Homeobox Gene IRX1 Is a Tumor Suppressor Gene in Gastric Carcinoma

Home , HIST2H2BE, IRX1

Oncogene (2010) 29, 3908–3920 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Homeobox gene IRX1 is a tumor suppressor gene in gastric carcinoma

X Guo1,3, W Liu1,3, Y Pan1,PNi2,JJi1, L Guo1, J Zhang1,JWu1, J Jiang1, X Chen1, Q Cai1, JLi1, J Zhang1,QGu1, B Liu1, Z Zhu1 and Y Yu1

1Department of Surgery of Shanghai Ruijin Hospital and Shanghai Institute of Digestive Surgery, Shanghai, PR China and 2Department of Clinical Biochemistry, Afﬁliated to Shanghai Jiao Tong University, School of Medicine, Shanghai, PR China

The IRX1 tumor suppressor gene is located on 5p15.33, a locatedonLOHloci.Ourgrouppreviouslycharacterized cancer susceptibility locus. Loss of heterozygosity of a high frequency of LOH at 5p15.33 in human gastric 5p15.33 in gastric cancer was identified in our previous cancer. We hypothesized that genes located on this locus work. In this study, we analyzed the molecular features may contribute to the pathogenesis of gastric cancer (Lu and function of IRX1. We found that IRX1 expression et al., 2005). IRX1 is harbored in this chromosome locus was lost or reduced in gastric cancer. However, no and belongs to the Iroquois homeobox gene family, which mutations were identified in IRX1-encoding regions. IRX1 has six members from IRX1 to IRX6. IRX1 is closely transcription was suppressed by hypermethylation, and the related to embryonic development, including foregut expression of IRX1 mRNA was partially restored in organs such as the lung (Ferguson et al.,1998;Becker gastric cancer cells after 5-Aza-dC treatment. Restoring et al., 2001; van Tuyl et al., 2006; Alarcon et al., 2008). IRX1 expression in SGC-7901 and NCI-N87 gastric Because the stomach is also of foregut origin, the high cancer cells inhibited growth, invasion and tumorigenesis frequency of LOH at the IRX1 locus in gastric cancer in vitro and in vivo. We identified a number of target genes suggested that the IRX1 gene may be involved in the by global microarray analysis after IRX1 transfection development of gastric cancer. Asaka et al. (2006) reported combined with real-time PCR and chromatin immunopre- that IRX3 is downregulated in breast cancer patients with cipitation assay. BDKRB2, an angiogenesis-related gene, shorter survival times. IRX3 was also downregulated in HIST2H2BE and FGF7, cell proliferation and invasion- androgen-insensitive prostate cancer cell lines (Zhao et al., related genes, were identified as direct IRX1 target genes. 2005). Lewis et al. (1999) reported that IRX2 is involved in The hypermethylation of IRX1 was not only detected in epithelial cell differentiation as well as ductal and lobular primary gastric cancer tissues but also in the peripheral proliferation of breast cells. The association of the IRX1 blood of gastric cancer patients, suggesting IRX1 could gene with gastric cancer has not previously been studied. potentially serve as a biomarker for gastric cancer. A typical tumor suppressor gene should have the Oncogene (2010) 29, 3908–3920; doi:10.1038/onc.2010.143; following features: the genetic alterations occur in both published online 3 May 2010 alleles (for example, one deleted and another mutated) or both alleles are deleted or mutated in cancer (Knudson, Keywords: epigenetic; gastric carcinoma; homeobox 1996, 2001). In the absence of an allelic mutation, reduced genes; IRX1; tumor suppressor gene expression in cancer may be caused by an epigenetic abnormality such as hypermethylation of the promoter region (Garinis et al., 2002; Esteller, 2007). To date, Introduction significant effort has been directed at identifying tumor suppressor genes related to gastric cancer; however, these Chromosome 5p15.33 is a cancer susceptibility locus. efforts have been unsuccessful thus far. In this study, we Significant genetic variants of 5p15.33 have been reported systematically analyzed the genetic structure, promoter in lung cancer in Western and Chinese populations activity and methylation status of the IRX1 gene. We also (McKay et al., 2008; Jin et al., 2009). Gastric cancer is analyzed its functional features after reconstitution of one of the common malignancies characterized by IRX1 expression in vitro and in vivo. IRX1 functions as a genomic instability with multiple genetic alterations, transcription factor, but the exact role IRX1 has in the including oncogene activation and tumor suppressor gene development of gastric cancer is currently unknown. The inactivation. Loss of heterozygosity (LOH) is one type of target genes and related pathways of IRX1 have not yet genetic variation, and tumor suppressor genes are always been identified. cDNA microarrays provide a powerful tool for Correspondence: Dr Y Yu or Dr Z Zhu, Department of Surgery, exploring complex gene expression profiles. Microarray Ruijin Hospital and Shanghai Institute of Digestive Surgery, Shanghai analysis of experimental samples, such as gene-trans- Jiao Tong University, School of Medicine, Ruijin er Road, No. 197, fected cells, has led to identification of valuable Shanghai 200025, PR China. molecular markers involved in tumor proliferation, E-mails: [email protected] or [email protected] 3These authors contributed equally to this work. angiogenesis, prognosis and therapeutic response Received 10 September 2009; revised 26 March 2010; accepted 2 April (Duggan et al., 1999; Quackenbush, 2006; Perez-Diez 2010; published online 3 May 2010 et al., 2007; Iorns et al., 2009). Thus, we used a global IRX1 gene in gastric carcinoma X Guo et al 3909

