). Thus, pretreatment with CuSO4 improved the cisplatin IC50 value via the

increase in intracellular platinum accumulation following cisplatin exposure (Figure 4C). Platinum accumulation was significantly higher in SK-LMS-7B-21 (8.8 pg/cell, P < .01) and SK-LMS-7B-33 cells (9.8 pg/cell, P < .01) than in SK-LMS-1 (1.9 pg/cell) and SK-LMSCV cells (1.2 pg/cell) without CuSO4 pretreatment (Figure 4C). However, platinum accumulation in pretreated SK-LMS-1 cells (8.6 pg/cell) was similar to that in SK-LMS-7B-21 and SK-LMS-7B-33 cells (8.8 pg/cell and 9.8 pg/cell; P ¼ 0.896 and P ¼ .927, respectively). These results suggest that pretreatment with CuSO4 in SK-LMS-1 cells mimics the improvement of platinum sensitivity in ATP7B knockdown cells (SK-LMS-7B-21 and SK-LMS-7B-33 cells).

Perinuclear ATP7B translocates to the cellular membrane in SK-LMS-1 cells after CuSO4 exposure Immunofluorescence analysis revealed that ATP7B localized to perinuclear and cytoplasmic regions in untreated

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FIGURE 3

ATP7B suppression in SL-LMS cells improves platinum resistance in vivo

Analysis of ATP7B as a platinum-resistant protein in vivo. A, To determine the cisplatin sensitivity of SL-LMS cells stably suppressing ATP7B to platinum in vivo, parent SK-LMS-1, SK-LMS-CV, SKLMS-7B-21, and SK-LMS-7B-33 cells were subcutaneously injected into nude mice (n ¼ 5 per group). After tumor xenografts were established, cisplatin (3 mg/kg) or PBS was administered i.p. twice weekly for 4 weeks. Average values (points) for 5 mice  SD (bars) are presented. Asterisk indicates, P < .05; double asterisk indicates P < .01. B, Thirty-nine days after implantation, tumors were removed and weighed. Values shown are the means (SD) of 5 mice. Asterisk indicates P < .05; double asterisk indicates P < .01; 1-way analysis of variance, followed by Dunnett’s analysis). ATP7A, adenosine triphosphatase copper transporting alpha; i.p., intraperitoneally; NS, not significant; PBS, phosphate-buffered saline. Kakuda et al. Novel CuSO4 therapy for leiomyosarcoma. Am J Obstet Gynecol 2019.

SK-LMS-1 cells, but after exposure to 10 mM cisplatin for 4 hours, ATP7B localized to the cellular membrane (Figure 4D). Moreover, 50 mM CuSO4 exposure for 3 hours also resulted in

cellular membrane localization. This suggests that ATP7B relocalizes to serve as an efflux transporter of cisplatin or CuSO4 following exposer to either compound.

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CuSO4 pretreatment followed by cisplatin exposure improves the platinum sensitivity of SK-LMS-1 cells in vivo On day 39 following the establishment of xenograft tumors, the average tumor volumes were 698.0  97.4 mm3 in group 2 mice and 676.1  110.3 mm3 in group 1 mice (Figure 5A, upper left panel; P ¼.895), indicating that cisplatin had no antitumor effect in the SK-LMS1-xenografted mice. In the low-dose groups, average day 39 tumor volumes were 412.2  57.8 mm3 in the cisplatin-treated and 715.5  108.5 mm3 in the PBS-treated groups (Figure 5A, upper right panel; P ¼ .054). In contrast, a significant decrease in tumor volumes was observed in the highdose groups between group 5 (259.8  58.5 mm3) and group 6 (696.7  90.7 mm3; P ¼ .006; Figure 5A, lower left panel). On day 39, significant differences were observed in tumor weight between the PBS- (523.8  40.5 mg) and cisplatintreated tumors (181.8  44.5 mg, P < .001) in the high-dose groups (Figure 5B). In contrast, no cisplatin antitumor effects were observed in the untreated (423.7  35.6 vs 411.6  75.23 mg; P ¼ .898) and low-dose groups (301.3  42.6 vs 502.3  79.2 mg; P ¼ .076) (Figure 5B). This indicates that dose-dependent pretreatment with CuSO4 induces cisplatin antitumor effects in vivo. The body weight of mice did not significantly change among groups 1, 3, and 5 (25.0  4.4 g vs 25.1  1.5 g vs 28.4  2.7; P > .05), and no abnormal macroscopic findings and diarrhea were observed in these mice. The value of hemoglobin did not significantly change among groups 1, 3, and 5 (10.2  3.0 g/ dL vs 11.4  0.8 g/dL vs 11.0  2.0 g/dL; P > .05).

