A novel Smac mimetic APG-1387 demonstrates potent antitumor activity in nasopharyngeal carcinoma cells by inducing apoptosis

Cancer Letters 381 (2016) 14–22

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

A novel Smac mimetic APG-1387 demonstrates potent antitumor activity in nasopharyngeal carcinoma cells by inducing apoptosis Ning Li a,1, Lin Feng a,1, Hui-Qiong Han a, Jing Yuan a, Xue-Kang Qi a, Yi-Fan Lian a, Bo-Hua Kuang a, Yu-Chen Zhang a, Cheng-Cheng Deng a, Hao-Jiong Zhang a, You-Yuan Yao a, Miao Xu a, Gui-Ping He a, Bing-Chun Zhao a, Ling Gao a, Qi-Sheng Feng a, Li-Zhen Chen a, Lu Yang a, Dajun Yang a,b,*, Yi-Xin Zeng a,c,* a Department of Experimental Research, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou 510060, China b Ascentage Pharma Group Corp. Limited, Taizhou 225309, China c Beijing Hospital, Beijing 100730, China

A R T I C L E

I N F O

Article history: Received 7 April 2016 Received in revised form 18 June 2016 Accepted 11 July 2016 Keywords: Smac mimetic Nasopharyngeal carcinoma Apoptosis NF-κB AKT

A B S T R A C T

Despite advances in the development of radiation against nasopharyngeal carcinoma (NPC), the management of advanced NPC remains a challenge. Smac mimetics are designed to neutralize inhibitor of apoptosis (IAP) proteins, thus reactivating the apoptotic program in cancer cells. In this study, we investigated the effect of a novel bivalent Smac mimetic APG-1387 in NPC. In vitro, APG-1387 in combination with TNF-α potently decreased NPC cell viability by inducing apoptosis in majority of NPC cell lines. The in vitro antitumor effect was RIPK1-dependent, whereas it was independent on IAPs, USP11, or EBV. Of note, the inhibition of NF-κB or AKT pathway rendered resistant NPC cells responsive to the treatment of APG-1387/TNF-α. In vivo, APG-1387 displayed antitumor activity as a single agent at well-tolerated doses, even in an in vitro resistant cell line. In summary, our results demonstrate that APG-1387 exerts a potent antitumor effect on NPC. These findings support clinical evaluation of APG-1387 as a potential treatment for advanced NPC. © 2016 Published by Elsevier Ireland Ltd.

Introduction Nasopharyngeal carcinoma (NPC) is a unique cancer arising from the epithelial lining of the nasopharynx, and is highly endemic in Southern Asia, with an age-standardized incidence rate of 6.4/ 100,000 for males and 2.4/100,000 for females [1]. Although earlystage NPC can be cured by radiotherapy alone, the survival of patients with metastatic NPC remains disappointing [2]. Resisting programmed cell death belongs to the hallmarks of cancer [3]. Many efforts have been made to exploit strategies to reactivate the apoptotic program in cancer cells. This has led to the development of Smac mimetics, which are designed to neutralize inhibitor of apoptosis (IAP) proteins. IAP proteins are overexpressed in various human malignancies and are associated with treatment resistance, disease progression and poor prognosis [4]. The signature Abbreviations: NPC, nasopharyngeal carcinoma; IAP, inhibitor of apoptosis; TNFα, tumor necrosis factor alpha; BIR, baculovirus IAP repeat; RIPK1, receptor interacting protein kinase 1; FADD, Fas-associated protein with death domain; PARP, poly (ADPribose) polymerase; EBV, Epstein–Barr virus; NF-κB, nuclear factor kappa B; PI3K, phosphoinositide 3-kinase. * Corresponding authors. Fax: +86 20 87343190. E-mail addresses: [email protected] (Y.-X. Zeng); [email protected] (D.J. Yang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.canlet.2016.07.008 0304-3835/© 2016 Published by Elsevier Ireland Ltd.

