metabotropic glutamate receptor 4 expressionin colorectal … · metabotropic glutamate receptor 4...

Metabotropic Glutamate Receptor 4 Expression in ColorectalCarcinoma and Its Prognostic SignificanceHeeJin Chang,1 Byong Chul Yoo,1 Seok-Byung Lim,1 Seung-Yong Jeong,1 Woo Ho Kim,2 andJae-Gahb Park1,2

Abstract Purpose: Metabotropic glutamate receptors (mGluR) play a variety of roles in both neuronaland nonneuronal cells. Recently, we reported that mGluR4 mediates 5-fluorouracil resistancein a human colon cancer cell line. In this study, we evaluated the nonneural expression ofmGluR4 and clarified the existence of mGluR4 in normal colon epithelium and colorectal carci-nomas. We also investigated the association of mGluR4 expression levels with various clinico-pathologic parameters.Experimental Design: mGluR4 expression was investigated in 21normal and 312 malignanttissues from various organs using immunohistochemistry. In addition, 241cases of colorectalcarcinomas were examined and correlations between mGluR4 expression and various clinico-pathologic parameters were then statistically analyzed.Results: Expression of mGluR4 was identified in the normal epithelia of the upper respiratorytract, gastrointestinal tracts, breast, uterine cervix, urinary bladder, and skin, whereas it was notdetected in the thyroid, lung alveoli, liver, testis, or prostate. In the corresponding malignant tis-sues, mGluR4 expression was frequently identified in colorectal carcinoma (68%), followed bymalignant melanoma, laryngeal carcinoma, and breast carcinomas. Expression of mGluR4 wasdetected in131 (54%)of 241colorectal carcinomas and12 (5%)cases among them showedover-expression in their cytoplasms. Loss of mGluR4 expression was negatively associated with tumordifferentiation (P = 0.028), whereas overexpression of mGluR4 was positively associated withrecurrence (P = 0.034) and poor disease-free survival (P = 0.017) in multivariate analyses.Conclusions: Our results suggest that mGluR4 signaling may play a role in colorectal carcinomasand that overexpression of mGluR4 is associated with poor prognosis.

Glutamate is an essential amino acid that plays importantroles in signaling as a major excitatory neurotransmitter atneuronal synapses. The action of glutamate is mediated by theglutamate receptors (1), which are divided into two majorgroups—ionotropic and metabotropic receptors. The iono-tropic receptors are cation-specific ion channels that mediaterapid synaptic transmission (1).

The metabotropic glutamate receptors (mGluR) are muchslower in their responses, which occur through a variety ofsecond messenger cascades via G proteins (1, 2). To date, eightmGluRs have been cloned and classified into three subtypesbased on sequence information and intracellular effectorsystems (3). Group I receptors (mGluR1 and mGluR5) are

coupled to phospholipase C and stimulate the production ofinositol (1,4,5)-triphosphate and diacylglycerol, leading toactivation of protein kinase C. Group II (mGluR2 and mGluR3)and group III receptors (mGluR4, mGluR6, mGluR7, andmGluR8) initiate the inhibitory cyclic AMP cascade (1–3).

Abnormal glutamate signaling has been linked to thepathogenesis of several human psychiatric and neurologicdisorders (4). In addition, glutamate is involved in a numberof important physiologic functions, including sensory percep-tion, memory, and learning; it is also involved in regulatingdevelopmental functions, such as proliferation, migration, andsurvival of neuronal progenitors and neurons (4–6).

Recently, glutamate receptors have also been identified inperipheral nonneuronal tissues, including bone (7), skin (8),and pancreas (9); glutamate signaling has been implicated indifferentiation of osteoblasts, proliferation of keratinocytes,and regulation of insulin secretion in these organs, respec-tively (7–9). Moreover, glutamate and its receptors have beenreported to play roles in development of melanoma in mice(10) and to promote growth of malignant glioma cells in vitro(11). In the previous study, we identified overexpression ofmGluR4 in a 5-fluorouracil–resistant colon cancer cell line,compared with its parental cell line SNU-769A (12). However,concepts such as the peripheral distribution of mGluR4 inhuman tissue and its roles in nonneuronal tissue are lesswell understood. Here, we report the expression of mGluR4

www.aacrjournals.orgClin Cancer Res 2005;11(9) May1, 2005 3288

Authors’Affiliations: 1Research Institute and Hospital, National Cancer Center,Goyang, South Korea and 2Cancer Research Institute and Cancer Research Center,Seoul National University, Seoul, South KoreaReceived 9/17/04; revised1/24/05; accepted 2/10/05.Grant support: National Cancer Center, South Korea.The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Note: H.J. Chang and B.C.Yoo contributed equally to this work.Requests for reprints: Jae-Gahb Park, Research Institute and Hospital, NationalCancer Center, 411-769 Goyang, Gyeonggi, South Korea. Phone: 82-2-760-3380;Fax: 82-2-742-4727; E-mail: [email protected].

