Targeting eIF4E inhibits growth, survival and angiogenesis in retinoblastoma and enhances efficacy of chemotherapy

Biomedicine & Pharmacotherapy 96 (2017) 750–756

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Original article

Targeting eIF4E inhibits growth, survival and angiogenesis in retinoblastoma and enhances efficacy of chemotherapy ⁎

Genguo Wang1, Zhi Li1, Zhuojun Li, Yi Huang, Xiaochun Mao, Chang Xu , Sha Cui

T ⁎

Department of Ophthalmology, Xiangyang Central Hospital, Hospital Affiliated to Hubei University of Arts and Science, Xiangyang, Hubei, 441021, People’s Republic of China

A R T I C L E I N F O

A B S T R A C T

Keywords: Ribavirin eIF4E Retinoblastoma Angiogenesis

Although the eukaryotic translation initiation factor 4E (eIF4E) has been shown to be critically involved in the transformation and progression of various tumors, little is known about the role of eIF4E in retinoblastoma. In this work, we report that ribavirin, a pharmacologic inhibitor of eIF4E function, effectively targets retinoblastoma and angiogenesis. Ribavirin treatment dose-dependently blocked the growth and stimulated apoptosis in various retinoblastoma cell lines, with IC50 values that are within the clinically achievable range. Ribavirin also significantly inhibited angiogenesis via disrupting capillary network formation and suppressing VEGF-induced migration, proliferation and survival of human retinal endothelial cells. In addition, ribavirin significantly augments chemotherapy agent’s inhibitory effects in retinoblastoma in vitro and in vivo. Mechanistically, ribavirin inhibited eIF4E function in retinoblastoma cells as shown by the decreased protein levels of Cyclin D1, cMyc and VEGF without affecting their mRNA expression. Overexpression of the wildtype and phosphormimetic but not the nonphosphorylatable form of eIF4E significantly abolished the inhibitory effects of ribavirin, further demonstrating eIF4E as the target of ribavirin. Genetic knockdown of eIF4E using two independent siRNAs mirrored ribavirin’s effects, confirming the role of eIF4E in retinoblastoma growth, survival and response to chemotherapy. Our findings provide a preclinical rationale to explore ribavirin as a strategy to treat retinoblastoma and highlight the therapeutic value of targeting eIF4E in retinoblastoma.

1. Introduction The eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in a wide variety of human solid tumors (eg, carcinomas of the breast, colon and brain) and blood cancers (eg, lymphoma and chronic myeloid leukemia) [1–3]. Overexpression of the eukaryotic translation initiation factor 4E (eIF4E) in cancer often correlates with poor clinical outcome. eIF4E is essential for growth, survival and angiogenesis of cancer cells by acting at a key step of cap-dependent translation and preferentially enhances the translation of carcinogenesis associated mRNAs, including c-Myc, cyclin D1 and vascular endothelial growth factor (VEGF) [4]. Various studies recently demonstrated that eIF4E upregulation is also involved in chemoresistance in cervical and brain cancers as it is a protective molecular mechanism in response to chemotherapy [5,6]. However, little is known about the role of eIF4E in retinoblastoma. Ribavirin is an antiviral drug with anti-cancer activities through inhibiting eIF4E [4,7–10]. Clinical trials with ribavirin in patients with

eIF4E overexpressing-acute myeloid leukemia show clinically beneficial without significant treatment-related toxicity [11]. It binds to eIF4E and subsequent inhibiting eIF4E’s association with the m7G cap protein and preventing RNA-dependent replication [12]. The mechanisms of action of ribavirin seem to vary across different types of cancers [7–9,11,13,14,15]. Various studies demonstrate that ribavirin inhibits eIF4E phosphorylation and subsequent protein translation in leukemia [9,16]. Others demonstrate that ribavirin inhibits mTOR signaling in tongue squamous carcinoma [17]. Ribavirin also targets a key enzyme in the guanosine biosynthetic pathway, inosine monophosphate dehydrogenase which may contribute to its anti-cancer activities [15]. Retinoblastoma is the most common ocular cancer in children and its clinical management is still challenging [18]. Besides mutation of tumor suppressor gene retinoblastoma 1, tumor angiogenesis is important in the progression of retinoblastoma and serves as a prognostic factor for disease dissemination [19,20]. Identification of agents that target both tumor cells and angiogenesis may represent an alternative therapeutic strategy for retinoblastoma. In this work, we evaluated

⁎ Corresponding authors at: Department of Ophthalmology, Xiangyang Central Hospital, Hospital Affiliated to Hubei University of Arts and Science, 39 Jingzhou Street, Xiangyang, 441021, People’s Republic of China. E-mail addresses: [email protected] (C. Xu), [email protected] (S. Cui). 1 These authors have contributed equally to this work.

http://dx.doi.org/10.1016/j.biopha.2017.10.034 Received 13 July 2017; Received in revised form 9 September 2017; Accepted 9 October 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.

