Eribulin Suppresses Clear Cell Sarcoma Growth by Inhibiting Cell

Published OnlineFirst December 3, 2019; DOI: 10.1158/1535-7163.MCT-19-0358

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Eribulin Suppresses Clear Cell Sarcoma Growth by Inhibiting Cell Proliferation and Inducing Melanocytic Differentiation Both Directly and Via Vascular Remodeling Sho Nakai1, Hironari Tamiya2, Yoshinori Imura2, Takaaki Nakai3, Naohiro Yasuda1, Toru Wakamatsu2, Takaaki Tanaka2, Hidetatsu Outani1, Satoshi Takenaka1, Kenichiro Hamada1, Akira Myoui1, Nobuhito Araki4, Takafumi Ueda5, Hideki Yoshikawa1, and Norifumi Naka1,2

ABSTRACT ◥ Clear cell sarcoma (CCS) is a rare but chemotherapy-resistant stimulated tumor cell melanocytic differentiation through ERK1/2 and often fatal high-grade soft-tissue sarcoma (STS) characterized inactivation (a MITF negative regulator) in vitro and in vivo. by melanocytic differentiation under control of microphthalmia- Moreover, tumor reoxygenation, probably caused by eribulin- associated transcription factor (MITF). Eribulin mesilate (eribulin) induced vascular remodeling, attenuated cell growth and inhibited is a mechanistically unique microtubule inhibitor commonly used ERK1/2 activity, thereby upregulating MITF expression and pro- for STS treatment, particularly liposarcoma and leiomyosarcoma. moting melanocytic differentiation. Finally, downregulation of In this study, we examined the antitumor efﬁcacy of eribulin on four MITF protein levels modestly debilitated the antiproliferative effect human CCS cell lines and two mouse xenograft models. Eribulin of eribulin on CCS cells. Taken together, eribulin suppresses CCS inhibited CCS cell proliferation by inducing cell-cycle arrest and through inhibition of cell proliferation and promotion of tumor apoptosis, shrunk CCS xenograft tumors, and increased tumor differentiation by acting both directly on tumor cells and indirectly vessel density. Eribulin induced MITF protein upregulation and through tumor reoxygenation.

Introduction The mainstay CCS treatment is complete surgical resection, although chemotherapy and/or radiotherapy are also used depending Clear cell sarcoma (CCS) is a rare but aggressive soft-tissue sarcoma on disease status. Despite multidisciplinary treatment, earlier reports (STS), appearing predominantly in adolescents and young adults. It found 5-year overall survival (OS) rates of only 30% to 67% (1, 2, 5, 6). usually arises in the lower extremities close to tendons, fasciae, and Notably, chemotherapy was reported to be largely ineffective in aponeuroses, although it occasionally occurs in the upper extremities patients at advanced stages, resulting in poor prognosis (7). Therefore, or the trunk (1, 2). CCS is termed “malignant melanoma of soft parts” novel therapeutic strategies including effective chemotherapy regi- due to melanocytic differentiation driven by expression of micro- mens are needed to improve patient prognosis. phthalmia-associated transcription factor (MITF), which upregulates MITF is a basic helix–loop–helix leucine zipper transcription factor tyrosinase (TYR), MART-1, and HMB45, and in some cases melanin that regulates key processes in several cell lineages, including mela- production (1). CCS is characterized cytogenetically by a t(12;22)(q13; nocytes, retinal pigment epithelial cells, osteoclasts, mast cells, and q12) chromosomal translocation and its resultant fusion oncogene melanoma cells (8). In melanoma, MITF controls not only differen- EWSR1–ATF1 (3). EWSR1–ATF1 fusion oncoprotein constitutively tiation but also proliferation (8). However, melanomas with extremely activates the MITF promoter, leading to melanocytic differentiation in high MITF levels exhibit greater susceptibility to cell-cycle arrest and CCS (4). differentiation, reducing tumorigenicity (9, 10). On the other hand, lower MITF activity increases the invasive and metastatic properties of melanoma cells by conferring a stem cell–like phenotype (9–11). In melanoma, MITF activity is regulated at transcriptional and posttran- 1Department of Orthopaedic Surgery, Osaka University Graduate School of scriptional levels, and these regulatory mechanisms include epigenetic 2 Medicine, Osaka, Japan. Musculoskeletal Oncology Service, Osaka International and microenvironmental signals (11, 12). However, the mechanic 3 Cancer Institute, Osaka, Japan. Department of Orthopaedic Surgery, Kawachi details of CCS are unclear and so as yet have not been exploited General Hospital, Kawachi, Japan. 4Department of Orthopaedic Surgery, Ashiya Municipal Hospital, Ashiya, Japan. 5Department of Orthopaedic Surgery, Osaka successfully for treatment. National Hospital, Osaka, Japan. Eribulin mesilate (eribulin), an analog of the natural marine compound halichondrin B, is a nontaxane synthetic microtubule Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). dynamics inhibitor currently used in many countries for advanced or metastatic breast cancer and STS (13, 14). Two phase III clinical Corresponding Author: Norifumi Naka, Osaka International Cancer Institute, 3- 1-69, Otemae, Chuo-Ku, Osaka 541-8567, Japan. Phone: 81-6-6945-1181; Fax: 81- trials of patients with metastatic breast cancer reported that eribulin 6-6945-1900; E-mail: [email protected] improved OS without corresponding effects on progression-free survival (PFS) compared with treatment of physician's choice or Mol Cancer Ther 2020;19:742–54 capecitabine (14–16). Similar ﬁndings of prolonged OS in the doi: 10.1158/1535-7163.MCT-19-0358 absence of effects on PFS were also reported in a phase III clinical 2019 American Association for Cancer Research. trial of patients with liposarcoma and leiomyosarcoma compared

