Insulin-like Growth Factor-binding Protein-7 (IGFBP7): A Promising Gene Therapeutic for Hepatocellular Carcinoma (HCC)

Transcription

1 originl rtile The Amerin Soiety of Gene & Cell Therpy Insulin-like Growth Ftor-inding Protein-7 (IGFBP7): A Promising Gene Therpeuti for Heptoellulr Crinom (HCC) Dong Chen 1, Ayesh Siddiq 2, Luni Emdd 2, Devrj Rjsekrn 2, Rhel Gredler 2, Xue-Ning Shen 2, Prsnn K Snthekdur 2, Jyoti Srivstv 2, Chdi L Roertson 2, Igor Dmitriev 3, Elen A Kshentsev 3, Dvid T Curiel 3, Pul B Fisher 2,4,5 nd Devnnd Srkr 2,4,5 1 Deprtment of Pthology, Virgini Commonwelth University, Shool of Mediine, Rihmond, Virgini, USA; 2 Deprtment of Humn nd Moleulr Genetis, Virgini Commonwelth University, Shool of Mediine, Rihmond, Virgini, USA; 3 Deprtment of Rdition Onology, Wshington University Shool of Mediine, St. Louis, Missouri, USA; 4 VCU Mssey Cner Center, Virgini Commonwelth University, Shool of Mediine, Rihmond, Virgini, USA; 5 VCU Institute of Moleulr Mediine, Virgini Commonwelth University, Shool of Mediine, Rihmond, Virgini, USA Heptoellulr rinom (HCC) is highly ftl disese mndting development of novel, trgeted therpies to eliit prolonged survivl enefit to the ptients. Insulinlike growth ftor-inding protein-7 (IGFBP7), sereted protein elonging to the IGFBP fmily, funtions s potentil tumor suppressor for HCC. In the present study, we evluted the therpeuti effiy of replitioninompetent denovirus expressing IGFBP7 () in humn HCC. profoundly inhiited viility nd indued poptosis in multiple humn HCC ell lines y induing retive oxygen speies (ROS) nd tivting DNA dmge response (DDR) nd p38 MAPK. In orthotopi xenogrft models of humn HCC in thymi nude mie, intrvenous dministrtion of profoundly inhiited primry tumor growth nd intrhepti metstsis. In nude mie suutneous model, xenogrfts from humn HCC ells were estlished in oth flnks nd only left-sided tumors reeived intrtumorl injetion of. Growth of oth left-sided injeted tumors nd right-sided uninjeted tumors were mrkedly inhiited y with profound suppression of ngiogenesis. These findings indite tht might e potent therpeuti erditing oth primry HCC nd distnt metstsis nd might e n effetive tretment option for terminl HCC ptients. Reeived 19 Otoer 212; epted 11 Deemer 212; dvne online pulition 15 Jnury 213. doi:1.138/mt INTRODUCTION Heptoellulr rinom (HCC) is mjor glol helth prolem nd is one of the five most ommon ners worldwide. 1 Although overll inidene of ners is deresing, the inidene of HCC is inresing signifintly nd it is the third highest use of ner-relted deths glolly. 1,2 In Asin ountries, heptitis B virus infetion is the predominnt use of HCC, wheres in Western ountries heptitis C virus infetion nd loholism ply the mjor role. 1,3 Dietry toxin, suh s fltoxin, is mjor use of HCC in su-shrn Afri. These diverse etiologies long with enumerle modultions in gene expression, signling pthwys, nd muttions hve posed mjor impediment in understnding the pthophysiology of this omplex disese nd developing effetive therpeuti strtegies. 4 The mortlity rte of HCC prllels tht of inidene euse HCC is highly resistnt to onventionl hemo- nd rdiotherpy nd no systemi therpy is ville for the dvned disese. 5 As suh developing novel therpies sed on underlying moleulr normlities is mndtory to effetively ountert this invrily ftl disese. Insulin-like growth ftor-inding protein-7 (IGFBP7) is sereted protein elonging to the IGFBP fmily. 6 The IGF xis plys key role in the growth, differentition, nd prolifertion of mmmlin ells nd onsists of two growth ftors (IGF-I nd IGF-II), their reeptors (IGF-IR nd IGF-IIR) nd group of IGFBPs (IGFBP1-7). 6,7 IGFBPs regulte the iovilility of IGFs y inding to IGFs with high ffinity therey limiting IGF ess to IGF-IR nd inhiiting IGF tivity. 6 However, IGFBPs lso exert IGF-independent tions. IGFBP7 differs from the other six memers of this fmily y lking the C-terminus nd hving 1 times lower ffinity for IGF-I. 8 A numer of studies, inluding ours, hve estlished IGFBP7 s potentil tumor suppressor gene for vriety of ners, inluding HCC Using oligonuleotide mirorry, we initilly identified IGFBP7 s the most downregulted gene y the onogene Astroyte-elevted gene-1, whih is overexpressed in >9% of HCC ptients nd its expression level negtively orreltes with overll survivl nd umultive reurrene rtes. 14 Using n immunohistohemil pproh in tissue mirorry, we doumented high IGFBP7 expression in norml humn liver while in 14 HCC ptients there ws sttistilly signifint grdul derese in IGFBP7 expression with the stges of HCC. 9 Moreover, in eh stge, IGFBP7 expression ws muh lower in poorly differentited grdes ompred with modertely differentited grdes. Fluoresene in-situ hyridiztion in tissue mirorry reveled loss of heterozygosity for IGFBP7 lous in 26% of HCC ptients. 9 In ddition, in humn HCC smples, Correspondene: Devnnd Srkr, Deprtment of Humn nd Moleulr Genetis, VCU Mssey Cner Center, 122 Est Brod St, PO Box 9835, Rihmond, Virgini 23298, USA. E-mil: dsrkr@vu.edu vol. 21 no. 4, pr. 213