F2005 American Association for Cancer Research.

Imaging, Diagnosis, Prognosis

Research. on August 8, 2020. © 2005 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

in nonneuronal tissues, such as normal and carcinoma tissuesof colon, and altered expression of mGluR4 associatedwith various clinicopathologic parameters in colorectalcarcinoma.

Materials and Methods

Patients and tissue samples. Twenty-one normal and 312 malignanttissue samples were obtained from 21 organs, including salivary gland,oral cavity, larynx, lung, esophagus, stomach, colon, liver, gallbladder,pancreas, breast, uterine cervix, endometrium, ovary, testis, prostate,kidney, urinary bladder, thyroid, adrenal gland, and skin. All tissuesamples were taken from the files of the Department of Pathology,Seoul National University Hospital, South Korea, with written informedconsent. The studied malignant tissues consisted of 287 carcinomas, 17germ cell tumors (12 seminoma of testis, 4 dysgerminoma, and 1 yolksac tumor of ovary), and 8 malignant skin melanomas.

In addition, 241 surgically resected colorectal carcinoma cases wereobtained from Seoul National University Hospital, Seoul, South Korea,in 1998. A review of clinical charts and pathologic reports was done toobtain clinicopathologic data. All cases were adenocarcinomas andwere classified according to WHO criteria (13) and staged according tothe criteria of the International Union Against Cancer (14). The clinicaloutcomes of the colorectal cancer patients were followed from the dateof operation until death or December 31, 2003. Mean follow-up timewas 43 months (range, 2-74 months). Recurrent disease was defined aseither local relapse or recurrence with distant metastases. Diseaserecurrence was detected by sonographic ultrasound, computed tomog-raphy, or magnetic resonance imaging, and was confirmed bypathologic examination. The microsatellite instability status of 230 ofthe enrolled cases had been previously reported (15); two cases wereadditionally analyzed for microsatellite instability at the BAT-25 andBAT-26 markers as previously described (15).

Immunohistochemistry. All tissues were routinely fixed in 10%buffered formalin and embedded in paraffin blocks. Core tissuebiopsies of 1 or 2 mm in diameter were taken from individualparaffin-embedded tissues (donor blocks) and arranged in newrecipient paraffin blocks (tissue array block) using a trephine apparatus(Superbiochips Laboratories, Seoul, South Korea). Because it hasalready been shown that a single sample from each tumor wassufficient to identify protein expression or molecular alteration relatedto clinical outcome, we sampled a tissue core from each case (16).

Immunostaining was done using the avidin-biotin peroxidasecomplex method. After antigen retrieval process using a citrate buffersolution (antigen unmasking solution; Vector Laboratories, Burlingame,CA) for 15 minutes in an 800 W microwave oven, polyclonal rabbit anti-mGluR4a (dilution 1:1,000; Upstate Biotechnology, Lake Placid, NY)and monoclonal mouse anti-p53 (clone DO7; dilution 1:100; VectorLaboratories) antibodies were applied. Cytoplasmic or membranousexpression was regarded as positive for mGluR4 expression and thepercentages of stained tumor cells were assigned the following scores: �,0%; +, 1 to 50%; ++, >50%. As for p53, staining at >10% of tumor cellnuclei was considered positive (17). Two pathologists (H.J. Chang andW.H. Kim), without knowledge of the clinicopathologic data, did blindanalysis of the immunostaining results.

Human colorectal cancer cell lines and Western blot analysis. Thehuman colorectal cancer cell lines, SNU-61, SNU-81, SNU-407, SNU-1033, SNU-1047, SNU-C2A, SNU-C4, and SNU-C5 (18, 19), and thehuman fibrosarcoma cell line, HT1080, were obtained from the KoreanCell Line Bank (Seoul, South Korea). Total homogenates from these celllines were analyzed by Western blot analysis as previously described(12) using either anti-mGluR4 (dilution 1:2,000; Upstate Biotechno-logy) or anti–h-actin (dilution 1:50,000; Sigma, Saint Louis, MO) asthe primary antibodies. To quantify the level of protein expression, theintensities of mGluR4- and actin-immunoreactive signals were calcu-lated using GelCompar II software (Applied Maths, Kortrijk, Belgium).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and cell

invasion assay. The effect of the mGluR4 agonist, L-2-amino-4-phosphonobutyric acid (L-AP 4; Tocris Cookson, Ltd., Avonmouth,United Kingdom), and the mGluR4 antagonist, (S)-amino-2-methyl-4-phosphonobutanoic acid (MAP 4, Tocris Cookson), on cell prolifera-tion was tested using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide colorimetric assay done as previously described(12). All experiments were done thrice, and the mean and SD ofincreased or decreased cell proliferation were calculated.

