Novel microtubule inhibitor MPT0B098 inhibits hypoxia-induced epithelial-tomesenchymal transition in head and neck squamous cell carcinoma

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1 Tsi et l. Journl of Biomedicl Science (2018) 25:28 RESEARCH Novel microtuule inhiitor MPT0B098 inhiits hypoxi-induced epithelil-tomesenchyml trnsition in hed nd neck squmous cell crcinom I-Ting Tsi 1,2, Ching-Chun Kuo 3,4,5, Jing-Ping Liou 6 nd Jng-Yng Chng 1,2,4,7* Open Access Astrct Bckground: Tumor hypoxi-induced epithelil mesenchyml trnsition (EMT) is criticl in promoting cncer metstsis. We recently discovered novel microtuule inhiitor, MPT0B098, tht employs novel ntitumor mechnism. It destilizes hypoxi-inducile fctor (HIF)-1α mrna y locking the function of humn ntigen R. Thus, we proposed tht MPT0B098 modultes hypoxi-induced EMT. Methods: In vitro IC 50 vlues were determined through the methylene lue dye ssy. To investigte moleculr events, reverse trnscriptse-polymerse chin rection, Western lotting, immunofluorescence stining, nd wound heling ssy were employed. Results: MPT0B098 significntly inhiited HIF-1α expression, epithelil-to-mesenchyml morphology chnges, nd migrtory ility in the humn hed nd neck squmous cell crcinom cell line OEC-M1. Furthermore, fter MPT0B098 tretment, the expression of two mesenchyml mrkers, vimentin nd N-cdherin, ws downregulted under hypoxic conditions. Moreover, MPT0B098 suppressed hypoxi-induced EMT in prt y inhiiting EMT-ctivting trnscription fctors, Twist nd SNAI2/Slug. In ddition, the inhiition of hypoxi-induced F-ctin rerrngement nd focl dhesion kinse phosphoryltion my hve contriuted to suppression of EMT y MPT0B098in OEC-M1 cells. MPT0B098 significntly inhiited trnsforming growth fctor(tgf)-β-induced phosphoryltion of receptorssocited Smd2/3 y downregulting TGF-β mrna nd protein expression. Conclusions: Tken together, this study provides novel insight into the role of MPT0B098 in inhiiting hypoxi-inducedemt,suggestingitspotentilusefortretinghedndneckcncers. Keywords: Hypoxi, Epithelil to mesenchyml trnsition, Microtuule inhiitor, TGF-β, Hed nd neck cncer Bckground In solid tumors, lood vessels form normlly nd re dysfunctionl, resulting in n inility to supply sufficient oxygen nd nutrients to the growing tumor mss [1]. Although decrese in oxygen tension cn e lethl for some cells, mny tumor cells cn survive under hypoxic conditions [2]. Tumor cells in hypoxi re resistnt * Correspondence: jychng@nhri.org.tw; z @emil.ncku.edu.tw Equl contriutors 1 Ntionl Institute of Cncer Reserch, Ntionl Helth Reserch Institutes, Tinn, Tiwn 2 Institute of Moleculr Medicine, College of Medicine, Ntionl Cheng Kung University, Tinn, Tiwn Full list of uthor informtion is ville t the end of the rticle to rdition nd chemotherpy; furthermore, hypoxic conditions cn promote tumor progression nd metstsis through numerous direct nd indirect mechnisms [2]. The most criticl fctor responding to hypoxic conditions is hypoxi-inducile fctor (HIF)-1α, sic helix loop helix trnscription fctor composed of n α suunit (regulted y the mount of oxygen tension) nd β suunit (constitutively expressed) [3]. Epithelil mesenchyml trnsition (EMT) is cellulr progrm in which epithelil cells lose their cell polrity nd cell cell dhesion ility nd ecome mesenchyml cells y cquiring firolstic morphology nd migrtory nd invsive fetures. First recognized s chrcteristic The Author(s) Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Tsi et l. Journl of Biomedicl Science (2018) 25:28 Pge 2 of 12 of emryogenic development, EMT is now indicted to e criticl in tumor mlignnt trnsformtion nd metstsis [4]. Ample evidence indictes tht hypoxi leds to chrcteristic chnges in cell morphology tht induce mesenchyml-like phenotype nd fcilitte tumor cell metstsis [5]. A hypoxic microenvironment is frequently found in hed nd neck squmous cell crcinom (HNSCC) nd is known s risk fctor for prognosis [6, 7]. In ddition, cute hypoxi cn cuse the development of ggressive cncers with high metsttic chrcteristics, resistnce to chemotherpy, nd higher tumor recurrence rtes in ptients with hed nd neck cncer [6, 8, 9]. We recently discovered novel indoline-sulfonmide compound, 7- ryl-indoline-1-enzene-sulfonmide (MPT0B098; Fig. 1), tht is potent microtuule inhiitor nd effective ginst pnel of humn cncer cell lines [10]. In contrst to other cliniclly used microtuule inhiitors, MPT0B098 is