Inhibition of hypoxia-inducible factor via upregulation of von Hippel-Lindau protein induces angiogenic switch off in a hepatoma mouse model

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1 Cittion: Moleculr Therpy Oncolytics (215) 2, 152; doi:1.138/mto All rights reserved /15 ARTICLE Inhiition of hypoxi-inducile fctor vi upregultion of von Hippel-Lindu protein induces ngiogenic switch off in heptom mouse model Hideki Iwmoto 1,2, Toru Nkmur 1,2, Hironori Kog 1,2, Jesus Izguirre-Cronell 3, Shinji Kmisuki 3, Fumio Sugwr 3, Mitsuhiko Ae 1,2, Kzuki Iwt 3, Yu Ikezono 1,2, Tkhiko Skue 1,2, Atsutk Msud 1,2, Hirohis Yno 4, Keisuke Oht 3, Mshito Nkno 1, Shigeo Shimose 1, Tomotke Shirono 1 nd Tkuji Torimur 1,2 Angiogenic switch off is one of the idel therpeutic concepts in the tretment of cncer. However, the specific molecules which cn induce ngiogenic switch off in tumor hve not een identified yet. In this study, we focused on von Hippel-Lindu protein (p) in heptocellulr crcinom (HCC) nd investigted the effects of sulfoquinovosyl-cylpropnediol (), novel synthetic sulfoglycolipid, for HCC. We exmined muttion rtio of gene in HCC using 3 HCC smples nd we treted the HCC-implnted mice with. Thirty clinicl smples showed no genetic muttion in HCC. significntly inhiited tumor growth y inhiiting ngiogenesis in heptom mouse model. induced tumor ngiogenic switch off y decresing hypoxi-inducile fctor (HIF)-1, 2α protein vi p upregultion. p upregultion decresed HIFα protein levels through different multiple mechnisms: (i) incresing p-dependent HIFα protein degrdtion; (ii) decresing HIFα synthesis with decrese of NF-κB expression; nd (iii) decrese of tumor hypoxi y vsculr normliztion. We confirmed these ntitumor effects of y the loss-of-function experiments. We found tht directly ound to nd inhiited trnsglutminse 2. This study provides evidence tht upregultion of tumor p is promising trget, which cn induce ngiogenic switch off in HCC. Moleculr Therpy Oncolytics (215) 2, 152; doi:1.138/mto.215.2; pulished online 2 Decemer 215 INTRODUCTION Angiogenesis is n essentil process for tumor growth nd progression. 1 Newly formed lood vessels in tumor re known to e norml nd immture structures, which result in high lekiness nd less perfusion. 2,3 Vsculr endothelil growth fctor (VEGF) fmily, firolst growth fctor (FGF) fmily, ngiopoietin, etc. mny prongiogenic fctors re secreted y not only helthy tissues, ut lso y cncer cells, which induce neovsculriztion in tumor. 4,5 Antingiogenic therpy hs een proposed in 197s 1 nd hs ecome one of the stndrd therpies for severl kinds of solid tumors. 6 Antingiogenic therpy using sorfeni is the only stndrd therpy for dvnced heptocellulr crcinom (HCC). 7 However, the eneficil effects of sorfeni for dvnced HCC re limited. 8 Additionlly, mny clinicl trils using other ntingiogenic drugs were performed for HCC ptients. But most of them hve filed so fr. 9,1 Therefore, further development of etter ntingiogenic therpies re needed to give more enefits for the ptients of dvnced HCC. One of the resons for the modest effects of sorfeni nd the other ntingiogenic drugs is cquired resistnce for ntingiogenic tretment in tumor. The previously developed ntingiogenic drugs trget the specific ngiogenic-relted molecules or their receptors. Inhiition of the specific molecules results in upregultion of lterntive ngiogenic fctors, so clled the escpe phenomenon in tumor. 11 For exmple, inhiition of VEGFR-2 induces lterntive upregultion of FGF-2 nd ngiopoietin Therpeutic strtegy, which trgets the specific downstrem proteins hs limittions in this point. Folkmn 13 who firstly proposed nti-ngiogenic therpy lso proposed the concept of ngiogenic switch in tumor. This concept is tht switching off tumor ngiogenic potentil leds to tumor dormncy nd is one of the most idel therpeutic strtegies for nticncer tretment. However, the molecules which cn induce ngiogenic switch off in tumor hve not een identified yet. Hypoxic microenvironments re common feture of solid tumors 14 nd cn rise due the prolifertive sttus of cncer cells or n uneven vsculr supply in tumor tissues. 15 Cncer cells dpt to hypoxic environments y ctivting numer of hypoxi-relted pthwys, e.g., ngiogenic proteins, prolifertion, survivl, nd energy metolism-relted pthwys. 14 Hypoxi-inducile fctors 1 Division of Gstroenterology, Deprtment of Medicine, Kurume University School of Medicine, Kurume, Jpn; 2 Liver Cncer Division, Reserch Center for Innovtive Cncer Therpy, Kurume University School of Medicine, Kurume, Jpn; 3 Deprtment of Applied Biologicl Science, Fculty of Science nd Technology, Tokyo University of Science, Tokyo, Jpn; 4 Deprtment of Pthology, Kurume University School of Medicine, Kurume, Jpn. Correspondence: H Iwmoto (iwmoto_hideki@med.kurume-u.c.jp) Received 5 August 215; ccepted 5 Octoer 215

