www.impactjournals.com/oncotarget/ Oncotarget, 2018, Vol. 9, (No. 1), pp: 743-754

Research Paper Organic cation transporter 3 mediates cisplatin and copper cross-resistance in hepatoma cells

Sarah Guttmann1,*, Gursimran Chandhok1,2,*, Sara Reinartz Groba1, Christoph Niemietz1, Vanessa Sauer1, Amanda Gomes1,3, Giuliano Ciarimboli4, Uwe Karst5, Andree Zibert1 and Hartmut H. Schmidt1 1Medizinische Klinik B für Gastroenterologie und Hepatologie, Universitätsklinikum Münster, Münster, Germany 2Present address: Monash Biomedicine Discovery Institute, and Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia 3Present address: Wilson Disease Clinic, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, India 4Universitätsklinikum Münster, Medizinische Klinik D, Experimentelle Nephrologie, Münster, Germany 5Institute of Inorganic and Analytical Chemistry, University of Münster, Münster, Germany *These authors contributed equally to this work Correspondence to: Hartmut H. Schmidt, email: [email protected] Keywords: OCT3; ATP7B; MT1; cisplatin; copper cross-resistance Received: June 30, 2017 Accepted: November 15, 2017 Published: December 12, 2017 Copyright: Guttmann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

ABSTRACT

Platinum-based drugs are first-line compounds in the treatment of many solid cancers. Major obstacles are tumors that become resistant and toxic side effects, both largely due to the expression of transporters that mediate the cellular processing of platinum. In this study, we addressed the establishment of cisplatin resistance in the absence of copper transporter ATP7B that has been previously found to be overexpressed in various resistant cells. Cisplatin sensitivity, induction of apoptosis, drug accumulation, and transporter gene expression were determined in hepatoma cell lines. Knockout or overexpression of copper transporter ATP7B did not affect cisplatin sensitivity. Cisplatin resistant cells showed a stably reduced cisplatin accumulation and a downregulation of organic cation transporter 3 (OCT3). In contrast, OCT3 overexpression could reverse resistance. Reduced MT1 expression was detected in the resistant cell line, however transient and highly dependent on the presence of cisplatin. Cross-resistance to copper was also associated with OCT3 downregulation. Our results suggest that a decreased level of OCT3 expression results in resistance to cisplatin and copper. OCT3 may represent a novel target for improved prognosis and anticancer therapy, including HCC.

