Tohoku J. Exp. Med., 2012, 226, 301-311The Newly Identified Expressed in Human ESCC 301

Identification of Distinct Expression Profiles between Esophageal Squamous Cell Carcinoma and Adjacent Normal Epithelial Tissues

Yisheng Tao,1,2,3 Damin Chai,2,3 Li Ma,2,3 Ting Zhang,4 Zhenzhong Feng,2,3 Zenong Cheng,2,3 Shiwu Wu,2,3 Yanzi Qin2,3 and Maode Lai1

1Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, P.R. China 2Department of Pathology, Bengbu Medical College, Bengbu, P.R. China 3Anhui Key Laboratory of Infection and Immunity, Bengbu Medical College, Bengbu, P.R. China 4Department of Pathology, Fuyang People’ s Hospital, Fuyang, P.R. China

Esophageal squamous cell carcinoma (ESCC) is a predominant type of esophageal cancer, which is a malignant tumor originating from the esophageal mucosa or gland and is aggressive with poor prognosis. Identification of new patterns would be helpful for providing new targets for the early detection and treatment of ESCC patients. In the present study, we employed cDNA array technology to compare gene expression profiles between ESCC tissues and adjacent normal epithelial tissues from ESCC patients. There was at least a 4-fold change in the expression levels of 72 genes that were significantly increased and 107 genes that were decreased in ESCC compared with normal esophageal epithelium. Among them, genes known to be involved in ESCC were found, including matrix metalloproteinases, transcription factors SOX-4 and SOX-17, the Wingless-type MMTV integration site family member 2, and cell cycle regulators. Moreover, we have newly identified the two genes that are down-regulated in ESCC: monoamine oxidase A, an enzyme that catalyzes monoamines oxidation and 15-hydroxyprostaglandin dehydrogenase [NAD+], a prostaglandin-synthesizing enzyme that physiologically antagonizes COX-2. Likewise, we found the three genes that are up-regulated in ESCC: CD7, a cell surface glycoprotein member of the immunoglobulin superfamily, LIM-domain kinase 1, a small subfamily with an unique combination of two N-terminal LIM motifs and a C-terminal kinase domain, and TTK , a previously unidentified member of the kinase family. These newly identified genes may be involved in the progression of the tumor and/or represent properties specific to ESCC.

Keywords: cDNA microarray; esophageal squamous cell carcinoma; gene expression; immunohistochemistry; normal epithelial tissue Tohoku J. Exp. Med., 2012, 226 (4), 301-311. © 2012 Tohoku University Medical Press

Esophageal cancer (EC) is a malignant tumor that there is an urgent need to clarify the molecular mechanism arises from the mucosa or gland of the esophagus. EC is underlying esophageal carcinogenesis. the sixth leading cause of cancer-related deaths, and one of cDNA microarray technology provides a powerful tool the most common cancer types around the world (Parkin et for large-scale gene expression studies (Hu et al. 2011). al. 2005; Jemal et al. 2010). Esophageal squamous cell car- Thus, cDNA microarrays allow simultaneous expression cinoma (ESCC) is a predominant type of EC worldwide, analysis of thousands of genes from tumor samples. Recent and comprised more than 90% of all EC cases (Daly et al. progress in the microarray-based assessment has the poten- 2000). Despite general advances in diagnosis and treat- tial to find clinical biomarkers in various cancers and to ment, long-term survival is still low with a 5-year survival predict patient outcomes, lymph node metastasis, and the rate of about 10–15% (Enzinger and Mayer 2003). One response to therapy (Schena et al. 1995; DeRisi et al. 1996; promising strategy to reduce ESCC mortality is early detec- Ramsay 1998). Several microarray studies have investi- tion and treatment. However, EC is an aggressive tumor gated the gene expression profiles in EC tissue and cell that is typically diagnosed only after the onset of symptoms, lines (Hu et al. 2001; Lu et al. 2001; Kan et al. 2001). which results in a poor prognosis (Jemal et al. 2010). Thus, However, useful information that can be derived from each

Received January 16, 2012; revision accepted for publication March 28, 2012. doi: 10.1620/tjem.226.301 Correspondence: Maode Lai, Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, 388 Yuhangtang Road, Hangzhou 310058, P.R. China. e-mail: [email protected]