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2.5. Western blot analyses

eIF4E as a therapeutic target in retinoblastoma. We also assessed the ability of clinically relevant concentrations of ribavirin to suppress growth, survival and angiogenesis of retinoblastoma, and correlated this with ribavirin’s ability in suppressing eIF4E activity.

Total proteins were extracted from 106 cells using RIPA lysis buffer (Thermo Scientific, US) supplemented with protease and phosphatase inhibitors (Thermo Scientific, US). Proteins were resolved by SDSPAGE, and blotted with anti-p-eIF4E, eIF4E, Cyclin D1, c-Myc and VEGF antibody (Invitrogen, US), respectively. Visualization was achieved by using Amersham ECL Western Blotting Detection Kit (GE HealthCare, UK).

2. Materials and methods 2.1. Cells and reagents Human retinoblastoma cell lines Y79 and WERI-Rb-1 (American Type Culture Collection) and RB116 (Kerafast, Inc) were culutred in RPMI medium with 1% HEPES (life technologies, US) and 10% FBS (Hycolon, UK). Human primary retinal microvascular endothelial cell (HREC) (Cell Systems Inc. US) were cultured in basal M131 medium (Invitrogen, CA) supplemented with microvascular growth supplement (Invitrogen, CA). Ribavirin (Kemprotec Ltd.), carboplatin (Sigma, US) and human recombinant vascular endothelial growth factor (VEGF, Abacam) was reconstituted in water and sterile-filtered before use. Reagents were stored in aliquots in −20 °C and thawed only once.

2.6. Transfection Retroviral MSCV-internal ribosome entry site (IRES) constructs containing eIF4E S209D, S209A and WT were kind gifts from Dr. Yi Tang’s laboratory and Y79 cells were transduced with constructs above to generate stable cell lines as previously described [17,21]. Specific knockdown of eIF4E were performed using siRNAs from IDT, Inc. 106 cells in 6-well pate were transfected with 100 nM specific eIF4E siRNAs and human scrabbled control siRNA via Dharmafect Transfection Reagent according to manufacture’s protocol. The target sequences of human eIF4E siRNAa and siRNAb are (AAG CAA ACC UGC GGC UGA UCU) and (ACA GCA GAG ACG AAG UGA C).

2.2. Measurement of proliferation and apoptosis 5 × 103 cell/wells in 96-well plate were treated with drug alone or combination for 72 h, after which 20 μl MTS (3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) was added to each well and incubated for 2–4 h. Proliferation activity was determined by measuring absorbance at 490 nm. Cell cycle was measured by labelling cells with propidium iodide DNA staining (Abcam, US) according to manufacture’s protocol followed by flow cytometry analysis on a Beckman Coulter FC500. 105 cells/well in 12-well plate were treated with drug alone or combination for 72 h. Apoptotic cells were labelled with Annexin V/7-AAD kit (BD Pharmingen, US) staining according to manufacture’s protocol. The percentage of Annexin V-positive cells were quantified by flow cytometry on a Beckman Coulter FC500. 106 cells/well in 6-well plate were treated with drug for 24 followed by flow cytometry analysis on a Beckman Coulter FC500. 105 cells/well in 12-well plate were treated with drug alone or combination for 72 h. Apoptotic cells were labelled with Annexin V/7-AAD kit (BD Pharmingen, US) staining according to manufacture’s protocol. The percentage of Annexin V-positive cells were quantified by flow cytometry on a Beckman Coulter FC500. 106 cells/well in 6-well plate were treated with drug for 24 h prior to measuring active caspase 3 activity using Caspase-3 Colorimetric Assay Kit (Biovision, US) according to manufacture’s instructions.