effective in suppressing tumor growth despite p-gp170/ MDR sttus [11], nd cn destilize HIF-1α mrna under hypoxic conditions y inhiiting the trnsloction of humn ntigen R (HuR) from the nucleus to the cytoplsm [12]. On the sis of the ility of MPT0B098 to modulte HIF-1α, we proposed tht this compound cn modulte hypoxi-induced EMT in hed nd neck cncers. Thus, this study investigted the effects nd underlying mechnisms of MPT0B098 on hypoxi-induced EMT in the highly invsive HNSCC cell line OEC-M1. Methods Chemicls nd ntiodies MPT0B098 ws synthesized y Prof. Jing-Ping Liou t the College of Phrmcy, Tipei Medicl University, Tipei, Tiwn. The detiled synthetic procedure ws descried previously [11]. Colchicine nd pclitxel were purchsed from Sigm-Aldrich (St. Louis, MO). Monoclonl ntiodies for Smd2, phospho-smd2 (Ser465/ 467), Smd3, phospho-smd3, Smd2/3, nd SNAI2/Slug nd polyclonl ntiodies for trnsforming growth fctor (TGF)-β, focl dhesion kinse (FAK), nd phospho-fak were purchsed from Cell Signling Technology (Dnvers, MA). Monoclonl ntiodies for HIF-1α nd N-cdherin were purchsed from BD Biosciences (Sn Jose, CA). A polyclonl ntiody for vimentin ws purchsed from Snt Cruz Biotechnology (Snt Cruz, CA). A polyclonl ntiody for Twist ws purchsed from GeneTex (Irvine, CA). A monoclonl ntiody for GAPDH ws purchsed from Merck Millipore (Drmstdt, Germny). All other chemicls of stndrd nlytic grde or higher were purchsed from E. Merck Co. (Drmstdt, Germny) or Sigm-Aldrich (St. Louis, MO). Cell lines nd cell culture OEC-M1 cells, derived from humn orl epidermoid crcinom, were estlished nd provided y Dr. Ching- Ling Meng from the Deprtment of Dentistry, Tri- Service Generl Hospitl, Ntionl Defense Medicl Center, Tipei, Tiwn [13]. The cells were cultured in RPMI 1640 medium, supplemented with 10% fetl ovine serum in humidified 5% CO 2 incutor t 37 C. For hypoxic conditions, cells were incuted in hypoxic chmer with gs mixture of 1% O 2 nd 5% CO 2 lnced with nitrogen. Cell morphology ssy Cells were stined with 0.5% crystl violet in 95% ethnol for 1 h nd then exmined through light microscopy. c Fig. 1 Antiprolifertive effect of MPT0B098 in OEC-M1 cells under normoxic nd hypoxic conditions. The chemicl structure of MPT0B098. The in vitro ntiprolifertive ctivity of MPT0B098 in OEC-M1 cells under normoxic nd hypoxic conditions. OEC-M1 cells were treted with MPT0B098, colchicine, or pclitxel under normoxic nd hypoxic conditions for 72 h. The IC 50 vlues of these compounds resulting from 50% inhiition of cell growth were clculted using the methylene lue dye ssy. Ech vlue represents the men ± SD of three independent experiments. c Hypoxi-induced drug resistnce is the IC 50 vlue of the test compounds in hypoxi divided y the equivlent in normoxi (* p <0.05)

3 Tsi et l. Journl of Biomedicl Science (2018) 25:28 Pge 3 of 12 Cell viility ssy Cells in logrithmic growth phse were cultured t density of 10,000 cells per well in 24-well plte. The cells were exposed to vrious concentrtions of the test drug for 72 h. The methylene lue dye ssy [14] ws used to evlute drug effect on cell growth. The IC 50 vlues resulting from 50% inhiition of cell growth were clculted grphiclly, compred with control group growth. Western lotting Cells were initilly seeded t density of cells in 100-mm 2 dishes. Following tretment, cell pellets were collected nd lysed in uffer (50 mm Tris-HCl, ph 8. 0, 150 mm NCl, 5 mm EDTA, 2 mm dithiothreitol, 2mMN 3 VO 4, 0.25 mm PMSF, 10 mm NF, 0.5% NP- 40, nd 20 μg/ml ech of protinin, leupeptin, nd pepsttin [proteinse inhiitors]). The superntnts were collected nd the mount of protein ws quntified. Equl mounts of protein from ech lyste were seprted y SDS-PAGE, lotted on polyvinylidenedifluoride memrnes, conjugted with vrious specific primry ntiodies, nd then proed with pproprite secondry ntiodies. The immunorective nds were detected using the enhnced chemiluminescent method nd then visulized on Kodk Bio-MAX MR film. Wound heling ssy Cells were grown to 80% confluence on culture dishes; then, wound ws creted in the center of the cell monolyer with plstic pipette tip. The migrtion of cells into the wound re ws ssessed fter 4, 6, 8, nd 18 h t 37 C under normoxic (5% CO 2, 21% O 2 ) nd hypoxic (5% CO 2,1%O 2, lnced with N 2 ) conditions. The re of denuded surfce ws quntified immeditely nd t the time points of 4, 6, 8 h fter wounding. The extent of wound closure ws determined