Oncogene (2010) 29, 5969–5975 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc SHORT COMMUNICATION EMX2 is epigenetically silenced and suppresses growth in human lung cancer J Okamoto1,2,7, T Hirata1,2,7, Z Chen1,3, H-M Zhou3, I Mikami1,2,HLi1, A Beltran1, M Johansson4,5, LM Coussens4,5, G Clement1, Y Shi1,6, F Zhang1, K Koizumi2, K Shimizu2, D Jablons1,5 and B He1,5 1Thoracic Oncology Program, Department of Surgery, University of California, San Francisco, CA, USA; 2Department of Surgery, Division of Thoracic Surgery, Nippon Medical School, Tokyo, Japan; 3Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China; 4Department of Pathology, University of California, San Francisco, CA, USA; 5Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA and 6Biosciences Division, SRI International, Menlo Park, CA, USA Lung cancer is a common cancer and the leading cause of than 80% of lung cancers are non-small cell lung cancer, cancer-related death worldwide. Aberrant activation of which includes distinct histological subtypes: adenocarci- WNT signaling is implicated in lung carcinogenesis. noma, squamous cell carcinoma and large cell carcinoma EMX2, a human homologue of the Drosophila empty (Travis, 2002). The mainstays of conventional treatments spiracles gene is a homeodomain-containing transcription have offered patients only a limited and generally short- factor. The function of EMX2 has been linked to the WNT term benefit. Overall, 5-year survival has remained at a signaling pathway during embryonic patterning in mice. dismal B15% for over two decades (Travis, 2002; Jemal However, little is known about the role of EMX2 in human et al., 2008). The molecular carcinogenesis of lung cancer is tumorigenesis. In this study, we found that EMX2 was characterized by multiple alterations of gene expression dramatically downregulated in lung cancer tissue samples and function. These arise from a series of molecular and and this downregulation was associated with methylation of morphological events affecting oncogenes such as K-ras theEMX2promoter.RestorationofEMX2expressionin and EGFR (Fong et al., 2003) and tumor suppressor genes lung cancer cells lacking endogenous EMX2 expression such as p53 and p16 (Zochbauer-Muller et al., 2000), all suppressed cell proliferation and invasive phenotypes, leading inexorably to abnormal changes in cell signaling inhibited canonical WNT signaling, and sensitized lung transduction pathways. cancer cells to the treatment of the chemo cytotoxic drug The homeobox gene family encodes transcription cisplatin. On the other hand, knockdown of EMX2 factors that regulate morphogenesis and cell differentia- expression in lung cancer cells expressing endogenous tion during embryogenesis by activating or repressing EMX2 promoted cell proliferation, invasive phenotypes the expression of target genes (Boersma et al., 1999). and canonical WNT signaling. Taken together, our study In addition, several homeobox genes have recently been suggests that EMX2 may have important roles as a novel shown to be associated with cancers (Raman et al., 2000; suppressor in human lung cancer. Abate-Shen, 2002; Samuel and Naora, 2005; Yoshida Oncogene (2010) 29, 5969–5975; doi:10.1038/onc.2010.330; et al., 2006). EMX2 is a human homologue of the published online 9 August 2010 Drosophila empty spiracles gene (ems), a homeodo- main-containing transcription factor with important Keywords: EMX2; methylation; lung cancer; WNT functions during early development. For example, mice signaling; tumor suppression harboring homozygous mutation of EMX2 (EMX2À/À) exhibit small cerebral hemispheres and olfactory bulbs (Dalton et al., 1989). EMX2 affects the proliferation of adult neural stem cells by regulating the frequency of The American Cancer Society lists lung cancer as the symmetric divisions that generate two stem cells within leading cause of cancer death in the United States, the adult neural stem cell population, and overexpres- exceeding all combined deaths from the next four most sion of EMX2 decreases the frequency of symmetric deadly cancers, that is breast, colon, prostate and divisions (Galli et al., 2002). EMX2 controls mamma- pancreatic cancers (American Cancer Society, 2008). More lian reproduction by adjusting endometrial cell prolif- eration without affecting differentiation (Taylor and Correspondence: Professor B He, Thoracic Oncology Program, Fei, 2005). Moreover, it has been reported that loss of Department of Surgery, University of California, 2340 Sutter Street, EMX2 function leads to ectopic WNT1 expression in Room N222, San Francisco, CA 94115, USA. the developing mammalian telecephalon, resulting in E-mail: [email protected] 7These authors contributed