2 2 Angiogenic switch off vi p regultion in HCC 1α nd 2α (HIFα proteins) ply centrl role in these pthwys. 16 HIFα proteins re regulted y prolyl hydroxylse-domin enzyme (PHD) nd degrded y Von Hippel-Lindu protein (p) under normoxi. However, hypoxi inhiits ctivity of PHD nd p, resulting in stiliztion of HIFα protein. 17 Therefore, efficient regultion of tumor hypoxi nd downregultion of HIFα proteins might e crucil key for ngiogenic switch off in tumor. The moleculr trgeting drug, which trgets p hs not een reported so much. Sulfoquinovosyl-cylglycerols (SQAG) re sulfoglycolipids tht were originlly derived from se urchin. 18 SQAGs cn e divided into two groups: monocyl (SQMG) nd dicyl (SQDG), which hve one nd two ftty cids, respectively. Shr et l. reported tht SQMG significntly inhiited tumor growth of rest or lung denocrcinoms trnsplnted in nude mice. Mori et l. 19 found tht the ntitumor effect of SQMG involved ntingiogenesis vi downregultion of Tie2 gene expression, ut the detiled mechnisms for SQAG ction remin uncler. Sulfoquinovosyl-cylpropnediol () is SQAG derivtive, which is rtificilly synthesized (Figure 1). Here we show tht the therpeutic strtegy tht trgets tumor p-hifα xis is promising for tretment of HCC. Upregultion of p due to drmticlly inhiits HIFα proteins in tumor even under hypoxic condition through multiple mechnisms, which leds ngiogenic switch off to HCC. RESULTS Somtic muttion of gene is rre in HCC ptients We investigted the muttion rte of the gene in 3 HCC ptients y DNA sequencing. All evluted HCC tissues showed wild-type gene profile, regrdless of tumor differentition stte (Tle 1). This result suggests tht gene could e promising therpeutic trget for tretment of HCC. upregultes tumor protein in HCC cell lines We evluted chnges in the mount of p y western lot nlysis using cells cultured in -contining medium. The expression of p ws significntly incresed y ddition of in dose-dependent mnner (Figure 1). inhiits tumor growth of nd in mice We exmined the ntitumor effects nd sfety of for HCC-ering mice. significntly inhiited tumor growth of nd in mice (Figure 1c). With respect to sfety, mnifested no overt toxicity in terms of weight loss nd myelotoxicity (Supplementry Tle S1). induces ngiogenic switch off in tumor y downregultion of HIFα proteins To investigte the mechnisms for the ntitumor effects of, we evluted the degree of tumor cell ngiogenesis, poptosis y immunohistochemistry. Tumor ngiogenesis ws significntly inhiited nd the numer of poptotic cells ws significntly incresed in oth nd treted with (Figure 1d,e nd Supplementry Figure S1,). We exmined prongiogenic nd ntingiogenic proteins in tumor treted with. The expression of representtive prongiogenic proteins (VEGF, FGF-2, nd Ang-2) ws significntly decresed, while the ntingiogenic protein TSP-1 significntly incresed in tumors treted with (Figure 2). Noteworthy, nd levels were significntly decresed in tumors treted with (Figure 2). And this decrese effect ws lso shown in tumors treted with (Supplementry Figure S2). To confirm whether could directly ffect HIFα proteins expression, we lso exmined the expression level of HIFα proteins in n in vitro ssy using nd cultured under hypoxi. VEGF expression, the downstrem protein of ws significntly decresed y ddition of in oth cell lines (Figure 2). And the expression of nd ws decresed y in dose-dependent mnner in oth cell lines (Figure 2). These results suggest tht cn switch off tumor ngiogenic potentil y decresing HIFα protein levels. degrdes HIFα proteins through upregultion of p To clrify whether p upregulted y re involved in decrese of HIFα, we ssessed the chnge of oth degrdtion nd synthetic pthwys in HIFα proteins. We performed n in vitro ssy using the protesoml inhiitor, MG-132 (Z-Leu-Leu-Leu-CHO, Cliochem, Sn Diego, CA). In the sence of MG-132, the mount of uiquitinted HIFα proteins under hypoxic conditions ws clerly diminished y tretment (Figure 3). However, HIFα protein levels recovered in the presence of MG-132 (Figure 3). These results suggest tht the protesoml degrdtion pthwy involving p upregultion is responsile for the -dependent decrese in HIFα protein levels. directly decreses HIFα synthesis nd downregultes the expression of NFκB To scertin whether directly decresed synthesis t trnscriptionl level, we performed quntittive rel-time polymerse chin rection (qrt-pcr) to mesure the ccumultion of mrna in. qrt-pcr nlysis indicted tht mrna levels in cells were significntly reduced y the ddition of in dose-dependent mnner (Figure 3). To clrify which fctors re involved in the reduction of HIFα synthesis, we exmined chnges of some hypoxi-independent ctivtors for HIFα synthesis using nd cultured with -contining medium under hypoxi. Then we found the presence of reduced NF-kppB (NFκB) expression in nd cells in dose-dependent mnner (Figure 3c). reduces tumor hypoxi y induction of vsculr normliztion, contriuting to indirect decrese of