A Novel 3P22.3 Gene CMTM7 Represses Oncogenic EGFR Signaling and Inhibits Cancer Cell Growth

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 link between CMTM7 methylation and its that it is a potential TSG. To determine if CMTM7 has tumor- silencing, we treated several silenced cell lines with a methyl- suppressive ability, we first analyzed the subcellular localization of transferase inhibitor Aza (A), alone or combined with a histone CMTM7 in HeLa cells. Overexpressed CMTM7 is located both on deacetylase inhibitor trichostatin A (T). The demethylation of the the cell membrane and in the cytoplasm, and shows a speckled promoter restored CMTM7 expression (Figure 1d). Bisulfite distribution pattern across the cytoplasm with partial colocaliza- genomic sequencing analysis further confirmed that CMTM7 tion to the Golgi-system (Supplementary Figure 5A). Cell counting promoter was heavily methylated in tumor cell lines, but assay and colony formation assay results suggest expression of demethylated to varying degrees after pharmacological treatment CMTM7 inhibits proliferation of HeLa cells (Supplementary Figures (Figure 1e). 5B, C and D). We further overexpressed CMTM7 by adenovirus We also performed multiplex differential genomic DNA PCR to infection in two esophageal carcinoma cell lines lacking CMTM7 investigate whether CMTM7 silencing/reduction is resulted from expression, KYSE410 and KYSE180, and two immortalized cell lines genetic deletion. Results revealed that no homozygous deletion with its expression, Het-1A and HEK293 (Figure 3a, Supplementary was detected in any silenced or downregulated tumor cell lines Figure 6A). Cell counting assay revealed that ectopic CMTM7 with or without methylation, as well as in expressing normal expression induced time-dependent and dose-dependent inhibi- epithelial cells and tissues (Supplementary Figure 3). Thus, tion on tumor cell proliferation, but not in immortalized cells epigenetic silencing is the major mechanism for CMTM7 silencing (Figure 3b, Supplementary Figure 6B). Furthermore, migration in these cell lines, although possible hemizygous deletions still assay found that CMTM7 inhibited the EGF-induced migration of cannot be excluded. ESCC cells, as showed by a significant decrease in the number of migrated cells in CMTM7-expressing cells (***Po0.001; Figure 3c). Wound-healing assay also showed that ectopic expression of CMTM7 is downregulated in primary tumors CMTM7 inhibited tumor cell migration but not in immortalized We next evaluated CMTM7 expression in primary esophageal cells (Figure 3d, Supplementary Figure 6C). tumor tissues and their paired surgical margins. CMTM7 was To confirm the in vivo tumor-suppressive ability of CMTM7, downregulated in 20 of 22 (90%) tumor tissues by real-time PCR tumor formation in nude mice was performed by injecting and semi-RT–PCR, but readily expressed in paired surgical margins KYSE180 cells infected with CMTM7 adenovirus or vector control. (Figures 2a and d). We further analyzed CMTM7 protein expression Within 5 weeks, solid tumors were readily visible in the control in representative samples by western blot and immunohisto- group, but only observed in one of three mice injected by CMTM7- chemistry. Consistent with the mRNA expression, CMTM7 was expressing cells. In addition, the size and weight of tumors caused downregulated in the protein level in ESCC tumor tissues but not by CMTM7-expressing cells was significantly smaller than