Clinical Significance of CD99 Down-Regulation in Gastric

Human Cancer Biology

Clinical Significance of CD99 Down-Regulation in Gastric Adenocarcinoma Jung Hyun Lee,1Seok-Hyung Kim,5 Li-Hui Wang,1, 6 Yo o n-L a C h o i, 4 Yo un g C hul K i m , 2 Jin Hee Kim,3 Tae Sung Park,2 Yun-Chul Hong,3 andYoung Kee Shin 1

Abstract Purpose: CD99 is a cell adhesion molecule associated with human tumors.The aim of the present study was to characterize its role in the development and progression of human gastric adenocarcinoma. Experimental Design:The expression of CD99 was investigatedin 283 gastric adenocarcino- mas andrelatedlesions and9 gastric carcinoma cell lines.We also analyzedthe methylation status of CD99 gene by using methylation-specific PCR andexaminedloss of heterozygosity (LOH) of this gene locus by using an intragenic marker. Moreover, we assessedwhether SP1, a positive transcription factor for CD99, is expressedin these samples. Results: We foundthat the decreasedexpression of CD99 was strongly associatedwith poor survival andunfavorable clinicopathologic variables. Promoter region methylation (15 of 89, 16.9%) andLOH (21 of 74, 28.4%) were observedandsignificantly associatedwith CD99 down-regulation (P < 0.05). In addition, most of the gastric adenocarcinoma cases with CD99 down-regulation had reduced expression of SP1 (47 of 103, 45.6%; P < 0.01). This relationship between CD99 andSP1was consolidatedby using SP1small interfering RNA transfection exper- iment and CD99 promoter luciferase assay. Furthermore, we showedthat CD99 down-regulation was associatedwith proliferation andmigration in gastric carcinoma cell line. Conclusion: These observations suggest that CD99 down-regulation is a critical event in the progression of gastric adenocarcinoma, and CD99 promoter methylation, CD99 LOH, andSP1 down-regulation were responsible for the down-regulation of CD99.

Gastric carcinoma is the second leading cause of cancer splicing variants CD44v6 and CD44v9 (2–6). A germline mortality in the world (1). A number of cell adhesion mutation in CDH1 has been frequently observed in hereditary molecules have been examined as potential clinical biomarkers gastric carcinoma, and multivariate analyses have revealed that of gastric adenocarcinoma, including CDH1 (E-cadherin), CDH1 down-regulation is an independent prognostic factor for FXYD5 (dysadherin), TACSTD1 (Ep-CAM), and the CD44 gastric cancer (7). In addition, increased expression of FXYD5, a CDH1 inactivator, has been significantly associated with poor prognosis (8, 9). TACSTD1 down-regulation and CD44v6 up- Authors’Affiliations: 1Department of Pharmacy, Research Institute of regulation have been also suggested to be prognostic markers Pharmaceutical Science, Seoul National University College of Pharmacy; for gastric cancer (5, 6). 2Department of Statistics, College of Nature Science, Seoul National University; 3Department of Preventive Medicine, Seoul National University College of Another interesting adhesion molecule is CD99, which is a Medicine; 4Department of Pathology, Samsung Medical Center, Sungkyunkwan sialomucin-type glycoprotein that is ubiquitously expressed, University School of Medicine, Seoul, Korea; 5Department of Pathology, albeit to varying degrees, by different cell types (10). Although Chungbuk National University College of Medicine, Cheongju, Chungbuk, Korea; its function and signaling pathway are not fully understood, it and 6Department of Pharmacology, Shenyang Pharmaceutical University College of Pharmacy, Shenyang, China is known that CD99 is involved in a number of cellular events Received7/21/06; revised1/25/07; accepted2/19/07. such as cell-cell adhesion (11, 12), maintenance of cellular Grant support: Seoul R&BD Program, Korea Health 21 R&D Project of the morphology (13), and cell death (14, 15). CD99 also regulates Ministry of Health andWelfare grant A050260 Korea, andNational Research the adhesion and diapedesis of leukocytes to inflamed vascular Laboratory Program of Korea Science andEngineering Foundation grant M10500000126 (T.S. Park). endothelium (16). Recently, it was proposed that CD99 down- The costs of publication of this article were defrayed in part by the payment of page regulation, induced by the EBV-encoded latent membrane charges. This article must therefore be hereby marked advertisement in accordance protein-1, in B cells is involved in the pathogenesis of with 18 U.S.C. Section 1734 solely to indicate this fact. Hodgkin’s disease (17). Furthermore, the increased or de- Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). creased expression of CD99 has been also suggested to act as a J.H. Lee andS.H. Kim contributedequally to this work. marker of various kinds of tumors, including Ewing’s sarcoma/ Requests for reprints: Young Kee Shin, Molecular Pathology Laboratory, primitive neuroectodermal tumor (18), lymphoblastic lympho- Department of Pharmacy, Seoul National University College of Pharmacy, San 56- ma/leukemia (19), some rhabdomyosarcomas (20), granulose 1, Sillim-dong, Gwanak-gu, Seoul, 151-742, Korea. Phone: 82-2-880-9126; Fax: cell tumor and sertoli-leydig cell tumor of the ovary (21), 82-2-872-1795; E-mail: [email protected]. F 2007 American Association for Cancer Research. pancreatic endocrine tumors (22), gallbladder carcinoma doi:10.1158/1078-0432.CCR-06-1785 (23), and stomach carcinoma (24). Based on these previous

