0008-5472.CAN-12-0673.Full.Pdf

Published OnlineFirst September 20, 2012; DOI: 10.1158/0008-5472.CAN-12-0673

Cancer Molecular and Cellular Pathobiology Research

Chemotherapeutic Sensitivity of Testicular Germ Cell Tumors Under Hypoxic Conditions Is Negatively Regulated by SENP1-Controlled Sumoylation of OCT4

Yu-Chih Wu6, Thai-Yen Ling7, Shing-Hwa Lu10, Hung-Chih Kuo3,11,12, Hong-Nerng Ho8,9, Shauh-Der Yeh5, Chia-Ning Shen3,6,11, and Yen-Hua Huang1,2,4

Abstract Testicular germ cell tumors (TGCT) generally respond well to chemotherapy, but tumors that express low levels of the transcription factor OCT4 are associated with chemoresistance and poor prognosis. Hypoxia is known to induce drug resistance in TGCTs; however, the mechanistic basis for reduced expression of OCT4 and drug resistance is unclear. Here we show that hypoxia reduces OCT4 levels and increases the resistance of embryonal carcinoma (EC) cells to cisplatin and bleomycin. Furthermore, we show that the loss of OCT4 expression under hypoxia can be triggered by sumoylation, which was regulated by SUMO1 and the SUMO1 peptidase SENP1. Under hypoxic conditions, overexpression of SUMO1gg (the active form of SUMO1) not only increased the level of sumoylated OCT4 (Su-OCT4), but also decreased the stability of OCT4 protein. In addition, overexpression of SENP1 reduced the Su-OCT4 level induced by SUMO1gg overexpression, thereby maintaining OCT4 levels and enhancing chemosensitivity. Mechanistic investigations revealed that OCT4 sumoylation occurred at K123, as overexpression of an OCT4-K123R mutant effectively reduced the level of Su-OCT4 under hypoxic conditions. Taken together, our results showed that hypoxia reduces OCT4 expression levels in ECs to increase drug resistance and that these effects could be countered to ablate the suppressive effects of hypoxia on chemosensitivity. Our ﬁndings also highlight SENP1 as a potential therapeutic target for drug resistant TGCTs. Cancer Res; 72(19); 1–11. 2012 AACR.

Introduction cinomas (with pluripotent embryonal carcinoma, EC), mature Testicular germ cell tumors (TGCT) are the most common teratomas, yolk sac tumors (YST), and choriocarcinomas (1). malignancies among human germ cell tumors and are his- In the clinic, pluripotent seminomas and ECs of TGCTs are tologically classified into seminomas and nonseminomas (1). able to differentiate into other tumor types (e.g., teratomas, Seminomas are generally histologically uniform and resemble YSTs, and choriocarcinomas; 2, 3). Feldman and colleagues a transformed state of primordial germ cells (PGC). Nonse- further proposed that differentiated TGCTs are more malig- minomas are typically heterogeneous and include teratocar- nant and display higher resistance to chemotherapy (4). We therefore presumed the identification of a therapeutic target involved in the differentiation process of pluripotent TGCTs Authors' Affiliations: 1Department of Biochemistry, 2Graduate Institute of could improve the prognosis of the drug-resistant germ cell Medical Sciences, School of Medicine, College of Medicine; 3Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical Univer- tumors. sity; 4Center for Reproductive Medicine, 5Department of Urology, Taipei Previous work has shown that drug resistance of TGCTs may Medical University Hospital and Taipei Medical University; 6Graduate be modulated by retinoic acid treatment (5) and/or hypoxic Institute of Life Sciences, National Defense Medical Center; 7Institute of Pharmacology, 8Graduate Institute of Clinical Genomics, College of Med- stresses (6). Hypoxia is also known to be an important stimulus icine, National Taiwan University; 9Department of Obstetrics and Gyne- for tumor progression and drug susceptibility. As embryos cology, Division of Reproductive Endocrinology and Infertility, National develop under hypoxic conditions, most notably during the Taiwan University and Hospital; 10Department of Urology, National Yang- Ming University School of Medicine, and Department of Urology, Taipei City peri-implantation period, hypoxia is also believed to be Hospital; 11Genomics Research Center, and 12Institute of Cellular and involved in modulating pluripotent cells during early embryo- Organismic Biology, Academia Sinica, Taipei, Taiwan genesis (7). In support of this, the transcription factor OCT4, a Note: Supplementary data for this article are available at Cancer Research master regulator controlling the pluripotency of embryonic Online (http://cancerres.aacrjournals.org/). stem (ES) cells (8), PGCs (9), and induced pluripotent stem Corresponding Authors: Yen-Hua Huang, Taipei Medical University, 250 (iPS) cells (10, 11), is upregulated under hypoxic conditions in Wuxing Street, Taipei 110, Taiwan. Phone: 886-2-27361661 ext. 3150; Fax: – 886-2-27356689; E-mail: [email protected]; and Chia-Ning Shen, ES and a wide variety of cancer cells (12 14). In TGCTs, OCT4 is Genomics Research Center, Academia Sinica, 128 Academia Road, Sec- upregulated in pluripotent seminomas and ECs (15) and a lack tion 2, Nankang, Taipei 115, Taiwan. E-mail: [email protected] of OCT4 in ECs has been shown to increase cisplatin resistance doi: 10.1158/0008-5472.CAN-12-0673 in vitro and in vivo (16, 17). These results highlight the role of 2012 American Association for Cancer Research. OCT4 in modulating the differentiation status and drug

