Expression of A-1,3-Fucosyltransferase Type IV and VII Genes Is Related to Poor Prognosis in Lung Cancer

ICANCERRESEARCH56.325-329.January15.19961 Expression of a-1,3-Fucosyltransferase Type IV and VII Genes Is Related to Poor Prognosis in Lung Cancer

Jun-ichi Ogawa,1 Hiroshi Inoue, and Shirosaku Koide First Department ofSurgerv. School ofMedicine, Tokai University, Bohseidai, Isehara, Kanagawa, 259-11, Japan

ABSTRACT MATERIALS AND METHODS

To date, five a-1,3-fucosyltransferasegenes(Fuc-Till, IV, V, VI, and Materials. Three hundred thirty-three consecutive patients with lung can VI!) have been cloned. To examine the role of a-1,3-fucosyltransferase in cer who hadundergonecurativetumor resectionandlymph nodedissection the synthesisofsialyl Lewisx and the prognosisoflung cancer,PCR from 1980 to 1993 were included in this study. Staging was according to the amplificationof five fucosyltransferasegenesandimmunohistochemical AmericanJoint Committeeon CancerThM classification(17). Patientswith staining of sialyl Lewis x wereperformed in 333patientswith lung cancer positive surgical margins were excluded. Procedures at the time of surgery whounderwentsurgicalresectionfrom1980to 1993.The frequenciesof were the same for all patients. Of the patients with non-small cell carcinoma, Fuc-TJIIIV, Fuc-TVI, Fuc-TIV, and Fuc-TVJI expression were 9%, 26%, none with stage I or II disease receivedadjuvanttherapy. Patientswith stage ifi 75%, and 66%, respectively. The frequency of sialyl Lewis x expression disease received postoperative chemotherapy or radiation therapy. Those with (75%) was comparable to Fuc-TIV and Fuc-TVII expression.However, smallcell carcinomareceivedpreoperativeandpostoperativechemotherapy. the grading of sialyl Lewis x staining correlated only with the grading of DNA Extraction. DNA was extracted from formaldehyde-fixed, paraffin Fuc-TVII gene amplification. Survival of the patients whose tumors embedded blocks (18). Three 3(L@m-thick sections were prepared from each blockaftercuttingawayasmuchof theparaffinaspossible.Thesectionswere showed strong expression of Fuc-TIV and/or FucT-Vil was significantly immersed in 1 ml xylene at room temperature for 30 mm and centrifuged for shorter than that of the patients whose tumors did not express either 5 mm at 15,000x g twice. After discardingthe supernatant,thepellet was Fuc-TIV or Fuc-TVII. These results suggest that Fuc-TIV and Fuc-T VII resuspendedin1 ml 100%ethanolandcentrifugedfor 5 mm at 15,000X g. expression may be of prognostic value among patients with lung cancer by Thepelletwaswashedagainwith 100%ethanol,driedthoroughly,and500 @l participating in the biosynthesisof sialyl Lewis x. buffer (50 mM Tris-HC1,1 mi@iEDTA,and 0.5% Tween20, pH 8.5) with proteinase K (200 @g/ml;WaKO Pure Chemicals, Osaka, Japan) were added. Incubationwasovernightat 48Â°C.Nucleicacid wasextractedtwice using 1 INTRODUCTION volumephenol:chloroform:isoamylalcohol(25:24:1)andthenextractedonce with 1 volume chloroform. The DNA was precipitated by the addition of 2.5 The SLex2 carbohydrate structure has been found on the surface of volumes 100%cold ethanol,incubationat â€”80Â°Cforat least30 mm, and various human cancer cells. Its expression plays an important role in centrifugation for 20 mm at 15,000 X g. The pellet was washed with 75% E-selectin-mediated adhesion to activated endothelium (1â€”3),leading ethanol,dried thoroughly,andresuspendedin20 mrsiTris-HC1solution(pH @ to hematogenous metastasis of cancers (4). Fuc-Ts are thought to be 7.5) to a final DNA concentrationof 1 The DNA concentrationwas key enzymes which regulate synthesis of SLex. Up to now, at least determinedspectrophotometrically. three distinct types of human Fuc-Ts can be distinguished by their PCR Analysis of Fuc-T. The primersequencespreparedfor PCR ampli enzymatic specificity (5), and some of their genes have been cloned