Addition of DHA Synergistically Enhances the Efficacy of Regorafenib for Kidney Cancer Therapy

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1 Pulished nlinefirst Ferury 26, 216; DI: / MCT Smll Moleule Therpeutis Addition of Synergistilly Enhnes the Effiy of Regorfeni for Kidney Cner Therpy Jeffrey Kim 1, Arzu Ulu 1, Dein Wn 2, Jun Yng 2, Brue D Hmmok 2,3,nd Roert H. Weiss 1,3,4 Moleulr Cner Therpeutis Astrt Kidney ner is the sixth most ommon ner in the United Sttes, nd its inidene is inresing. The tretment of this mlignny took mjor step forwrd with the reent introdution of trgeted therpeutis, suh s kinse inhiitors. Unfortuntely, kinse inhiition is ssoited with the onset of resistne fter 1 to 2 yers of tretment. Regorfeni, like mny multikinse inhiitors, ws designed to lok the tivities of severl key kinse pthwys involved in onogenesis (Rs/Rf/ MEK/ERK) nd tumor ngiogenesis (VEGF-reeptors), nd we hve reently shown tht it lso possesses solule epoxide hydrolse (seh) inhiitory tivity, whih my e ontriuting to its slutry effets in ptients. Beuse seh inhiition results in inreses in the -derived epoxydoospentenoi ids tht we hve previously desried to possess ntiner properties, we sked whether the ddition of to therpeuti regimen in the presene of regorfeni would enhne its enefiil effets in vivo. We now show tht the omintion of regorfeni nd results in synergisti effet upon tumor invsiveness s well s p-vegfr ttenution. In ddition, this omintion showed redution in tumor weights, greter thn eh gent lone, in mouse xenogrft model of humn renl ell rinom (RCC), yielding the expeted oxylipin profiles; these dt were supported in severl RCC ell lines tht showed similr results in vitro. Beuse is the predominnt omponent of fish oil, our dt suggest tht this nontoxi dietry supplement ould e dministered with regorfeni during therpy for dvned RCC nd ould e the sis of linil tril. Mol Cner Ther; 15(5); Ó216 AACR. Introdution 1 Division of Nephrology, Deprtment of Internl Mediine, University of Cliforni, Dvis, Cliforni. 2 Deprtment of Entomology, University of Cliforni, Dvis, Cliforni. 3 Cner Center, University of Cliforni, Dvis, Cliforni. 4 Medil Servie, Srmento VA Medil Center, Srmento, Cliforni. Note: Supplementry dt for this rtile re ville t Moleulr Cner Therpeutis nline ( Corresponding Author: Roert H. Weiss, Division of Nephrology, Deprtment of Internl Mediine, Genome nd Biomedil Sienes Building, Room 6311, 451 Helth Sienes Drive, University of Cliforni, Dvis, CA Phone: ; Fx: ; E-mil: rhweiss@udvis.edu doi: / MCT Ó216 Amerin Assoition for Cner Reserh. Renlellrinom(RCC)risesfromtherenltuulr epithelium (1, 2), is the most ommon mlignny of the kidney, nd is the sixth most ommon ner in the United Sttes. In ontrst to mny other ners, the inidene of RCC is inresing likely due to smoking s well s the inresed prevlene of the metoli syndrome in the Western world (3 7). When lolized to the kidney, surgil resetion is usully urtive; however, one the ner metstsizes, the survivl sttistis, even with urrently ville novel therpies, re disml. Among nonsurgil tretments of RCC, the immune modultors were historilly ssoited with very low suess rte (8), likely relted to immune suppression in the tumor miroenvironment, possily through lol genertion of tryptophn metolites (9, 1). Enter the er of trgeted therpeutis, whih hs resulted in the disovery of drugs possessing ntingiogeni tivity vi rogtion of vsulr endothelil growth ftor (VEGF) nd other tyrosine kinse reeptor signling pthwys involved in tumor growth nd ngiogenesis (11 14). However, while these pprohes represented mjor dvne in the field, they re unfortuntely ssoited with high level of resistne fter 1 to 2 yers of tretment (15, 16), nd furthermore, some re linked with troulingly high rte of systemi hypertension (17). Therefore, novel pprohes re urgently needed to improve the effiy of these drugs. In the urrent study, we exmined the use of the tyrosine kinse inhiitors in omintion with ompounds tht we hypothesized would ttenute tumor resistne. Regorfeni is seond-genertion multikinse inhiitor tht loks the tivity of kinses involved in the regultion of onogenesis (Rs/Rf/MEK/ERK) nd tumor ngiogenesis (VEGF-R1, VEGF-R2, nd VEGF-R3; ref.13). This drug is mrked improvement over the first-genertion ompounds (e.g., sorfeni) due to its higher speifi tivityledingto greter phrmologi poteny (13). The ntitumor tivity of regorfeni hs een demonstrted in vriety of prelinil models nd is ssoited with its kinse inhiitory effets, whih results in suppression of ell prolifertion, indution of poptosis, nd inhiition of tumor ngiogenesis (13, 18, 19), the lst eing key re of investigtion for therpies of highly ngiogeni RCC (2). We hve reently shown tht these multikinse inhiitors lok solule epoxide hydrolse (seh; ref. 21), key enzyme tht metolizes iotive lipids of 89 Mol Cner Ther; 15(5) My 216 Downloded from mt.rjournls.org on Deemer 14, Amerin Assoition for Cner Reserh.