HIFα proteins We then performed immunohistochemistry nd western lot nlysis of tumors dded the hypoxi mrker pimonidzole to investigte whether improves tumor hypoxic conditions. The mount of protein ound to pimonidzole ws significntly decresed in tissues treted with, suggesting tht tumor hypoxic conditions re improved y tretment with (Figure 3d,e). We lso performed doule stining ssy of α-smooth muscle ctin (SMA) nd CD31 in tissues. The control group hd mny tumor vessels with lcked α-sma-positive cells (Figure 3e). In contrst, tumor vessels were regressed in tumors treted with. Furthermore, most remining vessels in treted tumors exhiited α-sma-covered CD31 vessels, suggesting n increse in the numer of normlized vessels (Figure 3f). These results suggest tht improves tumor hypoxi y inducing vsculr normliztion, which cn lso contriute to indirect decrese of HIFα proteins. Moleculr Therpy Oncolytics (215) 152

3 Angiogenic switch off vi p regultion in HCC SO 3 3 HO O Normoxi Hypoxi Control Control 1 μmol/l 1 μmol/l Normoxi Hypoxi Control Control 1 μmol/l 1 μmol/l HO O OC = O (CH 2 ) 16 CH 3 c Tumor volume (mm 3 ) OH Moleculr weight: O-(6-sulfo--D-quinovopyrnosyl-1-O-steroyl-propne-1,3-diol) 1,2 3, 1, 2 mg/kg 2,5 8 2, * * * * * * * * Tumor volume (mm 3 ) 1,5 1, 5 2 mg/kg * * * * * * * Dy Dy d Numer of vessels (n/field) P <.1 e 5 P <.1 Numer of vessels (n/field) Figure 1 The chemicl structure of sulfoquinovosyl-cylpropnediol () nd the effects of for nd in mice. () The chemicl structure of. () increses the expression of p in nd. Western lots for oth cell lines exposed to (1 nd 1 μmol/l) under hypoxic conditions re shown. Cells were incuted with -contining medium for 24 hours, fter which the cells were moved to 3% O 2 hypoxic conditions for 24 hours nd then lysed with rdioimmunoprecipittion uffer. (c) Time course of tumor volume chnges for sucutneous tumorering mice treted with (n = 1). HAK-1B nd cell line (5 1 6 cells/mouse) were injected into the flnk region of the mice. The mice then received the following tretments y intrperitonel (i.p.) injection: phosphte-uffered sline (control group) or (2 mg/kg/dy, treted group). The tretments were continued for 21 dys. *P <.5 compred with the control group. All dt re represented y men ± stndrd devition (SD). (d) reduced vsculriztion produced in. (e) reduced vsculriztion produced in. Representtive Immunohistochemistry imges using CD31 stining in nd re shown. The numer of tumor vessels re represented s men ± SD (n = 1 per group). Scle r = 5 μm. Reductions in HIFα nd NFκB due to re restored y knockdown Upregultion of p y induced ccelertion of HIFα degrdtion nd reduction of HIFα synthesis. To confirm the effects of, we generted knockdown cells. In control sh- RNA cells, resulted in incresed p expression nd decresed HIFα nd NFκB expression (Figure 4). In contrst, the effect of on HIFα nd NFκB protein levels ws sent in Moleculr Therpy Oncolytics (215) 152

4 Angiogenic switch off vi p regultion in HCC 4 Tle 1 Genetic nlysis of the gene in 3 HCC ptients knockdown cells (Figure 4). This result suggests tht upregultion of y is essentil for the decrese of HIFα expression. Thirty HCC ptients Differentition (well/mode/poor) 8/22/ somtic muttion (Wt/Mt) /3 HCC, heptocellulr crcinom; ; von Hippel-Lindu. well = well, mode = modertely, poor = poorly. Wt = wild type, Mt = muttion. did not inhiit tumor growth, tumor ngiogenesis in knockdown in mice We lso performed in vivo ssys using knockdown cells. inhiited tumor growth, tumor ngiogenesis, nd improved tumor hypoxi in control sh-rna tumors. However, could not inhiit tumor growth, tumor ngiogenesis nd reduce tumor hypoxi in knockdown cells (Figure 4 d). VEGF Ang-2 FGF-2 TSP-1 VEGF signl/β ctin VEGF Ang-2 FGF-2.25 P <.5.6 P < P < Ang-2/β ctin... FGF-2/β ctin TSP-1/β ctin TSP-1 P <.5.7 P < /β ctin /β ctin HF2α P <.5 Normoxi Control Control Hypoxi Normoxi Control Control Hypoxi 1 μmol/l 1 μmol/l 1 μmol/l 1 μmol/l VEGF Figure 2 Sulfoquinovosyl-cylpropnediol () switches off tumor ngiogenic potentil y decresing HIFα protein levels. () Western lots of vsculr endothelil cell growth fctor, Ang-2, FGF-2, TSP-1,, nd in tumors treted with (2 mg/kg/dy, i.p injection for 21 dys). Whole tumors were collected from tumor-ering mice (n = 4 per group). The nd densities for ech protein were mesured nd clirted y. All dt re represented y men ± stndrd devition. () Western lots of vsculr endothelil cell growth fctor,, nd HIF 2α for nd exposed to under hypoxic conditions. Cells were incuted with -contining medium for 24 hours, fter which the cells were exposed to 3% O 2 hypoxic conditions for 24 hours nd then lysed with rdioimmunoprecipittion uffer. Moleculr Therpy Oncolytics (215) 152