2 The Amerin Soiety of Gene & Cell Therpy s Therpeuti for HCC d IGFBP7 mrna (A.U.) Ad.I7-1d Ad.I7-2d Ad.I7-3d IGFBP7 (ng/ml) 8 6 Ad.I7-1d Ad.I7-2d Ad.I7-3d 4 # 2 Ad.I7-1d Ad.I7-2d Ad.I7-3d DAPI IGFBP7 Merge 24 hours 48 hours 24 hours 48 hours Astroyte-elevted gene-1 overexpression nd IGFBP7 downregultion ould e sttistilly orrelted inditing tht genomi deletion s well s onogene-medited negtive regultion might e the moleulr mehnism of IGFBP7 downregultion in HCC ptients. 9,14 A seprte study nlyzing 14 ptients doumented negtive IGFBP7 stining in 35.6% HCC ptients whih sttistilly orrelted with lrge tumor size, inresed vsulr invsion, poor overll survivl, nd disese-free survivl rtes. 15 Thus IGFBP7 downregultion might e linilly relevnt prognosti mrker for ggressive HCC. Stle overexpression of IGFBP7 in ggressive humn HCC ells resulted in modest inhiition in prolifertion nd olony IGFBP7 Figure 1 genertes IGFBP7. (),, nd ells were either uninfeted (ontrol) or infeted with or t n MOI of 1 vp/ell. IGFBP7 mrna expression ws determined y quntittive RT-PCR 24 h post-infetion. GAPDH expression ws used s ontrol. IGFBP7 expression level in the uninfeted ells ws onsidered s one. The dt represent men ± SEM. P <.1. (),, nd ells were either uninfeted (ontrol) or infeted with or t n MOI of 1 vp/ell. Cells in the ontrol nd groups were hrvested post-infetion while those in group were hrvested 1, 2, nd post-infetion. The lystes were sujeted to western lot nlyses using nti-ha ntiody. EF1- ws used s loding ontrol. (),, nd ells were infeted with or t n MOI of 1 vp/ell. Conditioned medi were olleted 24 nd 48 hours post-infetion nd sujeted to IGFBP7 detetion y ELISA. The dt represent men ± SEM. # P <.5; P <.1. (d) ells were infeted with t 1 vp/ell. IGFBP7 expression ws deteted y immunofluoresene nlysis 24 hours postinfetion. Imges were nlyzed using Zeiss onfol lser snning mirosope. DAPI ws used to stin the nuleus. A.U., ritrry unit; DAPI, 4,6-dimidino-2-phenylindole; GAPDH, glyerldehyde 3-phosphte dehydrogense; HA, hemgglutinin; MOI, multipliity of infetion; RT-PCR, reverse trnsription-pcr; vp, virl prtile. (%) (%) (%) Colonies/25 ells # # # Ad.I7-1d Ad.I7-2d Ad.I7-3d formtion when ompred with the ontrol lones. 9 However, in suutneous xenogrft ssy using thymi nude mie, there ws profound inhiition of tumorigenesis y IGFBP7-overexpressing lones ompred with the ontrol lone, whih ould e orrelted with mrked inhiition of ngiogenesis nd indution of senesene. 9 These findings prompted us to evlute replition-inompetent denovirus-delivered IGFBP7 () s potentil therpeuti for HCC. Our in vitro nd in vivo studies doument tht potently indues poptosis in multiple HCC ell % Apoptoti ells % Apoptoti ells % Apoptoti ells PARP PARP PARP Figure 2 indues poptosis in humn HCC ells. (),, nd ells were either uninfeted (ontrol) or infeted with (1, vp/ell) or (1, 5 or 1, vp/ell). ws deteted y stndrd MTT ssy 1, 2, nd postinfetion. of the ontrol uninfeted ells t dy 1 ws onsidered s 1%. (),, nd ells were either uninfeted (ontrol) or infeted with (1, vp/ell) or (1 or 1, vp/ell) nd sujeted to olony formtion ssy. Colonies were sored fter 2 weeks. (),, nd ells were infeted with or t n MOI of 1, vp/ell. Apoptosis ws deteted y Annexin V stining followed y flow ytometry 1, 2, nd post-infetion. For, the dt represent men ± SEM of three independent experiments. # P <.5; P <.1. (d),, nd ells were either uninfeted (ontrol) or infeted with or t n MOI of 1, vp/ell. Cells in the ontrol nd groups were hrvested post-infetion while those in group were hrvested 1, 2, nd post-infetion. The lystes were sujeted to western lot nlyses using nti-parp ntiody. ws used s loding ontrol. HCC, heptoellulr rinom; MOI, multipliity of infetion; vp, virl prtile. d Moleulr Therpy vol. 21 no. 4 pr

