BRAF Inhibition Generates a Host Tumor Niche that Mediates Therapeutic Escape

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1 See relted ommentry on pg 9 ORIGINAL ARTICLE BRAF Inhiition Genertes Host Tumor Nihe tht edites Therpeuti Espe Inn V. Fedorenko 1, Jennifer A. Wrgo, Keith T. Flherty, Jne L. essin nd Keirn S.. Smlley 1, The urrent study defines firolst-derived nihe tht filittes the therpeuti espe of melnom ells from BRAF inhiition. rfeni tretment led to the relese of trnsforming growth ftor-β (TGF-β) from the melnom ells tht inresed the differentition stte of the firolsts, n ffet ssoited with fironetin deposition, inrese in α-smooth musle tin expression, nd the relese of neuregulin (NRG). At the sme time, vemurfeni diretly tivted the firolsts through prdoxil stimultion of the mitogen-tivted protein kinse pthwy, using them to serete heptoyte growth ftor (HGF). Tretment with the BRAF/EK inhiitor omintion reversed the relese of HGF. Adhesion of melnom ells to fironetin ws ritil in mplifying the firolst-derived NRG- nd HGF-medited PIK/AKT (phosphtidylinositol '-kinse/akt) survivl signling in the melnom ells following BRAF inhiition. In oulture studies, omintion tretment with inhiitors of BRAF/ET/HER kinse ws ineffetive t reversing the firolst-medited therpeuti espe from BRAF inhiition. Insted, it ws noted tht omined BRAF/PIK inhiition overme firolst-medited drug resistne in vitro nd ws ssoited with enhned ntitumor effets in n in vivo xenogrft model. Thus, we show tht melnom ells nd firolsts remodel their miroenvironment in response to BRAF inhiition nd tht these dpttions llow tumor ells to evde therpy through inresed PIK/AKT survivl signling. Journl of Investigtive Dermtology (1) 1, 11 1; doi:1.18/jid.1.9; pulished online 1 Septemer 1 IRODUCTION Our emerging understnding of therpeuti resistne suggests role for oth tumor utonomous mehnisms nd dptive prosurvivl signls from the host miroenvironment (Lito et l., 1; Strussmn et l., 1; Wilson et l., 1; Ael et l., 1). During progression, melnom ells lose ontt with their nturl inding prtners, the kertinoytes, nd insted intert with host endothelil ells nd firolsts (Hsu et l., ; Li et l., 1). Firolsts lso ontriute to the espe of melnom ells from vemurfeni therpy, in 1 The Deprtment of Tumor Biology, The offitt Cner Center nd Reserh Institute, Tmp, Florid, USA; Deprtment of Surgery, D Anderson Cner Center, Houston, Texs, USA; Deprtment of ediine, sshusetts Generl Hospitl, Boston, sshussetts, USA nd The Deprtment of Cutneous Onology, The offitt Cner Center nd Reserh Institute, Tmp, Florid, USA Correspondene: Keirn S.. Smlley, The Deprtment of Tumor Biology nd The Deprtment of Cutneous Onology, The offitt Cner Center nd Reserh Institute, 19 gnoli Drive, Tmp, Florid 61, USA. E-mil: keirn.smlley@moffitt.org Arevitions: C, onditioned medium; EC, extrellulr mtrix; EGF, epithelil growth ftor; ERK, extrellulr signl regulted kinse;, fironetin;, glyerldehyde -phosphte dehydrogense; GFP, green fluoresent protein; HGF, heptoyte growth ftor; APK, mitogen-tivted protein kinse; NRG, neuregulin; PARP, poly (ADP-riose) polymerse; PIK, phosphtidylinositol '-kinse; qrt-pcr, quntittive reltime reverse-trnsriptse PCR; RTK, reeptor tyrosine kinse; sirna, smll interfering RNA; TGF-β, trnsforming growth ftor-β;, vemurfeni; α-sa, α-mooth musle tin expression Reeived 6 rh 1; revised 1 July 1; epted 7 July 1; epted rtile preview online August 1; pulished online 1 Septemer 1 prt, through heptoyte growth ftor (HGF) signling (Strussmn et l., 1). In ddition, tumor-derived growth ftors, suh s epithelil growth ftor (EGF), neuregulin (NRG), nd IGF-I, ffet the responsiveness to BRAF inhiition nd my even drive BRAF inhiitor resistne (Villnuev et l., 1; Wilson et l., 1; Ael et l., 1). Other studies hve suggested tht dhesion to the extrellulr mtrix (EC) protets ner ells from poptosis following tretment with hemotherpeutis. This phenomenon, known s ell dhesion medited drug resistne, ws first desried for multiple myelom, with dhesion to fironetin (), deresing