fication of Fuc-T were as follows: Fuc-TllhIFuc-TV sense, 5'-CTGCTGGT GQCTGTGTGmCTfCTCCFAC-3' (positions 70-99/70-99) and anti (6â€”12).TheLewis type a-l,3/l,4-fucosyltransferase, found in kidney, sense, 5'-CAGCCAGCCGTAGGGCGTGAAGATGTCGGA-3' (positions gallbladder, and milk, is known to catalyze the transfer of fucose to 487â€”516/526â€”555). The size of the fragments amplified from Fuc-Till and both type 1 and type 2 chain-based structures. Fuc-TIII corresponds to Fuc-TV were 447 and 486 bp, respectively. Fuc-TIV sense, 5'-GCITGC this Lewis type. The myeloid type Fuc-T, found in leukocytes and CCGAAATI'GGQcTCCTGCACAC-3' (positions 807â€”833)and antisense, brain, catalyze the transfer of fucose to type 2 chain-based structures, 5'-CCAGAAGGAGGTGATGTGGACAGCGTA-3' (positions 1099-1125). but is much less active on sialylated substrates. Fuc-TIV corresponds The sizeof the fragmentamplifiedwas319 bp. Fuc-TVI sense,5'-CTCAA to this myeloid type. The plasma-type Fuc-T, found in plasma and GACGATCCCACTGTGTAC-3' (positions 110-132) and antisense,5'- liver, transfers fucose to type 2 chain-based structures whether they CAGCCAGCCGTAGGGCGTGAAGATGTCGGA-3' (positions 484-513). The sizeof the fragmentamplified was404 bp. Fuc-TVII sense,5'-CTCG are sialylated or not. Fuc-TV or Fuc-TVI, which are very similar in GACATC1TrGTGCCCTATG-3' (positions 438-460) and antisense,5'- their structures, correspond to this plasma type. In addition to the CGCCAGAA'1TFCTCCGTAATGTAG-3'(positions702-725). The size of aforementioned Fuc-T, a novel one designated Fuc-TVII recently has the fragmentamplifiedwas288bp. One @xggenomicDNA wasamplifiedin been cloned from human leukocytes (13, 14). This enzyme can a 50-pi reactionmixture containing25 pmol of primers, deoxynucleotide efficiently utilize sialylated type 2 chain-based acceptorsbut not those triphosphates (2.5 mM), 10 mM Tris-HC1 (pH 8.0), 50 mM KCI, 1.5 mM MgCI2, thatare not sialylated. 0.01%gelatin,and 1.5units Taq polymerase(TakaraSchuzo,Otsu,Japan). In a previous study, we have demonstrated that immunohistochem Forty cycles of amplification were performed with a thermal cycler (Iwaki Glass,Funabashi,Japan).Eachcycleconsistedof 1mmdenaturationat95Â°C, ical expression of SLex correlates with the prognosis in lung cancer 1mmannealingat60Â°C,and2 mmextensionat72Â°C.ElectrophoresisofPCR (15, 16). However, the question of which fucosyltransferase is related products (10 @tl)ona 2% agarosegel containing ethidium bromide (0.5 @g/ml) to the SLex expression and also to the correlation with prognosis wasdoneto evaluatethesizeof generatedfragments.Positivecontrolreactions remains to be answered. Therefore, we examined the expression of were performed with DNA template from the same patient whose DNA five Fuc-T genes in patients with lung cancer. templatestronglyexpressedallof the Fuc-Tgenes(Fig. 1,case1).Negative control reactions were done without DNA template. The grading of gene expression was as follows: (â€”),no expression; (+), weak expression in which Received 8/29/95; accepted I 1/7/95. The costsof publicationof this article weredefrayedin part by the paymentof page PCR productswere visible but much weakerthan the positive control; and charges. This article must therefore be hereby marked advertisement in accordance with (+ +), strongexpressioncomparabletothe positivecontrol. 18 U.S.C. Section 1734 solely to indicate this fact. InununohistochemicalExpressionof SLex. A streptavidin-biotinylper @ To whom requests for reprints should be addressed. Phone: 463-93-1 121; Fax: 463-95-7567. oxidase complex method using FH6 monoclonal antibody (kindly provided by 2 The abbreviations used are: SLex, sialyl Lewis x; Fuc-TIllâ€”Vil, a-1,3-fucosyltrans OtsukaPharmaceutical,Tokyo,Japan)againstsialyl dimeric Lewis x were ferasetypesIllâ€”VII. performed on thin sliced sections as previously reported (15). Immunohisto 325