The cell invasion assay was done using a cell invasion assay kit(Chemicon, Temecula, CA), according to the manufacturer’s instruc-tions. Briefly, the assay was done in an invasion chamber consisting of a24-well tissue culture plate with 12 cell culture inserts. A cell suspensionin serum-free RPMI 1640 (Invitrogen, Carlsbad, CA) containing 2 mg/mLsodium bicarbonate, 100 units/mL penicillin, and 0.1 mg/mL strepto-mycin was added to the inserts, and each insert was placed in the lowerchamber containing 10% bovine calf serum RPMI 1640. After 72-hourincubation in a cell culture incubator, invasiveness was evaluated bystaining of cells that migrated through the extracellular matrix layer andclung to the polycarbonate membrane at the bottom of insert.

Statistical analysis. The m2 and ANOVA tests were used todetermine the correlations between mGluR4 expression and eachclinicopathologic parameter. Disease-free survival was estimated by theKaplan-Meier method with the log-rank test. Multivariate analysis wasdone using the Cox proportional hazards model. P < 0.05 wasconsidered statistically significant. All statistical analyses were doneusing SPSS software (SPSS, Chicago, IL).

Results

Expression of metabotropic glutamate receptor 4 in normal andmalignant tissues of various organs. Table 1 summarizes theimmunohistochemical expression of mGluR4 in the testednormal and malignant tissues. Expression of mGluR4 wasidentified in the normal epithelia of the upper respiratory tract,gastrointestinal tract, breast, uterine cervix, urinary bladder, andskin, whereas expression was not detected in the thyroid, lungalveoli, liver, testis, or prostate. Normal colonic crypt epitheliashowed cytoplasmic expression of mGluR4 in the supranuclearportion. The myenteric plexus also expressed mGluR4 (Fig. 1).

In the corresponding malignant tissues, mGluR4 seemed tobe more specifically expressed than in normal tissues. mGluR4expression was frequently identified in colorectal adenocarci-nomas (68%), malignant melanomas of skin (63%), laryngealsquamous cell carcinomas (50%), and breast carcinomas(46%), whereas it was not detected in carcinomas of the salivarygland, esophageal squamous cell carcinomas, endometrialcarcinomas, prostatic adenocarcinomas, or seminomas of testis.

Expression of metabotropic glutamate receptor 4 and itsrelation to clinicopathologic features in colorectal carcinoma.Among the 241 cases of colorectal adenocarcinoma, 122 (51%)cases showed altered expression of mGluR4. One hundred ten(46%) cases showed loss of mGluR4 expression, whereas 12(5%) cases showed overexpression of mGluR4 with diffusecytoplasmic staining in >50% of tumor cells (Fig. 1).

When mGluR4 expression was examined compared withclinicopathologic parameters, we observed that loss of mGluR4expression was more frequently seen in moderately to poorlydifferentiated types than in well-differentiated types (24%versus 49%; P = 0.028). In addition, overexpression ofmGluR4 was more frequent in cases with recurrence than incases without recurrence (11% versus 3%; P = 0.034; Table 2),and overexpression of mGluR4 was identified in cases of Tstage 3 or 4 only (P = 0.199). In contrast, we observed no

www.aacrjournals.org Clin Cancer Res 2005;11(9) May1, 20053289

mGluR4 Expression in Colorectal Carcinoma



significant association between mGluR4 expression and otherclinicopathologic parameters, including tumor size, location,lymphatic or venous invasion, lymph node or distantmetastasis, stage, microsatellite instability, or p53 expression(Table 2).