5 Angiogenic switch off vi p regultion in HCC 5 MG-132 Normoxi + + Hypoxi Uiqutinted Uiqutinted mrna reltive expression level Control Normoxi P <.1 P <.5 Control 1 μmol/l 1 μmol/l Hypoxi c Normoxi Hypoxi Normoxi Hypoxi Control Control Control Control 1 μmol/l 1 μmol/l 1 μmol/l 1 μmol/l d NFkB e Normoxi Hypoxi Pimonidzole dducts Pimonidzole dducts/ P <.5 f CD31/α-SMA/DAPI Vessels intensity per re Tumor vessels density 5 P < α-sma-positive signl per re α-sma Pericyte covered vessesls P < P < Pericyte covered vessels (%) Figure 3 Multiple mechnisms decresing HIFα in sulfoquinovosyl-cylpropnediol (). () The presence of the protesome inhiitor MG-132 decresed the effect of on HIFα protein levels. cells incuted under either normoxi or 3% O 2 hypoxi were treted with 1 μmo/l for 24 hours nd further incuted for 4 hours in the presence of 1 μmo/l MG-132. () synthesis t the trnscriptionl level decresed upon ddition in dose-dependent mnner. HAK-1B cells were incuted with medium contining different dose of (1 nd 1 μmo/l). Cellulr RNA ws then extrcted nd nlyzed for mrna expression y quntittive rel-time polymerse chin rection, with GAPDH s control. (c) decreses NFκB expression in nd. Western lots for oth cell lines exposed to (1 nd 1 μmo/l) under hypoxic conditions re shown. Cells were incuted with -contining medium for 24 hours, fter which the cells were moved to 3% O 2 hypoxic conditions for 24 hours nd then lysed with rdioimmunoprecipittion uffer. (d) Representtive immunohistochemistry imges for pimonidzole in tumor treted with. Scle r = 2 μm. (e) Western lot nlysis of pimonidzole dducts in tumors treted with. Whole tumors were collected from tumor ering mice (n = 4 per group). Negtive control: cells cultured under normoxi in vitro. Positive control: cell cultured under 3% O 2 hypoxi in vitro. Pimonidzole dducts re shown in the indicted multiple nds. The nd densities for ech protein were mesured nd clirted y. Averge nds densities re represented s men ± stndrd devition (SD). (f) Representtive doule immunofluorescence imges using oth CD31 (green) nd α-sma (red) in tumors treted with. Nuclei were stined with DAPI. Scle r = 5 μm. Reltive numer of tumor vessels, α-sma-positive cells, nd tumor vessels covered with α-sma positive cells in tumors treted with re shown. The numer of tumor vessels nd the rtio of pericyte covered vessels re represented s men ± SD. DAPI, 4,6-dimidino-2-phenylindole, dihydrochloride. Moleculr Therpy Oncolytics (215) 152

6 Angiogenic switch off vi p regultion in HCC 6 Control sh-rna sh-rna Normoxi Hypoxi Normoxi Hypoxi (μmol/l) 1 1 Inhiition rte of tumor volume (%) P <.1 Tumor volume n.s. NFkB Control sh-rna sh-rna c control sh-rna sh-rna Numer of vessels (n/field) 7 P < Control sh-rna n.s. sh-rna d control sh-rna sh-rna Figure 4 The Effects of sulfoquinovosyl-cylpropnediol () in knockdown cell in the in vitro nd in vivo ssys. () Western lots of p,,, nd NFκB in knockdown cell exposed to under hypoxic condition in n in vitro ssy. The effect of on,, nd NFκB protein levels ws olished y knockdown. knockdown model ws mde y infecting cells with nd control sh-rna lentivirl prticles. Cells were incuted with -contining medium for 24 hours, fter which the cells were moved to 3% O 2 hypoxic conditions for 24 hours nd then lysed with rdioimmunoprecipittion uffer. () Decrese rte of tumor volume in tretment of mice ering tumors generted y cells expressing either control or sh-rna. Control HAK-1B nd knock down HAK-1B (5 1 6 cells/mouse) were injected into the flnk region of the mice (n = 1). The mice then received the following tretments y intrperitonel (i.p.) injection: phosphteuffered sline () (control group) or (2 mg/kg/dy, treted group). The tretments were continued for 21 dys. Tumor volume on scrifice ws mesured nd the verge tumor volume ws clculted in ech group. Averge tumor volume of treted group in ech group, i.e., control nd sh-rna, ws divided y tht of group in ech group. The decrese rte of tumor volume on scrifice is represented y men ± stndrd devition (SD). (c) The effect of to decrese numers of vessels in tumors rogted y downregultion of the gene. The numer of tumor vessels re represented s men ± SD (n = 1 per group). Scle r = 5 μm. (d) Representtive imges of pimonidzole in tretment of mice ering tumors generted y cells expressing either control or sh-rna. Scle r = 2 μm. did not show ny in vivo effects nd in vitro effects for cell line, nturlly mutted HCC cell line Since rtificil ltion of gene cused complete loss of effects in, next we evluted the effects of in nturlly mutted HCC cell line. We performed genetic muttion nlysis using direct DNA sequencing. Then we found hd point muttion of gene, while -sensitive cell lines oth nd cells were wild-type gene (Supplementry Tle S2). Although there ws no significnt difference in IC 5 vlue of mong,, nd (Supplementry Tle S3), could not inhiit tumor growth of in mice (Figure 5). Both did not inhiit tumor ngiogenesis nd did not increse poptosis in tumors (Figure 5,c). did not decrese the expression of in tumors (Figure 5d). Also in n in vitro ssy, VEGF, HIFα proteins, nd NFκB expression did not decrese y ddition of, in spite of incresing p Moleculr Therpy Oncolytics (215) 152 expression (Figure 5e). Loss-of-function experiments using rtificil ltion of gene nd the experiments using nturlly mutted HCC cell line clerly supported tht p is essentil for ntitumor effects of. directly inds with TG2 nd inhiits TG2 ctivity in dose-dependent mnner One remining question ws tht how upregultes the expression of p in cncer cells. Kim et l. 2 recently reported tht genetic ltion of TG2 resulted in upregultion of p. Therefore, we firstly performed the inding ssy of nd TG2. Then we found tht directly ound with TG2 in dosedependent mnner (Figure 6). Additionlly, in TG2 ctivity ssy, showed stronger inhiitory effect of TG2 ctivity in dosedependent mnner, compred with 5 -Nitroistin, TG2 inhiitor s positive control (Figure 6). Next, we exmined whether HCC

7 Angiogenic switch off vi p regultion in HCC Tumor volume (mm 3 ) 5, 4, 3, 2, 1, 2 mg/kg c Dy Numer of vessels (n/field) n.s. 1 n.s. d e VEGF Normoxi Hypoxi Control Control 1 μmol/l 1 μmol/l TUNEL-positive cell/ totl cell (%) signl/ n.s. NFkB Figure 5 The effects of sulfoquinovosyl-cylpropnediol () for, nturlly mutted HCC cell line. () Time course of tumor volume chnges for sucutneous -ering mice treted with (n = 1 per group). All dt re represented y men ± stndrd devition (SD). The mice then received the following tretments y intrperitonel (i.p.) injection: phosphte-uffered sline (control group) or (2 mg/kg/dy, treted group). The tretments were continued for 21 dys. () Representtive Immunohistochemistry imges of CD31 stining in re shown. The numer of tumor vessels re represented s men ± SD (n = 1 per group). Scle r = 5 μm. (c) Representtive immunohistochemistry imges of TUNEL stining in re shown. The numer of poptotic cells is represented s men ± SD. (d) Western lots of in tumors treted with. Whole tumors were collected from tumor ering mice (n = 4 per group). The nd densities for ech protein were mesured nd normlized y. The verge nd densities re represented s men ± SD. (e) Western lots of vsculr endothelil cell growth fctor,,,, nd NFκB for exposed to under hypoxic conditions re shown. Cells were incuted with -contining medium for 24 hours, fter which the cells were moved to 3% O 2 hypoxic conditions for 24 hours nd then lysed with rdioimmunoprecipittion uffer. TUNEL, TdT-medited dutp nick end leling. cell lines, which we used expressed TG2.,, nd expressed TG2 in western lot nlysis (Figure 6c). 5 -Nitroistin upregulted p expression in cell in dose-dependent mnner (Figure 6d). Similrly, upregulted p expression of in dose-dependent mnner (Figure 6d). DISCUSSION Muttions of the gene re often involved in tumorigenesis nd cncer progression, which is supported y the finding tht the gene shows somtic muttions in more thn 7% of cler-cell renl crcinoms. 21 However, s shown in Tle 1 nd Pio et l. 22, muttions in HCC re very rre. Therefore, cn e promising trget for moleculr trgeting therpy of HCC. In this study, we demonstrted tht inhiited tumor ngiogenesis y upregultion of p in tumors, which resulted in significnt ntitumor effect for HCC in mice without overt toxicity. decresed prongiogenic fctors nd incresed endogenous ngiognenic inhiitor through downregultion of HIFα proteins, which finlly reduced prongiogenic potentil of HCC cells. This phenomenon is clled ngiogenic switch off. Upregulting p y showed multiple mechnisms tht decrese the expression of HIFα proteins. Firstly, upregultion of p ccelerted HIFα degrdtion s direct mechnism. Second, upregultion of p Moleculr Therpy Oncolytics (215) 152

8 Angiogenic switch off vi p regultion in HCC 8 Response (RU) Binding kinetics of nd TG2 Conc. of (μmol/l) Reltive TG2 ctivity (%) Inhiitory effect of TG2 5 -Nitroistin Time (seconds) Drug concentrtion (μmol/l) 8 1 c TG2 d TG2 inhiitor positive control 5 -Nitroistin (μmol/l) (μmol/l) e TG2? Vsculr normliztion Improvement of tumor hypoxi Indirect effect decresing HIFα Prongiogenic fctor VEGF etc. Antingiogenic fctor TSP-1 etc. Tumor HIFα degrdtion NFkB HIFα synthesis Direct effects decresing HIFα Angiogenic switch off Figure 6 Sulfoquinovosyl-cylpropnediol () directly inds with nd inhiits TG2 ctivity. () Binding nlysis of TG2 with. An SPR nlysis for the inding etween nd humn TG2 ws performed. ws injected over flow cells on the immoilized TG2. The ckground resulting from the injection of running uffer ws sutrcted from the dt efore plotting. Resonnce units (RU) were generted y sutrction of the ckground signl generted simultneously on the control flow cell. () Inhiition of tissue trnsglutminse ctivity. (filled circle) strongly inhiited the ctivity of TG2 compred to 5 -Nitroistin (open circle). The horizontl xis indictes the concentrtion of drugs, nd the verticl xis indictes the reltive