Comment Principal findings We have demonstrated an association between ATP7B expression and platinum drug resistance in LMS cells. Our analysis showed that suppression of ATP7B expression improves platinum sensitivity in LMS and that ATP7B might

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FIGURE 4

Determination of IC50 value of cisplatin following CuSO4 pretreatment

Differences in cisplatin sensitivity were investigated with or without pretreatment with CuSO4. A, Cell viability after treatment with CuSO4 (15 mM) for 75 hours, cisplatin (2.5 mM) for 72 hours, and combined treatment with CuSO4 (15 mM) for 75 hours and cisplatin (2.5 mM) for 72 hours was investigated in SK-LMS-1 cells. Asterisk indicates P < .05; double asterisk indicates P < .01. B, The sensitivity to cisplatin, as determined by IC50, was investigated in SK-LMS-1 cells, SK-LMS-1 cells treated with CuSO4 (15 mM) before cisplatin exposure, SK-LMS-7B-21 cells, and SK-LMS-7B-33 cells. C, Intracellular platinum accumulation was investigated after treatment with 100 mM cisplatin for 60 minutes and further incubation with cisplatin-free medium for 180 minutes and was determined by ICP-MS (Agilent) analyses in SK-LMS-1, SK-LMS-CV, SK-LMS-7B-21, and SK-LMS-7B-33 cells, SK-LMS-1 cells with CuSO4 pre-treatment, and SK-LMS-CV cells with CuSO4 pretreatment. Pretreatment with Cu plus cisplatin indicates CuSO4 (15 mM) pretreatment with various concentrations of cisplatin to investigate the IC50 value of cisplatin. Asterisk indicates P < .05; double asterisk indicates P < .01. D, SK-LMS-1 cells were divided into 3 groups: no treatment, cisplatin exposure, and CuSO4 exposure. A, SK-LMS-1 cells with no treatment. B, SK-LMS-1 cells with cisplatin treatment for 3 hours. C, SK-LMS-1 cells with CuSO4 treatment for 3 hours. Cells were incubated with anti-ATP7B antibody (green). Nuclei were stained with DAPI (blue). In the no-treatment group for each cell, ATP7B was localized in perinuclear regions. In SK-LMS-1 cells, after exposure to cisplatin or CuSO4, ATP7B relocalized to the cell membrane. Scale bar ¼ 100 mm. ATP7B, adenosine triphosphatase copper transporting beta; Cu, copper; CuSO4, copper sulfate; DAPI, 40 ,60 -diamino-2-phenylindole; IC50, half-maximal inhibitory concentration; ICP-MS, inductively coupled plasma mass spectrometer; N.S., not significant. Kakuda et al. Novel CuSO4 therapy for leiomyosarcoma. Am J Obstet Gynecol 2019.

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FIGURE 5

Pretreatment of SL-LMS cells with CuSO4 improves platinum resistance in vivo

be a therapeutic target for the treatment of LMS. Moreover, our study showed that pretreatment of cells with CuSO4 before cisplatin administration increases the intracellular platinum accumulation and cellular platinum sensitivity in LMS cells. We considered that the CuSO4 pretreatment induced a decrease in the platinum efflux via ATP7B. Improvement of platinum resistance was also observed in vivo. We speculated that pretreatment of mice with CuSO4 before cisplatin administration might increase the blood concentration of Cu2þ and result in increased intracellular accumulation of Cu2þ at the time of cisplatin administration. Because of these changes, similar findings were observed in vivo.