the sensitivity to melphln (Dmino et l., 1999; Hzlehurst et l., ; Hzlehurst nd Dlton, 1). The extent to whih melnom ells nd their miroenvironment intert to provide protetive sntury tht llows the ner ells to evde therpy is not well understood. In the present study, we unovered previously unhrterized EC-derived protetive nihe tht drives therpeuti espe through the mplifition of host-derived survivl signls. Unexpetedly, inhiition of BRAF lso led to prdoxil mitogen-tivted protein kinse (APK) signling medited differentition nd EC deposition in norml skin firolsts (BRAF wild-type), suggesting tht off-trget effets of kinse inhiitors remodel the host environment. We propose role for i-diretionl signling etween the tumor nd host in the dptive responses to therpy nd demonstrte tht host ells re n tive plyer in the espe proess. Our dt suggest tht future therpeuti strtegies will require the trgeting of oth the tumor nd host responses. 1 The Soiety for Investigtive Dermtology 11

2 IV Fedorenko et l. Overly α-sa Plsti FF C μ TGF-β1 % Of ontrol C 1 1 FF7 μ C+ NS μ μ α-sa intensity per ell (totl fluoresene/ 1,) intensity per ell (totl fluoresene/ 1,) FF7 TGF-β C C+ vemu * TGF-β C C+ vemu Figure 1. BRAF V6E melnom ells nd vemurfeni (vemu) indue firolst differentition. (, left) Green fluoresent protein (GFP) tgged 1Lu melnom ells were plted on either tissue ulture plsti or onfluent monolyers of unleled FF firolsts nd treted with vemurfeni ( μ, 7 hours). (Right) Quntifition of GFP+ melnom ells, N =. () FF7 firolsts were treted with either onditioned medium (C) from 1Lu ells, C from 1Lu ells treted with vemurfeni, μ for 8 hours (C+vemu), 1. pg ml 1 trnsforming growth ftor (TGF-β1), or vemu efore eing stined for fironetin (, yellow) nd α-smooth musle tin (α-sa, red). Br = 1 μm. () Firolst differentition ws mesured y the level of nd α-sa expression. nd α-sa expression ws nlyzed using Definiens Developer v.. softwre suite; totl fluoresene intensity per nulei ws quntified. *P., P.1, nd P.1. RESULTS Plting of green fluoresent protein (GFP) tgged melnom ells onto onfluent firolst monolyer onveyed ner-totl protetion to the growth inhiitory effets of vemurfeni tretment ( μ, 7 hours) (Figure 1). ehnistilly, it ws found tht tretment of the firolsts using trnsforming growth ftor (TGF)-β, onditioned medi (C+vemu) from melnom ells treted with vemurfeni ( μ, 8 hours), or vemurfeni lone ( μ, 8 hours) inresed their differentition, s shown y the inresed expression of nd α-smooth musle tin expression (α-sa) (Figure 1 nd ). Although vemurfeni lone indued firolst differentition, the extent of this ws less thn either C+vemu or TGF-β lone. The stimultory effets of the melnom C on the firolsts ws TGF-β-dependent, with the ddition of the TGF kinse inhiitor SB1 found to prtilly inhiit firolst tivtion (Supplementry Figure 1 online). The inresed expression of ws required for firolst survivl, with its smll interfering RNA (sirna) medited knokdown ssoited with inresed firolst ell deth under serum-free onditions (Supplementry Figure online). Previous work from our l hs demonstrted BRAF inhiitor tretment to indue n epithelil mesenhyml trnsition like stte in melnom ells, phenotype often driven through TGF-β signling (Fedorenko et l., 1). As exogenous TGF-β, vemurfeni, nd C from vemurfeni-treted melnom ells indued the differentition of firolsts, we next sked whether BRAF inhiition led to the relese of TGF-β from the melnom ells. Tretment with vemurfeni inresed the protein expression of TGF-β in three out of six BRAF-mutnt melnom ell lines (Figure ). Also noted ws n inrese in mrna levels, s well s the relese of TGF-β, s mesured y quntittive rel-time reverse-trnsriptse PCR (qrt-pcr) nd ELISA ssys (Figure nd ). The potentil linil relevne of these findings ws suggested y the nlysis of pre- nd posttretment speimens from melnom ptients on BRAF inhiitor therpy, with inresed post-tretment levels of TGF-β mrna oserved in two out of four ptients nlyzed (Figure d). Although the origin of the TGF-β ould not e ttriuted exlusively to melnom ells (euse of the nture of ptient tissue, in whih mny ell types intert intimtely), these linil results re onsistent with our in vitro findings. Inresed reeptor tyrosine kinse (RTK) signling is known to medite the espe of melnom ells from BRAF inhiition (Nzrin et l., 1; Strussmn et l., 1; 116 Journl of Investigtive Dermtology (1), Volume 1