ctivity. (c) Western lots of TG2 in, nd. All HCC cell line we used expresses TG2. Cells were incuted under normoxi for 24 hours nd then lysed with rdioimmunoprecipittion uffer. (d) Western lots of p in exposed different concentrtion of 5 -Nitroistin nd. 5 -Nitroistin ws used s positive control for TG2 inhiitor. ws incuted with different dose of 5 -Nitroistin (1, 25, nd 75 μmol/l) nd (2.5, 5., nd 1 μmol/l) under normoxi for 24 hours nd then lysed with rdioimmunoprecipittion uffer. (e) Summry of the multiple mechnisms decresing HIFα of. upregultes tumor p. Upregulted p ccelertes HIFα degrdtion. Moreover, upregulted p decreses NFκB expression, resulting in decrese of HIFα synthesis. In ddition to these direct effects decresing HIFα, upregulted p improves tumor hypoxi y induction of vsculr normliztion, which contriutes to indirect decrese of HIFα proteins. All mechnisms contriute to induction of ngiogenic switch off in tumor. directly inds nd inhiits tumor TG2, implying tht upregultes p vi inhiition of TG2. downregulted NFκB expression, which reduced synthesis t trnscriptionl level. Finlly, upregultion of p improved tumor hypoxic microenvironment y induction of vsculr normliztion (Figure 6e). All these effects were olished y knockdown or genetic muttion, indicting tht p upregultion is criticl fctor for the effects of. Overexpression of HIFα proteins hs een shown in HCC. 23 HIFα proteins regulte the expression of mny ngiogenic fctors, which mkes HIFα centrl plyer in the ngiogenic switch. 24 In the present in vivo study, HIFα protein downregultion in switched off the ngiogenic potentil of tumors. Mny ntingiogenic gents such s sorfeni or evcizum produce ntitumor effects y inhiiting specific moleculr trgets in endothelil or cncer cells. 8 The inhiition of specific moleculr trgets cn induce upregultion of other growth fctors, inducing the escpe phenomenon. 11 This lterntive upregulted pthwys result in drug resistnce of HCC, which cuses less clinicl enefit. Inhiition of HIFα proteins cn downregulte multiple ngiogenic fctors expressed specificlly y cncer cells. Therefore, HIFα inhiitors cn e promising drugs, which induce the ngiogenic switch off. HIFα inhiitors cn e divided into two groups sed on their inhiitory mechnisms, with one group downregulting HIFα synthetic pthwys tht involve in inhiition of PI3K, RAS, or elf2α nd the other promoting HIFα degrdtion y enhncing the ctivity of HIF-degrding pthwys. 25,26 is unique gent tht hs qulities of oth types of HIFα inhiitors vi its upregultion of p. is centrl plyer in HIFα protein degrdtion nd could indeed decrese HIFα proteins levels y ctivting the Moleculr Therpy Oncolytics (215) 152

9 -protesome-dependent degrdtion pthwy. Additionlly, p upregultion ws lso le to downregulte NFκB. Rius et l. 27 hve reported tht NFκB regulted HIFα expression t the trnscriptionl level in hypoxi-independent mnner. And NFκB in turn is negtively regulted y p. 28 In this study, upregultion of p decresed NFκB expression nd genetic ltion of showed restortion of NFκB expression, suggesting tht p directly regultes the expression of NFκB. The results shown in the present study support these findings, indicting tht HIFα proteins cn e regulted y p not only through HIFα degrdtion, ut lso y ffecting HIFα synthesis. lso possessed n indirect effect wherey HIFα protein levels decrese in response to improved tumor hypoxi cused y vsculr normliztion. There hve een no reports descriing the role for p in inducing vsculr normliztion, lthough the results presented y Oht et l. 29 do support this evidence in tht the nlog SQMG lso showed vsculr normliztion in response to comintion of low-dose SQMG tretment nd rdition therpy. Improvement of tumor hypoxi y lso supports ccelertion of HIFα proteins degrdtion y p, indirectly. HIFα proteins re degrded y inding with PHDs nd p. 17 During hypoxi, PHD ctivity is inhiited, which leds to increse in the mount of stilized HIFα proteins. Therefore, improvement of tumor hypoxi y could indirectly increse degrdtion of HIFα proteins. Tken together, these findings indicte tht p decreses HIFα protein levels y multiple mechnisms, suggesting tht p upregultion y could e the promising trget, which cn induce tumor ngiogenic switch off. In this study, we showed tht directly inds with TG2 nd strongly inhiits TG2 ctivity. Kim et l. 2 reported tht TG2 cn polymerize, which results directly in the depletion of through uiquitintion nd protesoml degrdtion. They support our findings tht genetic ltion of TG2 resulted in upregultion of p. These findings imply tht we might e le to regulte ngiogenic switch from tumor cell surfce through inhiition of TG2 ctivity. We need further investigtion to know out the role of TG2 inhiition in induction of tumor ngiogenic switch off. In summry, we showed tht tumor p cn e promising trget to induce ngiogenic switch off in HCC nd hve demonstrted tht cn