Significance of survivin as a prognostic factor and a therapeutic target in endometrial cancer

Gynecologic Oncology 141 (2016) 564–569

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Significance of survivin as a prognostic factor and a therapeutic target in endometrial cancer Agapiti Hipoliti Chuwa a, Kenbun Sone a, Katsutoshi Oda a,⁎, Yuji Ikeda a, Tomohiko Fukuda a, Osamu Wada-Hiraike a, Kanako Inaba a, Chinami Makii a, Makoto Takeuchi a, Shinya Oki a, Aki Miyasaka a, Tomoko Kashiyama a, Takahide Arimoto a, Hiroyuki Kuramoto b, Kei Kawana a, Tetsu Yano c, Yutaka Osuga a, Tomoyuki Fujii a a b c

Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo Bunkyo, Tokyo 113-8655, Japan Kanagawa Health Service Association, Kanagawa, Japan Department of Obstetrics and Gynecology, National Center for Global Health and Medicine, Tokyo, Japan

H I G H L I G H T S • Survivin expression was associated with poor prognosis in endometrial cancer. • YM155, a survivin inhibitor, induced apoptosis in endometrial cancer cells. • Survivin may serve as a a promising therapeutic target for endometrial cancer.

a r t i c l e

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Article history: Received 2 February 2016 Received in revised form 31 March 2016 Accepted 2 April 2016 Available online 16 April 2016 Keywords: Survivin Prognosis Apoptosis Molecular targeted therapy Endometrial cancer

a b s t r a c t Introduction. Survivin is an anti-apoptotic protein encoded by the baculoviral inhibitor of apoptosis repeatcontaining (BIRC5) gene and is upregulated in 83% of endometrial cancers. We aimed to elucidate the prognostic importance of BIRC5 expression, and evaluate survivin as a therapeutic target for endometrial cancer, by knockdown of BIRC5 and using the survivin inhibitor-YM155. Methods. RNA sequencing data in 234 patients with endometrial carcinoma was obtained from The Cancer Genome Atlas database, and analyzed using Kaplan-Meier method, log-rank test and Cox proportional hazard model. Expressions of survivin in 16 endometrial cancer cell lines were analyzed by western blotting. Knocking down effect on survivin expression was evaluated using a small interfering RNA (siRNA). The anti-proliferative and pro-apoptotic effects of YM155 were assessed with cell viability, flow cytometry, and annexin V/propidium iodide assays. Results. High expression of BIRC5 was associated with poor progression free survival (P = 0.006), and shown to be an independent prognostic factor (HR = 1.97, 95% CI = 1.29–4.5, P = 0.045). Survivin was upregulated in 14 of 16 (87.5%) endometrial cancer cell lines, compared with endometrial immortalized cells. Apoptosis was induced by knockdown of BIRC5 in all 3 cell lines examined. YM155 showed increased population of sub-G1 cells (P b 0.001) in all 16 cell lines, and IC50 values to YM155 were b 50 nm in 15 cell lines. YM155 dosedependently and significantly increased the apoptotic cell population in all 16 cell lines (P b 0.001). Conclusions. Present study indicated that survivin expression is a significant prognostic factor and that survivin is a promising therapeutic target for endometrial cancer. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Endometrial cancer is the most common type of malignant tumor of the female reproductive system in developed countries, with an age⁎ Corresponding author at: Department of Obstetrics and Gynaecology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-8655, Japan. E-mail address: [email protected] (K. Oda).

http://dx.doi.org/10.1016/j.ygyno.2016.04.003 0090-8258/© 2016 Elsevier Inc. All rights reserved.

adjusted rate (per 100,000) approaching 14 [1]. Young women are increasingly at risk for developing endometrial cancer owing to rising prevalence of obesity and nulliparity [2]. Chemotherapeutic options are limited, and there are no approved molecular-targeted therapies to date. As such, novel strategies are needed in order to improve the prognosis of high-risk patients. Survivin is a member of the inhibitor of apoptosis (IAP) family of proteins which is overexpressed in many malignant tumors and almost