INTRODUCTION bases of DNA, mostly at N7 positions of guanosine and adenosine. Cross-links between two adjacent guanines as For decades, cisplatin (Cp), a platinum-based well as 1,2-d(GpG) interstrand cross-links represent major anticancer agent, is widely used in the clinic for therapy of lesions observed in the DNA after Cp treatment. Besides many solid tumors, including lung, ovary, testes, prostate, this genotoxicity, Cp results in a significant damage of breast, colorectal, and liver [1, 2]. Cisplatin has also been cytoplasm targets, prominently proteins. About half of the suggested to reduce the recurrence rate after curative patients respond well to platinum-based therapy, however surgical resection of hepatocellular carcinoma (HCC), intrinsic or acquired resistance to Cp is a major obstacle one of the most common cancers in the world [3]. Once [4]. The mechanisms of resistance have been classified into within the cell, the activated platinum reacts with purine several functional and hierarchical categories: (i) pre-target, www.impactjournals.com/oncotarget 743 Oncotarget (ii) on-target, (iii) post-target, and (iv) off-target. Pre-target RESULTS resistance covers the modification of target binding in the cytoplasma or to DNA, e.g. via decreased influx and/or ATP7B does not affect cisplatin sensitivity in increased efflux of the drug, whereas on-target resistance hepatoma cells reduces the molecular damage, e.g. by aggravated DNA repair. Post-target resistance includes an adaptive response Copper transporter ATP7B was reported to be of common signaling pathways, e.g. for a reduced rate of involved in the establishment of cisplatin (Cp) resistance regulated cell death, whereas modifications not directly in various cells [14–16]. In order to re-evaluate the impact associated to the chemotherapeutic agent are summarized of ATP7B for establishment of Cp resistance, a recently as off-targets [4]. Most resistance described in the literature generated human hepatoma HepG2 cell line lacking falls into the category of pre-targets resulting in reduced functional ATP7B (KO) was compared to parental cell platinum accumulation in tumor cells [1]. line expressing high levels of ATP7B. The ATP7B KO The cellular uptake of anticancer drugs, their tissue cell line was shown to lack expression of ATP7B protein distribution, toxicity, and the efficacy to erase tumor cells following zinc-finger induced mutagenesis ofATP7B [20]. largely depend on the expression and activity of transporters No significant differences with regard to cell survival [5, 6]. This group of carriers include the multidrug and toxin following Cp exposure were observed between the two extrusion transporters (MATEs; SLC47A), organic cation/ cell lines (Figure 1A). Variation of the time period of Cp carnitine transporters (OCTNs; SLC22A4 and 5), and exposure (5 minutes to 72 h) or Cp concentration (up to organic cation transporters (OCTs; SLC22A1-3). For OCTs, 200 µM) did not result in a different Cp sensitivity (data three different forms, OCT1, OCT2 and OCT3, have been not shown). In order to assess any differences in the characterized [6]. Most of the transporters display a high accumulation of the drug, intracellular Cp concentrations tissue and subcellular-specific expression translating into were determined in parental and ATP7B KO cells tumor-specific efficacy and cell type-specific side effects. (Figure 1B). The soluble cellular fraction of both cell lines Cisplatin is an excellent OCT2 substrate; however, it is a displayed almost identical levels of Cp suggesting that Cp poor substrate of either MATE1 or MATE2-K [7, 8]. Of uptake/storage was not altered by the KO of ATP7B. As note, the processing pathway of platinum drugs is shared ATP7B overexpression was implicated to confer resistance by the intracellular routes of metals, in particular copper, [14], the question was addressed whether retroviral vectors as exemplified by the family of copper transporters overexpressing ATP7B can confer improved Cp resistance (CTRs; SLC31A). CTR1, the principal copper importer of in hepatoma cell lines. However, overexpression of ATP7B mammalian cells, was reported to mediate the majority of in HepG2 and Huh-7 cells did not result in an increased platinum intake [9, 10]. Metallothionein 1 (MT1) located Cp resistance (Supplementary Figure 1). In contrast, both in the cytoplasm was proposed to bind cp resulting in a transduced cell lines displayed an increased resistance non-toxic storage [11]. In addition, increased expression of to copper suggesting that overexpression gives rise to copper transporting P-type adenosine triphosphatase ATP7A functional ATP7B. and ATP7B have been reported to be involved in platinum drug transport [12, 13]. In line, overexpression of ATP7B Hepatoma cells lacking ATP7B can achieve has been suggested to be of importance for establishment cisplatin resistance of Cp resistance in various tumors derived e.g. from ovary, oral squamous cell carcinoma and prostate [14–17]. Given Having shown that ATP7B expression does not the significant overlap between platinum and copper modulate Cp sensitivity and accumulation in hepatoma pathways, combination therapy involving platinum and cells, the question was addressed which other genes may copper chelator has been recently proposed [18]. result in an adaptation to toxic Cp concentrations. First, In terms of carrier expression that mediate the survival of ATP7B KO cells was determined following intracellular Cp transport, hepatocytes are of high interest, long-term Cp exposure. Exposure to 1.0 µM and 5.0 µM since transport of copper is one of the primary function Cp resulted in cell death after 7–21 days, while 0.1 µM of this well characterized cell type [19]. In this report, Cp did not disturb cell proliferation for more than we took advantage of a human hepatoma cell line that 23 days (Supplementary Table 1). To adapt the cells to carries a knockout (KO) of ATP7B [20]. We re-addressed toxic Cp concentrations, the cisplatin concentration was the role of ATP7B in hepatocytes [21] and characterized stepwise increased by 0.1 µM at a weekly basis. Using transporter expression after establishment of Cp resistant this protocol over a time period of several months, a Cp hepatoma cells. Our findings significantly increase the resistant cell line (CpR) was established that showed cell knowledge on the molecular role of individual transporters proliferation despite being continuously grown in high for the efficacy of platinum therapy, particularly ATP7B, Cp concentrations. Cp concentrations of up to 4 µM were MT1 and OCT3, and shed new light on the copper cross- well tolerated. CpR cells could be grown in the presence activity that may have significant clinical impact for the of high Cp for more than a year without evident changes development of improved platinum-based therapy. in cell morphology (Figure 2A). The morphology of CpR www.impactjournals.com/oncotarget 744 Oncotarget cells was similar to parental cell line ATP7B KO and copper homeostasis (ATP7A, CP, CTR1 and 2, GST, MT1) HepG2 cells (Supplementary Figure 2). The cumulative (Supplementary Table 2). RT-qPCR analysis showed that growth of CpR cells indicated similar proliferation rates the expression level of most genes was within a threshold as compared to untreated ATP7B KO cells (Figure 2B). of ±2 fold that was chosen to indicate similar expression Annexin V staining was used to characterize the induction as compared to ATP7B KO cells (Figure 3A). However, of apoptosis in CpR cells. Experiments were carried out the expression of organic cation transporter 3 (OCT3) using 10 µM Cp for 72 h, since extensive necrosis was was significantly downregulated (5.17 ± 2 fold), while observed at higher Cp concentrations (data not shown). metallothionein 1 (MT1) was significantly upregulated Induction of apoptosis was significantly reduced in the (8.91 ± 4 fold). For further validation, we assessed basal CpR cells as compared to ATP7B KO cells (Figure 2C). expression of OCT3 and MT1 in hepatoma cell line We next assessed the intracellular Cp concentration in the Huh-7 and primary human hepatocytes. Basal OCT3 nuclear and soluble fractions of CpR cells (Figure 2D). expression was almost identical in the samples, while MT1 While the nuclear fractions showed no differences of Cp expression was highly elevated in human hepatocytes accumulation, a significantly decreased level was observed relative to the two hepatoma cell lines (not shown). Next, in the soluble fraction of CpR cells as compared to ATP7B we determined whether the gene expression of OCT3 KO cells, corroborating that Cp is targeting cytoplasmic and MT1 in CpR cells is permanently affected even after compartments [4, 22]. drug removal. CpR cells were regrown in basal media for

at least one month (weaning) giving rise to CpRw cells.

Organic cation transporter 3 is stably Analysis of the cell viability in CpRw cells revealed an downregulated in cisplatin resistant hepatoma almost identical high level of resistance as compared to cells lacking ATP7B CpR cells suggesting that the acquired resistance is a stable modification (Figure 3B). Gene expression analysis

In order to address the molecular determinants showed that MT1 expression was normalized in CpRw cells governing the reduced Cp accumulation in CpR cells, to a level observed in the non-resistant ATP7B KO cells a RT-qPCR analysis was performed. A set of 16 genes (Figure 3C). However, OCT3 gene expression remained

was chosen from the literature that covers some of the downregulated in CpRw cells suggesting that expression is major transporters involved in transport of metals and/or stably compromised. copper homeostasis. The selected genes were implicated To further assess the adaptive role of MT1 and OCT3 to be involved in metal transport (DMT1, MATE1 and 2, gene expression for the establishment of Cp resistance, MDR1, MRP1 and 2, OCT1-3, OCTN1 and 2) and/or ATP7B KO cells were exposed to Cp for a short time