the in surgical margins (Figures 2b and c). control (**Po0.01; Figure 3e). These data showed that CMTM7 Bioinformatics analysis with GeneCards database (http://bioinfo. indeed had tumor-suppressive ability in ESCC. weizmann.ac.il/cards) indicates that CMTM7 is downregulated in cervix, testis, lung, thymus and lymph node tumors compared with normal tissues. We thus further analyzed CMTM7 expression CMTM7 induces G1-S cell cycle arrest in large-scale tissues using tissue microarrays, immunohisto- To elucidate the molecular mechanisms underlying the tumor cell chemistry results indicated that CMTM7 was also significantly growth inhibition by CMTM7, cell cycle status was studied by flow downregulated or absent in esophageal, gastric, lung, cervix, cytometry. When CMTM7 was expressed in KYSE410 and KYSE180 pancreatic and liver tumor tissues (Table 1, Supplementary cell lines, there were significant increases of G1 phase cells Figure 4). (*Po0.05), but no effect was observed for CMTM7-expressing Het- 1A and HEK293 cells (Figure 4a, Supplementary Figure 6D). These Promoter methylation and LOH of CMTM7 in primary tumors results suggest expression of CMTM7 could induce G1-S cell cycle arrest. We further examined several key cell cycle regulators by We further assessed epigenetic and genetic alterations of CMTM7 western blot and real-time PCR. Results showed that p27 in primary tumors. MSP showed that CMTM7 was methylated in expression was upregulated, whereas CDK2 and CDK6 were some primary carcinomas, including 12 of 39 (30.8%) nasophar- downregulated in CMTM7-expressing KYSE180 cells compared yngeal, 20 of 83 (24.1%) esophageal, 1 of 12 (8.33%) gastric, but with control (Figures 4b and c), consistent with features of G1 cell not in any of 20 breast and 11 colon tumor tissues, correlated with cycle arrest. its downregulation (Figures 2d and e; Supplementary Figure 2C). No methylation of CMTM7 promoter could be detected in 7 normal esophageal epithelial tissues, and only weak methylation CMTM7 promotes EGFR internalization and further downregulates was detected in 1 of 37 (2.7%) esophageal cancer surgical margins pAkt and 1 of 5 normal nasopharyngeal epithelial tissues (Figure 2d, Overexpression of EGFR has been reported in a number of human Supplementary Table 1). Detailed bisulfite genomic sequencing cancers, including ESCC. As ectopic CMTM7 expression could methylation analyses of individual CpG sites of the CMTM7 inhibit EGF-induced cell migration (Figure 3c), we analyzed surface promoter in two-paired esophageal tissues confirmed that CMTM7 and intra-cellular EGFR in KYSE180 cells by flow cytometry. As was heavily methylated in primary tumors but rare in paired shown in Figure 5a, cells transfected with CMTM7 (pink) contained surgical margins (Figure 2f). less surface and more intra-cellular EGFR at steady state than Chromosome 3p allelic losses are frequently present in many control cells (green). Furthermore, we used indirect immunofluor- types of cancers including esophageal carcinoma. Thus, two escence to observe the localization of EGFR (Figure 5b). At steady microsatellite markers, D3S2432 and D3S4525 located flanking state, EGFR was mainly localized on the plasma membrane in CMTM7, were selected to assess genetic alterations within CMTM7 control cells. However, in CMTM7-expressing cells, EGFR was locus 3p22.3. A panel of 31 paired primary esophageal tumors was internalized into cells, similar to flow cytometry results. These