2.7. RT-PCR Total RNA was prepared from 106 cells using TRIzol (Invitrogen, US). First-strand cDNA was synthesized from 1 μg of total RNA by using iScript cDNA Synthesis Kit (Bio-rad, CA). Cyclin D, C-Myc and VEGF mRNA expression were measured using CFX96 RT PCR machine with a SsoFast EvaGreen Supermix (Bio-rad, CA) using the primer as previously reported [16]. 2.8. Retinoblastoma tumor xenograft in SCID mouse 4–6 weeks old male NOD severe combined immunodeficient mice (NOD/SCID) were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences. All procedure were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. A total of 10 × 106 Y79 cells suspending in 100 μl were injected into the right flank of mice. Tumors were allowed to form and forty mice with palpable tumours were randomized into four groups (ten per each group). When tumors reach ∼200 mm3, mice were treated with vehicle (water), intraperitoneal carboplatin at 1 mg/kg once every three days, oral ribavirin at 50 mg/kg every day and combination of both. The tumor length and width were measured every three days. The tumor size was calculated using formula: (length)2 × (width)/2. Mice were euthanized when control tumor reached ∼1000 mm3.

2.3. In vitro capillary tube formation 100 μl of complete Matrigel (Chemicon International, Inc;, US) were plated onto the 96-well plate and incubated in 37 °C for 30 min for solidification. 2 × 104 HREC cells were suspended in 50 μl of basal M131 medium containing drug were gently plated onto the polymerized complete Matrigel in 96-well plate. Capillary network was formed after 6 ∼ 8 h incubation and documented using an inverted microscope (Zeiss, Germany).

2.9. Statistical analyses All data are expressed as mean and standard deviation. A paired ttest was used for Figs. 1, 2 A, B, 3 and 4 . These figures were obtained from three independent experiments conducted on separate days. For Fig. 2C, it shows changes in tumor size during sampling. Each data point shows the mean detected value from 10 different mice. For comparison against different treatment groups, a paired student t-test was used. A p-value < 0.05 was considered statistically significant.

2.4. Boyden chamber migration assay Migration assay was performed using the Boyden chamber which consists a Falcon cell culture insert. 5000 HREC cells, drug and basal M131 medium were placed in the gelatin-coated cell culture insert and M131 medium supplemented with VEGF were placed on the lower chamber in 24-well plate. After 6 h incubation, cells on the upper surface of the insert were removed with a cotton swab. Migratory cells move through the pores towards the lower surface of inserts were fixed, and stained with 0.4% Giemsa. The migrated cells were counted under using light microscope (Zeiss, Germany).

3. Results 3.1. Ribavirin effectively targets multiple biological functions of retinoblastoma To assess the anti-retinoblastoma activity of ribavirin, we firstly examined the effects of ribavirin in retinoblastoma growth and survival 751

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Fig. 1. Ribavirin is active against retinoblastoma tumor cells and retinal angiogenesis. Ribavirin at 10 μM, 20 μM, and 50 μM significantly inhibits proliferation (A), increases Annexin V percentage (B) and increases active caspase 3 activity (C) in multiple retinoblastoma cell lines: Y79, RB116 and WERI-Rb-1. (D) Ribavirin dose-dependently inhibits human retinal endothelial cell (HREC) capillary network formation. Complete Matrigel supplemented with various growth factors and cytokines were used for HREC tube formation. Ribavirin significantly inhibits migration (E), proliferation (F) and induces apoptosis (G) of HREC in the presence of human vascular endothelial growth factor (VEGF). 10 ng/ml VEGF was used. *p < 0.05, compared to control or VEGF.

chemotherapy, periocular and intra-arterial chemotherapy have fewer side effects from the chemo. To further assess the translational potential of ribavirin in retinoblastoma, we investigated whether combination of ribavirin with standard chemotherapy agent carboplatin can achieve greater efficacy than single drug alone. The concentrations of ribavirin or carboplatin used for combination studies are the concentrations that inhibits less than 50% growth and survival as single drug alone in retinoblastoma cells (Fig. 2A and B). The combination resulted in significantly more growth inhibition and apoptosis induction in a dosedependent manner (Fig. 2A and B). To assess the combinatory effect of ribavirin and carboplatin in vivo, we used Y79 to generate tumors in SCID mice. We did not observe significant toxicity in mice treated with oral ribavirin (50 mg/kg) or intraperitoneal carboplatin (1 mg/kg) alone as shown by mice body weight, appearance and behavior (Data not shown). Importantly, we observed greatly reduced tumor growth in the combination of ribavirin and carboplatin-treated group (Fig. 2C). This correlates well with in vitro data, demonstrating that ribavirin significantly enhances chemotherapy agent’s efficacy.