3 s Therpeuti for HCC The Amerin Soiety of Gene & Cell Therpy Fluoresene (%) No Rx NAC-5 NAC-1 Fluoresene (%) Fluoresene (%) Fluoresene (%) QGY QGY-773 # # No Rx NAC-5 NAC-1 Figure 3 indues retive oxygen speies (ROS). (),,, nd QGY-773 ells were infeted with or t n MOI of 1, vp/ell. ROS ws deteted y ommerilly ville kit s desried in Supplementry Mterils nd Methods. () The indited ells were treted s in nd either untreted or treted with N-etylysteine (NAC) t 5 or 1 mmol/l doses. ws determined y stndrd MTT ssy. The dt represent men ± SEM of three independent experiments. # P <.5; P <.1. MOI, multipliity of infetion; vp, virl prtile. lines nd profoundly inhiits primry tumor growth nd metstsis suggesting potentil linil pplition of in terminl HCC ptients. RESULTS inhiits humn HCC viility in vitro Adenovirus serotype 5 (Ad5), whih is routinely used s virl delivery vehile, trnsdues vi oxskie-denovirus reeptors. 16 Coxskie-denovirus reeptor is downregulted in mny ners, thus preventing effiient Ad trnsdution of tumor ells. 17 To irumvent this prolem, the fier kno of Ad5 is repled y Ad3 (Ad.5/3) tht llows inresed trnsdution of humn norml nd tumor ells independent of their oxskie-denovirus reeptor sttus. 18 We reted replition-inompetent denovirus expressing IGFBP7 in Ad5/3 kground (). An HA-tg ws dded to the COOH-terminus of IGFBP7 protein for esy detetion of the exogenous protein. We seleted three humn HCC ell lines,,, nd to evlute the effet of. ells hve mutnt p53 (ysteine to tyrosine muttion t mino id 22) nd wild-type R; ells re null for oth p53 nd R; nd ells, vrint of HepG2, re wild-type for oth p53 nd R. 19 We hose these ells to determine whether different geneti kgrounds might ffet the funtion of IGFBP7. The ells were either uninfeted, infeted with either (ontrol empty Ad) or 76 vol. 21 no. 4 pr. 213

4 The Amerin Soiety of Gene & Cell Therpy s Therpeuti for HCC Ad.I7- Ad.I7- Ad.I7- Ad.I7- Ad.I7- Ad.I7- p-atr ATR.8.4 No Rx SB-1 SB-2 p-atm ATM p-chk1 CHK1 p-chk2 CHK2 p-p53 p53 p-p38 p38 IGFBP QGY Figure 4 tivtes DNA dmge response pthwy.,, nd ells were either uninfeted or infeted with or t n MOI of 1, vp/ell. For ontrol nd groups, the smples were olleted t dy 2. Cell lystes were sujeted to western lot nlysis using the indited ntiodies. A representtive lot for ws presented s loding ontrol. MOI, multipliity of infetion; vp, virl prtile. t multipliity of infetion of 1 virl prtile (vp)/ell nd IGFBP7 mrna expression ws deteted y quntittive reverse trnsription-pcr. infetion did not ffet the sl IGFBP7 mrna expression. Upon infetion, mrked inrese in IGFBP7 mrna level ws deteted in ll the three ell lines with ells showing the most roust response (Figure 1). The mrna level findings were orroorted t the protein level y western lot nlysis in ell lystes using nti-ha ntiody (Figure 1). Compred with nd ells, ells demonstrted the most umultion of IGFBP7 protein. The level of the sereted IGFBP7 protein in the ulture medi were mesured y enzyme-linked immunosorent ssy using nti-igfbp7 ntiody (Figure 1) demonstrting the sme trend. It should e noted tht the sl IGFBP7 level in ells re higher thn tht in nd ells s suh the totl sereted protein level in ells, upon infetion, is higher thn tht in ells. Immunofluoresene nlysis deteted IGFBP7 protein in the ytoplsm upon infetion of humn HCC ells (Figure 1d).,, nd ells were infeted with t 1, vp/ell or with t 1, 5 or 1, vp/ell nd ell Figure 5 SB2358 protets from -indued inhiition of ell viility.,,, nd QGY-773 ells were infeted with or t n MOI of 1, vp/ell nd either untreted or treted with SB2358 (SB) t 1 or 2 μmol/l doses. ws determined y stndrd MTT ssy. The dt represent men ± SEM of three independent experiments. P <.1. MOI, multipliity of infetion; vp, virl prtile. viility ws monitored y stndrd MTT ssy over period of 3 dys (Figure 2). Even t 1 vp/ell, signifintly inhiited ell viility in ll the three ell lines. However, t higher doses there ws profound inhiition in ell viility in ll the ell lines. Even though ells produed muh more IGFBP7 ompred with the other two ell lines, the inhiitory effet of IGFBP7 were similr in ll the three ell lines. Interestingly, in ells derese in ell viility ws not oserved until dy 2. The long-term effet of on ell viility ws nlyzed y olony formtion ssy (Figure 2). In 2-week ssy, even with 1 vp/ell, omplete inhiition of olony formtion ws oserved in ll the three ell lines inditing potent growth inhiitory ility of IGFBP7. Anlysis of poptosis y nnexin V-inding ssy followed y flow ytometry demonstrted temporl indution of poptosis in ll the three HCC ell lines (Figure 2) whih were orroorted y detetion of leved PARP y western lot (Figure 2d). Correlting with MTT ssy, indution of poptosis ws delyed in ells nd not deteted until dy 3 fter infetion, wheres in nd ells there ws grdul indution of poptosis from dy 1 post-infetion onwrds. ells were the most sensitive showing the highest level of poptosis (Figure 2,d). Moleulr Therpy vol. 21 no. 4 pr