Figure 1: ATP7B expression does not affect cisplatin sensitivity in hepatoma cells. (A) Cell viability was determined by MTT assay relative to untreated cells (100%). Mean/SE are given (n = 5). (B) Intracellular cisplatin level was determined by TXRF in the soluble cellular fractions of the cells. Cells were incubated with cisplatin for 4 h. Mean/SE are given (n = 3). www.impactjournals.com/oncotarget 745 Oncotarget period (72 h) and subjected to RT-qPCR analysis. MT1 was Overexpression of OCT3 can reverse cisplatin significantly upregulated when toxic Cp concentrations of resistance 1.0 µM and 5.0 µM were used indicating that induction of MT1 represents a transient adaptive response to toxic Cp In order to further define the role of OCT3 for levels (Supplementary Figure 3A). In contrast, expression establishment of Cp resistance, the question was addressed of OCT3 was not affected corroborating that OCT3 whether overexpression of OCT3 in CpR cells can revert downregulation has been acquired during long-term Cp resistance. CpR cells were stably transfected with a vector

exposure of CpR cells. Of note, knockdown of MT1 by expressing OCT3 resulting in CpROCT3 cells. OCT3 mRNA

siRNA revealed no significant difference of Cp resistance was increased in CpROCT3 cells as compared to CpR cells suggesting that MT1 has a minor role for the establishment by a factor of ≈20 fold (Figure 4A). Using Western blot of Cp resistance (Supplementary Figure 3B and 3C). Also, analysis, OCT3 protein expression was found to be increased downregulation of copper and cisplatin transporter CTR1 to a lesser extent (≈2–3 fold) in the stably transfected cells did not affect Cp resistance (Supplementary Figure 4). (Figure 4B; Supplementary Figure 5). To assess the function

Figure 2: Human hepatoma cells lacking ATP7B can adapt to proliferation in toxic cisplatin. (A) Light microscopic image of CpR cells after continuous growth in toxic cisplatin concentration for more than a year. Cell morphology was identical to parental cells. Scale bar, 50 µm. (B) Cumulative growth of CpR cells in the presence (+Cp) and absence (-Cp) of cisplatin. Growth of parental ATP7B KO cells is depicted for comparison. Mean/SE are given (n = 3). (C) Determination of apoptosis by Annexin V staining in CpR cells and ATP7B KO cells. Mean/SE are given (n = 3). (D) Intracellular cisplatin concentration was determined in nuclear and soluble fractions of CpR and ATP7B KO cells. Mean/SE are given (n = 3). *P < 0.05. www.impactjournals.com/oncotarget 746 Oncotarget of OCT3, transport of 4-(4-Dimethylamino)styryl)-N- resistance in CpR cells. Following copper exposure, Methylpyridinium Iodide (ASP+), a well-known substrate of CpR cells displayed a significant higher cell viability OCT3 [23], was determined. OCT3 transport was increased as compared to ATP7B KO cells (Figure 5A). OCT3

in stably transfected CpROCT3 cells (Figure 4C). Importantly, overexpression increased copper induced toxicity to overexpression of OCT3 resulted in almost same levels of the level observed in ATP7B KO cells. In contrast, Cp sensitivity as observed in ATP7B KO cells (Figure 4D) OCT3 downregulation in ATP7B KO cells via siRNA suggesting that OCT3 is sufficient to revert Cp resistance (Supplementary Figure 6) was followed by an increased of CpR cells. copper resistance relative to vehicle treated cells (Figure 5B). To more directly assess the role of OCT3 for the OCT3 confers copper cross-resistance in transport of copper, the intracellular copper concentration hepatoma cells was determined. A significantly reduced intracellular copper concentration was found after OCT3 siRNA Cisplatin resistant cells were shown to also resist knockdown in ATP7B KO cells (Figure 5C) suggesting toxic copper by a phenomenon described as cross- that, in addition to mediate the uptake of Cp, OCT3 may resistance [24]. We addressed the question of cross- also support the uptake of copper.

Figure 3: Cisplatin resistance is a stable modification of hepatoma cells. (A) mRNA expression of CpR cells relative to ATP7B KO cells was calculated using the 2–ΔΔ method. Dotted line at a fold change of ±2 indicates significance. Mean/SE are given (n = 3). (B) Cell viability as determined by MTT assay relative to untreated cells (100%). Mean/SE are given (n = 5). *P < 0.05. (C) mRNA expression of CpRw cells is presented as fold change relative to ATP7B KO cells. Mean/SE are given (n = 3). www.impactjournals.com/oncotarget 747 Oncotarget DISCUSSION of the efficacy of Cp treatment. (i) ATP7B does not have an impact on Cp-induced toxicity, at least for hepatoma Growing evidence suggests that the efficacy of cells which represent the primary physiological expression platinum-based chemotherapy is closely related to the site of this transporter. Of note, the cells used in this study activity of transporters that mediate the intracellular allowed an analysis of Cp sensitivity in the absence of processing of the drugs both at the level of the targeted the second human P-type ATPAse as hepatocytes do not tumor cell for efficient induction of cytotoxicity as well express ATP7A [12]. (ii) MT1, ubiquitously expressed in as in non-tumor tissues for the reduction of unwanted side mammalian cells, was shown here to be highly induced effects. As primary hepatocytes do not show long-lasting by cisplatin, however induction can be transient and proliferation in tissue culture, we used most frequently normalizes after withdrawal of the drug. (iii) Expression employed human hepatoma cell lines. Our results shed level of OCT3 seems to be directly linked to cellular Cp new light on several major transporters and intracellular accumulation; conversely, its downregulation results in an carriers that have been implicated to be key determinants increased Cp resistance. Interestingly, OCT3 expression