Figure 1 Gene expression, gene copy numbers and promoter analysis of IRX1.(a) IRX1 mRNA detection in gastric cancer cell lines and immortalized gastric mucosa cell line GES-1 by real-time PCR (top) and gene copy numbers (bottom). IRX1 mRNA expression was significantly reduced or nearly absent in cancer cells but retained in GES-1 cells, and four out of seven gastric cancer cell lines showed IRX1 copy number reduction compared with the GES-1 cell line. (b) Putative motifs of the 50-flanking region of the IRX1 gene. Important putative motifs are identified and underlined. The transcriptional start site (TSS) is indicated as þ 1. (c) Progressive deletions and activity analysis of the IRX1 promoter by luciferase reporter. Progressive 50-deletions were constructed (left side) and transiently transfected into 293 T or GES-1 cells. The promoter fragments were linked to luciferase (black box). Bars on the right side denote luciferase activities, represented as mean±s.d. The data are representative examples taken from one of three experiments. cDNA microarray to identify downstream target genes cancer cell lines (Po0.001, Figure 1a, top). We of IRX1. We identified a number of target genes by examined the IRX1 copy numbers on all cell lines by global microarray analysis after IRX1 transfection real-time PCR, and four out of seven gastric cancer cell combined with real-time PCR and chromatin immuno- lines showed IRX1 copy number reduction compared precipitation assay. This work revealed that IRX1 is with the GES-1 cell line (Figure 1a, bottom). We downregulated in gastric cancer. Downregulation of sequenced PCR products for four exons of the IRX1 IRX1 due to LOH of 5p15.33, combined with epigenetic gene from seven gastric cancer cell lines and the GES-1 suppression of the retained allele, is the mechanism of control. No mutations were found except one single IRX1 inactivation in gastric cancer. nucleotide polymorphism within exon 2 in four out of seven cancer cell lines and the GES-1 cell line, which does not alter the encoded amino acid (rs844154, Results Supplementary Figure 1). (Sequencing results for other reported single nucleotide polymorphisms are shown in Analysis of IRX1 expression, mutation, gene copy Supplementary Table 1) We used online-accessible numbers and promoter region platforms to analyze the 50-franking from the transcrip- Compared with the GES-1 gastric mucosa cell line, tional start site of IRX1. The fragment À600 bp from the IRX1 mRNA was barely detectable in four gastric transcriptional start site was predicted as a probable cancer cell lines and was decreased in three gastric promoter region (Figure 1b). We amplified a 775-bp

Oncogene IRX1 gene in gastric carcinoma X Guo et al 3910

Figure 2 Methylation status of IRX1 CpG islands and the effects of 5-Aza-dC on IRX1 expression. (a) Schematic locations of CpG sites within the CpG islands that surround the IRX1 transcription start site by MSP and BSP analysis. (b) Upper: The core promoter region was methylated in all gastric cancer cell lines (marked as M) but unmethylated in the GES-1 cell line (marked as U). Lower: expression of IRX1 mRNA was restored in gastric cancer cell lines after 5-Aza-dC treatment (marked as E). (c) Methylated CpG site analysis by sequencing the PCR products. Compared with wild-type sequence (top letters), cytosines of methylated CpGs in gastric cancer cell lines are not converted, whereas unmethylated CpGs in GES-1 control cells are converted (indicated by arrow). (d) Schematic summary of 12 CpG sites in the promoter region from À519 to À679. Methylation analysis was performed in 10 clones for each cell line. Each row of circles represents a single clone, and each circle represents a single CpG site. Open circle represents unmethylated cytosine; ﬁlled circle represents methylated cytosine. All gastric cancer cell lines showed higher methylation levels compared with the GES-1 control. The methylation ratios of 10 clones for each cell lines are summarized in the lowest bar chart.

fragment and inserted the fragment into a luciferase bisulfate sequencing (BSP), which covered the regions of reporter vector (pGL3-Basic) for promoter activity À236 to À388 and À519 to À679, respectively (Figure 2a). analysis. A series of promoter/reporter fusion plasmids The core promoter region was methylated in all cancer cell containing progressive 50 deletions was constructed. lines according to MSP analysis. We treated cancer cell The different constructs were transiently transfected lines with 10 mM 5-Aza-20-deoxycytidine (5-Aza-dC) for 96 into 293T or GES-1 cell lines and luciferase activities or 120 h. The expression of IRX1 mRNA was restored or were measured. The fragment in pGL3-476 showed partially restored in some gastric cancer cell lines the highest activity compared with empty vector. The (Figure 2b). We examined the methylation status of 12 activity of the fragment in pGL3-248 was markedly CpG sites within the CpG island of IRX1 by sequencing decreased compared with pGL3-380 (Po0.001), which the PCR products in gastric cancer cell lines and the GES-1 suggested that the core promoter region of IRX1 is control after bisulfate treatment. The 12 CpG sites of situated on the inter-region of pGL3-248 and pGL3-380, the IRX1 promoter were methylated in gastric cancer accordant with the bioinformatics prediction cell lines compared with GES-1 (Po0.001, Figures 2c (Figure 1c). The fold-changes of the different constructs and d), and methylation correlated inversely with IRX1 compared with pGL3-Basic in 293T and GES-1 cells are mRNA expression levels. summarized in Supplementary Table 2.