Results

Analysis of the effect of CuSO4 pretreatment on platinum sensitivity in vivo. A, To determine the effect of pretreatment with CuSO4 on the cisplatin sensitivity of SL-LMS cells in vivo, parent SK-LMS-1 cells were subcutaneously injected into nude mice and 6 groups were established (n ¼ 5 per group). After tumor xenografts were established, the SK-LMS-1 group was treated with cisplatin (3 mg/kg) or PBS was administered i.p. twice weekly for 4 weeks. The SK-LMS-1 low-Cu group was administered PBS or cisplatin (3 mg/kg) i.p. 3 hours after the administration of CuSO4 (0.25 mg/kg) i.p. twice weekly for 4 weeks. The SK-LMS-1 high-Cu group was administered PBS or cisplatin (3 mg/kg) i.p. 3 hours after the administration of CuSO4 (1 mg/kg) i.p. twice weekly for 4 weeks. Average values (points) for 5 mice  SD (bars) are shown. Asterisk indicates P < .05; double asterisk indicates P < .01. B, Thirty-nine days after implantation, tumors were removed and weighed. Values are presented as the means (SD) of 5 mice. Asterisk indicates P < .05; double asterisk indicates P < .01; 1-way analysis of variance, followed by Dunnett’s analysis). Cu, copper; CuSO4, copper sulfate; i.p., intraperitoneally; NS, not significant; PBS, phosphate-buffered saline. Kakuda et al. Novel CuSO4 therapy for leiomyosarcoma. Am J Obstet Gynecol 2019.

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Suppression of ATP7A expression failed to improve cisplatin IC50, contrary to the suppression of ATP7B. The different effects of ATP7A and ATP7B silencing on cisplatin resistance may be linked to their different levels of expression and/or distinct trafficking.24 Previous studies showed that ATP7B is a stronger platinum efflux transporter compared to ATP7A.24 Therefore, the difference in the results between ATP7A and ATP7B might be caused by their differential ability to efflux platinum. Thus, we hypothesized that ATP7B is a potential therapeutic target in LMS. To investigate whether ATP7B silencing overcomes platinum resistance, a SK-LMS-1 ATP7B-suppressed cell line was established and used to demonstrate the amelioration of cisplatin resistance both in vitro and in vivo. The increase in cisplatin sensitivity was mirrored by an increase in cellular platinum accumulation. Although ATP7B is a well-known platinum efflux transporter, it was originally reported to be a Cu efflux transporter. ATP7B is expressed in the normal liver, kidney, and placenta tissues and in platinum-resistant ovarian and endometrial cancer and colorectal, lung, and gastric cancers.24,33-37 In this study, pretreatment with Cu and cisplatin was used to determine which compound was

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FIGURE 6

Schematic of copper homeostasis system and platinum drug efflux by ATP7B

The scheme shows the effects of CuSO4, platinum drug, and combination therapy of CuSO4 and platinum drug on leiomyosarcoma cells. Copper ions are mainly incorporated into the cytoplasm by CTR1 and platinum ions are incorporated into the cytoplasm in a concentrationdependent manner or influxed by CTR1. A, ATP7B is a copper-transporting adenosine triphosphatase that is localized within the cytoplasm; however, when the intracellular concentration of copper ions increases, ATP7B migrates into the cell membrane and copper ions are effluxed by ATP7B. B, ATP7B is known as a platinum transporter. Therefore, when platinum drugs were administered, ATP7B migrated into the cell membrane and platinum ions were effluxed by ATP7B in the same way as the efflux of copper ions. C, Our speculations are shown in this scheme. As mentioned in the previous text, ATP7B effluxes copper and platinum ions. When copper and platinum drugs are administered at the same time, little is known about whether ATP7B effluxes copper or platinum ions. Our results suggest that these ions coexist in the cytoplasm. Copper ions are effluxed by ATP7B with higher priority, whereas the platinum ions cannot be effluxed. As a result, the intracellular platinum accumulation increases and platinum sensitivity is significantly improved. ATP7B, adenosine triphosphatase copper transporting beta; CTR1, copper transporter receptor 1; CuSO4, copper sulfate. Kakuda et al. Novel CuSO4 therapy for leiomyosarcoma. Am J Obstet Gynecol 2019.

Original Research

effluxed by ATP7B. The changes in localization after cisplatin or CuSO4 exposure in SK-LMS-1 cells, investigated using immunofluorescence analysis, were consistent with those reported in previous studies.18,26 Therefore, we considered that copper ions and cisplatin might have been effluxed by ATP7B in SK-LMS-1 cells as reported previously. Initial experiments demonstrated that pretreatment with 15 mM CuSO4 successfully lowered the cisplatin IC50 in SK-LMS-1 cells and increased the intracellular platinum accumulation upon cisplatin exposure. Thus, CuSO4 pretreatment increased the intracellular platinum accumulation by blocking Cu transporters, particularly ATP7B. We hypothesized that in the presence of both Cu and cisplatin, ATP7B preferentially effluxes Cu, and, consequently, increases intracellular platinum accumulation. Based on the cellular accumulation of platinum drugs in the presence of both cisplatin and CuSO4, it appeared that ATP7B preferentially effluxed copper ions and that CuSO4 pretreatment weakened the efflux of cisplatin via ATP7B. We thus conclude that ATP7B may preferentially efflux Cu over platinum drugs and that pretreatment with Cu2þ may saturate ATP7B, resulting in increased intracellular platinum drug concentrations. A simplified scheme of our hypothesis is shown in Figure 6.

