Oncogene (2008) 27, 5267–5276 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ONCOGENOMICS Epigenetic disruption of -c response through silencing the tumor suppressor interferon regulatory factor 8 in nasopharyngeal, esophageal and multiple other carcinomas

KY Lee1,11, H Geng1,11,KMNg1,JYu2, A van Hasselt3, Y Cao4, Y-X Zeng5, AHY Wong1, X Wang1, J Ying1, G Srivastava6, ML Lung7, L-D Wang8, TT Kwok9, B-Z Levi10, ATC Chan1, JJY Sung2 and Q Tao1

1Cancer Epigenetics Laboratory, State Key Laboratory in Oncology in South China, Sir YK Pao Center for Cancer, Department of Clinical Oncology, Li Ka Shing Institute of Health Sciences, Prince of Wales Hospital, Chinese University of Hong Kong, Hong Kong, China; 2Department of Medicine and Therapeutics, Institute of Digestive Disease, Chinese University of Hong Kong, Hong Kong, China; 3Department of Surgery, Chinese University of Hong Kong, Hong Kong, China; 4Hunan Yale (Xiang Ya) School of Medicine, Central South University, Changsha, China; 5State Key Laboratory in Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, China; 6Department of Pathology, University of Hong Kong, Hong Kong, China; 7Department of Biology, Hong Kong University of Science and Technology, Hong Kong, China; 8Henan Key Laboratory for Esophageal Cancer, Zhengzhou University College of Medicine and Cancer Research Center of Xinxiang Medical College, Henan, China; 9Department of Biochemistry, Chinese University of Hong Kong, Hong Kong, China and 10Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel

16q24 is frequently deleted in multiple tumors including frequently silenced by epigenetic mechanism in multiple cancers of nasopharynx, esophagus, breast, prostate and carcinomas. liver. By array comparative genomic hybridization Oncogene (2008) 27, 5267–5276; doi:10.1038/onc.2008.147; (aCGH), we refined a 16q24 hemizygous deletion in published online 12 May 2008 nasopharyngeal carcinoma (NPC) lines. Semi-quanti- tative RT–PCR analysis revealed interferon regulatory Keywords: IRF8; methylation; CpG island; tumor factor 8 (IRF8) as the only downregulated within this suppressor gene; carcinoma deletion. IRF8 belongs to a family of interferon (IFN) regulatory factors that modulate various important physiologic processes including host defense, cell growth and differentiation and immune regulation. In contrast to the broad expression of IRF8 in normal adult and fetal Introduction tissues, transcriptional silencing and promoter methyla- tion of IRF8 were frequently detected in multiple Carcinogenesis is a multistep process with the accumu- carcinoma (except for hepatocellular) cell lines (100% lation of multiple genetic and epigenetic changes (Jones in NPC, 88% in esophageal and 18–78% in other and Baylin, 2002). In addition to the contribution of carcinoma cell lines) and in a large collection of oncogenes, inactivation of tumor suppressor primary carcinomas (78% in NPC, 36–71% in other (TSGs) is also frequently involved (Jones and Baylin, carcinomas). Methylation of the IRF8 promoter led to the 2002; Baylin and Ohm, 2006). Genetic mutations disruption of its response to IFN-c stimulation. Pharma- including deletions disrupt TSG functions (Knudson, cological and genetic demethylation could restore IRF8 2001), whereas epigenetic silencing also inactivates expression, indicating a direct epigenetic mechanism. TSGs through methylation of promoter CpG islands Ectopic expression of IRF8 in tumor cells lacking its (CGIs), which is a characteristic epigenetic feature of ONCOGENOMICS expression strongly inhibited their clonogenicity, confirm- tumor DNA (Jones and Baylin, 2002). ing its tumor suppressor function. Thus, IRF8 was Nasopharyngeal carcinoma (NPC) is prevalent in identified as a functional tumor suppressor, which is Southern China and Southeast Asia (Tao and Chan, 2007). Although its pathogenesis has been shown to be strongly associated with Epstein–Barr virus, its mole- Correspondence: Professor Q Tao, Cancer Epigenetics Laboratory, cular mechanism is still poorly elucidated (Lo et al., Department of Clinical Oncology, Sir YK Pao Center for Cancer, 2004; Tao and Chan, 2007). Numerous genetic altera- Prince of Wales Hospital, Chinese University of Hong Kong, tions have been detected in NPC cell lines and tumors, Hong Kong, China. including 3p14–22, 11q13.3–24, 13q14.3–22, 14q24.3–32.1 E-mail: [email protected] and 16q21–24 (Chen et al., 1999; Lo et al., 2000; 11These authors contributed equally to this work. Received 7 November 2007; revised 21 March 2008; accepted 1 April Tao and Chan, 2007). These regions thus represent 2008; published online 12 May 2008 the critical TSG loci for NPC. Previous searches for Methylation of IRF8 in multiple carcinomas KY Lee et al 5268 putative TSGs in these regions have identified only few clone to locus 16q24.1, spanning B160-kb, with three candidates, with tumor-specific promoter methylation, genes, COX4NB, COX4I1 and IRF8 residing at this such as BLU and RASSF1A at 3p21 (Lo et al., 2001; locus (Figure 1a). Semi-quantitative RT–PCR analysis Qiu et al., 2004), CADM1/TSLC1 at 11q23.1 (Lung revealed IRF8 silencing in 5 of 6 NPC cell lines with et al., 2006); THY1/CD90 at 11q22.3 (Lung et al., 2005), weak expression in C666-1, but not for the other two CDH1 at 16q22.1 (Chang et al., 2003), ADAMTS18 and genes COX4NB and COX4I1, suggesting that IRF8 is CDH13 at 16q23 (Sun et al., 2007; Jin et al., 2007b). the candidate TSG located at the 16q24.1 deletion These limited findings suggest that additional cancer- (Figure 1b). related genes are yet to be identified in NPC in the reported regions, or other unidentified loci. Frequent downregulation of IRF8 in multiple carcinomas In this study, we have refined a 16q24.1 deletion as We further determined IRF8 expression in a panel of one of the critical tumor suppressor loci in NPC cell normal adult and fetal tissues and multiple cell lines by lines using high-resolution, 1-Mb array comparative semi-quantitative RT–PCR. IRF8 expression was de- genomic hybridization (aCGH) analysis (Ying et al., tected in all normal tissues including nasopharynx and 2006). Loss of heterozygosity (LOH) at 16q24 is also esophagus (Figure 1d), as well as immortalized normal frequently present in multiple other solid tumors, such nasopharyngeal (NP69) and esophageal epithelial (NE1 as breast, prostate, hepatocellular and Wilm’s tumor and NE3) cell lines (Figure 2a). In contrast, IRF8 was (Lo et al., 2000; Paige et al., 2000; Jin et al., 2007b), frequently downregulated or silenced in carcinoma cell suggesting the presence of critical TSG(s) at this locus. lines of nasopharyngeal, esophageal, breast, lung, Several functional candidate TSGs have been identified cervical and colorectal carcinomas (Figure 2a), but less at this locus including CDH13 (Toyooka et al., 2001). frequently in gastric and seldom in hepatocellular Thus, it is needed to search thoroughly for more carcinoma cell lines (Supplementary Figure). Mean- TSGs at this locus and define their functions in while, expression of the IRF8 was confirmed by tumors. western blot in normal esophageal epithelial tissues and Among the genes resided at the 16q24.1 deletion, normal lymphoid cell line LCL-CCL256.1 (Figure 2b). silencing of interferon regulatory factor 8 (IRF8) was However, IRF8 was absent in HONE1 and HCT116 identified in several NPC cell lines. IRF8 is also known cell lines that are completely methylated and silenced for as interferon consensus sequence-binding protein this gene. (ICSBP), which belongs to the IRF family of transcrip- tion factors (Nguyen et al., 1997). IRF8 has been characterized as a central element of the interferon Methylation of the IRF8 promoter correlates (IFN)-g-signaling pathway, which modulates immune with its transcriptional silencing response and also regulates cell growth and differentia- We then examined the possible genetic/epigenetic tion (Tamura and Ozato, 2002). IRF8-deficient mice mechanisms of IRF8 downregulation. As predicted by developed a human chronic myelogenous leukemia online CpG Island Searcher (http://cpgislands.usc.edu/), (CML)-like disease (Holtschke et al., 1996). In patients there is a CGI spanning the transcription start site of with CML and acute myelogenous leukemia, IRF8 IRF8 (Figure 1c). Methylation-specific PCR (MSP) was expression was dramatically decreased, suggesting that thus performed to study the methylation status of this IRF8 could be a candidate TSG (Schmidt et al., 1998). CGI. Results showed that IRF8 was frequently methy- Here, we report the frequent epigenetic inactivation of lated in cell lines with downregulation (6 of 6 IRF8 in NPC, as well as other common carcinomas, nasopharyngeal, 14 of 16 esophageal, 7 of 9 breast, 3 such as esophageal, breast, cervical, lung and colorectal of 4 colorectal, 5 of 7 lung, 4 of 6 cervical and 3 of 17 carcinomas. Methylation of the IRF8 promoter gastric carcinoma cell lines) (Figure 2a, Supplementary abolishes its response to IFN-g. Moreover, ectopic Figure, Table 1), whereas no methylation was detected expression of IRF8 in silenced tumor cells strongly in the immortalized epithelial cell lines (NP69, NE1 and inhibited their clonogenicity, indicating that IRF8 is a NE3), indicating that methylation is tumor specific functional TSG. (Figure 2a). The presence of either unmethylated or methylated promoter alleles in virtually all cell lines including silenced ones indicates the absence of homo- Results zygous deletion of IRF8 promoter in these cell lines, except for LoVo with weak bands indicating possible Identification of IRF8 as a downregulated gene in NPC hemizygous deletion. Genetically altered regions pinpoint the loci of TSGs. To confirm the MSP results in higher resolution, We attempted to identify novel TSGs through bisulfite genomic sequencing (BGS) was performed to an integrative cancer epigenetic approach by coupling examine the methylation status of 43 individual CpG 1-Mb aCGH with expression analysis. A hemizygous sites (À596 to À175, relative to the transcription start deletion represented by a bacterial artificial chromo- site) within the IRF8 promoter CGI. The BGSresults some (BAC) clone bA478M13 (tumor/normal log2 were consistent with those of MSP in which dense signal ratio À0.45) was detected in 1 of 6 NPC cell methylation was only observed in methylated cell lines. Bioinformatics search using the Ensembl Genome lines, but not in unmethylated non-tumor cell line Browser (www.ensembl.org/index.html) mapped this NP69 (Figure 2c). Thus, the results revealed a strong