Methylation analysis and the effect of demethylation Functional analysis after restoring IRX1 expression treatment on cancer cell lines in vitro We examined the methylation status of the promoter We introduced IRX1 expression in SGC-7901 and NCI- region by means of methylation-speciﬁc PCR (MSP) and N87 gastric cancer cell lines by means of the pEGFP-N1

Oncogene IRX1 gene in gastric carcinoma X Guo et al 3911

Figure 3 Effect of IRX1 gene transfection on SGC-7901 gastric cancer cells. (a) Schematic figure for pEGFP-N1/IRX1 construction (top) and the effect of successful transfection of IRX1 (bottom). IRX1 transfection was confirmed by GFP uorescence in nuclei by fluorescence microscopy. (b) IRX1 mRNA and protein expression were induced after gene transfection and confirmed by qPCR, western blot and immunocytochemistry. The bar indicates 20 mm. (c) Colony formation (left) and cell growth activity (right) were suppressed after gene transfection based on soft agar and CCK-8 detection. (d) Cell migratory ability was suppressed by scratch wound healing assays. By wound distance analysis, cell migration ability was suppressed by IRX1 gene transfection. (e) Cell migration and invasive ability were suppressed in the transwell assay. These data are representative examples taken from one of three experiments. vector in which a reporter gene encoding enhanced green compared with the control (60.50±6.72 vs 134.80± fluorescence protein (GFP) was under the control of a 8.56, Po0.001). The amount of invasive cells in the constitutively active upstream promoter. Successful SGC-7901/IRX1 group was also significantly reduced transfection was determined by GFP fluorescence compared with the control (74.90±7.11 vs 169.20± tracing in nuclei (Figure 3a) and confirmed by qPCR 6.96, Po0.001). All experimental results in NCI-N87 (Po0.001), western blot and immunocytochemical gastric cancer cells by IRX1 transfection showed the analysis based on IRX1 re-expression (Figure 3b). same tendency observed in the SGC-7901 cell line and IRX1-transfected gastric cancer cells showed reduced are presented in Supplementary Figure 2. colony formation and cell proliferation in vitro compared with control-transfected cells (P ¼ 0.003 and Tumorigenicity after overexpressing IRX1 in nude mice P ¼ 0.007, respectively; Figure 3c). Vector-transfected We next examined the effect of IRX1 overexpression GFP þ control cells proliferated rapidly in the same by inoculating parental SGC-7901 or NCI-N87 culture conditions. We also examined cell migration cells, SGC-7901 or NCI-N87/vector and SGC-7901 or and invasive ability by scratch healing assay (Figure 3d), NCI-N87/IRX1 cells subcutaneously into the right flank transwell migration and transwell invasion assay regions of nude mice. Tumorigenicity was significantly (Figure 3e). The parental cells and empty vector- reduced in IRX1-transfected cells. Rapid tumor growth transfected cells nearly closed the wound 48 h after was observed in the control groups after 1 month scratch, whereas IRX1-transfected cells were unable to (Figure 4a). The tumor-inhibiting role of IRX1 transfec- heal the wound. The mean wound distances of tion was apparent with both cancer cell lines (P ¼ 0.001, the experimental sample and the control at 48 h were Figure 4b), with less mitosis and rare necrosis observed significantly different (326.844±64.08 mm vs 143.14± under the microscope (Po0.001, Figure 4c). These 8.41 mm; Po0.001). The amount of migrated cells in the findings imply that IRX1 suppresses the growth of IRX1-transfected sample was significantly reduced cancer cells.

Oncogene IRX1 gene in gastric carcinoma X Guo et al 3912

Figure 4 Tumorigenesis analysis in nude mice. (a) Re-expressing IRX1 in SGC-7901 and NCI-N87 cells inhibited tumorigenesis in nude mice. Top represents the SGC-7901 group; bottom represents the NCI-N87 group. (b) Tumor nodules resected (left) from the IRX1-transfection group were smaller than those from controls. Tumorigenesis curves for the SGC-7901 or NCI-N87/IRX1 group and controls are shown on the right. Rapid tumor growth was observed in the control groups (data represented as the mean±s.d.; P ¼ 0.001). (c) Histological examination found that mitosis of tumor cells was markedly reduced in the IRX1-transfection group compared with the control groups (top, indicated by arrows, the bar indicates 20 mm). Necrosis is apparent in controls due to their rapid growth (down, the bar indicates 50 mm).

Identification of target genes after IRX1 gene transfection genes for cluster mapping on the MeV microarray We analyzed the genome-wide transcriptome profile analysis platform (www.tm4.org/mev.html, Figure 5a). of SGC-7901, SGC-7901/vector and SGC-7901/ We also looked for genes related to molecular function IRX1 cells by Agilent oligo microarray (41 000 þ ). and picked up the top 20 gene sets that overlapped with The microarray data set has been deposited in the different function-clusters for exhibition (Figure 5b). GEO database (http://www.ncbi.nlm.nih.gov/geo/query/ We chose 17 upregulated genes and 19 downregulated acc.cgi?acc ¼ GSE17399). According to fold-change genes for real-time PCR verification and confirmed our (X2.0) screening between SGC-7901/vector and SGC- microarray findings for 8 upregulated genes and 11 7901/IRX1 cells, we found 224 upregulated genes and downregulated genes (Figures 6a and b). The gene 324 downregulated genes (Supplementary Table 3). We names and functional annotations are listed in Table 1. searched genes that overlapped with cancer-associated To determine whether these are direct target genes of and molecular function-related gene sets in MSigDB IRX1, we conducted chromatin immunoprecipitation (C4 and C5 gene sets; http://www.broad.mit.edu/gsea/ assays using IRX1 antibody and then analyzed the msigdb/index.jsp). We selected 42 cancer-associated pulled-down DNA. We identified three downregulated

