The Activity of Trabectedin As a Single Agent Or in Combination with Everolimus for Clear Cell Carcinoma of the Ovary

Published OnlineFirst May 27, 2011; DOI: 10.1158/1078-0432.CCR-10-2987

Clinical Cancer Cancer Therapy: Preclinical Research

The Activity of Trabectedin As a Single Agent or in Combination with Everolimus for Clear Cell Carcinoma of the Ovary

Seiji Mabuchi1, Takeshi Hisamatsu1, Chiaki Kawase1, Masami Hayashi1, Kenjiro Sawada1, Kazuya Mimura1, Kazuhiro Takahashi2, Toshifumi Takahashi2, Hirohisa Kurachi2, and Tadashi Kimura1

Abstract Purpose: The objective of this study was to evaluate the antitumor efficacy of trabectedin in clear cell carcinoma (CCC) of the ovary, which is regarded as an aggressive, chemoresistant, histologic subtype. Experimental Design: Using 6 human ovarian cancer cell lines (3 CCC and 3 serous adenocarcinomas), the antitumor effects of trabectedin were examined in vitro, and we compared its activity according to histology. We next examined the antitumor activity of trabectedin in both cisplatin-resistant and paclitaxel- resistant CCC cells in vitro. Then, the in vivo effects of trabectedin were evaluated using mice inoculated with CCC cell lines. Using 2 pairs of trabectedin-sensitive parental and trabectedin-resistant CCC sublines, we investigated the role of mTOR in the mechanism of acquired resistance to trabectedin. Finally, we determined the effect of mTOR inhibition by everolimus on the antitumor efficacy of trabectedin in vitro and in vivo. Results: Trabectedin showed significant antitumor activity toward chemosensitive and chemoresistant CCC cells in vitro. Mouse xenografts of CCC cells revealed that trabectedin significantly inhibits tumor growth. Greater activation of mTOR was observed in trabectedin-resistant CCC cells than in their respective parental cells. The continuous inhibition of mTOR significantly enhanced the therapeutic efficacy of trabectedin and prevented CCC cells from acquiring resistance to trabectedin. Conclusion: Trabectedin is a promising agent for CCC as a first-line chemotherapy and as a second-line treatment of recurrent CCC that had previously been treated with cisplatin or paclitaxel. Moreover, trabectedin combined with everolimus may be more efficacious for the management of CCC. Clin Cancer Res; 17(13); 4461–72. 2011 AACR.

Introduction 2–5). The lack of effective chemotherapy for recurrent CCC after frontline treatment is another important problem in Ovarian carcinoma is the fourth most common cause of the clinical management of CCC. Therefore, to improve the cancer death among women in the United States, with survival of patients with CCC, the development of novel more than 21,880 new cases diagnosed and 13,850 deaths treatment strategies in the setting of both first-line treat- estimated to have occurred in 2010 (1). ment and salvage treatment of recurrent disease are needed. Clear cell carcinoma (CCC) of the ovary has been known Trabectedin, which was formerly known as ecteinasci- to show poorer sensitivity to platinum-based frontline din-743 (ET-743), is an antineoplastic agent that was chemotherapy and to be associated with a worse prognosis originally derived from the Caribbean marine tunicate than the more common serous adenocarcinoma (SAC; refs. Ecteinascidia turbinate. It binds covalently to the minor groove of DNA, bending DNA toward the major groove and disrupting transcription, leading to G2–M cell-cycle Authors' Affiliations: 1Department of Obstetrics and Gynecology, Osaka arrest and ultimately apoptosis (6). Although the exact University Graduate School of Medicine, Osaka; and 2Department of mechanism of its action is not clear, it is thought that Obstetrics and Gynecology, Yamagata University, School of Medicine, the cytotoxic activity of trabectedin is based on the inhibi- Yamagata, Japan tion of transcription-dependent nucleotide-excision repair Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). (NER) via the trapping of the proteins responsible for NER, which induces cells to undergo apoptosis (7). S. Mabuchi and T. Hisamatsu contributed equally to this work. In a preclinical study, trabectedin showed significant Corresponding Author: Seiji Mabuchi, Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2 Yama- antitumor activity in vitro and in vivo against a range of daoka, Suita, Osaka 565-0871, Japan. Phone: 81-668-793-354; Fax: 81- solid tumor cells, including soft-tissue sarcoma (STS), 668-793-359; E-mail: [email protected] ovarian, breast, prostate, and renal cancers; melanoma; doi: 10.1158/1078-0432.CCR-10-2987 and non–small-cell lung cancer (8–11). On the basis 2011 American Association for Cancer Research. of the promising results of preclinical and clinical studies