301 302 Y. Tao et al. study was limited by the different resolutions of those stud- containing 37 μL 1.5 × Human OneArray Hybridization Buffer, 1 μL ies, and more gene expression studies are necessary to Sheared Salmon Sperm DNA (10 μg/μl), 17 μL target preparation plus obtain additional information from ESCC. nuclease-free ddH2O. The mixture was centrifuged for 5 min at max- In the present study, we employed a Human OneArray imum speed to eliminate potential debris. Then it was transferred to a microarray with 28,703 human genes to detect gene expres- PCR tube and denatured in the PCR machine at 95°C for 5 min. The sion changes in ESCC tissues and compared them with hybridization mixture was transferred onto the spotted region of OneArray DNA Microarray, being sure to avoid creating any bubbles. matched normal epithelial tissues from ESCC patients. The entire labeled target plus the microarray setup was placed into a Materials and Methods closable, chambered box, which was humidified by 2 × SSPE buffer in a 42°C oven for 16 hours. The slides were washed with an excess Tissue samples amount of pre-warmed 2 × SSPE, 0.1% SDS solution for 2 min at Surgically resected ESCC samples and their matched normal 42°C and then at room temperature. Each slide was dipped into 0.1 × epithelial tissue (taken 5.0 cm away from the tumor edge) were SSPE several times at room temperature and rinsed using a squeeze obtained from 90 patients with ESCC who provided informed consent bottle. The slides were spun dry in a centrifuge for at least 1 min, and at the First Affiliated Hospital of Bengbu Medical College in Bengbu, scanned data were extracted using GenePix 4100 from Molecular Anhui Province, PR China, from 1998 to 2001. The tissues were col- Devices. The data were normalized using Lowess normalization. We lected with institutional review board approval. The patients were all used 4-fold change in signal intensity as the criterion for significant previously untreated (i.e., no chemotherapy or radiotherapy) with change in gene expression. The fold change was presented as mean ± resectable primary ESCCs. The clinical and pathologic epidemiologi- SEM of the ratio value (n = 4). cal features of the patients are listed in Table 1. All samples were his- tologically confirmed as ESCC by pathologists at the First Affiliated Real-time PCR Hospital of Bengbu Medical College. The samples were immediately cDNA from 5 μg of total RNA was synthesized using AMV stored in Trizol reagent (Invitrogen, Carlsbad, CA) at −80°C. To ver- (Avian myeloblastosis virus) reverse transcriptase (Takara, Japan). ify cDNA microarray analysis data, 14 fresh samples and their Each single-stranded cDNA was diluted for subsequent PCR amplifi- matched normal epithelial tissues were confirmed using real-time cation by monitoring β- as a quantitative control. Real-time PCR, 90 paraffin embedded samples and 16 normal epithelial tissues RT-PCR was carried out using SYBR Premix Ex Taq kit (Takara Bio, were confirmed using immunohistochemistry (IHC) staining. Ohtsu, Japan) in the Rotor Gene 3000 system (Corbet Research, Sydney, Australia). The results were analyzed using Rotor Gene 5.0 RNA isolation and microarray analysis software. The total RNA was isolated using Trizol (Invitrogen, Carlsbad, USA) and then was purified using Qiagen RNeasy columns (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Immunohistochemistry Total RNA concentration was measured using NanoDrop spectropho- Paraffin-embedded samples (5 mm thickness) of ESCC and tometer (NanoDrop Technologies, Wilmington, DE, USA), and the matched normal tissues were used for histopathological analysis after quality was assessed by the A260/A280 and A230/A260 ratios. antigen retrieval. Briefly, tissues were de-paraffinized in xylene,

Next, 5 μg aliquot of RNA from ESCC or normal esophageal hydrated and incubated with 0.5% H2O2 in methanol for 20 min to epithelial tissue was reverse-transcribed into cDNA and labeled with block endogenous peroxidase activity. The slides were then washed Cy5-dCTP or Cy3-dCTP, respectively. The labeled cDNAs were re- with Tris-buffered saline and heated for 15 min at 100°C in 10 mM purified using Qiagen Mini-Elute PCR purification column (Qiagen, sodium citrate buffer (pH 6.0). The slides were cooled to 37°C, fol- Dusseldorf, Germany) according to the manufacturer’s protocol prior lowed by incubation with 1% BSA for 1 hour to block non-specific to hybridization. Human OneArray microarray with 28,703 human binding. The slides were incubated overnight at 4°C in a humidified genes (Phalanx Biotech Group Inc., Taiwan, China) was prehybrid- chamber with primary rabbit anti-human 34βE12 (high molecular ized in 25 mL pre-hybridization buffer containing 5 × SSPE, 0.1% weight , CKH, Maixin-Bio, Fuzhou), matrix metallopro- SDS solution, and 1% bovine serum albumin (BSA) for 2 hours at teinase (MMP)-3, MMP-9, minichromosome maintenance protein 2 42°C in the hybridization oven. The slides were transferred to dis- (MCM2), and WNT1 inducible signaling pathway protein 2 (WISP2) tilled water at room temperature for 2 min, and dried by centrifuga- monoclonal antibodies (all 1:100; Neomarkers, Fremont, CA, USA), tion. The slides were hybridized with 55 μL hybridization solution respectively. After rinsing with wash buffer, the slides were incu-

Table 1. Clinical characteristics and pathological data from the four patients with oesophageal squa- mous cell carcinoma who provided tissue samples for gene expression profiling assay.