Oncogene Methylation of IRF8 in multiple carcinomas KY Lee et al 5269

Log T/R 2 Chr. 16 1 Markers COX4NB COX4I1 IRF8 GAPDH

0 1-Mb aCGH Larynx CNE2 a Trachea

-1 C666-1 bA478M13 CNE1 84.00Mb 84.20Mb 84.40Mb 84.60Mb 84.80Mb 16q24.1 CNE2 COX4NB COX4I1 HK1 HNE1 IRF8 HONE1 F R

100-bp IRF8

Exon1 CpG sites MSP BGS1 BGS2 BGS region CpG island

Adult tissues Fetal tissues O 2 Markers Heart Sk .M. Liver Kidney Spleen Pancreas Esophagus Stomach Colon Rectum Larynx Trachea Lung Breast Cervix Ovary Placenta Testis Prostate Brain B.M. L.N. Sk .M. Skin Kidney Colon Bladder Brain Stomach Lung Liver H IRF8

GAPDH

Figure 1 Identification of IRF8 as a candidate (TSG) in nasopharyngeal carcinoma (NPC). (a) Upper panel: representative 1-Mb array comparative genomic hybridization (aCGH) result of CNE2. The deletion marked by bacterial artificial (BAC) clone bA478M13 is circled. Middle panel: bA478M13 is mapped to locus 16q24.1. Three genes, COX4I1, COX4NB and IRF8, located within this deletion. Lower panel: IRF8 transcript. Filled rectangle, coding exons; open rectangle, noncoding region. Reverse transcription (RT)–PCR primers are indicated by arrows. (b) RT–PCR showed that IRF8 is the only downregulated gene in the deletion. (c) The typical CpG island (CGI) spanning the exon 1 of IRF8. Each vertical bar represents a single CpG site. The transcription start site is indicated by a curved arrow. Methylation-specific PCR (MSP) primers and bisulfite genomic sequencing (BGS) region are shown. (d) IRF8 expression in human normal adult and fetal tissues, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control. SkM, skeletal muscle; BM, bone marrow; LN, lymph node.

correlation between IRF8 transcriptional silencing and suggest that epigenetic silencing of IRF8 disrupted its its promoter methylation. response to IFN-g.

IRF8 promoter methylation disrupts its response to IFN-g Pharmacological and genetic demethylation reactivates We further examine whether the IRF8 response to IRF8 expression IFN-g stimulation would be affected by the promoter To examine whether promoter methylation directly methylation of this gene. IFN-g treatment of normal mediates the silencing of IRF8, cell lines (BT549, and tumor cell lines with an unmethylated (HEK 293 MB231, T47D, CNE1, HK1, HONE1, KYSE510 and and HCT116-DKO) or weakly methylated (HepG2) KYSE520) were treated with demethylation agent 5-aza- promoter led to the induction of IRF8 expression. 2-deoxycytidine (Aza), with or without histone deacety- However, this response was abolished in cell lines lase inhibitor trichostatin A (TSA). After the treatment, with predominantly methylated promoter (HONE1, IRF8 expression levels were dramatically increased in all KYSE520 and HCT116) (Figure 3). These results cell lines examined (Figure 4a), together with the

Oncogene Methylation of IRF8 in multiple carcinomas KY Lee et al 5270 Esophageal Ca cell lines NPC cell lines HK1 HONE1 HNE1 NP69 C666-1 CNE2 CNE1 KYSE180 KYSE410 KYSE450 KYSE510 KYSE520 NE1 NE3 EC1 HKESC1 HKESC2 HKESC3 KYSE140 KYSE150 KYSE270 Markers EC18 KYSE30 KYSE70 SLMT-1 Markers IRF8

GAPDH Normal Carcinoma cell M Esophageal MSP tissues lines U Colorectal Ca Breast Ca cell lines Lung Ca Cervical Ca LF10N AY8N HCT116 HONE1 cell lines LCL-CCL256.1 cell lines cell lines IRF8

Markers BT549 MB231 MB468 MCF-7 T47D ZR-75-1 SK-BR-3 YCC-B1 YCC-B3 HCT116 HT-29 Lovo SW480 A427 A549 H292 H358 H1299 H1650 H1975 C33A CaSki SiHa tublin

IRF8 Western blot GAPDH 915 808 866 M MSP U

NP69

CNE1 BGS KYSE410

HCT116

Figure 2 Silencing of IRF8 by promoter methylation in tumor cell lines. (a) Expression and methylation of IRF8 in a panel of carcinoma cell lines. M, methylated; U, unmethylated. (b) Expression of IRF8 protein in normal esophageal epithelial tissues (LF10N and AY8N) and normal lymphoblastoid cell line (LCL) CCL256.1, and the absence of IRF8 protein in HONE1 and HCT116 carcinoma cell lines. (c) Bisulfite genomic sequencing (BGS) methylation analysis of the IRF8 promoter in normal epithelial cell line NP69, NPC cell line CNE1, esophageal cell line KYSE410 and colorectal cell line HCT116. Cloned BGS–PCR products were sequenced and each was shown as an individual row, representing a single allele of the promoter. Totally, 43 CpG sites (À596 to À175, relative to the transcription start site) within the CpG island (CGI) were analysed and each shown as a circle. Filled circle, methylated; open circle, unmethylated.