Analysis of disease-free survival was done in 218 patientswho had undergone curative resection (R0 according to theInternational Union Against Cancer guideline). Patients withmGluR4 overexpression showed significantly poorer disease-free survival than those without mGluR4 overexpression(P = 0.0176; Fig. 2). A multivariate Cox proportional regressionmodel significantly correlated stage (P = 0.001), overexpressionof mGluR4 (P = 0.0166), and venous invasion (P = 0.0181)with poor disease-free survival, independent of tumor differ-entiation, location, depth of invasion (pT stage), lymph node

or distant metastasis, lymphatic invasion, microsatellite insta-bility status, or p53 overexpression (Table 3).

Effect of metabotropic glutamate receptor 4 agonist andantagonist on cell proliferation and invasion of human colorectalcancer cell lines. To clarify whether mGluR4 signaling can affectthe proliferation and invasion of human colorectal cancer celllines, mGluR4 agonist (L-AP 4) and antagonist (MAP 4) wereused. Before this in vitro assessment, the expression of mGluR4in various human colorectal cancer cell lines was evaluated usingWestern blot analysis to select the cell lines (Fig. 3A). Eachhuman colorectal cancer cell line showed different intensity ofthe immunoreactive mGluR4 signals at 110 kDa. Among the celllines tested, the strongest immunoreactive signal of mGluR4 wasdetected in SNU-407, whereas both SNU-61 and SNU-1033showed moderate signal; the lowest signal was obtained from


Table1. Expression of mGluR4 in normal andmalignant tissues

Malignancy(expression rate)

OrganNormaltissue

No. positive cases/no. cases tested

Salivary gland +/�* 0/14 (0%)Adenoid cystic carcinoma 0/3Acinic cell carcinoma 0/3Mucoepidermoid carcinoma 0/2Carcinoma ex pleomorphic

adenoma0/5

Epithelial myoepithelialcarcinoma

0/1

Oral cavity and nasopharynx + 1/8 (13%)Squamous cell carcinoma 0/7Undifferentiated carcinoma 1/1

Larynx + 7/14 (50%)Squamous cell carcinoma 7/14

Lung +/�c 5/31 (16%)Squamous cell carcinoma 3/19Adenocarcinoma 1/10Large cell carcinoma 1/2

Esophagus + 0/3 (0%)Squamous cell carcinoma 0/3

Stomach + 10/30 (33%)Adenocarcinoma 10/30

Colon and rectum ++ 17/25 (68%)Adenocarcinoma 17/25

Liver � 5/16 (31%)Hepatocellular carcinoma 3/13Cholangiocarcinoma 2/3

Gall bladder + 1/6 (17%)Adenocarcinoma 1/6

Pancreas + 1/5 (20%)Adenocarcinoma 1/5

Breast + 6/13 (46%)Invasive ductal carcinoma 4/8Invasive lobular carcinoma 1/4Medullary carcinoma 1/1

Uterine cervix + 4/25 (16%)Squamous cell carcinoma 2/21

Table1. Expression of mGluR4 in normal andmalignant tissues (Cont’d)

Malignancy(expression rate)

OrganNormaltissue

No. positive cases/no. cases tested

Adenocarcinoma 2/4Endometrium +/�b 0/12 (0%)

Adenocarcinoma 0/12Ovary � 3/31 (10%)

Serous cystadenocarcinoma 0/12Mucinous

cystadenocarcinoma1/2

Endometrioidadenocarcinoma

0/5

Clear cell adenocarcinoma 0/3Undifferentiated carcinoma 2/4Dysgerminoma 0/4Yolk sac tumor 0/1

Testis � 0/12 (0%)Seminoma 0/12

Prostate � 0/4 (0%)Adenocarcinoma 0/4

Kidney +/�x 1/13 (8%)Renal cell carcinoma 1/11Transitional cell carcinoma 0/2

Urinary bladder ++ 3/15 (20%)Transitional cell carcinoma 3/15

Thyroid � 2/15 (13%)Papillary carcinoma 1/12Follicular carcinoma 0/2Medullary carcinoma 1/1

Adrenal gland + 1/12 (8%)Adrenal cortical carcinoma 1/12

Skin + 5/8 (63%)Malignant melanoma 5/8

*Focal expression of mGluR4 in duct of salivary gland.cFocal expression of mGluR4 in bronchus and no expression in alveoli.bWeak expression of mGluR4 in endometrial gland.xFocal expression of mGluR4 in collecting duct.