5 s Therpeuti for HCC The Amerin Soiety of Gene & Cell Therpy Before Rx After Rx ROI ounts Before Rx After Rx Before Rx After Rx d Metstsis sore ,2 e f N T -FP (ng/ml) Figure 6 inhiits orthotopi humn HCC xenogrfts nd intrhepti metstsis. QGY-lu ells were orthotopilly implnted y intrhepti injetion in thymi nude mie nd treted with intrvenous injetions of or (1 1 9 vp/injetion; totl of five injetions over 2-week period). () Bioluminesene imging of the mie efore nd fter tretment. () Quntifition of photon ounts from the nimls. The dt represent men ± SEM; P <.1. Sixteen nimls per group were used. () Photomirogrph of the livers t the end of the study. (d) Anlysis of intrhepti metstsis sore t the end of the study. (e) Anlysis of serum humn -fetoprotein ( -FP) levels t the end of the study. For d nd e, the dt represent men ± SEM; P <.1. (f) does not indue toxiity to norml mouse liver. Hemtoxylin nd eosin stining of setions ontining QGY-lu orthotopi tumor (T) nd the djent norml liver (N) from mouse treted with. Plese note the preservtion of norml rhiteture in the djent liver while the tumor shows hemorrhgi nerosis. The rrows indite the order etween the orthotopi tumor nd norml liver. HCC, heptoellulr rinom; ROI, region of interest; vp, virl prtile. We next heked the genertion of retive oxygen speies (ROS), meditor of oth senesene nd poptosis, upon infetion. In ddition to,, nd ells, we inluded highly ggressive humn ell line QGY-773 in these studies. infetion resulted in signifint umultion of superoxide rdils (Figure 3) nd n ntioxidnt N-etylysteine provided signifint protetion from -indued ell deth (Figure 3) in ll the four HCC ell lines. N-etylysteine provided protetion during the erly phse of ell deth until nd y post- infetion, the killing effets of overwhelms the protetive effets of N-etylysteine. Sine umultion of ROS might indue DNA dmge response (DDR), we nlyzed the tivtion of DDR pthwy. In ll the three ell lines, tivtion of txi telngietsi nd Rd3-relted (ATR), txi telngietsi mutted (ATM), hekpoint kinse-1 (CHK1), nd hekpoint kinse-2 (CHK2) ws oserved upon infetion y western lot nlysis of the phosphorylted forms of the forementioned proteins (Figure 4). Phosphoryltion of p53 ws oserved in nd ells, ut not in ells whih re p53-null, without ny signifint hnge in totl p53 level (Figure 4). The tivtion of DDR pthwy ws ssoited with the tivtion of the stressindued p38 MAPK (Figure 4). Indeed, inhiition of p38 MAPK y speifi inhiitor SB2358 provided signifint protetion from -medited ell deth in ll the four humn HCC ells (Figure 5). inhiits humn HCC xenogrfts in vivo QGY-773 ells develop ggressive tumors in nude mie nd for in vivo studies we used lone of QGY-773 ells stly expressing luiferse (QGY-lu). QGY-lu ells mintin the norml prolifertive potentil nd demonstrted similr suseptiility to when ompred with the prentl QGY-773 ells (Supplementry Figure S1). We performed orthotopi implnttion of QGY-lu ells in the livers of mle thymi nude mie nd monitored tumor development y ioluminesene imging. One week fter implnttion, when tumor is well estlished, the mie were treted with intrvenous injetions of or t dose of vp/injetion. A totl of five injetions were given over period of 2 weeks nd the nimls were srified 2 weeks fter the lst injetion. In -treted group, tumors grdully inresed in size s refleted y mrked inrese in ioluminesene intensity t the end of the experiment (Figure 6,). On the ontrry, in the -treted nimls, the tumors did not t ll grow in size. Rther in mjority of the nimls lmost no ioluminesene ws deteted t the end of the experiment (Figure 6). Mrosopilly, in -treted group, lrge tumors were oserved in the liver, while the livers of the -treted group preserved their norml morphology nd very smll tell-tle signs of the tumor ould e oserved (Figure 6). Mirosopi nlysis of the liver reveled signifint intrhepti metstsis in -treted group, while in the -treted group little to no intrhepti metstsis ws deteted (Figure 6d). Anlysis of mouse serum showed mrked derese in humn -fetoprotein level in -treted mie ompred with -treted mie (Figure 6e). injetion did not indue toxi effet in the djent norml mouse liver s reveled y mintenne of norml rhiteture upon histologil nlysis wheres the xenotrnsplnted tumor showed hemorrhgi nerosis (Figure 6f). In order to hek whether IGFBP7 is le to inhiit tumor formtion t distnt sites, we implnted QGY-lu ells suutneously on oth flnks of nude mie. After the estlishment of the tumors, requiring ~1 week, intrtumorl injetion of or ws dministered only to the tumors on the left flnk vol. 21 no. 4 pr. 213