using a panel of retinoblastoma cell lines: Y79, WERI-Rb-1 and RB116. Since the plasma ribavirin concentration at 88 μM have been detected in patients receiving ribavirin therapy [16], we treated retinoblastoma cells with ribavirin up to 50 μM. Three days of ribavirin treatment demonstrated that all cell lines were growth inhibited with IC50 at ∼20 μM (Fig. 1A), which is within the clinically achievable range. In addition, ribavirin significantly induced apoptosis of all cell lines tested as assessed by flow cytometry of Annexin V staining and caspase 3 activity (Fig. 1B and C). RB116 is more sensitive than WERI-Rb-1 and Y79 to ribavirin. Retinoblastoma is known to be angiogenesis-dependent tumor and anti-angiogenesis therapy has been shown to effectively target retinoblastoma [19,22]. We performed capillary network formation, a representative in vitro angiogenesis assay, using primary human retinal microvascular endothelial cell (HREC) on complete Matrigel supplemented with growth factors and cytokines [23]. HREC forms extensive capillary network within 6 h in control (Fig. 1D). However, HREC fails to form proper capillary network in the presence of 10 and 20 μM ribavirin. Hardly any tubular network was formed in the presence of 50 μM ribavirin (Fig. 1D), demonstrating that ribavirin inhibits retinal angiogenesis. Consistently, ribavirin significantly inhibits vascular endothelial growth factor (EVGF)-induced proliferation, migration and survival of HREC (Fig. 1E–G).

3.3. Ribavirin acts on retinoblastoma cells through inhibiting eIF4E activity Substantial evidence have shown that the anti-cancer activities of ribavirin are due to its ability to target eIF4E and its regulated protein translation [4,9]. We used several strategies to confirm that ribavirin also acts on retinoblastoma via targeting eIF4E. We observed the dosedependent decrease of phosphorylation of eIF4E at Ser209 in Y79 cells exposed to ribavirin (Fig. 3A). eIF4E specifically reduces translation of the transcripts that contain long and highly structured 5′ UTRs,

3.2. Ribavirin significantly enhances in vitro and in vivo efficacy of chemotherapy agent in retinoblastoma Carboplatin is a periocular chemotherapeutic drug which can also be used as via intra-arterial injection. Compared to systemic 752

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Fig. 2. Ribavirin significantly enhances the inhibitory effects of chemotherapy agent in retinoblastoma cells in vitro and in vivo. Combination of carboplatin with ribavirin is superior in inhibiting proliferation (A) and inducing apoptosis (B) than carboplatin or ribavirin alone in Y79, RB116 and WERI-Rb-1 cells. (C) Combination of carboplatin and ribavirin is superior in inhibiting Y79 tumor growth in xenograft mouse model than single drug alone. *p < 0.05, compared to carboplatin or ribavirin alone.

Fig. 3. Ribavirin acts on retinoblastoma cells via inhibiting eIF4E pathway. (A) Representative Western Blot photo showing the decreased levels of p-eIF4E, Cyclin D1, c-Myc and VEGF in Y79 cells treated with ribavirin at different concentrations. (B) mRNA expression level of Cyclin D1, c-Myc and VEGF are not affected by ribavirin in Y79 cells. Cells were treated with ribavirin for 24 h prior to western blot and gene mRNA expression analysis. Results shown are the relative to control. Overexpression of eIF4E S209D and WT but not S209A significantly reversed the anti-proliferative (C) and pro-apoptotic (D) effects of ribavirin in Y79 cells. *p < 0.05, compared to pVec (p-Vector control).

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Fig. 4. Genetic inhibition of eIF4E mirrors ribavirin’s inhibitory effects in retinoblastoma cells. (A) Representative Western Blot photo showing the decreased levels of p-eIF4E, eIF4E, Cyclin D1, c-Myc and VEGF in eIF4E-depleted Y79 cells. Two independent siRNAs specifically targeting eIF4E were used. (B) mRNA expression level of Cyclin D1, c-Myc and VEGF are not changed in eIF4E-depleted Y79 cells. Cells at 48 h post-transfection were harvested for western blot and mRNA expression analysis. eIF4E knockdown significantly enhances carboplatin’s effects in inhibiting proliferation (C) and inducing apoptosis (D) in Y79 cells. 0.2 μM of carboplatin was used. *p < 0.05, compared to SCRsiRNA (scramble siRNA).