3 IV Fedorenko et l. 1Lu W9 W79TR TGF-β. TGF-β1 P<.1 W16 W98A 1Lu C 8 7 C 8 7 C 8 7 TGF-β Strting mrna (ng) μ P-vlue =.7 d. TGF-β1 TGF-β1 (pg/ml) 1 1 Strting mrna (ng) Lu W79 W9 1Lu W79 μ W9. 1 Pre 1 Post 1 Pre Post Pre Post Ptient speimens Pre Post Figure. rfeni indues the relese nd seretion of trnsforming growth ftor-β (TGF-β) from some BRAF-mutnt melnom ells. () Western lot nlysis of six melnom ell lines treted with vemurfeni ( μ, 7 hours). Densitometry for TGF-β is depited in fold hnges ompred with eh respetive ontrol. Br = μm. () Quntittive rel-time reverse-trnsriptse PCR (qrt-pcr) for TGF-β1 shows vemurfeni-medited indution of TGF-β1 mrna expression in 1Lu. Dt were normlized to glyerldehyde -phosphte dehydrogense () nd 18S endogenous ontrols. () ELISA showing indution of TGF-β relese from BRAF V6E melnom ell lines following μ vemurfeni tretment (7 hours), expressed in pg ml 1.(d) Dt show qrt-pcr experiments mesuring the levels of TGF-β1 mrna in four mthed (efore nd fter tretment) pirs of melnom ptient speimens reeiving vemurfeni therpy (96 mg twie dily); error rs represent tehnil replites of single RNA extrtion. Ael et l., 1). A role for melnom- nd vemurfenitivted firolsts in the relese of prosurvivl melnom growth ftors ws demonstrted y ELISA ssys in whih TGF-β (.1 nd 1 ng ml 1 ) or vemurfeni ( μ) tretment inresed the firolst-medited relese of NRG nd HGF, respetively (Figure nd ). Interestingly, mximl firolst tivtion seemed to e dependent on dul TGF-β/vemurfeni tretment, with studies showing tht vemurfeni lone filed to indue NRG relese from firolsts, TGF-β1 lone filed to indue HGF relese from firolsts, nd the oservtion tht TGF-β inhiition did not fully suppress firolst tivtion (Supplementry Figure 1 online). Previous work hs shown tht BRAF inhiition tivtes APK signling in systems with either Rs muttions or upstrem RTK signling, s result of CRAF trnstivtion (Htzivssiliou et l., 1; Poulikkos et l., 1). To dte, the ility of BRAF inhiitors to tivte extrellulr signl regulted kinse (ERK) signling in norml, primry ells hs not een reported. We next determined the requirement for prdoxil ERK tivtion in the vemurfeni-medited relese of firolst HGF. Western lot nlyses showed single-gent vemurfeni to indue prdoxil APK signling in primry humn skin firolsts nd tht this ws reversed through omintion with the EK inhiitor trmetini (Figure ). A role for prdoxil ERK tivtion in firolst-medited melnom therpeuti espe ws demonstrted in ELISA ssys tht showed the omintion of trmetini with vemurfeni to ompletely suppress the vemurfeni-medited inrese in HGF expression (Figure d). Adhesion to EC proteins, suh s, n inrese ell survivl y mplifying RTK signls. To determine the role of expression in mplifying firolst-derived growth signls, we identified three melnom ell lines whose expression inresed following vemurfeni tretment (Supplementry Figure online). Knokdown of using sirna limited EGFR, -ET, nd ERBB reeptor phosphoryltion following lignd hllenge, n effet ssoited with impired PIK/AKT (phosphtidylinositol '-kinse/akt) signling (Figure nd ). An immunohistohemil nlysis of speimens from melnom ptients treted with vemurfeni (n = 9) ws then performed to vlidte tht melnom ells nd firolsts oexist in lose proximity, nd tht expression ws inresed t these sites of intertion. Exmintion y two 117