Clin Cancer Res 2007;13(9) May 1, 2007 2584 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. CD99 Down-Regulation in Gastric Carcinoma reports, we speculated that altered CD99 expression could also paraformaldehyde, and analyzed on a FACSCalibur (BDIS) by using the be a prognostic marker for gastric carcinoma patients. CellQuest software (BDIS). In the present study, we examined the expression of CD99 by Luciferase assay. CD99 promoter (-1641/+123) reporter and SP1 normal gastric samples, gastric carcinomas, and other gastric expression constructs were gifts from Dr. I.S. Lee (Konkuk Univer- sity, Korea) and Dr. M.W. Hur (Yonsei University School of Medicine, lesions by using immunohistochemistry-based tissue micro- Korea), respectively. Luciferase assay was done at SNU484, SNU638, array analysis and explored the clinical significance of CD99 MKN74, KATOIII, and AGS cells according to standard procedures down-regulation in gastric adenocarcinoma. In addition, we (see Supplementary Materials and Methods). evaluated the mechanism by which CD99 becomes down- SP1 and CD99 small interfering RNA transfection and real-time regulated in gastric carcinoma by assessing the methylation reverse transcription-PCR. The sense sequences of SP1 small interfer- status of the CD99 promoter, loss of heterozygosity (LOH) at ing RNA (siRNA; Santa Cruz Biotechnology), CD99 type I–specific the CD99 gene locus, and expression of SP1, which is a siRNA, and green fluorescent protein siRNA (control) are 5¶-AAUGA- transcription factor that induces CD99 expression. Further- GAACAGCAACAACUCC-3¶,5¶-GAAAGGCTGGCCATTATTA-3¶, and 5¶- ¶ more, we showed the pathophysiologic role of CD99 down- GGCUACGUCCAGGAGCGCACC-3 , respectively. Transfections were regulation in gastric carcinoma. done according to standard procedures (see Supplementary Materials and Methods). Real-time quantitative reverse transcription-PCR was carried out using the LightCycler Real-time PCR Detection system Materials and Methods (Roche Diagnostics). PCR primers for amplifying CD99 (Type I) and the control gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) are Patients, tissue samples, immunohistochemistry, and evaluation of shown in Supplementary Table S1. CD99 (Type I) and HPRT were results. Tissues from patients with gastric carcinoma and the other detected in the same tube at 530 and 705 nm by using FAM-conjugated gastric lesions, consisting of 114 gastric carcinomas, 20 high-grade Universal Probe Library probe 21 and DYXL-labeled HPRT probe (TIB dysplasias, 17 low-grade dysplasias, 48 intestinal metaplasias, 30 MOLBIOL GmbH) as the Taqman probe, respectively. chronic atrophic gastritis, and 54 normal gastric epithelium, were used Establishment of CD99-overexpressing stable cell line and migration for tissue microarray construction as previously described (25). Ethics assay. CD99 and empty vector were transfected into SNU216, committee permission for this study was obtained from the institu- SNU484, KATOIII, and AGS by using a LipofectAMINE LTX (Invitro- tional review board of Chungbuk National University Hospital. gen). Transfected cells were passaged after 1 day and selected with 400 Immunostaining of CD99 and SP1 was done on consecutive sections, Ag/mL G418 for 10 days. AGS and SNU484 were just established into using two anti-CD99 monoclonal antibodies (clones DN16 and YG32; CD99-overexpressing stable lines. Cell migration assay was done using DiNonA) and rabbit polyclonal anti-SP1 antibody (Santa Cruz established stable cell lines according to standard procedures (see Biotechnology, Inc.). The evaluation of both the intensity of immuno- Supplementary Materials and Methods). histochemical staining and the proportion of positively stained Statistical analysis. Means were compared by the nonparametric epithelial cells was done as previously