y clculting the rtio etween the surfce re of the wound for ech time point nd the surfce of the initil wound. Immunofluorescence nlysis of F-ctin OEC-M1 cells were fixed with 4% prformldehyde (Electron Microscopy Sciences, Htfield, PA), permeilized with 0.5% Triton X-100 in phosphte-uffered sline, treted with 0.1% sodium orohydride (Sigm-Aldrich, St. Louis, MO), locked with 5% horse serum, nd then incuted with phlloidin (Life Technologies, Githersurg, MD) for 30 min t room temperture in the drk. Nuclei were stined with DAPI. Cells were oserved with n OLYMPUS fluorescence microscope. Reverse trnscriptse-polymerse chin rection nlysis Ten microgrms of totl RNA, extrcted with Trizol regent (Life Technologies, Githersurg, MD), were treted with DNse nd converted to cdna using the SuperScript II RNse H Reverse Trnscriptse System (Invitrogen, Crlsd, CA). Reverse trnscriptse-poly merse chin rection (RT-PCR) ws performed using Perkin-Elmer GeneAmp PCR System 2400 (Applied Biosystems, Foster City, CA). PCR primers nd TqMn proes (5 -GACCTGGA-3 ) used to mplify the indicted genes were designed using Primer Express (version 1.0; Applied Biosystems) s follows: TGF-β1 forwrd 5 - cgggttgtgcggcgtggttg-3 nd reverse 5 -ggcgcccgggtt tgctggttgt-3 nd TGF-β2 forwrd 5 -gtcttggtgcggcct ttgc-3 nd reverse 5 -gctgctttgcgctttc-3. The rection mixture ws preheted t 95 C for 5 min, followed y 30 cycles of 95 C for 30 s, 55 C for 30 s, nd 72 C for 40 s; finl extension ws t 72 C for 7 min. In seprte rection, GAPDH ws mplified s the reference gene. PCR products of the trget genes were nlyzed through electrophoresis on 2% grose gel nd visulized using ethidium romide stining under UV light. Quntittive dt ws collected y mesuring the nds using ImgeJ (Bethesd, MD). Sttisticl nlysis Quntittive dt were reported s men ± SD. Sttisticl clcultions were completed on Microsoft Excel. P vlues for determining sttisticl significnce were clculted using n unpired two-tiled Student s t test. Results MPT0B098 exhiits low-level resistnce towrd OEC-M1 cell growth under hypoxic conditions We used the methylene lue dye ssy to exmine the ntiprolifertive efficcy of MPT0B098 nd other cliniclly used microtuule inhiitors, such s colchicine nd pclitxel, in OEC-M1 cells. As shown in Fig. 1, MPT0B098 inhiited the growth of OEC-M1 cells with IC 50 of 222 nd 265 nm under normoxic nd hypoxic conditions, respectively. This result indictes tht hypoxi leds to incresed low-level drug resistnce of MPT0B098 in OEC-M1 cells (Fig. 1c). In ddition, compred with MPT0B098, other microtuule inhiitors, including colchicine nd pclitxel, exhiited higher resistnce in OEC-M1 cells under hypoxic conditions thn under normoxic conditions. The IC 50 vlues of colchicine were 23 nd 37 nm under normoxi nd hypoxi, respectively, nd the IC 50 vlues of pclitxel were 4.4 nd 5.9 nm, respectively (Fig. 1). These results indicte tht MPT0B098 is more effective in overcoming hypoxi-induced drug resistnce thn colchicine nd pclitxel in OEC-M1 cells. MPT0B098 inhiits hypoxi-induced EMT in OEC-M1 cells Intrtumorl hypoxi induces EMT nd promotes cncer metstsis. HIF-1α plys criticl role in driving the

4 Tsi et l. Journl of Biomedicl Science (2018) 25:28 Pge 4 of 12 chrcteristic chnges in cell morphology cusing mesenchyml-like phenotype nd fcilitting the metstsis of tumor cells [5, 15]. Becuse MPT0B098 cn inhiit HIF-1α mrna nd protein expression in the humn lung denocrcinom cell line A549 [12], we speculted tht this compound inhiits HIF-1α expression nd suppresses EMT in OEC-M1 cells. Consistent with our previous findings, MPT0B098 demonstrted potent inhiition of HIF-1α expression in concentrtion-dependent mnner under hypoxic conditions in OEC-M1 cells (Fig. 2 nd ). In ddition, the inhiitory effect of MPT0B098 on HIF-1α ws found in nother humn HNSCC cell line, SCC-15 (Additionl file 1: FigureS1). On further exmining the role of MPT0B098 in hypoxi-induced EMT in OEC-M1 cells, we found tht OEC-M1 cells displyed epithelil chrcteristics under normoxic conditions, with round morphology nd linked cells (Fig. 2c, left pnel). However, under hypoxic conditions, cells displyed firolstic morphology nd lost their cell cell contct, which is mesenchyml chrcteristic, implying tht hypoxi cn trigger EMT (Fig. 2c, middle pnel). Notly, the trnsformtion from epithelil to mesenchyml cell type under hypoxic conditions could e inhiited y treting cells with