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RESEARCH ARTICLE

MPT0B098, a Microtubule Inhibitor, Suppresses JAK2/STAT3 Signaling Pathway through Modulation of SOCS3 Stability in Oral Squamous Cell Carcinoma Hsuan-Yu Peng1,2, Yun-Ching Cheng3, Yuan-Ming Hsu1, Guan-Hsun Wu1, ChingChuan Kuo4, Jing-Ping Liou5, Jang-Yang Chang1,6, Shiow-Lian Catherine Jin2☯, ShineGwo Shiah1☯*

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1 National Institute of Cancer Research, National Health Research Institutes, Miaoli, Taiwan, 2 Department of Life Sciences, National Central University, Taoyuan, Taiwan, 3 Department of Medical Research, Show Chwan Memorial Hospital, Changhua, Taiwan, 4 Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli, Taiwan, 5 School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan, 6 Division of Hematology and Oncology, Department of Internal Medicine, National Cheng Kung University Hospital, College of Medical, National Cheng Kung University, Tainan, Taiwan ☯ These authors contributed equally to this work. * [email protected]

OPEN ACCESS Citation: Peng H-Y, Cheng Y-C, Hsu Y-M, Wu G-H, Kuo C-C, Liou J-P, et al. (2016) MPT0B098, a Microtubule Inhibitor, Suppresses JAK2/STAT3 Signaling Pathway through Modulation of SOCS3 Stability in Oral Squamous Cell Carcinoma. PLoS ONE 11(7): e0158440. doi:10.1371/journal. pone.0158440 Editor: Jung Weon Lee, Seoul National University, REPUBLIC OF KOREA Received: May 4, 2016 Accepted: June 15, 2016 Published: July 1, 2016 Copyright: © 2016 Peng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract Microtubule inhibitors have been shown to inhibit Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signal transduction pathway in various cancer cells. However, little is known of the mechanism by which the microtubule inhibitors inhibit STAT3 activity. In the present study, we examined the effect of a novel small-molecule microtubule inhibitor, MPT0B098, on STAT3 signaling in oral squamous cell carcinoma (OSCC). Treatment of various OSCC cells with MPT0B098 induced growth inhibition, cell cycle arrest and apoptosis, as well as increased the protein level of SOCS3. The accumulation of SOCS3 protein enhanced its binding to JAK2 and TYK2 which facilitated the ubiquitination and degradation of JAK2 and TYK2, resulting in a loss of STAT3 activity. The inhibition of STAT3 activity led to sensitization of OSCC cells to MPT0B098 cytotoxicity, indicating that STAT3 is a key mediator of drug resistance in oral carcinogenesis. Moreover, the combination of MPT0B098 with the clinical drug cisplatin or 5-FU significantly augmented growth inhibition and apoptosis in OSCC cells. Taken together, our results provide a novel mechanism for the action of MPT0B098 in which the JAK2/STAT3 signaling pathway is suppressed through the modulation of SOCS3 protein level. The findings also provide a promising combinational therapy of MPT0B098 for OSCC.

Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors received no specific funding for this work. Competing Interests: The authors have declared that no competing interests exist.

Introduction The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signal transduction pathway is frequently dysregulated in various human cancer cells [1] and plays a critical role in oncogenesis including proliferation, apoptosis, drug resistance, migration, invasion and

PLOS ONE | DOI:10.1371/journal.pone.0158440 July 1, 2016

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MPT0B098 Modulates SOCS3 Stability