efficiently upregultes p in HCC, which resulted in significnt inhiition of tumor growth with inhiiting tumor ngiogenesis. These effects were cused y downregultion of HIFα proteins through multiple direct nd indirect mechnisms. could e next-genertion ntingiogenic drug in the tretment of HCC. MATERIALS AND METHODS Cell lines nd culture The humn HCC cells nd were provided y the Deprtment of Pthology, Kurume University School of Medicine, while cells were otined from Cmrex (Wlkersville, MD). Cells were mintined in Dulecco modified Egle medium (Gico Invitrogen Cell Culture, Aucklnd, New Zelnd) supplemented with 1% fetl ovine serum (Biowest, Nuille, Frnce). Humn umilicl vsculr endothelil cells were purchsed from Cmrex nd mintined in endothelil cell growth medium-2 (Clonetics, Sn Diego, CA) contining 1% fetl ovine serum. All cell lines were mintined t 37 C in humidified tmosphere with 5% CO 2. For in vitro hypoxic ssys, cells were cultivted in hypoxic conditions tht were mintined y hypoxi chmer (APM-3D, ASTEC, Fukuok, Jpn) flushed with gs mixture of 3% O 2, 5% CO 2, nd 92% N 2. Drugs (Figure 1) ws provided from CANGO (Tokyo, Jpn) nd dissolved in DMSO. Stock solutions were ppropritely diluted with phosphte-uffered sline so tht the finl DMSO concentrtion ws less thn.5%. Angiogenic switch off vi p regultion in HCC In vivo xenogrft ssy Mle 5-week-old nude mice (BALB/c nu/nu) were purchsed from Kyudo KK (Fukuok, Jpn) nd housed in specific pthogen-free conditions. All niml studies were pproved y the Kurume University Animl Cre nd Use Committee.,, nd cells (5 1 6 cells/mouse) suspended in phosphte-uffered sline were injected sucutneously into the mouse flnk region. The tumor volume (V = mm 3 ) nd mouse ody weight were mesured every 2 dys nd estimted using the following eqution: V =.5 length width 2. When the estimted tumor volume reched 15 2 mm 3, the mice received intrperitonel (i.p.) injections of either phosphte-uffered sline (control group) or (2 mg/kg/dy, treted group). The numer of mice in ech group ws 1. Tretments were continued for 21 dys nd on dy 22, tumor tissue nd lood smples were collected fter nesthesi using nemutl (.1 ml/g ody weight) y i.p. injection. Myelotoxicity ws evluted using the lood smples. Western lot nlysis Cells were lysed with rdioimmunoprecipittion uffer contining 1 mmol/l NF, 1 mmol/l N 3 VO 4, 1 mmol/l dithiothreitol, 1 mmol/l phenylmethylsulfonyl fluoride, nd phosphtse inhiitor (Thermo Scientific, Rockford, IL). Western lot nlyses were performed s previously descried. 3 For in vitro hypoxic ssys using western lot nlysis, cells were incuted with medium contining (1 nd 1 μmol/l) for 24 hours whereupon cells were moved to the hypoxic chmer with 3% O 2 for 24 hours nd then lysed with rdioimmunoprecipittion uffer. Rit primry ntiodies nd dilutions used were: nti-extrcellulr signl-regulted kinse 1/2 (ERK1/2) ntiody (1:2), nti-phosphorylted ERK1/2 ntiody (1:2), nti-mmmlin trget of rpmycin ntiody (1:2), nti-phosphorylted mmmlin trget of rpmycin ntiody (1:2), nti- ntiody (1:3), nd nti-nfκb ntiody (1:25). Rit nti-ngiopoietin-2 (Ang-2) nd FGF-2 ntiodies were from Snt Cruz Biotechnology (Snt Cruz, CA) nd used t 1:1 dilution. Rit nti-thromospondin-1 (TSP-1), nti-vegf, nd nti- ntiodies were from Acm (Tokyo, Jpn) nd used t dilutions of 1:3 nd 1:25, nd 1:25, respectively. Mouse nti- ntiody (1:5) ws from BD Biosciences (Tokyo, Jpn). Rit Anti-TG2 ntiody ws from Millipore (1:5; Merck Millipore, MA). Mouse Anti- ntiody (1:1,; Sigm, St Louis, MO) ws used s n internl loding control. Visuliztion of the protein signl ws chieved with horserdish peroxidse conjugted secondry ntiodies (1:5,; GE Helthcre UK, Buckinghmshire, UK) enhnced y chemiluminescence in western lot nlysis system (Amrshm Phrmci Biotech, Pisctwy, NJ) using LAS 4 mini (Fujifilm, Tokyo, Jpn). The mount of luminescence in ech smple ws quntified y multiguge softwre (Fujifilm). Immunohistochemistry Immunohistochemicl stining of CD31 nd TdT-medited dutp nick end leling stining ws performed s previously descried. 5,31 For doule immunofluorescence exmintion of CD31 nd α-smooth muscle ctin (α-sma), sections were incuted t 4 C overnight with rit nti-humn CD31 ntiody (Acm) nd fluorescein isothiocynte conjugted ntirit IgG ntiody (1:1; Invitrogen, Crlsd, CA) for 1 hour. After locking, sections were incuted with got nti-humn α-sma ntiody (1:1; Acm) t 4 C overnight nd Alex fluor 568-leled nti-got IgG ntiody (1:1; Invitrogen) for 1 hour. Nuclei were counterstined with TO-PRO-3 iodide (1:1,, Invitrogen). Imging ws performed in six-order zone region for ech smple (z-series Zeiss LSM-51 Met Confocl Microscope, Crl Zeiss, Jen, Germny) nd nlyzed with Zeiss LSM Imge Browser softwre version 3.5 (Crl Zeiss). RNA extrction nd qrt-pcr ssy Totl RNA ws extrcted using the RNA ssy kit (RNesy mini kit, QIAGEN, Tokyo, Jpn) ccording to the mnufcturer s instructions. qrt- PCR nlysis ws performed using power SYBR Green PCR Mster Mix (Applied Biosystems, Wrrington, UK). The primer sequences were s follows:, forwrd, 5 -TGCTCATCAGTTGCCACTTCC-3, nd reverse, 5 -CCAAATCACCAGCATCCAGAAGT-3 ; nd GAPDH forwrd, 5 -CATGAGAAGT ATGACAACAGCC-3, nd reverse, 5 -AGTCCTTCCACGATACCAAAG-3. The housekeeping gene GAPDH ws used s stndrd. qrt-pcr ws performed using the ABI PRISM 7 Sequence Detection System (Applied Biosystems) ccording to the mnufcturer s instructions. 9 Moleculr Therpy Oncolytics (215) 152