MPT0B098 (Fig. 2c, right pnel). These results suggest tht MPT0B098 my ply role in modulting hypoxiinduced EMT in OEC-M1 cells. MPT0B098 is more potent in inhiiting hypoxi-induced mesenchyml mrker expression thn cliniclly used microtuule inhiitors MPT0B098 could inhiit the expression of hypoxiinduced mesenchyml mrkers, including vimentin nd N-cdherin, in concentrtion-dependent mnner in OEC-M1 cells (Fig. 3 nd 3). Becuse MPT0B098 inhiited hypoxi-induced EMT, we nlyzed whether other cliniclly used microtuule inhiitors, such s colchicine nd pclitxel, lso suppress vimentin nd N- cdherin expression in OEC-M1 cells under hypoxic conditions. MPT0B098 cused reduction in vimentin nd N-cdherin expression in concentrtion dependent mnner, ut the sme effect ws not oserved in colchicine- or pclitxel-treted cells (Fig. 3 nd 3). MPT0B098 suppresses hypoxi-induced EMT prtilly y inhiiting the expression of EMT-ctivting trnscription fctors Twist nd SNAI2/slug During EMT, moleculr reprogrmming is triggered nd orchestrted y vrious EMT-ctivting trnscription fctors, including Twist nd SNAI2/Slug [16]. As shown c Fig. 2 MPT0B098 inhiits hypoxi-induced EMT in OEC-M1 cells. The effect of MPT0B098 onhypoxi-induced HIF-1αexpression. OEC-M1 cells were treted with vrious concentrtions, indicted s fold of IC 50 vlues, of MPT0B098 for 18 h under hypoxic conditions. At the end of the drug tretment, cell lystes were prepred nd nlyzed y SDS-PAGE nd Western lot. β-actin ws used s n internl control. Ech r depicts the men of the reltive intensity of HIF-1α from three independent experiments. c The effect of MPT0B098 on hypoxi-induced EMT.Cells were treted with MPT0B098 t concentrtion of 0.5-fold IC 50 for 48 h under hypoxic conditions nd then cell morphology ws exmined y crystl violet stining. Cells in normoxi were used s controls

5 Tsi et l. Journl of Biomedicl Science (2018) 25:28 Pge 5 of 12 Fig. 3 Effect of MPT0B098 nd other microtuule inhiitors on the expression of EMT-relted proteins in OEC-M1 cells under hypoxic conditions. Prllel comprison of the effects of MPT0B098, colchicine, nd pclitxel on the expression of vimentin, N-cdherin, nd E-cdherin in OEC-M1 cells.oec-m1 cells were treted with vrious concentrtions,indicted s fold of IC 50 vlues,of MPT0B098, colchicine, or pclitxel for 36 h under hypoxic conditions. At the end of the drug tretment, cell lystes were prepred nd nlyzed y SDS-PAGE nd Western lot. GAPDH ws used s n internl control. Ech r depicts the men of the reltive intensity of vimentin, N-cdherin, nd E-cdherin from three independent experiments (* p < 0.05, compred to hypoxi control). in Fig. 4 nd, we found tht the expression levels of Twist nd SNAI2/Slug were significntly suppressed in concentrtion-dependent mnner when cells were treted with vrious concentrtions of MPT0B098 under hypoxic conditions for 18 nd 36 h. These results indicte tht MPT0B098 suppresses hypoxi-induced EMT prtilly y inhiiting the expression of EMT-ctivting trnscription fctors, Twist nd SNAI2/Slug, in OEC-M1 cells. MPT0B098 significntly inhiits hypoxi-induced F-ctin rerrngement nd FAK phosphoryltion Actin filments (F-ctin) re orgnized in thin undles in epithelil cells; however, in trns-differentited mesenchyml cells, they re undled into thick contrctile stress fiers t the cell surfce. Thus, F-ctin rerrngement is ssocited with incresed cell movement during EMT; it is lso required for metsttic cncer cells to Fig. 4 MPT0B098 suppresses hypoxi-induced EMT y inhiiting the expression of EMT-ctivting trnscription fctors, Twist nd SNAI2/Slug. The effects of MPT0B098 on the expression of Twist nd SNAI2/Slug in OEC-M1 cells.oec-m1 cells were treted with vrious concentrtions, indicted s fold of IC 50 vlues, of MPT0B098 for 18 nd 36 h under hypoxic conditions. At the end of the drug tretment, cell lystes were prepred nd nlyzed y SDS-PAGE nd Western lot. GAPDH ws used s n internl control. Ech vlues depicts the men of the reltive intensities of Twist nd SNAI2/Slug from three independent experiments. Ech vlue depicts the men of the reltive intensities of Twist nd SNAI2/Slug from three independent experiments (* p < 0.05, compred to hypoxi control)