baculovirus IAP repeat (BIR) domain is shared by all IAP proteins, and some IAPs also possess a carboxy-terminal RING domain containing ubiquitin ligase (E3) activity [5]. There are eight human IAP proteins and Smac mimetics activate caspases by antagonizing XIAP, which directly inhibits key caspase proteins [5,6]. TNF-α is a pleiotropic cytokine involved in multiple biologic responses, including inflammation, cell survival, cell proliferation, and apoptosis [7,8]. TNF-α has shown to potently synergize with Smac mimetics in multiple cancer cells [9–14]. After TNF-α binding to its receptor TNF-RI, TNF-RI recruits the TNF-RI associated death domain (TRADD), which carries on recruiting TNF receptor-associated factor 2 (TRAF2), receptor interacting protein kinase 1 (RIPK1), and cIAP1/2 to form complex I [15,16]. RIPK1 then gets K63-polyubiquinated and promotes the formation of IKK complex and the activation of canonical and non-canonical NF-κB pathways [17]. Smac mimetics treatment leads to the auto-ubiquitination and proteasomal degradation of cIAP1/2 [10,11], and RIPK1 is then deubiquitinated and released from the complex I [18], and subsequently forms the pro-apoptotic complex IIa consisting of caspase-8, FADD and RIPK1, or complex IIb consisting of RIPK1 and RIPK3 to induce necroptosis [15,19,20]. Previously, our laboratory has reported that monovalent Smac mimetics AT-406 and SM-164 have potent antitumor effect on NPC stem-like cells [21]. APG-1387 is a novel potent bivalent Smac mimetic.

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This inhibitor is currently evaluated in a phase I trial in patients with solid tumors (ACTRN12614000268640). In this study, the effect of APG-1387 in NPC was investigated. The results showed that APG1387 has a potent antitumor effect on NPC both in vitro and in vivo by inducing apoptosis. These data suggest that APG-1387 may be a promising small-molecule drug in the treatment of NPC. Materials and methods Cells and culture conditions Human NPC cell lines, CNE1, CNE2, S18, S26, HONE1, HNE1, HK1, SUNE1, 5-8F, 6-10B, SUNE2, and C666 were maintained and conserved in Sun Yat-sen University Cancer Center. SUNE1, 5-8F, 6-10B, SUNE2 and an EBV-positive cell line CNE2EBV+ were kindly provided by Prof. Mu-Sheng Zeng in our center. S18 and S26 were kindly provided by Prof. Chao-Nan Qian in our center. These cells were cultured in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA) or RPMI-1640 (Invitrogen) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA). An immortalized nasopharyngeal epithelial cell line NP69 was maintained in keratinocyte serum-free medium (Gibco) supplemented with bovine pituitary extract. Cells were incubated in a 5% CO2 humidified incubator at 37 °C. Smac mimetic APG-1387 APG-1387 was kindly provided by the Ascentage Pharma Group Corp. Limited (Taizhou, China). For in vitro experiments, APG-1387 was dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO) at a concentration of 1 μmol/L, stored at −20 °C, and diluted in the corresponding culture medium just before use. For in vivo study, APG-1387 was dissolved in 12.5% Captisol (MedChem Express, HY17031; Shanghai, China). Cell viability assay Cell viability was performed with the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) following the manufacturer’s specifications. Cell viability was expressed graphically as mean ± SD of absorbance from treated cells vs. control cells in triplicate. Apoptosis analysis by flow cytometry Apoptosis was determined with an Annexin V− propidium iodide (PI) apoptosis detection kit (Keygen Biotech, KGA108; Nanjing, China) as described previously [21]. Western blots and coimmunoprecipitation Western blot analysis was performed as previously described [21]. For coimmunoprecipitation, cells were cultured on 10-cm culture plates, treated as indicated in the presence of 20 μM Z-VAD-FMK, and harvested in lysis buffer. After incubating on ice for 20 min, cells were centrifuged at 10,000 g for 10 min at 4 °C, and supernatant was collected. One milligram of cell lysates was immunoprecipitated overnight with 1 μg caspase-8 antibody (CST, #9746) and 20 microliters of Protein A/G PLUS-Agarose (Santa Cruz, sc-2003; Dallas, TX) at 4 °C. The next day, immunoprecipitates were washed 4 times with lysis buffer before proteins were eluted from beads by addition of 1 × SDS loading buffer and boiled for 5 min. Samples were then analyzed by western blot analysis. RIPK1 knockdown Transfections were done in 6-well culture dishes. siRNAs directed against human RIPK1 and control RNAs were obtained from Viewsolid Biotech (China). Lipofectamine RNAiMAX (Invitrogen, 13778-150) was used to transfect cells, as per manufacturer’s instructions. Xenograft experiments All animal experiments were performed in the Animal Center of Sun Yat-sen University. The animal protocol was approved by Sun Yat-sen University. Female BALB/c nude mice (3–5 weeks old) were purchased from Shanghai SLAC laboratory animal center (Animal experimental license no. SYXKyue2010-0102). Mice were then injected subcutaneously with 1 × 105 S26 cells or 1 × 107 HK1 cells (16 mice each) in the right axillary cavity. When tumors were measurable (100–180 mm3), mice were randomized into either APG-1387-treated arm or vehicle-treated arm (8 mice per arm) with approximately equivalent tumor volume. APG-1387 or 12.5% Captisol (vehicle control) was administered in about 200 μL intraperitoneally 3 times/week for 2 weeks. Body weights and tumor volumes (V) were measured every 2 days. Tumor volumes were calculated according to the equation V = (length × width2) /2.