Patients Gendar Age (years) TNM stage Clinical stage 1 Male 60 T1N0M0 IA 2 Male 65 T2N0M0 IIA 3 Male 68 T1N0M0 IIB 4 Male 71 T2N0M0 IA TNM, tumor, node, metastasis classification of malignant tumors. The Newly Identified Genes Expressed in Human ESCC 303 bated with biotin-labeled secondary antibody and avidin-biotin com- plex for 30 min at room temperature using the Elite Vectastain ABC kit (Vector Laboratories, Burlingame, CA) with 3,3’-diaminobenzi- dine as the chromogen. Negative controls were performed in all cases by omitting primary antibody. The degree of immunostaining was independently examined by two clinical pathologists who were unaware of patient outcomes. For each sample, five low-power fields (400 ×) were randomly selected. The staining intensity was scored as negative (0), mild (1+), moderate (2+), and strong (3+). The percentage of cancer cell staining was scored as 0 (less than 1% of cells stained), 1+ (1-10% of cells stained), 2+ (10-50% of cells stained), 3+ (50-75% of cells stained) and 4+ (greater than 75% of cells stained). A final score was calcu- lated by adding the scores for percentage and intensity, resulting in scores of 0 to 7. A score of 0 was considered negative, 2-3 was con- sidered weak, 4-5 was considered moderate, and 6-7 was considered strong.

Statistical analysis The association between protein expression and clinicopatho- logic characteristics was assessed using rank sum test, and the associ- ation between the was assessed using spearman rank correla- tion analysis. Values of P < 0.05 (two-tailed) were considered to be significant.

Results Identification of human esophageal squamous cell carci- noma (ESCC) All the samples were collected randomly and were diagnosed before surgical resection using gastroscopy Fig. 1. Characterization of esophageal squamous cell carcino- biopsy, and confirmed after surgical resection using hema- ma samples from gastroscope biopsy. A. Hematoxylin- eosin (HE) stain (× 400). B. Immunostaining for cyto- toxylin-eosin (HE, Fig. 1A) and high molecular weight (CKH) expression (brown). cytokeratin (CKH) IHC staining (Fig. 1B). Using HE stain- ing, we observed noticeable atypia in tumor cells and kera- tin pearl in tumor tissue structures, which are the typical and biochemical enzyme activity. markers of squamous cell carcinoma. Moreover, we veri- fied that CKH, which is used to identify squamous cell car- Verification of the microarray data using real-time PCR cinoma, was expressed in the cytoplasm of the tumor sam- To examine the reliability of the microarray data, we ples using IHC staining. Together, these results suggest that randomly selected seven up-regulated genes (MMP-3, 9,12, samples from ESCC tissues can be readily used in subse- MCM2, CD7, LIMK1 and TTK) and five down-regulated quent studies to detect differential gene expression. genes (WISP2, HIG1, HSPB2, MAOA and HPGD) to ver- ify their expression levels by real-time RT-PCR using the Identification of differentially expressed genes in ESCC same RNA samples that were used for microarray analysis, compared with the normal mucosae and 10 new RNA samples from other ESCC patients. The The profiles of primary ESCC tissues and their real-time RT-PCR results were fully consistent with those matched (adjacent) normal esophageal epithelium from four from the cDNA microarray data (Fig. 2). ESCC patients were compared using Human OneArray microarray. After performing paired t-test analyses, 179 Validation of differentially expressed proteins in clinical genes showed expression levels that differed significantly samples with ≥ 4-fold change. Among these genes, expression lev- To examine whether changes at the mRNA level reflect els of 72 genes were up-regulated (Table 2) and those of changes at the protein level, we focused on four candidate 107 genes were down-regulated (Table 3) in ESCC tumor genes that were implied in previous studies to play impor- tissues compared with adjacent normal tissues. We catego- tant roles in tumor development. MMP-3, MMP-9, MCM2 rized these genes into different classes based on their poten- and WISP2, were further tested using immunohistochemi- tial functions (Tables 2 and 3), including cell cycle regula- cal staining in formalin-fixed and paraffin-embedded tissue tors, cell growth/proliferation/differentiation factor, sections. Representative IHC patterns of these molecules extracellular matrix, DNA replication/repair/transcription, are shown in Fig. 3. We detected that MMP-3, MMP-9 and 304 Y. Tao et al.

Table 2. Upregulated gene expression in ESCC compared with normal esophageal mucosa tissue (n = 4).