SNU-81 (Fig. 3A). These four cell lines, and mGluR4-negativehuman hepatoma cell line Huh-7 (12), were used to verify theeffect of mGluR4 agonist and antagonist on cell proliferation.The proliferation of SNU-81 was increased by the addition ofmGluR4 agonist, L-AP 4 (Fig. 3B). However, the other threecolorectal cell lines and Huh-7 did not show any significantchange in proliferation. When cell lines were treated withmGluR4 antagonist, MAP 4, proliferation was suppressed butthis suppression was not observed in SNU-81 and Huh-7(Fig. 3B). Two cell lines, SNU-81 and SNU-407, showing mostdifferent levels of immunoreactive mGluR4 signal in Westernblot analysis, had been used in invasion assay. SNU-81, SNU-407, and Huh-7 cell lines did not show any invasive naturecompared with HT1080, a positive invasion control. However,

in the presence of L-AP 4, increased invasiveness was observed inboth SNU-81 and SNU-407 but not in Huh-7 (Fig. 3C).

Discussion

In addition to their expression in the central nervoussystem, glutamate receptors have been reported to be widelyexpressed in peripheral, neuronal, or nonneuronal tissues,such as bone (7), skin (8), and pancreas (9). Indeed, recentstudies have revealed that peripheral glutamate receptors maybe involved in a variety of physiologic functions (20–22). Forexample, glutamate receptors in the myenteric plexus of theguinea pig ileum were related to modulation of intestinalcontractility (20, 21), and mGluRs in the rat heart were


Fig. 1. Immunohistochemical expression ofmGluR4 in normal human tissues andcorresponding malignant tumors.A , normallaryngeal epithelium. B, laryngealsquamous cell carcinoma. C, normal breastduct and lobules. D, invasive ductalcarcinoma of breast showing partialcytoplasmic expression of mGluR4.E , normal colon epithelium showingcytoplasmic expression of mGluR4 in thesupranuclear portion. F, myenteric plexus ofcolon expressing mGluR4. G, colonadenocarcinoma showing diffusecytoplasmic expression of mGluR4.H, colon adenocarcinoma showing the lossof mGluR4 expression.




suggested to play an important role in cardiac function (22).Furthermore, glutamate-stimulated tumor growth and gluta-mate antagonist– induced suppression of cancer cell growthstrongly suggest that glutamate signaling is likely to beimportant in nonneuronal cancer cells (6).

In the present study, we found that mGluR4 was widelydistributed in a variety of normal tissues, including upper

respiratory epithelium and colon crypt. However, in thecorresponding malignant tissue, its expression was limited tospecific types of organ or histologic types. Especially, mGluR4expression was the most frequent in colon adenocarcinoma(Table 1). This finding suggests that mGluR4 may regulate thepathophysiology of normal and carcinoma cells originatingfrom the colon epithelium. In the normal colonic epithelium,mGluR4 was expressed in the cytoplasm rather than themembrane, perhaps due to receptor internalization, which isa mechanism for desensitization (23–25).

The physiologic role of mGluR4 is important in bothneuronal (26) and nonneuronal tissues (12). Decreasedexpression of the mGluR4 gene is associated with neuronalapoptosis and selective activation of mGluR4 protects againstexcitotoxic neuronal death (26). Consistent with these findings,our previous study showed that the mechanism underlying5-fluorouracil resistance in human cancer cells may involveincreased expression of mGluR4 (12). However, mGluR4expression in pretherapy biopsy samples was not associatedwith resistance to preoperative chemoradiation therapy in rectalcarcinoma (data not shown), and mGluR4 expression was notsignificantly different between groups with or without neo-adjuvant therapy in this study (Table 2). In contrast, mGluR4expression seemed to be significantly associated with differen-tiation (P = 0.028), recurrence (P = 0.034; Table 2), anddisease-free survival (P = 0.0176; Fig. 2). These results areconsistent with previously reported associations in oralsquamous cell carcinoma between glutamate receptor expres-sion and poor prognosis (27).

Our data indicate that mGluR4 expression is linked toprognosis of colorectal carcinoma, but it is not yet known howthis molecule executes this physiologic role to affect recurrenceand disease-free survival. Various regulation pathways arecoupled to mGluR signaling, including the mitogen-activatedprotein kinase, phosphatidylinositol-3-kinase, phospholipaseC, and inhibitory cyclic AMP pathways (28–30). All of thesepathways have been associated with cell proliferation, differ-entiation, and/or antiapoptotic survival (31), suggesting thatthe regulation and final effect of mGluR4 signaling caused by