4 IV Fedorenko et l. NRG-1 (pg/ml) ells FF FF7 FF7 FF FF7 FF7 8 1, 1, 1 * NS TGF-β1 1 pg/ml TGF-β1 1 ng/ml μ TGF-β1 1 pg/ml TGF-β1 1 ng/ml 1 n trm +trm perk ERK TGF-β1 1 pg/ml TGF-β1 1 ng/ml d Fold hnge in HGF seretion HGF (pg/ml) per ells FF *.. 1. FF7 * FF7 rfeni Trmetini Vem + Tr rfeni Trmetini Vem + Tr Figure. rfeni nd trnsforming growth ftor-β (TGF-β) o-operte to relese growth ftors from primry humn firolsts. () ELISA dt showing neuregulin (NRG) relese from three humn skin firolst ell lines, following tretment with TGF-β (1 pg ml 1 nd 1 ng ml 1 ) for 7 hours. () ELISA dt showing heptoyte growth ftor (HGF) relese from three humn skin firolst ell lines, following tretment with vemurfeni ( μ, 7 hours). () Western lot nlysis showing vemurfeni ( μ) to indue prdoxil mitogen-tivted protein kinse (APK) signling in primry humn skin firolsts tht ould e loked through omintion with trmetini (1 n). (d) ELISA dt showing HGF relese from two humn skin firolst ell lines, following 7-hour tretment with μ vemurfeni, 1 n trmetini, or the omintion. *P., P.1, *P.1, nd P.1. independent pthologists onfirmed res in the post-tretment speimens with high levels of stining, with infiltrting spindle-like ells, hrteristi of firolsts, noted in /9 smples (Figure ). Ares of strong stining were lso seen t the tumor strom interfe, t the sites of melnom ells nd firolst intertion (Figure ). Our studies thus fr demonstrted tht tivted firolsts inresed the o-opertive effets of RTKs nd upon PIK signling in melnom ells. We next sked whether inhiition of PIK/AKT signling ws suffiient to reverse the protetion onferred y the firolsts to the melnom ells. Quntifition of stining in GFP tgged W9 melnom ells reveled higher sl signling levels following plting upon firolsts ompred with plsti (Figure ). Upon tretment with vemurfeni ( μ, hours), signifint inreses in stining were oserved in the melnom ells following dhesion to three independent firolst ell lines (Figure nd ). rfeni ws not noted to indue AKT signling in the firolsts (Supplementry Figure online). In line with the oservtion tht mplifies AKT signling through multiple RTKs (Figure nd ), only limited inhiition of AKT signling ws seen when the oultures were treted with the omintion of rizotini nd lptini with vemurfeni (Figure nd d). As expeted, the inhiitory effets of the RTK inhiitors were stronger when the melnom ells were plted on firolsts s opposed to on plsti; however, these effets were still quite limited (Figure nd d). Interestingly, lthough the omintion of the BRAF inhiitor with lptini nd rizotini indued reltively smll derese in ell prolifertion, this triple omintion ws ineffetive t induing poptosis, s mesured y poly (ADP-riose) polymerse (PARP) levge possile refletion of the inomplete inhiition of PIK/AKT signling (Supplementry Figure 6 online nd Figure nd d). The omintion of vemurfeni with the PIK inhiitor GDC-91 ws ssoited with ner-omplete inhiition of the firolstmedited AKT signling in the melnom ells, n effet ssoited with mrked inrese in PARP levge (Figure nd d). Evidene for the role of PIK in filitting firolst-medited therpeuti espe ws demonstrted y the ility of the BRAF+PIK inhiitor omintion to enhne vemurfeni-medited poptosis in two dditionl melnom ells lines, eh plted on three individul primry humn skin firolst ultures (Figure 6). The extent of poptosis indution following BRAF+PIK inhiitor tretment 118 Journl of Investigtive Dermtology (1), Volume 1

5 IV Fedorenko et l. + pher AKT AKT perk perk perk ERK ERK ERK + + EGF (Ser7) AKT HGF (Ser7) long exp Fironetin pegfr pet W9 HGF NRG-1 EGF Lu 1Lu 1Lu + IHC ptient 1 + (Ser7) short exp AKT perk ERK NRG-1 IHC ptient IHC ptient Figure. Adhesion to fironetin () mplifies reeptor tyrosine kinse (RTK) signls in BRAFV6E mutnt melnom ells. () The 1Lu ells were treted with either nontrgeting () or smll interfering RNA (sirna) efore stimultion with heptoyte growth ftor (HGF), epithelil growth ftor (EGF), or neuregulin (NRG) (, 1, nd ng ml 1 respetively). () W9 ells were treted with either or sirna efore stimultion with HGF, EGF, or NRG (, 1, nd ng ml 1, respetively). () Representtive immunohistohemil stining of postvemurfeni filure melnom ptient speimens (N = 9). The speimens were exmined y two independent dermtopthologists who hve indited res tht re hrterized y high stining (red) nd spindle-like ells hrteristi of firolsts infiltrting the tumor tissue (rrows). Ares of strong