6 The Amerin Soiety of Gene & Cell Therpy s Therpeuti for HCC Tumor weight (mg) Tumor volume (u. mm) 2, 1,6 1, , Week 1 Lt Rt Lt Rt Week 2 Week 3 Week 4 Lt Rt Lt Rt d IGFBP7 (ng/ml) weeks 4 weeks e TUNEL-positive ells/field Lt Rt Lt Rt Figure 7 inhiits primry nd distnt tumors. QGY-lu ells (1 1 6 ) were suutneously implnted in oth flnks of thymi nude mie nd fter the estlishment of the tumors (~1 mm 3 ) only the left-sided tumors were injeted with either or (1 1 9 vp/ injetion; totl of five injetions over 2-week period). () Photogrph of the mie t the end of the experiment. () Mesurement of tumor volume during the ourse of the experiment. () Mesurement of tumor weight t the end of the experiment. Fifteen nimls per group were used. (d) IGFBP7 protein is deteted in the ser of mie reeiving intrtumorl injetion of. Blood ws olleted from the tip of the til t 2 weeks (i.e., t the end of the lst Ad injetion) nd t 4 weeks (i.e., t the end of the experiment). IGFBP7 ws mesured y ELISA s desried in the Mterils nd Methods. Five nimls were used per group. (e) Apoptoti ells were deteted in tumor setions y TUNEL ssy. For e, the dt represent men ± SEM; P <.1. Lt, left; Rt, right; vp, virl prtile. leving the right-flnk tumors untreted. A totl of five injetions (1 1 9 vp/injetion) were given over period of 2 weeks nd the nimls were srified 2 weeks fter the lst injetion. In treted group, there ws grdul inrese in size in oth the leftnd right-sided tumors (Figure 7 ). In mjority of the nimls, there ws omplete dissolution of the left-sided injeted tumors. More importntly, there ws lso signifint inhiition of growth of the right-sided uninjeted tumors. We mesured serum IGFBP7 level in these mie t 2 weeks (i.e., t the end of the lst Ad injetion) nd t 4 weeks (i.e., t the end of the experiment). The sl IGFBP7 level in the serum of -injeted nimls ws very low (Figure 7d). A signifint mount of IGFBP7 ws deteted in the serum of -injeted nimls t the end of the injetion yle (2 weeks timepoint). At the end of the experiment (t 4 weeks) signifint IGFBP7 ws still deteted in the serum lthough the level deresed from tht mesured t the 2 weeks timepoint. Signifint poptosis ws deteted in oth left- nd right-sided tumors upon tretment with s reveled y TUNEL ssy (Figure 7e). Histologil nlysis of the tumors showed res of nerosis in oth left- nd right-sided tumors in - treted group ut not in -treted group (Figure 8). Stining for IGFBP7 deteted undnt ytoplsmi IGFBP7 stining in the left-sided tumor injeted with. In the right-sided tumor of group, IGFBP7 stining ws not deteted when the stining retion ws stopped t timepoint when IGFBP7 stining in the left-sided tumor hs lredy developed (Figure 8). However, keeping the right-sided tumor setions of group longer in the stining solution resulted in detetion of low level IGFBP7 inditing tht IGFBP7 protein generted from the left side of the tumor might enter the irultion nd reh the tumor on the right side (Figure 8). There ws profound inhiition in prolifertion, deteted y Ki-67 stining, nd ngiogenesis, deteted y CD31 stining in oth left- nd right-sided -treted tumors ut not in -treted tumors (Figure 8). A lrger imge of Figure 8 is shown in Supplementry Figure S2 for etter visuliztion. To onfirm tht sereted IGFBP7 protein indues poptosis, QGY-lu ells were infeted with for 48 hours, the onditioned medi ws olleted, filtered, nd dded to freshly plted QGY-lu ells. The onditioned medi effiiently indued poptosis s reveled y detetion of PARP levge (Figure 8). Moleulr Therpy vol. 21 no. 4 pr

7 s Therpeuti for HCC The Amerin Soiety of Gene & Cell Therpy Lt Rt (rt-sided tumor) IGFBP7 DISCUSSION We previously demonstrted tht stle overexpression of IGFBP7 in humn HCC ell lines rogtes prolifertion nd indues senesene. 9 The present studies revel tht n denovirus-medited delivery of IGFBP7 indues poptosis in humn HCC ells without induing senesene. These differenes in outome might e ttriuted to the level of IGFBP7 delivered y eh system. The low level of IGFBP7, generted in the stle ell lines, indues DDR (s reveled y γ-h2ax stining) resulting in senesene. 9 The high level of IGFBP7, generted y, results in genertion of ROS nd lso DDR tht overwhelms the homeostti system leding to tivtion of p38 MAPK pthwy, known induer of poptoti sde, 2 nd indution of poptosis. ROSindued DNA doule-strnded reks tivte signling kinses, ATM nd its homologue ATR, tht phosphorylte CHK1 nd CHK2, respetively with susequent phosphoryltion of p53 tht might led to either ell yle rrest or poptosis. 