equally to this work. cortical dysplasia (Ligon et al., 2003). The WNT Received 3 February 2010; revised 11 June 2010; accepted 1 July 2010; pathway is well known to have important roles in published online 9 August 2010 embryogenesis, stem cell renewal and oncogenesis EMX2 in human lung cancer J Okamoto et al 5970 120 20 p < 0.001 100 10 80 60 Relative EMX2 Expression 0 10 40 Relative EMX2 Expression 5 20 Relative Methylation 0 0 Normal Tumor 1236910 45 78 (n=64) 1.2 10 1.0 8 0.8 6 0.6 4 0.4 2 0.2 Relative Methylation 0 0 Relative EMX2 Expression H460 H441 A549 A427 H522 H838 H322 H1703 H1299 H2170 H1666 H1975 H460 A549 H838 A427 H522 H322 H441 H1703 H1975 H2170 H1666 H1299 Normal EMX2 GAPDH DAC -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ -+ H460 A549H1703 H838 H1975 A427 H2170 H1666 H1299 H522 H322 H441 Figure 1 EMX2 expression was downregulated by methylation in lung cancer tissues and cell lines. Fresh samples (lung cancer tissue and its adjacent normal tissue) were collected from patients undergoing surgical resection with approval by the Committee on Human Research at the University of California, San Francisco (UCSF). Samples were promptly snap frozen in liquid nitrogen and stored at À170 1Cbefore use. Total RNA was extracted using TRIzol LS (Invitrogen, Carlsbad, CA, USA). Human lung cancer cell lines were all purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI 1640 with 10% fetal bovine serum, penicillin (100 IU/ml)/streptomycin (100 mg/ml)at371C in a humidified 5% CO2 incubator. cDNA synthesis and Taqman PCR were performed as previously described (Raz et al., 2008). Hybridization probes and primers (Supplementary Information Table S1) were purchased from Applied Biosystems (ABI, Foster City, CA, USA). EMX2 expression of samples was calculated by using the 2ÀddCt method (normalizing to their housekeeping gene GAPDH and then comparing to total RNA of adult normallungtissue(BioChain,Hayward,CA,USA)). Quantitative methylation-specific PCR (qMSP) was performed as previously described (Fackler et al., 2004; Grote et al., 2005, 2006). Genomic DNA was extracted with Qiagen DNeasy kits (Qiagen, Valencia, CA, USA) and bisulfite modification of genomic DNA was performed using EZ DNA Methylation-Gold Kits (Zymo Research, Orange, CA, USA). Primers and probes (Supplementary Table S1) were designed using Primer Express and Methyl Primer Express Software v1.0 (ABI) and purchased from Operon (Huntsville, AL, USA). Relative EMX2 methylation levels were determined by using the 2ÀdCt method (normalizing to the housekeeping gene ACTB (Raz et al., 2008)) and then calculating the ratio (tumor/matched normal for tissues; cell line/an adult normal lung tissue (BioChain) for cell lines). Both quantitative RT–PCR and qMSP were done in triplicate using an ABI 7300 Real-time PCR System. (a) Quantitative RT–PCR of 64 tumors and their matched adjacent normal lung tissues. The y axis represents normalized relative EMX2 mRNA expression (arbitrary unit). (b) Quantitative RT–PCR (upper panel) and quantitative MSP (lower panel) of 10 representative tumors (black bars) compared with their matched adjacent normal lung tissues (gray bars). (c) Quantitative RT–PCR analysis and (d) Quantitative MSP analysis in lung cancer cell lines. An adult normal lung tissue was used as a control. Results are means±s.d. (error bars). (e) DAC treatment of lung cancer cell lines. Treatment of cells lines with 5 mM DAC (Sigma, St Louis, MO, USA) was performed as previously described (Mazieres et al., 2004). Total RNA was isolated using Qiagen RNeasy kit 72 h after treatment, and EMX2 expression was examined by semiquantitative RT–PCR (primers (Supplementary Table S1) were purchased from Operon). GAPDH served as control for RNA quality and loading. Oncogene EMX2 in human lung cancer J Okamoto et al 5971 (Klaus and Birchmeier, 2008), including that in lung We first examined the mRNA levels of EMX2 in cancer (Mazieres et al., 2004; You et al., 2004; He et al., human lung cancer tissue samples and their matched 2005; Huang et al., 2008; Akiri et al., 2009). There have adjacent normal tissues obtained from 64 patients with been only a limited number of recent studies suggesting lung cancer. Upon comparison, 71.8% (46–64) lung possible involvement of EMX2 in human cancer. For cancer samples analyzed were found to have less EMX2 example, EMX2 may be anti-proliferative in the expression than their matched adjacent normal tissues endometrium, and its expression is decreased in (Figure 1a), and this downregulation was statistically endometrial tumors (Noonan et al., 2001, 2003). significant (mean values of EMX2 expression measured EMX2 also displays methylation but rarely in non- by quantitative