Clinical implications There are few effective chemotherapeutic agents available for patients with LMS, and because of their poor response rates, no standard chemotherapy regimens for advanced and recurrent forms of uterine LMS have been established. Various drugs such as cisplatin, etoposide, topotecan, paclitaxel, trabectedin, and pazopanib have failed to improve the overall survival of patients with LMS.38-43 These reports, which set uterine LMS apart from other gynecological cancers, demonstrate that uterine LMS requires a specific therapeutic approach. Although LMS has been reported as a platinum-resistant tumor, our results

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successfully showed improvement of platinum resistance in LMS cells both in vitro and in vivo. These results suggest that pretreatment with CuSO4 might change the strategy of chemotherapy for LMS because many drugs have some activity; however, in general, the prognosis remains poor. If the pretreatment with CuSO4 changes LMS to a platinumsensitive tumor, it is expected to improve the overall survival as in ovarian cancer. If we use this novel treatment in the clinical setting, pretreatment with CuSO4 before 3 hours of cisplatin administration can be considered as an example. Further research is needed to apply this treatment in clinical settings. However, the side effects of CuSO4 are a major concern of this treatment. Previous studies have shown that an excess or toxicity of Cu is associated with the pathogenesis of hepatic disorders, neurodegenerative changes, inhibition of erythropoiesis, and eye problems. Therefore, we discuss the side effects of this treatment in the following text.

Research implications The toxicity of CuSO4 in vivo has been comprehensively investigated in previous studies, and its median lethal dose in mice, 3 days after injection, is 29.5 mg/ kg.44 Another study reported that intraperitoneal injection of CuSO4 in single doses of 6.6, 13.2, or 19.9 mg/kg body weight produced no micronucleus induction in male CBA mice.45 Similar results were reported in another study that investigated the bone marrow of the animals treated with intraperitoneal injection of 6.6 mg/kg CuSO4 after 24 and 48 hours. Cytotoxic effects, such as the inhibition of erythropoiesis, were detected at 13.2 mg/kg, and the maximum tolerated dose was reported as 19.9 mg/kg.46 In our xenograft model, the treatment dose was lower than the toxic dose in mice, and the body weight of mice was not significantly different among the PBS group, low-Cu group, and high-Cu group. Moreover, inhibition of erythropoiesis was not observed in our xenograft model. Thus, our protocol

(administration of 1 mg/kg CuSO4 intraperitoneally twice weekly for 4 weeks) should be acceptable for reducing platinum resistance; however, further research is needed to determine the ideal dose of CuSO4 pretreatment. Previous studies have reported that the lowest dose of copper sulfate that has been toxic when ingested by humans is 11 mg/kg.47,48 According to the practice guide for dose conversion between animals and humans,49 our in vivo treatment with 1 mg/kg was estimated to be less than 0.1 mg/kg of the human equivalent; thus, we considered that this treatment is safe for humans and that it is possible to use CuSO4 pretreatment before cisplatin treatment.

Strengths and limitations We revealed that the pretreatment of cells with CuSO4 before the cisplatin administration increases cellular platinum sensitivity both in vitro and in vivo. To our knowledge, this is the first report demonstrating that cupric ions (Cu2þ) may serve as inhibitors of platinum drug efflux via ATP7B. Our analysis was performed only in LMS cells, and this is the limitation of our study. Therefore, further studies are needed to confirm the effectiveness of this treatment in ATP7B-expressing cancer.

Conclusions Our study demonstrates that ATP7B is associated with platinum drug resistance. Moreover, we found that CuSO4, which acts as a preferential ATP7B substrate over platinum, is a potential therapeutic agent in the treatment of LMS. Further studies are required to investigate the efficacy of CuSO4 pretreatment in ATP7Bexpressing cancers and to refine its use in clinical settings. We expect that pretreatment with CuSO4 before the administration of platinum drugs will sensitize ATP7B-expressing cancers to these drugs. n Acknowledgments The supplemental information was provided in the Supplementary Materials and Methods file.