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Translational Relevance everolimus has been approved for the treatment of renal cell carcinoma (19). In preclinical investigations, everoli- Clear cell carcinoma (CCC) of the ovary is a distinc- mus has been shown to inhibit the proliferation of ovarian tive subtype of epithelial ovarian cancer associated with cancer cells and enhance their sensitivity to cisplatin both a poorer sensitivity to platinum-based chemotherapy in vitro and in vivo (20–22), and its efficacy in ovarian and worse prognosis than the more common serous cancer patients is currently being evaluated in several phase adenocarcinoma. To improve survival, the identifica- I/II trials (23). However, no reports have addressed the role tion of a novel anticancer agent that more effectively of mTOR in the acquisition of resistance to trabectedin. targets CCC is necessary. Our results show that treat- In the current investigation, we evaluated the therapeutic ment with trabectedin significantly inhibited the growth efficacy of trabectedin as a single agent in vitro and in vivo. of CCC in vitro and in vivo. The growth-inhibitory effect We also examined the antitumor activity of trabectedin in of trabectedin on CCC cells was greater than those of both cisplatin-resistant and paclitaxel-resistant CCC cells. cisplatin, paclitaxel, and SN-38, which are clinically Finally, we investigated the role of AKT–mTOR signaling in employed as a part of the first-line chemotherapy for the mechanism of acquired resistance to trabectedin and this disease. This finding may be relevant for the plan- assessed the therapeutic potential of trabectedin in combi- ning of future clinical studies of first-line treatments of nation with everolimus in CCC cells. CCC of the ovary. Moreover, the significant antitumor activity of trabectedin against cisplatin- and paclitaxel- Materials and Methods resistant CCC shown in the current study may represent a novel treatment option for patients with recurrent Reagents/antibodies disease after first-line chemotherapy. Trabectedin was obtained from PharmaMar. Everolimus was obtained from Novartis Pharma AG. Cisplatin, paclitaxel, and 7-ethyl-10-hydroxycamptothecin (SN-38) were purchased from Sigma. LY294002 was purchased from Cell (10, 12, 13), trabectedin is now approved in Europe for the Signal Technology. Enhanced chemiluminescence (ECL) treatment of STS after anthracycline and ifosfamide failure Western blotting detection reagents were from Perkin and for treating patients who cannot receive these agents Elmer. Antibodies recognizing p70S6K, phospho-p70S6K (14). (Thr389), mTOR, phospho-mTOR (Ser2448), AKT, phos- In ovarian cancer, trabectedin has been studied in several pho-AKT (Ser473), and b-actin were obtained from Cell phase I and II clinical trials, showing a favorable toxicity Signaling Technology. The Cell Titer 96-well proliferation profile and promising activity in recurrent ovarian cancer assay kit was obtained from Promega. patients (15, 16). Following the encouraging results of these studies, a phase III trial investigating the activity of Drug preparation trabectedin plus liposomal doxorubicin versus liposomal Trabectedin was prepared as a 1 mg/mL stock solution doxorubicin in patients with recurrent ovarian cancer has in ethanol. Everolimus was diluted to the appropriate recently been conducted. As this study showed a survival concentration in double-distilled water just before its benefit of trabectedin-based combination chemotherapy, administration by gavage in the animal studies. For the trabectedin has become the focus of attention for research- in vitro analyses, everolimus was prepared in dimethyl ers investigating the treatment of epithelial ovarian cancer sulfoxide (DMSO) before being added to the cell cultures (17). as described previously (20–22). Cisplatin was dissolved As most ovarian cancer cell lines investigated in pre- in sterilized double-distilled water to a final concentra- vious preclinical studies of trabectedin have been from tion of 1 mmol/L. SN-38 and paclitaxel were dissolved in ovarianSAC(6,8,9)andonlyasmallnumberof DMSO to final concentrations of 10 and 100 mmol/L, patients with CCC histology were included in the pre- respectively. vious clinical studies (15–17), the therapeutic potential of trabectedin in patients with CCC is unknown. Because Cell culture of its nature as a newly developed anticancer agent, the The human ovarian CCC cell lines RMG1, RMG2, and mechanism of resistance to trabectedin is largely HAC2 were kindly provided by Dr. H. Itamochi (Tottori unknown. To further improve the prognosis of patients University, Tottori, Japan). These cells were cultured in with ovarian cancer, a deeper understanding of the phenol red–free Dulbecco’s modified Eagle’s medium mechanism of resistance to trabectedin and the devel- (DMEM Ham’s F-12; Gibco Ltd.) with 10% FBS, as reported opment of a novel treatment strategy to overcome this previously (20, 24–27). The human ovarian SAC cell lines resistance are needed. A2780, SKOV3, and Caov-3 cells were purchased from The mTOR is a serine/threonine kinase that plays a key American Type Culture Collection (ATCC). SKOV-3 cells role in cell growth, proliferation, survival, and tumor were cultured in McCoy’s 5A with 10% FBS. A2780 and angiogenesis (18). Everolimus, an orally bioavailable Caov-3 cells were maintained in DMEM (ATCC) with 10% mTOR inhibitor, acts as an allosteric inhibitor of mTOR. FBS. All 3 cell lines were maintained in a humidified On the basis of promising preclinical and clinical findings, incubator at 37 Cin5%CO2.