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susceptibility of EC cells. We therefore hypothesized that reagent (Fermentas) according to the manufacturer's hypoxia might increase the drug resistance of TGCT cells by instructions. To examine the OCT4 protein stability, plas- modulating the level of the pluripotent transcription factor mid-transfected HEK293T cells were cultivated at 37Cfor OCT4. 24 hours. Then the total cells were pooled together, split Sumoylation is a posttranslational modification involving equally into 6-cm plates to ensure the same cell transfection covalent linkage of the small ubiquitin-like modifier (SUMO) efficiency in each plate. After culture for an additional 24 to proteins, which regulates various cellular functions includ- hours, cycloheximide (CHX; 100 mg/mL, C4859, Sigma- ing nuclear-cytosolic transport, transcriptional regulation, Aldrich) was added to each plate, and cells were subse- apoptosis, stress responses, cell cycle progression, and protein quently harvested at the different indicated times for stability (18). The sumoylation process is reversible; sumoyla- SDS-PAGE separation and Western blot analysis. tion and desumoylation are controlled by ubiquitin-conjugating enzyme E2I (UBE2I or UBC9) and SUMO1/sentrin specific Xenograft tumor models peptidase (SENP) proteins (18). A previous report showed that For renal capsule grafting, 8-week-old nonobese diabetic/ the level of mouse Oct4 protein can be regulated by sumoyla- severe combined immunodeficient (NOD-SCID) mice were tion: the covalent linkage of SUMO1 at lysine 118 residue (K118) obtained from BioLasco Taiwan. NCCIT cells (106 cells) increased the stability of mouse Oct4 protein under normoxic infected with a lentivirus of an empty vector or HA-SENP1 conditions (19). However, whether the sumoylation process is were engrafted beneath the renal capsule of NOD-SCID mice involved in regulating the stability of human OCT4 protein in according to Szot and colleagues (20). At 8 weeks after implan- fi pluripotent germ cell tumors remains to be determined. tation, host mice were sacri ced by CO2 asphyxiation, and In the current work, we show that hypoxia reduces human sutures were dissected for histologic processing. For an in vivo OCT4 protein in EC cells via sumoylation at K123. Overexpres- drug susceptibility analysis, Empty- and HA-SENP1-NCCIT sion of SENP1 in hypoxic EC cells effectively increases the level cells (106 cells with Matrigel) were implanted into thymic nude of OCT4 protein and improves drug susceptibility in vitro and mice (BioLasco Taiwan) by a subcutaneous injection. The in vivo. These results indicate an important role for the SENP1- tumor volume (1/2 length width2) was measured every mediated desumoylation process in modulating the level of 3 or 4 days. On day 50, nude mice were injected intraperito- OCT4 protein and the drug sensitivity of EC cells under hypoxic neally with cisplatin (3 mg/kg/day) for 7 continuous days. conditions. Statistical analysis Materials and Methods All experiments were repeated at least 3 times with different Cell culture and hypoxic treatment individual samples. Data are expressed as the mean SD. NCCIT (CRL-2073), NT2 (CRL-1973), and HEK293T cells Statistical differences between sets of data were determined t P < were purchased from American Type Culture Collection. using paired 2-tailed Student test, with 0.05 considered fi NCCIT cells were maintained in RPMI-1640 medium (Invitro- signi cant. gen), and NT2 and HEK293T cells were maintained in Dulbec- co's Modified Eagle's Medium (Invitrogen). All cells were Results supplemented with 10% FBS at 37 C in a 5% CO2 humidified Hypoxia induces drug resistance and reduces OCT4 atmosphere. For hypoxic treatment, the cells were cultured at protein in EC cells 37 Cin5%CO2/1% O2/94% N2. Because hypoxia can promote the malignancy and drug resistance of EC cells (6), we initially examined whether In vitro sumoylation assay hypoxia reduces the drug susceptibility of EC cells by The cell-free in vitro sumoylation assay was carried out using altering the level of OCT4. Cisplatin and bleomycin, 2 of an in vitro sumoylation kit (UW8955, Enzo Life Sciences). HA- the most commonly used drugs for TGCT clinical chemo- tagged OCT4 expressed in HEK293T cells was harvested by therapy, were used to treat NCCIT and NT2 cells under immunoprecipitation with an anti-HA-agarose antibody normoxia (21% O2) and hypoxia (1% O2).AsshowninFig.1, (A2095, Sigma-Aldrich). In brief, the harvested HA-tagged hypoxia induced significant drug resistance to cisplatin and OCT4 protein was divided equally into 2 or 3 portions for bleomycin in both NCCIT and NT2 cells (Fig. 1A). Moreover, sumoylation reactions [SUMO activating enzyme (E1), SUMO hypoxia was found to decrease the level of OCT4 protein in a conjugating enzyme (E2, as UBC9), Mg-ATP] in the presence or time-dependent manner (Fig. 1B). Quantitative analysis absence of His6-SUMO1 protein (UW9195, Enzo Life Sciences) showed that hypoxia decreased the level of OCT4 protein and glutathione S-transferase (GST)-SENP1 (UW9760, Enzo by 60% in NCCIT cells and by 77% in NT2 cells after a 24- Life Sciences). Then the reactions were incubated at 37C for hour incubation (H24 of Fig. 1B, , P < 0.01). However, 60 minutes, washed extensively with PBS, and then stopped by hypoxia did not affect the mRNA level of OCT4 in EC cells adding sample buffer for SDS-PAGE separation and Western (Fig.1C).Thefinding that hypoxia decreased OCT4 protein blot analysis. level in NCCIT cells was confirmed using immunocytochemical staining combined with confocal microscopy (Supple- Protein stability assays mentary Fig. S1). These observations suggest that hypoxia HEK293T cells were transfected with either HA-OCT4-WT may regulate the stability of OCT4 protein via a posttrans- or HA-OCT4-K123R plasmids using a TurboFect transfection lational modification.