pigment epithelium-derived factor is active against retinoblastoma without producing significant systemic toxicity [22,27]. However, many angiogenesis inhibitors have less or no direct effects on retinoblastoma cells themselves. In this work, we demonstrate that clinically relevant concentrations of ribavirin, a FDA approved antiviral drug, inhibits not only retinoblastoma tumor cells but also angiogenesis, via suppressing eIF4E function. All retinoblastoma cell lines used for evaluation of ribavirin’s effects are sensitive to ribavirin treatment (Fig. 1A–C). Importantly, the IC50 of ribavirin in retinoblastoma is clinically achievable [16]. In addition, these cell lines express primitive stem cell and progenitor markers [28–30], suggesting that ribavirin might target retinoblastoma stem/ progenitor subpopulations. Using in vitro culture system as well as in vivo xenograft tumor model, we further demonstrate that combination of ribavirin and carboplatin at doses that are not toxic to mice results in greater efficacy than single drug alone in inhibiting retinoblastoma (Fig. 2). These findings are consistent with many studies showing that ribavirin synergistically cooperates with common chemotherapies in cancers [7,8,17]. Combination therapy consisting of two agents with different molecular targets can maximize potency and minimise toxicity [31]. The potent efficacy of ribavirin as a single agent and combination with chemotherapy at pharmacologically achievable doses suggests that ribavirin is an attractive candidate for retinoblastoma treatment. Besides targeting retinoblastoma cells, we show that ribavirin significantly inhibits retinal angiogenesis as shown by the in vitro HREC capillary network formation assay (Fig. 1D). Cell migration and proliferation are critical steps involved in endothelial cell capillary network formation. Ribavirin effectively inhibits HERC migration and growth, and induces HERC apoptosis (Fig. 1E–G). Although the antitumor activities of ribavirin have been shown in various tumors, its anti-angiogenesis activity is not well elucidated. Our findings demonstrate that ribavirin is a novel angiogenesis inhibitor. The ability of ribavirin in targeting cancer and cancer angiogenesis distinguishes it from anti-cancer compounds that only target cancer cells. As expected, retinoblastoma inhibition by ribavirin is associated with a decreased protein but not mRNA levels of Cyclin D1, c-Myc and VEGF (Fig. 3A and B). These suggest that ribavirin inhibits eIF4E function. Overexpression of the wildtype and phosphormimetic but not the nonphosphorylatable form of eIF4E significantly abolished the

including a number of mRNAs such as Cyclin D1, c-Myc and VEGF [4]. The reduced protein levels and unchanged mRNA levels of Cyclin D1, cMyc and VEGF in ribavirin-treated cells (Fig. 3A and B), demonstrating that ribavirin inhibits eIF4E activity in retinoblastoma cells. In addition, overexpression of the wildtype and phosphomimetic (S209D) but not nonphosphorylatable form (S209A) of eIF4E abolished the anti-proliferative and pro-apoptotic effects of ribavirin (Supplementary Fig. S1, Fig. 3C and D), further confirming eIF4E as the target of ribavirin in retinoblastoma cells. 3.4. eIF4E inhibition effectively target retinoblastoma and significantly sensitizes retinoblastoma cell response to chemotherapy agent eIF4E is critically involved in tumor transformation, progression and resistance [6,24]. However, the role of IF4E in retinoblastoma is not well understood. We have shown that inhibiting eIF4E via pharmacological method (eg. Ribavirin) effectively targets retinoblastoma in vitro and in vivo (Figs. 1–3). We asked whether inhibiting eIF4E via genetic method could mirror ribavirin’s effects. We specifically knockdown eIF4E using two siRNAs targeting different regions as shown by the decreased eIF4E levels in retinoblastoma cells (Fig. 4A). We observed the decreased levels of, Cyclin D1, c-Myc and VEGF whereas the mRNA levels of these eIF4E-sensitive transcripts were not affected in eIF4E-depleted Y79 cells (Fig. 4A and B). Importantly, decreased proliferation and increased apoptosis were detected in eIF4E-depleted cells (Fig. 4C and D). In addition, carboplatin is significantly more effective in inhibiting proliferation and inducing apoptosis in eIF4E-depleted compared to control cells (Fig. 4C and D), suggesting that eIF4E inhibition sensitizes retinoblastoma cell response to carboplatin. 4. Discussion Retinoblastoma is a heterogeneous disease with aberrant activation of oncogenes and suppression of the tumor suppressor genes, such as retinoblastoma 1 [25]. Angiogenesis, the growth of new blood vessels from pre-existing capillaries, is necessary for solid tumor growth and metastasis, in particular retinoblastoma [26]. Angiogenesis in retinoblastoma is the source of tumor survival and malignant progression. Studies have shown that angiogenesis inhibition by bevacizumab or 754