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undetectable in normal tissues [3,4]. Survivin was shown to be upregulated in various types of tumor, including oesophageal (80%), pancreatic (76.9%), colorectal (63.7%), oral (72%), gastric (35%), and bladder cancer (57.8%) [5]. Survivin expression level has been linked to human malignancies with poor prognosis, such as acute myeloid leukemia, gastric, prostate and breast cancer [3,6–8]. Higher levels of survivin were reported in 83% of endometrial adenocarcinomas, with expression increasing from the proliferative subtype to hyperplasia and carcinoma cases [9]. To our knowledge, no previous study showed prognostic importance of survivin in endometrial cancer. Survivin is encoded by the baculoviral IAP repeat-containing protein 5 (BIRC5) gene located on chromosome 17q25 [10]. Survivin interacts with x-linked inhibitor of apoptosis protein (XIAP), encoded by BIRC4, and the resultant complex binds to caspases 3, 7 and 9 to block apoptosis [11]. Survivin is also involved in a range of biological processes, including vascular development and cell proliferation [12], in part through interaction with c-H-RAS protein and activation of RAS/phosphatidylinositol 3-kinase (PI3K) pathway [13,14]. Silencing of BIRC5 using small interfering RNA (siRNA) induced apoptosis in three endometrial cancer cell lines (Ishikawa3-H-12, AN3CA, and KLE) [15], implying the potential of survivin as a therapeutic target for endometrial cancer. YM155 (sepantronium bromide) is an imidazolium-based small molecule agent that potently suppresses survivin via two mechanisms: firstly, by interfering with the binding of zinc finger transcription factor specificity protein 1 to the survivin promoter [16]; and secondly, by disrupting a coactivator complex of interleukin enhancer-binding factor 3 and the nuclear RNA-binding protein p54nrb [17], thereby suppressing survivin at nm concentrations [18]. The chemical structure of YM155 has revealed that it is a highly specific transcriptional suppressor of survivin [18]. Several clinical trials with YM155 have been conducted in solid tumors [19,20]. In phase I/II clinical studies, YM155 was reportedly well tolerated, with adverse effects that were mostly of grades 1 or 2 such as pyrexia, nausea, and stomatitis. In a phase II clinical trial for advanced lung cancer, 5.4% of patients achieved partial response and 37.8% achieved disease stabilization by YM155 monotherapy [21]. However, there have been no comprehensive assessments in multiple types of endometrial cancer cell lines, and it is still unclear whether survivin is suitable for therapeutic target. In the present study, we assessed the prognostic significance of BIRC5 expression in endometrial cancer, then evaluated survivin as a therapeutic target for endometrial cancer cells by silencing BIRC5 and using survivin inhibitor-YM155. 2. Materials and methods 2.1. Kaplan-Meier survival analysis using RNA sequencing data Kaplan-Meier analysis based on BIRC5 gene expression in endometrial cancer was performed using RNA sequencing data from The Cancer Genome Atlas (TCGA) [22]. We downloaded the data from TCGA data portal (https://tcga-data.nci.nih.gov/tcga/) on January 26, 2016, and normalized the data using RSEM (RNA-Seq by Expectation Maximization) [23]. The data was available in 234 clinical endometrial cancer samples, and these cases were classified into two groups (BIRC5-low and BIRC5-high) by using a median expression level as a threshold. 2.2. Endometrial cancer cell lines and culture conditions Ishikawa3-H-12 was provided by Dr. Masato Nishida (Kasumigaura Medical Center, Ibaraki, Japan). HHUA was purchased from the RIKEN Cell Bank (Tsukuba, Japan). RL95-2, HEC 1B, KLE, and AN3CA were purchased from the American Type Culture Collection (Manassas, VA, USA). The endometrial immortalized cell (EIC) line was a gift from Dr. Satoru Kyo (Shimane University, Japan) [24]. Other cell lines were established by Hiroyuki Kuramoto [25]. HHUA, KLE, and RL95-2 were cultured in