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Figure 1. Hypoxia decreases the drug susceptibility and OCT4 protein level in EC cells. A, drug susceptibility of NCCIT and NT2 cells against increasing concentrations of cisplatin and bleomycin under normoxia and hypoxia. Cell viability detected by the WST-1 assay is shown. Three individual experiments were carried out for each experimental condition. B, relative OCT4 protein expression levels in NCCIT and NT2 cells under hypoxia in different time periods analyzed by Western blot. H, hypoxia (H3, H6, H12, and H24 indicate that cells were incubated under hypoxic conditions for 3, 6, 12, and 24 hours; left); N, normoxia. Quantitative results are shown (right). C, OCT4 mRNA levels in NCCIT and NT2 cells analyzed by quantitative real-time PCR. Data are representative of at least triplicate experiments. , P < 0.05; , P < 0.01.

Hypoxia regulates the stability of the OCT4 protein in EC of 10 mmol/L MG132, a proteasome inhibitor, showed stronger cells through sumoylation colocalization of OCT4 protein (red) and EGFP-SUMO1 (green) Because sumoylation can modulate the stability of Oct4 in nuclei of NCCIT cells expressing SUMO1gg under hypoxic protein in mouse pluripotent cells (19), we examined whether conditions compared with either SUMO1aa overexpression or overexpression of EGFP-SUMO1gg (an active form of SUMO1) normoxic conditions (colocalization is indicated by yellow, could lead to sumoylation and destabilization of the OCT4 þMG132 vs. MG132 panel). Western blotting also showed protein in EC cells. that hypoxia enhanced the generation of high-molecular- Overexpression of EGFP-SUMO1gg resulted in a dose- weight sumoylated-OCT4 (Su-OCT4) in NCCIT cells expressing dependent decrease in the level of the OCT4 protein in NCCIT EGFP-SUMO1gg (Fig. 2D, lanes 5 and 6 vs. lane 4, indicated by cells (Figs. 2A and Supplementary Fig. S2). In the presence of an arrowhead), but not in NCCIT cells expressing EGFP- CHX, hypoxia further decreased the level of the OCT4 protein SUMO1aa (Fig. 2D, lanes 1–3). The unsumoylated OCT4 is in EGFP-SUMO1gg-NCCIT cells, compared with CHX treat- shown for both short- and long-exposure times (Fig. 2D, ment under normoxic conditions (Fig. 2B). These results indicated by arrows). These results further support OCT4 suggest that hypoxia-mediated sumoylation affects the stabil- sumoylation under hypoxia reducing the stability of the OCT4 ity of the OCT4 protein in NCCIT cells. protein in NCCIT cells. The finding that sumoylation of the OCT4 protein under hypoxia affects its stability was further supported by immu- Sumoylation of OCT4 protein occurs at K123 nofluorescent staining experiments. As shown in Fig. 2C, A previous study showed that sumoylation at K118 regulates immunofluorescent staining of NCCIT cells in the presence the stability of the Oct4 protein in mice (19). However, the