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All animal experiments were conducted in accordance with the Institutional Ethical Guidelines for Animal Experimentation at Osaka University (Osaka, Japan) and the Declaration of Helsinki. Their care was performed in accordance with institution guidelines at Osaka University. This study was approved by the Institutional Review Board and the Ethics Committee of the Osaka University Hospital (approval number 17217, approved on Sept. 29, 2017). The data set used and/or analyzed during the current study are available from the corresponding author on reasonable request. Authors’ contributions include the following: Mamoru Kakuda, Shinya Matsuzaki, Satoko Matsuzaki, Toshihiro Kimura, Masami Fujita, and Yutaka Ueda designed the study and wrote the manuscript. Mamoru Kakuda, Shinya Matsuzaki, Mayu Shiomi, Toshihiro Kimura, and Tomomi Egawa-Takata performed in vitro experiments. Mamoru Kakuda, Shinya Matsuzaki, and Toshihiro Kimura performed in vivo experiments. Eiji Kobayashi, Kiyoshi Yoshino, and Tomomi Egawa-Takata collected the human uterine leiomyosarcoma samples and performed immunohistochemistry experiments. Satoshi Serada and Tetsuji Naka provided antibodies and discussed the results. Mamoru Kakuda, Shinya Matsuzaki, and Mayu Shiomi performed Western blotting analysis of human parental cell lines. Mamoru Kakuda, Shinya Matsuzaki, and Tadashi Kimura discussed results. All authors reviewed and approved the final manuscript. The authors thank H. Abe and K. Sakiyama for administrative assistance in the preparation of this manuscript.

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22. Yang T, Chen M, Chen T, Thakur A. Expression of the copper transporters hCtr1, ATP7A and ATP7B is associated with the response to chemotherapy and survival time in patients with resected nonesmall cell lung cancer. Oncol Lett 2015;10:2584–90. 23. Martinez-Balibrea E, Martinez-Cardus A, Musulen E, et al. Increased levels of copper efflux transporter ATP7B are associated with poor outcome in colorectal cancer patients receiving oxaliplatin-based chemotherapy. Int J Cancer 2009;124:2905–10. 24. Mangala LS, Zuzel V, Schmandt R, et al. Therapeutic targeting of ATP7B in ovarian carcinoma. Clin Cancer Res 2009;15:3770–80. 25. Kuo MT, Fu S, Savaraj N, Chen HH. Role of the human high-affinity copper transporter in copper homeostasis regulation and cisplatin sensitivity in cancer chemotherapy. Cancer Res 2012;72:4616–21. 26. Kalayda GV, Wagner CH, Buss I, Reedijk J, Jaehde U. Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells. BMC Cancer 2008;8:175. 27. Ishida S, McCormick F, Smith-McCune K, Hanahan D. Enhancing tumor-specific uptake of the anticancer drug cisplatin with a copper chelator. Cancer Cell 2010;17:574–83. 28. Fu S, Hou MM, Wheler J, et al. Exploratory study of carboplatin plus the copper-lowering agent trientine in patients with advanced malignancies. Invest New Drugs 2014;32:465–72. 29. Fu S, Naing A, Fu C, Kuo MT, Kurzrock R. Overcoming platinum resistance through the use of a copper-lowering agent. Mol Cancer Ther 2012;11:1221–5. 30. Foster AW, Osman D, Robinson NJ. Metal preferences and metallation. J Biol Chem 2014;289:28095–103. 31. Hiramatsu K, Serada S, Enomoto T, et al. LSR Antibody therapy inhibits ovarian epithelial tumor growth by inhibiting lipid uptake. Cancer Res 2018;78:516–27. 32. Matsuzaki S, Serada S, Hiramatsu K, et al. Anti-glypican-1 antibody-drug conjugate exhibits potent preclinical antitumor activity against glypican-1 positive uterine cervical cancer. Int J Cancer 2018;142:1056–66. 33. Terada K, Schilsky ML, Miura N, Sugiyama T. ATP7B (WND) protein. Int J Biochem Cell Biol 1998;30:1063–7. 34. Li YQ, Chen J, Yin JY, Liu ZQ, Li XP. Gene expression and single nucleotide polymorphism of ATP7B are associated with platinum-based chemotherapy response in nonesmall cell lung cancer patients. J Cancer 2018;9:3532–9. 35. Katagiri H, Nakayama K, Rahman MT, et al. Is ATP7B a predictive marker in patients with ovarian carcinoma treated with platinum-taxane combination chemotherapy? Int J Gynecol Cancer 2013;23:60–4. 36. Komatsu M, Sumizawa T, Mutoh M, et al. Copper-transporting P-type adenosine

