Oncogene (2011) 30, 4498–4508 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE IRX1 inﬂuences peritoneal spreading and metastasis via inhibiting BDKRB2-dependent neovascularization on gastric cancer J Jiang1, W Liu1, X Guo, R Zhang, Q Zhi, J Ji, J Zhang, X Chen, J Li, J Zhang, Q Gu, B Liu, Z Zhu and Y Yu Department of Surgery, Shanghai Ruijin Hospital and Shanghai Institute of Digestive Surgery, Shanghai Key Laboratory of Diagnosis and Treatment for Gastric Cancer, Shanghai Jiao Tong University, School of Medicine, Shanghai, China The overexpression of IRX1 gene correlates with the growth arrest in gastric cancer. Furthermore, overexpres- sion of IRX1 gene suppresses peritoneal spreading and long distance metastasis. To explore the precise mechan- isms, we investigated whether restoring IRX1 expression affects the angiogenesis or vasculogenic mimicry (VM). Human umbilical vein endothelial cells (HUVECs) and chick embryo and SGC-7901 gastric cancer cells were used for angiogenesis and VM analysis. Small interfering RNA was used for analyzing the function of BDKRB2, a downstream target gene of IRX1. As results, the remarkable suppression on peritoneal spreading and pulmonary metastasis of SGC-7901 cells by IRX1 transfectant correlates to reduced angiogenesis as well as VM formation. Using the supernatant from SGC- 7901/IRX1 cells, we found a strong inhibiting effect on angiogenesis both in vitro and in chick embryo. SGC- 7901/IRX1 cells revealed strong inhibiting effect on VM formation too. By gene-speciﬁc RNA interference for BDKRB2, or its effector PAK1, we got an effective inhibition on tube formation, cell proliferation, cell migration and invasion in vitro. In conclusion, enforcing IRX1 expression effectively suppresses peritoneal spread- ing and pulmonary metastasis via anti-angiogenesis and anti-VM mechanisms, in addition to previously found cell growth and invasion. BDKRB2 and its downstream effector might be potential targets for anti-cancer strategy. Oncogene (2011) 30, 4498–4508; doi:10.1038/onc.2011.154; published online 23 May 2011 Keywords: gastric carcinoma; IRX1; angiogenesis; vasculogenic mimicry; BDKRB2 Correspondence: Dr Y Yu or Dr Z Zhu, Department of Surgery, Ruijin Hospital and Shanghai Institute of Digestive Surgery, ShanghaiJiao Tong University, School of Medicine, Ruijin er Road, No. 197, Shanghai 200025, PR China. E-mail: yingyan3y@yahoo.com.cn or surgeryzhu@yahoo.com.cn 1These authors contributed equally to this work. Received 10 October 2010; revised 26 March 2011; accepted 27 March 2011; published online 23 May 2011 Introduction IRX1 is a newly identiﬁed tumor suppressor gene on gastric cancer in our previous studies (Lu et al., 2005; Guo et al., 2010). Meanwhile, the tumor suppressor function of IRX1 was also conﬁrmed on head and neck squamous cell carcinoma by others (Bennett et al., 2008, 2009). IRX1 belongs to Iroquois homeobox gene family, which has six members of IRX1 to IRX6. IRX1 is closely related to embryonic development, including foregut organs such as lung (Becker et al., 2001; Alarcon et al., 2008). Our previous study revealed that restoring IRX1 in gastric cancer cells arrested cancer proliferation both in vitro and in vivo. In addition, we found that several downregulated genes involve in angiogenesis in IRX1-transfected gastric cancer cells by cDNA microarray analysis (GSE17399). BDKRB2 is one of the downregulated genes, which connects to angiogenesis in several tumors (Ishihara et al., 2002; Wu et al., 2002). Growth of solid tumor relies on blood vessels to supply oxygen and nutrients. Tumor progression is accompanied by ﬂourish neovascularization to satisfy its metabolic demands. Endothelium-dependent vessels are the predominant microcirculatory mechanism for solid tumors. Therefore, endothelium is the target of the anti-angiogenesis treatment (Matsuda et al., 2000; Getmanova et al., 2006). Vasculogenic mimicry (VM) of solid tumor is another microcirculatory mechanism, which is an endothelium-independent pattern. Various types of malignant tumors demonstrated internal blood supply network via VM formation, including malignant melano- ma (Maniotis et al., 1999), inﬂammatory breast cancer (Shirakawa et al., 2003) prostate cancer (Sharma et al., 2002), invasive ovarian cancer (Su et al., 2008), sarcoma (Hao et al., 2002), astrocytoma (Yue and Chen, 2005) and so on. VM is strongly associated with a poor prognosis in human tumors. So, VM becomes a potential target for anti-cancer strategy (Fujimoto et al., 2006; Zhang et al., 2007; Sun et al., 2008). Gastric cancer is one of the common solid malignancies characterized by accumulated genetic and epigenetic alterations. Peritoneal spreading and metastasis are the pivotal factors for its poor prognosis. Many efforts have been made to arrest the peritoneal spreading and long distance metastasis for gastric cancer. In this paper, we report a new ﬁnding that enforcing IRX1 expression disclosed anti-spreading and anti-metastasis efﬁcacies in