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with dacarbazine (13). Thus, eribulin is considered a promising with the experimental drugs or the vehicle (DMSO) for the indicated agent for other cancer types. time. The cell proliferation rate was assessed using the Premix WST-1 The anticancer mechanisms of eribulin appear to be unique, Cell Proliferation Assay System (TAKARA) according to the manu- although still not fully understood. Eribulin was shown to destabilize facturer's instructions. Absorbance at 450 and 650 nm (reference microtubules by inhibiting the growth parameters at the plus ends wavelength) was measured using a spectrophotometer. The relative without effect on microtubule shortening parameters, thereby giving cell proliferation rate was determined by subtracting the average value rise to irreversible mitotic disruption and apoptosis (17, 18). In of “time zero” measurements from sample measurements. addition, vascular remodeling by eribulin treatment, resulting in increased microvessel density (MVD) and enhanced tumor perfu- Flow cytometry sion, has been shown in human patients with breast cancer and All CCS lines were seeded at 1 106 per dish and cultured mouse xenograft models (19–21). Moreover, accumulating evidence overnight, followed by eribulin or vehicle treatment. After 0 to 24 hours indicates that eribulin inhibits the epithelial–mesenchymal transi- treatment at the indicated concentration, cells were harvested and tion of breast cancer cell lines and induces differentiation of STS cell stained with propidium iodide (PI) solution (25 mg/mL PI, 0.03% NP- lines in addition to regular cytotoxic effects, which have not been 40, 0.02 mg/mL RNase A, 0.1% sodium citrate) for 30 minutes at room reported for other microtubule-targeting agents such as taxanes and temperature. For cell-cycle analysis, we used the BD FACSVerse flow vinca alkaloids (22, 23). cytometer (BD Bioscience) and the BD FACSSuite Software Applica- In this study, we first report potent antitumor activity of eribulin tion (BD Bioscience) according to the manufacturer's protocol. against CCS cell lines and two mouse xenograft models. Further, eribulin induces melanocytic differentiation and vascular remodeling Western blot analysis in CCS tumors. At the molecular level, eribulin upregulates MITF For lysate preparation, cells were first washed with PBS and protein levels by inhibiting ERK1/2. In addition, we show that reox- lysed in RIPA (Thermo Fisher Scientific) supplemented with 1% ygenation through vascular remodeling potentially leads to tumor protease/phosphatase inhibitor cocktail. Protein concentrations differentiation and decreases cell proliferation. Finally, knockdown of were determined using the bicinchoninic acid (BCA) method MITF attenuates the cytotoxic effect of eribulin in CCS cells. (Thermo Fisher Scientific). Cell proteins were separated on 4% to 12% Bis-Tris gels (Life Technologies) and transferred to poly- vinylidene difluoride membranes (Nippon Genetics). The mem- Materials and Methods branes were blocked in tris-buffered saline (TBS) containing 5% Cell culture skim milk and Tween 20 (TBS-T) at room temperature and then We utilized four human CCS cell lines, Hewga-CCS, MP-CCS-SY, incubated with primary antibodies in Can Get Signal Solution 1 KAS, and SU-CCS1, to examine the antiproliferative properties of (TOYOBO) at 4C overnight, followed by incubation with sec- eribulin. The types of EWSR1–ATF1 chimeric transcripts in these CCS ondary antibodies in Can Get Signal Solution 2 (TOYOBO) at cells were confirmed as described previously (24). The Hewga-CCS room temperature for 1 hour. The primary antibodies are available line was established in our laboratory from a primary tumor (5), in Supplementary Table S1. whereas MP-CCS-SY and KAS lines were kindly provided by Dr. Moritake (Miyazaki University, Miyazaki, Japan) and Dr. Naka- In vivo mouse xenograft model mura (Japanese Foundation for Cancer Research, Tokyo, Japan), Five-week-old female BALB/c nu/nu mice (SLC) were housed at the respectively (25, 26), and SU-CCS1 was purchased from the ATCC. Institute of Experimental Animal Sciences, Osaka University Medical These cell lines were passaged soon after receipts, divided and School, in accordance with guidelines approved by the Animal Care stocked at 80C. All cells were maintained in DMEM (Nacalai and Use Committee of the Osaka University Graduate School of Tesque) supplemented with 100 mg/mL streptomycin sulfate, 100 Medicine. For the xenograft tumor growth assay, 1 107 CCS cells U/mL penicillin G (Life Technologies), and 10% heat-inactivated were injected subcutaneously into the left side of the back. Tumor FBS (Life Technologies) at 37Cinahumidified atmosphere of 5% dimensions were measured from the skin using a caliper and volume 2 CO2. When the cells reached subconfluence, they were detached calculated as (A B )/2, where A is the longest diameter and B is the using 0.25% trypsin plus EDTA (Life Technologies) and reseeded shortest diameter. When the average tumor diameter reached 5 mm, for experimental treatments. Cell lines were authenticated by the mice were randomized into eribulin- and vehicle-treated groups. examination of morphology, genotyping by PCR and growth char- Eribulin was intravenously injected through the tail vein at 1 or acteristics, and were mycoplasma free. Experiments were performed 3 mg/kg on days 1 and 8. Controls received equal-volume saline within 2 months of thawing. injections at the same time. Xenograft tumor volume and mouse body weight were measured twice weekly. When all mice were euthanized, Compounds the tumor weights were measured. All protocols were approved by the Eribulin was manufactured at and provided by Eisai. Co. Ltd. The Animal Care and Use Committee of the Osaka University Graduate ERK1/2 inhibitor SCH772984 was purchased from Cayman Chemical. School of Medicine. The drugs were prepared in DMSO before addition to cell cultures for in vitro examinations according to the manufacturer's instructions. Histology and IHC Eribulin was diluted in physiologic saline to the desired concentration Excised xenograft tumors were fixed in 10% neutral-buffered for in vivo experiments. formalin, embedded in paraffin, cut into 3-mm-thick sections, and examined by IHC, hematoxylin–eosin (HE) staining, and Fontana– WST-1 Cell proliferation assay Masson staining. For immunostaining, paraffin-embedded sections All CCS lines were seeded into 96-well plates at 2 103 cells/well in were first deparaffinized and dehydrated. Antigens were retrieved at triplicate, including three control wells to provide “time zero” for 95C for 10 minutes in a 10 mmol/L citrate buffer. After quenching absorbance readings. They were incubated overnight and then treated endogenous peroxidase activity for 10 minutes with methanol

