Aberrant Expression of OX1 Receptors for Orexins in Colon ...challenge. Most interestingly, orexin-A also promoted robust apoptosis in cells that are resistant to the most commonly

Therapeutics, Targets, and Chemical Biology

Aberrant Expression of OX1 Receptors for Orexins inColon Cancers and Liver Metastases: an Openable Gateto Apoptosis

Thierry Voisin, Aadil El Firar, Magali Fasseu, Christiane Rouyer-Fessard, V�eronique Descatoire,Francine Walker, Val�erie Paradis, Pierre Bedossa, Dominique Henin, Th�er�ese Lehy, and Marc Laburthe

AbstractResistance to apoptosis is a recurrent theme in colon cancer. We have shown previously that the 7-

transmembrane spanning receptor OX1R for orexins promotes robust apoptosis in the human colon cancercell line HT29 through an entirely novel mechanism involving phosphorylation of tyrosine-based motifs in OX1R.Here, we investigated the status of OX1R in a large series of human colorectal tumors and hepatic metastases. Allprimary colorectal tumors regardless of their localization and Duke's stages and all hepatic metastases testedexpressed OX1RmRNA and/or protein. In sharp contrast, adjacent normal colonocytes or hepatocytes as well ascontrol normal tissues were negative. Next, we showed that nine human colon cancer cell lines established fromprimary tumors or metastases expressed OX1R mRNA and underwent important apoptosis on orexin-Achallenge. Most interestingly, orexin-A also promoted robust apoptosis in cells that are resistant to the mostcommonly used drug in colon cancer chemotherapy, 5-fluorouracil. When human colon cancer cells werexenografted in nude mice, orexin-A administered at day 0 strongly slowed the tumor growth and even reversedthe development of established tumors when administered 7 days after cell inoculation. Orexin-A also acts bypromoting tumor apoptosis in vivo because caspase-3 is activated in tumors on orexin treatment of nude mice.These findings support that OX1R is an Achilles heel of colon cancers, even after metastasis or chemoresistance.They suggest that OX1R agonists might be novel candidates for colon cancer therapy. Cancer Res; 71(9); 3341–51.�2011 AACR.

Introduction

Colon cancer is a leading cause of cancer mortality in manycountries (1). Molecular genetic studies have identified keygenes whose mutations or altered expression can cause coloncancer (2). Many observations also indicated that colon cancergrowth is under the control of a variety of peptide growth factors(3) acting at tyrosine kinase receptors or G-protein–coupledreceptors (GPCR). Among those receptors, epidermal growthfactor receptor (EGFR) is prominent in colon cancers (4) due toits autoactivity and also because it can be transactivated byGPCRs (5). It is a therapeutic target because antibodies directedagainst EGFR have shown efficacy in colorectal cancer (4).

Much less is known about receptors and/or peptides thatinhibit growth and/or promote apoptosis of colon cancer cells.Some years ago, we reasoned that besides growth factors orpeptide hormones that promote colon cancer growth (3), weshould be able to find natural peptides behaving as suppres-sors of colon cancer growth. We tested this hypothesis byscreening the ability of a large series of peptide hormones andneuropeptides to inhibit the growth of the human coloncancer cell line HT29 cultured in the presence of fetal calfserum (FCS; ref. 6). Wemade 2 hits with orexin-A and orexin-Bwhich appear to be robust growth inhibitors (6). Orexins donot alter cell cycle but promote strong apoptosis by inducingcytochrome c release from mitochondria to cytosol andactivation of caspase-3 and caspase-7 (6).

Orexin-A and orexin-B (7), also named hypocretin-1 andhypocretin-2 (8), were discovered as hypothalamic peptidesthat are encoded in a prepro-orexin precursor. The 2 peptidesare present in hypothalamic neurons that regulate sleep,wakefulness, feeding, breathing, reward system, or drug addic-tion (9). Functions of orexins have been also described in a fewperipheral tissues (10, 11), but expression of orexins at theperiphery remains debatable (11–13). Classically, the actionsof orexins are mediated by two 7-pass transmembrane recep-tors OX1R and OX2R (7), the activation of which inducescellular calcium transients through Gq-dependent and -inde-pendent pathways (14).

Authors' Affiliations: INSERM, U773, Centre de Recherche Biom�edicaleBichat Beaujon CRB3; and Universit�e Paris Diderot, UMR S 773, Paris,France

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Thierry Voisin, INSERM U773, Centre deRecherche Biom�edicale Bichat Beaujon CRB3, F-75018, Paris, France.Phone: 33(0)157277486; Fax: 33(0)157277485; E-mail:[email protected] or Marc Laburthe, INSERM U773, Centre deRecherche Biomédicale Bichat Beaujon CRB3, F-75018, Paris. Tel: 33(0)157277486; Fax: 33(0)157277485; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-3473

�2011 American Association for Cancer Research.

