Inflammatory bowel diseases (IBD), such as ulcerative colitis

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1 Netrn-1 up-regulaton n nflammatory bowel dseases s requred for colorectal cancer progresson Andrea Parads a,1, Carne Masse a,1, Mare-May Cosseux a, Ncolas Gadot b, Floran Lépnasse c,célne Delloye- ourgeos a, Jean-Guy Delcros a, Magal Svrcek d, Clemens Neufert e, Jean-Franços Fléjou d, Jean-Yves Scoazec b,c,2, and Patrck Mehlen a,2,3 a Apoptoss, Cancer and Development Laboratory-Equpe labellsée La Lgue, Centre Natonal de la Recherche Scentfque, Unte Mxte de Recherche 5238, Unversté de Lyon, Centre Léon érard, Lyon, France; b ANIPATH, Faculté Laennec, Lyon Cedex 08, France; c Insttut Natonal de la Santé etdela Recherche Médcale, U865, Faculté Laennec, Lyon Cedex 08, France; d Assstance Publque-Hôptaux de Pars, Hôptal Sant-Antone, Servce d Anatome et Cytologe Pathologques and Insttut Natonal de la Santé et de la Recherche Médcale, Unte Mxte de Recherche S893, Team 13 Mcrosatellte Instablty and Cancers, Pars, France; and e Frst Medcal Clnc and Insttute for Molecular Medcne, Johannes Gutenberg Unversty of Manz, Manz, Germany Edted by ert Vogelsten, The Sdney Kmmel Comprehensve Cancer Center at Johns Hopkns, altmore, MD, and approved July 20, 2009 (receved for revew February 17, 2009) Chronc nflammaton and cancer are ntmately assocated. Ths s partcularly true for nflammatory bowel dseases (ID), such as ulceratve colts and Crohn s dsease, whch show a major ncreased rsk for colorectal cancer. Whle the understandng of the molecular pathogeness of ID has recently mproved, the mechansms that lnk these chronc nflammatory states to colorectal cancer development are n large part unknown. One of these mechansms s NF- pathway actvaton whch n turn may contrbute to tumor formaton by provdng ant-apoptotc survval sgnals to the epthelal cells. ased on the observaton that netrn-1, the ant-apoptotc lgand for the dependence receptors DCC and UNC5H s up-regulated n colonc crypts n response to NF-, we show here that colorectal cancers from nflammatory bowel dseases patents have selected up-regulaton of netrn-1. Moreover, we demonstrate that ths nflammaton-drven netrn-1 up-regulaton s causal for colorectal cancer development as nterference wth netrn-1 autocrne loop n a mouse model for ulceratve colts-assocated colorectal cancer, whle showng no effect on nflammaton, nhbts colorectal cancer progresson. Inflammatory bowel dseases (ID), such as ulceratve colts and Crohn s dsease, show a major ncreased rsk for colorectal cancer, but the mechansms that lnk these chronc nflammatory states to colorectal cancer development are n large part unknown. The man non-mmune lnk known so far s the actvaton of NF- (1). It was ndeed proposed that NF- pathway actvaton observed durng ID may contrbute to tumor formaton by provdng ant-apoptotc survval sgnals to the epthelal cells (1). Of nterest, netrn-1, a soluble proten ntally dscovered as an axon navgaton cue (2), was also proposed to play a crucal role durng colorectal tumorgeness by regulatng apoptoss (3). Indeed, netrn-1 receptors DCC and UNC5H that s, UNC5H1, UNC5H2, UNC5H3, and UNC5H4, also called UNC5A, UNC5, UNC5C, or UNC5D belong to the so-called dependence receptor famly (4 6). These dependence receptors, because of ther ablty to nduce cell death when dsengaged from ther lgands, create cellular states of dependence on ther respectve lgands (7) and, consequently, may behave as tumor suppressors because they elmnate tumor cells that would develop n settngs of lgand unavalablty (3, 8). Along ths lne, both overexpresson of netrn-1 or nactvaton of UNC5H3 n mce n the gastro-ntestnal tract are assocated wth ntestnal tumor progresson (3, 9). Even though accordng