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Figure 2. Hypoxia regulates endogenous OCT4 protein stability through sumoylation in EC cells. A, effect of increasing concentrations of EGFP-SUMO1gg on endogenous OCT4 protein level in NCCIT cells under hypoxia. Note the inverse correlation of expression levels of EGFP- SUMO1gg and endogenous OCT4 protein. B, effect of EGFP- SUMO1aa or EGFP-SUMO1gg on endogenous OCT4 protein level in NCCIT cells with cycloheximide treatment (CHX, 100 mg/mL) under normoxia or hypoxia (left). Quantitative results are shown (right). C, cellular localization of EGFP-SUMO1 and endogenous OCT4 in EGFP-SUMO1aa- and EGFP-SUMO1gg-NCCIT cells with or without MG132 (10 mmol/L) treatment (colocalization indicated by yellow). Blue, 40, 6-diamidino-2- phenylindole; red, OCT4; green, EGFP-SUMO1. D, Western blot analysis showing endogenous sumoylated OCT4 (Su-OCT4, indicated by arrowhead) in EGFP- SUMO1gg-NCCIT cells under hypoxia. Unmodiﬁed endogenous OCT4 is indicated by arrows. b-Actin was used as an internal control. DAPI, 40, 6-diamidino-2- phenylindole; IB, immunoblot.

sumoylation site of the human OCT4 protein is unknown. Position 123 of the HA-OCT4 protein. The high-molecular- Using the sumoylation motif of mouse Oct4 (yKxD/E) as a weight sumoylated forms of the HA-OCT4-WT and HA- reference, K123 and K222 were identified as potential sumoy- OCT4-K222R proteins are shown in lanes 2 and 7, respec- lation sites in the human OCT4 (Fig. 3A). To examine sumoyla- tively, and GST-SENP1 effectively reduced the levels of the tion at these sites in the human OCT4 protein, an HA-tagged Su-OCT4 protein (lane 3 vs. lane 2). wild-type OCT4 expression plasmid (HA-OCT4-WT) and 3 To examine the direct interaction of endogenous OCT4 and HA-tagged mutant OCT4 expression plasmids were con- SUMO1 in EC cells, pull-down assays with antibodies against structed. The K123 amino acid residue was mutated to R endogenous OCT4 were conducted to isolate the OCT4 protein in the peptide produced from the HA-OCT4-K123R mutant from NCCIT and NT2 cells. Western blotting with anti-OCT4, expression plasmid, and the HA-OCT4-K222R construct anti-SUMO1, and anti-rabbit immunoglobulin G antibodies produced a peptide that contained the same substitution showed a high-molecular-weight Su-OCT4 (Fig. 3C). at Position 222. The double-mutant expression plasmid, HA- To further confirm this result, plasmids of HA-OCT4-WT, OCT4-2KR, produced a peptide with both K to R substitu- -K123R, -K222R, or -2KR were cotransfected with FLAG- tions. The wild type and mutant expression plasmids were SUMO1gg and UBC9 into HEK293T cells for in vivo coimmu- transfected into HEK293T cells, and the HA-OCT4 proteins noprecipitation assays. As shown in Fig. 3D, the Su-OCT4 was were pulled down by anti-HA agarose beads for cell-free detected in WT- and K222R-HEK293T cells (lanes 2 and 7), and in vitro sumoylation assays. As shown in Fig. 3B, the in vitro coexpression of SENP1 significantly reduced the level of Su- sumoylation assay showed a unique sumoylation site at OCT4 (lane 3 vs. lane 2). Sumoylation of OCT4 was further

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Figure 3. Hypoxia regulates OCT4 protein stability through sumoylation modification at the K123 amino acid residue. A, the potential sumoylation sites of the human OCT4 protein is shown. B, the sumoylation site at K123 in the human OCT4 protein was determined using an in vitro sumoylation assay. C, the direct sumoylation of the endogenous OCT4 protein in NCCIT and NT2 cells was shown using in vivo coimmunoprecipitation experiments. D, the direct sumoylation of HA-OCT4 at K123 in HEK293T cells was shown using anti-FLAG antibodies in the in vivo coimmunoprecipitation experiments. E, levels of OCT4 and Su-OCT4 proteins in HEK293T cells cotransfected with HA-OCT4-WT, -K123R, -K222R, and -2KR and EGFP-SUMO1aa or EGFP-SUMO1gg. F, the levels of OCT4 and Su- OCT4 proteins in HEK293T cells cotransfected with HA-OCT4-WT, -K123R, -K222R, and -2KR and EGFP-SUMO1aa or EGFP-SUMO1gg under normoxic and hypoxic conditions (left). The expression ratios of Su-OCT4: OCT4 for cell separately expressing the HA-OCT4-WT and the HA-OCT4-K222R proteins were observed (right). G, the half-life of the OCT4 protein in the presence of cycloheximide (100 mg/mL) in HEK293T cells transfected with the HA-OCT4-WT or the HA- OCT4-K123R plasmid under normoxic or hypoxic conditions (top). Decreased levels of OCT4 proteins in cells were quantified (bottom). Arrow, unsumoylated OCT4 (OCT4); arrowhead, sumoylated OCT4 (Su-OCT4); asterisk, nonspecific signals; IB, immunoblot; IP, immunoprecipitation; , P < 0.05. confirmed by cotransfection of HEK293T cells with HA-OCT4 Because hypoxia significantly decreased the level of OCT4 (WT, K123R, K222R, or 2KR) and the EGFP-SUMO1aa or the protein in EC cells (Fig. 1), we examined whether hypoxia EGFP-SUMO1gg plasmid. As shown in Fig. 3E, EGFP-SUMO1gg regulates OCT4 protein stability through sumoylation. For this significantly increased the sumoylation of the wild-type OCT4 purpose, HEK293T cells were cotransfected with HA-OCT4 protein in HEK293T cells (lane 4 vs. lane 3), and the level of (WT, K123R, K222R, or 2KR) and the EGFP-SUMO1gg plasmids sumoylated HA-OCT4-WT was significantly reduced by coex- and were incubated under normoxia or hypoxia with addition pression of HA-SENP1 (lane 5 vs. lane 4). High-molecular- of MG132. As shown in Fig. 3F, under normoxic conditions, the weight Su-OCT4 was not detected in cells cotransfected with Su-OCT4 protein was detected in HA-OCT4-WT- and HA- the HA-OCT4-K123R (lane 7 vs. lane 4) and HA-OCT4-2KR OCT4-K222R-HEK293T cells (lanes 1 and 7, indicated by an mutant constructs (lane 11 vs. lane 4), respectively. These arrowhead). Cotransfection with HA-OCT4-K123R or HA- results show that sumoylation of the human OCT4 protein OCT4-2KR expression plasmids completely blocked Su-OCT4 occurs at K123. formation under normoxia (lanes 5 and 9). These results