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Dulbecco's Modified Eagle's Medium while the others were cultured in Eagle's Minimal Essential Medium (MEM). Both media contained 10% fetal bovine serum (FBS) and antibiotics. YM155 was purchased from Selleck (Houston, TX, USA) and stocked at − 80 °C as a 10-mm stock solution. The expression of estrogen receptor, and mutational status of PTEN, PIK3CA, and KRAS in these cell lines have been previously described (Supplementary Table 1) [26–30]. 2.3. Cell viability assay Cells (2 × 103/well) were seeded in 96-well plates with appropriate medium and allowed to attach for 24 h in a humidified incubator at 37 °C at 5% CO2. The medium was replaced with fresh medium containing various concentrations of YM155 (6.25 nm to 10 μm) for 72 h. A 10 μl volume of Cell Count Kit-8 solution (Dojindo, Tokyo, Japan) was added to each well for 3 h for the methyl thiazolyl tetrazolium (MTT) assay. The change in absorbance at 450 nm was measured on a microplate reader (BioTek, Winooski, VT, USA); cells treated with dimethyl sulfoxide were used for normalization to assess the growth-inhibitory effect of YM155. The experiment was repeated at least three times. 2.4. Cell cycle analysis Cells (4 × 105/well) were plated in 60-mm dishes. The medium was replaced after 24 h and cells were left untreated (control) or treated with different drug concentrations for 48 h. Cells were collected by trypsinization and washed twice with phosphate-buffered saline (PBS), then resuspended in pre-chilled 70% ethanol overnight at 4 °C. Cells were washed with PBS; 0.25 mg/ml RNase A (Sigma-Aldrich, St. Louis, MO, USA) was added, followed by incubation at 37 °C for 1 h. Cells were stained with propidium iodide (PI; 50 μg/ml) (Sigma-Aldrich) at 4 °C in the dark for 30 min. Cell cycle distribution was analyzed by flow cytometry on an Epics XL instrument (Beckman Coulter, Brea, CA, USA) using Cell Quest Prov.3.1 software (BD Biosciences, Franklin Lakes, NJ, USA). The experiment was repeated at least three times. 2.5. Detection of apoptosis Cells (4 × 105) were seeded in a 60-mm dish for 24 h; the medium was then replaced with fresh medium without drug or with 10 or 50 nm YM155. After 48 h, floating and trypsinized adherent cells were collected together, and resuspended in pre-diluted 1× binding buffer. Cells were stained with fluorescein isothiocyanate-conjugated (FITC) annexin V (BD Biosciences) solution and PI in the dark at room temperature for 15 min before analysis. Annexin V-FITC/PI double-positive cells were detected by flow cytometry and are expressed as a percentage of apoptotic cells. The experiment was repeated at least three times. 2.6. Western blotting Proteins were extracted from cells and analyzed by western blotting (BioRad, Hercules, CA, USA) as previously described [15] using primary antibodies against survivin, cleaved poly-ADP ribose polymerase (PARP), and cleaved caspase-7 (Cell Signaling Technology, Beverly, MA, USA). Antibody detecting β-actin (used as a loading control) was purchased from Sigma-Aldrich. Antibodies were used according to the manufacturers' recommendations. Protein bands were detected by Enhanced Chemiluminescence Select Solutions A and B (GE Healthcare Life Sciences, Piscataway, NJ, USA). 2.7. BIRC5 gene silencing Cells (8 × 105) were cultured in a 60-mm dish for 24 h. The medium was replaced with fresh medium containing 10% FBS without antibiotics. siRNA duplexes (siBIRC5-1 and -2), negative control siRNA, and lipofectamine-RNAiMax transfection reagent (Invitrogen, Carlsbad, CA,

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USA) complexes were separately prepared in Opti-MEM (Life Technologies, Carlsbad, CA, USA) and incubated for 15 min at room temperature, then added to the cells for 48 h before analysis. Gene knockdown and apoptosis were confirmed by western blotting and the annexin V-FITC/PI assay, respectively. 2.8. Statistical analysis Survival analysis was performed using Kaplan-Meier method and log rank test. Multi variate analyses were evaluated using the Cox proportional hazard model. For in vitro experiments, the significance of differences between groups was evaluated by t-test (one-way). Statistical analyses were performed using JMP v11 (SAS, Cary, NC, USA) and GraphPad Prism 6 (La Jolla, CA, USA). In all tests, differences were considered to be significant at P b 0.05. 3. Results 3.1. Survivin as an independent prognostic factor in endometrial cancer Firstly, we evaluated the impact of survivin on patient survival using RNA sequencing data from TCGA database. As shown in Fig. 1A, cases with high expression of BIRC5 showed significantly poor progression free survival (PFS) in endometrial cancer (P = 0.006). Although not statistically significant, cases with high expression of BIRC5 showed tendency to poor overall survival (OS) (P = 0.061) (Fig. 1B). Univariate analysis demonstrated that advanced stage (HR = 2.81, 95% CI = 1.56–5.02, P b 0.001), non-endometrioid histological subtype (HR = 2.05, 95% CI = 1.04–3.8, P = 0.04), and high expression of BIRC5 (HR = 2.34, 95% CI = 1.28–4.53, P = 0.005) were significantly associated with poor PFS (Table 1). Multivariate analysis identified expression of BIRC5 as an independent poor prognostic factor in endometrial cancer (HR = 1.97, 95% CI = 1.29–4.5, P = 0.045). 3.2. Survivin is overexpressed in endometrial cancer cell lines Then we explored the expression level of survivin in 16 endometrial cancer cell lines and immortalized endometrial cells by western blotting. Survivin expression was detected in all cell lines except for KLE and HHUA cells (Fig. 2). Relative expression of survivin in endometrial cancer cell lines revealed that 14 cell lines expressed survivin at a level higher than endometrial immortalized cells (Supplementary Table 1). No significant associations were found between survivin expression and mutational status of the RAS/PI3K pathway genes (PTEN, PIK3CA, and KRAS). 3.3. Inhibition of survivin induces apoptosis in endometrial cancer Next, we investigated whether silencing the BIRC5 induces apoptosis in three endometrial cancer cell lines, including HEC-180 which is