Figure 4: OCT3 mediates cisplatin resistance in hepatoma cells. (A) mRNA expression in CpROCT3 cells and CpR cells. Mean/ SE are given (n = 3). (B) Densitometric analysis of OCT3 protein expression as determined by Western blot. ATP7B KO cells were set to 100%. Mean/SE are given (n = 3). (C) OCT3 function was assessed by ASP+ uptake rates. Mean/SE are given (n = 3). (D) Cell viability was determined by MTT assay relative to untreated cells (100%). Mean/SE are given (n = 5). *P < 0.05. www.impactjournals.com/oncotarget 748 Oncotarget is relatively low in human hepatocytes and hepatoma cell does not actively expel significant amounts of Cp from the lines as compared to OCT1, while OCT2, the previously cell endorsing the previous proposal of an ATP7B-linked reported cisplatin transporter of kidney, is missing in these detoxification mechanism other than export [21]. However, cells [25]. OCT3 rather than OCT1 may therefore represent this conclusion is challenged by other reports where ATP7B the major target for establishment of Cp resistance when expression in non-hepatic cells resulted in decreased OCT2 expression is missing. (iv) OCT3 also contributed intracellular Cp levels [14]. In line, confocal microscopic to intracellular copper accumulation further corroborating analysis of ovarian cell lines expressing ATP7B suggested the clinical impact of the two intermingled transport Cp transport into vesicles [15]. Our observations therefore networks for efficient platinum-based therapy. do not exclude that elevated expression of ATP7B in cells ATP7B expression is mostly limited to hepatocytes other than hepatocytes may have clinical significance for [19]. However, induction of ATP7B has been suggested to the course of platinum drug therapy [28–30]. However, be associated to resistance [12, 13]. In order to explore an such mechanisms may involve molecular events unrelated establishment of Cp resistance in the absence of ATP7B, to export. we used a previously generated hepatoma KO cell line MT1 has been proposed to be capable of inactivating [20]. Expression of ATP7B in primary rat hepatocytes cisplatin [11]. MT1 but not GST, a second major thiol-rich was associated with increased Cp sensitivity as compared protein of cells, was identified here to be highly induced to Atp7b KO cells [21]. The previous finding indicates by Cp. Importantly, MT1 upregulation by Cp is transient that ATP7B may have a different role in hepatocytes as and its expression seems to be related to the immediate compared to other cells, including ovarian carcinoma cells presence of the drug. Our results suggest that MT1 and primary MNK fibroblasts, where overexpression of the induction may be a primary defense mechanism of the cell transporter was shown to rescue from Cp toxicity [14, 16, to cope with exposition to a metal-based drug. 26, 27]. However, primary hepatocytes rapidly undergo Molecular characterization of our cells indicates apoptosis and expression of ATP7B in non-hepatic cells may that OCT3 represents the dominant transporter for the not resemble the physiological function of the transporter. establishment of Cp resistance. Whereas it has been Our results that were derived in isogenic sublines of a previously demonstrated that expression of OCT1 and human hepatoma cell line, either after native expression, OCT2 was followed by intracellular accumulation of gene knockout or overexpression, suggest that ATP7B platinum drugs [31–33], analysis of OCT3 gave ambiguous does not affect Cp sensitivity. Our measurements presented results [34–36]. Studies involving resistant cell lines derived almost identical intracellular levels of Cp regardless whether from cervical cancer and oral squamous cell carcinoma ATP7B was expressed or not, suggesting that the transporter slowed downregulation of OCT3, corroborating our results

Figure 5: OCT3 confers cross-resistance. (A) Cell viability was determined by MTT assay relative to untreated cells (100%). Cells were exposed to 0.75 mM copper for 48 h. HepG2 cells expressing ATP7B are shown as control. Mean/SE are given (n = 3). (B) ATP7B KO cells were treated with siRNA directed against OCT3 and cell viability was assessed by MTT assay. Mean/SE are given (n = 3). (C) Intracellular copper concentration as determined by AAS. ATP7B KO cells served for comparison (100%). Mean/SE are given (n = 3). *P < 0.05. www.impactjournals.com/oncotarget 749 Oncotarget that this transporter is a major contributor for the generation Firstly, downregulation of OCT3 in parental cells of resistance after platinum drug treatment [37, 38]. resulted in increased copper resistance, whereas OCT3 Notably, OCT3 overexpression could almost fully reverse overexpression in Cp resistant cells was followed by a resistance in our cells suggesting that downregulation of higher sensitivity towards copper. Secondly, analysis of OCT3 is a single molecular event for establishment of the cellular copper accumulation suggested a significantly resistant cells. Other genes, e.g. DMT1 and CTR1, were reduced copper uptake after knockdown of OCT3. To only moderately (≤2 fold) modulated, however such gene our knowledge this is the first report on copper being a expression might contribute to resistance. For CTR1, substrate of OCTs. Typically, organic cations with one or the well-known importer of Cp [9, 10], our data using two positive charges represent a substrate of OCTs [6] knockdown experiments indicate that this transporter implying that the copper cation fits into this category. does not affect resistance. Our results do not provide strict OCT3 may therefore contribute to the activity of the evidence that Cp is directly transported by OCT3 across the universally expressed CTR1 transporter that accounts for a cell membrane, since other substrates unrelated to the drug significant import of copper [44]. Similarity, the impact of or secondary interacting molecules may critically influence CTR1 for the import of platinum was recently challenged the uptake [39]. In addition, we did not observe improved suggesting that other transporters, likely OCT3, might add cell viability after OCT3 knockdown in parental cells (data to the uptake of the drug [9, 10, 45]. Since the transport not shown) corroborating similar findings of others [37, 40]. of platinum drugs is partly overlapping with copper We therefore hypothesize that the role of downregulated homeostasis, an improved understanding of the molecular OCT3 with regard to Cp resistance may either be indirectly mechanisms seems mandatory for the identification of or modulated by other transporters, e.g. CTR1. networks that could improve the bioavailability of the Our study is limited since hepatoma cell lines like drug. Of note, established clinical procedures of copper HepG2 differ from primary human hepatocytes, although chelation have been recently proposed to overcome Cp we showed that the basal expression of OCT3 mRNA is resistance via enhanced CTR1 expression [46]. Induction similar. OCT3 may also have other roles in different cell of OCT3 expression, possibly via concomitant copper types [37]. It should be noted that oxaliplatin, which was chelation therapy might therefore also represent an not assessed in this study, can also be used for treatment alternative approach to improve current therapy involving of HCC, however as shown by others, subtle differences Cp for treatment of HCC [47, 48]. Thus, our data on between the uptake of both platinum compounds may exist OCT3-mediated cross-resistance further substantiate that [8, 35]. From the clinical point of view, it is interesting that more approaches to improve the efficacy of platinum drug OCT3 is widely expressed in tissues, including placenta, therapy by modulation of copper homeostasis could result intestine, heart, lung, brain, kidney and liver, whereas in a clinical benefit. In addition, it can be speculated that OCT1 and OCT2 show a very limited tissue distribution assessments of OCT3 expression in patients may be of and are most prominently expressed in the liver and the value to monitor the efficacy of anticancer therapy and to kidney, respectively [6]. Following oxaliplatin treatment, advance current prognosis [40]. the mRNA level of OCT3 was found to be downregulated in several cancerous tissues, including uterus, ovary, MATERIALS AND METHODS and lung where platinum treatment is most effective, suggesting that OCT3 downregulation might be operative Cell lines in patient tumor cells [34]. OCT1 and to a lesser extent OCT3 were found to be downregulated in HCC [41]. The cell lines HepG2 and Huh-7 were obtained Given the broad OCT3 expression amongst tissues and from American Type Culture Collection (ATCC). The tumors as well as the finding on OCT3-mediated Cp ATP7B knockout derivate of HepG2 (KO) were obtained resistance presented here and elsewhere [37], further as described [20]. HepG2 cells were cultured in RPMI analysis of resistant tumor cells could be of significant (Lonza). Huh-7 cells were cultured in DMEM High value to assess OCT3 as a novel marker for prognosis of Glucose (GE Healthcare). All media contained 10% platinum drug therapy. fetal bovine serum (FBS) supplemented with 100 U/mL One major finding of the study is the observed penicillin/streptomycin (Hyclone, Logan). Cell lines were