described (25). A detailed methods Wilcoxon’s rank sum test and Kruskal-Wallis test. The description of the analysis is provided in Supplementary Materials and association of the expression rate with clinicopathologic factors was 2 Methods. assessed by Fisher’s exact test and Pearson’s m test. Kaplan-Meier Methylation-specific PCR and LOH assay. Genomic DNA extraction analyses were done for the survival times of gastric adenocarcinoma from the tissues microdissected from slides, normal lymphocytes, and patients. Log-rank tests were used to compare the survival curves gastric cancer cells; bisulfate treatment of DNA; and methylation- between groups. The contribution of each mechanism to protein down- specific PCR were done as previously described (25, 26). DNA from regulation was determined by using the logistic regression model. normal lymphocytes methylated in vitro with SssI methylase was used Accuracy with which each mechanism predicts protein down-regulation as the positive control, and both water blanks and PCR mixtures was provided in Supplementary Materials and Methods. P < 0.05 was without template were used as negative controls. All methylated and regarded statistically significant. All statistical analyses were done by representative unmethylated PCR fragments were sequenced (for detail, using SPSS software (SPSS). see Supplementary Materials and Methods). Primers and PCR conditions used are also provided in Supplementary Table S1. Briefly, Results we designed two primer sets [i.e., (A) primers that amplify an area in the promoter region (-273 bp to -57 bp from the transcription initiation Expression of CD99 by gastric carcinoma and other gastric site) and (B) primers that amplify an area that is downstream of the lesions. The expression of CD99 by gastric adenocarcinoma transcription initiation site (-105 bp to +106 bp)]. and other gastric lesions was determined by immunohisto- LOH was assessed by subjecting tumor and normal DNA to PCR chemical analysis. In normal gastric epithelia, CD99 expression amplification of DYS403, which is a microsatellite locus that is located between exons 1 and 2 of the CD99 gene on the short arm of was strongest around the mucus neck cells and decreases chromosomes Xp22.3 and Yp11.3.7 PCR amplification and analysis gradually as these cells differentiate into mucus and glandular were done as previously described (for detail, see Supplementary cells (Supplementary Figure S1A). Notably, CD99 was consti- Materials and Methods; ref. 27). tutively expressed at the basolateral border of epithelial cells Cell lines, Western blotting, and flowcytometric analysis. Nine (Supplementary Figure S1B). CD99 expression was found to be human stomach cancer cell lines (i.e., SNU5, SNU16, SNU216, significantly decreased in the gastric carcinoma and intestinal SNU484, SNU638, MKN28, MKN74, KATOIII, and AGS) were metaplasia samples in terms of both the average immunoreac- maintained. Western blotting was done by using primary antibodies tive score and the expression rate (Fig. 1A; Supplementary against CD99 and SP1 and ACTB (see Supplementary Materials and Fig. S2). Comparing with normal gastric epithelia, there is no Methods), and CD99 and SP1 expression were semiquantified by significant change in low-grade dysplasia and high-grade scanning densitometry with ACTB normalization. For flow cytometric analysis, 106 cells were incubated with FITC-conjugated CD99 (YG32) dysplasia, but a slightly decease in early gastric cancer and an for 20 min at 4jC, washed three times with cold PBS, fixed with 1% obvious decrease in advanced gastric cancer (Fig. 1A; Supple- mentary Table S2). Only 34.5% (39 of 113) of the carcinoma cases showed CD99 expression, whereas almost 90% of the 7 Genome Database (http://www.ensembl.org/). normal gastric mucosa, chronic atrophic gastritis, and dysplasia