decrease in methylated alleles and increase in unmethy- Frequent IRF8 methylation in multiple primary lated alleles of the IRF8 promoter (Figure 4b). Com- carcinomas bined Aza and TSA treatment led to higher IRF8 We next investigated IRF8 methylation in a large expression levels (Figure 4a). collection of primary carcinomas (n ¼ 113), including We also found that the IRF8 could be activated in the NPC, esophageal, breast and cervical carcinomas, colorectal carcinoma cell lines HCT116 by genetic together with normal epithelial tissues as controls. demethylation through double knockout of DNA IRF8 methylation was detected in 35 of 46 (76%) methyltransferases (DNMTs) 1 and 3B (DKO cell line) endemic NPC tumors from Asian Chinese and all three (Figure 4a). Concomitantly, the IRF8 promoter was nude mice-passaged undifferentiated NPC tumors from dramatically demethylated in DKO cells as compared to North African (C15, C17 and C18) (Figure 5b) (Busson HCT116 cells (Figure 4b). Further high-resolution et al., 1988), with no methylation detected in any normal methylation analysis by BGSconfirmed the demethyla- nasopharyngeal tissue (Figure 5a). IRF8 methylation tion of IRF8 (Figure 4c), suggesting that the main- was also found in 25 of 43 (58%) esophageal carcino- tenance of IRF8 methylation is mediated by DNMT1 mas, whereas only two of the 43 paired surgical and 3B, such as other bona fide TSGs we and others marginal esophageal tissues showed methylation previously reported (Ying et al., 2006; Jin et al., (Figure 5b), and none of the 10 normal esophageal 2007a, b). Taken together, these results demonstrate tissues from healthy individuals had methylation that promoter methylation directly contributes to the (Figure 5a). Furthermore, IRF8 methylation was downregulation of IRF8 in multiple carcinomas. detected in 5 of 14 (36%) primary breast carcinomas,

Oncogene Methylation of IRF8 in multiple carcinomas KY Lee et al 5271 and 5 of 7 (71%) of primary cervical carcinomas transfected into NPC, esophageal and colorectal carci- (Figure 5b and Table 1). These results further demon- noma cells, which had completely methylated and strated that IRF8 methylation is frequent and tumor silenced IRF8 (CNE1, HONE1, KYSE510 and specific in multiple carcinomas. HCT116, Figures 6b and d). The colony formation efficiencies of each transfected cell line were evaluated by monolayer culture. Results showed that the ectopic IRF8 inhibited the anchorage-dependent growth expression of IRF8 significantly reduced the colony of carcinoma cells formation efficiency as compared to the vector only in The frequent downregulation of IRF8 in multiple these tumor cells (down to B23–48% of vector control, carcinoma cell lines and tumors but not in immortalized Po0.01, Figures 6a and c). Thus, IRF8 indeed possesses epithelial cell lines and normal tissues led to the growth inhibitory activities in tumor cells and is a postulation that IRF8 is likely a functional tumor functional tumor suppressor. suppressor. To investigate the effects of ectopic IRF8 expression on the growth of tumor cells in vitro, mammalian expression vector encoding IRF8 was Discussion