Table I Patientcharacteristics guished (Fuc-TIII 447 bp versusV 486 bp, Fuc-TIV 319bp versusVII patientsNo. No. of 288 bp), they were in separatelanes. ofpatients333Age Relationship between Fucosyltransferaseand SLex Expression. 64)SexMale231Female102Histological(yr)32â€”84 (median, The frequencies of expression (+) and (+ +) of Fuc-T were 9% for Fuc-TIII/V, 26% for Fuc-TVI, 75% for Fuc-TIV, and 66% for Fuc TVH, respectively. The frequencies of Fuc-TIV and Fuc-TVII expres typesAdenocarcinoma172Squamous sion were significantly greater than Fuc-TIII/V and Fuc-TVI. The

18Large cell carcinomaI frequency of expression (+) and (+ +) of SLex was 75%, which was cellcarcinoma24Small comparable to that of Fuc-TIV and Fuc-TVII. However, the grading cellcarcinoma15Adenosquamous of SLex staining correlated only with the grading of Fuc-TVII (Table carcinoma4Stageâ€•I162II40III131acell 2; P < 0.01). Survival According to SLex, Fuc-TIV, and Fuc-TVII Expres sion. Survival of patientswho had tumorswith SLex (+ +), Fuc-TIV (+ +), and Fuc-TVII (+ +) expression was significantly shorter than Stages were in accordance with the AJCC staging system (17). that in patients with SLex (â€”)or (+), Fuc-TIV (â€”)or (+), and Fuc-TVII (â€”)or (+) expression, respectively. Survival difference chemicalstainingwasexaminedinatleast1000cellsin five high-powerfields between the patients with (â€”)and (+) expression of SLex, Fuc-TIV, (magnification x 1000). The grading of SLex staining was as follows: (â€”), and Fuc-TVII was not significant (Fig. 2). The expression of Fuc <5% of cells had membraneousstaining;(+), 5â€”29%ofcells stained;and Till/V and Fuc-TVI did not significantly affect the survival of patients (+ +), 30%or morecellsstained.NegativecontrolsomittedtheFH6antibody. (data not shown). The 333 patients were divided into four groups Data Analysis. The frequenciesofgeneandSLexexpressionwereevalu according to their Fuc-TIV and Fuc-TVII expression: group 1, pa @ atedusingthe test.SurvivalcurveswerecalculatedusingtheKaplan-Meier method,and statisticalevaluationwasdoneusingthe log rank test.Patients tients with both Fuc-TIV(â€”) and Fuc-TVII(â€”) tumors (n = 54); who died of causes other than primary lung cancer were censored. group 2, patients with Fuc-TIV(+) and/or Fuc-TVII(+) tumors (n = 85); group 3, patients with either Fuc-TIV (+ +) or Fuc TVII(+ +) tumors (n = 62); and group 4, those with both Fuc-TIV RESULTS (+ +) and Fuc-TVII(+ +) tumors (n = 132). Group 3 and group 4 Clinical Characteristics of the Patients. The age, sex, histologi patients had significantly shorter survivals compared with group 1 or cal type, and stages of the patients are shown in Table 1. Of the 333 group 2 patients (P < 0.01). There was no significant survival patients, 131 died of primary lung cancer and 32 died of causesother difference between group 1 and group 2, nor between group 3 and than lung cancer. The follow-up period for the 152 patients who had group 4 (Fig. 3). Similar results were obtained when tumors were no evidence of tumor recurrence ranged from 21 to 185 (median, 65) subdivided according to stagesand histological types. In both stage I months. or stage IH disease, or in adenocarcinomas or squamous cell carcino Expression of Fucosyltransferase Genes. Prior to the experiment, mas, group 3 and 4 patients had significantly shorter survivals than we used four different initial amounts of genomic DNA from the group 1 and 2 patients (P < 0.01; Fig. 4). positive control patient: 1, 10, 100, and 1000 ng. Each specific Fuc-T gene product was amplified in a dose-dependentmanner by 30, 32, 35, DISCUSSION 37, 40, and 45 cycles of PCR. The specific product reached a plateau at 45 cycles when 1 @ggenomic DNA was used, whereas we esti In the biosynthesis of the SLex structure, sialylation precedes mated gene amplification with 1 @gDNAby 40 cycles of PCR (data fucosylation because a-2,3-sialyltransferase does not act on fucosy not shown). Gene amplification of five Fuc-T from eight representa lated substrates (19). Therefore, the final step in SLex synthesis is tive patients is shown in Fig. 1. Because amplified fragments of the catalyzed by Fuc-T. To date, five Fuc-T genes have been cloned and Fuc-TIII and -TV and Fuc-TIV and VII could not be easily distin their chromosomal locations determined (10, 14, 20). Previously, we case bp 1 2 3 4 5 6 7 8 1 2 3