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Fig. 1. Immunohistochemical analysis andsurvival analysis of CD99 expression by gastric carcinoma samples. A, gastric carcinoma (n =113),high-gradedysplasia(HD ; n = 20), low-grade dysplasia (LD ; n = 17), intestinal metaplasia (IM ; n = 48), chronic atrophic gastritis (CAG; n = 30), andnormal gastric epithelium ( NL ; n = 54) samples were subjectedto immunohistochemical analysis of CD99 expression. Left, average immunoreactive score ( IS) for each type of lesion. Right, frequency of samples positive for CD99 expression. A sample is defined as CD99+ if it has an immunoreactive score z1. B, relationship between the immunoreactive scores of individual gastric carcinoma sample and its stage. The gastric carcinoma patients were divided according to their stage (I-IV), and the immunoreactive scores of their samples are shown in colors. C, CD99 expression for representative gastric adenocarcinoma samples at each carcinoma stage. Membranous staining of CD99 was frequently observed in the early stages (I andII) but was infrequent in higher-stage (IIIandIV) samples. D, Kaplan-Meier curves of the overall survival of gastric carcinoma patients in early stages (I andII) and advanced stages (III and IV) whose cancer samples are positive (immunoreactive score z1) or negative (immunoreactive score <1) for CD99 expression. samples expressed CD99. Of the intestinal metaplasia samples, expression was significantly associated with better prognosis 47.9% (23 of 48) were positive for CD99 expression. In (risk ratio, 0.313; P < 0.01). Moreover, Kaplan-Meier survival addition, CD99 expression was completely absent in the analysis that was separately done for the early-stage (I and II) complete type of intestinal metaplasia but present in the and advanced stage (III and IV) groups showed that the absence incomplete type (Supplementary Figure S1C and D). of CD99 expression was significantly associated with shorter Down-regulation of CD99 in gastric carcinoma is associated with survival times in advanced stage patients (P < 0.05; Fig. 1D). unfavorable clinicopathologic variables and poor prognosis. Hypermethylation of the CD99 promoter region and LOH of How CD99 expression in gastric carcinoma relates to various the CD99 gene locus is associated with CD99 down-regulation. clinicopathologic variables was analyzed. Reduced expression To elucidate the mechanism by which CD99 is down-regulated of CD99 was significantly associated with all unfavorable in gastric carcinoma, we explored the methylation status of the variables, including greater tumor size, nodal metastasis, CD99 promoter region by using methylation-specific PCR and advanced gastric cancer, diffuse type, poor differentiation LOH of the CD99 gene locus. When the (A) primers were (high grade), deep tumor invasion, and advanced clinical employed, methylation was found in 15 of the 89 cases of staging (tumor-node-metastasis staging; Fig. 1B and C; gastric cancer (16.9%; Fig. 2A-C), and a significant correlation Supplementary Table S2). These findings were observed for between promoter methylation and the absence of CD99 both the expression rate and the average immunoreactive expression was observed (P < 0.05; Tables 1 and 2). In contrast, score. when the (B) primers were used, methylation was rarely Kaplan-Meier survival analysis revealed that, like other detected (4 of 68, 5.9%), and no significant association with unfavorable prognostic factors, reduced CD99 expression was CD99 down-regulation was found (Tables 1 and 2). In significantly associated with decreased 1-year and 3-year overall addition, methylation in the promoter was associated with survival (P < 0.01; Table 1). The mean survival time was 60.1 decreased 1-year/3-year survival (P < 0.01; Table 1), whereas months for CD99- gastric carcinoma patients (74 cases) and methylation in the downstream region showed no such 81.0 months for CD99+ patients (39 cases; P < 0.01). Analysis correlation. Despite these clinicopathologic implications of of the overall survival showed that the presence of CD99 CD99 promoter methylation, we could not detect similar