& 2014 Macmillan Publishers Limited Oncogene (2014) 3109 – 3118 CMTM7 as a tumor suppressor methylated in carcinomas HLiet al 3112

Figure 2. (a) Downregulation of CMTM7 was frequently detected in primary esophageal carcinomas by real-time PCR. N, paired surgical margin; C, carcinoma tissue. b-Actin was used as a loading control. RQ: relative expression level. (b) Downregulation of CMTM7 was detected in representative primary esophageal carcinomas by western blot. (c) Representatives of immunohistochemical staining for CMTM7 in one primary esophageal carcinomas tissue. Top, Â 100; bottom, Â 400. (d) CMTM7 expression by RT–PCR in representative ESCC and NPC tumors. N, normal control. (e) Representative analysis of CMTM7 methylation in primary carcinomas and normal tissues by MSP. EE, esophageal epithelial tissues; NPx, nasopharyngeal tissues; N, paired surgical margin; T, tumor; M, methylated; U, unmethylated. (f) Detailed BGS analysis of the CMTM7 promoter in two esophageal tissues. Filled circle, methylated CpG site; open circle, unmethylated CpG site. (g) LOH analysis in 31 paired esophageal tissues. White boxes, retention of heterozygosity; black boxes, LOH; grey boxes, not informative (homozygosity).