Oncogene IRX1 gene in gastric carcinoma X Guo et al 3913

Figure 5 Target gene identiﬁcation by global microarray analysis. (a) Clustering map of differentially expressed genes overlapped with cancer-associated genes set in the Molecular Signatures Database. Row represents gene, column represents experimental cells. Left column is IRX1-transfected cell; Middle column is SGC-7901 parental cell; Right is empty vector-transfected cell. Upregulated genes are shown in red and downregulated genes in green. (b) Fold-change map overlapped with molecular function-related genes set in the Molecular Signatures Database. Upregulated genes are listed on the left; and downregulated genes are listed on the right.

genes, BDKRB2 (NM_000623), FGF7 (NM_002009) curve (AUC) for methylated blood DNA detection were and HIST2H2BE (NM_003528) as direct IRX1 targets calculated. The AUC was 0.827±0.083 and was signifi- (Figure 6c). cantly higher than that of the null hypothesis (true area was 0.5, P ¼ 0.007, Figure 7e). Thus, the level of methylated IRX1 in plasma could serve as a molecular Methylation status of IRX1 in primary cancer tissues and marker for gastric cancer. To identify an optimal cut-off peripheral blood of gastric carcinoma patients point to detect gastric cancer, Youden’s index was used in We examined the methylation status of primary tumor this study. According to Youden’s index, the optimal tissues and associated plasma DNA from 15 gastric operating point of the blood methylation level of IRX1 carcinoma patients (Table 2) and 10 age-matched was 26.4%. At this cut-off point, the sensitivity was healthy controls. The mean methylated levels of IRX1 73.3% and specificity was 90%. in 15 primary tumor tissues and in plasma DNA were 51.90%±24.32 and 47.23%±35.93, respectively. The mean methylated level of plasma DNA in Discussion healthy control cases was 6.66%±11.71. (P ¼ 0.002; Figures 7a–d). Thus, significant methylation was ob- Gastric cancer is the second most common cancer in the served in the cancer group compared with healthy world. It is now apparent that multiple genetic altera- controls. We analyzed the methylation level using tions, including Helicobacter pylori infection, oncogene multiple clinicopathological parameters. Higher methy- activation and tumor suppressor gene inactivation, are lation levels were closely associated with increased age necessary steps in gastric cancer development (Telford (P ¼ 0.003) and TNM staging (P ¼ 0.051). The Receiver et al., 1994; Ushijima et al., 2006). In a previous study, Operating Characteristics curve and the area under we revealed a relevant genetic variant at a high

Oncogene IRX1 gene in gastric carcinoma X Guo et al 3914

Figure 6 Verification of IRX1 target genes by real-time PCR. (a) Upregulation of PTGS2, ANPEP, KDR, UGT8, INHBA, ERMAP, RALGPS1 and SPON1 was confirmed. (b) Downregulation of BDKRB2, PHYHIPL, HIST2H2BE, FGF7, PTGER1, NPTX1, EGR1, COL9A3, CUGBP2, DKK3 and BPI was confirmed. (c) Chromatin immunoprecipitation analysis was done with IRX1 antibody using lysates from GES-1 cells. Promoter pulldowns were assessed through quantitative PCR, which revealed amplified BDKRB2, FGF7, and HIST2H2BE compared with the negative IgG control.

frequency of LOH locus at 5p15.33 in gastric carcinoma. were analyzed. In addition, we reconstituted IRX1 The candidate gene IRX1 harbored on 5p15.33 was expression in gastric cancer cells. Our results suggest suspected as a tumor suppressor gene (Lu et al., 2005; that expression of IRX1 is signiﬁcantly decreased in Yu et al., 2006). gastric cancer. The inactivation of IRX1 genes via a In this study, we analyzed the expression level, combination of allelic loss and promoter methylation is regulating mechanism and biological function of IRX1 a common event in the development of gastric carcino- in gastric carcinoma. To examine the second hit on genesis. No sequence alterations (mutations) of IRX1 IRX1 inactivation in addition to gene copy number were found in gastric carcinoma. Our ﬁndings are deletion, sequence mutations and promoter methylation consistent with the Knudson two-hit hypothesis of

Oncogene IRX1 gene in gastric carcinoma X Guo et al 3915 Table 1 Veriﬁed genes and their functions by real-time PCR Genes Accession number Functions

Upregulated ANPEP NM_001150 (membrane) Aminopeptidase, CD13 KDR NM_002253 kinase insert domain receptor; transmembrane receptor protein tyrosine kinase-signaling pathway UGT8 NM_003360 Glycosphingolipid biosynthesis PTGS2 NM_000963 Prostaglandin-endoperoxide synthase 2; biosynthesis INHBA NM_002192 Inhibin b-A; cell cycle arrest; cell–cell signaling ERMAP NM_001017922 Erythroblast membrane-associated protein; integral to membrane RALGPS1 NM_014636 Ral GEF with PH domain and SH3-binding motif 1; small GTPase-mediated signal transduction SPON1 NM_006108 Spondin 1, extracellular matrix protein; protein binding