2 Pulished nlinefirst Ferury 26, 216; DI: / MCT Comintion of Regorfeni nd Inreses Effiy in RCC inflmmtion (22). Beuse inhiition of seh stilizes these lipids, therey prolonging their enefiil effets on ngiogenesis nd inflmmtion, we sked whether it is possile to pitlize on this enzymti tivity to enhne the slutry effets of these speifi kinse inhiitors in RCC. seh hydrolyzes epoxygented ftty ids generted y the P45 metolism of omeg-3 nd omeg-6 polyunsturted ftty ids (PUFA). Among these PUFAs, seh metolizes epoxyeiostrienoi ids (EET), whih re P45 produts of rhidoni id (ARA), nd epoxydoospentenoi id (EDPs), whih re lso P45 produts ut derive from dooshexenoi id (), to their less iotive diols (diols of EETs nd EDPs, dihydroxyeiostrienoi ids, DHETs) nd dihydroxydoospentenoi ids, DiHDPEs, respetively; Fig. 1; ref. 23). While EETs possess nti-inflmmtory (24) nd ntihypertensive (25) properties, they hve een shown to e prongiogeni (26 28), property tht n lerly e detrimentl in the tretment of highly ngiogeni tumors, suh s RCC. In ddition, reent studies hve suggested tht EETs n promote the progression of ner (29, 3), while other studies hve ontrdited these findings (31). In ontrst, EDPs, whih re lso stilized y seh inhiitors (Fig. 1), hve the opposite effet on ngiogenesis (32); hene, we fous on the metolites of seh in this study. We hypothesized tht the seh inhiitory tivity of regorfeni will result in mrked inreses in the nti-ngiogeni nd nti-hypertensive EDPs, whih will e enhned in the presene of exogenously dministered, usully the most undnt omponent of dietry fish oil supplements. We now show tht the omintion of nd regorfeni uses derese in HuVEC ell invsion s mesure of tumor ngiogenesis s well s synergistilly deresing ell viility ross three humn RCC lines. Furthermore, y using xenogrft model of RCC in thymi nude mie, we demonstrte derese in tumor mss in vivo ssoited with the expeted trget effets nd plsm oxylipin hnges. Thus, one vlidted in humn studies, novel therpy sed on the ddition of the dietry supplement to regorfeni hs the potentil to result in n enhned therpeuti effiy of this kinse inhiitor for tretment of dvned RCC. Mterils nd Methods Cell ulture Humn umilil vein endothelil ells (HuVEC; Lonz) were grown in endothelil sl medium (EBM-2) supplemented with growth ftors. The RCC ell lines 786-(VHL / ), Cki-1(VHL þ/þ ), nd Ren (VHL þ/þ ) were otined from the Amerin Type Culture Colletion nd the Renl proximl tuule epithelil ells (RPTEC or "norml humn kidney, NHK") were primry (i.e., non-immortlized) line quired from Lonz, whih were ultured in renl epithelil ell growth medium (REGM; Lonz). All ATCC nd Lonz ell lines undergo extensive uthentition tests during the essioning proess s desried on their We site; in ddition, ll ells were frequently tested for myoplsm in the uthor's lortory. The 786 nd Cki-1 nd Ren ells were mintined in RPMI, nd NHK ells were grown nd ultured in DMEM, oth supplemented with 1% FBS, 1 units/ml streptomyin, nd 1 mg/ml peniillin. Cells were mintined t 5% C2 nd t 37 C. All ell lines were used with pssge numer of two nd onfirmed to e free of myoplsm, per monthly lortory testing. Animls nd tretments All niml studies were pproved y the University of Cliforni Dvis Animl Use nd Cre Committee nd were performed in ordne with the NIH Guide for the Cre nd Use of Lortory Animls. Thirty-six 4-week-old mle thymi nude Nu/Nu mie (Hrln Lortories) were limted to housing onditions for 1 week nd were kept under 12-h light drk yle with free ess to wter nd food for the durtion of the experiment. Susequently, mie were injeted suspension ontining 786- ells t mixed in 3% of non-growth ftor redued Mtrigel (Corning In.) suutneously in the flnk region s previously desried (33). Tumor growth ws monitored twie week for eh mouse using digitl liper. Tumor volume (mm 3 ) ws lulted s length (width 2 /2). When tumor volume rehed pproximtely 1 mm 3 (round 3 4 weeks of inoultion), tretments nd diets egn. Mie were rndomly divided into two experimentl dietry groups: ontrol diet (5% orn oil) or 1% -enrihed diet (17.5 g nd 52.5 g orn oil/kg). ethyl ester repled orn oil to retin equl dietry ft etween oth isolori diets. The detiled omposition of the diets is desried in Supplementry Tle S1. Hlf of the mie in eh dietry group were given dily dministrtion of either 1 mg/kg regorfeni or vehile (PEG4/125 mmol/l queous methnesulfoni id; 8/2) vi orl gvge. Tretments ontinued for 3 weeks. Body weights nd tumor sizes were mesured every 2 dys. At the end of the experiment, plsm nd tissues were hrvested for immunohistohemistry nd oxylipin nlysis. Endothelil ell invsion ssy HuVECs were grown in 24-well pltes ontining Trnswell inserts of 8-mm pore polyvinylpyrrolidone-free polyronte filters oted with Mtrigel on the upper omprtment t density of 11 5 ells in EBM-2 medi ontining.1% BSA (34). EBM-2 medi onsisting of 1% BSA were dded in the ottom omprtment of the well s hemottrtnt. Both upper nd lower hmers ontined one of the following tretments: 1 mmol/l ARA, 1 mmol/l, 1 mol/l plus 1 mmol/l regorfeni, 1 mmol/l regorfeni, 1 mmol/l linolei id (LA) or DMS. Cells were inuted t 37 C for 2 hours to llow for migrtion. Afterwrd, Trnswells ontining ells were wshed in PBS, fixed in 5% glutrldehyde, nd stined with.5% Toluidine Blue. Next, the upper wells were gently srped to llow for imging nd quntifition of ells tht hd migrted towrd the lower omprtment of the Trnswell inserts. MTT ssy Cell viility ws ssyed y plting ells in 96-well pltes t densityof3 1 3 ells. After 24 hours, NHK, 786-, Cki-1, nd Ren ells were treted with 1 mmol/l of the ftty ids LA, ARA, eiospentenoi id (EPA), nd eh with the presene or sene of 1 mmol/l regorfeni nd DMS ontrol. After 24 hours of tretment, ells were quntified vi hemoytometer nd treted with medi ontining MTT solution (1 mg/ml thizolyl lue tetrzolium romide) for 3 hours. Afterwrd, the MTT solution ws removed, nd the lue Mol Cner Ther; 15(5) My Downloded from mt.rjournls.org on Deemer 14, Amerin Assoition for Cner Reserh.