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Determination of cell number trabectedin and everolimus (Fig. 6). Five- to 7-week-old RMG1 and RMG2 cells were seeded into 6-well plates at a nude mice (n ¼ 48) were inoculated s.c. with 5 106 density of 104/well. After each incubation, the monolayers RMG1 (n ¼ 24) or RMG2 (n ¼ 24) into their right flank. were washed once with PBS, the cells were detached with When the resultant tumors had reached about 50 mm3 in trypsin, and viable cells were counted by trypan blue dye size, the mice were assigned to 1 of 4 treatment groups, exclusion. which received PBS, trabectedin (0.2 mg/kg, weekly), everolimus (2.5 mg/kg, 3 times a week), or trabectedin (0.2 mg/ Cell proliferation assay kg, weekly) plus everolimus (2.5 mg/kg, 3 times a week). An MTS assay was used to analyze the effects of tra- Caliper measurements of the longest perpendicular tumor bectedin, cisplatin, paclitaxel, SN-38, and everolimus on diameter were done every week to estimate tumor volume cell viability. Trabectedin is reported to show cytotoxic by using the following formula: V ¼ L W D p/6, activity in various cancer cell lines at nmol/L concentra- where V is the volume, L is the length, W is the width, and D tions. In a previous study, the IC50 values of trabectedin in is the depth as described previously (30). breast cancer cell lines ranged from 0.1 to 3.7 nmol/L (28). It was also reported that the IC50 values of SN-38 in Establishment of chemoresistant cell lines RMG1 cells and RMG2 cells were 37 and 34 nmol/L, Trabectedin-resistant sublines from RMG1, RMG2, and respectively (29). On the basis of these findings, cell SKOV3 were developed in our laboratory by continuous viability was assessed after the addition of trabectedin, exposure to trabectedin. Briefly, cells of both lines were cisplatin, paclitaxel, or SN-38 at concentrations ranging exposed to stepwise increases in the trabectedin concentra- from 0 to 10 nmol/L. After 48 hours of incubation, the tion. The initial trabectedin exposure was at a concentra- number of surviving cells was assessed by determination tion of 0.1 nmol/L. After the cells had regained their of the A490 nm of the dissolved formazan product after the exponential growth rate, the trabectedin concentration addition of MTS for 1 hour, as described by the manu- was doubled and then the procedure was repeated until facturer (Promega). Cell viability is expressed as follows: selection at 10 nmol/L was attained. The resulting trabec- Aexp group/Acontrol 100. tedin-resistant sublines, called RMG1-YR, RMG2-YR, and SKOV3-YR, were subcultured weekly and treated monthly Western blot analysis with 10 nmol/L of trabectedin to maintain a high level of The cells were treated with 10 nmol/L trabectedin with or chemoresistance. without everolimus for 24 hours, washed twice with ice- Paclitaxel-resistant CCC sublines (RMG1-PR and RMG2- cold PBS, and lysed in lysis buffer for 10 minutes at 4C. PR) from CCC cells (RMG1 and RMG2) were also devel- The resultant lysates were centrifuged at 12,000 g at 4C oped by continuous exposure to paclitaxel, as previously for 15 minutes, and the protein concentrations of the reported (31). Cisplatin-resistant CCC sublines were devel- supernatants were determined using the Bio-Rad protein oped in our laboratory as reported previously (20, 24). assay reagent. Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Statistical analysis Blocking was conducted in 5% nonfat milk in 1 Tris- Cell proliferation was analyzed by Wilcoxon’s exact test. buffered saline. Western blot analyses were conducted with The tumor volume of the trabectedin-treated mice was various specific primary antibodies. Immunoblots were compared with that of the PBS-treated mice and analyzed visualized with horseradish peroxidase–coupled goat using Wilcoxon’s exact test. A P value of less than 0.05 was anti-rabbit or anti-mouse immunoglobulin, using the considered significant. ECL Western blotting system (Perkin Elmer). Results Subcutaneous xenograft model All procedures involving animals and their care were In vitro growth-inhibitory effects of trabectedin on approved by the Institutional Animal Care and Usage CCC and SAC cell lines Committee of Osaka University, in accordance with insti- To examine the effects of trabectedin on the proliferation tutional and NIH guidelines. Initial experiments were of ovarian cancer cells of CCC or SAC origin, we conducted conducted to examine the effects of trabectedin on ovarian MTS assays using 6 human ovarian cancer cell lines. As CCC (Fig. 3). Five- to 7-week-old nude mice (n ¼ 24) were shown in Figure 1A, 48 hours of treatment with trabectedin inoculated s.c. into the right flank either with 5 106 inhibited the proliferation of ovarian cancer cells in a dose- RMG1 (n ¼ 12) or RMG2 (n ¼ 12) cells in 200 mL of PBS. dependent manner, showing 40% to 80% growth inhibi- When the tumors reached about 50 mm3, the mice inocu- tion at the highest drug concentration tested. During his- lated with RMG1 or RMG2 were assigned into 2 treatment tology comparisons, a differential sensitivity to trabectedin groups. The first group (n ¼ 6) was i.v. administered PBS was shown. CCC cells (RMG1, RMG2, and HAC2) were weekly for 4 weeks. The second group (n ¼ 6) was i.v. more sensitive to trabectedin than SAC cells (A2780, Caov- administered trabectedin (0.2 mg/kg) weekly for 4 weeks. 3, and SKOV-3). A second set of experiments was conducted to examine Using RMG1 and RMG2 cell lines, which exhibited the the antitumor effect of combination treatment involving greatest sensitivity to trabectedin, as shown in Figure 1A, we

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A CAS CCC Figure 1. 1.2 SKOV-3 Caov-3 A2780 RMG-1 RMG-2 HAC-2 Trabectedin inhibits the proliferation of CCC cells. A, the 1 sensitivity of CCC and SAC cells * to trabectedin. CCC (RMG1, 0.8 * * * * * * * RMG2, and HAC2) and SAC

ration * * 0.6 * * * (A2780, Caov-3, and HAC2) cells * * were treated with the indicated 0.4 * Prolifer * * concentrations of trabectedin in 0.2 the presence of 5% FBS for 48 hours. Cell viability was assessed 0 using the MTS assay. Points, Trabectedin concentrations ( 0, 0.1, 1, 10nM ) mean; bars, SD (*, significantly different from the control; B P < 0.05). B, comparison of the (i) (ii) growth-inhibitory activities of 4 RMG1 RMG2 anticancer agents. CCC cells 1.2 1.2 (RMG1 and RMG2) were treated with the indicated concentrations 1 1 * of trabectedin, SN-38, cisplatin, or 0.8 0.8 * paclitaxel in the presence of 5% tion ** tion FBS for 48 hours. Cell viability was 0.6 0.6 ** assessed using the MTS assay.