21 ATM nd ATR might tivte p38 MAPK nd sustined tivtion of p38 MAPK might indue poptosis either y phosphorylting nd tivting p53 or y phosphorylting ell yle regultory proteins nd Lt Rt PARP H&E IGFBP7 Ki-67 CD31 Figure 8 IGFBP7 inhiits prolifertion nd ngiogenesis in primry nd distnt tumors. () Tumor setions were stined with hemtoxylin nd eosin (H&E) or stined for IGFBP7, Ki-67 nd CD31. Asterisk () represents neroti res. Arrows indite lood vessels. Originl mgnifition: 4. () IGFBP7 protein is deteted in the right-sided uninjeted tumor upon longer exposure. Setions from right-sided uninjeted tumors from -treted group were stined for IGFBP7. () Conditioned medi from -infeted ells indue poptosis. QGY-lu ells were either uninfeted (ontrol) or infeted with or t n MOI of 1 vp/ell for 48 hours. The onditioned medi ws olleted, filtered, nd dded to freshly plted QGY-lu ells for 48 hours. The lystes were sujeted to western lot nlyses using nti-parp ntiody. ws used s loding ontrol. Lt, left; MOI, multipliity of infetion; Rt, right; vp, virl prtile. tivting growth rrest nd DNA dmge induile proteins We oserve tivtion of ATM, ATR, CHK1, CHK2, nd p38 MAPK in ll the three HCC ell lines upon infetion. Ativtion of p53 is oserved depending upon the p53 sttus of the ells. These findings strongly suggest tht ROS-indued tivtion of DDR with susequent tivtion of p38 MAPK nd p53 medite IGFBP7-indued poptosis. The moleulr mehnism y whih indues ROS remins to e determined nd is urrently under study. The inhiition of insulin/igf signling nd MEK/ERK signling hs een suggested to e potentil moleulr mehnism of IGFBP7 tion. However, our studies unrvel the novel mehnism of tivtion of DDR pthwy in IGFBP7-indued poptosis. A potentil ell surfe reeptor for IGFBP7, similr to tht for IGFBP3, 23 might medite IGFBP7 effets independent of the known pthwys. Very interestingly, we did not oserve tivtion of either spse-8 or spse-9 during -indued poptosis suggesting tht IGFBP7-indued poptosis might e medited independent of lssil extrinsi or intrinsi pthwys of poptosis (Supplementry Figure S3). In melnom ells, reominnt IGFBP7 protein indued poptosis only in mutted BRAF-ontining ells y loking BRAF/MEK/ERK signling ut not in BRAF wild-type melnom ells. 1 In rest ner ells, it hs een proposed tht reominnt IGFBP7 tivtes p38 MAPK leding to p53/p21-dependent ell yle rrest nd poptosis. 24 BRAF muttion is rre event in humn HCC 25 nd we oserve poptosis indution y in four different humn HCC ell lines inditing tht in the ontext of HCC, IGFBP7-indued poptosis might e BRAF-independent. In ddition, p38 MAPK tivtion nd protetion from ell deth y p38 MAPK inhiitor were oserved in ll HCC ells, independent of their p53 sttus, inditing tht -indued poptosis my not e medited y p53 or in the sene of p53 n lternte pthwy is tivted to indue poptosis. We oserve differentil response to IGFBP7 depending upon the p53 sttus of the ell lines. In ll the three HCC ell lines,,, nd, the kinetis of the tivtion of the DDR pthwy nd p38 MAPK signling upon infetion were similr. However, even though ells showed the most roust expression of IGFBP7 upon infetion, ompred with nd ells, the kinetis of the killing ws delyed in ells. The ility of mutnt p53 to quire gin-of-funtion nd protet from poptosis hs een extensively studied. 26 Our findings rgue tht mutnt p53 in ells might onfer n initil protetive effet whih eomes overwhelmed when signifint mount of IGFBP7 umultes in the ells resulting in poptosis. Interestingly, IGFBP7 ws shown to e p53 downstrem gene in lung ner suggesting severl levels of ross-tlks etween p53 nd IGFBP7 tht need to e studied in detil. 27 tretment mrkedly inhiited oth primry orthotopi implnttion s well s intrhepti metstsis. Tretment of the left-flnked tumor with mrkedly inhiited growth of oth left- nd right-flnked tumors. While strong IGFBP7 stining ws deteted in the left-sided -injeted tumor, low level IGFBP7 stining ws deteted in the right-sided uninjeted tumor. These findings rgue tht IGFBP7 entering into vol. 21 no. 4 pr. 213

8 The Amerin Soiety of Gene & Cell Therpy s Therpeuti for HCC the irultion is le to inhiit tumors t distnt sites possily y interting with n s yet unidentified ell surfe reeptor on tumor ells. Reominnt IGFBP7 protein indues poptosis in melnom nd oloretl ner ells. 28 We lso oserve tht onditioned medi olleted from -infeted ells ould indue poptosis in humn HCC ells (Figure 8). As orollry, profound inhiition in ell prolifertion ws oserved in oth leftnd right-sided tumors in -treted group. In ddition, IGFBP7 tretment mrkedly inhiited ngiogenesis nd these dul effets, i.e., indution of poptosis nd inhiition of ngiogenesis, might provide the potent tumor inhiitory effets t distnt sites. There might lso e potentil role of IGFBP7 in induing n ntitumor immune response. Athymi nude mie re immunodefiient ut still hror nturl killer ells tht might onfer ntitumor response. 29 As yet, the role of IGFBP7 in immune system remins unexplored nd is potentil relm of study. One mjor detriment towrds Ad-medited gene therpy is the sequestrtion of Ad in the liver upon intrvenous dministrtion. 