Table 2. Expression of mGluR4 according toclinicopathologic features

Clinicopathologicfindings

Expression of mGluR4

P�� + ++

Size (mean, cm) 5.56 5.49 4.98 0.675Location

Proximal (n = 53) 23 29 1 0.465Distal (n = 188) 87 90 11

HistologyWell (n = 33) 8 23 2 0.028Moderate/poor* (n = 208) 102 96 10

Lymphatic invasionAbsent (n = 169) 74 85 10 0.467Present (n = 72) 36 34 2

Venous invasionAbsent (n = 230) 104 114 12 0.667Present (n = 11) 6 5 0

Depth of invasionT1/T2 (n = 36) 20 16 0 0.199T3/T4 (n = 205) 90 103 12

Lymph node metastasisAbsent (n = 129) 55 69 5 0.336Present (n = 112) 55 50 7

Distant metastasisAbsent (n = 191) 90 92 9 0.655Present (n = 50) 20 27 3

StageStage I + II (n = 117) 52 61 4 0.464Stage III + IV (n = 124) 58 58 8

Recurrencec

Absent (n = 154) 73 77 4 0.034Present (n = 53) 21 26 6

Microsatellite instabilityb


p53 expressionx


Adjuvant therapyNot done (n = 180) 81 89 10 0.764Done (n = 61) 29 30 2

Disease-free survival(mean duration, mo)

52 55 30 0.018

*Including16 cases of mucinous type.cRecurrence in 218 cases underwent curative resection; no staining results in11cases.bNo microsatellite instability analysis in nine cases.xNo staining results in five cases.

Fig. 2. Kaplan-Meier survival curve illustrating significantly different survival ratesfor patients having colorectal carcinoma with mGluR4 overexpression[mGluR4(++)] and those without mGluR4 overexpression [mGluR4(+) andmGluR4(�) P = 0.0176].




the overexpression of mGluR4 may affect the biologicalbehavior of colorectal carcinoma cells. Our in vitro experimentscould not examine the mGluR4 signaling network in detail(Fig. 3), but our results strongly support previous reportsdescribing the involvement of mGluR4 signaling in tumorgrowth and malignancy (6, 11). In the presence of the mGluR4agonist, SNU-81 cells showed increased proliferation (Fig. 3B),

whereas cell lines SNU-61, SNU-407, and SNU-1033, whichhad moderate to high mGluR4 immunoreactivities, were notaffected by addition of the mGluR4 agonist. In contrast, themGluR4 antagonist suppressed proliferation of cell linesSNU-61, SNU-407, and SNU-1033, whereas proliferation ofSNU-81 was not significantly affected (presumably becausethe level of mGluR4 signal was already low; Fig. 3B). Taken


Fig. 3. Effect of the mGluR4 agonist orantagonist on human colorectal cancer cellproliferation and invasion. A ,Western blotanalysis of various human colorectal cancercell lines. Immunoreactive signals ofmGluR4 were detected at110 kDa, with thevarious cell lines showing different signalintensities. Among the cells tested, thestrongest immunoreactive signal wasdetected in SNU-407, whereas the lowestwas detected in SNU-81. B, effect of themGluR4 agonist and antagonist on cellproliferation. Four human colorectal cancercell lines and the mGluR4-negative humanhepatoma cell line Huh-7 were tested todetermine whether the mGluR4 agonist orantagonist could alter cell proliferation.Theproliferation of SNU-81cells increasedfollowing addition of the mGluR4 agonist,L-AP 4, but theother threehumancolorectalcell lines did not show any change inproliferation.When the cell lines were treatedwith the mGluR4 antagonist, MAP 4,proliferationwas suppressed inall tested celllines except SNU-81. Neither L-AP 4 norMAP 4 affected the proliferation of Huh-7.C, increased cell invasiveness was inducedby the mGluR4 agonist, L-AP 4.The twocell lines showing the most variedimmunoreactive mGluR4 signals inWesternblot analysis (SNU-81and SNU-407)were tested in a cell invasion assay.Whenuntreated, neither cell line showed anyinvasiveness, compared with the positivecontrol cell line, HT1080. However, in thepresence of L-AP 4, both SNU-81andSNU-407 cells became invasive. Both L-AP4 and MAP 4 did not affect the invasivenessof Huh-7.The lower panel shows the relativemGluR4 signals among the four cell lines intheWestern blot analysis.




together, these results suggest that mGluR4 signaling may benecessary but not sufficient for colorectal cancer cellproliferation. Alteration of cell proliferation and invasivenessof colorectal cancer cell lines by the treatment of the mGluR4agonist and antagonist further supported the notion thatmGluR4 signaling plays a role in tumor growth andprogression and also provides a possible link between

mGluR4 overexpression and the prognosis of colorectalcarcinomas (Fig. 2; Table 3).