Table 1 Univariate/Multivariate analysis of BIRC5 expression in 234 endometrial cancers, using RNA sequencing data from TCGA. Univariate analysis

Multivariate analysis

Characteristics

HR

95% CI

P-value

HR

95% CI

P-value

Stage Advanced (III/IV) Early (I/II)

2.81

1.56–5.02

b0.001

2.42

1.29–4.5

0.006

Grade G3 G1/2

1.68

0.94–2.98

0.078

Histology Non-endometrioid Endometrioid

2.05

1.04–3.8

0.04

1.12

0.53–2.27

0.76

Age N63 ≤62

0.96

0.54–1.72

0.89

Muscle invasion N1/2 b1/2

1.22

0.62–2.3

0.56

BIRC5 expression High Low

2.34

1.28–4.53

0.005

1.97

1.29–4.5

0.045

derived from uterine papillary serous adenocarcinoma (UPSC). Cells were transfected with one of two different siRNAs targeting survivin (siBIRC5) or with negative control siRNA. Knockdown of BIRC5 was confirmed in all three examined cell lines (Fig. 3A). Suppression of survivin expression induced apoptosis in these three endometrial cancer cells (Fig. 3B), at rates which were statistically significant (Fig. 3C). These results imply an important role of survivin for cell growth and survival in endometrial cancer. 3.4. YM155 treatment inhibits endometrial cancer cell proliferation and increases the sub-G1 population Then we evaluated the anti-proliferative effect of survivin inhibitorYM155 against endometrial cancer cells. YM155 treatment inhibited growth in a dose-dependent manner in all 16 cell lines tested, with half-maximal inhibitory concentration (IC50) ranging from 14 to 150 nm (Fig. 4A and Supplementary Fig. 1). All cell lines except for Ishikawa3-H-12 were b50 nm of IC50. Notably, cell lines with low or without survivin expression, such as HEC-50B and KLE, showed similar sensitivity with other cells, therefore, no significant correlation was found between expression of survivin and IC50 of YM155 (Supplementary Fig. 2). To determine whether the growth-inhibitory effect of YM155 was due to cell cycle arrest or cell death, all 16 cell lines were treated with YM155 and analyzed by fluorescence-activated cell sorting. YM155 had a dose-dependent effect on cell cycling, inducing a

Fig. 1. Survival impact of BIRC5 expression. Significance of BIRC5 expression in endometrial cancer was identified using RNA sequencing data of 234 cases from TCGA database. The median expression level of BIRC5 was used as a threshold. Progression free survival (PFS) (A) and Overall survival (OS) (B) were analyzed using Kaplan-Meier analysis and log-rank test.

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Fig. 2. Overexpression of surviving in endometrial cancer cells. A total of 16 untreated endometrial cancer cell lines were examined for survivin expression by western blotting. Endometrial immortalized cell (EIC) line was used as a control.

significant increase in sub-G1 phase population in all 16 cell lines (P b 0.001: paired t-test) (Fig. 4B and Supplementary Fig. 3). Next, we evaluated the levels of cleaved caspase-7 and PARP to determine whether YM155 induces cell death via a caspase-dependent mechanism. Cleaved caspase-7 and PARP were activated in both HEC-180 (UPSC) and HEC-108 (endometrioid, Grade 3) cells by YM155 treatment, indicating the induction of apoptosis (Fig. 4C). Population of apoptotic cells was confirmed by the annexin V-FITC/PI assay. YM155 dose-dependently and significantly increased the apoptotic cell population in all 16 cell lines (P b 0.001: paired t-test) (Fig. 4D).

4. Discussion In this study, we showed that (i) high expression of BIRC5 was significantly associated with poor prognosis in endometrial cancer, and that (ii) inhibition of survivin exhibited anti-tumor effect, especially induction of apoptotic cell death, in 16 endometrial cancer cell lines.