3 Pulished nlinefirst Ferury 26, 216; DI: / MCT Kim et l. H 3 C H H 3 C H H 3 C w-6 PUFA Arhidoni id (ARA) C 2 H 32 2 H w-3 PUFA Dooshexenoi id () C 22 H 32 2 w-3 PUFA Eiospentenoi id (EPA) C 2 H 3 2 CYP 45 H 14,15-EET H 19,2-EDP 17, 18-EpETE H seh Inhiitors Solule epoxide hydrolse (seh) 14, 15-DHET H 19, 2-DiHDPE H 17, 18-DiHETE H H H H H H H Figure 1. EET nd EDP synthesis ours through ytohrome P45 fmily enzymes nturlly vi oxidtion of unsturted onds of preursor ftty ids. The EET nd EDP epoxygented produts re degrded y seh into their respetive diols. Inhiition of seh y regorfeni or other mens results in umultion of epoxygense metolites. rystlline preipitte internlized y the ells were dissolved with DMS. Finlly, pltes were pled in plte reder to mesure visile sorne t 57 nm. Immunolotting HuVECs were grown t density of ells in six-well pltes. After serum strvtion for 6 hours in EBM-2 medi ontining.1% ovine serum lumin (BSA), ells were treted with 1 mmol/l omeg-6 LA, 1 mmol/l LA þ regorfeni, 1 mmol/l, or 1 mmol/l þ 1 mmol/l regorfeni for 24 hours. Cells were then lysed, nd totl ell lystes were nlyzed for proteins of interest using ntiodies ginst phosphorylted VEGFR-2 nd -tin (Cell Signling Tehnology). Immunolotting of tumor tissue ws performed s previously desried (35). Briefly, fter the indited tretments, the tissues were wshed with PBS, lysed, nd sujeted to immunolotting. For the xenogrft tissue tumors, proteins were extrted with T-PER. The memrnes were loked in 5% nonft dry milk for 1 hour t room temperture, inuted with ntiodies (-tin, pvegfr-2, VEGFR-2, perk1/2, nd ERK1/2), nd then proed with HRP-tgged nti-mouse or ntirit IgG ntiodies. The signl ws deteted using ECL solutions (Thermo Fisher Sientifi). Densitometry ws performed using ImgeJ softwre. xylipin nlysis The quntittive profiling of oxylipin ws rried out s previously desried (36). Briefly, plsm smples were extrted using solid-phse extrtion rtridges. Smples were eluted through the rtridges, dried, nd then reonstituted y dding 2 nmol/l 1-ylohexyl-dodenoi id ure (CUDA) methnol solution. xylipins were then deteted using high-performne liquid hromtogrphy eletrospry ioniztion tndem mss spetrometry (HPLC-ESI-MS/MS). The optimized onditions of hromtogrphi seprtion hve een reported previously (37) s hve the instrument prmeters, inluding MRM trnsitions (ref. 36; Applied Biosystems, 4 QTRAP tndem mss spetrometer). Sttistil nlysis nd synergy lultions All dt were nlyzed for signifine in SAS version 9.3 (SAS Institute In.). Cell numers from invsion ssy, tumor weights, oxylipin quntifition, nd tumor volumes were nlyzed for signifine y one-wy ANVA t P <.5. Where signifint 892 Mol Cner Ther; 15(5) My 216 Moleulr Cner Therpeutis Downloded from mt.rjournls.org on Deemer 14, Amerin Assoition for Cner Reserh.