Fig. 1. PCR amplification of fucosyltransferase 486(Fuc-TV),447(ffl) genes in eight representative samples. Top, Fuc -L.N.4@$@ TI!!, V. VI, and IV genes; bottom, Fuc-TVII gene. Left, bp size markers; right, size of amplified prod 319@V) ucts in bp. Cases 1â€”3:all genes are strongly 1oo@ expressed. Fuc-TIII and Fuc-TV fragments can be distinguished in the latter three lanes at the top right. Cases4â€”6:Fuc-TVI, IV, and VII are strongly expressed. Case 7: Fuc-TIII, V. IV, and VII are strongly expressed. Case 8: Fuc-TIII, V. and IV are strongly expressed.

4-288(VU)

326

expressionFucosyltransferaseFuc-TIII,VFuc-TVIFuc-TIVTable 2Relationship betweenfucosyltransferase and sialyl Lewis X

(+)(++)Fuc-TVII(â€”)(+)(++)â€œ(â€”)(+)(++)(â€”)(+)(++)TotalSialyI(â€”) Lewis x (â€”)â€œ 25 (+) 108 3 7 85 18 14 28 31 58 40 28 49 118 (++) 121 8 2 97 17 18 30 26 76' 34 20 78d 131 Total75 3044 155 1464 2467 4213 4524 82 8235 16936 11418 6030 15984 333

a Grades of gene amplification are as follows: (â€”), no expression; (+), weak expression; (+ +), strong expression. b Grades of SLex staining are as follows: (â€”), <5% of cells had membranous staining; (+), 5â€”29%of cells stained; (+ +), 30% or more cells stained.

( P = 0.22. @@ d P values were obtained using a 3 X 3 x2 test.

100 found that SLex expression correlated with the prognosis of patients with lung cancer (15, 16). Results similar to the present report were also described in colon cancer (21). It was reported that highly 80 metastatic colon carcinoma cells, as compared to their low metastatic a2 counterparts, bind more efficiently to activated human endothelial @ 60 cells through the E-selectin-SLex interaction (22). These results sug gest that SLex may serve as a ligand for E-selectin and its expression

U correlates with the metastatic potential of cancer cells. In this study, .@ 40 we have shown that patients with tumors which strongly expressedthe Sialyl Lewis x (â€”X84) E Fuc-TIV and/or Fuc-TVJJ genes have a poor prognosis, and that a â€”â€”â€”.â€” SialylLewisx(+)(117) C.) 20 correlation between SLex and Fuc-TVII expression exists. This is in Sialyl Lewis x (4â€”f)(132) agreement with the finding that Fuc-TVII can efficiently use a-2,3- sialyllactosamines to form SLex (13, 14). It is not clear, however, 0 whether the correlation between Fuc-TIV expression and prognosis is 100 related to its role in the synthesis of SLex becauseFuc-TIV reportedly acts poorly on a-2,3-sialyllactosamines (8, 9). Goelz et al. (23) reported that, in some cultured cell line transfected with the Fuc-TIV 80 gene, sialyllactosamines can be fucosylated according to the range of carbohydrate structures of acceptors,and the SLex structure expressed I60 is likely to have more polylactosamine content with more than one fucosylated sites. In fact, the antigen defined by the FH6 monoclonal antibody used in this study is identified as a SLex carried by difuco ..@ 40 Fuc-TIV(--)(82) syl- or trifucosyl-type 2 chain as well as a long-type chain (24). It is E U 20 @-â€”.â€” Fuc-TIV(+)(82) Fuc-TIV(++)(169) 100 -@