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[2] S. Lim, T.Y. Saw, M. Zhang, M.R. Janes, K. Nacro, J. Hill, A.Q. Lim, C.T. Chang, D.A. Fruman, D.A. Rizzieri, S.Y. Tan, H. Fan, C.T. Chuah, S.T. Ong, Targeting of the MNK-eIF4E axis in blast crisis chronic myeloid leukemia inhibits leukemia stem cell function, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) E2298–2307. [3] O. Larsson, S. Li, O.A. Issaenko, S. Avdulov, M. Peterson, K. Smith, P.B. Bitterman, V.A. Polunovsky, Eukaryotic translation initiation factor 4E induced progression of primary human mammary epithelial cells along the cancer pathway is associated with targeted translational deregulation of oncogenic drivers and inhibitors, Cancer Res. 67 (2007) 6814–6824. [4] A. Kentsis, I. Topisirovic, B. Culjkovic, L. Shao, K.L. Borden, Ribavirin suppresses eIF4E-mediated oncogenic transformation by physical mimicry of the 7-methyl guanosine mRNA cap, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 18105–18110. [5] H. Xu, Z. Wang, L. Xu, G. Mo, G. Duan, Y. Wang, Z. Sun, H. Chen, Targeting the eIF4E/beta-catenin axis sensitizes cervical carcinoma squamous cells to chemotherapy, Am. J. Transl. Res. 9 (2017) 1203–1212. [6] M. Grzmil, J. Seebacher, D. Hess, M. Behe, R. Schibli, G. Moncayo, S. Frank, B.A. Hemmings, Inhibition of MNK pathways enhances cancer cell response to chemotherapy with temozolomide and targeted radionuclide therapy, Cell. Signal. 28 (9) (2016) 1412–1421, http://dx.doi.org/10.1016/j.cellsig.2016.06.005. [7] F. Shi, Y. Len, Y. Gong, R. Shi, X. Yang, D. Naren, T. Yan, Ribavirin inhibits the activity of mTOR/eIF4E, ERK/Mnk1/eIF4E signaling pathway and synergizes with tyrosine kinase inhibitor imatinib to impair bcr-Abl mediated proliferation and apoptosis in Ph+ leukemia, PLoS One 10 (2015) e0136746. [8] B.C. Kraljacic, M. Arguello, A. Amri, G. Cormack, K. Borden, Inhibition of eIF4E with ribavirin cooperates with common chemotherapies in primary acute myeloid leukemia specimens, Leukemia 25 (2011) 1197–1200. [9] K. Tan, B. Culjkovic, A. Amri, K.L. Borden, Ribavirin targets eIF4E dependent Akt survival signaling, Biochem. Biophys. Res. Commun. 375 (2008) 341–345. [10] A. Kentsis, L. Volpon, I. Topisirovic, C.E. Soll, B. Culjkovic, L. Shao, K.L. Borden, Further evidence that ribavirin interacts with eIF4E, RNA 11 (2005) 1762–1766. [11] S. Assouline, B. Culjkovic, E. Cocolakis, C. Rousseau, N. Beslu, A. Amri, S. Caplan, B. Leber, D.C. Roy, W.H. Miller Jr., K.L. Borden, Molecular targeting of the oncogene eIF4E in acute myeloid leukemia (AML): a proof-of-principle clinical trial with ribavirin, Blood 114 (2009) 257–260. [12] S. Crotty, C. Cameron, R. Andino, Ribavirin's antiviral mechanism of action: lethal mutagenesis? J. Mol. Med. (Berl.) 80 (2002) 86–95. [13] F. Volpin, J. Casaos, J. Sesen, A. Mangraviti, J. Choi, N. Gorelick, J. Frikeche, T. Lott, R. Felder, S.J. Scotland, T.S. Eisinger-Mathason, H. Brem, B. Tyler, N. Skuli, Use of an anti-viral drug, Ribavirin, as an anti-glioblastoma therapeutic, Oncogene 36 (21) (2017) 3037–3047, http://dx.doi.org/10.1038/onc.2016.457. [14] L. Teng, D. Ding, Y. Chen, H. Dai, G. Liu, Z. Qiao, R. An, Anti-tumor effect of ribavirin in combination with interferon-alpha on renal cell carcinoma cell lines in vitro, Cancer Cell Int. 14 (2014) 63. [15] K.L. Borden, B. Culjkovic-Kraljacic, Ribavirin as an anti-cancer therapy: acute myeloid leukemia and beyond? Leuk. Lymphoma 51 (2010) 1805–1815. [16] F. Pettersson, C. Yau, M.C. Dobocan, B. Culjkovic-Kraljacic, H. Retrouvey, R. Puckett, L.M. Flores, I.E. Krop, C. Rousseau, E. Cocolakis, K.L. Borden, C.C. Benz, W.H. Miller Jr., Ribavirin treatment effects on breast cancers overexpressing eIF4E, a biomarker with prognostic specificity for luminal B-type breast cancer, Clin. Cancer Res. 17 (2011) 2874–2884. [17] D. Dai, H. Chen, J. Tang, Y. Tang, Inhibition of mTOR/eIF4E by anti-viral drug ribavirin effectively enhances the effects of paclitaxel in oral tongue squamous cell carcinoma, Biochem. Biophys. Res. Commun. 482 (2017) 1259–1264. [18] C.L. Shields, J.A. Shields, Retinoblastoma management: advances in enucleation, intravenous chemoreduction, and intra-arterial chemotherapy, Curr. Opin. Ophthalmol. 21 (2010) 203–212. [19] E.F. Marback, V.E. Arias, A. Paranhos Jr., F.A. Soares, A.L. Murphree, C.M. Erwenne, Tumour angiogenesis as a prognostic factor for disease dissemination in retinoblastoma, Br. J. Ophthalmol. 87 (2003) 1224–1228. [20] K.A. Wikenheiser-Brokamp, Retinoblastoma family proteins: insights gained through genetic manipulation of mice, Cell. Mol. Life Sci. 63 (2006) 767–780. [21] M.G. Kharas, J.A. Deane, S. Wong, K.R. O'Bosky, N. Rosenberg, O.N. Witte, D.A. Fruman, Phosphoinositide 3-kinase signaling is essential for ABL oncogenemediated transformation of B-lineage cells, Blood 103 (2004) 4268–4275. [22] S.Y. Lee, D.K. Kim, J.H. Cho, J.Y. Koh, Y.H. Yoon, Inhibitory effect of bevacizumab on the angiogenesis and growth of retinoblastoma, Arch. Ophthalmol. 126 (2008) 953–958. [23] I. Arnaoutova, H.K. Kleinman, In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract, Nat. Protoc. 5 (2010) 628–635. [24] M. Bhat, N. Robichaud, L. Hulea, N. Sonenberg, J. Pelletier, I. Topisirovic, Targeting the translation machinery in cancer, Nat. Rev. Drug Discov. 14 (2015) 261–278. [25] B.L. Theriault, H. Dimaras, B.L. Gallie, T.W. Corson, The genomic landscape of retinoblastoma: a review, Clin. Exp. Ophthalmol. 42 (2014) 33–52. [26] R.S. Apte, J.W. Harbour, Inhibiting angiogenesis in retinoblastoma, Ophthalmic Res. 39 (2007) 188–190. [27] H. Yang, R. Cheng, G. Liu, Q. Zhong, C. Li, W. Cai, Z. Yang, J. Ma, X. Yang, G. Gao, PEDF inhibits growth of retinoblastoma by anti-angiogenic activity, Cancer Sci. 100 (2009) 2419–2425. [28] H. Hu, F. Deng, Y. Liu, M. Chen, X. Zhang, X. Sun, Z. Dong, X. Liu, J. Ge, Characterization and retinal neuron differentiation of WERI-Rb1 cancer stem cells, Mol. Vis. 18 (2012) 2388–2397. [29] A. Bejjani, M.R. Choi, L. Cassidy, D.W. Collins, J.M. O'Brien, T. Murray, B.R. Ksander, G.M. Seigel, RB116: an RB1+ retinoblastoma cell line expressing primitive markers, Mol. Vis. 18 (2012) 2805–2813. [30] S.A. Madreperla, R. Bookstein, O.W. Jones, W.H. Lee, Retinoblastoma cell lines Y79, RB355 and WERI-Rb27 are genetically related, Ophthalmic Paediatr. Genet. 12