Gene Title Gene Symbol Gene ID Fold change

Cell cycle regulators A2 CCNA2 NM_001237.2 4.14 ± 1.24 cyclinB2 CCNB2 NM_004701.2 12.68 ± 7.70 cyclin F CCNF NM_001761.1 5.83 ± 0.67 cell division cycle 2 CDC2 NM_001786.2 15.41 ± 14.89 BUB1 budding uninhibited by benzimidazoles 1 homolog beta BUB1β NM_001211.3 4.15 ± 0.60 CDC28 protein kinase regulatory subunit 1B CKS1B NM_001826.1 3.46 ± 0.64 CDC28 protein kinase regulatory subunit 2 CKS2 NM_001827.1 4.86 ± 2.90 centromere protein A 17kDa CENPA NM_001809.2 6.26 ± 2.60 centromere protein F, 350/400ka CENPF NM_016343.3 9.44 ± 1.42 guanine nucleotide binding protein GNGT1 NM_021955.2 4.72 ± 0.84 kinetochore associated 1 KNTC1 NM_014708.3 4.05 ± 0.45 kinetochore associated 2 KNTC2 NM_006101.1 6.31 ± 1.30 MLF1 interacting protein MLF1IP NM_024629.2 5.87 ± 0.74 Cell growth/proliferation/differentiation factors nucleolar and spindle associated protein 1 NUSAP1 NM_016359.1 6.40 ± 1.10 protein regulator of cytokinesis 1 PRC1 NM_003981.2 4.22 ± 0.92 family member 20A KIF20A NM_005733.1 4.04 ± 1.21 kinesin-like 7 KNSL7 NM_020242.1 9.30 ± 1.37 sperm associated antigen 5 SPAG5 NM_006461.2 8.82 ± 1.81 vascular endothelial growth factor VEGF NM_003376.3 4.11 ± 0.79 Extracellular matrix Matrix metalloproteinase 1 MMP1 NM_002421.2 60.62 ± 1.53 Matrix metalloproteinase 12 MMP12 NM_002426.1 7.51 ± 1.69 Matrix metalloproteinase 13 MMP13 NM_002427.2 55.26 ± 4.81 Matrix metalloproteinase 14 MMP14 NM_004995.2 6.8 ± 0.52 Matrix metalloproteinase 3 MMP3 NM_002422.2 8.25 ± 2.70 Matrix metalloproteinase 9 MMP9 NM_004994.1 5.72 ± 2.40 collagen, type I, alpha 1 COL1A1 NM_000088.2 8.63 ± 0.75 collagen, type V, alpha 2 COL5A2 NM_000393.2 4.7 0 ± 0.22 DNAreplication/repair/transcription Minichromosome maintenance 2 MCM2 NM_004526.2 6.22 ± 2.34 10 open reading frame 3 C10orf3 NM_018131.3 11.29 ± 2.76 chromosome 21 open reading frame 29 C21orf29 NM_144991.2 4.12 ± 0.51 chromosome 21 open reading frame 96 C21orf96 AK026743 5.70 ± 0.47 F-box protein 39 FBXO39 NM_153230.1 4.23 ± 0.63 helicase, lymphoid-specific HELLS NM_018063.3 6.58 ± 1.91 zinc finger protein 552 ZNF552 AK023769 4.91 ± 0.50 SRY (sex determining region Y)-box 4 SOX4 NM_003107.2 4.12 ± 0.90 origin recognition complex, subunit 6 homolog-like ORC6L NM_014321.2 10.91 ± 2.25 RAD51 homolog (RecA homolog, E. coli) RAD51 NM_002875.2 8.09 ± 4.31 ribonuclease H2, large subunit RNASEH2A NM_006397.2 4.88 ± 0.48 WD repeat and HMG-box DNA binding protein 1 WDHD1 NM_007086.1 5.25 ± 0.55 Cell membrane protein/ CD7 antigen CD7 NM_006137.5 4.14 ± 0.29 erythrocyte membrane protein band 4.1 EPB4.1 NM_004437.2 4.05 ± 0.64 SKI-like SKIL NM_005414.2 4.23 ± 0.61 wingless-type MMTV integration site family member 2 WNT2 NM_003391.1 4.66 ± 1.15 The Newly Identified Genes Expressed in Human ESCC 305

Table 2. Continued. Gene Title Gene Symbol Gene ID Fold change Biochemical enzymes activity ATPase family, AAA domain containing 2 ATAD2 NM_014109.2 8.25 ± 0.67 carbonic anhydrase IX CA9 NM_001216.1 4.10 ± 0.83 carboxypeptidase X CPXM NM_019609.2 6.24 ± 1.43 glucosamine-6-phosphate deaminase 1 GNPDA1 NM_005471.3 4.30 ± 0.65 glutathione peroxidase 2 GPX2 NM_002083.2 12.89 ± 8.90 LIM domain kinase 1 LIMK1 NM_002314.2 4.15 ± 0.49 TTK protein kinase TTK NM_003318.3 5.76 ± 2.57 T-LAK cell-originated protein kinase TOPK NM_018492.2 7.09 ± 2.92 maternal embryonic leucine zipper kinase MELK NM_014791.2 24.83 ± 4.12 protein tyrosine phosphatase, receptor-type, Z polypeptide 1 PTPRZ1 NM_002851.1 4.30 ± 0.87 serine/threonine kinase 6 STK6 NM_003600.2 5.86 ± 0.27 thymidine kinase 1, soluble TK1 NM_003258.1 4.04 ± 0.66 T-LAK cell-originated protein kinase TOPK NM_018492.2 7.09 ± 2.92 Protein binding/modify cation/transportation/protein chaperon homeo box B7 HOXB7 NM_004502.2 13.9 ± 3.87 IGF-II mRNA-binding protein 2 IMP2 NM_006548.3 9.34 ± 3.84 LSM4 homolog LSM4 NM_012321.2 9.34 ± 3.84 MyoD family inhibitor MDFI NM_005586.2 4.85 ± 0.52 SHC SH2-domain binding protein 1 SHCBP1 NM_024745.2 4.08 ± 1.23 solute carrier family 16, member 3 SLC16A3 NM_004207.1 4.42 ± 0.82 solute carrier family 2, member 1 SLC2A1 NM_006516.1 5.72 ± 0.81 UDP-glucose ceramide glucosyltransferase-like 1 UGCGL1 NM_020120.1 4.44 ± 0.32 others kinesin family member 4A TRIP13 NM_012310.2 4.65 ± 1.21 thyroid hormone receptor interactor 13 NFE2L3 NM_004237.2 5.78 ± 2.21 keratin 17 KRT17 NM_000422.1 12.45 ± 9.65 apolipoprotein C-I APOC1 NM_001645.3 6.98 ± 2.26 apolipoprotein E APOE NM_000041.2 4.34 ± 0.23 cadherin 11, type 2, OB-cadherin CDH11 NM_001797.2 5.43 ± 0.94 reticulocalbin 3, EF-hand calcium binding domain RCN3 NM_020650.2 4.01 ± 0.39 trophinin associated protein TROAP NM_005480.2 5.20 ± 0.81