10 1 Angiogenic switch off vi p regultion in HCC In vivo hypoxic ssys using pimonidzole To ssess in vivo hypoxic conditions in tumors, pimonidzole (6 mg/kg/ ody weight; Hypoxyproe-1, NPI, Belmont, MA) ws intrperitonelly injected into mice ering -treted (14 dys) -generted tumors (n = 4). Two hours fter injection, tumor tissues were collected under nesthesi. Immunohistochemistry using pimonidzole ws performed ccording to the mnufcturer s instructions. Pimonidzole dduct formtion ws evluted y western lotting. 4,32 Pimonidzole dducts were shown s the multiple nds, reflecting its cpcity to ind to vriety of proteins under hypoxic conditions. The western lot nlysis ws performed using LAS 4 mini (Fujifilm) nd the protein mounts in the nds for ech smple were evluted using multiguge softwre (Fujifilm). sh-rna knockdown ssy All experiments regrding genetic trnsformtion were pproved y the Kurume University Genetic Modifictions Sfety Committee. sh-rna knockdown ssys were performed s previously descried. 5,33 nd control sh-rna lentivirl prticles were purchsed from Snt Cruz Biotechnology nd cells were infected with these lentivirl prticles nd generted the stle knockdown nd control cells ccording to the mnufcturer s instructions. DNA extrction nd muttion nlysis The humn gene is locted on the short rm of chromosome 3 nd contins 3 exons. Genomic DNA ws extrcted from ech HCC cell line nd primry tissues from 3 HCC ptients using Nucleospin Tissue kit (Mcherey-Ngel, Duren, Germny) nd susequently PCR-mplified. Humn primry HCC smples were collected when heptectomy ws performed nd then freezed in liquid nitrogen. Informed consent ws otined from ll ptients nd the muttion nlyses were pproved y the ethicl committee of Kurume University. PCR products were ssessed y DNA sequencing performed y the Drgon Genomics Center, Tkr Bio (Otsu, Jpn). Surfce plsmon resonnce The inding kinetics of nd humn TG2 were nlyzed y surfce plsmon resonnce (SPR) iosensor (Bicore 3, GE Helthcre, Tokyo, Jpn). The surfce of CM5 sensor chip ws ctivted y injecting solution contining 37.5 mg/ml 1-ethyl-3-(3-dimethylminopropyl) crodiimide hydrochloride nd 5.8 mg/ml N-hydroxysuccinimide t 1 μl/minute rte for 7 minutes. Recominnt TG2 (Immundignostik AG) ws injected over the sensor chip nd cptured on the croxymethyl dextrn mtrix vi mine coupling rection. The surfce ws then locked y injecting 1 M ethnolmine hydrochloride t ph 8.5 for 7 minutes. Binding nlysis ws performed in HBS-P (1 mmol/l Hepes,.15M NCl,.5% Surfctnt P2, ph 7.4) uffer using flow rte of 2 μl/minute t 25 C. Vrious concentrtions of were successively injected upon the immoilized TG2 to detect the SPR response generted. BIAevlution 4.1 softwre (GE Helthcre) ws used to determine the kinetic prmeters. Tissue trnsglutminse ssy Trnsglutminse ssy ws performed using Trnsglutminse Assy Kit (Sigm-Aldrich, Tokyo, Jpn), followed the instruction. Briefly, 5 μl of queous solution ws dded to ech well t different concentrtions, nd the sme volume of ssy mixture contined in the kit ws lso dded. After incution t room temperture for 3 minutes, ech well ws wshed with ddh 2 O three times, nd then 1 μl of streptvidin peroxidse solution ws dded to ech well nd incuted t room temperture for 2 minutes. Ech well ws dded 18 μl of 3,3,5,5 -tetrmethylenzidine liquid sustrte system. Rection ws stopped with ddition of stop solution s soon s the color developed, nd ws mesured the sorption t 45 nm. 5 -Nitroistin (Sigm-Aldrich) ws used s positive control. 34 Sttisticl nlysis All experimentl dt re expressed s the men ± stndrd devition. Differences etween groups were exmined for sttisticl significnce using the Mnn Whitney U-test nd nonprmetric nlysis of vrince. If the one-wy nlysis of vrince ws significnt, differences etween the individul groups were estimted using Fisher s lest significnt difference test. Differences with P <.5 were considered sttisticlly significnt. CONFLICT OF INTEREST The uthors declre no conflict of interest. ACKNOWLEDGMENTS We would like to express our grtitude to Shr H., Reserch Institute of Biosciences, School of Veterinry Medicine, Azu University, Kngw, Jpn, for generously introducing us to CANGO tht provided. And I lso would like to express my grtitude to Shozo Iwmoto, who hs pssed wy in 214. He hs continuously supported me, mentlly. 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