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We showed that OX1R triggers apoptosis by an entirelynovel mechanism (15, 16) which is not related to Gq-mediatedphopholipase C activation and cellular calcium transients (13).Orexins induce tyrosine phosphorylation of 2 tyrosine-basedmotifs in OX1R, ITIM and ITSM, resulting in the recruitmentof the phosphotyrosine phosphatase SHP-2, the activation ofwhich is responsible for mitochondrial apoptosis (15, 16).

Given that (i) failure of tumor cells to undergo apoptosistranslates into tumor progression and chemotherapeuticresistance and (ii) apoptosis emerges as a potential targetfor cancer treatment (17, 18), it was important to determinethe status of orexin receptors and orexin-induced apoptosis incolon cancer. In the present work, we addressed this questionby studying the expression of orexin receptors in a large seriesof primary colonic tumors and also in hepatic metastases. Wealso investigated the ability of orexins to promote apoptosis ina large panel of colon cancer cell lines. We show that thereceptor OX1R is not present in normal colon or liver but isaberrantly expressed in all primary tumors and hepatic metas-tases tested. Futhermore, we show that orexins promoteapoptosis in most of the colon cancer cell lines tested includ-ing in cells which are resistant to 5-fluorouracil (5-FU), themost commonly used drug in colon cancer chemotherapy (4).Finally, we showed that orexins strongly reduced the devel-opment of tumors in nude mice xenografted with humancolon cancer cells. Because peripheral expression of orexins isdebatable (13), we also studied the expression of the orexinprecursor mRNA by quantitative real-time PCR (qRT-PCR) inprimary colon tumors, hepatic metastases, and normal colonand liver samples. No orexin precursor mRNA could bedetected in any samples. These data indicate that OX1Rmay be considered as a new valuable gate to apoptosis whichcould be openable by exogenous orexins or OX1R agonistsin colon cancer even after metastasis and chemoresistance to5-FU.

Materials and Methods

Tissue collectionThirty-eight archival specimens of formalin-fixed, paraffin-

embedded human colonic carcinomas were selected to offer asampling according to the severity of the disease and thedifferent sites of the large intestine (see Table 1). Biopsysamples obtained during endoscopy of normal adults sufferingfrom irritable bowel syndrome were also studied. Severalfrozen unfixed tissues were used for qRT-PCR: 10 coloniccarcinoma, 5 nontumoral colon tissues, 10 hepatic metasta-sectomies, and 7 nontumoral hepatic tissues. Tissues wereused in accordance with the requirements of the HumanResearch Committee of Hospital Bichat, Paris. HypothalamuscDNAs prepared from human normal brains were obtainedfrom Clontech.

Cell lines cultureThe human colon cancer cell lines were obtained from

American Type Culture Collection. The cell lines were estab-lished from resected tumors (19, 20) as follows: HT29, SW480,SW48, Caco-2, LS174T, and HCT116 from primary colorectal

adenocarcinomas; T84 from lung metastases; SW620 fromlymph node metastases; Colo205 from ascite metastases;and LoVo from left supraclavicular metastases. The HT29-FU cell line which resists to 1 mmol/L 5-FU was obtained asdescribed (21, 22). Cell lines were biannually verified bymorphology and growth characteristics and were mycoplasmafree (PCR). Cells were routinely cultured in 25-cm2 plasticflasks (Costar) and maintained at 37�C in humidified atmo-sphere of 5% CO2/air in Dulbecco's modified Eagle's medium(DMEM) containing 4.5 g glucose/L, supplemented with 10%FCS (Invitrogen). Caco-2 cells were grown in DMEM with 20%FCS and 1% nonessential amino acids. Colo205 cells weregrown in RPMI 1640, LoVo cells in Ham's F12, and T84 cells in50% DMEM/50% Ham's F12 supplemented with 10% FCS.

Immunohistochemical proceduresFive-micrometer thick sections were incubated at 37�Cwith

a rabbit polyclonal antibody raised against human OX1R(OX1R11-A; Alpha Diagnostic International) diluted 1:50 (20mg/mL). Thereafter, sections were incubated with biotinylatedsecondary antibody diluted 1:200, and finally with the avidin–biotin complex diluted 1:100 (Kit ABC Vectastain; VectorLaboratories). The peroxidase activity was revealed by thediaminobenzidine and nuclei were counterstained withMayer's hemalum. Negative controls were obtained by omit-ting the primary antibody, by replacing the latter by normalrabbit IgG, or by preincubation of the primary antibody withthe homologous immunogen peptide (OX1R11-P; Alpha Diag-nostic International). It was also verified that 50 mg of thehOX1R immunogen peptide per mL of diluted antiserumtotally abolished the signal. In separate experiments, we haveshown that the anti-OX1R antibody stains CHO cells expres-sing the recombinant OX1R, whereas no staining could beobserved in parental non-transfected CHO cells (not shown).The intensity of immunostaining was evaluated on the basis of3 criteria: the estimation of the percentage of immunoreactivecells on the totality of the section; the intensity of theimmunoreactivity expressed as negative (�), discrete to weakand moderate (þ), strong (þþ), or intense (þþþ); and theidentification of the cellular components which expressed theimmunoreactivity.