Downregulated NPTX1 NM_002522 Neuronal pentraxin I; synaptic transmission; calcium ion binding HIST2H2BE NM_003528 Histone 2; nucleosome assembly; DNA binding EGR1 NM_001964 Early growth response 1; regulation of transcription, DNA-dependent COL9A3 NM_001853 Collagen, type IX, a-3; extracellular matrix; structural molecule activity CUGBP2 NM_006561 RNA-binding protein 2 FGF7 NM_002009 Fibroblast growth factor 7; positive regulation of cell proliferation; growth factor activity BPI NM_001725 Bactericidal/permeability-increasing protein DKK3 NM_015881 Dickkopf homolog 3; development; morphogenesis; Wnt receptor-signaling pathway PTGER1 NM_000955 Prostaglandin E receptor 1; G-protein coupled receptor protein signaling pathway BDKRB2 NM_000623 Bradykinin receptor B2; vasodilation; transmembrane receptor protein tyrosine kinase signaling pathway PHYHIPL NM_032439 Phytanoyl-CoA hydroxylase interacting protein-like; integral to membrane

Table 2 Clinical information and blood methylation status of gastric cancer patients Case Age (years) Gender Location Differentiation TNM stage Diameter (cM) Tumor marker Methylation (%)

1 38 Female Body Low III 7 CA125m 5.6 2 55 Female Body Low III 5 Normal 77.8 3 47 Male Antrum Low IV 9 CA72-4m 30.6 4 60 Female Antrum Low IV 5 Normal 58.9 5 85 Male Antrum Middle I 4 Normal 88.9 6 58 Female Antrum Low II 7 Normal 41.7 7 72 Male Antrum High II 5 CA19-9m 83.3 8 54 Female Antrum Low II 4 CA72-4m 33.3 9 79 Female Antrum Low IV 7 CEA,CA125,CA19-9 and CA72-4m 91.7 10 60 Male Antrum Low II 10 CEAm 66.7 11 55 Female Antrum Low I 1 Normal 0 12 56 Female Antrum High I 0.5 Normal 0 13 42 Male Antrum Low III 8 Normal 33.3 14 64 Male Antrum Middle I 4 Normal 0 15 90 Male Antrum Middle IV 6 CA72-4m 91.7

tumorigenesis. Our study also revealed that transcrip- Hitherto, there has been very limited data on the tional silencing is the mechanism of IRX1downregulation functional association between the IRX1 gene and in human gastric cancer. The high level of methylation on human cancer. Introducing a tumor suppressor gene to CpG sites of the IRX1 promoterwasassociatedwitha cancer cells is an important method for cancer research transcriptional block. Epigenetic events, especially pro- (Tamura et al., 1998, 1999). Therefore, we constructed moter methylation, are now widely accepted causes of an IRX1-expressing vector and restored IRX1 expres- gene silencing (Kawakami et al., 2000; Robertson and sion in SGC-7901 and NCI-N87 gastric cancer cells. Jones, 2000; Garinis et al., 2002; Momparler, 2003; Choi These experiments revealed that restoring IRX1 can and Wu, 2005; Toyota and Issa, 2005; Esteller, 2007; Jee reduce growth and invasion of gastric carcinoma both et al., 2009). 5-Aza-dC is a methyltransferase inhibitor in vitro and in vivo. The tumor suppressing function of that is widely used to restore gene expression (Bergman the IRX1 gene was also conﬁrmed by Bennett et al. and Mostoslavsky, 1998; Tycko, 2000; Yamashita et al., (2008) recently. They found that introducing IRX1 2002; Shaker et al., 2004; Hellebrekers et al.,2007; expression in squamous cell carcinoma of the head and Schneider-Stock and Ocker, 2007; Hurtubise et al.,2008; neck disclosed the gene’s tumor suppressor potential. To Lemaire et al., 2008; Cairns, 2009). In the case of the clarify the precise mechanism of IRX1’s tumor-inhibit- IRX1 gene, introduction of 5-Aza-dC restored IRX1 ing function, we screened the target genes regulated by expression in most of the cancer cells. IRX1 overexpression.

Oncogene IRX1 gene in gastric carcinoma X Guo et al 3916

Figure 7 Methylation analysis of primary tumor tissues and peripheral blood from gastric carcinoma patients and healthy controls. (a) Methylation status of 12 CpG sites of the IRX1 promoter from primary tumor tissues from 15 gastric carcinoma patients. (b) Methylation status of 12 CpG sites of the IRX1 promoter from plasma DNA from 15 gastric carcinoma patients. Each row of circles represents an integrated methylation ratio from three clones, and each circle represents a single CpG site. Open circle represents unmethylated cytosine; filled circle or partially filled circle represents methylated ratio. (c) Methylation status of 12 CpG sites of the IRX1 promoter from plasma DNA from 10 age-matched healthy controls. (d) The mean methylated level of cancer tissues and plasma in 15 patients was 51.90%±24.32 and 47.23%±35.93 (upper and middle). The mean methylation level of healthy controls was 6.66%±11.71 (lower). (e) The Receiver Operating Characteristics curve of blood-methylated DNA detection for IRX1. The AUC was 0.827±0.083 (P ¼ 0.007), with sensitivity and specificity of 73.3 and 90%, respectively.

