Oncogene (2014) 33, 3109–3118 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE A novel 3p22.3 gene CMTM7 represses oncogenic EGFR signaling and inhibits cancer cell growth HLi1,2,JLi3,YSu1,YFan3, X Guo1,LLi3,XSu3, R Rong3, J Ying4,XMo1, K Liu1, Z Zhang5, F Yang6, G Jiang6, J Wang6, Y Zhang1, DMa1,QTao3 and W Han1 Deletion of 3p12-22 is frequent in multiple cancer types, indicating the presence of critical tumor-suppressor genes (TSGs) at this region. We studied a novel candidate TSG, CMTM7, located at the 3p22.3 CMTM-gene cluster, for its tumor-suppressive functions and related mechanisms. The three CMTM genes, CMTM6, 7 and 8, are broadly expressed in human normal adult tissues and normal epithelial cell lines. Only CMTM7 is frequently silenced or downregulated in esophageal and nasopharyngeal cell lines, but uncommon in other carcinoma cell lines. Immunostaining of tissue microarrays for CMTM7 protein showed its downregulation or absence in esophageal, gastric, pancreatic, liver, lung and cervix tumor tissues. Promoter CpG methylation and loss of heterozygosity were both found contributing to CMTM7 downregulation. Ectopic expression of CMTM7 in carcinoma cells inhibits cell proliferation, motility and tumor formation in nude mice, but not in immortalized normal cells, suggesting a tumor inhibitory role of CMTM7. The tumor-suppressive function of CMTM7 is associated with its role in G1/S cell cycle arrest, through upregulating p27 and downregulating cyclin-dependent kinase 2 (CDK2) and 6 (CDK6). Moreover, CMTM7 could promote epidermal growth factor receptor (EGFR) internalization, and further suppress AKT signaling pathway. Thus, our findings suggest that CMTM7 is a novel 3p22 tumor suppressor regulating G1/S transition and EGFR/AKT signaling during tumor pathogenesis. Oncogene (2014) 33, 3109–3118; doi:10.1038/onc.2013.282; published online 29 July 2013 Keywords: CMTM7; 3p22; tumor-suppressor gene; methylation; LOH INTRODUCTION squamous cell carcinoma (ESCC), nasopharyngeal carcinoma (NPC) Chemokine-like factor 1 (CKLF)-like MARVEL transmembrane and lung carcinoma, suggesting the presence of critical TSGs. domain-containing family (CMTM) is a novel family of proteins Several 3p TSGs have been identified, including RASSF1A (3p21.3), linking chemokines and the transmembrane-4 superfamily.1,2 DLEC1 (3p22.3), HYA22 (3p22.3), LIMD1 (3p21.3), BLU/ZMYND10 Human CMTM family consists of nine members, CKLF and (3p21.3), FHIT (3p14.2) and PLCD1 (3p22.3).22–28 TSGs exert tumor- CMTM1–8, having important roles in immune and male suppressive functions through inhibiting cell proliferation and reproductive systems as well as disease pathogenesis including migration, inducing cell apoptosis and cell cycle arrest. TSGs can tumorigenesis.3–17 Transmembrane-4 superfamily includes several be inactivated by both genetic and epigenetic mechanisms. types of proteins possessing the four transmembrane-helix Genetic deletion and point mutations disrupt TSG functions, structure, such as the classical transmembrane-4 superfamily whereas epigenetic mechanisms including promoter CpG (tetraspanin) and MARVEL domain-containing proteins. methylation and histone modifications, lead to TSG silencing Association with specialized membrane microdomains has been and functional loss thus also frequently involved in tumor reported for MARVEL domain-containing proteins.18 They development and progression.29,30 participate in a variety of cellular processes including In this study, we investigated the expression, function and tumorigenesis. Promoter methylation of MAL, a putative tumor- mechanism of a potential TSG—CMTM7, located at 3p22.3. We suppressor gene (TSG), contributes to gastric and colon found that CMTM7 is broadly expressed in normal tissues but carcinogenesis.19,20 Loss of occludin leads to the progression of frequently silenced or downregulated in some carcinoma types. breast cancer.21 Our previous study also showed that CMTM3 and Promoter methylation and LOH were both found to be mechan- CMTM5 possess tumor-suppressive functions with frequent isms contributing to CMTM7 downregulation. Ectopic CMTM7 epigenetic inactivation by promoter CpG methylation.9,10 expression led to the inhibition of cell proliferation and motility, CMTM6, CMTM7 and CMTM8 form a small gene cluster on through inducing G1/S cell cycle arrest by upregulating p27 and chromosome 3p22. Loss of heterozygosity (LOH) of 3p, especially downregulating cyclin-dependent kinase 2 (CDK2) and 6 (CDK6). 