6 Tsi et l. Journl of Biomedicl Science (2018) 25:28 Pge 6 of 12 spred from primry tumors [17, 18]. As shown in Fig. 5, f-ctin is locted within the cytoplsm under normoxic conditions. By contrst, hypoxi-induced F- ctin rerrngement showing incresed expression of stress fier ptterns nd concentrted t the plsm memrne on the edge of the cells. MPT0B098-treted OEC- M1 cells demonstrted reduction in the expression of hypoxi-induced stress fier ptterns nd memrne locliztion of F-ctin in concentrtion-dependent mnner. FAK is cytoplsmic tyrosine kinse, with criticl roles in the regultion of cell spreding, migrtion, nd survivl nd in the downstrem signling cscde ssocited with its own phosphoryltion [19, 20]. Chnges in the orgniztion of the ctin cytoskeleton, influenced y FAK, leds to remrkle chnges in the tyrosine phosphoryltion of severl signling proteins loclized t the focl dhesion complex [21]. To further explore the moleculr mechnisms responsile for MPT0B098 inhiition of hypoxi-induced ctin cytoskeletl rerrngement, we exmined the expression nd ctivtion of FAK protein. As shown in Fig. 5 nd c, FAK expression nd ctivtion were incresed under hypoxic conditions. Hypoxi-induced phosphoryltion of FAK decresed when the hypoxic cells were treted with MPT0B098. Tken together, these results suggest tht MPT0B098- inhiited FAK/ctin cytoskeletl rerrngement prtilly contriutes to EMT suppression. MPT0B098 downregultes TGF-β-induced phosphoryltion of receptor-ssocited Smds The reprogrmming of gene expression during EMT is initited nd controlled y signling pthwys responding to extrcellulr cues, including TGF-β, firolst growth fctor, epiderml growth fctor, heptocyte growth fctor, Wnt/β-ctenin, nd Notch [22]. Among these, TGF-β signling hs dominnt role in the initition of EMT progrms tht develop cncer progression [16, 22, 23]. We explored whether the TGF-β signling pthwy is involved in MPT0B098-medited inhiition of hypoxi-induced EMT. Our results indicted tht MPT0B098 suppressed the phosphoryltion of Smd2 nd Smd3 in hypoxi in OEC-M1 cells (Fig. 6 nd ). In ddition, MPT0B098 inhiited Smd signling in nother humn HNSCC cell line, SCC-15 (Additionl file 1: Figure S1). To further exmine the involvement of TGF-β in the hypoxi-induced ctivtion of Smd signling, TGF-β ws pplied to MPT0B098-treted hypoxic cells. TGF-β tretment significntly enhnced Smd2 nd Smd3 phosphoryltion even under hypoxic conditions (Fig. 6c nd d). Codministrtion of TGF-β c Fig. 5 MPT0B098 inhiits hypoxi-induced F-ctin rerrngement nd FAK phosphoryltion. Immunofluorescent nlysis of F-ctin. OEC-M1 cells were treted with vrious concentrtions, indicted s fold of IC 50, of MPT0B098 for 18 h under hypoxic conditions, stined with phlloidin to lel F-ctin (red), counterstined with DAPI (lue), nd then oserved using n OLYMPUS florescence microscope. Effect of MPT0B098 on FAK phosphoryltion nd expression. OEC-M1 cells were treted with vrious concentrtions of MPT0B098 for 18 h.at the end of the drug tretment, cell lystes were prepred nd nlyzed y SDS-PAGE nd Western lot. β-actin ws used s n internl control. c Ech r depicts the men of the reltive intensities of Twist nd SNAI2/Slug from three independent experiments (* p < 0.05, compred to hypoxi control)

7 Tsi et l. Journl of Biomedicl Science (2018) 25:28 Pge 7 of 12 c d Fig. 6 MPT0B098 downregultestgf-β/smd signlingin OEC-M1 cells. Cells were treted with vrious concentrtions,indicted s fold of IC 50 vlues, of MPT0B098 for 36 h under hypoxic conditions. Ech r depicts the men of the reltive intensities of phospho-smds nd Smds from three independent experiments (* p < 0.05, compred to hypoxi control). c Cells were treted with vrious concentrtions,indicted s fold of IC 50 vlues, of MPT0B098 for 18 h, followed y n ddition of 5 ng/ml TGF-β for nother 18 h under hypoxic conditions. At the end of the drug tretment, cell lystes were prepred nd nlyzed y SDS-PAGE nd Western lot. GAPDH ws used s n internl control. d Ech r depicts the men of the reltive intensities of phospho-smds nd Smds from three independent experiments (* p < 0.05, compred to hypoxi control) nd MPT0B098 significntly suppressed Smd2 nd Smd3 phosphoryltion in concentrtion-dependent mnner. In ddition, MPT0B098 slightly inhiited Smd2 nd Smd3 protein expression (Fig. 6c nd d), suggesting tht MPT0B098 influenced Smd inctivtion under hypoxic conditions through TGF-β modultion. MPT0B098 downregultes TGF-β signling y decresing expression levels of TGF-β mrna nd protein To further explore the reltionship etween MPT0B098 nd TGF-β signling, we exmined the expression levels of TGF-β mrna nd protein in MPT0B098-treted hypoxic cells. We found tht MPT0B098 suppressed