0.4 Paclitaxel Paclitaxel Points, mean; bars, SD Proliferat Proliferat 0.4 Cisplatin *** Cisplatin *** (*, significantly different from 0.2 SN-38 0.2 SN-38 cisplatin; **, significantly different Trabectedin Trabectedin from paclitaxel; and ***, 0 0 00.1110 00.1110 significantly different from SN-38; P < 0.05). Trabectedin concentrations (nmol/L) Trabectedin concentrations (nmol/L)

next compared the antitumor effect of trabectedin with In vivo growth-inhibitory effect of trabectedin on the those of cisplatin, paclitaxel, and SN-38 (Fig. 1B). As growth of ovarian CCC shown, both RMG1 and RMG2 cells showed poor sensi- To examine the in vivo growth-inhibitory effect of tra- tivity to cisplatin at the concentrations tested in the current bectedin, we employed an s.c. xenograft model in which study, which is consistent with the findings of a previous athymic mice were s.c. inoculated with RMG1 or RMG2 investigation (32). As previously reported, the activity of cells. When the tumors reached approximately 50 mm3, the SN-38 was greater than those of cisplatin and paclitaxel mice were randomized into 2 treatment groups, receiving (32). Importantly, the activity of trabectedin was signifi- PBS or trabectedin, as described in "Material and Methods." cantly greater than that of SN-38. These results suggest that Overall, drug treatment was well tolerated with no appar- trabectedin is a promising agent for the treatment of CCC ent toxicity throughout the study. Tumor volume was of the ovary. measured weekly after the start of treatment (Fig. 3B and D). The appearance of tumors 4 weeks from the first day of Effect of trabectedin on cisplatin- or paclitaxel- treatment is also shown in Figure 3A and C. The mean resistant CCC in vitro RMG1-derived tumor burden in mice treated with trabec- We next examined the growth-inhibitory effect of tra- tedin was 110.1 mm3 compared with 410.2 mm3 in the bectedin on chemoresistant CCC cells by using the MTS PBS-treated mice, and the mean RMG2-derived tumor assay. For this purpose, we employed cisplatin-resistant, burden in the animals treated with trabectedin was 98.2 paclitaxel-resistant, and their representative parental CCC mm3 compared with 330.6 mm3 in the placebo-treated cells, as described in "Materials and Methods." As shown in mice. Overall, treatment with trabectedin decreased the Figure 2, treatment with trabectedin inhibited the prolif- RMG1- and RMG2-derived tumor burden by 73% and eration of all of these CCC cells in a dose-dependent 70%, respectively, compared with PBS. These results indi- manner. The antitumor effects of trabectedin in the cispla- cate that trabectedin has significant antitumor effects as a tin- and paclitaxel-resistant CCC cells were slightly milder single agent on CCC. than those observed in their respective parental cells at lower concentrations (0.1–1 nmol/L). However, at a con- Increased AKT–mTOR activation in trabectedin- centration of 10 nmol/L, the antitumor effects of trabecte- resistant CCC cell lines din in the cisplatin- and paclitaxel-resistant CCC cells were We have previously reported that AKT–mTOR signal- equivalent to those observed in their respective parental ing is involved in the resistance of ovarian CCC cells to cells. cisplatin (20, 24). To examine whether AKT–mTOR

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A RMG1 B RMG2 1.2 1.2

1 1 *** 0.8 0.8

0.6 * 0.6 ** * 0.4 0.4 ** Proliferation RMG1 Proliferation RMG2 2 0. 2 RMG1-CR 0.20 2 RMG2-CR RMG1-PR RMG2-PR 0 0 0 0.1 1 10 0 0.1 1 10 Trabectedin concentrations (nmol/L) Trabectedin concentrations (nmol/L)

Figure 2. Effect of trabectedin on the growth of chemoresistant CCC cells. Cisplatin- and paclitaxel-resistant sublines were established as described in "Materials and Methods." A and B, parental (RMG1 and RMG2), cisplatin-resistant variant (RMG1-CR and RMG2-CR), and paclitaxel-resistant variant (RMG1-PR and RMG2-PR) cells were treated with the indicated concentrations of trabectedin in the presence of 5% FBS for 48 hours. Cell viability was assessed using the MTS assay. Points, mean; bars, SD (*, significantly different from RMG1-CR or RMG1-PR; **, significantly different from RMG2-CR or RMG2-PR; and ***, significantly different from RMG2-CR; P < 0.05). signaling is involved in trabectedin resistance in CCC, we mTOR in both trabectedin-resistant sublines and paren- established trabectedin-resistant sublines (RMG1-YR and tal chemosensitive cells by Western blotting. As shown in RMG2-YR) from RMG1 and RMG2 cells, as described in Figure 4C and D (i), significantly higher phosphoryla- "Material and Methods." To examine whether these tion of AKT and mTOR was observed in both trabecte- sublines had acquired resistancetotrabectedin,wefirst din-resistant cell lines than in their respective parental evaluated the sensitivities of these cell lines to trabecte- cell lines. Moreover, p70S6K, a downstream effector of dinbyusingtheMTSassay.AsshowninFigure4A,clear mTOR, was also hyperphosphorylatedinbothtrabecte- differential sensitivity to trabectedin was observed din-resistant cell lines. As shown in Figure 4C (ii) and D between the trabectedin-sensitive parental and respective (ii), the increased phosphorylation of AKT, mTOR, and trabectedin-resistant sublines. We next examined trabec- p70S6K was inhibited by treatment with a phosphoino- tedin-induced apoptosis in these cell lines. Treatment sitide 3-kinase (PI3K) inhibitor, LY294002. Moreover, with trabectedin induced cleavage of PARP in parental the increased phosphorylation of p70S6K was inhibited cells but not in trabectedin-resistant sublines (Fig. 4B), by treatment with everolimus. These results may indicate indicating that these sublines had acquired resistance to the involvement of AKT–mTOR signaling in trabectedin trabectedin. We next investigated the activity of AKT– resistance.