3p12-22, is common in many cancer types including esophageal CMTM7 also promoted epidermal growth factor receptor (EGFR) 1Department of Immunology, Center for Human Disease Genomics, Key Laboratory of Medical Immunology, Ministry of Health, School of Basic Medical Sciences, Peking University, Beijing, China; 2Department of Clinical Laboratory, Peking University People’s Hospital, Beijing, China; 3Cancer Epigenetics Laboratory, State Key Laboratory of Oncology in South China, Department of Clinical Oncology, Sir YK Pao Center for Cancer and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong and CUHK Shenzhen Research Institute, Hong Kong; 4Department of Pathology, Cancer Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China; 5Department of Otolaryngology/Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, China and 6Department of Thoracic Surgery, Peking University People’s Hospital, Beijing, China. Correspondence: Professor Q Tao, Cancer Center, PWH, Department of Clinical Oncology, The Chinese University of Hong Kong, Shatin, Hong Kong or Professor W Han, Peking University Center for Human Disease Genomics, 38 Xueyuan Road, Beijing 100191, China. E-mail: [email protected] or [email protected] Received 16 October 2012; revised 11 April 2013; accepted 29 April 2013; published online 29 July 2013 CMTM7 as a tumor suppressor methylated in carcinomas HLiet al 3110 internalization and further downregulated pAkt. Thus, CMTM7 acts (50%), 1 hypopharyngeal, 5 of 9 breast (55.5%), 2 of 4 cervical as a novel functional TSG. (50%), 1 of 16 gastric (6.25%) carcinoma cell lines and 1 of 3 glioma cell lines (33%; Figure 1c, Supplementary Figure 2B). In contrast, no silencing of CMTM6 was detected in any cell line, RESULTS whereas CMTM8 is expressed in most cell lines, except for its Only CMTM7 is frequently silenced by promoter methylation in silencing in several lymphoma cell lines as well as occasional some carcinoma types carcinoma cell lines (Supplementary Figure 1). CMTM6, CMTM7 and CMTM8 form a gene cluster on 3p22, a The putative CMTM7 promoter identified by bioinformatics chromosomal region rich in TSGs (Figure 1a). We examined their promoter analysis (http://www.genomatix.de) contains a typical expression profiles in a panel of normal adult tissues and tumor CpG island, with common control elements-TATA and CAAT boxes cell lines. Semiquantitative reverse transcription (RT)–PCR showed (Figure 1a). We thus analyzed CMTM7 promoter methylation by that CMTM6, CMTM7 and CMTM8 were all widely expressed in methylation-specific PCR (MSP). The specificity of our MSP system normal tissues (Figure 1b; Supplementary Figure 1). However, was validated using DNA not bisulfite treated (Supplementary CMTM7 was downregulated or silenced in multiple carcinoma cell Figure 2A). Frequent methylation of CMTM7 promoter was lines, including 8 of 18 esophageal (44.4%), 3 of 6 nasopharyngeal observed in ESCC and NPC cell lines with reduced or silenced Figure 1. (a) CMTM6, CMTM7 and CMTM8 form a gene cluster on 3p, a region rich in TSGs. A typical CpG island (CGI) spans the promoter to exon 1 of CMTM7. Vertical bar: a single CpG site. Curved arrow: the transcription start site. MSP and bisulfite genomic sequencing (BGS) regions are shown. (b) CMTM7 is broadly expressed in normal human adult tissues. (c) CMTM7 is frequently silenced in esophageal (ESCC) and nasopharyngeal (NPC) carcinoma cell lines with promoter methylation, but expressed and unmethylated in immortalized normal epithelial cell lines (underlined). GAPDH was used as an internal control as shown before. M, methylated; U, unmethylated. (d) Pharmacologic demethylation with 5-aza-2’-deoxycytidine alone (A) or combined with trichostatin A (A þT) restored CMTM7 expression in methylated and silenced carcinoma cell lines. (e) Detailed BGS analysis of the CMTM7 promoter in KYSE410 and C666-1 cells with or without A þT treatment. Circles, CpG sites analyzed; row of circles, an individual promoter allele that was cloned, randomly selected, and sequenced; filled circle, methylated CpG site; open circle, unmethylated CpG site. Oncogene (2014) 3109 – 3118 & 2014 Macmillan Publishers Limited CMTM7 as a tumor suppressor methylated in carcinomas HLiet al 3111 CMTM7, but not in 12 immortalized epithelial cell lines and other analyzed and 45% esophageal tumor tissues were observed LOH tumor cell lines (Figure 1c, Supplementary Figure 2B). We (Figure 2g), suggesting that genetic alteration is also involved in also noted that no methylation was detected in several cell CMTM7 disruption in tumor tissues. lines including KYSE180, in which CMTM7 was barely detectable (Figure 1c, Supplementary Figure 2B), suggesting that CMTM7 exerts tumor-suppressive functions other genetic alterations or histone modification may also account for CMTM7 downregulation. The above expression and methylation results of CMTM7 indicate To further assess a