TGF-β mrna (Fig. 7) nd protein expression (Fig. 7) in dose-dependent mnner under hypoxic conditions in OEC-M1 cells. In ddition, we found tht MPT0B098 inhiited TGF-β protein expression under normoxic conditions (Fig. 7c). These results suggest tht MPT0B098 regultes the EMT process in prt through reduction in TGF-β mrna nd protein levels followed y downregultion of the TGF-β/Smd signling cscdes. MPT0B098 inhiits hypoxi-induced cell migrtion in OEC-M1 cells Since EMT is ssocited with tumor migrtion, especilly under hypoxic conditions [4, 24], we investigted whether MPT0B098 inhiits the EMT progrm, which then leds to suppression of the migrtory cpility of OEC-M1 cells in hypoxi. In the wound heling ssy we oserved tht hypoxi induces greter cell migrtion compred with normoxi, wheres MPT0B098 inhiits hypoxi-induced cell migrtion with 8 h tretment in OEC-M1 cells (Fig. 8). From quntifiction of the wounded re we found tht MPT0B098 effectively inhiited hypoxic cell migrtion in time-dependent mnner (Fig. 8, left pnel), without impiring cell viility (Fig. 8, right pnel), compred with control cells with tretment durtion of 4 18 h. Moreover, we

8 Tsi et l. Journl of Biomedicl Science (2018) 25:28 Pge 8 of 12 c Fig. 7 MPT0B098 downregultes TGF-β signling y decresing the expression of TGF-β mrna nd protein in OEC-M1 cells under hypoxic conditions. Effect of MPT0B098 on the expression levels of TGF-β1ndTGF-β2 mrna. Cells were treted with vrious concentrtions of MPT0B098 in hypoxi. After incution for 36 h, totl RNA ws extrcted, reverse trnscried into cdna, nd sujected to PCR for detection of TGF-β1 nd TGF-β2. GAPDH ws used s n internl control. Dt re represented s men ± SD in triplicte. **P < 0.01 nd ***P < 0.001for comprison etween the control nd tretment groups using n unpired two-tiled Student sttest. Effect of MPT0B098 on the expression level of TGF-β protein. Cells were treted with vrious concentrtions of MPT0B098 for 36 h in hypoxi. Quntifiction of TGF-β protein ws determined y normliztion with GAPDH (* p < 0.05, compred to hypoxi control). c The inhiitory effect of MPT0B098 on TGF-β under normoxic conditions. Cells were treted with MPT0B098 in normoxi t the indicted concentrtions for 36 h. Quntifiction of TGF-β protein ws determined y normliztion with GAPDH (* p < 0.05, compred to control) compred the ntimigrtory effect of MPT0B098 with other microtuule inhiitors, including colchicine nd pclitxel, nd found tht the wound heling inhiitory ctivity of MTP0B098 ws significntly higher thn colchicine nd pclitxel t drug concentrtion of 0.5-fold IC 50, under hypoxic conditions (Fig. 8c). Discussion Microtuule-trgeting drugs, such s txotere, epothilone B, discodermolide, vincristine, 2-methoxyestrdiol, nd colchicine, cn downregulte the expression of HIF- 1α protein ut not HIF-1α mrna [25]. MPT0B098, novel indoline-sulfonmide-sed microtuule inhiitor, cn destilize HIF-1α mrna in hypoxi y inhiiting the trnsloction of HuR from the nucleus to cytoplsm [12]. This ility distinguishes MPT0B098 from other microtuule inhiitors. MPT0B098 is effective ginst pnel of humn cncer cell lines, regrdless of the p- gp170/mdr sttus [10 12]. Becuse MPT0B098 hs unique ility to modulte HIF-1α, we proposed tht MPT0B098 lso suppresses hypoxi-induced mlignnt trnsformtion. Hypoxi cn induce EMT nd enhnce the migrtion nd invsion ility of tumor cells [26]. EMT is key step for tumor metstsis. Hypoxi leds to chrcteristic chnges in cell morphology, cusing mesenchyml-like phenotype, distinct chrcteristic of the EMT process [4]. In this study, we selected highly invsive humn HNSCC cell line, OEC-M1 [27], to clrify the role of MPT0B098 in EMT regultion. We oserved tht MPT0B098 could inhiit expression of HIF-1α protein nd the hypoxiinduced mesenchyml-like phenotype in OEC-M1 cells (Fig. 2c). In ddition, this compound ws more effective, s indicted y low hypoxi-induced drug resistnce, thn colchicine nd pclitxel in inhiitingoec-m1 cell growth under hypoxic conditions (Fig. 1c). As determined y expression of the two mesenchyml mrkers vimentin nd N-cdherin, we noted tht MPT0B098 ws more potent in inhiiting hypoxi-induced vimentin nd N-cdherin upregultion thn colchicine nd pclitxel in OEC-M1 cells (Fig. 3). These results suggest tht MPT0B098 is distinct from other cliniclly used microtuule inhiitors in its inhiition of hypoxi-induced EMT. Unlike the clssic reversl of the EMT phenomenon, E-cdherin protein ws slightly suppressed y MPT0B098 (Fig. 3 nd 3). Cdherins hve een reported to regulte the orgniztion nd dynmics of microtuules. This ehvior my lso ffect cdherin iology through microtuule-sed vesiculr trffic [28]. Thus, the interply etween E-cdherin nd microtuules my e disrupted y the inhiition of microtuule