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noma is due to altered expression of SP1 was assessed by Table 1. Univariate analysis of the overall survival immunohistochemical analysis because the promoter region of of stomach cancer patients by Kaplan-Meier CD99 contains several SP1-binding sites (Fig. 2A; refs. 28, 29). survival analysis Down-regulation of SP1 in gastric carcinoma was found in 47

Variables No. Overall P* of 113 cases (41.6%) and was significantly associated with the patients survival (%) down-regulation of CD99 expression (P < 0.05; Table 2). However, decreased expression of SP1 was not associated with 1y 3y decreased 1-year/3-year survival (Table 1). Sex 0.176 When the protein expression of CD99 and SP1 were Male 74 84.96 69.70 semiquantified, the SNU5, SNU16, MKN28, and MKN74 cell Female 40 82.50 55.00 Age 0.251 lines showed high expression of both CD99 and SP1 (Fig. 3A <60 54 85.1968.22 and B). However, the amount of CD99 transcript in SNU484, z60 60 83.07 61.03 SNU216, KATOIII, and AGS did not correlate with the protein Tumor stage <0.0001 expression of Sp1 (Fig. 3B). When CD99 promoter assay was I-II 52 98.04 98.04 III 36 83.33 52.78 done, the discrepancy of SP1 and CD99 observed in SNU484 IV 26 57.69— and KATOIII could be explained. In the case of SNU484, CD99 Tumor size 0.0004 promoter was activated by itself, and SP1 inhibited CD99 <5.0 4989.6283.32 promoter activity (Fig. 3C). On the other hand, CD99 promoter z 5.0 65 80.00 50.77 was not activated by Sp1 in KATOIII (Fig. 3C). In SNU638, Gross type <0.0001 AGC 82 78.05 51.22 MKN74, and AGS, SP1 activated CD99 promoter (Fig. 3C). EGC 32 100 100 Furthermore, when SP1 siRNA was introduced into in the high Grade 0.0003 CD99- and SP1-expressing line MKN74, SP1 siRNA dramati- Sig to 1 42 97.62 85.16 cally down-regulated CD99 transcription (Fig. 3D-F). 2-3 72 76.3952.78 Histologic type 0.504 CD99 down-regulation was associated with proliferation and Intestinal 66 84.64 61.24 migration in gastric carcinoma cell line. CD99-overexpressing Diffuse 46 82.61 69.57 AGS stable cell line showed reduced migration and cell Mixed 2 100 100 proliferation (Fig. 3G-I). In accordance with, CD99 knockdown Chemotherapy 0.123 in MKN74 increased migration (Fig. 3J and K). However, No 65 82.83 71.83 Yes 4985.71 55.10 apoptosis was not induced in transiently CD99-transfected CD99 0.002 KATOIII (data not shown). These results showed that CD99 <1 74 79.73 54.05 may have essential role in migration and proliferation in gastric z1 3992.1184.13 carcinoma. MSPCR (A) 0.006 - 74 85.14 61.90 Contribution of SP1 down-regulation, LOH, and promoter + 15 60.00 33.33 methylation to CD99 down-regulation in gastric adenocarcinoma. MSPCR (B) 0.975 To assess the effect of promoter methylation, LOH, and SP1 - 64 77.80 48.82 down-regulation on CD99 expression, we did multivariate +4—— logistic regression analysis using these variables. We found that MSPCR (A/B) 0.210 Both - 52 80.77 53.37 all variables were significantly associated with CD99 expres- Either + 17 64.71 41.18 sion (Table 2). Notably, promoter methylation and CD99 LOH (total) 0.043 LOH showed high odds ratio for CD99 down-regulation - 53 84.68 69.00 (Table 2). These changes were not observed in most of cases + 21 66.67 — SP1 0.339 without showing CD99 down-regulation (1 of 28, specificity = <2 47 82.98 59.57 96.4% for promoter methylation and 2 of 22, specificity = z2 56 83.64 68.87 96.4% for CD99 LOH), suggesting that these mechanisms are specifically related to CD99 down-regulation (Supplementary Abbreviations: EGC, early gastric cancer; AGC, advanced gastric Materials and Methods). In addition, SP1 down-regulation cancer; MSPCR, methylation-specific PCR. were responsible for more cases showing CD99 down- *P of the log-rank test. regulation than methylation and LOH (Supplementary Materi- als and Methods). Furthermore, decreased expression of SP1, CD99 LOH, and CD99 promoter methylation can together promoter methylation in nine gastric adenocarcinoma cell lines predict about 81% of the gastric carcinoma cases with CD99 (data not shown). down-regulation, suggesting that these constitute the major LOH of the CD99 gene locus was found in 21 of 74 cases mechanism of inducing it (Supplementary Materials and (28.4%) and was significantly associated with down-regulation Methods). of CD99 expression (P < 0.05; Table 2). In addition, LOH of the CD99 gene locus was associated with decreased 1-year/3- Discussion year survival (P < 0.05; Table 1). Analysis of nine cell lines revealed that SNU638 and AGS also showed LOH of the CD99 Here, we studied the expression of CD99 in gastric carcinoma gene locus (data not shown). samples and determined its clinical significance, particularly SP1 expression in gastric carcinoma was associated with CD99 with regard to the prognosis of the patients. We found that the expression. Whether CD99 down-regulation in stomach carci- expression of CD99 was clearly down-regulated in gastric