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Statistical analysis IC50 values were calculated by nonlinear regression analysis with GraphPad Prism 5. Two-tailed Student t-test was used to compare the means between groups. Twosided P value of less than 0.05 was considered to be statistically significant.

Results APG-1387 in combination with TNF-α inhibits growth potently in most NPC cell lines The structure of the novel bivalent Smac mimetic APG-1387 is shown in Fig. 1a. We first examined the effect of APG-1387 as a single agent in a panel of 12 NPC cell lines and the immortalized nasopharyngeal epithelial cell line NP69. Only one cell line, S26, displayed an obvious response with a half maximal inhibitory concentration (IC50) value at a high nanomolar concentration of 1934 nmol/L (Table 1). However, when APG-1387 was administered in the presence of TNF-α (1 ng/mL), most cell lines were responsive to relative low-dose APG-1387 (Table 1). All of these cell lines were nonresponsive to TNF-α alone. Nonetheless, two NPC cell lines, i.e. HK1 and C666, were resistant to the combination therapy. NP69 was also resistant to the combination (Fig. S1). Therefore, four cell lines, including CNE1, CNE2, S26 and HK1, were chosen for further analysis based on their response to APG-1387 alone or in combination with TNF-α: CNE1 and CNE2, which were sensitive to APG-1387 only when cotreated with TNF-α; S26, which was responsive to APG1387 alone; and HK1, which was resistant to APG-1387 in combination with TNF-α (Fig. 1b). APG-1387 alone or in combination with TNF-α induced apoptosis in sensitive cell lines Apoptosis detection by Annexin V and PI staining showed that when treated with APG-1387 alone, the dramatic increase of Annexin V positive cells was seen in S26 only (Fig. 2a,b). However, when APG1387 was coadministered with TNF-α, the dramatic increase of Annexin V positive cells could now be observed in CNE1, CNE2 and S26 (Fig. 2a,b). The increase of Annexin V positive cells could not be observed in HK1, even when the combination therapy was given (Fig. 2a,b). We further photographed these cells using a phase contrast microscope and the results demonstrated that CNE1 and CNE2 cells were killed by APG-1387 in the presence of TNF-α, S26 cells were killed by APG-1387 alone or with TNF-α, and HK1 cells could not be killed by APG-1387 with or without TNF-α (Fig. S2). The above results of these cell lines were in consistency with their response pattern that was determined by cell viability assay. Colony formation

Table 1 IC50 for APG-1387 alone or with TNF-α (1 ng/mL) in 12 NPC cell lines and the NP69 cell line. IC50 (nM)

CNE1 CNE2 S18 S26 HONE1 HNE1 SUNE1 5-8F 6-10B SUNE2 HK1 C666 NP69

APG-1387

APG-1387 + TNF-α

38725 1934 29735 24040 -

10 26 8 2 8 133 44 54 103 67 -

NPC: nasopharyngeal carcinoma. “-” means unresponsiveness.

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Fig. 1. APG-1387 induces TNF-α-dependent cell death in nasopharyngeal carcinoma cell lines. (a) The structure of the bivalent Smac mimetic APG-1387. (b) Selected cell lines were treated with indicated concentrations of APG-1387, alone or in combination with TNF-α (1 ng/mL) for 72 hours and then analyzed for cell viability. Four cell lines were chosen: CNE1 and CNE2, response to APG-1387 only when cotreatment with TNF-α; S26, response to APG-1387 alone; HK1, resistant to APG-1387 in combination with TNF-α. Data were represented as mean ± standard deviation (SD) of 3 independent experiments.