Table 1 Summary of IRF8 methylation in cell lines and primary In this report, we used an integrative epigenetic carcinomas approach combining aCGH with RT–PCR analysis to Samples Promoter methyla- screen for downregulated genes in NPC, and identified tion (%) an IFN regulatory factor, IRF8, as a candidate TSG. We further showed that IRF8 is frequently silenced by Carcinoma cell lines promoter methylation in a tumor-specific manner, not Nasopharyngeal 6/6 (100) Esophageal 14/16 (88) only in NPC, but also in other common cancers, such as Breast 7/9 (78) esophageal, breast, cervical, lung and colorectal carci- Colorectal 3/4 (75) nomas. The response of IRF8 to IFN-g treatment was Lung 5/7 (71) abolished in those cell lines with methylated promoter. Cervical 4/6 (67) In addition, we showed that ectopic expression of IRF8 Gastric 3/17 (18) in silenced carcinoma cell lines dramatically inhibits Primary carcinomas their clonogenicity. Thus, IRF8 behaves as a functional Nasopharyngeal Ca (NPC from North African) 3/3 (100) TSG in multiple cancers, and might be important in Nasopharyngeal Ca (endemic NPC of Asian 35/46 (76) their pathogenesis. Chinese) Cervical Ca 5/7 (71) Epigenetic gene silencing is associated with the onset Esophageal Ca 25/43 (58) and progression of various cancers (Jones and Baylin, Breast Ca 5/14 (36) 2002). Multiple studies tried to identify TSGs silenced by DNA methylation. Compared to other common Immortalized normal epithelial cell lines carcinomas, such as breast, colon and lung cancers, NP69, NE1, NE3 0/3 NPC is not well studied and its molecular pathogenesis Normal tissues poorly known. Thus, we used an integrated genomic and Normal nasopharyngeal tissues 0/3 epigenetic approach to identify NPC-related TSGs, and Normal esophageal epithelial tissues 0/10 successfully refined a hemizygous deletion at 16q24.1 in Surgical-margin esophageal tissues from eso- 2/43 (5) phageal Ca patients NPC cell lines, We further identified IRF8 as the only downregulated gene within this deleted region. The Abbreviations: Ca, carcinoma; NPC, nasopharyngeal carcinoma. downregulation of IRF8 is well correlated with its

HCT116 293 HepG2HONE1 KYSE520 HCT116 DKO γ Markers IFN : -+ -+ -+ -+ -+ -+ +ve control

IRF8 RT-PCR GAPDH

IRF8 methylation: U U+(M) M+(U) M+(U) M U

Figure 3 Epigenetic silencing of IRF8 results in the disruption of its response to interferon (IFN)-g. Reverse transcription (RT)–PCR showed that in response to IFN-g treatment (250 U/ml, 24 h), IRF8 expression is upregulated in HEK 293, HepG2 and HCT116-DKO cell lines which are unmethylated for IRF8, but not in HONE1, KYSE520 and HCT116 cell lines with predominantly methylated promoter. IRF8 promoter methylation status in each cell line is shown within the rectangles (bottom panel). þ , IFN-g treated; À, control without treatment; (M), weak methylation; (U), weak unmethylation.

Oncogene Methylation of IRF8 in multiple carcinomas KY Lee et al 5272 Genetic Pharmacological demethylation demethylation BT549 MB231 T47D CNE1 HK1 HONE1 KYSE510 KYSE520 HCT116 Az a: - + - + - + - + - + - + + - + + - + TSA:

- + - + - + - + - + - - - - Markers M + - + - DKO IRF8 RT-PCR GAPDH

BGS KYSE520 HCT116 KYSE510 CNE1 Aza A+T A+T A+T DKO Markers

M HCT116 MSP U DKO

Figure 4 Pharmacological demethylation with 5-aza-2-deoxycytidine (Aza) (or combined with trichostatin A, TSA) and genetic demethylation restored IRF8 expression in methylated and silenced cell lines. (a) Reverse transcription (RT)–PCR analysis of IRF8 expression. M, 100-bp DNA ladder. Right panel: genetic demethylation by double knockout of DNA methyltransferases (DNMTs) 1 and 3B in HCT116 cell line (DKO cell line) induced IRF8 expression. (b) Representative methylation-specific PCR (MSP) analyses of IRF8 promoter in tumor cell lines after demethylation. (c) BGSmethylation analysis confirmed the demethylation of IRF8 in HCT116- DKO cell line.

Normal tissues

Normal Normal esophageal nasopharyngeal tissues epithelial tissues NEE1 NEE2 NEE3 NEE4 NEE5 NEE6 NEE7 NEE63 NEE64 NEE68 Markers VC6 VC10 VC12 M MSP U

NPC

Asian Chinese North African Cervical Ca C15 C17 C18 Markers 88 89 90 91 92 93 95 97 99 100 102 104 105 12 6 20 21I 29I 50 M MSP U