4 Pulished nlinefirst Ferury 26, 216; DI: / MCT Comintion of Regorfeni nd Inreses Effiy in RCC differenes were found, Tukey multiple omprison test ws performed t proility of ¼.5. The dt re presented s mens SEM. Different letters ppering ove rs in r grphs designte tht signifint differenes were found, while rs shring the sme letter indite tht signifine ws not hieved. Brs hving two letters (suh s '') indite tht signifine ws not hieved ompred with group '' or group ''. Synergy ws ssessed y lulting the omintion index (CI) vlues using CluSyn softwre, whih provides quntittive definition for dditive effet (CI ¼ 1), synergism (CI < 1), nd ntgonism (CI > 1) in omintion tretments. Results nd Disussion Codministrtion of regorfeni nd suppresses vsulr endothelil ell invsion nd is ssoited with ttenuted ngiogenesis mrkers Beuse the ddition of in the presene of seh inhiition provided y regorfeni would e expeted to inrese A lol EDP levels (Fig. 1) nd therey ttenute ngiogenesis (32), we first evluted this property in n in vitro model of ngiogenesis (38, 39). Beuse we previously reported n inrese in HuVEC prolifertion nd infiltrtion when treted with EETs, speifilly 11,12-EET nd 14,15-EET, whih re generted from ARA (32), we utilized the omeg-6 PUFA LA, the predominnt PUFA found in orn oil, s n dditionl ontrol. HuVEC were grown on mtrigel in Trnswell pltes, in whih ells tht infiltrted the mtrigel were enumerted in order to ssy for invsive potentil (see Mterils nd Methods). After tretment with 1 mmol/l ARA, 1 mmol/l, 1 mmol/l þ 1 mmol/l regorfeni, 1 mmol/l regorfeni, 1 mmol/l LA or DMS for 2 hours, invding HuVEC were imged (Fig. 2A) nd quntitted (Fig. 2B). The ells treted onurrently with nd regorfeni were found to e the lest invsive of ll onditions tested, with redution of 6% ompred with DMS ontrol. This omintion likely resulted in higher mount of EDPs, whih omes out with high vilility of in onert with the inhiition of seh DMS LA (1 µmol/l) (1 µmol/l) ARA (1 µmol/l) Regorfeni (1 µmol/l) Regorfeni (1 µmol/l) + (1 µmol/l) LA (Control) C LA +Regorfeni +Regorfeni pvegfr-2 B Atin VEGFR-2 Numer of ells 14, 12, 1, 8, 6, 4, 2, DMS (ontrol) LA ARA Regorfeni Regorfeni+ d pvegfr-2 reltive expression LA (ontrol) LA + Regorfeni + Regorfeni Figure 2. Endothelil ell invsive potentil ws ssyed using n in vitro Mtrigel model with HuVECs. A, HuVECs were grown on Mtrigel nd treted with 1 mmol/l LA, 1 mmol/l ARA, 1 mmol/l, 1 mmol/l þ 1 mmol/l regorfeni, 1 mmol/l regorfeni, or DMS for 2 hours. The ells trnsiting the Mtrigel were photogrphed (see Mterils nd Methods). B, quntifition of ells ws performed y ounting the ells in wells (n ¼ 3 for eh tretment, repeted in triplites). C, in seprte prllel experiment, HuVEC ells were grown to onfluene nd treted with the indited ompounds for 24 hours nd immunolotted for pvegfr-2 nd -tin s loding ontrol. VEGFR-2 ws lso immunolotted under the sme onditions in seprte lot. These experiments were eh repeted t lest three times. Brs in grphs indite men SEM. Different letters ove rs indite sttistil differene mong groups (P <.5). ARA, rhidoni id. Mol Cner Ther; 15(5) My Downloded from mt.rjournls.org on Deemer 14, Amerin Assoition for Cner Reserh.

5 Pulished nlinefirst Ferury 26, 216; DI: / MCT Kim et l. fforded y regorfeni. To onfirm trget inhiition y regorfeni, we evluted its kinse tivity on the phosphorylted (kinse-tive) form of VEGFR-2 (4) nd showed tht pvegfr-2 expression ws lower in ells treted with regorfeni fter oth LA nd tretments lone, ut more pronouned with the omintion (Fig. 2C), therey onfirming trget enggement with regorfeni. Regorfeni lone demonstrted nonsignifint derese in VEGFR-2 expression (dt not shown). These results re onsistent with previous dt showing tht EDPs inhiit VEGF-indued ell migrtion in HuVEC fter eing treted with 19,2-EDP (32). Further, these findings demonstrte synergisti effet of nd regorfeni on endothelil ells to suppress ngiogenesis, primrily vi suppression of endothelil ell migrtion likely vi high levels of EDP. The omintion of regorfeni nd synergistilly dereses survivl of kidney ner ells in vitro We next ssessed ell viility in vivo utilizing two humn kidney ner lines (786- nd Cki-1) nd the mouse kidney ner ell line Ren, s well s primry (non-immortlized) norml humn kidney epithelil (NHK) ells s ontrols. All ells were treted with 1 mmol/l of the ftty ids LA, ARA, EPA, nd eh, in the presene or sene of 1 mmol/l regorfeni nd DMS ontrol. LA, whih is the mjor polyunsturted ftty id omprising orn oil, served s the in vitro ontrol tretment tht would est mimi the onditions of orn oil dministrtion in vivo suh tht the two experiments ould e ompred. EPA ws used to disern if the mitigtion of ell viility ws due to n omeg-3 effet or speifilly to. After 24 hours of tretment, oth regorfeni þ nd lone deresed ell viility in ll three of the ner lines with no signifint effet on NHK ells; however, greter derese in ell viility ws found with the former tretment (Fig. 3). Furthermore, ells were quntified from n experiment performed in prllel to the MTT ssy on the four ell types (inset, Fig. 3); these dt demonstrte tht the omintion of regorfeni with resulted in synergisti responses fter 24 hours of inution. The therpeuti effiy ws ssessed y lulting CI vlues using CluSyn softwre (41). Anlysis of omintion therpeuti indexes reveled synergisti effets y demonstrting the CI vlues in the rnge of.61 to.85 (synergy defined s CI <1) with the omintion of regorfeni nd lone mong the three RCC lines. Antgonisti intertions (CI>1) were found with LA nd ARA with CI lultions 1.14 nd 1.23, respetively. These findings demonstrte tht the omintion of regorfeni nd produed synergisti derese in severl RCC, ut not in norml renl epithelil ell, viility. 12% 1% Viility (%) 8% 6% 4% NHK 786- Cki-1 Ren 2% % DMS LA ARA EPA Regor Regor + LA Regor + ARA Regor + EPA Regor + + Regorfeni # of ells CI NHK 6, ,82.61 Cki-1 5,14.68 Ren 5,59.85 Figure 3. Cell viility ws ssyed vi MTT in NHK, 786-, Cki-1, nd Ren ells. NHK, 786-, Cki-1, nd Ren ells were treted with 1 mmol/l of the ftty ids LA, ARA, EPA, nd eh in the presene or sene of 1 mmol/l regorfeni nd DMS ontrol. After 24 hours of tretment, n MTT ssy ws performed, nd ells were ounted vi hemoytometer. The nd regorfeni CI ws lulted using CluSyn softwre s disussed in Mterils nd Methods. These experiments were eh repeted t lest three times. Brs in the grph indite men SEM., sttistil differene ompred with DMS tretment of the identil ell line (P <.5). 894 Mol Cner Ther; 15(5) My 216 Moleulr Cner Therpeutis Downloded from mt.rjournls.org on Deemer 14, Amerin Assoition for Cner Reserh.