Microarray is a power technology that is able to a variety of biological processes, including tumor perform genome-wide analysis in one experiment. Gene- growth and invasion. FGF7 is a potent epithelial cell- expressing profiles can characterize genes that are specific growth factor, and is involved in tumorigenesis differentially regulated in different experimental condi- through stimulating proliferation and invasion (Ropi- tions. In this study, we compared changes in gene quet et al., 1999; Shaoul et al., 2006). These findings expression profiles in gastric cancer cells with or without raised the possibility that IRX1 overexpression down- IRX1 gene transfection and identified a set of IRX1 regulated expression of BDKRB2, FGF7 and target genes. Using real-time PCR analysis, we con- HIST2H2BE, consequently inhibiting angiogenesis, cell firmed 8 upregulated genes and 11 downregulated genes. proliferation and invasion in gastric cancer. Of course, Subsequently, we identified three direct IRX1 target other indirect target genes, such as the transgelin- genes by chromatin immunoprecipitation assay: encoding gene TAGLN, have been reported to have a BDKRB2, FGF7 and HIST2H2BE. HIST2H2BE growth-promoting effect on gastric carcinogenesis encodes H2B histone, which serves as a cell proliferation (Huang et al., 2008). marker and has been found overexpressed in serum and In clinical practice, gastric cancer patients are often gastric cancer tissues by our group and others (Kamei not symptomatic in the early stage. Endoscope biopsy is et al., 1992; Hao et al., 2008). BDKRB2 was reported as still the golden standard for gastric cancer diagnosis. an enhancer of angiogenesis in cancer (Plendl et al., However, endoscope examination is uncomfortable and 2000; Ishihara et al., 2001, 2002; Ikeda et al., 2004). invasive. Serum specimen screening is a promising FGF7 encoded a member of the fibroblast growth factor diagnostic tool because it is easy and inexpensive. Some (FGF) family. FGF family members possess broad serum tumor markers have been used in gastrointestinal mitogenic and cell survival activities, and are involved in cancer screening and monitoring. Most of them are

Oncogene IRX1 gene in gastric carcinoma X Guo et al 3917 glycoprotein antigens such as carcinoembryonic antigen addition to gene copy number deletion. Restoring IRX1 (CEA), carbohydrate antigen 19–9 (CA19-9) and carbo- expression impairs cell proliferation, migration, invasion hydrate antigen 72–4 (CA72-4). However, the early and tumorigenesis both in vitro and in vivo. The diagnostic role of CEA, CA19-9 and CA72-4 for gastric mechanism of the tumor suppressor effect of IRX1 over- cancer remains controversial. The sensitivity of a single expression could be explained by a set of target genes biomarker in tumor diagnosis is low (usually less than identified by global microarray analysis and confirmed 40%) with a higher false-positive rate. Some authors by real-time PCR and chromatin immunoprecipita- proposed that these biomarkers can be useful for tion assay. Downregulation of the direct target genes therapeutic response monitoring and cancer relapse BDKRB2, FGF7 and HIST2H2BE contributed to detection but not for reliable diagnostics (Tocchi et al., decreased angiogenesis, cell proliferation and invasion. 1998; Ychou et al., 2000; Lai et al., 2002). Identifying Hypermethylation of IRX1 detected in the peripheral novel serological biomarkers with higher specificity and blood of gastric cancer patients suggests a biomarker sensitivity is eagerly desired for gastric cancer. Recent potential for IRX1. data showed that the presence of tumor-associated alterations (such as promoter hypermethylation) have been described in the plasma DNA from patients with Materials and methods various kinds of cancer (Kawakami et al., 2000; Duffy et al., 2009). DNA methylation may provide a new Cell lines generation of cancer biomarkers in the next decade Gastric cancer cell lines NCI-N87, SNU-1, SNU-16, AGS and (Duffy et al., 2009; Tommasi et al., 2009). However, no KATO-III were obtained from American Type Culture studies have looked into the relationship between the Collection (Manassas, VA, USA). Gastric cancer cell lines IRX1 molecular marker and gastric cancer. We are the MKN-45 and SGC-7901, immortalized human gastric mucosa first to investigate the correlation between methylated cell line GES-1 and embryonic kidney cell line 293T were IRX1 and gastric cancer using peripheral blood samples. preserved in our institute. The methylation levels in gastric cancer patients were significantly increased compared with controls Patients (P ¼ 0.002). In addition, we found that the IRX1 Gastric cancer tissues and peripheral blood were collected methylation level in blood increased with advanced from patients and healthy controls at Shanghai Ruijin TNM staging and aging. This finding implies that Hospital, Shanghai Jiao Tong University, School of Medicine, circulating methylated IRX1 is a promising molecular after obtaining the subjects’ informed consent and with marker for gastric cancer. However, as a methylated institutional review board approval of the hospital. All patients obtained a confirmed diagnosis of gastric carcinoma marker its specificity and sensitivity for early diagnosis after resection. of gastric cancer require further verification. In conclusion, we outlined the functions of the IRX1 tumor suppressor gene during gastric carcinogenesis in Mutation, copy number and promoter analysis Figure 8. IRX1 expression is markedly downregulated Primers covering exons and splicing regions were designed. A total of seven fragments overlapping four exons were in gastric cancer cells. The expression level of IRX1 in amplified. Mutations were analyzed by sequencing the PCR gastric cancer correlates with promoter methylation in products (Sangong, Shanghai, China). Gene copy numbers of IRX1 for all cell lines were analyzed by real-time PCR. Three pairs of primers targeting different regions were used (P1 for exon one and intron one junction, P2 for exon two and intron two junction and P3 for intron two and exon three junction). We choose the single-copy gene RPP30 (ribonuclease P/MRP 30 kDa subunit) as internal control. The reactions were run with the following conditions: one cycle at 50 1C for 2 min, 95 1C for 10 min and then 40 cycles (95 1C for 15 s and 56 1C for 30 s). Target gene copy number was determined by the relative quantitative comparative threshold cycle (DDCt) method. In this method, the relative copy-number difference of the target gene in tumor sample against reference sample is given by 2ÀDDCt described by Kuga et al. (2008). Promoter fragments were amplified from human genomic DNA by two-step nested PCR. The fragments of the 50- flanking region were inserted into the MluI and BglII sites of the promoterless firefly luciferase reporter vector pGL3-Basic to construct luciferase reporter pGL3-775 bp. By progressive Figure 8 Schematic summary of the IRX1 tumor suppressor gene 50deletion, we obtained PCR products for seven additional on carcinogenesis of gastric cancer. IRX1 is inactivated via a combination of LOH and hypermethylation, which may be induced plasmids: pGL3-747, pGL3-644, pGL3-476, pGL3-410, by radiation stress or an inflammatory reaction to pathogens. pGL3-380, pGL3-248 and pGL3-152. The primers used are Restoring IRX1 expression impairs cell proliferation, migration, listed in Supplementary Table 4. One hundred nanograms invasion and tumorigenesis both in vitro and in vivo, which could be of each human IRX1-luciferase reporter gene construct was explained by the downregulation of specific target genes identified transfected into cells using Lipofectamine 2000 (Invitrogen, by global microarray analysis. Carlsbad, CA, USA). The pRL-TK vector (5 ng) (Promega,