Quantification of apoptotic cells by Annexin V labelingApoptotic cells were determined using the Guava Nexin Kit

(Guava Technologies) which discriminates between apoptoticand nonapoptotic cells as described (6). Cells were incubatedin the absence or presence of fresh medium containing 1mmol/L orexin-A. After 48 hours, apoptotic cell staining wasanalyzed with a Guava PCA system (15). Results are expressedas the percentage of apoptotic Annexin V/PE (phycoerythrin)-positive cells and are the means of 3 independent analyses.

qRT-PCR procedureTotal RNAs in cultured cells and in frozen unfixed tissue

sections were extracted using NucleoSpin RNA II kit(Macherey-Nagel). Quality and integrity of RNA were evalu-ated using an Agilent 2100 bioanalyzer (Agilent Technologies,Inc.). Real-time PCR amplification was carried out using a

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Cancer Res; 71(9) May 1, 2011 Cancer Research3342




LightCycler 480 PCR system (Roche Diagnostics). The qPCRmedium included 4 mL of cDNA, 10 mL of Light Cycler 480SYBR Green I master mix (Roche Diagnostics), and 2 mL ofeach forward and reverse primer in a final reaction volume of20 mL. Primer sets (QuantiTect Primer Assays) were designedagainst the complete nucleotide sequences, as deposited onGENE GLOBE, using Qiagen QT00058912 (Hs_HCRTR1_1_SG)for the human OX1R, QT02288356 (Hs_HCRTR2_2_SG) forthe human OX2R, and QT01192646 (Hs_GAPDH_2_SG) for

the calibrator glyceraldehyde-3-phosphate dehydrogenase(GAPDH). For prepro-orexin, the primers were 50-CCAAG-CACCATGAACCTTCC-30 (sense) and 50-GCAAGAGCAAGT-CTTTTGACG-30 (antisense). Assays were run using thefollowing protocol: 95�C for 5 minutes, 95�C for 10 seconds,gene-specific annealing temperature 60�C for 30 seconds,72�C for 30 minutes for 40 cycles, followed by a gradualincrease in temperature from 50�C to 97�C during the meltingstage. The OX1R, OX2R, prepro-orexin, and GAPDH amplicon

Table 1. OX1R immunoreactivity in cancers of the proximal and distal colon

Colon cases Duke's stage Tumor site Percentage of stained cells Staining intensity

Proximal1 A Cecum 100 � to þþþ2 A Right 80 þ to þþ3 A Right 95 þþþ4 A Right 90 � to þ5 B Cecum 85 þ to þþ6 B Cecum 85 �7 B Cecum 60 þ to þþ8 B Cecum 70 � to þ9 B Right 50 � to þþ10 B Right 65 � to þ11 B Right 50 �12 B Right 80 � to þþþ13 C Cecum 90 þ14 C Cecum 60 �15 C Cecum 90 þ to þþ16 C Right 90 � to þ17 C Right 100 þ to þþ18 C Right 95 þ to þþþ19 C Right 100 þ20 C Right 80 � to þþ21 C Right 100 þ to þþþ

Distal1 A Left 100 �2 A Left 70 � to þ3 A Sigmoid 100 þþ to þþþ4 A Sigmoid 90 þ to þþþ5 A Rectosigmoid 50 �6 B Left 95 þþ7 B Left 85 þ to þþþ8 B Sigmoid 50 �9 B Sigmoid 80 �10 B Sigmoid 80 � to þ11 B Rectum 100 þ12 C Left 95 � to þþ13 C Left 100 þ to þþ14 C Left 100 þþ15 C Sigmoid 50 �16 C Sigmoid 70 � to þ17 C Sigmoid 90 � to þþ

NOTE: Staining intensity was variable from place to place and scored as: �, discrete to weak; þ, moderate; þþ, strong; þþþ,intense.

Orexin Receptor in Colon Cancers and Liver Metastases

www.aacrjournals.org Cancer Res; 71(9) May 1, 2011 3343




sizes were 85, 103, 148 and 119 bp, respectively. Ct (thresholdcycle value) values were converted into quantities relative(Qrel) and corrected for PCR amplification efficiency, using thefollowing formula: Qrel ¼ 2�[DCt (sample) �DCt (sample)].