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triphosphatase (ATP7B) is associated with cisplatin resistance. Cancer Res 2000;60: 1312–6. 37. Kuo MT, Chen HH, Song IS, Savaraj N, Ishikawa T. The roles of copper transporters in cisplatin resistance. Cancer Metastasis Rev 2007;26:71–83. 38. Rose PG, Blessing JA, Soper JT, Barter JF. Prolonged oral etoposide in recurrent or advanced leiomyosarcoma of the uterus: a gynecologic oncology group study. Gynecol Oncol 1998;70:267–71. 39. Miller DS, Blessing JA, Kilgore LC, Mannel R, Van Le L. Phase II trial of topotecan in patients with advanced, persistent, or recurrent uterine leiomyosarcomas: a Gynecologic Oncology Group study. Am J Clin Oncol 2000;23:355–7. 40. Sutton G, Blessing JA, Ball H. Phase II trial of paclitaxel in leiomyosarcoma of the uterus: a gynecologic oncology group study. Gynecol Oncol 1999;74:346–9. 41. Cui RR, Wright JD, Hou JY. Uterine leiomyosarcoma: a review of recent advances in molecular biology, clinical management and outcome. BJOG 2017;124:1028–37. 42. Hensley ML, Patel SR, von Mehren M, et al. Efficacy and safety of trabectedin or dacarbazine in patients with advanced uterine leiomyosarcoma after failure of anthracycline-based chemotherapy: subgroup analysis of a phase 3, randomized clinical trial. Gynecol Oncol 2017;146:531–7. 43. van der Graaf WT, Blay JY, Chawla SP, et al. Pazopanib for metastatic soft-tissue sarcoma (PALETTE): a randomised, double-blind, placebo-controlled phase 3 trial. Lancet 2012;379: 1879–86. 44. Shiraishi N, Taguchi T, Kinebuchi H. Copper-induced toxicity in the macular mutant mouse: an animal model for Menkes’ kinky-hair disease. Toxicol Appl Pharmacol 1991;110: 89–96. 45. Copper and its inorganic compounds [MAK Value Documentation, 2006]. The MAKCollection for Occupational Health and Safety. p. 44-72. https://doi.org/10.1002/3527600418. mb744050e0022. 46. Ashby J, Tinwell H, Callander RD. Activity of urethane and N,N-dimethylurethane in the mouse bone-marrow micronucleus assay: equivalence of oral and intraperitoneal routes of exposure. Mutat Res 1990;245:227–30. 47. Roychoudhury S, Massanyi P, Bulla J, et al. In vitro copper toxicity on rabbit spermatozoa motility, morphology and cell membrane integrity. J Environ Sci Health A Tox Hazard Subst Environ Eng 2010;45: 1482–91. 48. Sweet DV, Fang J, Shiang D. Registry of Toxic Effects of Chemical Substances (RTECS), 1986 computer tape. Data files.; National Inst. for Occupational Safety and Health, Cincinnati, OH; 1986. Report No.: PB87-900300/XAB United States NTIS Subscription.

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49. Nair AB, Jacob S. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm 2016;7:27–31.

Author and article information From the Department of Obstetrics and Gynecology, Osaka University, Osaka (Drs Kakuda, Sh. Matsuzaki, Ueda, Shiomi, Fujita, Kobayashi, Yoshino, and Ta. Kimura); the Department of Obstetrics and Gynecology, Sakai Medical Center, Osaka (Dr Kakuda); the Department of Gynecology, Otemae Hospital, Osaka (Dr Sa. Matsuzaki); the

Department of Gynecology, Osaka International Cancer Institute, Osaka (Dr To. Kimura); the Department of Obstetrics and Gynecology, Osaka Keisatsu Hospital, Osaka (Dr Egawa-Takata); the Center for Intractable Immune Disease, Kochi Medical School, Kochi University, Kochi (Drs Serada and Naka); and the Department of Obstetrics and Gynecology, University of Occupational and Environmental Health, Fukuoka (Dr Yoshino), Japan. 1 These authors contributed equally to this work. Received April 28, 2019; revised July 12, 2019; accepted July 18, 2019.

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ajog.org This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (17K11278 to Shinya Matsuzaki, 16K11139 to Toshihiro Kimura, and 16K11137 to Masami Fujita) and Japan Society of Gynecologic Oncology (no number, to Shinya Matsuzaki). The authors report no conflict of interest. Corresponding author: Shinya Matsuzaki, MD. [email protected]; or Yutaka Ueda, MD. [email protected]

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Supplemental Materials and Methods Western blotting

used to categorize clinical staging. Patient characteristics are shown in Table 1.