3 This disdvntge serves s potentil dvntge for HCC where Ad-medited delivery of therpeuti gene might e extremely effiious in erditing primry tumor nd distnt metstsis. Our studies estlish s potent therpeuti for humn HCC nd might usher in Phse I/II linil tril for terminl HCC ptients hving no effetive therpeuti option. Sine IGFBP7 shows tumor suppressor properties in other ners, the urrent strtegy might e potentil therpeuti modlity for rod spetrum of ners s well. MATERIALS AND METHODS Genertion of. Replition-inompetent E1/E3-deleted ws generted in Ad.5/3 kone in whih the fier kno of Ad5 ws repled y tht of Ad3. Ad.5/3 expressing luiferse (Ad.5/3- Lu1) ws previously generted. 18 The genome of ws generted y homologous DNA reomintion in Esherihi oli etween linerized plsmid pshcmv-igfbp7 (shuttle vetor ontining IGFBP7 open-reding frme under the ontrol of CMV promoter) nd BstBI-digested genomi DNA of Ad.5/3-Lu1 vetor. The resultnt p plsmid, ontining the Ad.5 genome enoding the IGFBP7 gene in ple of the erly virl E1 region nd the reominnt F5/3 fier gene, ws seleted with knmyin. ws resued y trnsfeting HEK-293 ells with pad. IGFBP7 plsmid DNA leved with PI to relese the virl inverted terminl repets. The viruses were titered y stndrd plque ssy. Cell lines, ulture ondition, viility, nd poptosis ssys. Humn HCC ell lines,,,, nd QGY-773, were ultured s desried. 9,14 ws determined y stndrd MTT ssy s desried. 14 For olony formtion ssys, ells (25) were plted in 6-m dishes nd olonies >5 ells were ounted fter 2 weeks. Apoptosis ws determined y nnexin V-inding ssy followed y flow ytometry s desried. 2 Enzyme-linked immunosorent nlysis. The reominnt IGFBP7 protein used s stndrd ws otined from R&D (Minnepolis, MN; tlog no B7). The wells of 96-well plte were oted with the stndrd or with onditioned medi (1 μl) overnight t 4 C. The wells were wshed four times with phosphte-uffered sline (PBS) nd then loked with 5% non-ft milk in PBS for 1 hour. Following nother PBS wsh, nti-ig- FBP7 ntiody (1:1,) ws dded nd inuted overnight t 4 C without shking. After nother four times of PBS wsh, nti-mouse seondry ntiody (1:1,) ws dded nd inuted t room temperture for 2 hours with gentle shking. The wells were wshed with PBS four times nd 1 μl Glo Sustrte Regent (R&D; DY993) ws dded to eh well for 15 minutes nd the retion ws stopped y dding 1 μl Stop Solution (R&D; DY994). The plte ws red t 56 nm using multiplte reder (Turner Biosystems, Sunnyvle, CA). Orthotopi xenogrft studies in nude mie. All niml studies hve een pproved y the Institutionl Animl Cre nd Use Committee of Virgini Commonwelth University. QGY-lu ells (QGY-773 ells stly expressing luiferse) were orthotopilly implnted y intrhepti injetion in thymi nude mie (6 8 weeks of ge). Tumor growth ws monitored y ioluminesene imging with Xenogen IVIS imger (PerkinElmer, Wlthm, MA) one week. One week fter implnttion, when tumor ws well estlished, the mie were treted with intrvenous injetions of or t dose of vp/injetion. A totl of five injetions were given over period of 2 weeks nd the nimls were srified 2 weeks fter the lst injetion. Two loes of liver were slied into totl of 4 6 strips nd emedded in prffin. For eh mouse, 1 setions t 3 5 μm distne were hemtoxylin nd eosin stined nd mirosopilly nlyzed for metstses. Metstti lesions were lssified in miro, smll, nd lrge, nd urden ws lulted y the sum of ll metstti lesions multiplied 1 (miro), 2 (smll), nd 3 (lrge). Sixteen nimls per group were used for these studies. Nude mie xenogrft studies. QGY-lu ells (1 1 6 ) were suutneously implnted in oth the flnks of thymi nude mie. After the estlishment of the tumors (~1 mm 3 ), requiring ~1 week, intrtumorl injetion of or ws dministered only to the tumors on the left flnk leving the right-flnk tumors untreted. A totl of five injetions (1 1 9 vp/injetion) were given over period of 2 weeks nd the nimls were srified 2 weeks fter the lst injetion. Tumor volume ws mesured with lipers using the formul: π/6 lrger dimeter (smller dimeter) 2. Fifteen nimls per group were used for these studies. Sttistil nlysis. Dt were represented s the men ± SEM nd nlyzed for sttistil signifine using one-wy nlysis of vrine followed y Newmn Keuls test s post ho test. A P vlue of <.5 ws onsidered s signifint. SUPPLEMENTARY MATERIAL Figure S1. QGY-773 nd QGY-lu ells show similr suseptiility to. Figure S2. Enlrged version of Figure 8. Figure S3. indues poptosis in spse-8 nd spse- 9 independent pthwy. Mterils nd Methods. ACKNOWLEDGMENTS The present study ws supported in prt y grnts from the Jmes S. MDonnell Foundtion nd Ntionl Cner Institute grnt R1 CA13854 (D.S.), the Smuel Wxmn Cner Reserh Foundtion (SWCRF) grnt (D.S. nd P.B.F.) nd Ntionl Institutes of Helth grnt R1 CA (P.B.F.). D.S. is the Hrrison Endowed Sholr in Cner Reserh nd Blik sholr. P.B.F. holds the Thelm