Tumorigenicity assay in nude miceExponentially growing LoVo, HT29, or HCT116 cells were

harvested, washed with PBS, and resuspended at a concentra-tion of 1� 107/mL. Mice were anesthetized by intraperitonealinjection of Rompun 2%, Clorketam 1000 in 140 mL of 0.9% (w/v) NaCl. Cells (106/100 mL) were then inoculated subcuta-neously into the flank of mice. All nude mice developedtumors following injection of 106 LoVo, HT29, or HCT116cells. Tumor development was followed by caliper measure-ments in 2 dimensions (L and W), and the volume (V) of thetumor was calculated by the formula for a prolate ellipsoid(V ¼ L � W2� 3.14/6) as reported (23). For treatment withorexin-A, the peptide was dissolved at a concentration of 1mg/mL in PBS and 0.112, 1.12, or 11.2 mmoles/kg of bodyweight were administered daily by intraperitoneal injection.Control mice received PBS. The body weight of mice wassimilar after 30 days of treatment with and without orexin-A,that is, 24.7 � 1.4 g (n ¼ 6) and 23.2 � 0.6 g (n ¼ 6),respectively. No adverse effect of orexin could be observedduring treatment. Cleaved caspase-3 (C92 605; BD Pharmin-gen; 1:200 dilution) expression was studied by immunohisto-chemistry on selected paraffin sections of tumors developed innude mice (24).

Results

OX1R is aberrantly expressed in human colon tumorsex vivo

We investigated OX1R expression in colonic adenocarcino-mas versus normal colonic epithelium by immunohistochem-istry. All the 38 tumors tested (Table 1) were immunoreactivefor OX1R (Fig. 1). The immunostaining was specific, as itdisappeared after incubation of the OX1R antibody with itshomologous immunogen peptide (Fig. 1) or when the primaryantibody was omitted (not shown). On sections, 50% to 100%of cells were immunoreactive (Table 1). In contrast, normalproximal and distal mucosae of patients with irritable bowelsyndrome, taken as controls, did not express OX1R (Fig. 1A).This is in good agreement with qRT-PCR experiments whichalso failed to detect any OX1R mRNA in the same tissuesamples (Table 2). The mean percentages of immunoreactivecells were similar in the proximal and distal colon, that is, 82%and 83%, respectively. In these 2 sites, there was no correlationbetween the numbers of cells showing OX1R expression andthe severity of the tumor. The same holds true for the intensityof immunostaining which was also independent of the severityof the tumor, variable from one tumor to another and betweencells within a tumor. Immunostaining was observed in neo-plastic glands, whereas normal glands and still normal colo-nocytes remained negative for immunostaining (Fig. 1C andD). OX2R mRNA was not detectable by qRT-PCR in any coloncancer tested (Table 2). We conclude that OX1R is aberrantlyexpressed in epithelial cells during colon carcinogenesis.

The immunoexpression of OX1R in colon cancer cells wasdetected predominantly intracellularly in cytoplasm and/ornucleus as well as at the plasma membrane with a variabilitydepending on the tumor (Fig. 1). For the whole of coloniccancers, the immunoreactivity predominated for 58% of themin the cytoplasm, 47% in nuclei, whereas membranes wereseen in 34%. Dysplatic mucosae neighboring cancers showed avariable immunolabeling. Because the main paradigm of 7-transmembrane spanning receptors (25), including orexinreceptors (13), is activation at the plasma membrane byexternal ligands, we further explored the cellular distributionof OX1R in HT29 cells in which external orexins promoteapoptosis (6). When immunostaining was performed on HT29cell pellets under the same procedure as that used for colontumors, very similar data were obtained, that is, labeling ofintracellular compartments and the plasma membrane (Sup-plementary Fig. S1).

Because orexins were previously claimed to be present inthe gut (26), though this view is now debatable (13), we testedthe possible presence of the orexin precursor mRNA in 10colonic tumors. No orexin precursor transcript could bedetected in any colon tumor (Table 2) under conditions inwhich specific amplification products were detected in humanhypothalamus taken as a control tissue where orexins aremainly produced (7, 8). These data indicate that colon tumorsexpress the orexin receptor OX1R but not the orexin peptides.

OX1R is aberrantly expressed in human hepaticmetastases from colorectal cancers ex vivo

Next, we raised the question of whether OX1R present inprimary colon tumors were still expressed after migration intheir main site of metastasis (e.g., the liver). A collection of10 human hepatic metastases was investigated for theexpression of OX1R transcripts using qRT-PCR. Seven sam-ples of normal liver tissue which are adjacent to metastaseswere used as controls. As shown in the Figure 2A, expressionof OX1R mRNA was obtained in all hepatic metastases. Incontrast, qRT-PCR experiments failed to detect any OX1RmRNA in normal liver samples (Fig. 2A). The expression ofOX1R mRNA was similar in liver metastases and primarycolon tumors (Fig. 2 and Table 2). We confirmed these databy investigating OX1R expression by immunohistochemistryin hepatic metastases (Fig. 2B). All metastatic tumors stu-died expressed OX1R, whereas adjacent normal hepatocytesremained negative. It is also worth noting that no orexinprecursor mRNA could be detected by qRT-PCR in humanliver (Table 2). Altogether, these data indicated that aftercolon cancer cell migration to the liver, metastatic nests stillexpress OX1R.