results suggested that CMTM7 overexpression may promote the 3 kinase (PI3K)/Akt signaling pathways are two major downstream internalization of EGFR. pathways initiated by the activation of EGFR. We subsequently The Ras/Raf/mitogen-activated protein kinase kinase (MEK)/ examined the effect of CMTM7 on Akt and ERK activation extracellular signal-regulated kinase (ERK) and phosphatidylinositol (Figure 5c). After CMTM7 expression in KYSE180 cells,

Oncogene (2014) 3109 – 3118 & 2014 Macmillan Publishers Limited CMTM7 as a tumor suppressor methylated in carcinomas HLiet al 3113 Table 1. CMTM7 is downregulated or absent in some primary carcinoma tissues using tissue microarrays

ÀþþþP-value

Esophageal cancer (77) 21 (27%) 41 (53%) 15 (20%) o0.0001a Normal esophageal (77) 7 (9%) 27 (35%) 43 (56%) Gastric cancer (30) 2 (7%) 23 (76%) 5 (17%) o0.0001a Normal gastric (30) 0 10 (33%) 20 (67%) Lung cancer (19) 3 (16%) 16 (84%) 0 o0.0001a Normal lung (19) 1 (5%) 7 (37%) 11 (58%) Cervical cancer (31) 9 (29%) 14 (45%) 8 (26%) o0.009a Normal cervix (31) 1 (3%) 24 (77%) 6 (20%) Liver cancer (188) 29 (15%) 146 (78%) 13 (7%) o0.0001a Normal liver (47) 0 34 (72%) 13 (28%) Pancreatic cancer (175) 51 (29%) 122 (70%) 2 (1%) o0.0001a Normal pancreas (35) 0 16 (46%) 19 (54%)

aSigniﬁcant difference.

Figure 3. Tumor-suppressive functions of CMTM7 in ESCC. (a) Expression of CMTM7 was conﬁrmed by western blot. (b) Cell Counting Kit-8 assay showed that cell growth rates were inhibited by CMTM7 in KYSE410 and KYSE180 cells, but not in immortalized Het-1A cells. The results are expressed as mean±s.d. of three independent experiments. (c) Migration assay showed that the ectopic expression of CMTM7 could inhibit EGF-induced cell migration. The results are expressed as mean±s.d. of three independent experiments. ***Po0.001. (d) For wound- healing assay, representative of images were photographed at time 0 and 24 h after scratching. (e) CMTM7 effectively suppressed tumorigenicity in nude mice. The average tumor size and weight of CMTM7-expressing KYSE180 cells versus mock cells are expressed as mean±s.d. in 3–4 inoculated sites for each group. **Po0.01. MOI, multiplicity of infection.

& 2014 Macmillan Publishers Limited Oncogene (2014) 3109 – 3118 CMTM7 as a tumor suppressor methylated in carcinomas HLiet al 3114

Figure 4. (a) Flow cytometry was used to compare the DNA content between mock and CMTM7-expressing cells (right). Summary of cell proportions in different phases of cell cycle (left). The results were expressed as mean±s.d. of three independent experiments. *Po0.05. (b) Protein expression of p27, p21, p53, CDK2, CDK4, CDK6, cyclin E1 and cyclin D1 were detected by western blot. (c) mRNA expressions of p27, CDK2 and CDK6 were detected by real-time PCR. b-Actin was used as an internal control. *Po0.05.