6 Pulished nlinefirst Ferury 26, 216; DI: / MCT Comintion of Regorfeni nd Inreses Effiy in RCC A Corn Corn + Regor + Regor B Body weight (g) Dys 23 C pvegfr-2 perk1/2 ERK1/2 Atin Corn Control Corn +Regorfeni +Regorfeni 3 Tumor weights (mg) Corn Corn + Regor + Regor Reltive expression Corn (Control) Corn + Regor + Regor pvegfr-2 perk1/2 ERK1/2 Figure 4. The omintion of regorfeni nd redues tumor weight with the expeted on-trget effets. Mle thymi Nu/Nu mie (n ¼ 8 per group) were trnsplnted with 786- ells nd susequently dministered either orn oil or -enrihed diet (see Mterils nd Methods). Where indited, mie were given 1 mg/kg of regorfeni or vehile dministered y orl gvge dily for 18 dys. A, ody weights were mesured t the indited times. B, tumor weights were determined. C, terminl tumors were immunolotted with pvegfr-2, perk1/2, or ERK1/2, nd -tin in ws used s loding ontrol. Lines nd rs in grphs indite men SEM. Different letters ove rs indite sttistil differene mong groups (P <.5). The omintion of regorfeni nd dereses tumor growth in vivo In light of previous dt from one of our lortories demonstrting tht tretment with EDP onurrently with seh inhiition ttenuted oth tumor growth nd ngiogenesis (32), we next sked whether the onurrent ddition of regorfeni nd synergizes in n in vivo xenogrft model of humn RCC using the 786- (VHL / ) humn RCC ell line used in severl previous studies (4, 42, 43). Mle thymi Nu/Nu mie were strted on the diets nd phrmologi tretments fter the 786- xenogrfts hieved volume of 1 mm 3. The mie were given free ess to either diet with ft originting from orn oil, whih is nturlly high in the omeg-6 PUFA LA, or 1% enrihed diet. The onentrtion in the diet ws determined y metoli ody size using n verge dily food intke of 5 g/ dy/mouse, whih trnsltes to 3.1 g/dy of in 7-kg humn. This mount is hievle through onsuming fish oil supplementtion nd in ft hs een reommended to derese progression in IgA nephropthy, ommon renl disese (44). Mie were given either regorfeni (1 mg/kg/dy) or vehile ontrol dministered y orl gvge. Tumors nd terminl plsm were olleted fter 18 dys of intervention for immunolot nd oxylipin nlysis, respetively. There ws no signifint differene etween tretment groups in ody weights fter 18 dys, inditing lk of generl toxiity (Fig. 4A); tumor weights (Fig. 4B) nd volume (Supplementry Fig. S1) were found to e the smllest in the mie treted with regorfeni while ingesting the diet (1.9-fold derese) nd there ws synergisti derese of the omintion s ompred with or regorfeni dministered lone. To evlute the trget effets of regorfeni in the xenogrfted nimls, we evluted the MAPK nd VEGFR pthwys, whih re known reeptor tyrosine kinse trgets (13). Immunolotting of the tumors for pvegfr-2 demonstrted the most drmti redution in the tumors from the þregorfenitreted mie with miniml effets upon these proteins in the other nimls (Fig. 4C), inditing tht regorfeni ttenutes the tive forms of oth MAPK nd VEGFR speies, onsistent with the HuVEC dt (see Fig. 2C). Beuse we hve previously shown n seh inhiitory effet of regorfeni similr to sorfeni (45), the influene of regorfeni nd in the in vivo model is likely speifi to this omintion. Mol Cner Ther; 15(5) My Downloded from mt.rjournls.org on Deemer 14, Amerin Assoition for Cner Reserh.

7 Pulished nlinefirst Ferury 26, 216; DI: / MCT Kim et l. A Plsm [EDP] (nmol/l) d Corn Corn + Regor + Regor 19(2)-EpDPE 16(17)-EpDPE 13(14)-EpDPE 1(11)-EpDPE Plsm [EDP] (nmol/l) B Plsm [DiHDPE] (nmol/l) 2,5 2, 1,5 1, ,2-DiHDPE Sum plsm [DiHDPEs] (nmol/l) (8)-EpDPE 16,17-DiHDPE Sum plsm [EDPs] (nmol/l) Corn Corn + Regor + Regor d Corn oil Corn oil+ Regor Corn Corn + Regor + Regor 13,14-DiHDPE 1,11-DiHDPE +Regor C Epoxide to diol rtio (EDP:DiHDPE) Sum epoxide to diol rtio 12 19(2) Corn oil Corn oil + Regor + Regor 16(17) 13(14) 1(11) Corn oil Corn oil + Regor + Regor d d Figure 5. Plsm oxylipin nlysis shows inresed levels of EDPs in mie fed with the diet ompred with the other mie. xylipins were mesured y HPLC-ESI- MS/MS on terminl plsm. A, EDPs. B, -derived diols. C, the epoxide-to-diol rtio s mesure of seh inhiition. Brs in grphs indite men SEM. Different letters ove rs indite sttistil differene mong groups (P <.5). The diet resulted in n inrese in ll CYP45 metolites of in murine plsm While the irulting plsm oxylipin profile n suggest the mehnism of the oservtions, these dt do not lwys orrelte with wht is ourring t the lol (i.e., tissue) level (46). The EDP speies re rpidly metolized to their diol onstituents due to the tions of seh; however, the inhiitory tions on this toli enzyme from n seh-inhiitor, s we hve shown for sorfeni (21, 47), were evident in the plsm (32). Terminl plsm oxylipin nlysis showed the expeted higher levels of 7(8)-EDP, 1(11)-EDP, 13(14)-EDP, 16(17)-EDP, nd 19(2)- EDP in mie treted with the diet ompred with the orn oil diet groups (Fig. 5A). An inrese in the orresponding diols ws lso oserved s 1(11)-DiHDPE, 13(14)-DiHDPE, 16(17)- DiHDPE, nd 19(2)-DiHDPE in the -fed mie (Fig. 5B). The prodution of these diols ws ntiipted due to the enrihed dietry. To ssess in vivo seh inhiition, we exmined the rtio of epoxide to their orresponding diol produts in the plsm. The sum epoxide-to-diol rtio ws found to e 2.2-fold in the orn oil diet þ regorfeni tretment group ompred with the orn oil