to the dependence receptor hypothess, a loss of netrn-1 receptors should represent a smlar selectve advantage for tumor growth than ganng autocrne expresson of netrn-1, n sporadc colorectal cancer the vast majorty of tumors dsplay a loss of DCC or/and UNC5H (9 12). In these tumors wth decreased receptors expresson, netrn-1 s not up-regulated and s barely detectable wthn the tumors [(Fg. 1A) and (13)]. Interestngly, the fracton of colorectal cancer showng netrn-1 upregulaton rather than netrn-1 receptors losses frequently dsplay hgh expresson of markers of NF- actvaton such Cox-2 and I (13). Together wth the observaton that netrn-1 gene s a transcrptonal target for NF- (13) and wth the vew of ID-assocated colorectal cancer beng lnked to NF- and survval (1), we nvestgated whether netrn-1 may be nvolved n progresson of ID-assocated colorectal cancer. Results and Dscusson Frst, we analyzed by mmunohstochemstry netrn-1 expresson n the colonc mucosa of 30 patents wth ID (15 wth ulceratve colts, 15 wth Crohn s dsease). In all cases, a strong expresson of netrn-1 was detected n epthelal cells, especally n areas of nflammaton (Fg. 1 and not shown); the apparent expresson levels were markedly hgher than n the normal colonc mucosa (Fg. 1 A and ). We then compared the expresson of netrn-1 n a panel of colorectal adenocarcnomas from 30 patents wth ID (24 wth ulceratve colts, sx wth Crohn s dsease), and n a sample of 52 sporadc colorectal adenocarcnomas. Whle only seven out of 52 (13.5%) sporadc colorectal adenocarcnomas dsplayed hgh netrn-1 levels as compared to the nternal controls, 21 out of the 30 tumors (70%) from ID patents were shown to express hgh netrn-1 levels as compared to the same controls (Fg. 1 A C and data not shown) ( 2 test, P 01). A smlar netrn-1 up-regulaton n tumors from ID patents was also observed at the mrna level. Indeed, RNA from seven pars tumor/normal tssue were extracted and netrn-1 level was analyzed by Q-RT-PCR analyss. Four out of the seven pars tested showed at least a 3.3-fold ncrease of netrn-1 n the tumor, wth an ncrease range from 3.3- to 18.4-fold. In ID patents, comparable levels of netrn-1 expresson were observed n both leberkuhnan and collod adenocarcnomas, rrespectve of ther clncal stage (Fg. 1 v and v). We fnally tested the expresson of netrn-1 n the spectrum of dysplastc lesons of the colon observed n surgcal specmens from 25 patents wth ID (21 wth ulceratve colts, four wth Crohn s dsease). In flat dysplasa, netrn-1 was constantly detected; the Author contrbutons: A.P., C.M., J.-Y.S., and P.M. desgned research; A.P., C.M., M.-M.C., N.G., F.L., C.D.-., and J.-G.D. performed research; M.S., C.N., and J.-F.F. contrbuted new reagents/analytc tools; A.P., C.M., C.D.-., J.-Y.S., and P.M. analyzed data; and J.-Y.S. and P.M. wrote the paper. The authors declare no conflct of nterest. Ths artcle s a PNAS Drect Submsson. 1 A.P. and C.M. contrbuted equally to ths work. 2 J.-Y.S. and P.M. contrbuted equally to ths work. 3 To whom correspondence should be addressed. E-mal: mehlen@lyon.fnclcc.fr. Ths artcle contans supportng nformaton onlne at /DCSupplemental. MEDICAL SCIENCES cg do pnas PNAS Early Edton 1of6

2 A A N T m N T v C 1.8 p<01 v C Apparent expresson level of netrn-1 (% of nternal controls) v >100 Total number of cases ID cancers Sporadc cancers Fg. 1. Netrn-1 expresson n neoplastc lesons from ID and control patents. (A) Expresson of netrn-1 n the normal colonc mucosa () and n a sporadc colon adenocarcnoma (). In the normal colonc mucosa (), a fant labelng s vsble n epthelal cells lnng the crypts (arrows); there s a decreasng gradent of expresson from the bottom of the crypts to the epthelum surface. In an example of well dfferentated adenocarcnoma (), only a few cells are postve wthn the tumor (T), whereas the adjacent pertumoral mucosa (N) retans a readly vsble stanng. () Expresson of netrn-1 n representatve samples from nflamed mucosa () and neoplastc lesons ( v) from ID patents. In the nflamed mucosa (), a strong netrn-1 labelng s vsble, from the bottom of the crypts (arrows) to the surface; the gradent of expresson vsble n the normal colonc mucosa s partly preserved (m, musculars mucosae). Netrn-1 labelng s well vsble n epthelal cells at hgh magnfcaton vews of flat low grade dysplasa () and flat hgh grade dysplasa (). Agan, netrn-1 s strongly detected n an example of DALM (v) wth hgh grade dysplasa; the expresson levels are comparable n the neoplastc area (T) and the adjacent pertumoral mucosa (N). Fnally, netrn-1 s readly detected n two examples of colon adenocarcnoma: a typcal leberkuhnan adenocarcnoma (v) and a collod mucous carcnoma (v), characterzed by the presence of massve mucous secreton (). Indrect mmunoperoxdase technque; orgnal magnfcatons: (A), 400;, 180; (), 220;, 400;, 350; v, 250; v, 120; v, 100. (C) Semquanttatve analyss of netrn-1 expresson n colorectal cancer. Quantfcaton of the apparent netrn-1 expresson level was evaluated as descrbed n the methods secton va a comparson to an nternal control. In ID patents, netrn-1 apparent expresson levels were comparable or hgher to the nternal control, whle n sporadc cancer patents netrn-1 levels were much lower than the nternal control. apparent expresson level was hgher n hgh-grade lesons (n 6) than n low grade lesons (n 15). Netrn-1 was constantly detected n DALM (Dysplasa Assocated Leson or Mass) (n 9), all of hgh grade but one (Fg. 1 v). In contrast, netrn-1 was 2 of 6 兩 Relatve netrn-1 expresson (arbtrary unt) Ctrl AOM/DSS Fg. 2. Netrn-1 expresson n a mouse model for nflammaton-medated colorectal tumor progresson. (A) Representatve colonc lesons n mce submtted to the DSS/AOM treatment. The flat mucosa () dsplays sgns of chronc nflammaton, wth dstorted crypts scattered wthn a lamna propra () contanng numerous lymphocytes and plasma cells. Neoplastc lesons are llustrated by an example of hgh grade adenoma (), formed by numerous rregular tubular structures lned by markedly atypcal cells, and by an example of focally nvasve adenocarcnoma (), characterzed by straght tumoral glands separated by an abundant stroma; note the presence of a neoplastc gland wthn the musculars propra (arrow). These tumors fulflled the dagnostc crtera for low grade and hgh grade adenomas and for adenocarcnomas (24); some adenocarcnomas showed sgns of local nvason, such as the nfltraton of the musculars propra by neoplastc glands. () Immunodetecton of netrn-1 n the non neoplastc nflamed mucosa (), n neoplastc epthelal cells of a hgh grade adenoma () and n neoplastc glands nfltratng the musculars propra wthn an nvasve adenocarcnoma (). Indrect mmunoperoxdase technque. Orgnal magnfcatons:, 480;, 380;, 300. (C) Relatve expresson of netrn-1 mrna n the colon of DSS/AOM-treated mce. Results from Q-RT-PCR are shown. usually fantly expressed n the four cases of adenoma-lke mass avalable for the study. Interestngly, whle DCC has been descrbed to be down-regulated n sporadc cancer n assocaton wth the chromosome 18q LOH (14), 28 out of the 30 tumors from ID patents show a DCC expresson that albet detected at low level was not decreased n tumoral tssues compared to adjacent normal mucosa (data not shown). Thus, strong netrn-1 expresson appears to be a characterstc feature of the whole spectrum of neoplastc lesons observed n ID patents. We then moved to an anmal model for ID-assocated colorectal cancer. Adult mce were submtted to the DNA alkylaton agent azoxymethane (AOM) and the pro-nflammaparads et al.