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containing 3% H2O2, the sections were blocked for 1 hour with TBS Wound healing assay containing 2% BSA at room temperature. The sections were then Monolayers of CCS cells were scraped (wounded) in a straight line incubated with primary antibodies at 4C overnight, followed by with a 10 mL pipette tip. The detached cells were removed by washing 1 hour incubation with HRP-conjugated secondary antibodies. with medium and the remaining CCS cells incubated under 3% Finally, label sections were stained with 3,30-diaminobenzidine oxygen, 5% oxygen, normoxia. Images of each sample culture were tetrahydrochloride (Dako) and counterstained using hematoxylin. obtained at 0 and 24 hours using a microscope and wound width The primary antibodies are available in Supplementary Table S1. measured as an index of cell motility. Fontana–Masson staining was performed to identify melanin synthesis as described previously (24). siRNA transfection CCS cells were seeded at a density of 3 103 cells/well in 96-well Measurement of melanin content plates and grown overnight. Cells were transfected with 1 nmol/L CCS cell lines were seeded in six-well plates at 2 105 cells/well and siRNAs for 24 hours using Lipofectamine RNAiMAX Transfection incubated for 24 hours. The plating medium was then exchanged with Reagent (Life Technologies). Two kinds of siRNAs targeting MITF fresh medium containing 10 nmol/L eribulin or vehicle. Cells were (constructs I and II; #s8791 and #s8972) and a nontargeting negative incubated for an additional 96 hours, washed twice in PBS, detached control siRNA (#4390844) were purchased from Thermo Fisher using 0.25% trypsin plus EDTA, and transferred to 1.5 mL tubes. After Scientific. centrifugation for 10 minutes at 14,000 rpm, the cell pellets were dissolved in 1 mol/L NaOH (100 mL) for 60 minutes at 80C. The Statistical analysis optical density of the supernatant was measured at 405 nm using a All data are expressed as mean SD. Two-tailed Student t test or spectrophotometer, and the result was compared with a standard curve one-way ANOVA was used to determine statistical differences. Values of synthetic melanin (Sigma-Aldrich). of P < 0.05 were considered significant and the specific values are indicated in legends to figures as P < 0.05 (), P < 0.01 (). Intracellular TYR activity assay CCS cells were cultured in six-well plates at 2 105 cells/well. After 24 hours, the plating medium was exchanged for fresh medium Results including 10 nmol/L eribulin or vehicle. Cells were incubated for an Eribulin exerts the cytotoxic effect by inducing G2–M cell-cycle additional 96 hours, washed with PBS, and lysed in RIPA buffer arrest and apoptosis in CCS cells supplemented with 1% protease/phosphatase inhibitor cocktail. Pro- To assess the potential antiproliferative efficacy of eribulin against tein concentrations were determined using the BCA method. The CCS, we examined the growth rates of four CCS cell lines following lysate was centrifuged, and supernatant samples containing 50 mg total exposure to therapeutic concentrations of eribulin (0–30 nmol/L) for protein were transferred to 96-well plates and mixed with 100 mL 96 hours by WST-1 assay. Eribulin dose-dependently reduced viable samples of 0.2% 3,4-dihydroxy-L-phenylalanine (Sigma-Aldrich) in cell numbers of all four lines (Fig. 1A), with the highest potency against ¼ phosphate buffer. After incubation at 37 C for 1 hour, the absorbance the Hewga-CCS line (IC50 0.69 nmol/L), followed by KAS (1.91 at 475 nm was measured using a microplate reader. nmol/L), MP-CCS-SY (2.21 nmol/L), and SU-CCS1 (2.21 nmol/L). Thus, the proliferation of all four lines exhibited high sensitivity to qRT-PCR analysis eribulin, in accordance with the effects on other sarcoma cell lines Total RNA was purified using the RNeasy Mini Kit (Qiagen) and including fibrosarcoma (HT-1080), liposarcoma (SK-UT-1), and leio- reverse transcribed using the High-Capacity cDNA Reverse Tran- myosarcoma (SK-LMS-1; refs. 23, 27). Next, flow cytometry was scription Kit (Life Technologies). Real-time PCR was performed using performed to assess the effects of eribulin on the cell cycle. At 10 a StepOnePlus Real-Time PCR System (Life Technologies) and SYBR nmol/L, eribulin treatment induced G2-M cell-cycle arrest in all four Green Realtime PCR Master Mix (TOYOBO). Expression values were CCS lines beginning as early as 3 hours after exposure and increasing normalized to that of b-actin. The PCR primers used (forward and with time thereafter (Fig. 1B). Moreover, at 100 nmol/L, eribulin 0 reverse) were as follows: b-actin (5 -ATTGCCGACAGGATGCA- treatment induced more potent G2–M cell-cycle arrest (Fig. 1C). In GAA-30 and 50-GCTGATCCACATCTGCTGGAA-30), MITF (50- addition, the cellular level of cleaved caspase-3, the major effector of GAGGCAGTGGTTTGGGCTT-30 and 50-AATTCTGCACCCGG- apoptosis, was enhanced after 72 hours exposure to more than 10 GAATC-30), EWSR1–ATF1 (type1, type3) (50-GAGGCATGAGCA- nmol/L eribulin as evidenced by Western blot analyses (Fig. 1D). GAGGTGG-30 and 50-GAAGTCCCTGTACTCCATCTGTG-30), These experiments suggest that eribulin exerts its cytotoxic effect by EWSR1–ATF1 (type2) (50-CCTACAGCCAAGCTCCAAGTC-30 and blocks of metaphase stage in CCS cells. 50 -GCCTGGACTTGCCAACTGTA-30). Eribulin suppresses the growth of CCS xenograft tumors, MITF degradation assays increases tumor melanin synthesis, and induces tumor To analyze the degradation kinetics of MITF, CCS cells were seeded vasculature remodeling in 10 cm dishes at 5 105 cells/dish for cycloheximide chase assays. We next evaluated the antitumor effects of eribulin against After 24 hours, the medium was exchanged with fresh medium Hewga-CCS and MP-CCS-SY xenograft tumors in nude mice. Mice containing 10 nmol/L eribulin or vehicle. Cells were then incubated were injected subcutaneously with each of cells and tumors allowed for an additional 48 or 96 hours, treated with cycloheximide (60 mg/ to grow until the average diameter reached 5 mm. Mice were then mL) (Wako Pure Chemical Industries) for the indicated times, and treated intravenously with eribulin at 1 or 3 mg/kg or with equal- harvested. Equal amounts of cell protein were separated by 4% to 12% volume vehicle once every 7 days for two cycles. Treatment with Bis-Tris gel electrophoresis and MITF expression analyzed by immu- both 1 and 3 mg/kg eribulin markedly suppressed tumor growth noblotting. The integrated optical densities of protein bands were compared with the vehicle control in both xenograft models (Fig. 2A quantified by TotalLab Quant software (Nonlinear Inc.). and B; Supplementary Fig. S1A). In 3 mg/kg eribulin-treated mice,