diet lone, with gretest differene eing 3.6-fold inrese found in the 16(17)-EDP-to-16(17)-DiHDPE (Fig. 5C). Surprisingly, the epoxide-to-diol rtio in the plsm of the -fed mie did not reflet seh inhiition s the onentrtions were found to e out the sme for ll of the mesured speies, nd even lower in the 1(11)-EDP-to-1(11)-DiHDPE nd 13(14)-EDP-to-13(14)-DiHDPE. This ws lso oserved in n erlier experiment performed with sorfeni rther thn regorfeni tretments (dt not shown). Reently, it hs een identified tht the omeg-3 derived EDPs re turned over more rpidly thn the orresponding omeg-6 derived EETs s seh hs preferene for these -derived epoxygented metolites (47). Thus, it is oneivle tht due to this preferene in sustrte nd the undne of EDP in the lood, seh enzyme levels my e upregulted nd higher in the -diet fed mie, resulting in greter mount of epoxide turnover to diols, leding to derese in the epoxide-to-diol rtio in the plsm, lthough this my not e representtive of tissue. Future investigtions my eluidte this oservtion y mesuring 896 Mol Cner Ther; 15(5) My 216 Moleulr Cner Therpeutis Downloded from mt.rjournls.org on Deemer 14, Amerin Assoition for Cner Reserh.

8 Pulished nlinefirst Ferury 26, 216; DI: / MCT Comintion of Regorfeni nd Inreses Effiy in RCC Dietry Figure 6. Proposed mehnism of omining regorfeni tretment with dietry to inhiit RCC growth. seh inhiition y regorfeni redues degrdtion of -derived epoxides nd leds to inresed EDP iovilility resulting in ttenution of ngiogenesis nd likely n inrese in vsodiltion (whih ws not mesured in this study). The kinse inhiitor tivity of regorfeni impedes VEGF nd MAPK/Rf signling, whih likely further dereses ngiogenesis nd onomitnt RCC tumor growth. Cirulting Vsodiltion EDPs Regorfeni Angiogenesis H seh H DiHDPE H H VEGFR-2 MAPK/Rf Signling irulting EET nd EDP onentrtions or mesuring oxylipins in other tissues. Conlusion We hve shown tht omintion tretment of with regorfeni results in synergisti effiy over regorfeni or lone in inhiiting growth in n in vivo xenogrft model of VHL-mut RCC. We further show tht there is derese in mrkers of ngiogenesis nd tht this growth inhiition is ompnied y the expeted trget effets. We provide evidene tht tumor growth ttenution likely ours s result of inresing levels of EDPs due to the seh inhiitory property of regorfeni (Fig. 6). Until linil tril is omplished, ptient use of the ommon nd redily ville dietry supplement, fish oil, n therefore e reommended in individuls undergoing regorfeni tretment for dvned RCC. Dislosure of Potentil Conflits of Interest No potentil onflits of interest were dislosed. Authors' Contriutions Coneption nd design: J. Kim, A. Ulu, B.D. Hmmok, R.H. Weiss Development of methodology: J. Kim, A. Ulu, B.D. Hmmok Aquisition of dt (provided nimls, quired nd mnged ptients, provided filities, et.): J. Kim, A. Ulu, J. Yng Anlysis nd interprettion of dt (e.g., sttistil nlysis, iosttistis, omputtionl nlysis): J. Kim, A. Ulu, D. Wn, J. Yng, R.H. Weiss Writing, review, nd/or revision of the mnusript: J. Kim, A. Ulu, D. Wn, J. Yng, B.D. Hmmok, R.H. Weiss Administrtive, tehnil, or mteril support (i.e., reporting or orgnizing dt, onstruting dtses): J. Kim, D. Wn, B.D. Hmmok, R.H. Weiss Study supervision: J. Kim, R.H. Weiss ther (worked with Dr. Weiss to develop originl hypothesis sed on previously pulished ollortive work): B.D. Hmmok Grnt Support This work ws supported y NIH grnts 1R1CA A1, 1R3CA , nd 1R1DK8269-1A1, the Medil Servie of the US Deprtment of Veterns' Affirs, nd Dilysis Clinis, In. (DCI; ll to R.H. Weiss). Prtil support ws provided y NIEHS R1 ES271 nd NIEHS Superfund Progrm P42 ES4699 (to B.D. Hmmok.) The osts of pulition of this rtile were defryed in prt y the pyment of pge hrges. This rtile must therefore e herey mrked dvertisement in ordne with 18 U.S.C. Setion 1734 solely to indite this ft. Reeived toer 19, 215; revised Ferury 11, 216; epted Ferury 22, 216; pulished nlinefirst Ferury 26, 216. Referenes 1. Esudier B, Ktj V, Group EGW. Renl ell rinom: ESM Clinil Prtie Guidelines for dignosis, tretment nd follow-up. Ann nol 21;21:v Cohen HT, MGovern FJ. Renl-ell rinom. N Engl J Med 25;353: Luo J, Mrgolis KL, Admi H, Lopez AM, Lessin L, Ye W, et l. Body size, weight yling, nd risk of renl ell rinom mong postmenopusl women: the Women's Helth Inititive (United Sttes). Am J Epidemiol 27;166: Hggstrom C, Rpp K, Stoks T, Mnjer J, Bjorge T, Ulmer H, et l. Metoli ftors ssoited with risk of renl ell rinom. PloS ne 213;8: e Chow WH, Dong LM, Deves SS. Epidemiology nd risk ftors for kidney ner. Nt Rev Urol 21;7: Hunt JD, vn der Hel L, MMilln GP, Boffett P, Brennn P. Renl ell rinom in reltion to igrette smoking: met-nlysis of 24 studies. Int J Cner 25;114: Weiss RH, Lin PY. Kidney ner: identifitionof novel trgets for therpy. Kidney Int 26;69: Belldegrun AS, Kltte T, Shuh B, LRohelle JC, Miller DC, Sid JW, et l. Cner-speifi survivl outomes mong ptients treted during the ytokine er of kidney ner ( ): enhmrk for emerging trgeted ner therpies. Cner 28;113: Wettersten HI, Hkimi AA, Morin D, Binhi C, Johnstone ME, Donohoe DR, et l. Grde-dependent metoli reprogrmming in kidney ner reveled y omined proteomis nd metolomis nlysis. Cner Res 215;75: Mol Cner Ther; 15(5) My Downloded from mt.rjournls.org on Deemer 14, Amerin Assoition for Cner Reserh.