3 A DCC-EC H2N HOOC NH2 netrn-1 4 netrn bndng (%) Relatve netrn-1 netrn-4 Kd = 5.5 ± 2.0 nm Kd = 35.1 ± 2.2 nm COOH netrn (nm, Log) C Ctrl IKKβ IKKβ+ p<01 D p<01 Caspase-3 actvty (fold over ctrl) Caspase-3 actvty (fold over ctrl) MEDICAL SCIENCES pcdna UNC5H2 netrn netrn E HA-caspase-9 myc-dcc netrn IP α-ha DCC IgG caspase-9 Input DCC caspase-9 Fg. 3. trggers apoptoss va nterference wth netrn-1-medated nhbton of netrn-1 receptors death sgnalng. (A) Schematc representaton of netrn-1 and one of ts receptor DCC. Netrn-1 bnds the extracellular doman of DCC (DCC-EC) va nteracton wth fbronectn type III domans (1 6). The fourth doman () used to nduce cell death s ndcated. () drect bndng to netrn-1. Increasng concentratons of flag-tagged netrn-1 were ncubated wth 5 g/ml and bound netrn-1 was detected by mmuno-labelng. s able to bnd netrn-1 wth a K d of nm. Note that s able to weakly bnd netrn-4, another member of the netrn famly, wth a consstent low K d ( nm). The values represent the mean standard devaton of three ndependent experments. (C) Caspase-3 actvty measured n UNC5H2-transfected HEK293T cells, ncubated wth condtoned medum from NF- actvated (IKK ) or control HL100 cells, n presence or not of (1 g/ml). Medum from IKK -transfected cells was able to block caspase-3 actvaton nduced by UNC5H2. Ths effect s due to the presence of netrn-1 produced and secreted by HL100 cells n response to NF- actvaton (13)., added to condtoned medum, was able to restore caspase-3 actvaton n UNC5H2-transfected HEK293T cells ncubated wth IKK medum by bndng to, and nactvatng secreted netrn-1. (D) Caspase-3 actvty of HCT116 cells treated wth. HCT116 cells that express endogenous netrn-1 were treated or not wth (1 g/ml) n the presence or absence of an excess of netrn-1 or netrn-4. (E) Actvaton of DCC apoptotc pathway by. HEK293T cells were co-transfected wth HA-caspase-9 and myc-dcc constructs and treated wth netrn-1 and/or. Interacton of DCC and caspase-9 was assessed by pull-down usng -HA antbody and by revealng DCC usng -myc antbody. In presence of netrn-1, DCC bndng to caspase-9 was decreased, whle the addton of (1 ng/ml) restored the nteracton. Moreover, a hgh dose of netrn-1 (excess) was able to nhbt acton. Fg. S2 show expresson level of netrn-1, netrn-3, and netrn-4 n a seres of cancer cell lnes. tory reagent dextran sodum sulfate (DSS), leadng to the development of nflammaton drven colorectal tumors (15). After 10 weeks of treatment, sgns of dffuse nflammaton were present n the colonc mucosa; epthelal crypts were dstorted and rregularly dstrbuted n the lamna propra, whch contaned hgh numbers of nflammatory cells, ncludng lymphocytes and plasma cells (Fg. 2 A). Netrn-1 was strongly expressed n epthelal cells of nflamed mucosa (Fg. 2 ). Neoplastc lesons progressvely developed n the nflamed mucosa (Fg. 2 A and A). All neoplastc lesons, ncludng adenomas and adenocarcnomas, showed evdence of netrn-1 expresson at mmunohstochemcal examnaton (Fg. 2 and ). Ths up-regulaton of netrn-1 was not only detected at the proten level but was also observed at the RNA level by Q-RT-PCR analyss (Fg. 2C). Of nterest, n agreement wth the dependence receptor noton whch predcts that a tumor selects Parads et al. PNAS Early Edton 3of6