Cells and clinical samples were lysed in radioimmunoprecipitation assay buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% protease-inhibitor cocktail [Nacalai Tesque] and 1% phosphataseinhibitor cocktail [Nacalai Tesque]). After centrifugation (13,200 rpm, 4 C, 15 minutes), soluble proteins in the supernatant were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes treated with chicken polyclonal anti-ATP7A antibody (ab13995; Abcam) as in our previous study17 or with rabbit monoclonal anti-ATP7B antibody (SAB1403592; Sigma-Aldrich). Negative expression of ATP7A was confirmed in an ATP7A-silenced endometrial carcinoma cell line in our previous study.17 Subsequently, the Western blots were incubated with a 1:5000 dilution of HRP-conjugated goat antichicken IgG (Amersham Biosciences UK, Buckinghamshire, United Kingdom) or a 1:5000 dilution of donkey antirabbit HRP-conjugated secondary antibodies (GE Healthcare Bio-Sciences, Piscataway, NJ) to determine ATP7A and ATP7B expression levels. For a loading control, the Western blots were reprobed with an antibody against beta-actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Immunohistochemistry

Patients Informed consent was obtained from all donors, and all studies involving human subjects were approved by the Institutional Review Boards No. 17217 of Osaka University Hospital. Board-certified pathologists confirmed the diagnoses of all tumors as leiomyosarcoma following histological review. TNM 7th edition (Union for International Cancer Control) criteria were used to categorize pathological staging and International Federation of Gynecology and Obstetrics staging of cervical cancer (2008) were

Surgically resected LMS tissues were obtained from patients with uterine leiomyosarcoma. Patients characteristics are shown in Table 1. Sections were prepared from formalin-fixed, paraffinembedded tissue specimens, deparaffinized, and rehydrated in graded alcohols. Immunohistochemical staining for ATP7B was performed using the ABC method with a rabbit monoclonal anti-ATP7B antibody (SAB1403592; Sigma-Aldrich) and the Vectastain ABC kit (PK-4001; Vector Laboratories) according to the manufacturer’s protocol. Images of immunostained sections were obtained using an Olympus FSX100 (Tokyo, Japan). The immunostained images were scored according to the intensity of staining as follows: 0, no staining; 1, weak staining; and 2, strong staining. The density of staining was scored as follows: 0, 0e9% positivity; 1, 10e40% positivity; 2, 41e70% positivity; and 3, 71e100% positivity. The final IHC score was determined by multiplying the intensity score (0, 1, 2) with the density score (0, 1, 2, or 3), resulting in a maximum score of 6. Three independent gynecological oncologists (Y.U., K.Y., and S.M.) who were blinded to the histological data analyzed the stained sections using an Olympus BH2 microscope. In cases of disagreement, the staining results were reevaluated by careful discussion until a consensus was reached. Staining of placental tissues was used as the positive control of ATP7B because they are reported to show strong expression of ATP7B.33 Uterine myometrium was used as a negative control because no staining was observed.

Effect of pretreatment with CuSO4 on platinum sensitivity in vitro Cells were suspended in DMEM supplemented with 10% FBS and were seeded in 96-well plates (1000 cells per well; Costar, Corning) for 24 hours. They were then exposed to various concentrations of CuSO4 (0e200 mM) for 72 hours. Cell proliferation was

Original Research

evaluated using the WST-8 assay (Cell Counting Kit-SF; Nacalai Tesque) after treatment at the time points indicated by the manufacturer. The absorption of WST-8 was measured at a wavelength of 450 nm (reference wavelength, 630 nm) using a Model 680 Microplate Reader (Bio-Rad Laboratories). Absorbance values for treated cells indicative of proliferation rates were expressed as percentages relative to values for untreated controls, and the drug concentrations resulting in a 50% inhibition of cell growth (IC50 values) were calculated.