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Fig. 2. Methylation status of the CD99 promoter region andLOH status of CD99 in gastric carcinoma. A, schematic depiction of the region of the CD99 gene between -400 and+200 bp relative to the transcription start site. Brown arrows, primary PCR primers; black arrows, secondary PCR primers; purple arrows, SP1-bin ding sites; black circles, CpGs. B, methylation-specific PCR (MSPCR)oftheCD99 promoter in representative gastric carcinoma cases. U, nonmethylated; M, methylated; PC, positive control; DW, distilled water. Methylation-specific PCR was done by using sequences upstream (A) and downstream (B) of the transcription start site.C, representative sequences of the methylation-specific PCR (A) and(B) products. Arrows, unmethylatedandmethylatedsites. Although the sequence from the unmethylatedDNA samp le shows the C-to-Tconversion (arrows), the corresponding sequence of the methylated case shows the retention of Cs (arrows). D, schematic depiction of the CD99 gene locus showing the location of the microsatellite marker DYS403. E, representative results after assessing normal gastric muscosa (N)andtumorspecimens(T) from two gastric carcinoma patients. Arrow, LOH. carcinoma, particularly for advanced stage (P < 0.001). In chemical analysis, CD99 promoter assay, and an in vitro siRNA addition, the loss of CD99 expression was strongly associated transfection study. with a poor prognosis (P < 0.001). We also analyzed the Although CD99 has been suggested to participate in various potential mechanisms that could be responsible for CD99 physiologic processes, its exact functions, particularly those in down-regulation in gastric carcinoma and found that methyl- epithelial cells, remain unclear. Unlike lymphoid cells, epithe- ation of the CD99 promoter and CD99 LOH can account for lial cells have been rarely reported to express CD99. Here, we the cases with CD99 down-regulation. One of supporting found that in normal gastric epithelia, CD99 is constitutively evidence for this is our observation that the SNU638 cell line, expressed at their basolateral border. The location and which shows CD99 LOH, also expresses CD99 at low levels. expression pattern of CD99 and its down-regulation in gastric Finally, we found that loss of SP1 expression, which is a potent carcinoma progression suggest that this molecule could act as positive regulator of CD99, accounts for many of the CD99- an intercellular adhesion molecule. The role of CD99 as an gastric carcinoma cases. This was revealed by immunohisto- intercellular adhesion molecule has already been previously

Table 2. Analysis of the effect of promoter methylation, LOH, and SP1 down-regulation on CD99 expression by using the logistic regression model

Predictor Univariate analysis Multivariate analysis* B (SE) P Odds ratio B (SE) P Odds ratio

MSPCR (-) vs (+) 2.11 (1.06) 0.048 8.22 2.23 (1.090) 0.051 9.33 LOH (-) vs (+) 1.18 (0.796) 0.025 3.25 2.20 (0.859) 0.010 9.06 SP1 (z2) vs (<2) 1.15 (0.435) 0.008 3.16 1.68 (0.701) 0.017 5.35

*Goodness of fit, 61.5 (P = 0.385).

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Fig. 3. Transfection of gastric cell lines with SP1siRNA decreases their CD99 expression. A, CD99 andSP1protein expression profiles of nine gastric carcinoma cell lines.The Western blot bands were analyzed by scanning densitometry. B, comparison of CD99 mRNA andSP1protein levels in nine gastric carcinoma cell lines. CD99 mRNA levels were analyzedby real-time PCR with HPRT1normalization. SP1protein levels were estimatedas describedin ( A). C, effect of SP1on the promoter activity of CD99. D, effect of SP1siRNA on CD99 expression by MKN74. E, fluorescence-activatedcell sorting analysis of the CD99 expression of MKN74 transfectedwith green fluorescent protein (GFP) or SP1siRNA. Gray line, anti-CD99 antibody ^ stained cells; black profile, control antibody ^ stained cells; R.I., relative intensity. F, immunoblot analysis of the CD99 andSP1expression of MKN74 cells transfectedwith green fluorescent protein or SP1siRNA. G, fluorescence-activatedcell sorting analysis of the CD99 expression in CD99-overexpressing stable AGS. Gray line, CD99; black profile, control. H, migration assay of CD99-overexpressing stable AGS. I, effect of CD99 in proliferation rate. J, quantitative real-time PCR analysis of CD99 expression. K, effect of CD99 knockdown on migration by MKN74. documented in T cells (11, 12). Moreover, upon CD99 supporting the suggestion that gastric carcinoma cells arise stimulation, an Ewing’s sarcoma cell line forms cell junctions not from metaplastic cells but from gastric cells and then lose that resemble adherens junctions (30). It is known that CDH1 their identity as tumor progression (31). First, the loss of down-regulation is critical for the formation of adherens CD99 expression was also observed in intestinal metaplasias junctions and gastric carcinogenesis (7). Thus, it is possible of complete type, in which the gastric cell identity was lost, that CD99 signaling is involved in epithelial cell-epithelial cell through the gastritis-dysplasia-carcinoma sequence, suggesting adhesion, similarly to CDH1. that the intestinal metaplasia should be recognized as a With regard to gastric carcinoma progression, the loss of paraneoplastic phenomenon (31). Second, this late event CD99 expression can be considered as a later event, may be responsible for the loss of intercellular adhesion