angiogenesis [2]. The STAT family member STAT3 has been reported to possess oncogenic potential as constitutive activation in oral squamous cell carcinoma (OSCC) and transduce signals elicited by various cytokines leading to regulation of specific target genes that contribute to a malignant phenotype [3–5]. Furthermore, targeting STAT3 with dominant negative mutants of STAT3 or antisense oligonucleotides specific for the STAT3 DNA sequence causes reversion of the malignant phenotype of squamous cell carcinoma [6, 7], suggesting that STAT3 is a key mediator for the pathogenesis of these cancers. There are two classical negative feedback regulators for the JAK/STAT signaling pathway, the protein inhibitors of activated STATs (PIAS) and the suppressors of cytokine signaling (SOCS), through which the STAT pathway is silenced by masking STAT binding sites on the receptors, by binding to JAKs to inhibit their kinase activity, or by targeting proteins for proteasomal degradation through ubiquitination [8, 9]. Among these negative regulators, SOCS3 is known to attenuate interleukin-6 (IL-6) induced STAT3 activation [10, 11]. An in vivo study has shown that Socs3-deficient mice produced a prolonged activation of STAT3 after IL-6 treatment [10], indicating a crucial role of SOCS3 in IL-6/JAK/STAT signaling axis. Moreover, loss of SOCS3 expression has been described in head and neck squamous cell carcinoma (HNSCC) [12]. Experimental overexpression of SOCS protein in cancer cells results in growth suppression and apoptosis induction [12], strongly suggesting that SOCS proteins may function as tumor suppressors. Thus, SOCS3 is regarded as a useful diagnostic molecule and a potential therapeutic target for HNSCC. To date, more than 90% of HNSCC belongs to OSCC in the South-East Asia, including Taiwan [13]. Despite the fact that most patients who are readily amenable to clinical examination and diagnosed at an early stage have an excellent survival rate, the 5-year survival rate for those patients with loco-regional recurrences and neck lymph metastasis has not significantly improved over the past years [14]. Thus, there is a need for a better understanding of the biological nature of oral cancers in order to develop novel strategies to improve the efficacy of the treatment. At present, the usage of chemotherapy drugs available for oral cancers, such as 5-fluorouracil (5-FU) and cisplatin, is limited due to their side effects, drug resistance and nonspecificity [15, 16]. As a result, more attention has been drawn to the combinational approach aiming to improve the efficacy of the chemotherapeutic drugs on OSCC tumorigenesis and progression [17–19]. In the present study, we used a novel small-molecule microtubule inhibitor, 7-aryl-indoline-1-benzene-sulfonamide (MPT0B098) [20], to examine whether a microtubule-based chemotherapy modulates the JAK2/STAT3/SOCS3 signal pathway. We found that MPT0B098 could delay the turnover of SOCS3 protein in OSCC cell lines and resulted in JAK2/STAT3 inactivation and induction of apoptosis. Inhibition of endogenous SOCS3 significantly reduced the MPT0B098-induced apoptosis in oral cancer cells, whereas overexpression of SOCS3 induced the apoptosis. Furthermore, treatment with MPT0B098 in combination with cisplatin or 5-FU caused significantly apoptosis as compared to the treatment with a single agent or the combination of cisplatin and 5-FU. Taken together, our results uncover a novel mechanism for the action of the microtubule inhibitor MPT0B098 on SOCS3 modulation in OSCC cells. They also provide a promising therapeutic strategy for OSCC.

Materials and Methods Cell culture and chemicals Human oral keratinocytes (HOK) were purchased from ScienCell (Carlsbad, CA, USA) and cultured according to the manufacturer’s instructions. The culture conditions for all the OSCC cell lines are summarized as previously described [21]. All cells were cultured at 37°C in a 5% CO2 atmosphere and maintained in the presence of 10% fetal bovine serum (FBS, Kibbutz


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BeitHaemek, Israel) for approximately 3 months after resuscitation from the frozen aliquots. The cells used for the experiments were lower than 20 passages. All culture media were purchased from Invitrogen. Sodium pyruvate was obtained from Merck and hydrocortisone from Sigma-Aldirch. The chemical synthetic procedure of MPT0B098 was described previously [22].

Protein extraction and Western blot analysis The OSCC cells were lysed in a lysis buffer containing 50 mM Tris-HCl, 1% NP-40, 150 mM NaCl, 0.1% SDS, 1 mM PMSF, 1mM Na3VO4 and protease inhibitor cocktail (1:1000) (SigmaAldrich, Inc.). Protein concentrations then were determined by the BCA assay kit (Thermo, USA) according to the manufacturer’s instructions. Equal amount of protein lysates were loaded onto 10~12% SDS polyacrylamide gels and subjected to electrophoresis, followed by transferring the proteins to poly-vinylidene fluoride membrane (Pall Life Sciences, Glen Cove, NY). The membranes then were probed with antibodies specific for Bcl-2 (sc-509) (Santa Cruz, Heidelberg, Germany), phosphor-JAK2 (Millipore, MA, USA), JAK1 (#E021119), phosphorJAK1 (#E021149) (EnoGene, NY, USA), Mcl-1 (#GTX102026) (GeneTex, Inc, Irvine, CA, USA), Survivin (#2463), Pim-1 (#2409), JAK2 (#2863), phosphor-STAT3 (#2236) (Epitomics Inc., Burlingame, CA), STAT3 (#610189) (BD Biosciences, NJ, USA), α-tubulin (#MS-581-P) (Thermo, CA, USA), caspases-3 (#9662), PARP (#5625), TYK2 (#9312), phosphor-TYK2 (#9321), SOCS1 (#3950), SOCS2 (#2779), SOCS3 (#2932), PIAS1 (#3550), and PIAS3 (#4164) (Cell signaling, USA). The anti-GAPDH antibody (Thermo, CA, USA) was used as an internal control. Signals from HRP-coupled secondary antibodies were visualized by the enhanced chemiluminescence (ECL) detection system (PerkinElmer, Waltham, MA) and the chemiluminescence was exposed onto Kodak X-Omat film (Kodak, Chalon/Paris, France).

RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) assay Total RNA was isolated from OSCC cells with the TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions. First strand cDNA was synthesized using random hexamer primers and SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA). PCR was run on a Biometra T3000 thermocycler (Biometra GmbH, Germany). The reaction cycles included an initial denaturation step at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s and one cycle of final extension for 10 min at 72°C. The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The sequences of the primers used for PCR were as follows: SOCS3 5’-TATG CGGC CAGC AAAG AATC A-3’ (forward), 5’-CG GG CAAT CTCC ATTG GCT-3’ (reverse); GAPDH 5’-GAA GGT GAA GGT CGG AGT-3’ (forward), 5’-GAA GAT GGT GAT GGG ATT TC -3’ (reverse). PCR products were subjected to electrophoresis on 2% agarose gel and visualized on UVP GDS-8000 Bioimaging System (UVP, CA, USA) with 0.01% of SYBR1 Safe (Invitrogen, Carlsbad, CA, USA) staining.

Plasmids and transfection To construct the human SOCS3 expression vector, a 677-base pair fragment of SOCS3 was amplified by PCR using the SOCS3 cloning primers: 5’-GGGG ATCC GCCA CCAT GGTC ACCC ACAG CAAG-3’ (forward) and 5’-GGGA ATTC TTAA GCGG GGCA TCGT AC-3’ (reverse), and then cloned into the BamHI/EcoRI sites of pcDNA3.1+ vector (Invitrogen, Gaithersburg, MD). For gene knockdown experiments, the shRNA clones for SOCS3, STAT3 and their control pLKO_TRC vector (NS) were obtained from the National RNAi Core Facility


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(Academia Sinica, Taiwan). The plasmids were transiently transfected into OSCC cells using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s protocol. Briefly, Lipofectamine and plasmid were dissolved in Opti-MEM1 I Reduced Serum Medium (Gibco, NY, USA) separately and incubated at room temperature for 5 min. The two reagents then were mixed and incubated for additional 20 min at room temperature. Subsequently, the Lipofectamine-plasmid mixture was added drop-wise onto the OSCC cells grown in the culture medium containing 10% FCS After incubation for 48 hrs, the cells were harvestedfor further analysis.

Preparation of monomer and polymer fractions of microtubule The monomer and polymer fractions of microtubule were prepared following the previously described methods with slight modifications [23]. Briefly, cells were seeded in 60-mm plastic petri dishes and treated with testing drugs for indicated times. Cells then were washed twice with cold phosphate-buffer saline (PBS), followed by extraction with microtubule-stabilizing buffer (MSB) (85 mM PIPES, pH 6.93, 1 mM EGTA, 1 mM MgCl2, 2 M Glycerol, and Sigma protease inhibitor cocktail) containing 0.5% Triton X-100. After 3 min, the Triton extract (monomer fraction) was gently removed from the dish and transferred to an eppendrof tube, and 1/5 volume of 5x SDS sample buffer (10% SDS, 325 mM Tris-HCl, pH 6.8, 30% glycerol, 250 mM DTT and 1 mM phenylmethylsulfonyl fluoride) was added into the tube. The remaining part (polymer fraction) in the petri dish was gently washed twice with cold MSB. The polymer fraction then was extracted with MSB and mixed into 1/5 volume of 5x SDS sample buffer. Both extracts were boiled for 10 min and stored at -80°C till western blot analysis.

Cell viability assay The OSCC cells were harvested in the exponential growing phase and seeded into 96-well plates (3000 cells/well). After overnight incubation, 200 μl of culture medium containing the test compound was dispensed into each well. Following 72-h treatment, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye (Calbiochem, CA, USA) was added. After 2 hrs of incubation, the medium and MTT dye were removed by slow aspiration and 100μl of dimethyl sulfoxide (DMSO) was added to dissolve the remaining MTT formazan crystals. The absorbance at 550 nm was measured using a 96-well plate SpectraMax 250 reader (Molecular Devices, CA, USA).

Cell cycle analysis The OSCC cells were seeded in 12-well plates (2x105 cells/well) and treated with drugs for 24 hrs. Cells then were trypsinized, washed with PBS, and fixed in 70% ethanol at -20°C. After fixation, cells were washed twice with PBS and incubated in 0.5% Triton X-100/PBS containing 1 mg/ml RNase A at 37°C for 30 min. Subsequently, the cells were stained with propidium iodide (PI) at the final concentration of 30 μg/ml. Samples were measured using the FACscan flow cytometer (Becton Dickinson).