phosphorylated Akt was downregulated, whereas no change in other epigenetic alterations such as histone modifications would ERK activation was observed. These results suggested that the also likely contribute to CMTM7 downregulation in tumor cells. PI3K/Akt pathway is the EGFR downstream target inhibited The tumor-suppressive function of CMTM7 was investigated by CMTM7. in vitro and in vivo. Ectopic expression of CMTM7 effectively suppresses esophageal carcinoma cell growth, motility and tumor formation in nude mice, but with no effect in immortalized normal DISCUSSION cells, suggesting a tumor-specific suppression of CMTM7. CMTM7 CMTM is a novel family of proteins linking chemokines and is also able to inhibit G1-S phase transition through upregulating transmembrane-4 superfamily, having important roles in the p27 and downregulating CDK2 and CDK6. G1-S phase transition is immune and male reproductive systems as well as tumorigenesis. known to be a major checkpoint for cell cycle progression. Cyclin- Our previous studies indicate that CMTM3 and CMTM5 exert dependent kinase inhibitors, serving as critical negative mediators tumor-suppressive functions with frequent epigenetic inactivation during this transition through CDK inhibition, regulate the activity by promoter CpG methylation.9,10 CMTM6, CMTM7 and CMTM8 of cyclin E-CDK2 and cyclin D-CDK4/6 complex, resulting in Rb form a gene cluster on 3p22.3, an important TSG locus associated phosphorylation and Rb-E2F binding destruction. The released E2F with the pathogenesis of multiple tumors. In this study, we found activates the transcription of genes necessary for S-phase entry that CMTM7 is broadly expressed in human normal tissues and and cell cycle progression.31 It is thus reasonable to speculate that normal epithelial cell lines, while silenced or downregulated in CMTM7 is able to induce p27 expression, in turn, resulting in some carcinoma cell lines and primary tumors because of inhibition of CDK2 and CDK6 activity, and thus inhibit G1-S phase promoter methylation and LOH. In primary tumors, the presence transition. of various infiltrating normal tissue cells would also result in Expression of EGFR is common in various normal cells including detectable expression of CMTM7 found in some cases. Moreover, cells of epidermal, mesenchymal and neurogenic origins. When

Oncogene (2014) 3109 – 3118 & 2014 Macmillan Publishers Limited CMTM7 as a tumor suppressor methylated in carcinomas HLiet al 3115

Figure 5. (a) EGFR expression in KYSE180 cells was detected by ﬂow cytometry. Cells transfected with CMTM7 (pink) contained less surface and more intra-cellular EGFR at steady state than control cells (green). Isotype immunoglobulin G was used as the negative control (purple). (b) EGFR expression in KYSE180 cells was detected by immunoﬂuorescence. EGFR was internalized into cytoplasm in CMTM7-expressing cells. (c) Protein expression of phosphorylated and total Akt and ERK in KYSE180 cells were detected by western blot. b-Actin was used as an internal control.

EGF binds to EGFR, signaling pathways are activated; this can lead EGFR-PI3K/Akt signaling pathway, resulting in the upregulation of to cell proliferation and differentiation. EGFR has an important role p27 and further inhibition of CDK2 and CDK6. CMTM7 may inhibit in cell differentiation, development, proliferation and mainte- cell proliferation and migration via the EGFR-PI3K/Akt pathway. nance. With EGFR overexpression because of mutation or Miyazaki et al. also reported that CMTM7 binds to BLNK in structural alteration, carcinogenesis, invasion and metastasis are mouse B lymphoma Bal17 cells and negatively regulates BCR facilitated.32 MARVEL domain protein families may be part of the endocytosis through activation of ERK and c-Jun N-terminal machinery of membrane apposition events, such as transport kinase.39 BLNK functions as a tumor suppressor in pre-B-cell vesicle biogenesis, neurotransmitter secretion and polarized leukemia, which can upregulate p27 and induce cell cycle arrest membrane trafﬁcking. CD82/KAI1, a metastasis tumor suppressor and apoptosis.40 Our preliminary results show that BLNK is and a classical tetraspanin with one internalization consensus downregulated in esophageal carcinoma tissues compared with sequence YXXF in its cytosotic loop domain, is associated with non-tumorous tissues. Endogenous CMTM7 is partially localized EGFR and regulates EGFR internalization in several different types with BLNK in A549 cells, indicating that BLNK may also interact of epithelial cells.33 The tyrosine-based YXXF motif can bind with human CMTM7 in non-leukemia cell line (data not shown). directly to the adaptor protein 2 m2-subunit. The adaptor protein 2 Further studies are needed to assess the direct interaction associates with clathrin and is involved in budding from the between CMTM7 and BLNK, and whether CMTM7 and BLNK plasma membrane.34 In CMTM family, CMTM8 contains two YXXF have synergistic effect on the upregulation of p27. motifs and may be an attenuator of EGF-induced signaling via Our preliminary studies also showed that CMTM6 and CMTM8 facilitating EGFR endocytosis and subsequent desensitization.35 are downregulated in some esophageal tumors at the mRNA and CMTM7 also contains one YXXF motif. In this study, we found that protein levels (data not shown). It has been reported that CMTM6 overexpressed CMTM7 is partially colocalized with the Golgi- is one of candidate deleted genes in lung adenocarcinoma system and promotes EGFR internalization at steady state. Ras/Raf/ with bronchioloalveolar carcinoma features,41 although CMTM6 mitogen-activated protein kinase kinase (MEK)/ERK and PI3K/Akt expression remained high in most cell lines examined except for pathways are two important downstream signaling pathways of some marginal downregulation in few cell lines. In HeLa cells, EGFR.36 The PI3K/Akt pathway is linked to cell cycle regulation and overexpression of CMTM6 has no effect on cell proliferation frequently activated in many tumors.37 In G1-S transition, (Supplementary Figure 6), indicating that CMTM6 may not activated PI3K/Akt signaling inhibits p27 activity.38 Our studies possess tumor-suppressive functions, which needs further studies. indicated that CMTM7 inhibits phosphorylation of Akt, but has no For CMTM8, it can induce cell apoptosis through a mitochondria- effect on ERK. Thus, CMTM7 is associated with the inhibition of mediated pathway and function as a negative regulator of