fironetin stining re shown t the tumor strom interfe where melnom ells nd firolsts lso intert (dotted line). Br = 1 μm. ws slightly inresed on firolsts ompred with plsti in the W79 ell line ut not the 1Lu (Supplementry Figure 7 online). The in vivo relevne of miroenvironmentmedited PIK/AKT signling in the espe response of melnom ells ws demonstrted in humn melnom mouse xenogrft model, in whih dosing with the omintion of the BRAF inhiitor PLX7 nd the PIK inhiitor GDC-91 used signifint levels of tumor regression ompred with either PLX7 or GDC-91 lone (Figure 6). A model showing the proposed intertion of the host melnom ells under vemurfeni tretment is shown in Figure 6. DISCUSSION Although there is some evidene tht host firolsts lso medite resistne to BRAF inhiition through inresed HGF relese, the mehnisms underlying the melnom ell/firolst intertion remin poorly desried 119

6 IV Fedorenko et l. 1 d FF 1 1 FF 6 Plsti on BR trol AF ET HE i T ET HE i + ER i T H i + E i E i H Ri + B R ER + R i i BRAF + A i PI B F K RA i i + PI Fi B K C RA i on Fi BR tro AFl ET E i H T i ET E + ER i i + HE Ti HE i H R + B R ER i + R i i B AF + RA i B F PI K RA i i + PI Fi BR K AFi i 1, on BR trol AF ET E i H ET E i + ERTi H H T i + E i E i H R + B R ER i + R i i B AF + RA i B F PI K RA i i + PI Fi B K C RA i on Fi BR tro A l Fi E E T ET E i + HE Ti H i + E Ti HER H R + B Ri ER i + R i i B AF + RA i B F PI K RA i i + PI Fi BR K AFi i , On FF C 1 On plsti C PIKi + BRAFi Cleved PARP+ BRAFi 1 1 ETi + HERi + BRAFi 7 st i Pl * FF * 1 FF FF7 GFP FF7 plsti plsti Fold hnge in intensity ompred with ells treted on plsti Plsti FF Figure. Firolsts protet melnom ells from vemurfeni (vemu)-medited ytotoxiity through phosphtidylinositol '-kinse/akt (PIK/AKT). () Green fluoresent protein (GFP) tgged W9 melnom ells were plted on plsti or firolst monolyers nd treted with μ vemurfeni ( hours) efore eing stined for (Ser7). Br = μm. () Fold hnges in vemurfeni-indued from were lulted. () elnom ells treted with single gent or with omintions of μ vemurfeni (BRAFi), μ GDC-91 (PIKi), n rizotini (ETi), nd 1 μ lptini (HERi). Anlysis of (Ser7) nd leved poly (ADP-riose) polymerse (PARP) on individul GFP tgged ells ws performed using flow ytometry. Histogrms show levels of, with n AKT+ gte drwn sed on μ GDC-91 tretment on plsti. (d) Column grphs show the perentge of melnom ells from tht re in the AKT+ nd leved PARP+ popultions. P.1 nd *P.1. (Strussmn et l., 1). Firolst survivl is dependent on tthment to n pproprite EC, with dhesion to onstituting mjor survivl signl (Zhng et l., 199; Ili et l., 1998; Almeid et l., ; Lin et l., 11). is lso potent hemottrtnt for firolsts tht stimultes their motility (Postlethwite et l., 1981). Our dt suggest tht the relese of TGF-β from melnom ells treted with BRAF inhiitor nd the effets of the BRAF inhiitor itself hve 1 Journl of Investigtive Dermtology (1), Volume 1 ritil roles in the tivtion of host firolsts. Tretment with TGF-β, vemurfeni, or C from vemurfeni-treted melnom ells inresed firolst differentition. At the sme time, either exogenous TGF-β or vemurfeni enhned NRG nd HGF relese from firolsts, respetively. Reent reports hve suggested tht TGF-β relesed from melnom ells upon BRAF inhiition my lso inrese RTK expression in melnom ells (Sun et l., 1). This, long with the dt

7 IV Fedorenko et l. % Of ells nnexin V+ 1Lu W * * * 6 * GDC +GDC GDC +GDC GDC +GDC +GDC +GDC +GDC FF FF7 FF7 FF FF7 FF7 Tumor volume 8 6 1Lu xenogrfts P=.17 P=.16 GDC P=. +GDC RTK signl mplifition PIK/AKT pthwy tivtion Survivl rfeni TGF-β EC growth ftor Firolsts Figure 6. Comined BRAF/phosphtidylinositol '-kinse (BRAF/PIK) inhiition reverses firolst-medited drug resistne nd leds to tumor regression in vivo. () Green fluoresent protein (GFP) tgged melnom ells (W79, 1Lu) were seeded on top of firolst ell lines (FF, FF7, FF7) overnight efore eing treted with either vehile ontrol or μ vemurfeni (vemu) nd μ GDC-91 for 7 hours efore eing stined for Annexin-V nd nlyzed y flow ytometry. P-vlues were lulted etween vemurfeni only