Cp and copper cross-resistance both linked to OCT3 maintained in 5% CO2 at 37°C in a humidified chamber. downregulation. A copper resistant human hepatoma Huh-7 cell subline was shown before to have increased Growth in cisplatin containing medium cell survival after Cp exposure [42]. Whereas some of the main copper transporters, including CTR1, ATP7A Cisplatin solutions (TEVA Pharma) were prepared and ATP7B, have been described to mediate Cp and from a 3 mM stock in standard cell culture medium. Cells copper cross-resistance [24, 26, 43], our data add to the were cultured in the presence of 0.1 µM Cp for one week. candidates mediating cross-resistance. The implication The cell growth was monitored by trypan blue staining of OCT3 mediating copper cross-resistance is twofold. (Sigma-Aldrich). Passaging of cells was performed twice www.impactjournals.com/oncotarget 750 Oncotarget a week at 70–90% confluency. Images were obtained (Roche Annexin-V-FLUOS kit) and analyzed using flow with an Olympus CKX41-X10 microscope using cellSens cytometry (Epics XL.MCL, Beckman Coulter). Standard 1.11 imaging software. Cisplatin accumulation Transfection 106 cells were incubated in standard cell culture Stable ATP7B overexpressing cell lines were medium with Cp at 37°C for 4 h. After incubation, cells generated using retroviral vectors harboring wild were washed with ice cold PBS and resuspended in Lysis type ATP7B. Wildtype ATP7B cDNA was cloned into Buffer (15 mM NaCl, 0.5% Triton X-100). Cells were pGCsamEN retroviral vector encoding blasticidin centrifuged at 14,000 rpm for 5 min at 4°C. Supernatant resistance (kind gift of O. Wildner, Ruhr Universität (soluble fraction) and the cell pellet (nuclear fraction) were Bochum). Cells expressing empty vector pGCsamEN collected. The platinum concentration was determined were used as controls. Cells were selected in media by means of total reflection X-ray analysis (TXRF, S2 containing 6 µg/mL blasticidin (Invitrogen). Stable OCT3 Picofox, Bruker Nano). Quantification was performed by overexpressing cell line was generated using pcDNA3.1_ the Bruker Spectra software (version 6.1.5.0) and based on hOCT3 (kind gift of Prof. Koepsell, University Würzburg, the known concentration of the internal arsenic standard Germany). Cells expressing OCT3 were selected with solution (Fluka Chemie GmbH). 1.5 µg/ml Geneticin (Gibco). Knockdown experiments were performed using 25 pmol siRNA directed against Copper determination OCT3 (Ambion; #109426), MT1 (Ambion; #215639), and antisense oligonucleotide (ASO) directed against 5 × 105 cells were seeded in 12 wells and incubated

CTR1 (kindly provided by Ionis Pharmaceuticals, Inc.). with 0.01 mM copper prepared from a 10 mM CuCl2 Reverse transfection of siRNA was carried out following stock solution (Sigma) in standard cell culture medium the protocol of the manufacturer using 4 µl Lipofectamine for 4 h. After two washings using PBS, cells were lysed RNAiMax (Invitrogen). All transfection experiments were to determine total protein by Bradford assay (BIO RAD). confirmed via RT-qPCR after 24 h post transfection. Samples were dried at 65°C for 24 h dissolved in 300 µl 65% nitric acid (Suprapure; Merck, Germany). Analysis Cumulative cell growth was performed by atomic absorption spectroscopy (Shimadzu AA-6300). 5 × 105 cells were seeded in a 6 well plate (Cellstar) and cultured in cell culture medium with and without Cp Real-time quantitative PCR addition. The cell number was determined every three days using trypan blue and 5×105 cells were reseeded. The PCR analysis was performed as described previously cumulative cell number was calculated. Each cell count [49]. RNA derived from primary human hepatocytes was independently repeated at least three times. after whole liver perfusion and cryopreservation was used as control. Total RNA was isolated by RNeasy Cytotoxicity assay kit (Qiagen). 1 µg of RNA was transcribed using SuperScript II (Invitrogen) according to the instructions 104 cells were seeded in triplicates in a 96 well of the manufacturer. For quantitative real time PCR plate (Falcon) and cultivated in 100 µl RPMI media (qPCR) the RT product was incubated with SYBR Green lacking phenol red (Lonza). The next day, different Cp PCR Core Plus (Eurogentec, Belgium) and 150 nM of or copper concentrations were added. For Cp and copper, primers. PCR was analyzed on the ABI Prism 7900 HT incubation times of 72 h and 48 h were used, respectively. Sequence Detection System (PE Applied Biosystems). Cell viability was determined by addition of MTT (3-[4, Each sample was tested in three independent experiments 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide; using all primers in the same experiment. Ct values were Sigma) that was added at 1mg/ml for 2 h. Absorbance at normalized to the expression of the house-keeping gene 560 nm was measured after addition of sodium dodecyl (ΔΔct method) and fold change was calculated using the sulfate (SDS; Roth) and dimethyl sulfoxide (DMSO; 2ΔΔCt-method. Roth). Viability was determined as a percentage of untreated control cells (100%). OCT3 protein determination