OX1R mRNA expression and orexin induced apoptosisin human colon cancer cell lines including5-FU–resistant cells

A large collection of colon cancer cell lines was investi-gated for the expression of OX1R transcripts using qRT-PCR.Expression of OX1R mRNA could be detected in all coloncancer cell lines but one (i.e., HCT116; Fig. 3A). No mRNA forthe other orexin receptor subtype OX2R could be detected in

Voisin et al.





any cell line tested (not shown). Orexin-A (1 mmol/L)induced apoptosis (Fig. 3B) and subsequent reduction incell number (Supplementary Fig. S2) in all colon cancer celllines tested but one (i.e., HCT116). Similar results wereobtained with orexin-B (data not shown). It is worth notingthat these effects were observed in culture medium contain-ing FCS which promotes robust cell growth. The extents ofapoptosis were variable from one cell line to another (Fig. 3).Quite interestingly, they are strongly correlated with theamounts of OX1R mRNA detected in the various celllines (Fig. 3C). Altogether, these data indicated that func-tional OX1 receptors that are able to promote apoptosisare readily expressed in most of the colon cancer cell lines.

The most commonly used drug in colon cancer che-motherapy 5-FU is able to induce apoptosis in colon cancercells, but development of drug resistance is a primary causeof failure of chemotherapy (4). To investigate the function-ality of OX1R in 5-FU–resistant colon cancer cells, we usedthe HT29-FU cell line in which a long-term 5-FU exposureselected cells resistant to the drug (21). The expression ofOX1R mRNA was similar in HT29-FU cells and parentalHT29 cells (Fig. 3A). We verified that 5-FU up to 1 mmol/L did not promote apoptosis or reduction in cell number(Supplementary Fig. S3) in HT29-FU cells, whereas it wasquite active in the parental HT29 cells (Fig. 4). Orexin-A wasable to induce strong apoptosis in the HT29-FU cells which

Figure 1. Indirect immunostainingof OX1R in human colon tumors.A, paraformaldehyde-fixedsigmoid fromapatientwith irritablebowel. No immunoreactivesignal was observed. B–G,paraformaldehyde-fixed colontumors. B, strong immunostainingin neoplastic glands, whereasnormal glands remained negative.C, detail of this tumor showing thedifference of immunostaining inthe same glands betweenneoplastic cells and still normalcolonocytes. D, invasive cancer ofthe sigmoid. E, same tumor,negative control after incubation ofthe OX1R antibody with itshomologous immunogen peptide.F, invasive cancer of the rightcolon. G, same tumor, the signalwas negative after incubation ofthe OX1R antibody with itshomologous immunogen peptide.Bar, 200 mm for (B), 50 mm for (Aand C–E), and 20 mm for (F and G).

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are resistant to 5-FU action (Fig. 4). When we considered theparental HT29 cells which are sensitive to 5-FU (21), it wasalso very interesting to observe that orexin-A induced

apoptosis not only in the absence of 5-FU (see Figs. 3Aand 4) but also in the presence of 1 or 10 mmol/L 5-FU(Fig. 4). Similar data were obtained with orexin-B (not

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Figure 2. Expression of OX1RmRNA and OX1R immunostainingin liver metastasis from colorectaltumors. A, relative OX1R mRNAexpression in 10metastases and 7adjacent liver tissues wasdetermined by qRT-PCR andcalculated as described inMaterials and Methods. Formetastases, individual values areshown in a scatter plot and themean value is 51 arbitrary units. B,indirect immunostaining of OX1Rin paraformaldehyde-fixed livermetastasis of a coloniccarcinoma. C, negative controlobtained by omitting the primaryantibody. Bar, 20 mm.

Table 2. Relative OX1R, OX2R, and prepro-orexin mRNA expression in various colon tumor tissues

Localization Duke's stage OX1R OX2R Prepro-orexin

T1 Left distal colon A 58 � 7 nd ndT2 Left distal colon B 69 � 9 nd ndT3 Left distal colon C 43 � 7 nd ndT4 Left distal colon C 58 � 3 nd ndT5 Left distal colon D 143 � 16 nd ndT6 Left distal colon D 32 � 6 nd ndT7 Sigmoid distal colon A 137 � 18 nd ndT8 Sigmoid distal colon C 187 � 23 nd ndT9 Right proximal colon A 48 � 9 nd ndT10 Right proximal colon B 70 � 11 nd ndNTC1 Distal colon nd nd ndNTC2 Distal colon nd nd ndNTC3 Proximal colon nd nd ndNTC4 Proximal colon nd nd ndNTC5 Proximal colon nd nd ndNTL Liver nd nd ndControl tissue Hypothalamus 169 � 12 75 � 6 190 � 21

NOTE: T1 to T10 represent proximal and distal colonic tumors from 10 different patients. NTC1 to NTC5 represent nontumoralproximal and distal colonic mucosae from 5 different patients with irritable bowel syndrome. NTL is a representative nontumoral liversample adjacent to a colorectal metastasis. All RNAswere prepared from frozen collected tissues. Human hypothalamus cDNAswereused as positive controls. Results are expressed as means � SE (4 determinations).Abbreviation: nd, not detected.

Voisin et al.





shown). These data show that orexins promote apoptosis in5-FU–resistant cells.