assays showed that APG-1387 inhibits the clonogenic growth of CNE1 and S26 cells, and can synergize with ionizing radiation (IR) in these cells (Fig. S3). We then confirmed the results of Annexin V/PI staining by western blot analysis of antibodies against PARP, caspase-8, and cleaved caspase-3. The levels of cleaved PARP, cleaved caspase-8, and cleaved caspase-3 were increased in S26 with APG-1387 alone, and in CNE1, CNE2 and S26 with APG-1387/TNF-α treatment, but not in HK1 cells (Fig. 2c). APG-1387 leaded to the increase of cleaved PARP, cleaved caspase-8, and cleaved caspase-3 in S26 in a concentration- and time-dependent manner (Fig. 2d). To determine whether the cell death induced by APG-1387 in combination with TNF-α was caspase-dependent, APG-1387 and TNF-α were coadministered to CNE1 and CNE2 with or without addition of Z-VAD-FMK, which is a pan caspase inhibitor. In both cell lines, the growth inhibition was completely blocked by addition of Z-VAD-FMK (Fig. 2e). We further determined which caspase ultimately mediates APG-1387-induced apoptosis with a panel of caspase inhibitors. In both CNE1 and CNE2, the inhibition of pan caspase completely restored cell growth, while the inhibition of

caspase-1, caspase-2, caspase-3, caspase-6 and caspase-8 also had a rescue effect to some extent on cell viability (Fig. 2f). The coimmunoprecipitation by caspase-8 antibody displayed that the treatment of APG-1387 alone or with TNF-α promoted the formation of RIPK1-FADD-Caspase-8 complex in S26 cells (Fig. 2g). The addition of Z-VAD was to enhance the formation of complex IIa. A time course of RIPK1-FADD-Caspase-8 complex formation showed that upon the treatment of APG-1387 and TNF-α, the complex IIa was more evident after 4 hours, whereas upon single agent APG1387 treatment, the complex IIa was more evident after 8 hours (Fig. 2g). In contrast, the treatment of APG-1387 alone or with TNF-α did not induce the formation of RIPK1-FADD-Caspase-8 complex in HK1 cells (Fig. 2h). APG-1387-induced cell death is not IAP-dependent, whereas dependent on RIPK1 To investigate whether the observed drug sensitivity is IAPdependent, we examined the protein levels of cIAP1, cIAP2, and XIAP in 12 NPC cell lines and the NP69 cell line. Western blot analysis

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Fig. 2. APG-1387 alone or in combination with TNF-α kills nasopharyngeal carcinoma cells by inducing apoptosis. (a) CNE1, CNE2, S26 and HK1 cells were treated with DMSO, TNF-α (1 ng/mL), APG-1387 (1 μM), or combination of APG-1387 (1 μM) and TNF-α (1 ng/mL) for 48 hours, and then double stained with Annexin V and propidium iodide (PI) and analyzed using flow cytometry. D: DMSO; T: TNF-α; A: APG-1387. Experiments were repeated 3 times and representative images are shown. (b) Quantification of Annexin V positive cells shown in (a). Data were represented as mean ± SD of 3 independent experiments. *P < 0.05, #P < 0.005, t-test, all comparisons made to DMSO-treated group. (c) The same four cell lines were treated with DMSO, TNF-α (1 ng/mL), APG-1387 (1 μM), or combination of APG-1387 (1 μM) and TNF-α (1 ng/mL) for 24 hours and were harvested. Changes in PARP, caspase-8 and cleaved caspase-3 levels were analyzed by western blot. D: DMSO; T: TNF-α; A: APG-1387. (d) Western blot analysis of the effect of APG-1387 on PARP, caspase-8 and cleaved caspase-3 levels at 5 different concentrations and 5 different time points in the single-sensitive cell line S26. (e) CNE1 and CNE2 cells were treated with APG-1387 (1 μM) and TNF-α (1 ng/mL) in the presence or absence of 10 μM Z-VAD-FMK. Cell viability was determined by cell viability assay using CCK-8. A: APG-1387; T: TNF-α. Data were represented as mean ± SD of 3 independent replicates. (f) CNE1 and CNE2 cells were treated with DMSO, TNF-α (1 ng/mL), APG-1387 (1 μM), APG-1387 (1 μM) in combination with TNF-α (1 ng/mL), or APG-1387/TNF-α with 10 μM of Z-VAD-FMK, Z-YVAD-FMK, Z-VDVADFMK, Z-DQMD-FMK, Z-VEID-FMK, or Z-IETD-FMK. Cell viability was determined by cell viability assay using CCK-8. Data were displayed as mean ± SD of 3 independent experiments. (g,h) S26 (g) and HK1 (h) cells were treated as indicated and lysates were immunoprecipitated with a caspase-8 antibody. RIPK1, FADD, and caspase-8 levels in the caspase-8 immunocomplex and the cell lysates (1% input) were measured by western blot analysis. T: TNF-α; A: APG-1387; Z, Z-VAD-FMK. Experiments were repeated 3 or more times with similar results.