& 2014 Macmillan Publishers Limited Oncogene (2014) 3109 – 3118 CMTM7 as a tumor suppressor methylated in carcinomas HLiet al 3116 4,14,35 EGF-induced signaling. CMTM8 is also downregulated in 5 min. The tissue chips were incubated with 3% (v/v) H2O2 at room some cell lines. Future studies of roles of these genes in human temperature for 10 min, rinsed twice and blocked in 10% normal goat diseases are needed. serum for 30 min. The slides were then incubated at 37 oC for 1 h with anti- CMTM7 monoclonal antibody. Mouse monoclonal antibody against CMTM is a novel family of proteins, with very limited studies 33 conducted thus far for their molecular functions. In this study, we CMTM7 was prepared and purified in our laboratory. After thorough washing, Dakocytomation Envision System HRP (DakoCytomation, provided first evidences that CMTM7 is a potent TSG and has Carpinteria, CA, USA) was applied for 30 min. After rinsing in phosphate- important suppressive role in cancer development and progres- buffered saline, all slides were visualized with 0.05% (w/v) 3,3’- sion. A better understanding of the tumor-suppressive role of diaminobenzidine, and then counter-stained with hematoxylin. Mouse CMTM7 will significantly improve our knowledge about the roles immunoglobulin G was used as a negative control. of CMTM family proteins in carcinogenesis, and may lead to more The immunoreactive score of Remmele and Stegner (IRS)48 was effective management of cancers with CMTM7 alterations. calculated according to immunostaining intensity (none ¼ 0; weak ¼ 1; moderate ¼ 2; strong ¼ 3) and percentage of positive tumor cells (none ¼ 0; o10% ¼ 1; 10–50% ¼ 2; 51–80% ¼ 3; 480% ¼ 4). For final MATERIALS AND METHODS statistical analysis, the IRS value of 0 was ranked as 0 (negative), the IRS Cell lines, treatment and tumor samples values of 1–3 as 1 (weak expression), the IRS values of 4–8 as 2 (moderate expression) and the IRS values of 9–12 as 3 (strong expression). Tumor cell lines studied include esophageal, nasopharyngeal, hypopharyngeal, gastric, hepatocellular, pancreatic, colorectal, breast, cervical, lung, prostate, bladder and renal carcinomas as well as glioma and lymphomas, Cell infection with immortalized, non-transformed normal epithelia cell lines used as Cells were infected with CMTM7 adenovirus (ad-CMTM7) or vector- 9,31,32 controls. These cell lines were obtained either from the American containing adenovirus (ad-mock) as described.11 Multiplicity of infection Type Culture Collection (ATCC, Manassas, VA, USA) or our collaborators. All of 100, 200 and 400 were used, and the efficiency of infection was esophageal and NPC cell lines used in this study were of squamous cell monitored by ad-pEGFP. Cells with 490% infection efficiency were used 42 43 type, with only one NPC cell line C666-1 is Epstein–Barr virus-positive. for further analysis. Some cell lines were treated with 10 mM of 5-aza-2’-deoxycytidine (Aza) (Sigma-Aldrich, St Louis, MO, USA) for 3 days, with or without further treatment with 100 nM trichostatin A (Cayman Chemical Co., Ann Arbor, MI, Cell proliferation assay USA) for additional B16 h. Primary tumor tissues and paired non-tumor Cells infected with CMTM7 or vector control were harvested and plated in tissues were obtained from patients under primary surgery at the Peking 96-well plates at a density of 2000 cells per well, then incubated at 37 oC. University People’s Hospital, with patients’ consent and institutional ethics Cell proliferation was analyzed using the Cell Counting Kit-8 (CCK-8, approval. Other archival normal and tumor DNA samples have been Dojindo Molecular Technologies, Kumamoto, Japan). At indicated time described.9,31 Tissue microarrays were purchased from Cybrdi Co. points, 10 ml of the CCK-8 solution was added into each well and incubated (Chaoying Biotechnology Co., Xi’an, China) and Outdo Biotechnology Co. for 2 h. Absorbance at 450 nm was measured to calculate the number of (Shanghai Outdo Biotech Co., Shanghai, China). viable cells. Results were obtained from