MCM2 proteins were highly expressed in ESCC. applied to large-scale molecular profiling. In the present Moreover, MMP-3 and MMP-9 were located in the cyto- study, we identified differential gene expression profiles plasm and MCM2 was located in the nucleus of tumor cells. between ESCC and matched normal esophageal mucosa tis- The positive rates of MMP-3, MMP-9 and MCM2 in ESCC sue samples from four cases using cDNA microarray tech- were up to 84.44% (76/90), 83.33% (75/90) and 82.22% nology. We found a total of 179 genes that showed at least (74/90), respectively, which are significantly higher than a 4-fold expression change in ESCC compared with normal that of normal mucosa (25%, 4/16, P < 0.05; 18.75%, 3/16, tissues. Among these genes, 71 were up-regulated and 108 P < 0.05; 37.50%, 6/16, P < 0.05, respectively). However, down-regulated. The microarray results were further con- the positive rate of WISP2 detected in the nucleus was sig- firmed using real-time PCR and immunohistochemistry, nificantly lower in ESCC (33.33%, 30/90) when compared which suggested that the microarray data were reliable. with normal mucosa (68.75%, 11/16, P < 0.05). These These genes can be categorized into several different results were consistent with real-time PCR results and thus classes based on their function (see Tables 2 and 3). Some further support the microarray results at the protein level. genes, especially the up-regulated ones, may participate in the occurrence and development of ESCC, while others Discussion may constitute the molecular identity of ESCC. The development of microarray technology enabled Cell cycle progression is carefully regulated and there the analysis of hundreds to thousands of genes and can be is a delicate balance between proliferation and cell death, 306 Y. Tao et al.

Table 3. Downregulated gene expression in ESCC compared with normal esophageal mucosa tissue (n = 4).