www.aacrjournals.org 2589 Clin Cancer Res 2007;13(9) May 1, 2007 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology capability. CD99 down-regulation was clearly associated with relationship between CD99 and SP1, we did CD99 promoter increased direct tumor invasion and nodal metastasis. This assay and verified that SP1 may be critical for CD99 promoter agrees well with our previous clinicopathologic study for the activity in several cancer cell lines. Furthermore, the combina- loss of CD99 expression in gallbladder carcinomas (23). tion of CD99 promoter methylation, CD99 LOH, and SP1 Notably, the CD99-overexpressing AGS cell line showed down-regulation can account for nearly 81% of the gastric reduced migration and proliferation, and CD99 knockdown cancer cases with CD99 down-regulation. It is likely that the in MKN74 increased migration. In accordance with, the remaining 19% of cases may be explained by additional forced expression of CD99 in the osteosarcoma cell lines mechanisms that lead to the loss of CD99 expression, such as significantly inhibited cell migration and abrogated their mutations, posttranslational modifications, and additional metastatic ability (32). Third, it is possible that CD99 down- derangements of other transcription factors. We also suggest regulation is related to the loss of cellular identity or that CD99 mutations could be responsible to some degree for differentiation. We found that loss of CD99 expression by CD99 down-regulation as the KATOIII cell line contains a point tumor cells was clearly associated with their loss of cellular mutation or an unknown single nucleotide polymorphism in polarity and their failure to maintain a glandular structure in its 5¶ untranslated region and expresses little CD99 mRNA. It is gastric carcinoma cases. It has also been shown that the possible that such CD99 alteration may decrease mRNA CD99 down-regulation leads to the loss of normal morphol- stability (Supplementary Materials and Methods; Supplemen- ogy in Hodgkin’s disease (13, 17). Fourth, because the mouse tary Table S3). CD99 orthologue has been found to specifically activate In conclusion, this study revealed that loss of CD99 natural killer cells and dendritic cells by binding to PILRB expression in gastric carcinoma is clearly associated with poor (33, 34), the loss of CD99 in gastric carcinoma may serve as prognosis and unfavorable clinicopathologic variables. Most of an immune escape mechanism. the cases with loss of CD99 expression can be explained by a When the contributions of CD99 promoter methylation, combination of loss of SP1 expression, LOH of the CD99 gene CD99 LOH, and SP1 down-regulation to CD99 down- locus, and methylation of the CD99 promoter region. We also regulation in gastric carcinoma were analyzed by using a showed that CD99 down-regulation was associated with logistic regression model, it was found that the CD99 down- proliferation and migration in gastric carcinoma cell line. regulation can be explained much better by all three potential These observations suggest that the loss of CD99 expression is a causes together than by just one. It was shown that CD99 critical event in gastric carcinoma progression. promoter methylation and LOH specifically occur when CD99 is down-regulated. In contrast, whereas over half of the cancer Acknowledgments cases with CD99 down-regulation had decreased expression of + SP1, SP1 down-regulation was also observed in some CD99 We thank Jung Sun Lee andMeeYoung Sim for their technical assistance, inclu- cases. To confirm the clinicopathologic study about the ding slide cutting and immunohistochemistry.

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Jung Hyun Lee, Seok-Hyung Kim, Li-Hui Wang, et al.

Clin Cancer Res 2007;13:2584-2591.

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