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showed that the sensitivity of APG-1387 was IAP-independent (Fig. 3a). In fact, as long as APG-1387 was administered, alone or with TNF-α, the protein levels of cIAP1, cIAP2, XIAP and livin were decreased in both sensitive and resistant cell lines (Fig. 3b,h). A concentration and time course of CNE2 cells’ response to APG-1387 treatment showed that the degradation of cIAP1, cIAP2, XIAP and livin was in a concentration and time-dependent manner (Fig. 3c,d). As RIPK1 is a key regulator of a cell’s life-or-death and an essential component in complex IIa [22], we examined the level of RIPK1 in the same panel of cell lines. As shown in western blotting and qRT-PCR, the protein and mRNA levels of RIPK1 were found to be relatively low in resistant cell lines HK1 and C666 (Fig. 3e,f). Correlation analysis in responsive cell lines showed that IC50 values did not significantly correlate with RIPK1 protein or mRNA levels (Fig. S4). Knockdown of RIPK1 rescued S26 cells from death to a great extent (Fig. 3g). The effect of RIPK1 knockdown was evaluated by 3 different siRNAs (Fig. 3i). Knockdown of RIPK1 reversed APG1387-induced apoptosis (Fig. 3j). Besides, there was no detectable TNF-α present in all the cell lines (data not shown). Ubiquitinspecific protease 11 (USP11) was reported to be a barrier to Smacmimetic-induced cell death by stabilizing cIAP2. [23] qRT-PCR analysis showed that the levels of USP11 were not higher in the resistant cell lines than in the sensitive ones (Fig. S5). Since NPC is associated with Epstein–Barr virus (EBV), we investigated the effects of EBV infection on the levels of IAPs and Smacmimetic-induced apoptosis. Western blot analysis showed that the expression levels of cIAP1, cIAP2 and XIAP were elevated in CNE2EBV+ cells compared with CNE2-EBV− cells, although livin was decreased (Fig. 3k). However, IAPs were decreased as long as APG1387 and TNF-α treatment was given no matter what the status of EBV infection was (Fig. 3k). Of note, the formation of apoptosis was more evident upon 6-hour combination treatment of APG-1387 and TNF-α in CNE2-EBV+ cells, as shown by western blot analysis (Fig. 3l). However, there was little difference in cell viability between CNE2EBV− and CNE2-EBV+ cells upon APG-1387/TNF-α treatment (data not shown). Taken together, our data indicate that APG-1387 induced cell death is RIPK1-dependent, whereas independent on IAPs, USP11, or EBV. Inhibition of NF-κB or AKT pathway sensitizes HK1 cells to the combination treatment of APG-1387 and TNF-α Previous reports have demonstrated that the inhibition of NFκB or AKT pathway leads to the response of resistant cells to Smac mimetics/TNF-α treatment [24,25]. To test whether chemical inhibition of these pathways can overcome the resistance to APG-1387/ TNF-α induced apoptosis in HK1 cells, the IKK2 inhibitor, BMS345541, and the PI3K inhibitor, LY294002, were used. HK1 cells were pretreated with increasing concentration of BMS345541 for 1 hour followed by APG-1387/TNF-α treatment for 72 hours. The results of cell viability assay showed that BMS-345541 synergized with APG-1387/TNF-α treatment in a concentrationdependent manner to inhibit cell growth (Fig. 4a). The IKK inhibition by BMS-345541 with 5 μM or 20 μM did not affect the levels of cIAP1, cIAP2 and XIAP significantly (Fig. 4b). BMS-345541 in the concentration of 5 μM slightly attenuated the accumulation of phosphorylated IκBα, whereas 20 μM severely block phosphorylation (Fig. 4c). Nonetheless, 5 μM BMS-345541 and APG-1387/TNF-α acted in concert to inhibit cell viability (Fig. 4a). Coimmunoprecipitation by caspase-8 antibody demonstrated that upon the stimulation of BMS-345541 (5 μM) followed by APG-1387/TNF-α, RIPK1 and FADD were detected in the caspase-8 immunocomplex (Fig. 4g). In a similar fashion, pretreatment with LY294002 for 1 hour then followed by APG-1387/TNF-α treatment induced cell growth inhibition in a concentration-dependent manner (Fig. 4d). The addition of LY294002 had little effect on levels of cIAP1, cIAP2 and XIAP