Significance of survivin expression on endometrial cancer has not been fully clarified in endometrial cancer. There have been a few reports suggesting co-expression of multi-proteins, including survivin, as a prognostic factor in endometrial cancer [31,32]. However, one report suggested that survivin expression itself was not a prognostic factor in 62 endometrial cancer patients [33]. In our study, expression of BIRC5 was significantly associated with poor PFS in endometrial cancer. It is often difficult to correlate OS with genes' expressions in endometrial cancer because of the favorable prognosis [34], however, we found that high expression of BIRC5 showed strong tendency to poor OS in our study. To our knowledge, this is the first study that identified survival significance of survivin (BIRC5) expression itself in endometrial cancer. We also found that survivin was overexpressed in 14 of 16 endometrial cancer cell lines, and that survivin knockdown or pharmacological inhibition had anti-tumorigenic effects exerted via induction of caspase-mediated apoptosis. Moreover, we observed that the sensitivity to YM155 was not dependent on tumor grade, histological type, or

Fig. 3. Suppression of BIRC5 gene induces apoptosis in endometrial cancer. Cells were transfected with one of two siRNAs targeting BIRC5 (siBIRC5) or with negative control siRNA (siCT). (A) Knockdown of survivin by siBIRC5 was confirmed by western blotting. (B) Apoptosis was detected by the annexin V-FITC/PI assay. (C) The ratio of apoptotic cells was statistically compared between siCT and siBIRC5-1/siBIRC5-2 by using t-test.

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Fig. 4. YM155 suppresses endometrial cancer cell proliferation and induces apoptosis. (A) IC50 values to YM155 in 16 endometrial cancer cell lines. (B) Population of sub-G1 by treatment of YM155. Paired t-test was used for statistical analysis. (C) Induction of caspase-mediated apoptosis by YM155, evaluated by cleaved caspase-7 and cleaved PARP in western blotting. (D) Population of apoptotic cells in 16 cell lines. Paired t-test was used for statistical analysis.

survivin expression levels. Our observation that survivin is highly overexpressed in endometrial cancer cell lines is consistent with previous findings [12]. Survivin is known to be regulated by variety of oncogenes and tumor suppressor genes, including the PI3K/mTOR pathway genes [35]. The PI3K/mTOR signaling is frequently activated in endometrial cancer as a result of mutations in the pathway components, such as phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphatase and tension homolog (PTEN), and KRAS [36,37]. However, no significant correlation was found between survivin/KRAS/ PTEN/PIK3CA status and IC50 values to YM155. In our study, increase of sub-G1 phase population in cell cycle and induction of apoptosis were observed in all of 16 cell lines treated with YM155, irrespective of endogenous levels of survivin. These results suggest that survivinindependent effects of YM155 may be important for endometrial cancer inhibition. Although the specific capability of YM155 was shown to suppress survivin but not other members of the IAP family (or other antiapoptotic Bcl-2 family) proteins in prostate cancer cells [18], survivinindependent effects of YM155 have recently been reported. YM155 was shown to cause DNA-damaging effects with induction of γH2AX and pKAP1 [38,39]. In addition, anti-apoptotic proteins, Mcl-1 and specificity protein 1, were also suppressed by YM155 in oral cancer cells [40]. Further studies are needed in order to identify biomarkers that can predict sensitivity to YM155. This study has several limitations. First, biomarkers for predicting survivin expression and sensitivity to YM155 were not identified. Second, the experiment using xenograft model is required to clarify the anticancer effect of survivin inhibitor. Third, although clinical trials are underway in other types of tumor, the effect and tolerability of YM155 in endometrial carcinomas require further study. Finally, as YM155 was sensitive to endometrial cell lines with low survivin expression, survivin-independent effects of YM155 should be taken into consideration. In conclusion, we have demonstrated for the first time that BIRC5 gene expression levels are significantly associated with poor PFS and

show strong tendency to poor OS in endometrial cancer. Survivin is frequently overexpressed in endometrial cancer and its inhibition induces apoptosis even in cell lines derived from high-grade tumors. These findings suggest that survivin itself has significant prognostic importance in endometrial cancer and may be a suitable therapeutic target. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ygyno.2016.04.003. Disclosure The authors of this work have no competing interests to disclose. Acknowledgments The authors thank Otoe Hagiwara and Kaori Tomita for their support and assistance; Masato Nishida and Satoru Kyo for generously providing Ishikawa cells and EIC, respectively; and Editage for English language editing (http://englishediting.editage.com/). This work was financially supported by a Grant-in-Aid for Scientific Research (C) (grant number: 26462515 to K. Oda); Grants-in-Aid for Young Scientific Research (B) (15K20128 to K. Sone and 25861471 to T. Arimoto); a Grant-inAid for Research Activity Start-up (25893229 to Y. Ikeda and 15H06173 to T. Kashiyama) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. This study was also supported by a research program from the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct), Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T. Yano). References [1] C.E. DeSantis, C.C. Lin, A.B. Mariotto, R.L. Siegel, K.D. Stein, J.L. Kramer, R. Alteri, A.S. Robbins, A. Jemal, Cancer treatment and survivorship statistics, 2014, CA Cancer J. Clin. 64 (2014) 252–271. [2] A.S. Anderson, T.J. Key, T. Norat, C. Scoccianti, M. Cecchini, F. Berrino, M.C. BoutronRuault, C. Espina, M. Leitzmann, H. Powers, M. Wiseman, I. Romieu, European code