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confirm that the sumoylation of human OCT4 protein occurs group, NCCIT cells that expressed HA-SENP1 had more intense at K123. We also found that hypoxia significantly increased the OCT4 staining (Fig. 4D; HA-SENP1 panel vs. Empty vector Su-OCT4 level in cells that were cotransfected with HA-OCT4- panel). Lower cytokeratin expression was correlated with WT (lane 2 vs. lane 1) or HA-OCT4-222R (lane 8 vs. lane 7). The intense OCT4 staining and was observed in NCCIT cells that unsumoylated OCT4 protein level is shown in both long and expressed HA-SENP1 compared with empty vector control short exposures (indicated by arrows). Quantitative analysis cells. Quantitative analysis confirmed that there were more showed that the ratio of Su-OCT4/OCT4 in HA-OCT4-WT- and OCT4-positive cells in the tumor generated from transplanta- HA-OCT4-K222R-HEK293T cells was significantly increased tion of SENP1-overexpressed NCCIT cells than in the tumor under hypoxia (Fig. 3F, right panel). generated from transplantation of control NCCIT cells. The To further examine whether hypoxia decreased the stability finding that OCT4 staining was negatively correlated with of OCT4, HEK293T cells were transfected with HA-OCT4-WT cytokeratin staining shows that SENP1 suppresses the differ- and HA-OCT4-K123R expression plasmids before treatment entiation of NCCIT cells in vivo via maintaining the level of the with CHX, and cells were harvested at various time points. As OCT4 protein (Fig. 4E). shown in Fig. 3G, hypoxia decreased the half-life of OCT4 in the presence of CHX, compared with similar normoxic conditions SENP1 increases the drug sensitivity of EC cells in (WT panel), and the wild-type OCT4 protein displayed a longer hypoxic conditions half-life than did the mutant HA-OCT4-K123R protein under Low levels of OCT4 are known to be associated with higher hypoxic conditions. These results collectively show that hyp- drug resistance in ECs (16, 17). Because SENP1 can increase oxia enhances sumoylation of the human OCT4 protein at OCT4 protein stability in hypoxic conditions (Fig. 4), we K123, which leads to a reduction in the stability of the human examined the effect of SENP1 on the drug sensitivity of EC OCT4 protein. cells. As shown in Fig. 5, higher drug sensitivity was observed in SENP1-overexpressing NCCIT cells compared with control SENP1 suppresses OCT4 sumoylation and increases NCCIT cells (Fig. 5A; , P < 0.05; , P < 0.01). The IC50 value OCT4 stability under hypoxic conditions of HA-SENP1-overexpressing NCCIT cells in hypoxic condi- SENP1 is a negative regulator of protein sumoylation. Ini- tions (21.63 0.12 mmol/L for cisplatin and 7.33 1.04 mmol/L tially, we found that the level of SENP1 transcription was lower for bleomycin) was greatly decreased to a level similar to that of in NCCIT/NT2 cells compared with human ES cells (Supple- control or HA-SENP1-overexpressing NCCIT cells in normoxic mentary Fig. S3). We further found that overexpression of conditions (16.9 0.83 mmol/L for cisplatin and 3.72 0.52 SENP1 in NCCIT cells suppressed hypoxia-induced reduction mmol/L for bleomycin; Supplementary Table S3). There was no of the OCT4 protein. NCCIT cells were infected with a lentivirus significant difference in cell viability between control and HA- carrying an empty vector (vector control) or HA-SENP1-IRES- SENP1-overexpressing NCCIT cells in either normoxic or tRFP (HA-SENP1). As shown in Fig. 4A, immunofluorescent hypoxic conditions (Supplementary Fig. S5). Xenograft experi- staining showed that higher expression of SENP1 (indicated by ments using nude mice (n ¼ 35) were conducted to examine tRFP, red fluorescence) correlated with the more intense OCT4 the effect of SENP1 on the drug susceptibility of EC cells in vivo. staining (indicated by fluorescein isothiocyanate, green fluo- In these experiments, Control NCCIT cells (infected with rescence). Western blotting further confirmed that SENP1 can empty lentiviral vector) and SENP1-overexpressing NCCIT suppress hypoxia-induced reduction of the OCT4 protein in cells (infected with lenti-HA-SENP1) were injected into nude NCCIT cells. Quantitative analysis showed that overexpression mice, and the tumor volume was determined 50 days postin- of HA-SENP1 significantly increased the OCT4 protein level in jection (Fig. 5B). The tumor-bearing mice were then treated hypoxic conditions (Fig. 4B; , P < 0.05, n ¼ 3). The increased with cisplatin for 7 continuous days (3 mg/kg/day, via stability of the endogenous OCT4 protein resulting from intraperitoneal injection) and tumor size was measured on coexpression of SENP1 was further confirmed by CHX treat- days 1, 4, and 7. When compared with the day 1 control ment. The HA-SENP-NCCIT cells contained a higher level of group, cisplatin significantly decreased the size of the tumor the OCT4 protein after the 12 hours incubation under nor- in the HA-SENP1 group (35% vs. 73% decrease; Fig. 5C). moxic conditions, compared with the vector control group Hematoxylin and eosin (H&E) staining showed that the (Supplementary Fig. S4). tumors in the HA-SENP1 group were poorly differentiated In contrast, knockdown of UBC9, a SUMO-conjugating compared with the control group tumors (Fig. 5D). The enzyme, in cells by shRNAs blocked hypoxia-induced reduction positive hypoxia-inducible factor-1a (HIF-1a)expression of the OCT4 protein (Fig. 4C, H24, shLuc vs. shUBC9). These indicates the hypoxic microenvironment in tumor regions results support the assertion that sumoylation of the OCT4 (Fig.5D).Importantly,fewertRFP-labeledandp21-expres- protein under hypoxia affects its stability in NCCIT cells. sing cells were observed in the HA-SENP1 groups, suggesting Regulation of the OCT4 protein stability by SENP1 was that SENP1-overexpressing NCCIT cells were sensitive to further examined using tumor-bearing mice models. Control cisplatin treatment (Fig. 5D). Positive staining of cleaved NCCIT cells (infected with an empty lentiviral vector) and caspase-3 in the HA-SENP1 groups (Fig. 5D) further sup- SENP1-overexpressing NCCIT cells (infected with lenti-HA- ported the higher cisplatin susceptibility of SENP1-overex- SENP1) were transplanted into renal capsules of NOD-SCID pressing NCCIT cells. Quantitative analysis confirmed the mice. As shown in Fig. 4D, tRFP staining marked grafted tumor observation and showed that SENP1 increased the suscep- tissues (Fig. 4D, tRFP panel). Compared with the empty vector tibility of NCCIT cells to cisplatin treatment in vivo (Fig. 5E).