Gene Title Gene Symbol Gene ID Fold change Cell growth/proliferation/differentiation factors butyrylcholinesterase BCHE NM_000055.1 0.15 ± 0.10 BTG family, member 2 BTG2 NM_006763.2 0.10 ± 0.05 cyclin D2 CCND2 NM_001759.2 0.15 ± 0.03 calponin 1, basic, smooth muscle CNN1 NM_001299.3 0.05 ± 0.01 carboxypeptidase E CPE NM_001873.1 0.21 ± 0.14 endothelin 3 EDN3 NM_000114.2 0.17 ± 0.04 erythrocyte membrane protein band 4.1-like 3 EPB41L3 NM_012307.2 0.21 ± 0.09 EPH receptor A3 EPHA3 NM_005233.3 0.25 ± 0.10 , light polypeptide kinase MYLK NM_005965.3 0.18 ± 0.10 monoamine oxidase A MAOA NM_000240.2 0.17 ± 0.04 LIM and senescent cell antigen-like domains 2 LIMS2 NM_017980.2 0.13 ± 0.05 15-hydroxyprostaglandin dehydrogenase [NAD+] HPGD NM_000860.3 0.09 ± 0.03 likely ortholog of mouse hypoxia induced gene 1 HIG1 NM_014056.1 0.25 ± 0.05 Cdc42 guanine nucleotide exchange factor (GEF) 9 ARHGEF9 NM_015185.1 0.16 ± 0.08 Arg/Abl-interacting protein ArgBP2 ARGBP2 NM_021069.2 0.08 ± 0.02 DNAreplication/repair/transcription betaine-homocysteine methyltransferase 2 BHMT2 NM_017614.3 0.06 ± 0.02 cocaine- and amphetamine-regulated transcript CART NM_004291.2 0.14 ± 0.07 cartilage intermediate layer protein CILP NM_003613.2 0.14 ± 0.04 creatine kinase, brain CKB NM_001823.3 0.15 ± 0.05 dermatopontin DPT NM_001937.3 0.14 ± 0.05 early B-cell factor 2 EBF2 NM_022659.1 0.18 ± 0.07 four and a half LIM domains 1 FHL1 NM_001449.3 0.04 ± 0.03 hypothetical protein FLJ12895 FLJ12895 NM_023926.3 0.16 ± 0.07 SRY (sex determining region Y)-box 17 SOX17 NM_022454.2 0.10 ± 0.01 transforming, acidic coiled-coil containing protein 1 TACC1 NM_006283.1 0.18 ± 0.09 tachykinin receptor 2 TACR2 NM_001057.1 0.18 ± 0.07 zinc finger and BTB domain containing 16 ZBTB16 NM_006006.3 0.04 ± 0.014 immunoglobulin domain protein (myotilin) TTID NM_006790.1 0.08 ± 0.02 SH3 domain binding glutamic acid-rich protein SH3BGR NM_007341.2 0.06 ± 0.04 nuclear receptor subfamily 4, group A, member 3 NR4A3 NM_006981.2 0.18 ± 0.07 nuclear receptor subfamily 4, group A, member 1 NR4A1 NM_002135.3 0.19 ± 0.07 nuclear receptor subfamily 3, group C, member 2 NR3C2 NM_000901.1 0.09 ± 0.01 myosin, light polypeptide 9, regulatory MYL9 NM_006097.3 0.13 ± 0.11 matrix Gla protein MGP NM_000900.2 0.06 ± 0.02 myocyte enhancer factor 2C) MEF2C NM_002397.2 0.21 ± 0.05 Kruppel-like factor 9 KLF9 NM_001206.1 0.20 ± 0.20 Kruppel-like factor 2 (lung) KLF2 NM_016270.2 0.09 ± 0.1 (amyloidosis, Finnish type) GSN NM_000177.3 0.23 ± 0.07 aldehyde dehydrogenase 1 family, member A2 ALDH1A2 NM_003888.2 0.13 ± 0.06 actin, gamma 2, smooth muscle, enteric ACTG2 NM_001615.2 0.17 ± 0.08 acyl-Coenzyme A oxidase 2, branched chain ACOX2 NM_003500.2 0.16 ± 0.09 ribonuclease, RNase A family, 4 RNASE4 NM_002937.3 0.24 ± 0.08 protein S (alpha) PROS1 NM_000313.1 0.20 ± 0.07 Cell membrane protein/signal transduction claudin 5 CLDN5 NM_003277.2 0.15 ± 0.03 sorbin and SH3 domain containing 1 SORBS1 NM_015385.1 0.04 ± 0.02 transforming growth factor, beta receptor III TGFBR3 NM_003243.1 0.09 ± 0.02 WNT1 inducible signaling pathway protein 2 WISP2 NM_003881.2 0.07 ± 0.05 small muscle protein, X-linked SMPX NM_014332.1 0.06 ± 0.05 serum deprivation response SDPR NM_004657.3 0.08 ± 0.02 ribonuclease, RNase A family, 4 RGS2 NM_002937.3 0.15 ± 0.09 Ras interacting protein 1 RASIP1 NM_017805.2 0.25 ± 0.07 RAS guanyl releasing protein 2 RASGRP2 NM_005825.2 0.16 ± 0.03 RAP1A, member of RAS oncogene family RAP1A NM_002884.1 0.19 ± 0.09 protein tyrosine phosphatase-like, member a PTPLA NM_014241.2 0.14 ± 0.06 The Newly Identified Genes Expressed in Human ESCC 307