4 I A Iκα Cox p< p<5 R elatve κα expresso n Relatve Cox-2 expresson Ctrl AOM/DSS AOM/DSS/ Ctrl AOM/DSS AOM/DSS/ Fg. 4. Interference wth netrn-1 does not affect chronc nflammaton n a mouse model of ID-assocated colorectal cancer. (A) Representatve colonc lesons n mce submtted to DSS/AOM treatment for 10 weeks and admnstered ntra-pertoneally three tmes per week wth. The flat mucosa (Left) dsplays sgns of chronc nflammaton, as n control anmals (Left). Neoplastc lesons are llustrated by an example of low grade adenoma (Rght), formed by numerous regular tubular structures lned by slghtly atypcal cells. ()I and Cox-2 expresson n DSS/AOM treated mce admnstered wth. I and Cox-2 expresson was measured by Q-RT-PCR. ether a gan of netrn-1 or a loss of netrn-1 receptors (16, 17), n AOM/DSS-assocated tumors, the analyss of the netrn-1 receptors expresson by Q-RT-PCR shows that UNC5H1, UNC5H2, UNC5H3, UNC5H4, or DCC were not downregulated that s, whle UNC5H1 and UNC5H2 level was not sgnfcantly changed, UNC5H3, UNC5H4, and DCC were actually up-regulated- (Fg. S1). To show whether ths hgh netrn-1 level s causal to the cancer pathology, we analyzed whether ttraton of netrn-1 n mce treated wth DSS/AOM could be assocated wth preventon of tumor progresson. We used for ths study as a netrn-1 nterferng agent. s the 4 th fbronectn doman of DCC ectodoman (Fg. 3A) and has been shown to nteract wth netrn-1 (18). To confrm the /netrn-1 nteracton, an ELISA was performed coatng and revealng a possble nteracton wth ncreased concentratons of ether netrn-1 or netrn-4. As shown n Fg. 3, the dssocaton constant of netrn-1 was estmated to 5.5 nm, a K d that s 6.4 lower than the K d detected for /netrn-4. Thus, DCC- 4Fbn specfcally nteracts wth netrn-1. Ths recombnant compound, even though t fals to prevent the nteracton of netrn-1 wth ts receptors (data not shown), nhbts the ablty of netrn-1 to block netrn-1 receptors death sgnalng n vtro. Ths was shown by the followng experments. Frst, n agreement wth recent publshed observatons (13), forced expresson of UNC5H2 n HEK293T cells s assocated wth apoptoss unless a condtoned medum from NF- actvated cells s added. In these settngs, addton of reversed the ant-apoptotc effect provded by the condtoned medum (Fg. 3C). Second the HCT116 colorectal cancer cells express endogenous netrn-1 that has been shown to consttutvely block UNC5H-nduced apoptoss (19). As shown n Fg. 3D, the treatment wth DCC- 4Fbn of HCT116 cells trggers apoptoss unless an excess of netrn-1 s added to the culture medum. To further support the specfcty of n ttratng netrn-1, netrn-4 was added n excess nstead of netrn-1. As shown n Fg. 3D, netrn-4 was not able to nhbt -nduced apoptoss. Thrd, netrn-1 has been shown to nhbt DCC-medated apoptoss by preventng DCC nteracton wth the apcal caspase-9 (20, 21). To nvestgate whether could block the nhbtory actvty of netrn-1 on netrn-1 receptors death sgnalng, HEK293T cells were then co-transfected wth DCC and caspase-9 and mmunoprecptatons were performed n the presence or absence of netrn-1 and/or. As shown n Fg. 3E, whle caspase-9 nteracts wth DCC unless netrn-1 s added, the addton of allows DCC/caspase-9 nteracton even when netrn-1 s present. Altogether these data support the vew that acts as a netrn-1 ttratng agent that trggers tumor cell death apoptoss by actvaton of netrn-1 receptors apoptotc pathway. We thus analyzed whether ttraton of netrn-1 wth DCC- 4Fbn n mce treated wth DSS/AOM could be assocated wth 4of6 cg do pnas Parads et al.