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Figure 4. SENP1 rescues hypoxia- induced OCT4 instability in EC cells in vitro and in vivo.A, immunocytochemical staining showing the colocalization of HA- SENP1-IRES-tRFP (tRFP, red), OCT4 (green), and 40, 6-diamidino-2- phenylindole (blue) in hypoxic NCCIT cells. An arrowhead indicates an NCCIT cell with low SENP1 and OCT4 protein expression. Dotted lines show the cells with high expression of both SENP1 and OCT4 proteins. DAPI, 40, 6-diamidino-2- phenylindole. B, relative OCT4 protein levels in Empty-NCCIT and HA-SENP1-NCCIT cells after 12 and 24 hours of hypoxic incubation. The dashed line indicates the OCT4 protein level in cells after 24 hours of normoxic incubation. Three individual experiments were carried out for each experimental condition. C, OCT4 protein levels in shLuc- or shUBC9-NCCIT cells under hypoxia. shLuc, shRNA of luciferase; shUBC9, shRNA of UBC9 (left). Right, quantitative results. D, immunohistochemical staining of tRFP, OCT4, and cytokeratin in the tumor part of Empty- and HA- SENP1-NCCIT cells. Bar, 100 mm; K, kidney regions; T, tumor regions. E, quantitative results of D using ImageScope software in different methods. n ¼ 3; , P < 0.05; , P < 0.01.