Apoptosis For lysis of cells, RIPA buffer (60 mM tris-HCl, 150 mM NaCl, 2% Na-deoxycholate, 2% Triton X-10, 0.2% 106 cells were incubated with 10 µM Cp at 37°C SDS, and 15 mM EDTA) and protease inhibitors (Roche, for 72 h. Supernatants and cells were collected and Basel, Switzerland; Complete Mini, EDTA-free) were subjected to Annexin-V and propidium iodide staining used. 50 µg protein lysate was loaded on a 12.5% SDS gel.

www.impactjournals.com/oncotarget 751 Oncotarget Polyclonal anti-rabbit OCT3 antibody (1:1,000; Abcam; REFERENCES ab124826) was added overnight. β-Actin was assessed for protein loading control (1:1,000; sc-47778 HRP, Santa 1. Kelland L. The resurgence of platinum-based cancer Cruz Biotechnology, Santa Cruz, CA, USA). chemotherapy. Nat Rev Cancer. 2007; 7:573–84. 2. Ishikawa T, Kubota T, Abe S, Watanabe Y, Sugano T, OCT3 functional assay Inoue R, Iwanaga A, Seki K, Honma T, Yoshida T. Hepatic arterial infusion chemotherapy with cisplatin before radical 6 10 cells were seeded in 96 wells and grown to local treatment of early hepatocellular carcinoma (JIS score 80–90% confluency. Cell culture medium was removed 0/1) improves survival. Ann Oncol. 2014; 25:1379–84. and HCO -free Ringer-like solution (pH 7.4) was 3 3. Tanaka S, Shimada M, Shirabe K, Maehara S, Harimoto N, added. Cellular uptake was performed as described Tsujita E, Sugimachi K, Maehara Y. A novel intrahepatic before [50] using 5 µM 4-(4-Dimethylamino)styryl)-N- arterial chemotherapy after radical resection for advanced + Methylpyridinium Iodide (ASP ). A fluorescence plate hepatocellular carcinoma. Hepatogastroenterology. 2005; reader (Infinity M200; Tecan, Crailsheim, Germany) 52:862–5. with a monochromator system (excitation at 450 nm and 4. Galluzzi L, Vitale I, Michels J, Brenner C, Szabadkai G, emission at 590 nm) was used for analysis. Harel-Bellan A, Castedo M, Kroemer G. Systems biology of cisplatin resistance: past, present and future. Cell Death Statistical analysis Dis. 2014; 5:e1257. 5. Ciarimboli G. Membrane transporters as mediators of Statistical analysis was performed by Kruskal- cisplatin side-effects. Anticancer Res. 2014; 34:547–50. Wallis 1-way ANOVA and Wilcoxon Mann-Whitney- test using SPSS 22.0 software. Data are given as mean ± 6. Nies AT, Koepsell H, Damme K, Schwab M. Organic cation standard error of the mean (SE). A p < 0.05 value was used transporters (OCTs, MATEs), in vitro and in vivo evidence for the importance in drug therapy. Handb Exp Pharmacol. to indicate significance. 2011; 201:105–67. 7. Ciarimboli G, Deuster D, Knief A, Sperling M, Holtkamp M, Abbreviations Edemir B, Pavenstadt H, Lanvers-Kaminsky C, am Zehnhoff-Dinnesen A, Schinkel AH, Koepsell H, Jurgens H, CpR, cisplatin resistant; CpR , cisplatin resistant w Schlatter E. Organic cation transporter 2 mediates cisplatin- and weaned¸ CpR , OCT3 overexpressing cisplatin OCT3 induced oto- and nephrotoxicity and is a target for protective resistant cells; interventions. Am J Pathol. 2010; 176:1169–80. 8. Yokoo S, Yonezawa A, Masuda S, Fukatsu A, Katsura T, Author contributions Inui K. Differential contribution of organic cation transporters, OCT2 and MATE1, in platinum agent-induced Conception and design: SG, GC, VS, HS, AZ. nephrotoxicity. Biochem Pharmacol. 2007; 74:477–87. Acquisition of data: SG, GC, SRG, AG, CN, UK, GCi. 9. Holzer AK, Manorek GH, Howell SB. Contribution of Analysis and interpretation of data: SG, GC, SRG, VS, the major copper influx transporter CTR1 to the cellular UK, GCi, HS, AZ. Writing, review, and/or revision of the accumulation of cisplatin, carboplatin, and oxaliplatin. Mol manuscript: SG, GC, HS, AZ. Pharmacol. 2006; 70:1390–4. 10. Kuo MT, Fu S, Savaraj N, Chen HH. Role of the human ACKNOWLEDGMENTS high-affinity copper transporter in copper homeostasis regulation and cisplatin sensitivity in cancer chemotherapy. We are grateful to M. Holtkamp and O. Nadzemova Cancer Res. 2012; 72:4616–21. for technical assistance. 11. Kelley SL, Basu A, Teicher BA, Hacker MP, Hamer DH, Lazo JS. Overexpression of metallothionein confers CONFLICTS OF INTEREST resistance to anticancer drugs. Science. 1988; 241:1813–5. 12. Dmitriev OY. Mechanism of tumor resistance to cisplatin The authors declare no potential conflicts of interest. mediated by the copper transporter ATP7B. Biochem Cell Biol. 2011; 89:138–47. GRANT SUPPORT 13. Samimi G, Safaei R, Katano K, Holzer AK, Rochdi M, Tomioka M, Goodman M, Howell SB. Increased expression S.R.G. was supported by a scholarship from the of the copper efflux transporter ATP7A mediates resistance Faculty of Medicine of the University of Münster (MedK to cisplatin, carboplatin, and oxaliplatin in ovarian cancer 15-0055). cells. Clin Cancer Res. 2004; 10:4661–9.