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Figure 1.

Eribulin reduced viable CCS cell number by induction of G2–M cell-cycle arrest and apoptosis. A, Four CCS cell lines, Hewga-CCS, MP-CCS-SY, KAS, and SU-CCS1 were treated with 0–30 nmol/L eribulin for 96 hours and viable cell number estimated by WST-1 assay. The calculated IC50 values are shown in the table. B and C, Cell lines were treated with 10 (B)and100(C) nmol/L eribulin for 0 to 24 hours, stained with PI, and analyzed for cell-cycle stage by flow cytometry. D, Cell lines were treated with 0.1 to 1,000 nmol/L eribulin or vehicle for 72 hours, and expression of the apoptosis marker cleaved caspase-3 was evaluated by Western blotting. Data in A are presented as mean SD, n ¼ 3. mean body weight loss was approximately 7.5% and 17.5% at 11 days tary Fig. S1D and S1E). These results confirm that eribulin sup- in Hewga-CCS and MP-CCS-SY xenografts, respectively, and recov- presses tumor cell proliferation and induces apoptosis in vivo as well ered within about 10 days after the second injection (Supplementary as in culture. Fig. S1B), suggesting moderate nontarget toxicity. Hematoxylin and Given that eribulin triggers phenotypic alterations in breast cancer, eosin staining revealed reduced cell density and increased fibrotic liposarcoma, and leiomyosarcoma cell lines (22, 23), we next examined area in eribulin-treated tumors compared with vehicle-treated whether eribulin altered CCS cell phenotype. Specifically, we assessed tumors (Supplementary Fig. S1C). IHC analyses also revealed fewer if eribulin-induced melanocytic features, including expression of cells immunopositive for the proliferating cell marker PCNA and proteins involved in melanin production and actual melanin accu- increased numbers expressing the apoptosis marker cleaved caspase- mulation. Indeed, Fontana–Masson staining revealed the increased 3 in eribulin-treated tumors compared with controls (Supplemen- number of melanin-positive cells in eribulin-treated xenografts

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Figure 2. Eribulin inhibited growth, elevated melanin synthesis, and induced vascular remodeling of Hewga-CCS and MP-CCS-SY xenograft tumors. A and B, Hewga-CCS and MP-CCS-SY cells were engrafted in nude mice. Mice were then treated with either eribulin (1 or 3 mg/kg) or vehicle by intravenous injection (5 mice/group). The size of Hewga-CCS (A) and MP-CCS-SY (B) tumors during treatment are shown. C, Fontana–Masson staining for melanin and quantitative evaluation of melanin-positive cell number (% of total cells) in both xenograft tumors of all three treatment groups (five fields counted/group) are shown. D, IHC staining for CD31 and quantification of CD31-positive cell number in both xenograft tumors of all three treatment groups (five fields counted/group) are shown. E, IHC staining for HIF1a in both xenograft tumors from all three treatment groups is shown. F, Tumor tissues from Hewga-CCS and MP-CCS-SY xenografts at the endpoint were collected. Whole-cell proteins were extracted and subjected to Western blot analyses. Scale bars are 100 mm. Data in A, B, C,andD are presented as mean SD. , P < 0.05; , P < 0.01 vs. the vehicle group.