(Fig. 4e). Pretreatment with LY294002 for 1 hour significantly decreased the level of phosphorylated AKT (Fig. 4f). Similarly, pretreatment with LY294002 at the optimal concentration of 10 μM followed by APG-1387/TNF-α treatment induced the formation of the RIPK1-Caspase-8-FADD complex (Fig. 4g). APG-1387 inhibits tumor growth as a single agent in xenografted NPC models Finally, to evaluate the therapeutic efficacy of APG-1387 in vivo, two NPC xenotransplantation models were examined based on their response patterns in vitro: S26 and HK1. S26 did respond to APG1387 as a single agent in vitro, whereas HK1 was resistant to the combination treatment of APG-1387 and TNF-α in vitro. In both models, treatment with intraperitoneal injection of APG-1387 inhibited NPC tumor growth (Fig. 5a,b). The difference between these two models was that the tumor growth inhibition in S26 was shown upon the starting of treatment, whereas the effect in HK1 could be observed after 10 days of treatment (Fig. 5a,b). APG-1387 treatment also resulted in reduced tumor weight in both models (Fig. 5c-f). Examination of harvested tumors showed that compared with vehicle-treated controls, APG-1387 increased the number of cleaved-caspase-3-positive tumor cells in both S26 xenografts and HK1 xenografts (Fig. 5g). Although APG-1387 treatment resulted in a weight loss in about 6–14 days after treatment, the body weight changes recovered well after the termination of treatment, suggesting APG-1387 was well tolerated (Fig. S6). Discussion In the present study, we showed that the bivalent Smac mimetic, APG-1387, in combination with TNF-α, could inhibit cell viability by inducing apoptosis in majority of NPC cell lines. The in vitro antitumor effect is RIPK1-dependent, whereas independent on IAPs, USP11, or EBV. Of interest, the inhibition of NF-κB or AKT pathway rendered NPC cells responsive to the treatment of APG-1387/TNFα. Furthermore, we demonstrated that APG-1387 displayed antitumor activity as a single agent at well-tolerated doses in vivo. The effect of Smac mimetics is now being evaluated in many clinical trials in patients with solid tumors and lymphoma [26]. APG1387 is a new potent bivalent small-molecule mimetic of Smac. Evidence suggested that bivalent Smac mimetics have higher antitumor activity and higher potency to degrade IAPs than monovalent Smac mimetics [4]. Although autocrine TNF-α renders cancer cells sensitive to Smac mimetic treatment [13], our results showed that the levels of TNF-α were not detectable in any of the NPC cell lines including the single sensitive S26 cell line. Besides, none of the cell lines were responsive to the treatment of exogenous TNF-α alone in our study. Most NPC cells were sensitive to APG-1387 only when cotreated with TNF-α. This was in consistency with previous reports, where Smac mimetic and TNF-α act in concert to induce cell apoptosis [9–13]. Several immune cytokines, such as TNF-α, TRAIL and interleukin 1 beta (IL-1β) can synergize with Smac mimetics in a variety of cancer cell lines in vitro [5,9–13,27,28]. Of note, stimulating the innate immune system by nonpathogenic oncolytic viruses was tested to be effective and safe in tumors treated with Smac mimetics [29], suggesting that Smac mimetics in combination with pathogen mimetics is a promising strategy in cancer therapy. Ten of the 12 NPC cell lines tested had a response to combined APG-1387/TNF-α treatment, while 2 cell lines (HK1 and C666) did not respond to the combination therapy. Several reports showed that cIAP2 renders cancer cells resistant to cell death that is induced by Smac mimetic/TNF-α [12,24,30]. However, our results demonstrated that IAP levels were not associated with the resistance of APG1387 in NPC cell lines, although the cIAP2 level of C666 was high. In our study, the expression level of RIPK1 tended to be low in the