0 @ -i_ 100 80 -@--.@

80 60

C,) 60 5)

C,,) 40 U Fuc-TIV (â€”)and VII (â€”)(54)

40 @-@- Fuc-TJV (+) and/or VII (+) (85) Fuc-TVII (â€”)(110) U 20 â€”...â€”...â€” Fuc-TJV (++) or VII (++) (62) U â€”â€”â€”.@ â€”Fuc-TVII(+Ã·)(157)Fuc-TVll(+)(6@) @â€”Fuc-TIV(+-,-)andVII(++)(132) 0â€” . . . . 0 0 1 2 3 4 5 5 Year Year Fig. 3. Survival according to Fuc-TIV and Fuc-TVII expression. Each tick mark Fig. 2. Survival according to sialyl Lewis x. Fuc-TIV, and Fuc-TVII expression. representsa patient who is alive or died of causesother than primary lung cancer. Group Survival curves were calculated using the Kaplan-Meier method, and statistical evaluation 1: Fuc-TIV(â€”)andFuc-TVII(â€”);Group2: Fuc-TIV(+) and/orFuc-TVII(+); Group3: wasdoneusingthe log rank test.P < 0.01: SLex(+) versus(+ +), Fuc-TIV(â€”)versus Fuc-TIV(+ +) or Fuc-TVH(+ +); Group 4: Fuc-TIV (+ +) and Fuc-TVII(+ +). (+ +) Fuc-TIV(+) versus(+ +), Fuc-'I'VII(â€”)versus(+ +); P < 0.02: SLex(â€”)versus P < 0.01: group 1 versus3, group 1 versus4, group 2 versus3, group 2 versus4; not (++); P < 0.03: Fuc-TVII(+) versus(++). statistically significant: group I versus 2, group 3 versus 4. 327

100 pression in our study may reflect the retrodifferentiation of cancer cells. Expression of the Fuc-TIV and Fuc-TVII genes correlate with 80 poor prognosis by participating in the synthesis of SLex and may be of prognostic value in lung cancer. It will be important to understand the role of fucosyltransferases in the formation of 60 SLex, since this might lead to anticancer strategies based on the down-regulation of SLex expression via inhibition of fucosyltrans C,) 5) ferase activity. 40

Stage I :Group 1 and 2 (78) REFERENCES U 20 ______StageI :Gmup3and4 (84) 1. Phillips,M.S.,andPaulson,J.C.ELAM-lmediatescelladhesionbyrecognitionofacarbohy L., Nudelman,E.,Gaeta,F.C. A., Perez,M., Singhal,A. K., Hakomori, â€” â€”- Stage III :Group 1 and 2 (46) drate ligand, sialyl-LeÂ°.Science (Washington DC), 250: 1130â€”1132,1990. Stage III :Group 3 and 4 (85) 2. Walz, G., Aruffo, A., Kolanus, W., Bevilacqua, M., and Seed, B. Recognition by 0 . . . . @. ELAM-lof thesialyl-LeÂ°determinantonmyeloidandtumorcells.Science(Wash 0 1 2 3 4 5 ingtonDC),250: 1132â€”1135,1990. @ Year Tiemeyer,M.,Swiedler,S.J.,Ishihara,M.,Moreland,M.,Schweingruber,H., Hirtzer, P., and Brandley, B. Carbohydrate ligands for endothelial-leukocyte adhesion molecule-l. Proc. Natl. Acad. Sci. USA, 88: 1138â€”1142,1991. 4. Hoff, S. D., Matsushita, Y., Ota, D. M., Cleary, K. R., Yamori, T., Hakomori, S., and 100 â€” Irimura,T.Increasedexpressionofsialyl-dimericLeÂ°antigenin livermetastasesof human colorectal carcinoma. 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Jun-ichi Ogawa, Hiroshi Inoue and Shirosaku Koide

Cancer Res 1996;56:325-329.

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