Effect of orexin-A on the growth of tumors developed byxenograft of colon cancer cells in nude miceSubcutaneous inoculation of 106 LoVo cells into the flank

of nude mice resulted in the development of tumors at thesite of inoculation (Fig. 5). Tumor development was followeduntil 15 or 30 days and autopsy of mice did not revealed any

metastatic site. Daily intraperitoneal injection of orexin-A(1.12 mmoles/kg) beginning the day colon cancer cells werexenografted into mice resulted in strong decrease of tumorvolumes in LoVo cells. As for control mice, no metastatic siteof tumor development was detected in orexin-A–treatedmice. The effect of orexin-A in reducing tumor volumewas also observed with other doses of peptide (Fig. 5A,inset). After a 15-day treatment with 0.112, 1.12, and 11.2mmoles orexin-A/kg, the tumor volumes were decreased by62%, 78%, and 81%, respectively. In another set of experi-

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Figure 3. Expression of OX1R mRNA and effect of orexin-A on cellapoptosis in human colon cancer cell lines. A, relative OX1R mRNAexpression in cell lines was determined by qRT-PCR and was calculatedas described in Materials and Methods. B, the proapoptotic effect oforexin-A in cells was investigated as described in Materials and Methods.Results are expressed as means � SE (3 determinations). ***, P < 0.001;ns, nonsignificant versus untreated cells. C, correlation between theproapoptotic response to orexin challenge and the expression of OX1RmRNA in colon cancer cell lines as determined in (A). r ¼ 0.86.

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Figure 4. Effect of orexin on apoptosis and cell number in HT29-FU cells inthe presence or absence of 5-FU. Cells, grown in the presence of 10%FCS, were treated with (black bars) or without (open bar) orexin-A for 48hours in the presence of 5-FU at the indicated concentrations. A,proapoptotic effect of orexin-A (1 mmol/L) in parental HT29-FU cells.Results are expressed as the percentage of orexin-A–induced apoptoticcells. Background apoptosis without 5-FU and orexin-A was 4.4%� 0.3%and was subtracted from the values shown in the figure. B, antigrowtheffect of orexin-A (1 mmol/L) in HT29-FU (B) cells. Cells excluding trypanblue were counted. ***, P < 0.001 versus untreated cells.






ments, treatment of LoVo tumors in nude mice was initiated7 days after inoculation of LoVo cells. It appeared thatorexin-A (1.12 mmoles/kg) very rapidly and strongly reversedthe development of established tumors (Fig. 5B). Immuno-histologic examination of LoVo tumors developed in controland orexin-A–treated mice revealed that they were poorlydifferentiated adenocarcinomas in both cases (not shown).Characterization of apoptosis in LoVo tumors developed innude mice was followed by immunochemical staining ofactivated caspase-3 in paraffin sections of tumors (Supple-mentary Fig. S4). Weak and intense stainings of activatedcaspase-3 were observed in control and orexin-A–treatedmice, respectively. Quite interestingly, the amount of OX1RmRNA measured by qRT-PCR in resected LoVo tumors wasvery similar in control and orexin-A–treated mice (notshown), suggesting that long-term treatment with orexindid not downregulate OX1R mRNA levels.

Two other human colon cancer cell lines which expressedor not OX1R (Fig. 3) were also xenografted in nude mice (e.g.,HT29 and HCT116, respectively). Treatment of mice develop-ing HT29 tumors with orexin-A resulted in drastic reduction of

tumor volume (Fig. 5C), whereas no effect could be observedwith HCT116 tumors (Fig. 5D).

Discussion

In this work, we showed that ectopic expression of theorexin receptor OX1R is a very frequent event in colontumorigenesis regardless of the location of primary cancerin proximal or distal large intestine and also of their severityfrom Duke's A to Duke's C grade. Indeed, OX1R is expressed inall 38 colorectal tumors tested but not in normal glands,still showed negative expression in normal colonocytes andcontrol nontumoral colonic mucosa of patients with irritablebowel syndrome. This aberrant expression is maintained aftermetastatic migration of colon cancer cells to the liver whichdoes not itself express OX1R in healthy conditions. We alsoshowed that activation of OX1R by exogenous orexins in coloncancer cell lines established from primary tumors or metas-tases results in robust cell apoptosis and subsequent reduc-tion in cell number. The proapoptotic action of orexins is alsoinstrumental in colon cancer cells which have been selected

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700HCT116HT29

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C D

Figure 5. Effect of dailyinoculation of orexin-A on thegrowth of tumors developed byxenografting human colon cancercells in nude mice. LoVo cells (Aand B), HT-29 cells (C), or HCT116cells (D) were inoculated in theflank of nude mice at day 0. Micewere daily injectedintraperitoneally with 100 mL oforexin-A solutions starting atday 0 (*) or day 7 (triangle) or with100 mL of PBS (*) for controls. Thedaily treatment corresponded to1.12 mmoles of orexin-A/kg (A–D),0.112, or 11.2 mmoles/kg (A, inset).The development of tumors wasfollowed by caliper measurement.Data are means � SE of 6 tumorsin each group. *, P < 0.01 versuscontrol.