In conclusion, the role of mGluR4 in colon epithelium andits regulatory mechanisms still remain to be clarified. However,our results confirm the existence of mGluR4 signaling in thecolon and indicate its significance as a possible poor prognosticfactor in colorectal carcinoma.


Table 3. Multivariate analysis for disease-free survival

Parameters Case no. DFS (mean, mo) Univariate analysis (P)

Multivariate analysis

Hazard ratio (95% CI) P

Age (y) NS NSV59 106 54>59 112 54

Sex NS NSFemale 81 55Male 137 51

Size (cm) NS NSV3.3 34 52>3.3 184 53

Site NS NSProximal 48 55Distal 170 52

Histology NS NSWell 32 55Moderate/poor 186 52

Tstage NS NST1/T2 38 60T3/T4 180 51

N stage <0.0001 NSN0 130 60N1/N2 88 42

M stage 0.0001 NSM0 194 55M1 24 34

Stage <0.0001 2.6472 (1.6363-4.2826) 0.0001Stage I + II 122 61Stage III + IV 96 42

Lymphatic invasion 0.0116 NSAbsent 164 55Present 54 44

Venous invasion 0.0168 2.5837 (1.1758-5.6778) 0.0181Absent 208 54Present 10 34

MGluR4 IHC* 0.0056 2.6188 (1.1910-5.7582) 0.0166�/+ 196 53++ 9 30

p53 IHCc NS NS� 87 54+ 128 52

MSIb 0.0434 NSAbsent 189 52Present 22 62

Abbreviations: DFS, disease-free survival; MSI, microsatellite instability; 95% CI, 95% confidence interval; NS, statistically not significant; IHC, immunohistochemistry.*No staining of mGluR4 in11cases.cNo staining results in three cases.bNo microsatellite instability results in seven cases.





References1.TanabeY, Masu M, Ishii T, Shigemoto R, et al. A family

of metabotropic glutamate receptors. Neuron 1992;8:169^79.

2. Aramori I, Nakanishi S. Signal transduction and phar-macological characteristics of a metabotropic gluta-mate receptor, mGluR1, in transfected CHO cells.Neuron1992;8:757^65.

3. SkerryTM, Genever PG. Glutamate signalling in non-neuronal tissues. Trends Pharmacol Sci 2001;22:174^81.

4. Hollmann M, Heinemann S. Cloned glutamate recep-tors. Annu Rev Neurosci1994;17:31^108.

5. Komuro H, Rakic P. Modulation of neuronal migrationby NMDA receptors. Science1993;260:95^7.

6. RzeskiW,Turski L, Ikonomidou C. Glutamate antago-nists limit tumor growth. Proc Natl Acad Sci U S A2001;98:6372^7.

7. Patton AJ, Genever PG, Birch MA, et al. Expressionof an N-methyl-D-aspartate-type receptor by humanand rat osteoblasts and osteoclasts suggests a novelglutamate signaling pathway in bone. Bone 1998;22:645^9.

8. Genever PG, Maxfield SJ, Kennovin GD, et al. Evi-dence for a novel glutamate-mediated signaling path-way in keratinocytes. J Invest Dermatol 1999;112:337^42.

9. Brice NL,Varadi A, Ashcroft SJ, et al. Metabotropicglutamate and GABA(B) receptors contribute to themodulation of glucose-stimulated insulin secretion inpancreatic h cells. Diabetologia 2002;45:242^52.

10. Pollock PM, Cohen-Solal K, Sood R, et al. Melano-ma mouse model implicates metabotropic glutamatesignaling in melanocytic neoplasia. Nat Genet 2003;34:108^12.

11. TakanoT, Lin JH, Arcuino G, et al. Glutamate releasepromotes growth of malignant gliomas. Nat Med2001;7:1010^5.

12. Yoo BC, Jeon EK, Hong SH, et al. Metabotropic

glutamate receptor 4-mediated 5-fluorouracil resis-tance in human colon cancer cell line. Clin CancerRes 2004;10:4176^84.

13. Hamilton SR,Vogelstein B, Kudo S, et al. Carcinomaof the colonand rectum. In: Hamilton SRand AaltonenLA, editors.WHO classification of tumors: pathologyand genetics of tumors of the digestive system. Lyon(France): IARCPress; 2000. p.103^41.