A.H. Chuwa et al. / Gynecologic Oncology 141 (2016) 564–569

[3] [4]

[5] [6] [7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

against cancer 4th edition: obesity, body fatness and cancer, Cancer Epidemiol. 39 (Suppl. 1) (2015) S34–S45. J. Song, H. Su, Y.Y. Zhou, L.L. Guo, Prognostic value of survivin expression in breast cancer patients: a meta-analysis, Tumour Biol. 34 (2013) 2053–2062. S. Khan, H. Ferguson Bennit, M.M. Asuncion Valenzuela, D. Turay, C.J. Diaz Osterman, R.B. Moyron, G.E. Esebanmen, A. Ashok, N.R. Wall, Localization and upregulation of survivin in cancer health disparities: a clinical perspective, Biologics 9 (2015) 57–67. P.K. Jaiswal, A. Goel, R.D. Mittal, Survivin: a molecular biomarker in cancer, Indian J. Med. Res. 141 (2015) 389–397. S. Fulda, Inhibitor of apoptosis proteins in hematological malignancies, Leukemia 23 (2009) 467–476. J. Waligorska-Stachura, A. Jankowska, R. Wasko, W. Liebert, M. Biczysko, A. Czarnywojtek, D. Baszko-Błasyzk, V. Shimek, M. Ruchała, Survivin—prognostic tumor biomarker in human neoplasm—review, Ginekol. Pol. 83 (2012) 537–540. J.L. Liu, W. Gao, Q.M. Kang, X.J. Zhang, S.G. Yang, Prognostic value of survivin in patients with gastric cancer: a systematic review with meta-analysis, PLoS One 8 (2013), e71930. S. Erkanli, F. Kayaselcuk, E. Kuscu, T. Bagis, F. Bolat, A. Haberal, B. Demirhan, Expression of survivin, PTEN and p27 in normal, hyperplastic, and carcinomatous endometrium, Int. J. Gynecol. Cancer 16 (2006) 1412–1418. C.H.A. Cheung, C.C. Huang, F.Y. Tsai, J.Y.C. Lee, S.M. Cheng, Y.C. Chang, Y.C. Huang, S.H. Chen, J.Y. Chang, Survivin – biology and potential as a therapeutic target in oncology, Onco Targets Ther 6 (2013) 1453–1462. Z. Song, Y. Xuebiao, M. Wu, Direct interaction between survivin and Smac/DIABLO is essential for the anti-apoptotic activity of survivin during taxol-induced apoptosis, J. Biol. Chem. 278 (2003) 23130–23140. J. Bhattacharyya, K. Mihara, M. Ohtsubo, S. Yasunaga, Y. Takei, K. Yanagihara, A. Sakai, M. Hoshi, Y. Takihara, A. Kimura, Overexpression of BMI-1 correlates with drug resistance in B-cell lymphoma cells through the stabilization of survivin expression, Cancer Sci. 103 (2012) 34–41. K.W. Sommer, C.J. Schamberger, G.E. Schmidt, S. Sasgary, C. Cerni, Inhibitor of apoptosis protein (IAP) survivin is upregulated by oncogenic c-H-Ras, Oncogene 22 (2003) 4266–4280. J. Pallares, Ljl Martinez-Guitar, X. Docket, D. Llobet, M. Rue, J. Palacios, J. Prat, X. Matias-Guiu, Survivin expression in endometrial carcinoma: a tissue microarray study with correlation with PTEN and STAT-3, Int. J. Gynecol. Pathol. 