Voisin et al.





(21) for their ability to resist to the most commonly used drugin colon cancer chemotherapy (i.e., 5-FU; refs. 4, 27). Finally,orexins also strongly decrease the development of tumors invivo in 2 models of colon cancer cells xenografted in nudemice. Altogether our data suggested that the orexin receptorOX1R represents a new promising gate to apoptosis in coloncancers which could be openable by exogenous orexins orOX1R agonists.The molecular mechanisms whereby OX1R is aberrantly

expressed during colon cancer progression are still unknown.The gene encoding OX1R maps to the human chromosomalregion 1p33 (7). Chromosome 1p and the 1p32-36 loci havebeen shown to undergo genetic changes including deletions inhuman colorectal tumorigenesis (28). It could be hypothesizedthat deletions in chromosome 1 may hinder the repression ofOX1R gene that occurs in normal colon.OX1Rs are expressed in all hepatic metastases of colorectal

cancer (Fig. 2) and in colon cancer cell lines established fromlymph nodes, ascite, or lung metastases (Fig. 3A). This is animportant point for 2 reasons: (i) The status of a given receptormay be different in primary tumors and metastases. Forexample, the EGFR status in primary colorectal tumors doesnot correlate with EGFR expression in related metastatic sites(29). A loss of EGFR expression was observed in a significantnumber of lymph node and liver metastases (29, 30), an eventwhich has evident implication for treatment with EGFR-targeted antibodies (29). The status of OX1R appears to besimilar in primary colorectal tumors and liver metastases. Thisis crucial if we consider that OX1R could represent a newtarget in colon cancer therapy or for metastases imaging.Indeed, the possibility to use OX1R-targeted agonists toinduce apoptosis of cancer cells is more relevant in metas-tases, including micrometastases, than in the primary tumorwhich can be resected. (ii) The liver is the main site ofcolorectal cancer metastasis. The status of OX1R in hepaticmetastases appears to be suitable for imaging of microme-tastases. The high affinity of OX1R for orexins makes thistarget convenient for radioisotopic imaging with 111In-labeledOX1R agonists as described previously for in vivo imaging oftumors with radiolabeled peptides (31). For imaging, it is alsocrucial that normal liver cells do not express OX1R (Fig. 2). Inthat respect, OX1R appears to be much more interesting forimaging than vasoactive intestinal peptide (VIP) receptors(32) which are not only expressed in colon cancers (33) butalso highly expressed throughout the normal human gut (34,35) and liver (36).The current view of the 7-pass transmembrane OX1R

mediating apoptosis follows a mechanism that involves orexinbinding at the plasma membrane triggering tyrosine phos-phorylation of 2 tyrosine-based motifs ITIM (15) and ITSM(16) in OX1R and subsequent recruitment and activation ofthe tyrosine phosphatase SHP-2 (13, 15, 16). The first steps ofOX1R-driven apoptosis appear to occur at the plasma mem-brane level in agreement with the localization of OX1R at thecell surface of tumors and cultured cells. However, OX1R wasalso detected intracellularly in cytoplasm and/or nucleus incolon tumors and HT29 cells. The reason why OX1R isexpressed intracellularly in colon cancers and its biological

significance, if any, are not known. Many GPCRs have beenshown to be resident at intracellular sites (37–39). The pre-sence of intracellular GPCRs in colon cancer cells has beenpreviously reported for other peptide receptors (40) andalteration of receptor trafficking in cancer cells has beenreported (41).

Failure of tumor cells to undergo apoptosis translates intomalignant potential and chemotherapeutic resistance (18).Any means to selectively induce apoptosis of cancer cellsversus normal cells should be considered as of tremendousinterest (42). The ability of orexins to promote apoptosis incolon cancer cells in culture is well correlated to the expres-sion of OX1R mRNA supporting the idea that OX1R level isdeterminant for the induction of apoptosis. Because all color-ectal tumors and hepatic metastases express OX1R, the tumorcells should be amenable to apoptosis in vivo if simulated byOX1R agonists. Most of the colon cancer cell lines establishedfrom primary tumors or metastases undergo apoptosis onchallenge with orexins. In contrast, normal colonic epithelialcells and liver cells do not express OX1R and do not undergoorexin-induced apoptosis thereby (6). This represents animportant feature in view of the possible use of OX1R as atherapeutic target. OX1Rs induce apoptosis through theintrinsic or mitochondrial pathway (6). Colon cancer cellsalso express Fas receptors (43) which, as death receptors,induce apoptosis through the extrinsic apoptosis pathway (17,18, 42). Unfortunately, most colon cancer cell lines are resis-tant to Fas ligand–mediated apoptosis even if they are positiveto FasR (18). In contrast, normal colonic cells and hepatocytesremain exquisitely sensitive to Fas-mediated apoptosis (18)strongly limiting the potential use of FasR agonists as candi-dates for chemotherapeutic intervention (18). Attempts to useTNF and Fas ligand as an apoptosis-based therapy in coloncancer have also been thwarted by induction of NF-kB–mediated inflammation and fulminant hepatic failure, respec-tively (42). The OX1R clearly does not suffer from such limits,as orexins do decrease tumor development in vivo in models ofhuman colon cancer cells xenografted in nude mice and donot bring about any observable adverse effect upon long-termtreatment whatever the doses of orexin-A used up to 11.2mmoles/kg.