nd vemurfeni/gdc-91 omintion tretments. () Xenogrft tumor volume ws lulted using the modified ellipsoid formul (tumor volume =½ L W ). P-vlues were lulted etween ontrol nd tretment groups. () odel showing the intertion of the host melnom ells under vemurfeni tretment. *P., P.1, nd *P.1. ontined herein, suggests TGF-β relese to set the stge for omplex growth ftor medited ross-tlk etween melnom ells nd firolsts. Anlysis of tumor speimens from melnom ptients on BRAF inhiitor therpy suggest tht melnom ells nd firolsts exist in lose proximity in vivo, suggesting the likelihood of this ross-tlk ourring. The ility of vemurfeni to stimulte HGF relese from norml primry skin firolsts ws unexpeted, nd it ws linked to the ility of vemurfeni to indue prdoxil APK signling in norml humn firolsts. Prdoxil APK signling ours when BRAF inhiitors trnstivte CRAF s result of upstrem signls emnting from either Rs muttions or inresed levels of growth ftor signling (Htzivssiliou et l., 1; Joseph et l., 1; Poulikkos et l., 1; Giney et l., 1). Although the BRAF/EK inhiitor omintion ws noted to suppress the relese of HGF from the firolsts, this would not e expeted to fully reverse host-medited resistne. Other signls, suh s melnom-derived TGF-β, would still e le to tivte the host firolsts, perhps prtly explining why the drfeni/trmetini omintion n dely ut not prevent the onset of quired resistne in melnom ptients (Flherty et l., 1; Lrkin et l., 1). Although TGF-β relesed lolly from the BRAF inhiitor treted melnom ells ppered to onstitute n importnt mehnism of firolst tivtion, it is worth noting tht melnom ells relese other ftors known to stimulte firolsts, inluding pltelet-derived growth ftor nd stroml-derived ftor (Orimo et l., ; Willenerg et l., 1; Whipple nd Brinkerhoff, 1). There is lso evidene tht the introdution of mutnt BRAF into melnom ells inreses their seretion of IL-1α tht uses tumor-ssoited firolsts to indue immune suppression (Khlili et l., 1). The oservtion tht drug-treted melnom ells tivted firolsts, inresing NRG nd HGF relese, ws suggestive of role for host ells in mediting therpeuti espe. Under seline onditions, BRAF-mutnt melnom ells exhiit high levels of feedk inhiition in the APK signling pthwy tht suppresses the ility of RTKs to tivte ERK (Lito et l., 1). Following vemurfeni tretment, the feedk inhiition of the APK pthwy eomes deregulted, inresing the responsiveness to growth ftors suh s EGF, NRG, HGF, nd firolst growth ftor (Lito et l., 1). There is lredy evidene tht oth HGF 11

8 IV Fedorenko et l. nd NRG limit responses to vemurfeni nd its nlog PLX7 (Shrm et l., 1; Strussmn et l., 1; Ael et l., 1). For ll growth ftors evluted, the expression of ws required for the mximl tivtion of PIK/AKT signling, suggesting ritil role for environmentl remodeling in the mplifition of these espe signls. In this instne, the seemed to e derived from the tivted firolsts nd from the melnom ells themselves. It is lredy known in lung ner tht o-opertion etween integrin nd RTK signling is required for the optiml tivtion of the downstrem PIK/AKT pthwy (orello et l., 11). Similr findings hve lso een reported for α β 1 -integrin, in whih funtionl ssoition etween the integrin with the VEGFR reeptor in lymphti vessels nd EGFR nd ERBB in intestinl epithelil ells is required for effiient signling (Lee nd Julino, ; Zhng et l., ). In some experimentl systems, integrin α β 1 lso omplexes with -ET, with the presene of either vitronetin or found to signifintly inrese the level of ET reeptor phosphoryltion (Rhmn et l., ). When oultured with firolsts, vemurfeni mrkedly enhned inresed AKT signling in the melnom ells. The tivtion of AKT ws medited through multiple RTKs nd y diret melnom/firolst dhesion, with the BRAF/ PIK inhiitor omintion found to e signifintly more effetive t reversing the dptive survivl ompred with BRAF inhiitor omined with multiple RTK inhiitors. It ws lso found tht omined BRAF/PIK inhiition ws signifintly more effetive t reduing the growth of melnom xenogrfts ompred with either BRAF or PIK inhiitor