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compared with vehicle-treated controls (Fig. 2C), indicating that tional activation nor an interaction between eribulin and EWSR1– eribulin induces melanocytic differentiation in CCS cells. ATF1. Furthermore, it has been reported that eribulin can remodel tumor On the basis of these results, we speculated that eribulin modulates vasculature, so tumor sections were stained with anti-CD31 as a the posttranscriptional regulation of MITF protein in CCS cells. To test vascular endothelial cell marker and/or with anti-a-SMA as a pericyte this, we measured MITF protein degradation kinetics by cyclohexi- marker (19, 21). Immunostaining for CD31 revealed that eribulin mide (60 mg/mL) chase assay in the presence of 10 nmol/L eribulin or treatment significantly increased mean vessel density (MVD) in vehicle. The rate of MITF protein decline in the presence of cyclo- eribulin-treated xenograft tumors and living tumor cells were absent heximide was significantly reduced in Hewga-CCS cells treated with in perivascular areas of 3 mg/kg eribulin-treated tumors (Fig. 2D). eribulin for 48 or 96 hours compared with vehicle-treated control cells, Double staining for CD31 and a-SMA was also performed to quantify suggesting that eribulin prolongs MITF protein half-life (Fig. 4C). the extent of pericyte coverage, which is crucial for vessel maturation In melanoma, it is well established that downregulation of MITF and stabilization. The vessel maturity index, calculated as the ratio of protein results from ERK1/2-stimulated phosphorylation at Ser 73 and the a-SMA-positive area to the CD31-positive area, was significantly recruitment of p300/CBP, which ultimately leads to ubiquitination and greater in both eribulin dose groups compared with control (Supple- proteasome-mediated degradation (12). Thus, we examined the acti- mentary Fig. S1F), suggesting that eribulin promotes the normaliza- vation (phosphorylation) status of ERK1/2 in response to eribulin tion of vessel morphology and induces tumor reperfusion. To inves- treatment of CCS cells. Intriguingly, ERK1/2 phosphorylation was tigate whether these morphologic changes in tumor vessels cause time-dependently diminished in eribulin-treated CCS cell lines functional changes, we examined the expression of HIF1a, which (Fig. 4D), suggesting that ERK1/2 inactivation may reduce MITF contributes to the cellular hypoxia response. Indeed, IHC and Western protein degradation, thereby enhancing MITF protein levels and blot analyses revealed a dramatic decrease in HIF1a expression by activity in eribulin-treated CCS cells. To examine if ERK1/2 inacti- eribulin-treated tumors compared with vehicle-treated controls, dem- vation contributes to eribulin-induced melanocytic differentiation, we onstrating that eribulin promotes reoxygenation (i.e., mitigates intra- treated Hewga-CCS and MP-CCS-SY cells with SCH772984, a specific tumoral hypoxia; Fig. 2E and F). These in vivo findings suggest that inhibitor of ERK1/2 signaling, and measured the effects on melano- eribulin induces both melanocytic differentiation and vascular remo- cytic differentiation markers and melanin synthesis (29). Consistent deling in CCS xenografts. with eribulin-induced melanocytic differentiation through ERK1/2 inactivation, SCH772984 alone blocked ERK1/2 activation and time- Eribulin enhances melanin synthesis, TYR activity, and dependently upregulated the melanocytic differentiation markers, melanocytic differentiation markers in CCS cells in vitro MITF, TYR, TRP1, and TRP2, without any effect on EWSR1–ATF1 To determine whether eribulin directly induces melanocytic dif- expression, in both CCS cell lines (Fig. 4E). Melanin synthesis was also ferentiation in CCS cells, we assessed the quantity of cellular melanin in elevated in SCH772984-treated cells compared with untreated cells cultured CCS cells treated with 10 nmol/L eribulin or vehicle for (Supplementary Fig. S2A). Moreover, SCH772984 inhibited the pro- fi 96 hours. Melanin synthesis was signi cantly elevated in all eribulin- liferation of both CCS cell lines with an IC50 of about 60 nmol/L treated lines compared with vehicle-treated control cells (Fig. 3A). (Supplementary Fig. S2B). Because TYR is the rate-limiting enzyme in melanin production, we Tumor lysates extracted from Hewga-CCS and MP-CCS-SY xeno- also measured TYR activity by the conversion of uncolored 3,4- grafts were also examined for expression of ERK1/2 and melanocytic dihydroxy-L-phenylalanine substrate to darkly colored dopaquinone differentiation markers. Consistent with in vitro data, eribulin treat- product in CCS cell extracts. Coincident with increased melanin ment markedly decreased ERK1/2 phosphorylation and increased content, treatment with 10 nmol/L eribulin for 96 hours increased MITF, TYR, and TRP2 expression levels with no change in TYR activity significantly compared with vehicle-treated control cells EWSR1–ATF1 (Fig. 4F). These findings support the notion that (Fig. 3B). These observations suggest that the melanin synthesis inhibition of ERK1/2 phosphorylation causes increased MITF expres- pathway is activated by eribulin exposure in CCS cells. sion, at least in part, by direct effect of eribulin on CCS cells, which MITF is the most important transcription factor for melanogenesis evokes melanocytic differentiation. and is regulated in response to modulation of EWSR1–ATF1 activity in CCS (28). MITF also upregulates the expression levels of TYR, TRP1, Mitigation of tumor hypoxia by vascular remodeling elevates and TRP2, which catalyze melanin biosynthesis (8). We thus examined MITF protein via ERK1/2 inhibition and attenuates CCS cell the protein expression levels of EWSR1–ATF1, MITF, TYR, TRP1, proliferation and TRP2 by Western blot analysis. Consistent with TYR activity It has been reported that hypoxia in melanoma leads to transcrip- and melanin measurements, 10 nmol/L eribulin time-dependently tional suppression of MITF (11), suggesting that tumor reoxygenation enhanced the expression levels of MITF, TYR, TRP1, and TRP2, but concomitant with eribulin-induced vascular remodeling may mediate not of EWSR1–ATF1, in CCS cells (Fig. 3C), indicating induction of MITF protein upregulation and ensuing melanocytic differentiation. melanocytic differentiation by eribulin treatment. To test this notion, Hewga-CCS and MP-CCS-SY cells were grown in 1% oxygen, 3% oxygen, atmospheric oxygen, or atmospheric oxygen “ ” Eribulin induces melanocytic differentiation via inhibition of with the hypoxia mimetic CoCl2, which suppresses prolyl hydrox- ERK1/2 in CCS cells in vitro and in vivo ylase activity. As predicted, MITF protein expression was reduced in To dissect the mechanisms underlying MITF protein upregulation CCS cells under hypoxia (Fig. 5A and B). However, unexpectedly, in eribulin-treated CCS cells, we first investigated the effects of eribulin MITF transcription was not reduced in either cell line by hypoxia on MITF gene transcription in CCS cells. Surprisingly, MITF mRNA (Fig. 5C). Similar results were obtained in both cell lines following – fi expression was not induced by eribulin exposure (Fig. 4A), and CoCl2 treatment (Supplementary Figs. S3A S3C). These ndings EWSR1–ATF1 mRNA expression was not altered (Fig. 4B), suggesting suggest that hypoxia-mediated reduction of MITF expression in CCS that MITF protein upregulation is attributable to neither transcrip- cells occurs via a posttranscriptional mechanism.