A C

Figure 3. Effect of trabectedin on the growth of CCC-derived tumor cells in vivo. Athymic nude mice were s.c. inoculated with RMG1 or RMG2 cells. When the tumors B D reached a mean size of about 50 RMG1 RMG2 mm3, the mice were i.v. 500 500 ) ) administered PBS or 0.2 mg/kg 3 Control 3 Control trabectedin weekly for 4 weeks. A 400 Trabectedin 400 Trabectedin and C, appearance of s.c. tumors. B and D, graphs depicting weekly 300 300 tumor volumes (mm3) for each treatment group. Points, mean; 200 200 bars, SD (*, significantly different from the PBS-treated mice; Tumor volume (mm 100 * Tumor volume (mm 100 * P < 0.05). * * 0 * * 0 * * 210 3 4 0 1 32 4 Weeks after start of treatment Weeks after start of treatment

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A B RMG1 (i) 1.2

0.8 *

0.6 * RMG1-YR RMG1 RMG2-YR RMG2

0.4 Proliferation * 0.2 RMG1 RMG1-YR 0 0 0.1 1 10 Trabectedin concentrations (nmol/L)

(ii) RMG2 Trabectedin 10 nmol/L 10 Trabectedin Trabectedin 10 nmol/L 10 Trabectedin Control Control Trabectedin 10 nmol/L 10 Trabectedin Control Control nmol/L 10 Trabectedin 1.2 Full-length PARP 1 Cleaved PARP 0.8 * 0.6 * 0.4 β-Acn Proliferation

0.2 RMG2 * RMG2-YR 0 0 0.1 1 10 Trabectedin concentrations (nmol/L)

C (i) (ii) RMG1-YR D (i) (ii) RMG2-YR Everolimus Control LY294002 RMG1 RMG1-YR Everolimus Control LY294002 RMG2 RMG2-YR

P-AKT P-AKT P-AKT P-AKT

AKT AKT AKT AKT

P-mTOR P-mTOR P-mTOR P-mTOR

mTOR mTOR mTOR mTOR

P-p70S6K P-p70S6K P-p70S6K P-p70S6K

p70S6K p70S6K p70S6K p70S6K

Actin Actin Actin Actin

Figure 4. The increased activation of AKT–mTOR signaling in trabectedin-resistant CCC cells. A and B, establishment of trabectedin-resistant variant cell lines. Trabectedin-resistant sublines were established as described in "Materials and Methods." A, trabectedin-sensitive parental (RMG1 and RMG2) and trabectedin-resistant variant (RMG1-YR and RMG2-YR) cells were treated with the indicated concentrations of trabectedin in the presence of 5% FBS for 48 hours. Cell viability was assessed using the MTS assay. Points, mean; bars, SD (*, P < 0.05). B, effect of trabectedin on the cleavage of PARP in trabectedin- sensitive parental and trabectedin-resistant variant cell lines. RMG1, RMG1-YR, RMG2, and RMG2-YR treated with 10 nmol/L trabectedin for 24 hours. The cells were harvested and then the lysates were subjected to Western blotting by using anti-PARP or anti-b-actin antibody. C and D, AKT/mTOR signaling activation in trabectedin-sensitive parental and trabectedin-resistant variant cells in vitro. RMG1, RMG1-YR, RMG2, and RMG2-YR cells were serum starved overnight in the presence or absence of 20 mmol/L LY294002 or 20 nmol/L everolimus. The cells were harvested, and equivalent amounts (30 mg) of protein were subjected to SDS-PAGE and blotted with anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-AKT (Ser473), anti-AKT, anti-phospho- p70S6K (Thr389), anti-p70S6K, or anti-b-actin antibodies.

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A RMG1 RMG1-YR (i) (ii) RMG1 (iii) Trabectedin Trabectedin + Everolimus Trabectedin

Trabectedin 96 h72 h48 h24 hC96 h72 h48 h24 hC h24 h48 h72 hC96 h24 h48 96 h72 -++ + hC h24 h48 96 h72 -+- - LY294002 P-AKT P-AKT --- + Everolimus

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B RMG2 RMG2-YR (i) (ii) RMG2 (iii) Trabectedin Trabectedin + Everolimus Trabectedin 96 h72 h48 h24 hC h24 h48 96 h72 96 h72 h48 h24 hC96 h72 h48 h24 hC h24 h48 h72 hC96 h24 h48 96 h72 -++ + Trabectedin -- + - LY294002 P-AKT P-AKT --- + Everolimus

AKT AKT P-p70S6K

P-mTOR P-mTOR p70S6K

mTOR mTOR Actin

P-p70S6K 4321 P-p70S6K

p70S6K p70S6K

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Figure 5. Treatment with trabectedin induces the prolonged activation of mTOR in CCC cells. A and B (i and iii), trabectedin induces the activation of mTOR signaling in an AKT-dependent manner. Trabectedin-sensitive parental (RMG1 and RMG2) and trabectedin-resistant variant (RMG1-YR and RMG2-YR) cells were treated with 10 nmol/L trabectedin in the presence or absence of 20 nmol/L of everolimus for 24 hours. Then, the medium was removed and replaced with medium with or without 10 nmol/L of everolimus. After an additional 0, 24, 48, or 72 hours of incubation, the cells were harvested. A and B (ii), RMG1 and RMG2 cells were treated with 1 nmol/L trabectedin in the presence or absence of 20 mmol/L LY294002 or 20 nmol/L of everolimus for 24 hours. Then, cells were harvested. Equivalent amounts (30 mg) of protein were subjected to SDS-PAGE and blotted with anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-AKT (Ser473), anti-AKT, anti-phospho-p70S6K (Thr389), anti-p70S6K, or anti-b-actin antibodies.