three independent experiments in triplicates. Semiquantitative RT–PCR and real-time PCR analysis Human multiple tissue RNA samples were purchased from Clontech Cell migration assay (Mountain View, CA, USA) or Stratagene (La Jolla, CA, USA). Reverse The migration assay was performed in a 48-well chemotaxis chamber transcription using random hexamer and RT–PCR using Go-Taq (Promega, (Neuro Probe, Cabin John, MD, USA) as previously described.10 The top and Madison, WI, USA) were performed with primers for CMTM7, CMTM6 and bottom compartments of the chambers were separated by polycarbonate CMTM8 (Supplementary Table 2). Detection of control glyceraldehyde 3- filters (8 mm pore size) coated with rat tail collagen I (BD Biosciences, phosphate dehydrogenase (GAPDH) in all the studied samples has been San Jose, CA, USA). Briefly, serum-starved cells (1 Â 106 cells/ml) were confirmed before.44,45 9 resuspended in serum-free RPMI 1640 containing 0.1% bovine serum Real-time PCR for p27 and CMTM7 were done as previously described, albumin and added to the upper well of the chamber. 20 ng/ml human according to the dissolving curve and electrophoresis, the samples with EGF (Peprotech, Rockville, NJ, USA) was added to the lower wells. The cells unspecific amplification were regarded as zero. For CDK2 and CDK6,we o were incubated for 7 h (KYSE410 and KYSE180) at 37 C in a 5% CO2 used Universal Probelibrary-based assays for quantitative RT–PCR. Quanti- humidified atmosphere. After incubation, nonmigrated cells were scraped tative PCR was run on ABI PRISM 7000 Sequence Detection System off and migrated cells were identified through fixing and staining. At least (Applied Biosystems, Foster City, CA, USA). The reactions were performed five random fields of vision per well were counted for quantitation of with TaqMan Gene Expression Master Mix, 200 nM primers and 100 nM o migrated cells. Triplicate wells were performed in each assay, and the assay probe. Amplification conditions were 10-min initial denaturation at 95 C, was repeated at least three times. followed by 40 cycles of each 15 s at 95 oC and 1 min at 60 oC. Samples were run in triplicate. All samples were normalized against GAPDH using comparative Ct method (ddCt). Primers were shown in Supplementary Wound-healing assay Table 2. Cells infected with CMTM7 or vector control were cultured until confluent; the cell layers were then carefully scratched using sterile tips.9,45 After DNA bisulfite treatment and promoter methylation analysis incubation for 24 or 36 h, the cells were photographed under a phase Bisulfite modification of DNA, MSP and bisulfite genomic sequencing were contrast microscope. carried out as previously described.46,47 MSP and bisulfite genomic sequencing primers were listed in Supplementary Table 2. Cell cycle analysis Cells infected with CMTM7 or vector control were harvested and fixed in Microsatellite analysis 70% ethanol and stained with propidium iodide, and DNA content was Two microsatellite markers, D3S2432 and D3S4525, on chromosome analyzed by FACSCalibur (BD Biosciences). The results were analyzed by 3p22.3 were used. Sequence information was obtained from the NCBI ModFit software (Verity Software House, Topsham, ME, USA). database. The ABI PRISM 3100 Genetic Analyzer was used to analyze the PCR amplicon using GeneScan and Genotyper software (Applied Tumor formation in nude mice Biosystems). Microsatellite analysis primers were listed in Supplementary Table 2. In vivo tumor-suppressive ability of CMTM7 was investigated by tumor xenograft experiment. In all, 4 Â 106 KYSE180 cells infected with CMTM7 or vector control were injected subcutaneously into the left dorsal flank of 3 Immunohistochemistry or 4 nude mice (4–6 weeks old), respectively. Tumor formation in nude Human tissue slides were deparaffinized and rehydrated. Antigen retrieval mice was monitored over a 5-week period. The tumor volume was 2 was done by heating a solution in 0.01 M citrate buffer (pH 6.0) twice for calculated by the formula V ¼ 0.5 Â L Â W .