Apoptosis assay Caspase-3 protease activity was measured by the Caspase-3/CPP32 activity Colorimetric Assay kit (Biovision Incorporate, CA, USA). In brief, cells were seeded in 6-well plates (2x106 cells/ well) and treated with drugs for 24 hrs. The cells then were harvested and centrifuged at 4°C for 5 min. The cytosolic extracts were prepared by repeated cycles of freezing and thawing of the cells in 50 μl of lysis buffer. The lysates were centrifuged at 10,000 xg for 5 min. Protein


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concentrations were determined by the BCA assay kit (Thermo, USA) according to the manufacturer’s instructions. Cell lysates (100 μg) were then diluted with 50μl of lysis buffer followed by addition of 50 μl of the 2X reaction buffer. After incubation in the presence of the fluorescence substrate Ac-DEVD-pNA (200 μM) at 37°C for 2 hrs in dark, the absorbance at 405 nm was determined using a SpectraMax 250 reader (Molecular Devices, CA, USA). For the Annexin V-fluorescein isothiocyanate (FITC)/PI assay, testing cells were washed with cold PBS twice, and then resuspended in the buffer containing Annexin V-FITC and PI. The mixture was kept in dark for 15 min at room temperature before analysis by flow cytometry.

Immunofluorescent staining and microscopy For immunofluorescence microscopy, the cells were fixed with freshly prepared 3% paraformaldehyde (Merck, Whitehouse Station, NJ) for 10 min, permeabilized with 0.1% Triton-X/ PBS for 10 min, followed by blocking with 3% bovine serum albumin (BSA) in PBS at 37°C for 1 hr. The cells then were incubated with a mouse α-tubulin monoclonal antibody (MS-581-P, Thermo; diluted in 3% BSA/PBS) for 1 hr, followed by incubation with a FITC-conjugated secondary antibody (Santa Cruze) at room temperature for 1 hr in dark. Finally, the nuclei were counterstained with 40 , 6-diamidino-2-phenylindole (DAPI) at room temperature for 5 min. The coverslips were mounted with ProLong Gold anti-fade reagent (Invitrogen, Carlsbad, CA). The images were captured using the Leica TCS SP5 Confocal Microscopy Workstation (Leica, Wetzlar, Germany).

Statistical analysis The data were expressed as the mean ± standard error (SE) from at least three independent experiments. Differences between various treatment groups were assessed by ANOVA and Student's t-test. A p-value < 0.05 was considered as significant. Calculations were performed using Graph Pad Prism Ver. 4.01 (San Diego, CA).

Results MPT0B098 inhibits the proliferation and tubulin polymerization in OSCC cells In an initial study, the effect of MPT0B098 (Fig 1A) on cell viability was assessed in various human OSCC cells using the MTT assay. As shown in Fig 1B, MPT0B098 inhibited cell growth of all OSCC cell lines tested in a dose-dependent manner, and the IC50 concentrations for these cell lines, including OEC-M1, HSC-3 SCC-25, Tu183, DOK and YD-15, were ranging from 0.14 to 0.45 μmol/L (Table 1). On the other hand, the normal human oral keratinocytes (HOK) exhibited less susceptibility to the inhibitory effect of MPT0B098 with the IC50 of 6.3 μmol/L (Fig 1B and Table 1). The differential toxicity of MPT0B098 on normal keratinocytes and oral cancer cells indicates a potential of the compound for clinical use. We further examined whether tubulin depolymerization was induced by MPT0B098 in the OSCC cells. For this purpose, we isolated soluble non-polymerized tubulin and insoluble polymerized tubulin from OEC-M1 and HSC-3 cells after MPT0B098 treatment. The tubulin polymerization assay showed that MPT0B098 inhibited tubulin polymerization in a time- and concentration-dependent manner (Fig 1C and S1 Fig). The effect of MPT0B098 on microtubule structure then was determined by immuno-fluorescence microscopy using anti-α-tubulin antibody. The results showed that MPT0B098 disrupted microtubule network in a time-dependent manner similar to that of tubulin polymerization (Fig 1D). Furthermore, to test the capacity of MPT0B098 on depolymerization of the tubulin network, we treated


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Fig 1. MPT0B098 inhibits the proliferation and induces microtubules depolymerization in OSCC cells. (A) Chemical structure of MPT0B098. (B) OSCC cells were treated with increasing concentrations of MPT0B098 for 72 hrs and the cell viability was assessed by MTT assay. Data are presents as mean ± SE relative to DMSO vehicle control (indicated as 0 μM) from three replicate experiments. *, p