www.impactjournals.com/oncotarget 752 Oncotarget 14. Komatsu M, Sumizawa T, Mutoh M, Chen ZS, Terada K, 26. Katano K, Safaei R, Samimi G, Holzer A, Rochdi M, Furukawa T, Yang XL, Gao H, Miura N, Sugiyama T, Howell SB. The copper export pump ATP7B modulates the Akiyama S. Copper-transporting P-type adenosine cellular pharmacology of carboplatin in ovarian carcinoma triphosphatase (ATP7B) is associated with cisplatin cells. Mol Pharmacol. 2003; 64:466–73. resistance. Cancer Res. 2000; 60:1312–6. 27. Samimi G, Katano K, Holzer AK, Safaei R, Howell SB. 15. Katano K, Safaei R, Samimi G, Holzer A, Tomioka M, Modulation of the cellular pharmacology of cisplatin and Goodman M, Howell SB. Confocal microscopic analysis of its analogs by the copper exporters ATP7A and ATP7B. Mol the interaction between cisplatin and the copper transporter Pharmacol. 2004; 66:25–32. ATP7B in human ovarian carcinoma cells. Clin Cancer Res. 28. Aida T, Takebayashi Y, Shimizu T, Okamura C, 2004; 10:4578–88. Higasimoto M, Kanzaki A, Nakayama K, Terada K, 16. Yoshizawa K, Nozaki S, Kitahara H, Ohara T, Kato K, Sugiyama T, Miyazaki K, Ito K, Takenoshita S, Yaegashi N. Kawashiri S, Yamamoto E. Copper efflux transporter Expression of copper-transporting P-type adenosine (ATP7B) contributes to the acquisition of cisplatin- triphosphatase (ATP7B) as a prognostic factor in human resistance in human oral squamous cell lines. Oncol Rep. endometrial carcinoma. Gynecol Oncol. 2005; 97:41–5. 2007; 18:987–91. 29. Nakayama K, Kanzaki A, Ogawa K, Miyazaki K, 17. Moinuddin FM, Shinsato Y, Komatsu M, Mitsuo R, Neamati N, Takebayashi Y. Copper-transporting P-type Minami K, Yamamoto M, Kawahara K, Hirano H, adenosine triphosphatase (ATP7B) as a cisplatin based Arita K, Furukawa T. ATP7B expression confers multidrug chemoresistance marker in ovarian carcinoma: comparative resistance through drug sequestration. Oncotarget. 2016; analysis with expression of MDR1, MRP1, MRP2, LRP and 7:22779–90. https://doi.org/10.18632/oncotarget.8059. BCRP. Int J Cancer. 2002; 101:488–95. 30. Martinez-Balibrea E, Martinez-Cardus A, Musulen E, 18. Ishida S, McCormick F, Smith-McCune K, Hanahan D. Gines A, Manzano JL, Aranda E, Plasencia C, Neamati N, Enhancing tumor-specific uptake of the anticancer drug Abad A. Increased levels of copper efflux transporter cisplatin with a copper chelator. Cancer Cell. 2010; ATP7B are associated with poor outcome in colorectal 17:574–83. cancer patients receiving oxaliplatin-based chemotherapy. 19. Lutsenko S. Copper trafficking to the secretory pathway. Int J Cancer. 2009; 124:2905–10. Metallomics. 2016; 8:840–52. 31. Ciarimboli G, Ludwig T, Lang D, Pavenstadt H, Koepsell H, 20. Chandhok G, Schmitt N, Sauer V, Aggarwal A, Bhatt M, Piechota HJ, Haier J, Jaehde U, Zisowsky J, Schlatter E. Schmidt HH. The effect of zinc and D-penicillamine in a Cisplatin nephrotoxicity is critically mediated via the stable human hepatoma ATP7B knockout cell line. PLoS human organic cation transporter 2. Am J Pathol. 2005; One. 2014; 9:e98809. 167:1477–84. 21. Leonhardt K, Gebhardt R, Mossner J, Lutsenko S, Huster D. 32. Zhang S, Lovejoy KS, Shima JE, Lagpacan LL, Shu Y, Functional interactions of Cu-ATPase ATP7B with cisplatin Lapuk A, Chen Y, Komori T, Gray JW, Chen X, Lippard SJ, and the role of ATP7B in the resistance of cells to the drug. Giacomini KM. Organic cation transporters are determinants J Biol Chem. 2009; 284:7793–802. of oxaliplatin cytotoxicity. Cancer Res. 2006; 66:8847–57. 22. Yu F, Megyesi J, Price PM. Cytoplasmic initiation of 33. More SS, Li S, Yee SW, Chen L, Xu Z, Jablons DM, cisplatin cytotoxicity. Am J Physiol Renal Physiol. 2008; Giacomini KM. Organic cation transporters modulate the 295:F44–52. uptake and cytotoxicity of picoplatin, a third-generation 23. Massmann V, Edemir B, Schlatter E, Al-Monajjed R, platinum analogue. Mol Cancer Ther. 2010; 9:1058–69. Harrach S, Klassen P, Holle SK, Sindic A, Dobrivojevic M, 34. Yokoo S, Masuda S, Yonezawa A, Terada T, Katsura T, Pavenstadt H, Ciarimboli G. The organic cation transporter Inui K. Significance of organic cation transporter 3 3 (OCT3) as molecular target of psychotropic drugs: (SLC22A3) expression for the cytotoxic effect of oxaliplatin transport characteristics and acute regulation of cloned in colorectal cancer. Drug Metab Dispos. 2008; 36:2299–306. murine OCT3. Pflugers Arch. 2014; 466:517–27. 