Oncogene IRX1 gene in gastric carcinoma X Guo et al 3918 Madison, WI, USA) harboring the Renilla luciferase gene HRP-conjugated secondary antibody. Protein bands were was co-transfected as an internal control for transfection visualized by the ECL detection system (Amersham Bio- efficiency in mammalian cells. Each construct was trans- sciences, Uppsala, Sweden). Autoradiograms were quantified fected in triplicate. Luciferase activity was determined with by densitometry (Quantity One software; Bio-Rad, Hercules, an analytical luminometer (TD-20/20, Turner Designs, CA, USA). GAPDH-specific antibody was used for a loading Sunnyvale, CA, USA). control. Relative protein levels were calculated by reference to the amount of GAPDH protein. Mouse anti-GFP (1:800, Sigma) and mouse anti-GAPDH (1:1000, Santa Cruz, CA, Methylation analysis and 5-Aza-20-deoxycytidine treatment USA) were used as the primary antibodies. Genomic DNA from cell lines, gastric cancer tissues and plasma (0.2 ml) was purified using DNAzol (Invitrogen), treated with sodium bisulfate (Sigma, Phoenix, AZ, USA), Immunocytochemical analysis and then analyzed by MSP or bisulfate-sequencing PCR SGC-7901 cells were cultured in RPMI-1640 medium supple- (BSP). The PCR products were confirmed by agarose gel mented with 10% fetal calf serum on cover slides and electrophoresis and visualized using ethidium bromide staining maintained at 37 1C in an atmosphere containing 5% CO2 for MSP. A negative control (without DNA) was included in and 95% air for 24 h. Then the slides were fixed in 4% every set of PCR experiments. Amplified bisulfate PCR paraformaldehyde for 30 min and treated with 1% Triton–PBS products were subcloned into the TA vector system (Promega) solution for 15 min at room temperature. Next, protein according to the manufacturer’s instructions. DNA sequencing expression of IRX1 in the SGC-7901 cell line with or without was performed on three to ten individual clones (Sangong). gene transfection was examined using polyclonal anti-human Cell lines were treated with 10 mM of 5-Aza-20-deoxycytidine IRX1 (Abnova, H00079192-A01, Taiwan, working dilution: (5-Aza-dC; Sigma) for 96 h or 120 h; the control group was 1:600) by the EnVision two-step procedure. treated with 1 Â PBS. Total RNA was extracted for assessment of IRX1 mRNA levels. The primers used are summarized in Cell growth and colony formation assay Supplementary Table 4. Cell growth was assayed using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Soft agar colony formation was per- mRNA expression analysis formed using the cell transformation detection kit (Chemicon, Total RNA was extracted using Trizol solution. Reverse Temecula, CA, USA) according to the manufacturer’s transcription (RT) was performed in a 20-ml reaction system instructions. After 21 days of culture, the colonies were according to the manufacturer’s recommendation. RT–PCR stained and counted under an inverted phase-contrast micro- primers for IRX1 (260 bp) were 50-GCTCTTCGGCAGCG scope. A dot with cells 450 was counted as a colony. These ACAC-30 (F) and 50-GCTCTGGGGCCTCCTTTG-30 (R). data were reported as the means±s.d. by counting 10 fields Primers for b-actin (428 bp) were 50-CCACTGGCATCGTG randomly. ATGGAC-30 (F) and 50-GCGGATGTCCACGTCACACT-30 (R). Each PCR was performed for 30 thermal cycles and then Invasion, migration and scratch healing assays the PCR products were observed by electrophoresis on a 1.5% Cell invasion and cell migration assays were performed. For agarose gel and visualized after staining with ethidium the scratch assay, cells were treated with 10 mg/ml mitomycin C bromide. QRT–PCR was carried out with the IRX1 primers (Sigma) for 3 h and then wounded with a pipette tip. Fresh, full 50-CGCGGATCTCAGCCTCTTC-30 (F) and 50-CCCCAGG medium was added, and the wound closing procedure was GTTGTCCTTCAGT-30 (R). Primers for GAPDH were observed for 48 h. Photographs were taken every 6 h. For 50-GGACCTGACCTGCCGTCTAG-30 (F) and 50-GTAGCC migration and invasion assays, cell culture was performed in CAGGATGCCCTTGA-30(R). Quantitative measurement of Transwell chambers (8 mm, 24-well format; Corning, NY, IRX1 mRNA levels was performed by ABI Prism 7000 USA). For the invasion assay, the insert membranes were (Applied Biosystems, Foster city, CA, USA). These data were coated with diluted Matrigel (BD Biosciences, San Jose, CA, analyzed by the comparative C method. t USA). Cells (1 Â 105) were added to the upper chamber and cultured for 48 h. For the migration assay, the insert IRX1 overexpression membranes were not coated with Matrigel but were cultured IRX1 overexpression vector pEGFP-N1–IRX1 was constructed under the same conditions. Finally, the insert membranes were using primers 50-ATACTCGAGTCCTTCCGGCAGCTGG cut and stained with Crystal violet (0.04% in water; 100 ml) and GC-30 (F) and 50-ATAGAATTCCGGACGGGAGGGCTG the permeating cells were counted under the inverted micro- CTA-30 (R). SGC-7901 and NCI-N87 gastric cancer cell lines scope and photographed. At least three independent experi- were used for the overexpression studies. We obtained stably ments were performed for all conditions. The data are shown transfected clones by G418 selection (Promega). A stable as the means±s.d. transfectant of the pEGFP-N1 empty vector was used as a control. Transfection was verified by fluorescence microscopy Tumor growth in nude mice (Olympus, Japan). Cells (100 ml, 1 Â 106 cells) from parental cell lines SGC-7901 or NCI-N87, or from transfected lines cancer cells/vector or Western blot analysis cancer cells/IRX1 were collected and inoculated subcuta- Protein from treated cell lines was extracted by mammalian neously into the right flank regions of 4-week-old male protein extraction reagent (Pierce, Appleton, WI, USA) BALB/c nude mice (Institute of Zoology, Chinese Academy supplemented with protease inhibitors cocktail (Sigma). Fifty of Sciences, Shanghai, China). Experimental and control micrograms protein samples were resolved by 10% SDS– groups had five mice each. Tumor nodules were measured PAGE and then transferred to PVDF membranes. The every 4 days with calipers. Mice were killed after 1 month. membranes were blocked with TBST buffer (TBS plus 0.1% Tumor growth curves and inhibiting rates were calculated. Tween-20) containing 5% w/v non-fat milk and hybridized After tumor excision, the tissues were fixed in 10% buffered with primary antibody, followed by incubation with specific formalin. All formalin-fixed and paraffin-embedded samples