9 Tsi et l. Journl of Biomedicl Science (2018) 25:28 Pge 9 of 12 c Fig. 8 MPT0B098 inhiits hypoxi-induced cell migrtion in OEC-M1 cells. Effect of MPT0B098 on cell motility. OEC-M1 cells were treted with 0.5- fold IC 50 of MPT0B098 for 8 h nd then cell motility ws determined y the wound heling ssy. Quntifiction of cell migrtion ws crried out y mesuring the wound re (left pnel) nd clculting cell viility (right pnel) fter cells were treted with 0.5-fold IC 50 of MPT0B098 for 4, 6, 8, nd18 h. c Prllel comprison of the effect of MPT0B098 with other microtuule inhiitors, including colchicine nd pclitxel, t the drug concentrtion of 0.5-fold IC 50 on cell motility (left pnel) nd viility (right pnel) t tretment durtion of 8 h. Dt re represented s men ± SD in triplicte. **P < 0.01 nd ***P < for comprison etween the control nd tretment groups using n unpired two-tiled Student s ttest polymeriztion under MPT0B098 tretment. The underlying mechnisms merit further investigtion. Moleculr reprogrmming occurring during EMT is triggered nd orchestrted y vrious EMT-ctivting trnscription fctors, including Twist nd SNAI2/Slug [16]. Yng et l. found tht HIF-1α regulted the expression of Twist y inding directly to the hypoxiresponse element in the Twist proximl promoter. In ddition, silencing of Twist in HIF-1α-overexpressing or hypoxic cells reversed EMT nd metsttic phenotypes [29]. Cheng et l. reported tht tretment with HIF-1α sirna diminished the upregultion of SNAI2/ Slug expression [30]. Becuse MPT0B098 is effective in suppressing HIF-1α expression, we investigted the effect of MPT0B098 on modultion of Twist nd SNAI2/ Slug nd found tht the expression levels of hypoxiinduced Twist nd SNAI2/Slug were significntly decresed in concentrtion-dependent mnner when cells were treted with vrious concentrtions of MPT0B098 (Fig. 4). Trnscription progrm switching in EMT is induced y signling pthwys medited y TGF-β, one morphogenetic protein, Wnt-β-ctenin, Notch, Hedgehog, nd receptor tyrosine kinses. These pthwys re ctivted y vrious dynmic stimuli from the locl microenvironment. Of note, TGF-β signling hs dominnt role in EMT [22, 30 32]. Here, we demonstrted tht MPT0B098 inhiits hypoxi-induced EMT y downregulting the expression levels of TGF-β mrna (Fig. 7) nd protein (Fig. 7). We previously demonstrted tht MPT0B098 inhiited HuR trnsloction to the cytoplsm [12]. HuR, n mrna-stilizing protein,

10 Tsi et l. Journl of Biomedicl Science (2018) 25:28 Pge 10 of 12 Fig. 9 Proposed pthwy of MPT0B098-medited EMT suppression. Blue solid line: proposed working models; ornge dotted line: literture reported shuttles etween the nucleus nd cytoplsm through severl export pthwys. Moilizing HuR from the nucleus to the cytoplsm leds to mintennce of the stility of HuR-medited trget mrnas. Incresed stility of TGFβ mrna y HuR promotes EMT in pncretic cncer [33]; therefore, whether MPT0B098 ffect the stility of TGF-β mrna through HuR dependent or independent mechnisms which needs to e further investigted. TGF-β signling towrd EMT is medited y oth Smd-dependent nd -independent pthwys. The Smd pthwy is unique to TGF-β signling [34]. Our further evlution demonstrted tht MPT0B098 inhiited hypoxi-induced EMT y locking TGF-β-dependent Smd signling (Fig. 6). Becuse TGF-β signling occurs not only in hypoxi ut lso in normoxi, we found tht MPT0B098 decresed the expression of TGF-β protein under normoxic conditions (Fig. 7c), suggesting tht the inhiitory effect of MPT0B098 on TGF-β signling ws not restricted to the hypoxic condition. Zhng et l. reported tht exposure of cells to hypoxi resulted in phosphoryltion of Smd2 nd Smd3 s well s stimultion of trnscriptionl ctivities of HIF-1α nd upregultion of TGF-β2 expression, suggesting tht utocrine regultion of TGF-β2 production in hypoxi my involve crosstlk etween Smd3 nd HIF-1α signling pthwys [35]. The interply etween ech molecule in response to MPT0B098 needs further elucidtion. In ddition to TGF-β/Smd signling, Cicchini et l. reported tht TGF-β induces Src-dependent ctivtion of FAK protein [36]. The results shown in Fig. 5 show