In vivo experiments with human colon cancer cellsxenografted in nude mice show that orexin-A is veryefficient in slowing the tumor growth. It works at a doseas low as 0.112 mmoles/kg and reaches a maximal effect at1.12 mmoles/kg. Not only orexin-A is able to drastically slowtumor growth when administered at the same time as cellinoculation (day 0), but most interestingly, it also reversesvery rapidly the growth of already established tumors (seeFig. 5B). It has been shown previously that orexins inhibitcultured colon cancer cell growth by inducing apoptosiswithout altering cell cycle (6). It is likely that orexins alsoreduced tumor growth in vivo by promoting apoptosis,as we showed by immunostaining (see SupplementaryFig. S4) that caspase-3 is activated in tumors upon orexintreatment.

The orexin receptor OX1R in colon cancer might beconsidered as a new type of gene in cancer, as it is aberrantly






expressed as a functional protein whose function, whenactivated by an agonist, is to promote apoptosis of thecancer cell. The colon cancer cells unexpectedly provide anew gate to promote their death by apoptosis which was notpresent in the normal colonocyte from which they derive. Inthis context, an important question arises: are OX1Rs acti-vated in colorectal primary tumors or metastases in vivo?Several lines of evidence support that the answer is no: (i)Colon tumors do not express the orexin precursor mRNA,suggesting that OX1Rs in colon cancer cells might not beactivated by an autocrine loop. (ii) The major source oforexins is the hypothalamus. The sites of synthesis of orexinsin the periphery are still debatable (11, 13). Although earlyimmunohistochemical studies detected orexins in smallintestine, stomach and pancreas in rodents (26), furtherqRT-PCR experiments identified orexin precursor mRNAin rat testis but not in most other peripheral tissues includ-ing gut and liver (12). In this article, we have shown thatnormal human colonic mucosa and liver tissues do notexpress the orexin precursor mRNA. (iii) A few studiesreported the radioimmunologic detection of very low con-centrations of orexin-A in human plasma but resultsbetween laboratories probably need to be interpreted withcaution (11). Anyway, the reported levels of orexin-A inhuman plasma are very low, in the range between 2 and40 pmol/L, in comparison of the Kd of the human OX1R (i.e.,7 nmol/L; ref. 16), making very unlikely that the elusiveblood-borne orexins can activate OX1R in colon cancer.Altogether, our data and the literature suggest that theOX1Rs aberrantly expressed in colon cancer cells are mostprobably not activated by endogenous orexins in vivo. There-fore, it may be suggested that OX1Rs in colorectal cancerconstitute a gate to apoptosis which probably remainsunopened in vivo but could be openable by therapeuticadministration of exogenous orexins or OX1R agonists.

The development of long-lived peptide agonists or nonpep-tide agonists of orexin receptors will represent an importantadvance not only in neuroscience (44) but also in coloncancer research. The OX1R, orexins and forthcoming OX1Ragonists might be novel candidates for colorectal cancertherapy.

In conclusion, we provide evidence that OX1R is aberrantlyexpressed in all primary colorectal tumors andmetastases andthat its activation by exogenous orexins result in robustapoptosis of colon cancer cells in culture and strong decreaseof tumor development in mice xenografted with colon cancercells in vivo. Another remarkable property of OX1R-mediatedapoptosis in colon cancer cells is that it remains efficient inHT29-FU cells that have been selected for their ability to resistto 5-FU. In this context, the orexin receptor OX1R representsan Achilles heel of colon cancer and is a new promisingtherapeutic target.

Disclosure of Potential Conflict of Interest

No potential conflicts of interest were disclosed.

Acknowledgment

The authors thank Th�ecla Lesuffleur from UMR-S 938 INSERM (France) forher generous gift of the HT29-FU cell line.

Grant Support

A. El Firar is supported by Minist�ere de la Recherche et de l’EnseignementSup�erieur, and Fondation pour la Recherche M�edicale (FRM).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 24, 2010; revised February 4, 2011; accepted February 28,2011; published OnlineFirst March 17, 2011.

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2011;71:3341-3351. Published OnlineFirst March 17, 2011.Cancer Res Thierry Voisin, Aadil El Firar, Magali Fasseu, et al. ApoptosisCancers and Liver Metastases: an Openable Gate to Aberrant Expression of OX1 Receptors for Orexins in Colon

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Aberrant Expression of OX1 Receptors for Orexins in Colon ...challenge. Most interestingly, orexin-A also promoted robust apoptosis in cells that are resistant to the most commonly