Oncogene IRX1 gene in gastric carcinoma X Guo et al 3919 were carefully examined after staining with hematoxylin–eosin change. The threshold used to screen up- or downregulated (HE) and photographed. genes was fold-change X2.

Global cDNA microarray analysis and target gene verification Accession number The whole human genome oligo microarray (Agilent, Santa Microarray data reported herein have been deposited at Clara, CA, USA) was used. After hybridization and washing, the NCBI Gene Expression Omnibus (http://www.ncbi.nlm. the microarray slides were scanned with an Agilent DNA nih.gov/geo/query/acc.cgi?acc ¼ GSE17399) with the accession microarray scanner. The resulting text files extracted from number GSE17399. Agilent Feature Extraction Software (version 9.5.3) were imported into the Agilent GeneSpring GX software (version 7.3) for further analysis. Differentially expressed genes were identified through fold-change screening. For target gene Conflict of interest verification we used real-time PCR and chromatin immunoprecipitation assay. The primers for the target genes are listed The authors declare no conflict of interest. in Supplementary Table 4.

Statistics Data from cell growth, luciferase assays and mice tumorigen- Acknowledgements esis experiments were analyzed with one-way ANOVA. The data from plasma methylation detection of IRX1 were This work was supported, in part by grants from the National analyzed by independent-sample T-test. Receiver Operating Natural Science Foundation of China (30572127, 30770961 and CharacteristicsROC graph was performed. Quantitative 30973486), the Chinese National High Tech Program (863- values were expressed as the means±s.d. SPSS 15.0 software 2006AA02A402, 2006AA02A301), the Shanghai Pu Jiang Project (SPSS Inc., Chicago, IL, USA) was used for all statistical (PJ200700367), Key Research Project from Shanghai Science and analyses, and a P-value o0.05 was considered signiﬁcant. Technology Commission (09DZ1950101 and 09JC1409600), and Gene expression proﬁling of cell lines was analyzed by fold- Shanghai Charity Foundation for Cancer Research.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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