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Figure 3. Eribulin upregulated melanin synthesis, TYR activity, and melanocytic differentiation markers in CCS cells. A and B, CCS cells were cultured for 96 hours with 10 nmol/L eribulin or vehicle. The melanin content (A; proportion of total protein relative to control) and TYR activity (B; relative to control) in CCS cells are shown. C, CCS cells were treated with 10 nmol/L eribulin for 0, 24, 48, 72, or 96 hours. EWSR1–ATF1, MITF, TYR, TRP1, and TRP2 protein expression levels were estimated by Western blotting. Data in A and B are presented as mean SD, n ¼ 3. , P < 0.01 vs. the control group.

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Figure 4. Eribulin induces melanocytic differentiation through inhibition of ERK1/2. A and B, CCS cells were treated with 10 nmol/L eribulin for 0, 24, 48, 72, or 96 hours. MITF (A) and EWSR1–ATF1 (B) mRNA levels in CCS cells were quantiﬁed using qRT-PCR (normalized to b-actin). C, Hewga-CCS cells were cultured for 48 or 96 hours in the presence of 10 nmol/L eribulin or vehicle. Cells were then treated with the protein synthesis inhibitor cycloheximide for 0, 3, 6, 12, or 24 hours, and protein lysates were prepared. MITF and b-actin protein expression levels were examined by Western blot analysis. Quantiﬁcation of relative MITF expression (normalized to b-actin) is shown. D, CCS cells were treated with 10 nmol/L eribulin for 0, 24, 48, 72, or 96 hours. Phosphorylation of ERK1/2 (p-ERK1/2) was assessed by Western blot analysis. E, Hewga-CCS and MP-CCS-SY cells were treated with the selective ERK1/2 inhibitor SCH772984 at 100 nmol/L for 0, 24, 48, 72, or 96 hours and then subjected to Western blot analyses with the indicated antibodies. F, Tumor tissues from Hewga-CCS and MP-CCS-SY xenografts at the endpoint were harvested, and lysates were prepared for Western blot analyses using the indicated antibodies. Data in A, B,andC are presented as mean SD, n ¼ 3. , P < 0.05 vs. the control group.

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Figure 5. Reoxygenation elevates MITF protein levels through inhibition of ERK1/2 signaling and reduces CCS cell growth. A and B, Hewga-CCS and MP-CCS-SY cells grown under 1% oxygen, 3% oxygen, or atmospheric (21%) oxygen in the absence (A and B, left) or presence (A and B, right) of 100 nmol/L SCH772984. HIF1a, MITF, and p-ERK1/2 protein expression levels were assessed by Western blot analysis. C, MITF mRNA levels in both CCS cells grown under 1%, 3%, or 21% oxygen for 24 hours were quantiﬁed using qRT-PCR. D, Cell proliferation in both CCS cells grown under 3%, 5%, or 21% oxygen for 96 hours was estimated by WST-1 assay. E, Wound healing assays were conducted under 3%, 5%, or 21% oxygen. Data in C, D,andE are presented as mean SD, n ¼ 3. , P < 0.05 and , P < 0.01 vs. the vehicle group.