Oncogene (2014) 3109 – 3118 & 2014 Macmillan Publishers Limited CMTM7 as a tumor suppressor methylated in carcinomas HLiet al 3117 Flow cytometry 7 Shi S, Rui M, Han W, Wang Y, Qiu X, Ding P et al. CKLFSF2 is highly expressed in Cells infected with CMTM7 or vector control were harvested and washed testis and can be secreted into the seminiferous tubules. Int J Biochem Cell Biol with phosphate-buffered saline, fixed in 4% paraformaldehyde and/or 2005; 37: 1633–1640. permeabilized in 0.1% Triton-X-100, then blocked and incubated with anti- 8 Wang Y, Li T, Qiu X, Mo X, Zhang Y, Song Q et al. CMTM3 can affect the tran- EGFR antibody and fluorescein isothiocyanate-conjugated secondary scription activity of androgen receptor and inhibit the expression level of PSA in antibody. Cells were counted by flow cytometry (FACS Calibur) with an LNCaP cells. Biochem Biophys Res Commun 2008; 371: 54–58. excitation of 488 nm and analyzed with CELLQuest software (BD 9 Wang Y, Li J, Cui Y, Li T, Ng KM, Geng H et al. CMTM3, located at the critical tumor Biosciences). suppressor locus 16q22.1, is silenced by CpG methylation in carcinomas and inhibits tumor cell growth through inducing apoptosis. Cancer Res 2009; 69: 5194–5201. Immunofluorescence 10 Shao L, Cui Y, Li H, Liu Y, Zhao H, Wang Y et al. CMTM5 exhibits tumor suppressor Cells infected with CMTM7 or vector control were harvested and fixed with activities and is frequently silenced by methylation in carcinoma cell lines. Clin 4% paraformaldehyde, permeabilized in 0.1% Triton-X-100, and blocked Cancer Res 2007; 13: 5756–5762. with 3% fetal bovine serum. The cells were then treated with anti-EGFR 11 Guo X, Li T, Wang Y, Shao L, Zhang Y, Ma D et al. CMTM5 induces apoptosis of antibody for 1 h and incubated with fluorescein isothiocyanate-conjugated pancreatic cancer cells and has synergistic effects with TNF-alpha. Biochem secondary antibody for 1 h. This experiment was replicated three times. Biophys Res Commun 2009; 387: 139–142. Cells were imaged with Leica TCS SP5 confocal microscope (Leica 12 Shao L, Guo X, Plate M, Li T, Wang Y, Ma D et al. CMTM5-v1 induces apoptosis in Microsystems, Wetzlar, Germany). cervical carcinoma cells. Biochem Biophys Res Commun 2009; 379: 866–871. 13 Li H, Guo X, Shao L, Plate M, Mo X, Wang Y et al. CMTM5-v1, a four-transmem- Western blot analysis brane protein, presents a secreted form released via a vesicle-mediated secretory pathway. BMB Rep 2010; 43: 182–187. Western blot was done according to the standard protocol as described 9 14 Jin C, Wang Y, Han W, Zhang Y, He Q, Li D et al. CMTM8 induces caspase- previously. Signals were detected by Odyssey Infrared Imager (LICOR dependent and -independent apoptosis through a mitochondria-mediated Bioscience, Lincoln, NE, USA). pathway. J Cell Physiol 2007; 211: 112–120. Antibodies used were: EGFR, p53, p27, p21, CDK2 (Santa Cruz 15 Li T, Zhong J, Chen Y, Qiu X, Zhang T, Ma D et al. Expression of chemokine- Biotechnology, Santa Cruz, CA, USA), cyclin D1 (NeoMarkers, Fremont, like factor 1 is upregulated during T lymphocyte activation. Life Sci 2006; 79: CA, USA), cyclin E1 (Bioworld Technology, Inc., St Louis Park, MN, USA), 519–524. CDK4, CDK6, phospho- AktSer473, total-Akt and total-ERK (Cell Signaling, Thr202/Tyr204 16 Shao L, Li T, Mo X, Majdic O, Zhang Y, Seyerl M et al. Expressional and functional Beverly, MA, USA), phospho-ERK (KangChen Bio-Tech, Kang- studies of CKLF1 during dendritic cell maturation. Cell Immunol 2010; 263: Chen, Shanghai, China), b-actin (Sigma-Aldrich) and IRDye secondary 188–195. antibodies against mouse or rabbit immunoglobulin G (Li-Cor Biosciences). 17 Plate M, Li T, Wang Y, Mo X, Zhang Y, Ma D et al. Identification and character- The anti-CMTM7 mouse monoclonal antibody and anti-CMTM6 rabbit ization of CMTM4, a novel gene with inhibitory effects on HeLa cell growth polyclonal antibody were made in our lab. through Inducing G2/M phase accumulation. Mol Cells 2010; 29: 355–361. 18 Sanchez-Pulido L, Martin-Belmonte F, Valencia A, Alonso MA. MARVEL: a con- Statistical analysis served domain involved in membrane apposition events. Trends Biochem Sci 2002; 27: 599–601. Statistical analysis was carried out using Statistical Package for Social 19 Lind GE, Ahlquist T, Kolberg M, Berg M, Eknaes M, Alonso MA et al. Hyper- Sciences 14.0 for Windows (SPSS Inc., Chicago, IL, USA). Student’s t-test was used to analyze the results expressed as mean±s.d. Differences were methylated MAL gene - a silent marker of early colon tumorigenesis. J Transl Med 2008; 6:13. considered significant when Po0.05. 20 Buffart TE, Overmeer RM, Steenbergen RD, Tijssen M, van Grieken NC, Snijders PJ et al. MAL promoter hypermethylation as a novel prognostic marker in gastric cancer. Br J Cancer 2008; 99: 1802–1807. CONFLICT OF INTEREST 21 Martin TA, Mansel RE, Jiang WG. Loss of occludin leads to the progression of The authors declare no conflict of interest. human breast cancer. Int J Mol Med 2010; 26: 723–734. 22 Hu XT, Zhang FB, Fan YC, Shu XS, Wong AH, Zhou W et al. Phospholipase C delta 1 is a novel 3p22.3 tumor suppressor involved in cytoskeleton organization, with its ACKNOWLEDGEMENTS epigenetic silencing correlated with high-stage gastric cancer. Oncogene 2009; 28: 2466–2475. We thank Bert Vogelstein, George Tsao and Sun Young Rha for some tumor cell lines 23 Ying J, Poon FF, Yu J, Geng H, Wong AH, Qiu GH et al. 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2014) 3109 – 3118 & 2014 Macmillan Publishers Limited