Esophageal Ca (T) +paired normal tissues (N) Breast Ca 24 25 26 27 29 30 31

Markers N TN TN TN TN TNTNT 13 14 16 17 18 19 21 22 23 M MSP U

Figure 5 Representative analyses of IRF8 methylation in normal tissues and primary tumors by methylation-specific PCR (MSP). M, methylated; U, unmethylated. (a) Normal epithelial tissues. (b) Nasopharyngeal carcinoma (NPC); cervical and breast carcinoma; esophageal carcinoma (T) and their surgical marginal tissues (N).

epigenetic silencing (promoter methylation) but not any silenced by either genetic mutations including deletion, gross genetic deletion of this gene, although we still or promoter methylation or both mechanisms (Knud- could not exclude the possibility of its possible micro- son, 2001). IRF8 methylation was detected in both deletions. These results are well in line with the revised endemic and sporadic NPC, but not in normal Knudson ‘two-hit’ hypothesis that a TSG could be nasopharyngeal tissues, indicating that the epigenetic

Oncogene Methylation of IRF8 in multiple carcinomas KY Lee et al 5273 pcDNA3.1 IRF8 Markers pcDNA3.1 IRF8 IRF8 CNE1 CNE1 GAPDH NPC

IRF8 HONE1 HONE1 GAPDH

IRF8 KYSE510 GAPDH KYSE510 Esophageal

IRF8 HCT116 Colon

HCT116 GAPDH

Transfected cells HONE1 HCT116 (+ve control) BJAB pcDNA3.1 IRF8 pcDNA3.1 IRF8

IRF8

tublin

Figure 6 Inhibition of carcinoma cell clonogenicity by ectopic IRF8 expression in monolayer culture. (a) Cells were transfected with pcDNA3.1-IRF8 or control vector, and selected with G418 for 10–14 days, and stained with Gentian violet. (b) Reverse transcription (RT)–PCR analysis showed IRF8 expression after transfection. (c) Quantitative analysis of colony formation. The numbers of G418- resistant colonies in each vector-transfected control were set to 100%, whereas IRF8-expressed cells were presented as mean±s.d. Three independent experiments were carried out in triplicate. Asterisk indicates statistically significant difference (*Po0.01). (d) Western blot analysis of IRF8 protein in pcDNA3.1-IRF8-transfected cells, BJAB lymphoma cell line was used as a positive control.

inactivation of IRF8 could be a common and important its growth inhibitory effect on tumor cells through IFN- step during NPC tumorigenesis. signaling pathway (STATs) and the induction of IRF1 IRF8 is a of the IRF gene family, and 8 in mouse model (Egwuagu et al., 2006). We found which includes IRF1-8 and IRF9/ISGF3g (Nguyen that IRF8 promoter methylation abolishes its response et al., 1997). Among them, IRF1 and IRF5 have been to IFN-g in tumor cells. When inspecting the IRF8 shown to exert tumor suppressor activities in multiple promoter, an IFN-g activation site (GAS) with a single carcinomas and induce tumor cell (Bouker CpG site is present at À145 to À131 (GATTTCTCG et al., 2005; Hu et al., 2005; Watson et al., 2006). The GAAAGC). It is very likely that methylation of this expression of IRF8 is regulated by IFN-g, a proin- CpG site would affect the binding of transcription flammatory (Yang et al., 2007b). IFN-g exerts factors and the IRF8 response to IFN-g. IRF8 promoter