Activation of ERK1/2 has been observed in several cancer cell and CoCl2, demonstrating that ERK1/2 signaling suppresses MITF lines under hypoxia and is associated with cellular migration, expression in hypoxic CCS cells (Fig. 5A and B; Supplementary angiogenesis, and enhanced resistance to apoptosis (30–32). Figs. S3A and S3B). Our data suggest that reoxygenation via Accordingly, we investigated whether ERK1/2 signaling was acti- eribulin-induced vascular remodeling (i.e., reversal of tumor hyp- vated in CCS cells under hypoxic conditions. Indeed, both hypoxic oxia) reduces ERK1/2 activity, leading to MITF upregulation and and CoCl2-treated CCS cells exhibited elevated ERK1/2 phospho- further melanocytic differentiation of CCS cells. Collectively, eri- activation (p-ERK1/2 expression; Fig. 5A and B; Supplementary bulin facilitates melanocytic differentiation through both direct Figs. S3A and S3B). The reciprocal relationship between MITF and effects on CCS cells and indirect effects through vascular remodel- ERK1/2 expression levels under hypoxia was further conﬁrmed ing, resulting in tumor reperfusion, ERK1/2 inactivation, reduced using SCH772984. As expected, blockade of ERK1/2 signaling MITF phosphorylation, greater MITF stability, and upregulation of reversed the attenuation of MITF protein levels induced by hypoxia melanocytic MITF target genes.

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Eribulin Effects on Clear Cell Sarcoma

Figure 6. Knockdown of MITF debilitates the antiproliferative effect of eribulin on CCS cells. KAS and MP-CCS-SY cells were transfected with MITF-speciﬁc siRNAs or a nontargeting negative control siRNA. A, MITF protein expression levels of both cells 48 hours after MITF knockdown were evaluated by Western blot analysis. B, Proliferation of both cells was measured by WST-1 assay during 1 to 3 days of culture. C, Cells were treated with different concentrations of eribulin for 48 hours and growth inhibition was measured by WST-1 assay. D, Cells were treated with 10 nmol/L eribulin for 48 hours, and expression of cleaved caspase-3 was assessed by Western blot analysis. E, Schematic presentation of the antitumor effects of eribulin on CCS cells. Data in B and C are presented as mean SD, n ¼ 3. , P < 0.05 and , P < 0.01 versus the control group.

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To examine if this indirect process influences other aspects of tumor Intratumoral hypoxia is an indicator of tumor aggression and poor behavior, we examined the effects of hypoxia on CCS cell proliferation prognosis (37). Indeed, numerous studies have documented a corre- and motility, behaviors necessary for tumor growth and invasion/ lation between intratumoral hypoxia and tumor progression. Eribulin metastasis. Both 3% and 5% oxygen significantly increased Hewga- was previously shown to increase tumor vessel density and tumor CCS and MP-CCS-SY cell proliferation (Fig. 5D). Moreover, both cell reoxygenation, resulting in enhanced antitumor activities compared lines showed increased migration under 3% and 5% oxygen in the with other cytotoxic agents (19, 20). Our in vivo data also demon- wound healing assay (Fig. 5E). These results suggest that reoxygen- strated that eribulin treatment enhanced MVD and normalized vessel ation affects CCS cell proliferation and motility, both of which may morphology, thus mitigating intratumoral hypoxia. These findings contribute to the therapeutic efficacy of eribulin. raise the possibility that eribulin can reduce tumor aggression by modifying the tumor microenvironment. Cheli and colleagues pro- Silencing of MITF rescues the antiproliferative effect of eribulin posed that a hypoxic microenvironment promoted melanoma aggres- on CCS cells sion by reducing MITF expression through the HIF1a-controlled To investigate whether MITF protein expression is involved in the transcriptional repressor chondrocytes protein 1 (DEC1), which sub- cytotoxic effect of eribulin on CCS cells, MITF was silenced employing sequently binds and suppresses the MITF promoter (11, 38). Contrary siRNA transduction in KAS and MP-CCS-SY cells. Two kinds of MITF to initial expectations, however, MITF gene transcription was not siRNAs efficiently silenced MITF in both cells (Fig. 6A). This caused reduced in hypoxic CCS cells, although MITF protein level was no significant difference in cell proliferation of both cells (Fig. 6B). downregulated. We then demonstrated that hypoxic CCS cells exhibit Importantly, MITF knockdown partly rescued the antiproliferative increased ERK1/2 phosphorylation (activation) and reduced MITF effect of eribulin in both cells (Fig. 6C). In addition, cleaved caspase-3 protein expression, suggesting that ERK1/2 is a post-transcriptional expression was decreased compared with the negative controls in both mediator of hypoxia-dependent MITF suppression in CCS cells. These cells by silencing of MITF (Fig. 6D). These findings suggest that MITF findings indicate that MITF modulation in CCS cells is clearly different upregulation, at least in part, contributes to the antiproliferative effect from modulation in melanoma cells, supporting the notion that CCS is of eribulin in CCS cells. a distinct disease from melanoma, despite designation as malignant melanoma of soft parts. The tumor vascular remodeling in response to eribulin is intriguing. Discussion Prior preclinical studies suggested eribulin's ability to improve delivery Phenotypic transition of cancer cells and alteration of the tumor of subsequently administered drugs (19, 20). Eribulin (1.0 mg/kg) microenvironment to a state less conducive to aggression are synergistically enhanced antitumor effects of the subsequent Doxil (a important factors affecting treatment outcome. In this study, we liposomal anticancer agents) in a non–small cell lung cancer xenograft demonstrate that eribulin exerts both direct and indirect anticancer model compared with vinorelbine (20). Likewise, prior treatment with effects on CCS cells, resulting in suppression of cell proliferation, 1.0 mg/kg eribulin significantly augmented the antitumor activity of facilitation of tumor differentiation, reduced invasive capacity, and capecitabine or paclitaxel in breast cancer xenograft models (19, 20). ultimately tumor shrinkage. Eribulin-induced CCS cell differenti- This could be beneficial in the case of residual tumor remaining after ation in vitro and in vivo was driven by augmentation of MITF treatment with eribulin. Furthermore, because conventional chemo- expression mediated through ERK1/2 inactivation. Furthermore, therapy is largely ineffective in patients with CCS, it would be useful for reoxygenation caused by eribulin-induced vascular remodeling CCS treatment to utilize eribulin to exert the effect of subsequently inhibited CCS cell proliferation and ERK1/2 signaling, leading to administered drugs more intensely. tumor differentiation (Fig. 6E). In this study, the high dose of 3 mg/kg eribulin was employed to Cancer can be regarded as a disease of cell differentiation. Cancer evaluate the efficacy of eribulin in CCS xenografts refractory to cells exhibit suspended differentiation properties compared with conventional chemotherapies in addition to 1 mg/kg eribulin. The normal cells, and maintaining this undifferentiated state is critical dose of 1 mg/kg eribulin is well used in preclinical studies and more for tumorigenesis (33, 34). Several agents with the ability to induce relevant to clinical practice. In our results, treatment with 1 mg/kg tumor differentiation, such as retinoic acid and 1a,25-dihydroxyvi- eribulin demonstrated remarkable inhibition of tumor growth, fi tamin D3, have been identi ed and tested over the last few years, with increased differentiation, and vascular remodeling compared with the some already in clinical use (35, 36). Therefore, targeting impaired vehicle control in both xenograft models, consistent with 3 mg/kg terminal differentiation could be a promising general therapeutic eribulin. Thus, Eribulin would be a promising therapeutic agent for strategy for malignancy. In CCS, we demonstrate that eribulin- patients with CCS. induced upregulation of MITF expression leads to tumor differenti- Collectively, this study demonstrates that eribulin exerts potent ation, and that silencing of MITF modestly attenuates the antiproli- anticancer activities on CCS cells both directly and via vascular ferative effect of eribulin on CCS cells. Thus, differentiation status of remodeling. Eribulin gives rise to nonmitotic effects on residual tumor tumor controlled by MITF expression levels may contribute somewhat such as alteration of differentiation status and tumor microenviron- to maintaining the sensitivity to eribulin in CCS. In addition, whether ment, following antimitotic, cytotoxic activity. The complexity of these MITF as an oncogene is intimately involved in CCS proliferation is not antineoplastic mechanisms suggests beneficial therapeutic possibilities clear in this study, whereas we found that MITF expression was of combination anticancer regimens including eribulin. implicated in CCS tumor differentiation and the cytotoxic effect of eribulin, and that eribulin remarkably inhibited CCS growth. These Disclosure of Potential Conflicts of Interest findings suggest that CCS growth is not dependent solely on MITF No potential conflicts of interest were disclosed. expression levels, and that other mechanisms, including mitotic and nonmitotic effects, as well as MITF upregulation may be potentially Authors’ Contributions important for eribulin activity in CCS cells. Further investigation into Conception and design: S. Nakai, T. Nakai, T. Wakamatsu, T. Tanaka, S. Takenaka, other mechanisms of action for eribulin is required. A. Myoui, H. Yoshikawa, N. Naka

