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.