5 A lesons colonc Nb neoplastc AOM/DSS AOM/DSS/ Low-grade adenomas Hgh-grade adenomas Adenocarcnomas AOM/DSS AOM/DSS/ Fg. 5. Interference wth netrn-1 prevents tumor progresson n a mouse model of ID-assocated colorectal cancer. (A) Number of neoplastc lesons (low grade adenomas, hgh grade adenomas or ntra-mucosc and nvasve adenocarcnomas) n each of the 14 mce treated wth AOM and DSS, n presence or not of. Note that fve out of seven treated mce were adenocarcnoma free anmals whle seven out of seven show adenocarcnoma n the control treated mce. () Representatve mages of colons removed from mce treated wth AOM and DSS n presence (Rght) or not (Left) of. preventon of tumor progresson. A seres of mce were then submtted to the DSS/AOM treatment for 10 weeks whle admnstered ntra-pertoneally three tmes per week wth DCC- 4Fbn. repeated njecton n DSS/AOM mce had only a modest effect on the number of early neoplastc lesons (61 versus 95) suggestng that netrn-1 nhbton has only a moderate, f any, effect on colorectal tumor ntaton. Moreover, the nflammatory aspect of the epthelum appears smlar n control versus -treated mce (Fg. 4A). Along ths lne, DCC- 4Fbn fals to have any sgnfcant effect on the level of NF- actvaton as montored by the measurement of I and Cox-2 mrna levels (Fg. 4). Contrarly, had a dramatc effect on tumor progresson as repeated njecton of trggers an ncreased frequency of low grade adenoma assocated wth a decreased frequency of hgh-grade adenoma and adenocarcnoma (Fg. 5, 2 test, P 05). Thus netrn-1 up-regulaton n response to nflammaton nduced by DSS s a causal mechansm for colorectal cancer progresson. Together wth the observaton that colorectal cancer from patents wth ID shows up-regulaton of netrn-1, wth the fact that NF- has been proposed to contrbute to ID assocatedcolorectal cancer formaton by provdng ant-apoptotc survval sgnals to the epthelal cells and wth the observaton that forced expresson of netrn-1 n gastrontestnal tract s assocated wth ntestnal tumor progresson n mce (3), we propose the followng sequence for colorectal tumor progresson n ID patents. In response to chronc nflammaton assocated wth ID, NF- actvaton trggers netrn-1 up-regulaton not only n the normal but also n the altered epthelum, and ths netrn-1 up-regulaton, probably n assocaton wth other tumor ntaton mechansms, provdes the suffcent survval sgnal to the epthelal cells for tumor progresson. ecause drug compounds targetng netrn-1 are currently under development, t may then be temptng to nvestgate the relevance of complementng the actual treatments of ID, that, untl now, have focused manly on preventng nflammaton n general, wth such ant-netrn-1 drugs that would more specfcally nhbt progresson of early neoplastc lesons toward aggressve colorectal cancer. Ths s partcularly mportant n lght of the dffculty detectng and treatng ID-assocated lesons by conventonal technques at an early stage and because of the current absence of preventve treatment, apart from coloproctectomy n ulceratve colts whch s often assocated wth negatve sde effects and poor lfe qualty. Materals and Methods Netrn-1 Immunodetecton n Human Tssues. For mmunodetecton of netrn-1 n human tssues, archval, formaln-fxed, paraffn-embedded tssue materal was used. The materal selected for study ncluded: samples from 52 cases of sporadc colorectal adenocarcnomas (wth ther correspondng pertumoral mucosa), 30 cases of colorectal adenocarcnomas from patents wth ID (24 wth ulceratve colts, sx wth Crohn s dsease), and 30 samples of nflamed mucosa from patents wth ID (15 wth ulceratve colts, 15 wth Crohn s dsease). All lesons were classfed accordng to