9 Pulished nlinefirst Ferury 26, 216; DI: / MCT Kim et l. 1. Gnti S, Tylor SL, Au Aoud, Yng J, Evns C, sier MV, et l. Kidney tumor iomrkers reveled y simultneous multiple mtrix metolomis nlysis. Cner Res 212;72: Molin AM, Motzer RJ. Clinil prtie guidelines for the tretment of metstti renl ell rinom: tody nd tomorrow. nologist 211; 16: Hutson TE, Dvis ID, Mhiels JP, De Souz PL, Rottey S, Hong BF, et l. Effiy nd sfety of pzopni in ptients with metstti renl ell rinom. J Clin nol 21;28: Wilhelm SM, Dums J, Adnne L, Lynh M, Crter CA, Shutz G, et l. Regorfeni (BAY ): new orl multikinse inhiitor of ngiogeni, stroml nd onogeni reeptor tyrosine kinses with potent prelinil ntitumor tivity. Int J Cner 211;129: Chng YS, Adnne J, Tril PA, Levy J, Henderson A, Xue D, et l. Sorfeni (BAY 43 96) inhiits tumor growth nd vsulriztion nd indues tumor poptosis nd hypoxi in RCC xenogrft models. Cner Chemother Phrmol 27;59: Motzer RJ, Mihelson MD, Rosenerg J, Bukowski RM, Curti BD, George DJ, et l. Sunitini effiy ginst dvned renl ell rinom. J Urol 27;178: Sheprd DR, Gri JA. Toxiity ssoited with the long-term use of trgeted therpies in ptients with dvned renl ell rinom. Expert Rev Antiner Ther 29;9: Wu S, Chen JJ, Kudelk A, Lu J, Zhu X. Inidene nd risk of hypertension with sorfeni in ptients with ner: systemti review nd metnlysis. Lnet nol 28;9: Mross K, Frost A, Steinild S, Hedom S, Buhert M, Fsol U, et l. A phse I dose-esltion study of regorfeni (BAY ), n inhiitor of onogeni, ngiogeni, nd stroml kinses, in ptients with dvned solid tumors. Clin Cner Res 212;18: Eisen T, Joensuu H, Nthn PD, Hrper PG, Wojtukiewiz MZ, Niholson S, et l. Regorfeni for ptients with previously untreted metstti or unresetle renl-ell rinom: single-group phse 2 tril. Lnet nol 212;13: Folkmn J.Tumor ngiogenesis: therpeuti implitions. N Engl J Med 1971;285: Liu JY, Prk SH, Morisseu C, Hwng SH, Hmmok BD, Weiss RH. Sorfeni hs solule epoxide hydrolse inhiitory tivity, whih ontriutes to its effet profile in vivo. Mol Cner Ther 29;8: Morisseu C, Hmmok BD. Impt of solule epoxide hydrolse nd epoxyeiosnoids on humn helth. Annu Rev Phrmol Toxiol 213; 53: Spetor AA, Fng X, Snyder GD, Weintru NL. Epoxyeiostrienoi ids (EETs): metolism nd iohemil funtion. Prog Lipid Res 24;43: Node K, Huo Y, Run X, Yng B, Spieker M, Ley K, et l. Anti-inflmmtory properties of ytohrome P45 epoxygense-derived eiosnoids. Siene 1999;285: Yu Z, Xu F, Huse LM, Morisseu C, Drper AJ, Newmn JW, et l. Solule epoxide hydrolse regultes hydrolysis of vsotive epoxyeiostrienoi ids. Cir Res 2;87: Weler AC, Mihelis UR, Popp R, Bros-Sird E, Murugn A, Flk JR, et l. Epoxyeiostrienoi ids re prt of the VEGF-tivted signling sde leding to ngiogenesis. Am J Physiol Cell Physiol 28;295: C Pozzi A, Mis-Perez I, Air T, Wei S, Su Y, Zent R, et l. Chrteriztion of 5,6- nd 8,9-epoxyeiostrienoi ids (5,6- nd 8,9-EET) s potent in vivo ngiogeni lipids. J Biol Chem 25;28: Wng Y, Wei X, Xio X, Hui R, Crd JW, Crey MA, et l. Arhidoni id epoxygense metolites stimulte endothelil ell growth nd ngiogenesis vi mitogen-tivted protein kinse nd phosphtidylinositol 3-kinse/Akt signling pthwys. J Phrmol Exp Ther 25; 314: Cheng LM, Jing JG, Sun ZY, Chen C, Dkor RT, Zeldin DC, et l. The epoxyeiostrienoi id-stimulted phosphoryltion of EGF-R involves the tivtion of metlloproteinses nd the relese of HB-EGF in ner ells. At Phrmol Sin 21;31: Pnigrhy D, Edin ML, Lee CR, Hung S, Bielenerg DR, Butterfield CE, et l. Epoxyeiosnoids stimulte multiorgn metstsis nd tumor dormny espe in mie. J Clin Invest 212;122: Wng D, Duois RN. Epoxyeiostrienoi ids: doule-edged sword in rdiovsulr diseses nd ner. J Clin Invest 212;122: Zhng G, Pnigrhy D, Mhkin LM, Yng J, Liu JY, Stephen Lee KS, et l. Epoxy metolites of