SENP1 enhances the drug sensitivity of EC cells by NCCIT control cells (Fig. 6B, lanes 1–6), clearly demon- maintaining the level of the OCT4 protein strating an inverse relationship between the level of endog- To examine whether the SENP1 protein increases the enous OCT4 protein and the IC50 value (Fig. 6B). These drug sensitivity of EC cells by maintaining the OCT4 protein results show that SENP1 regulates susceptibility to cisplatin stability, we knocked down the expression of the endoge- and bleomycin through its effects on the level of OCT4 nous OCT4 protein in NCCIT cells in which HA-SENP1 was proteininECcells. simultaneously overexpressed in the presence of cisplatin or bleomycin. Knockdown of OCT4 and the overexpression of HA-SENP1 in NCCIT cells are shown in Fig. 6A. Under both Discussion normoxic and hypoxic conditions, OCT4 silencing signiﬁ- OCT4 is a key transcription factor involved in regulating the cantly increased IC50 tocisplatinandbleomycininboth self-renewal and pluripotency of ES cells and PGCs (8, 9). the HA-SENP1-NCCIT cells (Fig. 6B, lanes 7–12) and the Recent work has also shown that OCT4 has a key role in

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Figure 5. SENP1 increases the drug susceptibility of EC cells under hypoxia in vitro and in vivo. A, drug susceptibility of Empty- or HA- SENP1-NCCIT cells against increasing concentrations of cisplatin and bleomycin under normoxia (left) or hypoxia (right). Cell viability detected by the WST-1 assay is shown. Three individual experiments were carried out for each experimental condition. B, the tumor size of xenograft Empty- and HA-SENP1- NCCIT cells in nude mice for 50 days. C, percentage of the tumor size in nude mice with cisplatin treatment (3 mg/kg/day). D, H&E staining for differentiation level, immunohistochemical staining of HIF-1a, tRFP, cleaved caspase-3, and p21 in the Empty and HA- SENP1 groups are shown. E, quantitative results of D using ImageScope software in different methods. n ¼ 3. , P < 0.05; , P < 0.01.

reprogramming somatic cells to a pluripotent stage (11). Most posttranslational sumoylation (19), and hypoxic oxygen ten- TGCTs express OCT4, suggesting that it plays a critical role in sion (25). Hypoxia is known to affect the level of OCT4 germ cell neoplasia and that it may be useful as a marker for expression in ES cells (12), PGCs (25), iPS cells (26), and a preinvasive and invasive TGCTs (21, 22). A portion of TGCTs wide variety of cancer stem cells (CSC; 13, 14). For example, display resistance to chemotherapy, and this chemoresistance hypoxia can increase the level of OCT4 expression in ES cells is associated with several genetic elements (23) including the through stabilizing the hypoxia-inducible protein HIF-2a.It loss of OCT4 expression. Recent evidence has showed that loss has been shown that HIF-2a binds to the hypoxia response of OCT4 in EC cells increases resistance to cisplatin treatment element of the OCT4 promoter, and then activates OCT4 (16, 17). However, the mechanism underlying the loss of OCT4 expression (25). We also found that hypoxia increased the in EC cells is less clear. OCT4 is known to be regulated by HIF-2a level in ES cells (Supplementary Fig. S6A), but not in EC multiple processes, including epigenetic methylation (24), cells (Supplementary Fig. S6B). This result may explain why

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SENP1 Increases OCT4 Stability and Drug Susceptibility of EC

its ubiquitination and protein degradation (30). Removing a SUMO1 group from the nuclear HIF-1a by SENP1 activates HIF-1a and HIF-1b complex formation, thus driving the transcriptional activation of EPO, VEGF, and Glut-1 (30). Moreover, a sumoylation modification at the specific K118 residue increases mouse Oct4 protein stability under normoxic conditions (19). In our results, hypoxia downregulated SENP1 protein level (Supplementary Fig. S7A) and upregulated the RSUME mRNA level (a sumoylation enhancer; Supplementary Fig. S7B), and decreased human OCT4 stability in EC cells through sumoylation modification at K123 (Figs. 2 and 3). Overexpression of SENP1 in EC cells removed SUMO1 from OCT4 (Fig. 3B, D–F), and increased OCT4 protein stability and drug susceptibility (Figs. 4 and 5). Loss of OCT4 in EC cells has been shown to increase resistance to cisplatin treatment (23), but the mechanism behind this remains to be determined. In several types of CSCs, hypoxia is known to increase the OCT4 protein level, thereby enhancing drug resistance (31, 32). For example, signaling of AKT and ABCG2 is known to mediate OCT4-induced drug resistance in hepatocellular carcinomas (33). However, OCT4 seems to play an opposite role in EC cells. Reduction of OCT4 via hypoxia or retinoic acid treatment in EC cells has been shown to be associated with increase in drug resistance (5, 6). The mechanisms that lead to increase in drug resistance in differentiated EC cells have been proposed to involve epigenetic remodeling and p21 regulation (34, 35). In addition to the role of epigenetic remodeling, our current work shows that overexpression of SENP1 in EC cells under hypoxia significantly restores the level of OCT4 protein and increases drug susceptibility both in vitro and in vivo (Figs. 4 and 5). The role of SENP1 in enhancing the drug susceptibility of EC Figure 6. The expression of SENP1 enhances the drug sensitivity of EC cells is supported by the lower expression of cytoplasmic p21 cells by maintaining the level of the OCT4 protein. A, OCT4 silencing and and the higher level of cleaved caspase-3 found in the HA- SENP1 overexpression in NCCIT cells was observed. B, half maximal SENP1-xenograft tumors (Fig. 5D and E). These results are inhibitory concentration (IC50) values of NCCIT cells to cisplatin and bleomycin were determined. shLuc, shRNA of Luciferase; shOCT4, consistent with previous work showing that OCT4 negatively shRNA of OCT4. , P < 0.05; , P < 0.01. hypoxia increased the OCT4 expression in ES cells, but decreased the OCT4 protein level in EC cells (Supplementary Fig. S6). The discrepancy of hypoxia effect on the levels of HIF- 2a and OCT4 in ES and EC cells may be because of the different expression levels of SENP1 in these cells. SENP1 has been showed to be a regulator of HIF-2a protein stability (27). Compared with ES cells, the SENP1 level in EC cells is extremely low (around 20% of the ES cells; Supplementary Fig. S3). This low expression of SENP1 may explain the low HIF-2a and OCT4 protein levels in EC cells under hypoxia (Supplementary Figs. S3 and S6). SENP1 can cause desumoylation by removing the SUMO group from target proteins. The current work showed that SENP1 can reduce the sumoylation of OCT4 protein induced by hypoxia in EC cells, leading to increased OCT4 protein stability and enhancement of drug sensitivity. Hypoxia is known to increase protein sumoylation-associated gene expression, SUMO1 RSUME including and (a small RWD-containing pro- Figure 7. The proposed mechanism of hypoxia-regulated OCT4 / tein that is a sumoylation enhancer; 28, 29). In a SENP1 sumoylation and drug susceptibility in pluripotent embryonal carcinoma mice model, hypoxia induces sumoylation of HIF-1a and drives cells. Su, SUMO1.