the recommendatons of the Venna classfcaton (22). After deparaffnzaton and dehydraton, 4- m thck tssue sectons were boled n ctrate buffer ph9 usng a water bath at 97 C for 50 mn. For blockng endogenous peroxdases, tssue sectons were ncubated n 5% hydrogen peroxde n sterle water. They were then ncubated at room temperature for one hour wth a polyclonal goat antbody to netrn-1 (R&D Systems). Ths antbody was dluted n antbody dluent soluton (ChemMate, Dako) at 1/800. After rnsng n phosphate buffer salne, sectons were ncubated wth a botnylated secondary antbody bound to a streptavdn peroxdase conjugate (Lsab Kt, Dako). Peroxdase actvty was revealed usng DA as a chromogen. Semquanttatve Analyss of Netrn-1 Expresson n Colorectal Cancers. A denstometrc evaluaton of netrn-1 expresson n tumoral and pertumoral tssues was performed as prevously descrbed (23). Fve felds were dgtzed and converted onto a gray scale. The apparent expresson level of netrn-1 n ganglon cells of submucosal and myenterc plexuses was used as an nternal control and the correspondng gray level was normalzed to 100%. The apparent expresson netrn-1 level n adjacent neoplastc and non neoplastc epthelal cells was expressed as a percentage of the control value n the same secton. Anmal Model: Hstologcal and Immunohstochemcal Analyss. To nduce colorectal carcnogeness assocated wth chronc colts, mce were treated as descrbed prevously (15). refly, pathogen-free 8-week-old female wld-type alb/c mce were njected.p. wth 10 mg/kg body weght of AOM dssolved n PS. The day after, 2.5% DSS was gven n the drnkng water over 1 week, followed by 2 weeks of regular water. Mce were treated wth DSS for 1 week every 2 weeks untl the 10 th week of the experment and were njected three tmes per week wth 30 g purfed or wth PS. The anmals were klled at the begnnng of the tenth week and the colon was removed for hstologcal analyss or total RNA extracton. The ntestne was preserved, open and fxed n buffered formaln. After macroscopcal examnaton under a magnfyng lens, all vsble lesons were taken; n addton, random tssue samples were performed at regular ntervals to detect non-macroscopcally vsble lesons. Four-mcometer thck sectons were prepared from formalnfxed, paraffn-embedded tssue samples and staned wth hematoxyln-eosnsaffron. All lesons were dentfed and classfed accordng to nternatonal recommendatons (24). The hstologcal comparson between treated and non-treated anmals was performed blndly. Immunodetecton of netrn-1 MEDICAL SCIENCES Parads et al. PNAS Early Edton 5of6

6 was performed from the same tssue materal, after selecton of representatve samples. The technque was the same as descrbed above. Quanttatve RT-PCR. Total RNA was extracted usng NucleoSpn RNA II Kt (Macherey Nagel) accordng to manufacturer s protocol. RT-PCR reactons were performed wth Scrpt cdna Synthess Kt (o-rad). One mcrogram total RNA was reverse-transcrbed usng the followng program: 25 C for 5 mn, 42 C for 30 mn, and 85 C for 5 mn. For expresson studes, the target transcrpts were amplfed n LghtCycler 2.0 apparatus (Roche Appled Scence), usng the LghtCycler FastStart DNA Master SYR Green I Kt (Roche Appled Scence). Expresson of target genes n AOM/DSStreated mce was normalzed to acdc rbosomal phosphoproten PO (RPLPO), used as housekeepng gene. Expresson of Netrn-1, -3, and -4 genes n human tumoral cell lnes was normalzed to hypoxanthneguanne phosphorbosyltransferase (HPRT) gene. Netrn-1 expresson n human ID patents was normalzed to the ubqutously expressed retnod X receptor alfa (RXR ) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes, that showed less varablty n expresson between