Newmeyer Cormn Chir in Cner Reserh nd is SWCRF Investigtor. The uthors delred no onflit of interest. REFERENCES 1. El-Serg, HB nd Rudolph, KL (27). Heptoellulr rinom: epidemiology nd moleulr rinogenesis. Gstroenterology 132: Siegel, R, Nishdhm, D nd Jeml, A (212). Cner sttistis, 212. CA Cner J Clin 62: El-Serg, HB, Dvil, JA, Petersen, NJ nd MGlynn, KA (23). The ontinuing inrese in the inidene of heptoellulr rinom in the United Sttes: n updte. Ann Intern Med 139: Llovet, JM nd Bruix, J (28). Moleulr trgeted therpies in heptoellulr rinom. Heptology 48: Llovet, JM, Burroughs, A nd Bruix, J (23). Heptoellulr rinom. Lnet 362: Hw, V, Oh, Y nd Rosenfeld, RG (1999). The insulin-like growth ftor-inding protein (IGFBP) superfmily. Endor Rev 2: Moleulr Therpy vol. 21 no. 4 pr

9 s Therpeuti for HCC The Amerin Soiety of Gene & Cell Therpy 7. Pollk, M (28). Insulin nd insulin-like growth ftor signlling in neoplsi. Nt Rev Cner 8: Ymnk, Y, Wilson, EM, Rosenfeld, RG nd Oh, Y (1997). Inhiition of insulin reeptor tivtion y insulin-like growth ftor inding proteins. J Biol Chem 272: Chen, D, Yoo, BK, Snthekdur, PK, Gredler, R, Bhuti, SK, Ds, SK et l. (211). Insulin-like growth ftor-inding protein-7 funtions s potentil tumor suppressor in heptoellulr rinom. Clin Cner Res 17: Wjpeyee, N, Serr, RW, Zhu, X, Mhlingm, M nd Green, MR (28). Onogeni BRAF indues senesene nd poptosis through pthwys medited y the sereted protein IGFBP7. Cell 132: Burger, AM, Zhng, X, Li, H, Ostrowski, JL, Betty, B, Vennzoni, M et l. (1998). Down-regultion of T1A12/m25, novel insulin-like growth ftor inding protein relted gene, is ssoited with disese progression in rest rinoms. Onogene 16: Suzuki, H, Igrshi, S, Nojim, M, Mruym, R, Ymmoto, E, Ki, M et l. (21). IGFBP7 is p53-responsive gene speifilly silened in oloretl ner with CpG islnd methyltor phenotype. Crinogenesis 31: Vizioli, MG, Sensi, M, Mirnd, C, Cleris, L, Formelli, F, Anni, MC et l. (21). IGFBP7: n onosuppressor gene in thyroid rinogenesis. Onogene 29: Yoo, BK, Emdd, L, Su, ZZ, Villnuev, A, Ching, DY, Mukhopdhyy, ND et l. (29). Astroyte elevted gene-1 regultes heptoellulr rinom development nd progression. J Clin Invest 119: Tomimru, Y, Eguhi, H, Wd, H, Koyshi, S, Mrushi, S, Tnemur, M et l. (212). IGFBP7 downregultion is ssoited with tumor progression nd linil outome in heptoellulr rinom. Int J Cner 13: Bergelson, JM, Cunninghm, JA, Droguett, G, Kurt-Jones, EA, Krithivs, A, Hong, JS et l. (1997). Isoltion of ommon reeptor for Coxskie B viruses nd denoviruses 2 nd 5. Siene 275: Küster, K, Koshel, A, Rohwer, N, Fisher, A, Wiedenmnn, B nd Anders, M (21). Downregultion of the oxskie nd denovirus reeptor in ner ells y hypoxi depends on HIF-1lph. Cner Gene Ther 17: Murkmi, M, Ugi, H, Wng, M, Belousov, N, Dent, P, Fisher, PB et l. (21). An denovirl vetor expressing humn denovirus 5 nd 3 fier proteins for trgeting heterogeneous ell popultions. Virology 47: Puisieux, A, Glvin, K, Trolen, F, Bress, B, Mris, C, Glun, E et l. (1993). Retinolstom nd p53 tumor suppressor genes in humn heptom ell lines. FASEB J 7: Srkr, D, Su, ZZ, Leedev, IV, Sune, M, Goplkrishnn, RV, Vlerie, K et l. (22). md-7 (IL-24) Medites seletive poptosis in humn melnom ells y induing the oordinted overexpression of the GADD fmily of genes y mens of p38 MAPK. Pro Ntl Ad Si USA 99: Roos, WP nd Kin, B (26). DNA dmge-indued ell deth y poptosis. Trends Mol Med 12: Hn, J nd Sun, P (27). The pthwys to tumor suppression vi route p38. Trends Biohem Si 32: Ingermnn, AR, Yng, YF, Hn, J, Mikmi, A, Grz, AE, Mohnrj, L et l. (21). Identifition of novel ell deth reeptor mediting IGFBP-3- indued nti-tumor effets in rest nd prostte ner. J Biol Chem 285: Bentr, T, Yng, W, Amemiy, Y, Evdokimov, V, Khn, H, Hollowy, C et l. (212). IGFBP7 redues rest tumor growth y indution of senesene nd poptosis pthwys. Brest Cner Res Tret 133: Tnnpfel, A, Sommerer, F, Benike, M, Ktlini, A, Uhlmnn, D, Witzigmnn, H et l. (23). Muttions of the BRAF gene in holngiorinom ut not in heptoellulr rinom. Gut 52: Muller, PA, Vousden, KH nd Normn, JC (211). p53 nd its mutnts in tumor ell migrtion nd invsion. J Cell Biol 192: Chen, Y, Cui, T, Knösel, T, Yng, L, Zöller, K nd Petersen, I (211). IGFBP7 is p53 trget gene intivted in humn lung ner y DNA hypermethyltion. Lung Cner 73: Wjpeyee, N, Kpoor, V, Mhlingm, M nd Green, MR (29). Effiy of IGFBP7 for tretment of metstti melnom nd other ners in mouse models nd humn ell lines. Mol Cner Ther 8: Smyth, MJ, Hykw, Y, Tked, K nd Ygit, H (22). New spets of nturl- killer-ell surveillne nd therpy of ner. Nt Rev Cner 2: Wu, Q, Moyn, T nd Xing, J (21). Cner gene therpy y denovirus-medited gene trnsfer. Curr Gene Ther 1: vol. 21 no. 4 pr. 213