inhibitory effects of ribavirin, further confirming that eIF4E inhibition is required for the action of ribavirin in retinoblastoma. Although other molecular mechanisms of anti-cancer activities of ribavirin have been reported, such as ERK/MNK inhibition [7], the majority of studies together with our results indicate that eIF4E inhibition is a common mechanism of ribavirin in cancer [4,8,16]. We and others have shown that VEGF is an essential growth factor for tumor angiogenesis (Fig. 1D and F)[32]. Although we only demonstrated the inhibitory effects of ribavirin in in vitro angiogenesis, the ability of ribavirin in blocking VEGF protein translation suggests that ribavirin is highly likely to inhibit angiogenesis in vivo. A significant findings of our study is that specific inhibition of eIF4E via genetic approach mirrors ribavirin’s effects in retinoblastoma, including inhibiting growth, inducing apoptosis and enhancing carboplatin’s effects in retinoblastoma cells (Fig. 4C and D). This is the first direct evidence demonstrating that eIF4E is important in biological activities of retinoblastoma and its response to chemotherapy. Consistently, the decreased protein but not mRNA levels of Cyclin D1, cMyc and VEGF are observed in eIF4E-depleted retinoblastoma cells. Interestingly, phosphorylation of eIF4E at Ser209 are decreased in both cells treated with ribavirin and eIF4E depletion (Figs. 3 A and 4 A). eIF4E phosphorylation is essential for chemoresistance [5,6,33], which might explain our results on the enhancement between eIF4E inhibition and carboplatin in retinoblastoma. Our work adds retinoblastoma to a lists of eIF4E-regulated cancers and further supports a crucial role of eIF4E and its inhibition in cancer therapy. In addition, eIF4E phosphorylation is also an important step in control of mRNA transport and cell transforming properties [34]. It would be interesting to examine whether eIF4E inhibition plays any role in carcinogenesis in retinoblastoma. However, little information is known on the expression levels of eIF4E in retinoblastoma compared to normal retinal tissue. A large cohort of retinoblastoma tissues from patients and normal retinal tissues from healthy donors would be idea to investigate whether eIF4E is upregulated in retinoblastoma. We would like to clarify that targeting eIF4E is effective in inhibiting many cancers regardless of eIF4E level [13,17]. We speculate that greater efficacy of eIF4E inhibition would be achieved in those cancers with elevated eIF4E. The expression patterns of eIF4E in retinoblastoma and normal counterparts warrants future investigation in order to fully understand the role of eIF4E in retinoblastoma. In conclusion, we have confirmed ribavirin’s activity against retinoblastoma and its angiogenesis using three different cell lines and xenograft mouse model. Ribavirin is a clinically available generic drug with limited toxicity [35]. Our findings provide a rational for the clinical study evaluating ribavirin in the setting of retinoblastoma. Our findings also reveal the critical role of eIF4E in retinoblastoma. Conflict of interest All authors declare no conflict of interest. Acknowledgement This work was supported by a research grant provided by Natural Science Foundation of Hubei (2014CFB214). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.biopha.2017.10.034. References [1] I.B. Rosenwald, J.J. Chen, S. Wang, L. Savas, I.M. London, J. Pullman, Upregulation of protein synthesis initiation factor eIF-4E is an early event during colon carcinogenesis, Oncogene 18 (1999) 2507–2517.