lone. These dt re in greement with reent prelinil studies demonstrting tht the omintion of BRAF nd PIK inhiitor indues more rpid regression of tumors in BRAF V6E/PTEN-null GEs ompred with BRAF inhiitor lone (rsh Durn et l., 1). In our xenogrft model, PLX7 ws reltively wek s single gent. This is likely onsequene of the 1Lu melnom ell line eing oth BRAF mutnt nd null for PTEN. There is lredy evidene from our l nd others tht PTEN loss n e meditor of intrinsi BRAF inhiitor resistne, nd there is evidene tht errnt PTEN funtion is ssoited with shorter PFS in melnom ptients reeiving BRAF inhiitor therpy (Priso et l., 11; Xing et l., 11; Nthnson et l., 1). The oserved heterogeneity in TGF-β seretion (highest in PTEN-null ell lines) supports these findings. The requirement for PIK signling in miroenvironmentmedited therpeuti espe ws demonstrted y the ility of omined BRAF/PIK inhiition to overome this protetion. Tken together, these dt suggest tht the PIK/ AKT signling pthwy integrtes multiple host-derived signls required for therpeuti dption. This ide is lso supported y reent work suggesting tht muttions in AKT1 re involved in tumor-intrinsi therpeuti dpttion (Shi et l., 1). Loss of PTEN expression nd/or funtion on BRAF inhiitor therpy hs een suggested s mehnism of therpeuti espe (Vn Allen et l., 1). Although previous studies showed vrile ytokine prodution in estlished firolst lines, the urrent study used erly-pssge primry ultures from three helthy humn donors nd is likely to etter represent firolst iology. A more extensive hrteriztion of linil smples my improve our understnding of the ytokines produed y the strom in ptients treted with BRAF inhiitors (Khlili et l., 1; Strussmn et l., 1). Our urrent understnding of BRAF inhiitor espe suggests role for short-term dpttion in whih ells evde the immedite effets of the drug. The dt ontined herein suggest role for host tumor ross-tlk in the erliest phses of dpttion; however, it is lso likely tht pressure from the host my lso help to selet for esping lones or muttions. Long-term tretment of melnom ptients with smll-moleule inhiitors suh s drfeni nd vemurfeni will depend on their ility to suppress the esping popultion of ells tht ultimtely repopulte the tumor. This study presents evidene tht dptive hnges in norml host ells filitte the espe of melnom ells from BRAF inhiition. It is likely tht omintion therpies suh s BRAF +PIK inhiition my e one strtegy to limit the protetion onveyed y the host. ATERIALS AND ETHODS Cell ulture nd regents The 1Lu, W9, W79, W16, W98A, nd 1Lu melnom ells lines nd FF, FF7, nd FF7 humn primry skin firolsts were gift from Dr eenhrd Herlyn (The Wistr Institute, Phildelphi, PA). W9-GFP ws from Dr Peter Forsyth (offitt Cner Center, Tmp, FL). The identities of ll ell lines were onfirmed y Biosynthesis (Lewisville, TX) through STR vlidtion nlysis. Cell lines were mintined in % fetl ovine serum/rpi-16. Conditioned medium ws prepred y dding fresh medium to 1Lu ells for 8 hours in the presene of vehile or μ vemurfeni. Then, the medium ws olleted nd diluted 1:1 with fresh medium, mthing the onentrtions of drugs/vehile. SB1 ws otined from Sellek Chemils (Houston, TX). Western lotting Proteins were extrted nd western lotting ws performed s desried previously (Fedorenko et l., 1). Uniform protein loding ws onfirmed y lotting for glyerldehyde -phosphte dehydrogense (). The ntiodies to S7, totl AKT, perk, totl ERK, pet (Tyr1/1), pegfr (Y117), pher (Y189), nd TGF-β were from Cell Signling Tehnology (Beverly, A). The α-sa ntiodies were from Am (Cmridge, A), wheres the ntiody ginst ntiody ws from BD (Sn Jose, CA), ws from Sigm (St Louis, O), nd phlloidin ws from Invitrogen (Life Tehnologies, Crlsd, CA). Immunofluoresene stining elnom nd primry skin firolst ells were plted on glss overslips overnight efore tretment. Cells were then fixed, stined, nd imged s desried previously (Fedorenko et l., 1). Imges were nlyzed using the Definiens Developer v.. (Definiens AG, unih, Germny) softwre suite. The totl fluoresene intensity ws normlized to the numer of nulei (stined y DAPI (',6-dimidino--phenylindole)) for monoultures or to the numer of GFP positive ells in oultures. For oulture experiments, 1 Journl of Investigtive Dermtology (1), Volume 1