Table 3. Continued. Gene Title Gene Symbol Gene ID Fold change PDZ domain containing RING finger 4 PDZRN4 NM_013377.2 0.08 ± 0.01 mesenchyme homeo box 1 MEOX1 NM_004527.2 0.21 ± 0.08 gremlin 2 homolog, cysteine knot superfamily GREM2 NM_022469.2 0.20 ± 0.07 aspartoacylase (aminoacylase 2, Canavan disease) ASPA NM_000049.1 0.07 ± 0.02 armadillo repeat containing, X-linked 1 ARMCX1 NM_016608.1 0.16 ± 0.02 folate receptor 2 (fetal) FOLR2 NM_000803.2 0.23 ± 0.01 growth hormone receptor GHR NM_000163.1 0.23 ± 0.04 phosphodiesterase 2A, cGMP-stimulated PDE2A NM_002599.1 0.21 ± 0.08 Biochemical enzymes activity calcium/calmodulin-dependent protein kinase II gamma CAMK2G NM_001222.2 0.16 ± 0.04 cytochrome b reductase 1 CYBRD1 NM_024843.2 0.22 ± 0.04 superoxide dismutase 3, extracellular SOD3 NM_003102.1 0.07 ± 0.01 WAP four-disulfide core domain 12 WFDC12 NM_080869.1 0.23 ± 0.06 1, regulatory (inhibitor) subunit 1A PPP1R1A NM_006741.2 0.05 ± 0.03 protein phosphatase 1, regulatory subunit 12B PPP1R12B NM_002481.2 0.14 ± 0.04 protein phosphatase 1, catalytic subunit, beta isoform PPP1CB NM_002709.2 0.22 ± 0.09 3-oxoacid CoA transferase 1 OXCT1 NM_000436.2 0.25 ± 0.07 natriuretic peptide receptor A/ A NPR1 NM_000906.2 0.21 ± 0.04 kallikrein B, plasma (Fletcher factor) 1 KLKB1 NM_000892.2 0.18 ± 0.06 acetyl-Coenzyme A acetyltransferase ACAT1 NM_000019.2 0.21 ± 0.09 hypothetical protein FLJ23033 FLJ23033 NM_024686.3 0.14 ± 0.014 hypothetical protein FLJ13639 FLJ13639 NM_024705.1 0.24 ± 0.06 glutamate decarboxylase-like 1 GADL1 AL832766 0.04 ± 0.03 glutathione peroxidase 3 (plasma) GPX3 NM_002084.2 0.17 ± 0.04 protein transportation \protion binding complement component 7 C7 NM_000587.2 0.03 ± 0.01 cytoplasmic polyadenylation element binding protein 3 CPEB3 NM_014912.3 0.23 ± 0.03 DES NM_001927.3 0.03 ± 0.01 Kell blood group precursor (McLeod phenotype) XK NM_021083.2 0.18 ± 0.05 smoothelin SMTN NM_006932.3 0.22 ± 0.14 heat shock 27kDa protein family, member 7 HSPB7 NM_014424.3 0.04 ± 0.02 heat shock 27kDa protein 2 HSPB2 NM_001541.2 0.12 ± 0.04 hemoglobin, alpha 1 HBA1 NM_000558.3 0.20 ± 0.07 ATPase, Na+/K+ transporting, beta 2 polypeptide ATP1B2 NM_001678.3 0.13 ± 0.05 Cdc42 guanine nucleotide exchange factor (GEF) 9 ARHGAP10 NM_015185.1 0.16 ± 0.08 repeat domain 25 ANKRD25 NM_015493.3 0.10 ± 0.03 scrapie responsive protein 1 SCRG1 NM_007281.1 0.25 ± 0.06 C, gamma (actin binding protein 280 FLNC NM_001458.1 0.04 ± 0.01 FYVE and coiled-coil domain containing 1 FYCO1 NM_024513.1 0.22 ± 0.05 Calcium ion binding/transport calcium channel, voltage-dependent, beta 2 subunit CACNB2 NM_000724.2 0.11 ± 0.07 calmodulin 1 (phosphorylase kinase, delta) CALM1 NM_006888.2 0.25 ± 0.09 calcium/calmodulin-dependent protein kinase II CaMKIINalpha NM_018584.4 0.21 ± 0.10 Down syndrome critical region gene 1-like 1 DSCR1L1 NM_005822.1 0.09 ± 0.05 FK506 binding protein 5 FKBP5 NM_004117.2 0.21 ± 0.10 junctophilin 2 JPH2 NM_020433.3 0.14 ± 0.06 chromosome 2 open reading frame 23 2orf23 NM_022912.1 0.23 ± 0.04 2 (beta) TPM2 NM_003289.3 0.07 ± 0.04 thioredoxin interacting protein TXNIP NM_006472.1 0.20 ± 0.08 TU3A protein TU3A NM_007177.1 0.06 ± 0.02 solute carrier family 25, member 4 SLC25A4 NM_001151.2 0.25 ± 0.14 ADP-ribosylation-like factor 6 interacting protein 5 ARL6IP5 NM_006407.3 0.26 ± 0.06 others hypothetical protein DKFZp434F0318 DKFZP434F0318 NM_030817.1 0.11 ± 0.01 DKFZP586A0522 protein DKFZP586A0522 NM_014033.2 0.21 ± 0.08 tetranectin (plasminogen binding protein) TNA NM_003278.1 0.09 ± 0.02 prostaglandin-endoperoxide synthase 1 PTGS1 NM_000962.2 0.05 ± 0.01 308 Y. Tao et al.

Fig. 2. Confirmation of relative gene expression levels using real-time RT-PCR. Differentially expressed genes from micro- array data were confirmed using real time RT-PCR (n = 14). Target gene expression levels were standardized with β-actin expression levels. The results from real-time RT-PCR showed that expression levels of all selected gene were consistent with the microarray data.