dooshexenoi id () inhiit ngiogenesis, tumor growth, nd metstsis. Pro Ntl Ad Si U S A 213; 11: Inoue H, Kuffmn M, Shhm S, Lndesmn Y, Yng J, Evns CP, et l. CRM1 lokde y seletive inhiitors of nuler export ttenutes kidney ner growth. J Urol 213;189: Nithiptikom K, Endsley MP, Isell MA, Flk JR, Iwmoto Y, Hillrd CJ, et l. 2-rhidonoylglyerol: novel inhiitor of ndrogen-independent prostte ner ell invsion. Cner Res 24;64: Kim J, Crlson ME, Kuhel GA, Newmn JW, Wtkins BA. Dietry reduesdownstrem endonninoid nd inflmmtory gene expression nd epididyml ft mss while improving spets of gluose use in musle in C57BL/6J mie. Int J es 216;4: Yng J, Shmelzer K, Georgi K, Hmmok BD. Quntittive profiling method for oxylipin metolome y liquid hromtogrphy eletrospry ioniztion tndem mss spetrometry. Anl Chem 29;81: Nording ML, Yng J, Georgi K, Hegedus Krowski C, Germn JB, Weiss RH, et l. Individul vrition in lipidomi profiles of helthy sujets in response to omeg-3 Ftty ids. PloS ne 213;8:e Koh AE, Polverini PJ, Kunkel SL, Hrlow LA, DiPietro LA, Elner VM, et l. Interleukin-8 s mrophge-derived meditor of ngiogenesis. Siene 1992;258: Skovseth DK, Kuhler AM, Hrldsen G. The HUVEC/Mtrigel ssy: n in vivo ssy of humn ngiogenesis suitle for drug vlidtion. Methods Mol Biol 27;36: Guo D, Ji Q, Song HY, Wrren RS, Donner DB. Vsulr endothelil ell growth ftor promotes tyrosine phosphoryltion of meditors of signl trnsdution tht ontin SH2 domins. Assoition with endothelil ell prolifertion. J Biol Chem 1995;27: Chou TC.Drug omintion studies nd their synergy quntifition using the Chou-Tlly method. Cner Res 21;7: Ivnov SV, Kuzmin I, Wei MH, Pk S, Geil L, Johnson BE, et l. Downregultion of trnsmemrne roni nhydrses in renl ell rinom ell lines y wild-type von Hippel-Lindu trnsgenes. Pro Ntl Ad Si U S A 1998;95: Kurn G, Hudon V, Dupln E, hh M, Puse A. Chrteriztion of von Hippel Lindu pthwy involved in extrellulr mtrix remodeling, ell invsion, nd ngiogenesis. Cner Res 26;66: Dondio JV, Jr., Bergstrlh EJ, fford KP, Spener DC, Holley KE. A ontrolled tril of fish oil in IgA nephropthy. Myo Nephrology Collortive Group. N Engl J Med 1994;331: Hwng SH, Weksler AT, Zhng G, Morisseu C, Nguyen LV, Fu SH, et l. Synthesis nd iologil evlution of sorfeni- nd regorfeni-like seh inhiitors. Bioorg Med Chem Lett 213;23: She NH, stermnn AI, Yng J, Hmmok BD, Hhn A, Shuhrdt JP. Comprison of the effets of long-hin omeg-3 ftty id supplementtion on plsm levels of free nd esterified oxylipins. Prostglndins ther Lipid Medit 214; : Morisseu C, Ineoglu B, Shmelzer K, Tsi HJ, Jinks SL, Hegedus CM, et l. Nturlly ourring monoepoxides of eiospentenoi id nd dooshexenoi id re iotive ntihyperlgesi lipids. J Lipid Res 21; 51: Mol Cner Ther; 15(5) My 216 Moleulr Cner Therpeutis Downloded from mt.rjournls.org on Deemer 14, Amerin Assoition for Cner Reserh.

10 Pulished nlinefirst Ferury 26, 216; DI: / MCT Addition of Synergistilly Enhnes the Effiy of Regorfeni for Kidney Cner Therpy Jeffrey Kim, Arzu Ulu, Dein Wn, et l. Mol Cner Ther 216;15: Pulished nlinefirst Ferury 26, 216. Updted version Supplementry Mteril Aess the most reent version of this rtile t: doi:1.1158/ mct Aess the most reent supplementl mteril t: Cited rtiles This rtile ites 47 rtiles, 18 of whih you n ess for free t: E-mil lerts Sign up to reeive free emil-lerts relted to this rtile or journl. Reprints nd Susriptions Permissions To order reprints of this rtile or to susrie to the journl, ontt the AACR Pulitions Deprtment t pus@r.org. To request permission to re-use ll or prt of this rtile, use this link Clik on "Request Permissions" whih will tke you to the Copyright Clerne Center's (CCC) Rightslink site. Downloded from mt.rjournls.org on Deemer 14, Amerin Assoition for Cner Reserh.