14. Greene FL, Page DL, Fleming ID, et al. AJCC cancerstaging manual. NewYork: Springer-Verlag; 2002.

15. JeongSY,ShinKH,ShinJH,etal.Microsatelliteinsta-bility and mutations in DNA mismatch repair genes insporadic colorectal cancers. Dis Colon Rectum 2001;46:1069^77.

16.TorhorstJ, Bucher C, KononenJ, et al.Tissue micro-arrays for rapid linking of molecular changes to clinicalendpoints. AmJPathol 2001;159:2249^56.

17. Nishio M, KoshikawaT, Kuroishi T, et al. Prognosticsignificance of abnormal p53 accumulation in primary,resected non-small-cell lung cancers. J Clin Oncol1996;14:497^502.

18. Park JG, Oie HK, Sugarbaker PH, et al. Characteris-tics of cell lines established from human colorectalcarcinoma. Cancer Res1987;47:6710^8.

19. Oh JH, Ku JL, Yoon KA, et al. Establishment andcharacterizationof12 human colorectal-carcinomacelllines. IntJCancer1999;81:902^10.

20. Shannon HE, Sawyer BD. Glutamate receptors ofthe N-methyl-D-aspartate subtype in the myentericplexus of the guinea pig ileum. J Pharmacol Exp Ther1989;251:518^23.

21. Sinsky M, DonnererJ. Evidence for a neurotransmit-ter role of glutamate in guinea pig myenteric plexusneurons. Neurosci Lett1998;258:109^12.

22. Gill SS, Pulido OM, MuellerRW, et al. Immunochem-ical localization of the metabotropic glutamate recep-tors in the rat heart. Brain Res Bull1999;48:143^6.

23. Ferguson SS. Evolving concepts in G protein-coupled

receptor endocytosis: the role in receptor desensitiza-tion and signaling. Pharmacol Rev 2001;53:1^24.

24. Dale LB, Babwah AV, Ferguson SS. Mechanisms ofmetabotropic glutamate receptor desensitization: rolein the patterning of effector enzyme activation.Neuro-chem Int 2002;41:319^26.

25. Mundell SJ, Pula G, Carswell K, et al. Agonist-induced internalization of metabotropic glutamate re-ceptor 1A: structural determinants for protein kinaseC- and G protein-coupled receptor kinase-mediatedinternalization. JNeurochem 2003;842:294^304.

26. Borodezt K, D’Mello SR. Decreased expression ofthemetabotropic glutamate receptor-4 gene is associ-ated with neuronal apoptosis. J Neurosci Res 1998;53:531^41.

27. Choi SW, Park SY, Hong SP, et al.The expression ofNMDA receptor 1is associated with clinicopathologicparameters and prognosis in the oral squamous cellcarcinoma. J Oral Med Pathol 2004;33:533^7.

28. Peavy RD, Chang MS, Sanders-Bush E, et al.Metabotropic glutamate receptor 5-induced phos-phorylation of extracellular signal-regulated kinasein astrocytes depends on transactivation of the epi-dermal growth factor receptor. J Neurosci 2001;21:9619^28.

29. Iacovelli L, Bruno V, Salvatore L, et al. Native group-III metabotropic glutamate receptors are coupled tothe mitogen-activated protein kinase/phosphatidyli-nositol-3-kinase pathways. J Neurochem 2002;82:216^23.

30. Thandi S, Blank JL, John Challiss RA. Group-Imetabotropic glutamate receptors, mGlu1a andmGlu5a, couple to extracellular signal-regulated ki-nase (ERK) activation via distinct, but overlapping,signallingpathways. JNeurochem 2002;83:1139^53.

31. Marinissen MJ, Gutkind JS. G-protein-coupledreceptors and signaling networks: emerging para-digms.Trends Pharmacol Sci 2001;53:531^41.




2005;11:3288-3295. Clin Cancer Res Hee Jin Chang, Byong Chul Yoo, Seok-Byung Lim, et al. Carcinoma and Its Prognostic SignificanceMetabotropic Glutamate Receptor 4 Expression in Colorectal

Updated version

http://clincancerres.aacrjournals.org/content/11/9/3288

Access the most recent version of this article at:

Cited articles

http://clincancerres.aacrjournals.org/content/11/9/3288.full#ref-list-1

This article cites 28 articles, 8 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/11/9/3288.full#related-urls

This article has been cited by 7 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://clincancerres.aacrjournals.org/content/11/9/3288To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/content/11/9/3288.full#ref-list-1

http://clincancerres.aacrjournals.org/content/11/9/3288.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



metabotropic glutamate receptor 4 expressionin colorectal … · metabotropic glutamate receptor 4...

Documents