Fig. 3. Expression of MMP-3, MMP-9, MMC2 and WISP2 proteins in tumor tissues from esophageal cancer patients using immunohistochemical staining. Normal esophageal epithelial tissues were used as the control. Images were taken under 200 × magnification. Brown staining indicates positive signals. The Newly Identified Genes Expressed in Human ESCC 309

Table 4. Immunohistochemical assay for the expression of MMP-3, MMP-9, MCM and WISP2 in human esophageal squa- mous cell carcinoma and normal oesophageal epithelium. MMP-3 MMMP-9 MCM2 WISP2 Tissue type Case No. − + − + − + − + ESCC 90 14 76 15 75 16 74 10 6 Normal 16 12 4 13 3 10 6 5 11 X2 25.93 29.15 14.67 7.18 P 0.000 0.000 0.00013 0.000753 which maintains a steady state in adult tissue. Disruption -3, -9, -12, -13 and -14, were markedly up-regulated in of this equilibrium by loss of cell cycle control may eventu- ESCC compared with normal esophageal mucosa. These ally lead to tumor development (Sandal 2002). It has been well-understood genes were highly expressed in ESCC, well known that , a family of proteins that control suggesting that they may play an important role in the inva- cell cycle progression in cells, are abnormally overex- sion and metastasis of ESCC. pressed in various human cancers (Egloff et al. 2006). In Both SOX-4 and SOX-17 are members of the SRY- the present study, we detected several cell cycle regulators related HMG-box (SOX) family of transcription factors that were up-regulated markedly in ESCC tissues compared involved in the regulation of embryonic development and with normal esophageal mucosae, including cyclin B1, the determination of cell fate (Smith and Sigvardsson cyclin A2, cyclin F, cell division cycle 2 (CDC2), CDC28 2004). It has been reported that SOX factors impact tumor- protein kinase regulatory subunit 1B (CKS1B) and CDC28 igenesis by modulating β-/T cell factor (TCF) activ- protein kinase regulatory subunit 2 (CKS2). ity and the expression of oncogenic Wnt target genes such L-Monoamine oxidases (MAOs) are a family of as Cyclin-D1 and c-Myc. For example, SOX-17 represses enzymes that catalyze monoamines oxidation (Tipton et al. β-catenin/TCF, while SOX4 enhances their transcriptional 2004). It has been recently reported that high expression of activity (Kormish et al. 2010). Our cDNA microarray result monoamine oxidase A (MAOA) was found in normal basal demonstrated up-regulation of SOX-4 and down-regulation prostatic epithelium and high-grade primary prostate can- of SOX-17 in ESCC, which is similar to Kormish’s report cer. On the contrary, both normal secretory prostatic epi- and suggests that Sox-Wnt interactions may be involved in thelium and low-grade prostate cancer had low expression ESCC development. MCM proteins are DNA replication of MAOA (Flamand et al. 2010). In this study, we also licensing factors and obvious markers for proliferation found that MAOA was down-regulated markedly in ESCC (Forsburg 2004). It has been reported that increased levels when compared with normal esophageal mucosae. We of MCMs marked not only proliferative malignant cells, but speculate that MAOA may also play an important role in also precancerous cells and the potential for recurrence inhibition of ESCC development. 15-Hydroxyprosta­ ­glan­ (Alison et al. 2002; Going et al. 2002). In this study, din dehydrogenase [NAD+] (HPGD) is a prostaglandin- MCM2 was found to increase more than 6-fold in ESCC synthesizing enzyme that physiologically antagonizes compared with normal esophageal mucosae, indicating that COX-2, and may play an important role in diverse physio- MCM2 may be necessary for ESCC development. logical aspects ranging from inflammation to cancer (Tai et Wingless-type MMTV integration site family member al. 2006). A recent study has found that HPGD was down- 2 (WNT2) is a member of the WNT family. The WNT sig- regulated in a majority of lung, colon, breast and bladder naling pathway is a network of proteins best known for cancers (Mohamed et al. 2011). In this study, we found a their roles in embryogenesis and cancer, but is also involved significant down-regulation of HPGD in ESCC, which sug- in normal physiological processes in adult animals (Lie et gests a possibility that HPGD may act as a tumor suppres- al. 2005). In the present study, the observation of high sor of ESCC. WNT2 gene expression in ESCC indicates that WNT2 may The MMPs are a family of zinc-dependent proteolytic transduce a growth-regulatory signal in these cells. CD7 is enzymes capable of degrading the extracellular matrix. a cell surface glycoprotein member of the immunoglobulin They are important regulators of many physiological pro- superfamily and is found on thymocytes and mature T-cells. cesses such as embryonic development, morphogenesis, tis- It plays an essential role in T-cell and T-cell/B-cell interac- sue remodeling and reproduction. They are also known to tions during early lymphoid development (Stillwell and be involved in some pathological processes including Bierer 2001). It has been reported that CD7 expression is arthritis, cardiovascular disease and cancer (Murphy and up-regulated in chronic myeloid leukemia (CML) cells and Nagase 2008). It has been reported that MMPs are involved in other leukemias and lymphomas (Rogers et al. 2010). In in tumor cell invasion and metastatic dissemination the present study, we showed that CD7 had an increased (Fishman et al. 2001; Deryugina and Quigley 2006). As expression in ESCC, suggesting that CD7 could be involved shown in Table 2, several MMPs genes, including MMP-1, in crosstalk between tumor and immune cells. 310 Y. Tao et al.

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