Quantification of intracellular platinum accumulation with pretreated CuSO4 To investigate the effect of pretreatment with CuSO4, 6 treatment groups were established: SK-LMS-1, SK-LMS-1 with CuSO4 pretreatment, SK-LMS-CV, SK-LMS-CV with CuSO4 pretreatment, SK-LMS-ATP7B-7B-21, and SK-LMSATP7B-7B-33. Cells (4  106) from each group were seeded into 2 150 mm tissue culture dishes and incubated for 24 hours. One group of SK-LMS-1 and SK-LMS-CV cells were each pretreated with 15 mM CuSO4 3 hours before cisplatin administration. All groups were then exposed to 100 mM cisplatin for 60 minutes at 37 C, washed twice with PBS, and incubated in cisplatin-free DMEM supplemented with 10% FBS. Three hours following this, whole extracts were prepared and cisplatin accumulation in cells was analyzed as described in main text.

Immunofluorescence for ATP7B Immunofluorescence staining was performed 2 days after cells were seeded on coverslips (3000 cells/well). Before staining, cells in the treatment groups were pretreated with 10 mM cisplatin or 50 mM CuSO4 for 3 hours. Cells were then analyzed for the localization of ATP7B. After rinsing with PBS, cells were fixed with 4% formaldehyde in PBS for 10 minutes at room temperature. After fixation, cells were washed with PBS and permeabilized with 0.01% Triton X-100 in PBS for 2 minutes. Next, cells were then rinsed with PBS, blocked for 1 hour

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in Blocking One (Nacalai Tesque) and incubated overnight at 4 C with the primary antibody for ATP7B (ab124973, Abcam, Cambridge, MA). Subsequently, cells were washed with PBS and incubated for 1 hour at 37 C with the secondary antibody. For ATP7B, a 1:100 dilution of Alexa Fluor 647-donkey antirabbit IgG (711-606-152; Jackson ImmunoResearch Laboratories, West Grove, PA) was used as a secondary antibody. Antibody solutions were diluted in Can Get Signal Solution A (NKB-501; TOYOBO, Osaka, Japan). After the final washing, coverslips were mounted in Vecta Shield with DAPI (VECTOR Laboratories). Fluorescence images were obtained using a fluorescence microscope (model BZ-9000; Keyence, Osaka, Japan). Merged fluorescence images were generated with the software. Fluorescence signal from Alexa Fluor 647 is represented in green.

In vivo model of cisplatin resistance after pretreatment with CuSO4 All animal experiments were conducted in accordance with the Institutional Ethical Guidelines for Animal Experi-

mentation of our Osaka University (Osaka, Japan). Four week old female ICR nu/nu mice were obtained from Charles River Japan (Yokohama, Japan). For subcutaneous xenograft experiments, 2.5  106 SK-LMS-1 cells were suspended in 100 mL of PBS and injected subcutaneously into the backs of the ICR nu/nu mice (n ¼ 5 per group). At 10 days after xenograft establishment, mice were randomly divided into 6 groups (groups 1e6). In group 1, xenograft mice were administered cisplatin (3 mg/kg) i.p., without premedication twice weekly for 4 weeks. In group 2, xenograft mice were administered PBS i.p. without premedication twice weekly for 4 weeks. In group 3, xenograft mice were administered cisplatin (3 mg/kg) i.p. 3 hours after the administration of CuSO4 (0.25 mg/kg) i.p. twice weekly for 4 weeks. In group 4, xenograft mice were administered PBS i.p. 3 hours after the administration of CuSO4 (0.25 mg/kg) i.p. twice weekly for 4 weeks. In group 5, xenograft mice were administered cisplatin (3 mg/kg) i.p. 3 hours after the administration of CuSO4 (1 mg/kg) i.p. twice weekly for 4 weeks. In group 6, xenograft mice were administered PBS

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ajog.org i.p. 3 hours after the administration of CuSO4 (1 mg/kg) i.p. twice weekly for 4 weeks. Groups 1 and 2 were labeled no premedication groups, group 3 and 4 were labeled low-dose groups, and groups 5 and 6 were labeled high-dose groups. Tumor volumes were determined twice weekly according to the following formula: tumor volume (cubic millimeters) ¼ (width2  length)/2. Forty-nine days after tumor implantation, mice were killed and tumors were removed and weighed. The body weight of mice were recorded, blood samples were taken from the heart, and hemoglobin was measured using VetScan HMII (Abaxis, Union City, CA). ABC, avidin-biotin-peroxidase complex; ATP7A, adenosine triphosphatase copper transporting alpha; ATP7B, adenosine triphosphatase copper transporting beta; CuSO4, copper sulfate; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HRP, horseradish peroxidase; IHC, immunohistochemistry; i.p., intraperitoneally; LMS, leiomyosarcoma. Kakuda. Novel CuSO4 therapy for leiomyosarcoma. Am J Obstet Gynecol 2019.