35. Yonezawa A, Masuda S, Yokoo S, Katsura T, Inui K. 24. Katano K, Kondo A, Safaei R, Holzer A, Samimi G, Cisplatin and oxaliplatin, but not carboplatin and nedaplatin, Mishima M, Kuo YM, Rochdi M, Howell SB. Acquisition are substrates for human organic cation transporters of resistance to cisplatin is accompanied by changes in (SLC22A1-3 and multidrug and toxin extrusion family). J the cellular pharmacology of copper. Cancer Res. 2002; Pharmacol Exp Ther. 2006; 319:879–86. 62:6559–65. 36. Kitada N, Takara K, Minegaki T, Itoh C, Tsujimoto M, 25. Nies AT, Koepsell H, Winter S, Burk O, Klein K, Kerb R, Sakaeda T, Yokoyama T. Factors affecting sensitivity to Zanger UM, Keppler D, Schwab M, Schaeffeler E. antitumor platinum derivatives of human colorectal tumor Expression of organic cation transporters OCT1 (SLC22A1) cell lines. Cancer Chemother Pharmacol. 2008; 62:577–84. and OCT3 (SLC22A3) is affected by genetic factors and 37. Li Q, Peng X, Yang H, Rodriguez JA, Shu Y. Contribution cholestasis in human liver. Hepatology. 2009; 50:1227–40. of organic cation transporter 3 to cisplatin cytotoxicity www.impactjournals.com/oncotarget 753 Oncotarget in human cervical cancer cells. J Pharm Sci. 2012; mechanisms for copper homeostasis. Am J Physiol 101:394–404. Gastrointest Liver Physiol. 2009; 296:G356–64. 38. Negoro K, Yamano Y, Nakashima D, Saito K, Nakatani K, 45. Bompiani KM, Tsai CY, Achatz FP, Liebig JK, Howell SB. Shiiba M, Bukawa H, Yokoe H, Uzawa K, Wada T, Copper transporters and chaperones CTR1, CTR2, Tanzawa H, Fujita S. Cross-resistance of platinum derivatives ATOX1, and CCS as determinants of cisplatin sensitivity. in H-1R, a cisplatin-resistant cell line. Oncol Rep. 2009; Metallomics. 2016; 8:951–62. 21:443–9. 46. Chen HH, Chen WC, Liang ZD, Tsai WB, Long Y, Aiba I, 39. Ciarimboli G. Organic cation transporters. Xenobiotica. Fu S, Broaddus R, Liu J, Feun LG, Savaraj N, Kuo MT. 2008; 38:936–71. Targeting drug transport mechanisms for improving 40. Hsu CM, Lin PM, Chang JG, Lin HC, Li SH, Lin SF, platinum-based cancer chemotherapy. Expert Opin Ther Yang MY. Upregulated SLC22A3 has a potential for Targets. 2015; 19:1307–17. improving survival of patients with head and neck 47. Hsiao JH, Tsai CC, Liang TJ, Chiang CL, Liang HL, squamous cell carcinoma receiving cisplatin treatment. Chen IS, Chen YC, Chang PM, Chou NH, Wang BW. Oncotarget. 2017; 8:74348–58. https://doi.org/10.18632/ Adjuvant hepatic arterial infusion chemotherapy is oncotarget.20637. beneficial for selective patients with Hepatocellular 41. Heise M, Lautem A, Knapstein J, Schattenberg JM, Hoppe- carcinoma undergoing surgical treatment. Int J Surg. 2017; Lotichius M, Foltys D, Weiler N, Zimmermann A, Schad A, 45:35–41. Grundemann D, Otto G, Galle PR, Schuchmann M, et al. 48. Ikeda M, Okusaka T, Sato Y, Furuse J, Mitsunaga S, Downregulation of organic cation transporters OCT1 Ueno H, Morizane C, Inaba Y, Kobayashi T, Arai Y. A (SLC22A1) and OCT3 (SLC22A3) in human hepatocellular Phase I/II trial of continuous hepatic intra-arterial infusion carcinoma and their prognostic significance. BMC Cancer. of 5-fluorouracil, mitoxantrone and cisplatin for advanced 2012; 12:109. hepatocellular carcinoma. Jpn J Clin Oncol. 2017; 42. Safaei R, Katano K, Samimi G, Naerdemann W, 47:512–9. Stevenson JL, Rochdi M, Howell SB. Cross-resistance to 49. Siaj R, Sauer V, Stoppeler S, Spiegel HU, Kohler G, cisplatin in cells with acquired resistance to copper. Cancer Zibert A, Schmidt HH. Dietary copper triggers onset of Chemother Pharmacol. 2004; 53:239–46. fulminant hepatitis in the Long-Evans cinnamon rat model. 43. Song IS, Savaraj N, Siddik ZH, Liu P, Wei Y, Wu CJ, World J Gastroenterol. 2012; 18:5542–50. Kuo MT. Role of human copper transporter Ctr1 in the 50. Mehrens T, Lelleck S, Cetinkaya I, Knollmann M, transport of platinum-based antitumor agents in cisplatin- Hohage H, Gorboulev V, Boknik P, Koepsell H, Schlatter E. sensitive and cisplatin-resistant cells. Mol Cancer Ther. The affinity of the organic cation transporter rOCT1 is 2004; 3:1543–9. increased by protein kinase C-dependent phosphorylation. 44. Kim H, Son HY, Bailey SM, Lee J. Deletion of hepatic Ctr1 J Am Soc Nephrol. 2000; 11:1216–24. reveals its function in copper acquisition and compensatory

www.impactjournals.com/oncotarget 754 Oncotarget