normal and colorectal tumoral tssues (9). The amount of target transcrpts, normalzed to the housekeepng gene, was calculated usng the comparatve C T method. A valdaton experment was performed, to demonstrate that effcences of target and housekeepng genes were approxmately equal. The sequences of the prmers are avalable upon request. Cell Lnes, Cell Death Assay, and Immunoprecptaton. Human tumor cell lnes HEK293T, HCT116, and HL100 were cultured n DMEM (Invtrogen) supplemented wth 10% FS and gentamycn (50 g/ml). Transent transfectons of HL100 cells were performed usng Fugene 6 Transfecton Reagent (Roche Appled Scence), whle HEK293T cells were transfected wth Lpofectamne Plus Reagent (Invtrogen), accordng to the manufacturer s nstructons. For caspase-3 actvty assay, HL100 cells were transfected wth IKK or empty vector and serum starved for 3 h before ther culture medum was added on HEK293T cells, transfected wth UNC5H2 or empty vector. HEK293T cells were harvested after 3 h ncubaton n the presence or absence of 1 g/ml and caspase-3 actvty assay was performed usng the Caspase 3/CPP32 Fluormetrc Assay Kt (Gentaur ovson), accordng to the manufacturer s nstructons. Caspase actvty (actvty/mn/mcrogram of proten) was calculated from a 1-h knetc cycle readng on a spectrofluormeter (405 nm/510 nm, Vctor, Wallac). Caspase actvaton s presented as the rato between the caspase actvty of the sample and that measured n HEK293T cells transfected wth the mock vector and ncubated wth the control HL100 medum. HCT116 cells were treated wth 1 ng/ml for 24 h n combnaton or not wth 150 ng/ml purfed netrn-1 or netrn-4. DCC and caspase-9 co-mmunoprecptaton analyss was performed as descrbed prevously (20). Purfcaton. DNA fragment was generated by PCR usng pdcc-cmv-s as a template and nserted nto the vector by annealng to promote the expresson of n fuson wth an N-termnal 6 Hs tag. producton was then performed usng a standard procedure. refly, was expressed n L21 cells n response to IPTG at 25 C and the L21 lysate was subjected to affnty chromatography usng Hs purfcaton on N-NTA columns (Qagen). /Netrn-1 and -4 ndng Assay. DCC-Fbn4 (5 g/ml) n PS was adsorbed on 96-well maxsorp plate (Nalge Nunc Internatonal) for 1 h at 37 C. After blockng n PS-5% SA for 1 h at 37 C, Flag-tagged netrn-1 or netrn-4 (APOTECH Corporaton) (rangng from 0 to 0.5 g/ml) dluted n PS-0.1% Tween-20 (PS-T) 5% SA was then added. After a 1-h ncubaton at 37 C, plates were extensvely washed, and bound netrns were detected by mmunolabelng usng an ant-flagm2 antbody (Sgma-Aldrch) and a HRPgoat-ant-mouse (Jackson ImmunoResearch) usng OPD as colormetrc substrate. Absorbance measurement was performed on the multlabel Vctor staton (Wallac). Statstcal Analyss. The data reported are the mean SD of at least three ndependent determnatons, each performed n trplcate. Statstcal analyss was performed by the nonparametrc Mann Whtney U test unless ndcated. ACKNOWLEDGMENTS. We thank M. Susag for text correctons. Ths work was supported by nsttutonal grants from Centre Natonal de la Recherche Scentfque, Centre Léon érard (P.M.), and the Lgue Contre le Cancer (P.M.), Insttut Natonal du Cancer (P.M.), Agence Natonale de la Recherche (P.M.), the European Unon Specfc Targeted Research Projects Hermone and APOSYS (P.M.), and Assocaton pour la Recherche contre le Cancer-Insttut Natonal du Cancer (P.M. and J.Y.S.) A.P. and C.M. are supported by fellowshps from Assocaton pour la Recherche contre le Cancer. 1. Greten FR, et al. (2004) IKKbeta lnks nflammaton and tumorgeness n a mouse model of colts-assocated cancer. 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