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Fig. 3. APG-1387 induced apoptosis is RIPK1-dependent, whereas independent on IAPs or EBV. (a) A pane of 12 nasopharyngeal carcinoma cell lines and the nasopharyngeal epithelial cell line NP69 were analyzed for cIAP1, cIAP2 and XIAP levels by western blot analysis. (b) CNE1, CNE2, S26 and HK1 cells were treated with DMSO, TNF-α (1 ng/mL), APG-1387 (1 μM), or combination of APG-1387 (1 μM) and TNF-α (1 ng/mL) for 24 hours and were harvested. Changes in cIAP1, cIAP2, XIAP and livin protein levels were analyzed by western blot. D: DMSO; T: TNF-α; A: APG-1387. (c) Western blot analysis of the effect of APG-1387 on cIAP1, XIAP and livin levels at 5 different concentrations in CNE2. (d) Western blot analysis of the effect of APG-1387 on cIAP1, XIAP and livin levels at 6 different time points in CNE2. (e) Western blot analysis of RIPK1 level in 12 nasopharyngeal carcinoma cell lines and the NP69 cell line. (f) Relative expression of the mRNAs encoding RIPK1 in the same panel of cells by the method of quantitative RT-PCR. Results were normalized to GAPDH. The data are reported as mean ± SD of 3 independent repeats. (g) Cell viability as measured by CCK-8 cell viability assay of S26 cells with RIPK1 knockdown via siRNA or negative control (NC) siRNA treated with indicated concentrations of APG-1387 plus TNF-α (1 ng/mL). Data were represented as mean ± SD of 3 independent experiments. (h) C666 cells were treated with DMSO, TNF-α (1 ng/mL), APG-1387 (1 μM), or combination of APG-1387 (1 μM) and TNF-α (1 ng/mL) for 24 hours and changes in cIAP1, cIAP2 and XIAP protein levels were analyzed by western blot. D: DMSO; T: TNF-α; A: APG-1387. (i) Western analysis of RNAi efficiency of RIPK1 siRNA in control (C) S26 cells and those transfected with RIPK1-targeting siRNA or negative control (NC) siRNA. Cell lysates were collected 48 hours post transfection. (j) Control (C) S26 cells and those transfected with RIPK1-targeting siRNA or negative control (NC) siRNA were treated with APG-1387 (1 μM)/TNF-α (1 ng/mL) for 12 hours and changes in PARP, caspase-8 and cleaved caspase-3 levels were analyzed by western blot. (k) CNE2 and CNE2-EBV+ cells were treated with or without APG-1387 (1 μM)/TNF-α (1 ng/mL) for 6 hours and changes in cIAP1, cIAP2, XIAP and livin levels were analyzed by western blot. AT: APG-1387/TNF-α. (l) CNE2 and CNE2-EBV+ cells were treated with or without APG1387 (1 μM)/TNF-α (1 ng/mL) for 6 hours and changes in PARP, caspase-8 and cleaved caspase-3 levels were analyzed by western blot. AT: APG-1387/TNF-α.

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Fig. 4. Inhibition of NF-κB or PI3K/AKT pathway renders HK1 cells responsive to APG-1387/TNF-α treatment. (a) HK1 cells were pretreated with BMS-345541 at indicated concentrations for 1 hour and then treated with DMSO, TNF-α (1 ng/mL), APG-1387 (1 μM), or combination of APG-1387 and TNF-α for 72 hours. Cell viability was measured by cell viability assay with CCK-8. Data were represented as mean ± SD of 3 independent experiments. (b,c) HK1 cells were pretreated with DMSO, 5 μM BMS-345541 or 20 μM BMS-345541 for 1 hour, and then treated with the combination of APG-1387 (1 μM) and TNF-α (1 ng/mL) for the indicated times. Cell lysates were harvested for western blot analysis of cIAP1, cIAP2 and XIAP levels in (b), and phospho-IκBα and IκBα levels in (c). A: APG-1387; T: TNF-α; B: BMS-345541. (d) HK1 cells were pretreated with increasing concentrations of LY294002 for 1 hour and then treated with DMSO, TNF-α (1 ng/mL), APG-1387 (1 μM), or combination of APG-1387 and TNF-α for 72 hours. Cell viability was measured by cell viability assay with CCK-8. Data were displayed as mean ± SD of 3 independent experiments. (e,f) HK1 cells were pretreated with DMSO or 10 μM LY294002 for 1 hour, and then treated with the combination of APG-1387 (1 μM) and TNF-α (1 ng/mL) for the indicated times. cIAP1, cIAP2 and XIAP levels in (e), and phospho-AKT and AKT levels in (f) were measured by western blot analysis. A: APG-1387; T: TNF-α; LY: LY294002. (g) HK1 cells were pretreated with DMSO, 5 μM BMS-345541 or 10 μM LY294002 for 1 hour, and then treated as indicated for 4 hours. Lysates were immunoprecipitated with a caspase-8 antibody. RIPK1, FADD, and caspase-8 levels in the caspase-8 immunocomplex and the cell lysates (1% input) were measured by western blot analysis. T: TNF-α; A: APG-1387; Z, Z-VAD-FMK. Experiments were repeated 3 or more times with similar results.

resistant cell lines as compared with the sensitive ones, and knockdown of RIPK1 rescued NPC cells from death. Upon TNF-α stimulation, RIPK1 plays a critical role in mediating NF-κB activation, apoptosis and necroptosis [22]. Although apoptosis induced by cycloheximide/ TNF-α does not require RIPK1 [18], RIPK1 is a necessity for apoptosis upon Smac/TNF-α treatment [13,18,31]. The possible reason is that