tht MPT0B098 significntly suppressed hypoxi-induced FAK phosphoryltion. Becuse FAK is criticl modultor in regulting ctin cytoskeleton orgniztion [19 21], we further oserved tht MPT0B098 inhiited hypoxiinduced expression of the stress fier pttern nd memrne locliztion of F-ctin (Fig. 5). Accordingly, we proposed tht MPT0B098 inhiits hypoxi-induced EMT in HNSCC y (1) suppressing HIF-1α expression, (2) inhiiting the EMT-ctivting trnscription fctors Twist nd SNAI2/Slug, (3) locking TGF-β/Smd signling, nd (4) interfering with FAK-medited ctin cytoskeleton rerrngement (Fig. 9). Further evlution to clrify the interply etween MPT0B098 nd the prticulr molecules is wrrnted. Conclusions Tken together, we demonstrted for the first time tht the novel microtuule Inhiitor MPT0B098 inhiits hypoxi-induced EMT in HNSCC through modultion of HIF-1α, Twist, SNAI2/Slug, TGF-β/Smd signling, nd FAK/ctin cytoskeleton rerrngement. The results presented here my provide novel insight into the mechnism of ction of microtuule inhiitors for inhiiting EMT. MPT0B098 hs gret potentil for clinicl tretment of hypoxic tumors nd merits further investigtion. Additionl file Additionl file 1: Supplementl mterils. (DOCX 739 k) Arevitions EMT: epithelil-mesenchyml trnsition; FAK: focl dhesion kinse; GAPDH: glycerldehyde 3-phosphte dehydrogense; HIF-1α: hypoxiinduced fctor 1 lph; HNSCC: hed nd neck squmous cell crcinom; HuR: humn ntigen R; TGF-β: trnsforming growth fctor et

11 Tsi et l. Journl of Biomedicl Science (2018) 25:28 Pge 11 of 12 Acknowledgements All uthors sincerely cknowledge Ms. Hsin-Yi Pn for ssisting grphics processing, nd thnk the funding support. Funding This study ws supported y following grnts from the following gencies: the Ministry of Helth nd Welfre (CA105-SP-01), the Ntionl Helth Reserch Institutes (CA-105-PP-22), the Ministry of Science nd Technology (MOST B ; MOST B MY3), nd Ministry of Eduction (Aim for the Top University Project t Ntionl Cheng Kung University) of Tiwn. Avilility of dt nd mterils No pplicle Authors contriutions JYC nd CCK provided the conception nd design the study. ITT performed the experiments. ITT nd CCK nlyzed dt. CCK, ITT, nd JYC wrote the min mnuscript text. JPL Designed nd synthesized MPT0B098. All uthors red nd pproved the finl mnuscript. Ethics pprovl nd consent to prticipte No pplicle Consent for puliction No pplicle Competing interests The uthors declre tht they hve no competing interests. Pulisher s Note Springer Nture remins neutrl with regrd to jurisdictionl clims in pulished mps nd institutionl ffilitions. Author detils 1 Ntionl Institute of Cncer Reserch, Ntionl Helth Reserch Institutes, Tinn, Tiwn. 2 Institute of Moleculr Medicine, College of Medicine, Ntionl Cheng Kung University, Tinn, Tiwn. 3 Institute of Biotechnology nd Phrmceuticl Reserch, Ntionl Helth Reserch Institutes, Zhunn, Tiwn. 4 Institute of Clinicl Phrmcy nd Phrmceuticl Sciences, College of Medicine, Ntionl Cheng Kung University, Tinn, Tiwn. 5 Grdute Progrm for Aging, Chin Medicl University, Tichung, Tiwn. 6 College of Phrmcy, Tipei Medicl University, Tipei, Tiwn. 7 Division of Hemtology/ Oncology, Deprtment of Internl Medicine, Ntionl Cheng Kung University Hospitl, College of Medicine, Ntionl Cheng Kung University, Tinn, Tiwn. Received: 1 Novemer 2017 Accepted: 24 Mrch 2018 References 1. Brown JM, Gicci AJ. The unique physiology of solid tumors: opportunities (nd prolems) for cncer therpy. 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12 Tsi et l. Journl of Biomedicl Science (2018) 25:28 Pge 12 of Heldin CH, Lndstrom M, Moustks A. Mechnism of TGF-et signling to growth rrest, poptosis, nd epithelil-mesenchyml trnsition. Curr Opin Cell Biol. 2009;21(2): Zhng H, Akmn HO, Smith EL, Zho J, Murphy-Ullrich JE, Btumn OA. Cellulr response to hypoxi involves signling vi Smd proteins. Blood. 2003;101(6): Cicchini C, Luddio I, Citrell F, Corzzri M, Steindler C, Conigliro A, Fntoni A, Amicone L, Tripodi M, TGFet-induced EMT. Requires focl dhesion kinse (FAK) signling. Exp Cell Res. 2008;314(1): Sumit your next mnuscript to BioMed Centrl nd we will help you t every step: We ccept pre-sumission inquiries Our selector tool helps you to find the most relevnt journl We provide round the clock customer support Convenient online sumission Thorough peer review Inclusion in PuMed nd ll mjor indexing services Mximum visiility for your reserch Sumit your mnuscript t