24 (2005) 247–253. T. Fukuda, O. Wada-Hiraike, K. Oda, M. Tanikawa, C. Makii, K. Inaba, Miyamoto Y. MiyasakaA, T. Yano, D. Maeda, T. Sasaki, K. Kawana, M. Fukayama, Fujii T. OsugaY, Putative tumor suppression function of SIRT6 in endometrial cancer, FEBS Lett. 589 (2015) 2274–2281. Q. Cheng, X. Ling, A. Haller, T. Nakahara, K. Yamanaka, A. Kita, H. Koutoku, M. Takeuchi, M.G. Brattain, F. Li, Suppression of survivin promoter activity by YM155 involves disruption of Sp1-DNA interaction in the survivin core promoter, Int. J. Biochem. Mol. Biol. 3 (2012) 179–197. N. Nakamura, T. Yamauchi, M. Hiramoto, M. Yuri, M. Naito, M. Takeuchi, K. Yamanaka, A. Kita, T. Nakahara, I. Kinoyama, A. Matsuhisa, N. Kaneko, H. Koutoku, M. Sasamata, H. Yokota, S. Kawabata, K. Furuichi, Interleukin enhancer-binding factor 3/NF110 is a target of YM155, a suppressant of survivin, Mol. Cell. Proteomics 11 (2012) (M111.013243). T. Nakahara, A. Kita, K. Yamanaka, M. Mori, N. Amino, M. Takeuchi, F. Tominaga, S. Hatakeyama, I. Kinoyama, A. Matsuhisa, M. Kudoh, M. Sasamata, YM155, a novel small-molecule survivin suppressant, induces regression of established human hormone-refractory prostate tumor xenografts, Cancer Res. 67 (2007) 8014–8021. R.J. Kelly, A. Thomas, A. Rajan, G. Chun, A. Lopez-Chavez, E. Szabo, S. Spencer, C.A. Carter, U. Guha, S. Khozin, S. Poondru, C. Van Sant, A. Keating, S.M. Steinberg, W. Figg, G. Giaccone, A phase I/II study of sepantronium bromide (YM155, survivin suppressor) with paclitaxel and carboplatin in patients with advanced non-small-cell lung cancer, Ann. Oncol. 24 (2013) 2601–2606. M.R. Clemens, O.A. Gladkov, E. Gartner, V. Vladimirov, J. Crown, J. Steinberg, F. Jie, A. Keating, Phase II, multicenter, open-label, randomized study of YM155 plus docetaxel as first-line treatment in patients with HER2-negative metastatic breast cancer, Breast Cancer Res. Treat. 149 (2015) 171–179. G. Giaccone, P. Zatloukal, J. Roubec, K. Floor, J. Musil, M. Kuta, R.J. van Klaveren, S. Chaudhary, A. Gunther, S. Shamsili, Multicenter phase II trial of YM155, a smallmolecule suppressor of survivin, in patients with advanced, refractory, non-smallcell lung cancer, J. Clin. Oncol. 27 (2009) 4481–4486.