Trabectedin induces the activation of mTOR in an we next examined whether AKT–mTOR signaling is acti- AKT-dependent manner vated by trabectedin treatment in CCC cells. As shown in To further examine whether AKT–mTOR signaling is Figure 5A and B (i), treatment with trabectedin-induced involved in the mechanism of resistance to trabectedin, prolonged phosphorylation of AKT, mTOR, and p70S6K in

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Treatment schedule A Day 01234567 Group 1 PBS Medium change

Day 01234567

Group 2 Everolimus Medium change

Day 6543210 7

Group 3 Trabectedin Medium change

Day 6543210 7

Group 4 Trabectedin Everolimus Medium change

Day 6543210 7

Group 5 Trabectedin Everolimus Medium change

(i) RMG1 D 500 B (i) (ii) Control 2,500 200 RMG1 RMG1 Everolimus )

3 400 Trabectedin ) 3 2,000 * Trabectedin + Everolimus 150 Group 1 300 Group 3 1,500 Group 2 Group 4 Group 3 100 * Group 5 200 * 1,000 Group 4 ** Group 5 * * (mm volume Tumor * ** 50 100

Cell number (1 x 10 Cell number 500 ** * ** * 0 **** 0 ** 0 ** ** 3528211470 4942 1470 21 28 35 42 49 0 7 14 21 28 startafterDays of treatment Days after start of treatment Days after start of treatment C (i) (ii) (ii) RMG2 2,500 50 400 Control RMG2 RMG2 Everolimus 2,000 40 )

) Trabectedin 3 3 300 Group 1 * Trabectedin + Everolimus 1,500 Group 2 30 Group 3 ** Group 4 Group 3 200 Group 4 Group 5 * 1,000 Group 5 20 * * * ** * 100 500 10 (mm volume Tumor Cell number (1 x 10 Cell number * ** * ** 0 0 ** **** 0 ** ** ** 3528211470 4942 1470 21 28 35 42 49 0 7 14 21 28 startafterDays of treatment Days after start of treatment Days after start of treatment

Figure 6. Inhibition of mTOR activity by everolimus prevents CCC cells from acquiring resistance to trabectedin. A–C, in vitro experiments. A, treatment schedule. Trabectedin-sensitive parental cells (RMG1 or RMG2) were seeded into 6-well plates at a density of 104/well. Then, the cells were treated with 1 nmol/L trabectedin and/or 20 nmol/L everolimus in the presence of 5% FBS in accordance with the treatment schedule. The first group was treated with PBS (control). Then, the medium was removed and replaced with normal growing medium. The second group was treated with 20 nmol/L everolimus for 24 hours. Then, the medium was removed and replaced with normal growing medium. The third group was treated with 1 nmol/L trabectedin for 24 hours. Then, the medium was removed and replaced with normal growing medium. The fourth group was treated with 1 nmol/L trabectedin plus 20 nmol/L everolimus for 24 hours. Then, the medium was removed and replaced with normal growing medium. The fifth group was treated with 1 nmol/L trabectedin plus 20 nmol/L everolimus for 24 hours. Then, the medium was removed and replaced with medium containing 20 nmol/L everolimus. After 72 hours of incubation, more everolimus was added. All treatments were repeated every 7 days for 7 weeks. B and C (i), cell growth was monitored by weekly cell counting, as described in "Materials and Methods," and the results are shown. Points, mean; bars, SD (*, significantly different from group 2; **, significantly different from group 3, 4, or 5). (ii), the magnified graphs for groups 3 to 5 are shown. Points, mean; bars, SD (*, significantly different from group 3; **, significantly different from group 4). D, in vivo experiments. Athymic nude mice were s.c. inoculated with RMG1 or RMG2 cells. When the resultant tumors reached a mean size of about 50 mm3, the mice were treated with PBS, 0.2 mg/kg trabectedin once a week, 2.5 mg/kg everolimus 3 times a week, or weekly 0.2 mg/kg trabectedin plus 2.5 mg/kg everolimus 3 times a week, for 4 weeks. i and ii, graphs depicting weekly tumor volumes (mm3) for each treatment group. Points, mean; bars, SD (*, significantly different from the PBS-treated mice; **, significantly different from the trabectedin-treated mice; P < 0.05).

