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 beta–expressing 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 beta–suppressed cell line (SK-LMS-7B cells) was generated, 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 μM; SK-LMS-7B cells, half-maximal inhibitory concentration, 4.2 μM, 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 μM 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.
Uterine leiomyosarcoma (LMS) arises from uterine smooth muscles and is a relatively rare tumor, constituting only 2–3% of all uterine malignancies. 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. Five year survival estimates as per the International Federation of Gynecology and Obstetrics stage are 76%, 60%, 45%, and 29% for stages I–IV, respectively.
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 (CuSO 4 ) 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 CuSO 4 pretreatment before cisplatin administration.
What does this study add to what is already known?
We demonstrated that CuSO 4 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.
The first treatment should be surgical management. However, the recurrence rate of LMS has been reported as approximately 60–70%, with poor prognosis despite complete surgical resection. 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.
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%. To improve the survival rate of LMS, addition of trabectedin to doxorubicin, a phase III trabectedin trial, and a phase II pazopanib trial have been reported. 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.
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%. However, their efficacy is only 3% in LMS, which is frequently resistant to platinum drugs, leading to poor prognosis. 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. These studies demonstrated that Annexin A4 and ATP7A are therapeutic targets in platinum-resistant cancers, as are various other platinum transporters.
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.
In particular, previous reports have shown that ATP7B and CTR1 are strongly associated with platinum resistance. 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.
Previous studies have reported that CTR1 is a high-affinity copper (Cu)-uptake transporter and a major uptake transporter of platinum drugs. Cu 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 al treated 5 patients showing platinum-resistant tumors, with Cu-lowering 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 = 13], non–small-cell lung [n = 10], and epithelial ovarian [n = 8]) cancer that had failed to respond to standard treatments (45 had failed to respond to platinum drugs). 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 Cu 2+ because Cu 2+ is considered an outlier in the Irving-Williams series, suggesting that it has a strong competitive edge over cisplatin for the same binding site ; thus, copper sulfate (CuSO 4 ) may favor intracellular platinum accumulation.
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 μg/mL streptomycin (Nacalai Tesque, Kyoto, Japan) at 37°C under a humidified atmosphere of 5% CO 2 . SKN cells were maintained in Ham’s F12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/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 μg/mL streptomycin.
Western blotting
Cells were prepared, as described previously. Proteins were transferred to polyvinylidene difluoride membranes treated with chicken polyclonal anti-ATP7A 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 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.
Immunohistochemical staining for ATP7B was performed using the avidin-biotin-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-LMS-C, 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 (IC 50 ) 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 (0–50 μM) 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 proliferation rates were expressed as percentages relative to values for untreated controls, and the drug concentrations resulting in a 50% inhibition of cell growth (IC 50 values) were calculated.
Quantification of intracellular platinum accumulation
Cisplatin accumulation in cells was analyzed according to a previously established method, with minor modifications. In brief, 4 × 10 6 cells (SK-LMS-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 μM 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). Transfected cells were selected with 0.5 μg/mL puromycin (Invitrogen). Clones were maintained in 0.3 μg/mL puromycin to obtain stable suppression. Two stable ATP7B-suppressing cell lines were established and designated SK-LMS-ATP7B-7B-21, and SK-LMS-ATP7B-33. A control cell line, SK-LMS-1, 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 obtained from Charles River Japan (Yokohama, Japan). For subcutaneous xenograft experiments, 2.5 × 10 6 SK-LMS-1, SK-LMS-CV, SK-LMS-7B21, and SK-LMS-7B33 cells were suspended in 100 μL of PBS and injected subcutaneously into the backs of the ICR nu/nu mice (n = 5 per group).
Ten days after xenograft establishment, tumors measured 100 mm 3 . 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 × length)/2. Forty-nine days after tumor implantation, mice were killed and tumors were removed and weighed.
Effect of pretreatment with CuSO 4 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 CuSO 4 before the administration of cisplatin (451657; Sigma-Aldrich). In particular, 15 μM CuSO 4 was administrated and cisplatin was added at various concentrations after 3 hours of CuSO 4 treatment. Cells were then exposed to various concentrations of cisplatin (0–200 μM) for 72 hours, and the IC 50 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 CuSO 4
To investigate the effect of pretreatment with CuSO 4 , 6 treatment groups were established: SK-LMS-1, SK-LMS-1 with CuSO 4 before treatment, SK-LMS-CV, SK-LMS-CV with CuSO 4 before treatment, SK-LMS-ATP7B-7B-21, and SK-LMS-ATP7B-7B-33. Cells (4 × 10 6 ) from each group were investigated for the intracellular platinum accumulation. Additional information can be found in the Supporting Information on Material and Methods.
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 μM cisplatin or 50 μM CuSO 4 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 CuSO 4
For subcutaneous xenograft experiments, 2.5 × 10 6 SK-LMS-1 cells were suspended in 100 μL of PBS and injected subcutaneously into the backs of the ICR nu/nu mice (n = 5 per group). At 10 days after xenograft establishment, the mice were randomly divided into 6 groups (groups 1–6).
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 CuSO 4 (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 CuSO 4 (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 CuSO 4 (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 CuSO 4 (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 RMG-I) 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, SK-UT1, A2780, SKOV3, and OVISE cells ( Figure 1 A).
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 1 B). Cisplatin IC 50 values were also determined for both control and knockdown cell lines. The cisplatin IC 50 values in 2 types of ATP7A-silenced SK-LMS-1 cells (SK-LMS-si7A2, IC 50 = 11.3 μM, P = .35; SK-LMS-si7A3, IC 50 = 11.7 μM, P = 0.30) were not significantly different compared with control cells (IC 50 = 16.0 μM). However, cisplatin IC 50 values in 2 types of ATP7B-silenced SK-LMS-1 cells (SK-LMS-si7B1, IC 50 = 4.3 μM, P < .01; SK-LMS-si7B2, IC 50 = 4.4 μM, P < .01) were significantly lower than the controls (IC 50 = 16.8 μM; Figure 1 C).
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-LMS-ATP7B-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 1 D). 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
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 2 A. Among the 14 specimens, 8 cases (57.2%) scored more than 4 points (high group), 3 cases (21.4%) scored 1–3 points (low group), and 3 cases (21.4 %) scored 0-1 points (negative group).
| Variables | Leiomyosarcoma |
|---|---|
| Number of cases | 14 |
| Age, median (range) | 51 (38–60) |
| FIGO stage | |
| I | 7 |
| II | 2 |
| III | 5 |
| IV | 0 |
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