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[22] Cancer Genome Atlas Research Network, C. Kandoth, N. Schultz, A.D. Cherniack, R. Akbani, Y. Liu, H. Shen, A.G. Robertson, I. Pashtan, R. Shen, C.C. Benz, C. Yau, P.W. Laird, L. Ding, W. Zhang, G.B. Mills, R. Kucherlapati, E.R. Mardis, D.A. Levine, Integrated genomic characterization of endometrial carcinoma, Nature 497 (2013) 67–73. [23] B. Li, C.N. Dewey, RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome, BMC Bioinformatics 12 (2011) 323. [24] S. Kyo, M. Nakamura, T. Kiyono, Y. Maida, T. Kanaya, M. Tanaka, M. Inoue, Successful immortalization of endometrial glandular cells with normal structural and functional characteristics, Am. J. Pathol. 163 (2003) 2259–2269. [25] H. Kuramoto, M. Nishida, T. Morisawa, M. Hamano, H. Hata, Y. Kato, E. Ohno, T. Iida, Establishment and characterization of human endometrial cancer cell lines, Ann. N. Y. Acad. Sci. 622 (1991) 402–421. [26] B.A. Lessey, A.E. Vendrov, L. Yuan, Endometrial cancer cells as models to study uterine receptivity, in: H. Kuramoto, M. Nishida (Eds.), Cell and Molecular Biology of Endometrial Carcinoma, Springer-Verlag, Tokyo 2003, pp. 267–279. [27] T. Fujisawa, M. Hamano, H. Hata, Y. Kamata, J. Watanabe, R. Sekimoto, H. Kuramoto, Establishment and characterization of two different types of new human endometrial adenocarcinoma cell lines (HEC-251 and HEC-265), Eur. J. Gynaecol. Oncol. 25 (2004) 299–304. [28] K. Oda, J. Okada, L. Timmerman, P. Rodriguez-Viciana, D. Stokoe, K. Shoji, Y. Taketani, H. Kuramoto, Z.A. Knight, K.M. Shokat, F. McCormick, PIK3CA cooperates with other phosphatidylinositol 3′-kinase pathway mutations to effect oncogenic transformation, Cancer Res. 68 (2008) 8127–8136. [29] K. Shoji, K. Oda, T. Kashiyama, Y. Ikeda, S. Nakagawa, K. Sone, Y. Miyamoto, H. Hiraike, M. Tanikawa, A. Miyasaka, T. Koso, Y. Matsumoto, O. Wada-Hiraike, K. Kawana, H. Kuramoto, F. McCormick, H. Aburatani, T. Yano, S. Kozuma, Y. Taketani, Genotype-dependent efficacy of a dual PI3K/mTOR inhibitor, NVPBEZ235, and an mTOR inhibitor, RAD001, in endometrial carcinomas, PLoS One 7 (2012), e37431. [30] A. Miyasaka, K. Oda, Y. Ikeda, O. Wada-Hiraike, T. Kashiyama, A. Enomoto, N. Hosoya, T. Koso, T. Fukuda, K. Inaba, K. Sone, Y. Uehara, R. Kurikawa, K. Nagasaka, Y. Matsumoto, T. Arimoto, S. Nakagawa, H. Kuramoto, K. Miyagawa, T. Yano, K. Kawana, Y. Osuga, T. Fujii, Anti-tumor activity of olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, in cultured endometrial carcinoma cells, BMC Cancer 14 (2014) 179. [31] S. Erkanli, F. Bolat, F. Kayaselcuk, B. Demirhan, E. Kuscu, COX-2 and survivin are overexpressed and positively correlated in endometrial carcinoma, Gynecol. Oncol. 104 (2007) 320–325. [32] M. Lambropoulou, N. Papadopoulos, G. Tripsianis, G. Alexiadis, O. Pagonopoulou, A. Kiziridou, V. Liberis, S. Kakolyris, E. Chatzaki, Co-expression of survivin, c-erbB2, and cyclooxygenase-2 (COX-2): prognostic value and survival of endometrial cancer patients, J. Cancer Res. Clin. Oncol. 136 (2010) 427–435. [33] R.T. Aksoy, A.T. Turan, N. Boran, A. Tokmak, B.Z. Isikdogan, H.G. Tulunay, M. Dogan, Lack of relation of survivin gene expression with survival and surgical prognostic factors in endometrial carcinoma patients, Asian Pac. J. Cancer Prev. 15 (2014) 6905–6910. [34] P. Morice, A. Leary, C. Creutzberg, N. Abu-Rustum, E. Darai, Endometrial cancer, Lancet 387 (2016) 1094–1108. [35] D.C. Altieri, Survivin, cancer networks and pathway-directed drug discovery, Nat. Rev. Cancer 8 (2008) 61–70. [36] K. Oda, Y. Ikeda, K. Kawana, Y. Osuga, T. Fujii, mTOR signaling in endometrial cancer: from a molecular and therapeutic point of view, Curr. Obstet. Gynecol. Rep. 4 (2015) 1–10. [37] K. Oda, Y. Ikeda, T. Kashiyama, A. Miyasaka, K. Inaba, T. Fukuda, K. Asada, K. Sone, O. Wada-Hiraike, K. Kawana, Y. Osuga, T. Fujii, Characterization of TP53 and PI3K signaling pathways as molecular targets in gynecologic malignancies, J. Obstet. Gynaecol. Res. (2016) (in press). [38] T.G. Glaros, L.H. Stockwin, M.E. Mullendore, B. Smith, B.L. Morrison, D.L. Newton, The “survivin suppressants” NSC 80467 and YM155 induce a DNA damage response, Cancer Chemother. Pharmacol. 70 (2012) 207–212. [39] B.H. Chang, K. Johnson, D. LaTocha, J.S. Rowley, J. Bryant, R. Burke, R.L. Smith, M. Loriaux, M. Muschen, C. Mullighan, B.J. Druker, J.W. Tyner, YM155 potently kills acute lymphoblastic leukemia cells through activation of the DNA damage pathway, J. Hematol. Oncol. 8 (2015) 39. [40] K. Sachita, H.J. Yu, J.W. Yun, J.S. Lee, S.D. Cho, YM155 induces apoptosis through downregulation of specificity protein 1 and myeloid cell leukemia-1 in human oral cancer cell lines, J. Oral Pathol. Med. 44 (2015) 785–791.