Smac mimetics lead to the formation of the pro-apoptotic RIPK1 containing complex II. The results for RIPK1 in the current study are in line with what was demonstrated in previous reports. Both NF-κB and PI3K/AKT signaling pathways regulate various cellular processes, such as proliferation, growth and apoptosis [32,33]. Previous studies demonstrated that inhibition of NF-κB or AKT

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Fig. 5. APG-1387 exhibits antitumor activity in xenografted nasopharyngeal carcinoma models. Mice bearing S26 or HK1 tumors were treated with APG-1387 (10 mg/kg) or vehicle intraperitoneally 3 times per week for 2 consecutive weeks. (a,b) Changes in tumor volume between APG-1387 group and vehicle group of S26 tumor-bearing mice and HK1 tumor-bearing mice. Data are shown as mean tumor volume ± SD (eight mice/group). (c,d) Tumor weight between APG-1387 group and vehicle group of S26 tumor-bearing mice and HK1 tumor-bearing mice. *P < 0.05, t-test. (e,f) Photographs of harvested tumors of S26 tumor-bearing mice and HK1 tumor-bearing mice. (g) Tumor sections from APG-1387-treated and vehicle-treated, S26 tumor-bearing mice and HK1 tumor-bearing mice were immunostained with cleaved caspase-3 antibody. Nuclear was stained with DAPI. All images were collected at the same magnification. Scale bar = 100 μm.

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signaling sensitizes resistant cells to Smac mimetic/TNF-α by promoting significant formation of complex IIa [24,25]. Likewise, by this strategy, we could sensitize the combination treatmentresistant cell line HK1 in our study by inducing the complex IIa. However, there was still a portion of cells that did not respond, indicating that NF-κB and PI3K-AKT pathways are responsible for a part of the resistance in NPC cells, and additional signaling pathways may operate on anti-apoptosis. In addition to NF-κB and PI3KAKT, IAPs also play an essential role in many other signaling pathways, such as MAPK, JNK, and Myc [34]. For example, a recent report showed that inhibition of p38/MK2 axis sensitized acute myeloid leukemia to Smac mimetics [35]. Future studies involving different signaling pathways of sensitive and resistant cells are needed to further investigate the reason of their different responses to Smac mimetic/TNF-α treatment, and the optimal combinational scheme. Although APG-1387 was not a potent drug as a single agent in vitro, we have found that it was effective as a single agent in in vivo models. Even the resistant cell line HK1 showed a slower tumor growth in vivo with APG-1387 alone. This may be largely related to the complexity of microenvironment in vivo [14]. As we mentioned above, many immune cytokines can synergize with Smac mimetics. These observations indicate that APG-1387 may be effective as a single agent in patients with NPC. In summary, this research demonstrated that APG-1387 has a potent antitumor effect on NPC both in vitro and in vivo by inducing apoptosis. We showed that APG-1387 induced cell death is RIPK1dependent, whereas it was independent on IAPs, USP11, or EBV. In addition, we showed that the inhibition of NF-κB or AKT pathway could sensitize the resistant NPC cells to Smac mimetic/TNF-α treatment. These findings suggest that APG-1387, a Smac mimetic, should be evaluated in clinical trials for patients with advanced NPC. Acknowledgments This work was supported by the National Basic Research Program of China (973 Program: 2013CB910303), the National High Technology Research and Development Program of China (863 Program: 2012AA02A206 and 2012AA02A501), the National Natural Science Foundation of China (81172107), and the Health & Medical Collaborative Innovation Project of Guangzhou City, China (No. 201400000001, 201508020250). We thank Prof. Mu-Sheng Zeng (Sun Yat-sen University Cancer Center, Guangzhou, China) and Prof. Chao-Nan Qian (Sun Yat-sen University Cancer Center, Guangzhou, China) for providing NPC cell lines. Conflict of interest None. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2016.07.008. References [1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, CA Cancer J. Clin. 65 (2015) (2012) 87–108. [2] F. Wu, R. Wang, H. Lu, B. Wei, G. Feng, G. Li, et al., Concurrent chemoradiotherapy in locoregionally advanced nasopharyngeal carcinoma: treatment outcomes of a prospective, multicentric clinical study, Radiother. Oncol. 112 (2014) 106–111. [3] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144 (2011) 646–674. [4] S. Fulda, D. Vucic, Targeting IAP proteins for therapeutic intervention in cancer, Nat. Rev. Drug Discov. 11 (2012) 109–124.

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