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Wu et al.

regulates p21, which is highly expressed in chemoresistant Writing, review, and/or revision of the manuscript: Y.-C. Wu, T.-Y. Ling, S.-H. Lu, H.-N. Ho, C.-N. Shen, Y.-H. Huang germ cell tumors and protected against cisplatin-induced Administrative, technical, or material support (i.e., reporting or orga- apoptosis (35). nizing data, constructing databases): T.-Y. Ling, S.-H. Lu, H.-C. Kuo, H.-N. Ho, In conclusion, the current work shows that hypoxia S.-D. Yeh, Y.-H. Huang Study supervision: S.-H. Lu, C.-N. Shen, Y.-H. Huang decreases the level of OCT4 protein in EC cells via sumoylation at K123 (Fig. 7). Overexpression of SENP1 in EC cells under hypoxia can effectively restore the level of OCT4 protein and Acknowledgments in vitro in vivo ﬁ The authors thank Professor Hsiu-Ming Shih (Institute of Biomedical improve drug sensitivity and . These ndings Sciences, Academia Sinica, Taipei, Taiwan) for providing plasmids and sugges- suggest that SENP1 may be a promising therapeutic target for tions, and Hsu-Liang Chiang (Department of Biochemistry, Taipei Medical drug-resistant TGCTs. University, Taipei, Taiwan) for technical assistance.

Disclosure of Potential Conﬂicts of Interest Grant Support ﬂ No potential con icts of interest were disclosed. This work was supported in part by the National Science Council grants NSC99-2628-B-038-009-MY3, NSC99-3111-B-038-001, and NSC100-2321-B-038- Authors' Contributions 003 to Y.-H. Huang, and NSC-99-2632-B-038-001-MY3 to Y.-H. Huang and Conception and design: Y.-C. Wu, T.-Y. Ling, S.-H. Lu, H.-N. Ho, C.-N. Shen, Y.-H. C.-R. Tzeng; Intramural Grant of Academia Sinica and National Science Council Huang grants NSC98-3111-B-001-005 to C.-N. Shen; and Taipei City Hospital grants Development of methodology: Y.-C. Wu, H.-C. Kuo, Y.-H. Huang 10001-62-037 to S.-H. Lu. The costs of publication of this article were defrayed in part by the payment of Acquisition of data (provided animals, acquired and managed patients, advertisement provided facilities, etc.): Y.-C. Wu, T.-Y. Ling, S.-H. Lu, H.-C. Kuo, S.-D. Yeh, Y.- page charges. This article must therefore be hereby marked in H. Huang accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Analysis and interpretation of data (e.g., statistical analysis, biosta- tistics, computational analysis): Y.-C. Wu, T.-Y. Ling, S.-H. Lu, C.-N. Shen, Received February 27, 2012; revised June 28, 2012; accepted July 9, 2012; Y.-H. Huang published OnlineFirst September 20, 2012.

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Chemotherapeutic Sensitivity of Testicular Germ Cell Tumors Under Hypoxic Conditions Is Negatively Regulated by SENP1-Controlled Sumoylation of OCT4

Yu-Chih Wu, Thai-Yen Ling, Shing-Hwa Lu, et al.

Cancer Res Published OnlineFirst September 20, 2012.

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