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survival of human hepatocellular carcinoma, Biomed. Pharmacother. 84 (2016) 237–243. [34] I. Topisirovic, M. Ruiz-Gutierrez, K.L. Borden, Phosphorylation of the eukaryotic translation initiation factor eIF4E contributes to its transformation and mRNA transport activities, Cancer Res. 64 (2004) 8639–8642. [35] F. Pettersson, S.V. Del Rincon, A. Emond, B. Huor, E. Ngan, J. Ng, M.C. Dobocan, P.M. Siegel, W.H. Miller Jr., Genetic and pharmacologic inhibition of eIF4E reduces breast cancer cell migration, invasion, and metastasis, Cancer Res. 75 (2015) 1102–1112.

(1991) 49–56. [31] S. Kummar, H.X. Chen, J. Wright, S. Holbeck, M.D. Millin, J. Tomaszewski, J. Zweibel, J. Collins, J.H. Doroshow, Utilizing targeted cancer therapeutic agents in combination: novel approaches and urgent requirements, Nat. Rev. Drug Discov. 9 (2010) 843–856. [32] R. Ronca, M. Benkheil, S. Mitola, S. Struyf, S. Liekens, Tumor angiogenesis revisited: regulators and clinical implications, Med. Res. Rev. 37 (6) (2017) 1231–1274, http://dx.doi.org/10.1002/med.21452. [33] Y. Liu, L. Sun, X. Su, S. Guo, Inhibition of eukaryotic initiation factor 4E phosphorylation by cercosporamide selectively suppresses angiogenesis, growth and

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