4468 Clin Cancer Res; 17(13) July 1, 2011 Clinical Cancer Research

Trabectedin for Clear Cell Carcinoma of the Ovary

both RMG1 and RMG2 cells (lanes 1–5). As shown in chemotherapy failed to prevent the development of trabec- Figure 5A and B (ii), trabectedin-induced p70S6K phos- tedin-resistant cells. However, importantly, when everoli- phorylation in RMG1 and RMG2 cells was inhibited by mus was administered every 3 days to continuously inhibit cotreatment with everolimus or LY294002 (lane 3). More- the activity of mTOR (group 5), the combination of trabec- over, trabectedin-induced p70S6K phosphorylation in tedin and everolimus resulted in the complete disappear- RMG1 and RMG2 cells was inhibited by cotreatment with ance of CCC cells. These results indicate that the continuous everolimus and trabectedin [Fig. 5A and B (i), lanes 6–10, inhibition of mTOR during trabectedin treatment is and Fig. 5A and B (ii), lane 4], suggesting that trabectedin required to prevent CCC cells from acquiring resistance induces P70S6K phosphorylation via the AKT–mTOR sig- to trabectedin. We further examined the in vivo growth- naling cascade. In contrast, in trabectedin-resistant CCC inhibitory effect of trabectedin in the setting of combination cells (RMG1-YR and RMG2-YR), treatment with trabecte- therapy with everolimus (Fig. 6C). In mice inoculated with din did not induce the phosphorylation of AKT, mTOR, or RMG1 cells and then treated with PBS, trabectedin, and p70S6K [Fig. 5A and B (iii)]. These results indicate that the everolimus, the mean tumor burden was 382.2, 240.7, and AKT–mTOR activation induced by trabectedin treatment in 141.5 mm3, respectively. The tumors that developed in the trabectedin-sensitive parental cells plays an important role mice treated with a combination of trabectedin and ever- during the acquisition of trabectedin resistance in CCC olimus were very small (mean tumor burden ¼ 48.1 mm3). cells. Similarly, in the mice inoculated with RMG2 cells and then We also investigated whether the activation of AKT– treated with PBS, trabectedin, everolimus, and trabectedin mTOR signaling is involved in the mechanism of resistance and everolimus, the mean tumor volume was 276.2, 184.0, to trabectedin in SAC cells. As shown, treatment with 87.2, and 32.2 mm3, respectively. Collectively, these results trabectedin induced prolonged activation of the AKT– indicate that everolimus enhanced the growth-inhibitory mTOR signaling pathway in SKOV3 cells(Supplementary effect of trabectedin in this model. Fig. S1A). Moreover, hyperactivation of AKT–mTOR signaling was also observed in the trabectedin-resistant SKOV3 Discussion cells (SKOV3-YR; Supplementary Fig. S1B). These results indicated that the effect of trabectedin on AKT–mTOR CCC of the ovary was recognized as a distinct histologic signaling is not unique to CCC cells. subtype of epithelial ovarian tumors by the World Health Organization in 1973 (33). The precise incidence of CCC is Continuous inhibition of mTOR by everolimus unknown, but CCC is the second most frequent histologic prevents CCC cells from acquiring resistance to subtype in Japan, with an incidence of more than 20%, trabectedin which is more frequent than the 5% incidence observed in Given the elevated basal activation of AKT–mTOR sig- Western countries (34, 35). naling found in trabectedin-resistant CCC cell lines In the setting of frontline chemotherapy, the response rate (Fig. 4C and D) as well as the prolonged activation of of CCC to conventional platinum-based chemotherapy AKT–mTOR signaling induced by trabectedin treatment in involving a platinum agent alone or in combination with trabectedin-sensitive parental cells (Fig. 5A and B), we cyclophosphamide and doxorubicin was reported to be as considered that the inhibition of AKT–mTOR signaling low as 11%. In contrast, SAC patients displayed a response in CCC cells hold promise as a method for overcoming rate of 72% (3). The efficacy of carboplatin–paclitaxel, the the resistance to trabectedin. Thus, we next examined the current standard regimen, against CCC has not been fully effect of mTOR inhibition by everolimus during the acqui- investigated.Aretrospectivereviewof6randomizedphaseIII sition of trabectedin resistance. clinical trials showed that patients with stage III CCC who Trabectedin-sensitive parental cells (RMG1 or RMG2) were treated with carboplatin–paclitaxel displayed shorter were treated with 1 nmol/L trabectedin and/or 20 nmol/ survival than those with other histologic subtypes of epithe- L everolimus in accordance with the treatment schedule lial ovarian cancer (2). However, a recent retrospective ana- shown in Figure 6A. On the basis of our in vitro experiment, lysis suggested that clear cell histology is not associated with which showed that the inhibition of mTOR activity by inferior survival (4). In the setting of salvage treatment of everolimus lasted for 72 hours [Fig. 5A and B (i), lanes recurrent CCC after frontline treatment,the response rates of 6–10], in the fifth group, everolimus was administered every various regimens were extremely low (5). Collectively, these 3 days for 1 month to continuously inhibit mTOR activity. previous findings suggested that CCC is associated with a As shown in Figure 6B, 24 hours of treatment with trabec- diminished sensitivity to chemotherapy and a worse prog- tedin or everolimus significantly decreased the number of nosis than other epithelial ovarian cancers. To improve viable CCC cells (group 1 vs. group 2 and group 1 vs. group survival, the development of new treatment strategies that 3); however, after roughly 3 weeks of exposure, the surviving target CCC more effectively is necessary. cells started to proliferate again as a result of acquired On the basis of a previous preclinical study suggesting resistance to trabectedin. When trabectedin was adminis- that SN-38, an active metabolite of irinotecan, is more tered in combination with everolimus for 24 hours (group effective in CCC cells than any other anticancer agent 4), the antitumor activity of trabectedin was significantly including cisplatin (32), the activity of irinotecan-based enhanced (group 3 vs. group 4); however, this combination combination chemotherapy had been investigated in

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Mabuchi et al.