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Development of methodology: S. Nakai, H. Yoshikawa, N. Naka Yukiko Eguchi, and Mari Shinkawa for their technical support and Enago for Acquisition of data (provided animals, acquired and managed patients, provided providing high-quality editing service. This study was supported by grants from the facilities, etc.): S. Nakai, N. Yasuda, H. Outani, S. Takenaka, N. Naka Japan Society for the Promotion of Science, JSPS KAKENHI [Grant Nos.: Analysis and interpretation of data (e.g., statistical analysis, biostatistics, JP16H05448 (to N. Naka), JP16K20050 (to S. Takenaka), JP18K16639 (to H. Tamiya) computational analysis): S. Nakai, Y. Imura, K. Hamada, N. Naka and JP18K16679 (to T. Tanaka)], the Osaka Medical Research Foundation for Writing, review, and/or revision of the manuscript: S. Nakai, Y. Imura, H. Outani, Intractable Diseases (to S. Nakai), the Japan Orthopaedics and Traumatology S. Takenaka, H. Yoshikawa, N. Naka Research Foundation, Inc. 372 (to Y. Imura) and 378 (to H. Tamiya), and the Administrative, technical, or material support (i.e., reporting or organizing data, Practical Research for Innovative Cancer Control from Japan Agency for Medical constructing databases): S. Nakai, H. Tamiya, N. Yasuda, T. Wakamatsu, Research and Development, AMED (to N. Naka). S. Takenaka, N. Naka Study supervision: S. Nakai, T. Wakamatsu, N. Araki, T. Ueda, H. Yoshikawa, The costs of publication of this article were defrayed in part by the payment of page N. Naka charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Acknowledgments We are grateful to Drs. Hiroshi Moritake and Tohru Sugimoto for kindly providing the human CCS cell line MP-CCS-SY, Dr. Takuro Nakamura for supplying the KAS Received April 2, 2019; revised September 16, 2019; accepted November 27, 2019; cell line, and Eisai Co., Ltd. for providing eribulin. We also thank Ryota Chijimatsu, published ﬁrst December 3, 2019.

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754 Mol Cancer Ther; 19(3) March 2020 MOLECULAR CANCER THERAPEUTICS

Eribulin Suppresses Clear Cell Sarcoma Growth by Inhibiting Cell Proliferation and Inducing Melanocytic Differentiation Both Directly and Via Vascular Remodeling

Sho Nakai, Hironari Tamiya, Yoshinori Imura, et al.

Mol Cancer Ther 2020;19:742-754. Published OnlineFirst December 3, 2019.

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