9 IV Fedorenko et l. melnom ells were GFP leled, plted on unleled firolsts, nd imged using fluoresene mirosopy. elnom nd firolst ells were differentited y the GFP lel. In resue experiments, the numer of GFP+ ells were ounted for three field of view imges per tretment (N = ). Firolst differentition ws mesured y the level of nd α-sa expression. RNA interferene Cells were plted nd left to grow overnight. The % fetl ovine serum/rpi medium ws repled with Opti-E (Invitrogen). pool sirnas in omplex with Lipofetmine (Invitrogen) were dded. Srmled, nontrgeting sirnas were used s ontrols. Cells were trnsfeted for 7 hours efore tretment. ELISA ssys The TGF-β1 nd HGF ELISA Kits were purhsed from R&D Systems (innepolis, N). The NRG ELISA Kit ws from Am (Cmridge, A). Animl studies All niml studies were rried out under pproved protools y the Institutionl Animl Cre nd Use Committee t the University of South Florid. Femle SHO mie (Chrles River) were suutneously injeted with. 1 6 ells per mouse. Tumors were llowed to grow to ~ 1 mm. ie were dministered D11 ontrol how, AIN-76A 17 mg kg 1 PLX7-formulted how (Reserh Diets, New Brunswik, NJ), vehile (.% methylellulose,.% Tween-8) orl gvge, or GDC-91 orl gvge (1 mg kg 1 ) dily for 8 dys. ouse tumor volumes (1 L W) were mesured. Sttistil nlysis Results re reported s men vlues, error rs inditing ± SE. GrphPd Prism 6 softwre ws used to lulte sttistil signifine of mgnitude of hnges etween different onditions using the prmetri unpired t-test with P-vlues depited s follows: *P., P.1, *P.1, nd P.1. qrt-pcr for ell lines nd ptient speimens Cells were treted for 7 hours, nd then totl RNA ws isolted using Qigen s RNesy ini Kit (Hilden, Germny). DNA from ohort of ptient speimens ws generously shred y Keith Flherty from sshussetts Generl Hospitl. Ptient speimens were otined with written, informed ptient onsent ording to pproved protools y the Institutionl Review Bord t the sshussetts Generl Hospitl. The pretretment iopsies were performed etween nd dys efore inititing therpy, nd on-tretment iopsies were olleted etween 7 nd dys fter the initition of therpy. The following Tqn Gene Expression Assys primer/proes were used: Hs6_m1 (), P/N 191E (18S), nd Hs999999_m1 (). The 18S nd dt were used to normlize TGF-β1, ounting for ellulrity. qrt-pcr retions were performed s desried previously (Priso et l., 11). Flow ytometry Cells were grown overnight, nd then treted with vehile (DSO), μ vemurfeni, μ GDC-91, or the two drugs in omintion (7 hours). Cells were stined for Annexin-V nd TR (tetrmethylrhodmine methyl ester), s desried previously (Fedorenko et l., 1). For nlysis of nd leved PARP, FF humn primry skin firolsts (. 1 ells) were plted in 6-well pltes overnight. GFP tgged W9 melnom ells (. 1 ells) were then plted either on plsti or on FF firolsts nd inuted overnight. Cells were then treted with μ vemurfeni (BRAFi), μ GDC-91 (PIKi), n rizotini (ETi), nd/or 1 μ lptini (HERi) for hours efore eing olleted y srping, fixed with % prformldehyde (1 minutes, room temperture), nd permeilized with 1% old methnol (1 hour, room temperture). Cells were stined with onjugted to AlexFluor 67 (Ser7; Cell Signling Tehnology, Beverly, A) nd leved PARP onjugted to phyoerythrin (BD) in.% ovine serum lumin in phosphte-uffered sline (1 hour, room temperture). Cells were nlyzed y flow ytometry. CONFLICT OF IEREST JAW is memer of the dvisory ord for GlxoSmithKleine, Rohe, nd Genenteh. She is lso pid speker for Genenteh nd memer of the Spekers Bureu for Dv Onology. ACKNOWLEDGES Work in the Smlley l is supported y R1 CA16117 from the Ntionl Institutes of Helth with Core Support provided y P-CA769. SUPPLEEARY ATERIAL Supplementry mteril is linked to the online version of the pper t REFERENCES Ael EV, Bsile KJ, Kugel CH III et l. 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