several clinical studies in Japan (36–40). Following the tumor effects of trabectedin in the cisplatin- and paclitaxel- encouraging activity of irinotecan-based chemotherapy resistant CCC cells were slightly milder than those observed shown in these retrospective studies, the Japanese Gyne- in the respective parental cells, at the higher concentration cologic Oncology Group (JGOG) recently conducted a (10 nmol/L), the antitumor effects of trabectedin in the randomized phase II study comparing the activity of iri- cisplatin- or paclitaxel-resistant CCC cells were similar to notecan plus cisplatin versus carboplatin plus paclitaxel in those observed in their respective parental cells. Collectively, patients with CCC, in protocol JGOG3014 (41). In this these results suggest that cisplatin- and paclitaxel-refractory study, treatment with irinotecan plus cisplatin failed to CCCs are also candidates for trabectedin treatment. show its superiority over carboplatin plus paclitaxel with In this study, we investigated the role of the AKT–mTOR regard to progression-free survival, indicating the urgent signaling pathway in trabectedin resistance and found that need for the development of new antineoplastic agents for trabectedin-resistant CCC cell lines exhibit enhanced activa- patients with CCC of the ovary. tion of AKT–mTOR signaling compared with the corre- In the current study, we have shown that trabectedin sponding trabectedin-sensitive parental cell lines (Fig. 4C possesses significantly greater antitumor activity than SN- and D). Moreover, treatment with trabectedin induced pro- 38, which was reported to be the most effective agent for longed activation of AKT–mTOR signaling in CCC cells the treatment of CCC of the ovary. The in vitro growth- (Fig. 5). To the best of our knowledge, this is the first report inhibitory effect of trabectedin was greater than those of to show the involvement of AKT in the mechanism of other agents tested in the current studies (Fig. 1B). We also trabectedin resistance. AKT is known to be involved in evaluated the efficacy of trabectedin in vivo by using s.c. chemoresistance to various chemotherapeutic agents (46, xenograft models (Fig. 3). As previously reported, i.v. 47). As it has been previously reported that the inhibition of treatment with trabectedin was well tolerated with no AKT activity sensitizes human ovarian cancer cells to con- apparent toxicity. In mice that were s.c. inoculated with ventional anticancer agents (46, 48), we consider that the RMG1 or RMG2 cells, treatment with trabectedin signifi- inhibition of AKT–mTOR signaling holds promise for over- cantly inhibited tumor growth. Collectively, these findings coming trabectedin resistance. As AKT inhibitors have not indicate that trabectedin has significant clinical activity as a been convincingly proven to be clinically efficacious, we single agent for CCC in a setting of frontline therapy. employed a more clinically practical approach—the use of In previous clinical studies of ovarian cancer, trabectedin mTOR inhibitor, which is now approved for the clinical has usually been administered at concentrations ranging treatment of renal cell carcinoma, 90% of which shows a from 1 to 1.5 mg/m2 every 3 to 4 weeks (15–17). The clear cell histology. In the current study, we employed ever- maximum serum trabectedin concentration in these olimus as an mTOR inhibitor, as it showed significant in vitro patients was reported to be 1.8 ng/mL, which is equivalent and in vivo antitumor activity toward CCC cells (20). Impor- or higher than that observed in mice when trabectedin was tantly, treatment with everolimus in combination with tra- administered at a dose of 0.2 mg/kg (42, 43). The con- bectedin enhanced the efficacy of trabectedin in our centration of trabectedin used in the present in vitro experi- experimental model. Moreover, the continuous inhibition ments (1–10 nmol/L) has also been achieved in patients in of mTOR by everolimus prevented CCC cells from acquiring phase I clinical trails (43). On the basis of these previous resistance to trabectedin (Fig. 6). Because the RMG1-YR and findings, we consider that our treatment schedule is reason- RMG2-YR cells used in this study mimictheclinicalsituation able and clinically achievable. of resistance development in trabectedin-treated patients, The reason why CCC cells showed greater sensitivity to our results suggest that administering trabectedin in combi- trabectedin than SAC cells in the current study remains nation with mTOR inhibitor is a promising treatment of unknown (Fig. 1A). In a preclinical study, experiments with patients with CCC of the ovary. In addition, the finding that synchronized cells showed that cancer cells in the G1 phase the effect of trabectedin on AKT–mTOR signaling was also are more sensitive to trabectedin than cells in the G2 or S- observed in SAC cells indicates that treatment with trabecte- phase (44). As reported previously (45), in the current din in combination with everolimus might be efficacious for study, the CCC cells used in the current study showed a epithelial ovariancancer in general (Supplementary Fig. S1). longer doubling time than SAC cells. Moreover, when the We have to recognize the potential weakness of our cell-cycle distribution was determined by flow cytometry, experimental design; that is, we used a s.c. xenograft model. we found that the percentage of cells in the G1 phase was As peritoneal dissemination is the main process responsible significantly higher in the CCC cell lines than in the SAC for the progression of human ovarian cancer, the intraper- cells (data not shown). This may explain why CCC cells itoneal injection of cancer cells might more accurately showed greater sensitivity to trabectedin than SAC cells. model advanced disease. Therefore, further investigation An additional important finding of our study is the sig- using an intraperitoneal model or a genetically engineered nificant antitumor activity of trabectedin in cisplatin- and mouse model of ovarian cancer would be useful. paclitaxel-resistant CCCs (Fig. 2) because the lack of effective In conclusion, our findings indicate that trabectedin is a chemotherapy for recurrent CCC after frontline platinum- promising agent for treating CCC of the ovary both as a based combination chemotherapy is a major clinical pro- frontline treatment and as a salvage treatment of recurrence blem in the management of CCC. In the current study, after platinum- or paclitaxel-based chemotherapy. We although at lower concentrations (0.1–1 nmol/L), the anti- believe that our preclinical data provide significant

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Trabectedin for Clear Cell Carcinoma of the Ovary

support for future clinical trials of trabectedin in this Grant Support patient population. This work was supported in part by a grant-in-aid for Young Scientists (B), no. 21791554, from the Ministry of Education, Culture, Sports, Science Disclosure of Potential Conflicts of Interest and Technology of Japan and a grant-in-aid for General Scientific Research (B), no. 22390308. No potential conflicts of interest were disclosed. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Acknowledgments this fact.

We thank Ayako Okamura for her technical assistance. We also thank Received November 9, 2010; revised May 1, 2011; accepted May 7, 2011; Remina Emoto for her secretarial assistance. published OnlineFirst May 27, 2011.

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4472 Clin Cancer Res; 17(13) July 1, 2011 Clinical Cancer Research

The Activity of Trabectedin As a Single Agent or in Combination with Everolimus for Clear Cell Carcinoma of the Ovary

Seiji Mabuchi, Takeshi Hisamatsu, Chiaki Kawase, et al.

Clin Cancer Res Published OnlineFirst May 27, 2011.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-10-2987

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