Oncogene Methylation of IRF8 in multiple carcinomas KY Lee et al 5274 methylation could then enable tumor cells to carcinoma cells. Considering the high incidence of escape IFN-g-mediated apoptosis, thus involved in epigenetic silencing of IRF8 in NPC, cervical and carcinogenesis. esophageal carcinomas, it would thus be worthy As a transcription factor, IRF8 probably functions as exploring further the possible use of IRF8 methylation a tumor suppressor through inducing the transcription as an epigenetic biomarker for the molecular diagnosis of TSGs and apoptotic genes and repressing the and prognosis predictions of these tumors. expression of oncogenic genes. For examples, IRF8 forms a complex with PU.1 to activate the transcription of p15INK4B/CDKN2B,abona fide TSG, in murine Materials and methods myeloid cells in response to IFNaˆ treatment (Schmidt et al., 2004). IRF8 also induces the expression of Cell lines, tumor and normal tissue samples multiple TSGs such as NF1 (Zhu et al., 2004; Tamura A series of carcinoma cell lines were studied, including et al., 2005) and apoptotic genes such as FAS(Yang nasopharyngeal, esophageal, breast, lung, cervical and color- et al., 2007a, b), and represses the expression of the ectal cancers (Ying et al., 2006; Jin et al., 2007a, b). oncogenic BCR/ABL and antiapoptotic BCL2 (Tamura Immortalized normal epithelial cell lines NP69, NE1 and NE3 (Tsao et al., 2002), HEK 293 and the lymphoblastoid cell et al., 2003; Burchert et al., 2004). line (LCL) CCL256.1 (Tao et al., 2002) were used as controls. The expression and function of IRF8 has long been Colon HCT116 cell lines with double knockout of DNMTs: thought to be limited to hematopoietic cells (Tamura HCT116 DNMT1À/À DNMT3BÀ/À (DKO) cells (gifts of Bert and Ozato 2002; Kanno et al., 2005). The tumor Vogelstein, Johns Hopkins) were used (Rhee et al., 2002). suppressor function of IRF8 in both chronic and acute Human normal adult and fetal tissue RNA samples were myelogenous leukemia has been further verified in purchased commercially (Stratagene, La Jolla, CA, USA or IRF8À/À mice, which developed a CML-like syndrome Millipore Chemicon, Billerica, MA, USA) (Ying et al., 2006). (Holtschke et al., 1996). However, IRF8 is much less Samples of normal nasopharyngeal tissues and normal characterized in epithelial tumors. Very recently, Yang esophageal epithelial tissues were described previously et al. (2007b) reported that IRF8 is repressed by (Srivastava et al., 2000; Wong et al., 2006). DNA samples from various primary carcinoma samples, 46 Asian Chinese methylation in a single colon cell line (SW620), which NPC, three nude mice-passaged NPC tumors derived from is related to apoptotic resistance and tumor metastasis. North Africans, 43 esophageal squamous cell carcinomas (T) Consistently, we detected frequent IFR8 downregulation and their corresponding surgical marginal normal tissues (N), by promoter methylation in other colon cell lines, seven cervical carcinomas and 14 breast carcinomas were HCT116, HT-29 and LoVo. Furthermore, genetic described previously (Steenbergen et al., 2004). demethylation (through double knockout of DNMT1 and 3B in HCT116) restored IRF8 expression, providing Cell treatments direct evidence that IRF8 silencing in HCT116 is due to Cell lines were treated with Aza (Sigma, St Louis, MO, USA) its aberrant promoter methylation governed by both and TSA as described previously (Ying et al., 2006). For the DNMT1 and 3B. We also analysed IRF8 expression in treatment of IFN-g, cells were seeded in six-well plate the day multiple carcinomas, and demonstrated that IRF8 acts before treatment, medium was changed and recombinant as a tumor suppressor in NPC, esophageal and color- human IFN-g (Millipore) was added to the culture to a final ectal tumor cells. Our study thus greatly extends the concentration of 250 U/ml. Cells were harvested 24-h post- treatment for analysis (Yang et al., 2007b). current knowledge of IRF8 functions in normal and tumorous nonhematopoietic cells. LOH of 16q24 was frequently detected in a variety of Array-CGH Whole-genome arrays (1 Mb resolution) with 3040 BAC/PAC cancers (Lo et al., 2000; Paige et al., 2000; Jin et al., clones were used (Wellcome Trust Sanger Institute, Cam- 2007b), suggesting that IRF8 may be a critical tumor bridge, UK), (www.ensembl.org/Homo_sapiens/index.html). suppressor in multiple carcinomas. Our studies revealed aCGH was performed and analysed as described previously that the epigenetic inactivation of IRF8 is common in (Ying et al., 2006). other multiple cancers, such as esophageal, breast, cervical and lung carcinomas. We confirmed that IRF8 Semi-quantitative RT–PCR analysis functions as a general tumor suppressor by colony Reverse transcription–PCR (RT–PCR) was performed for 36 formation assay in NPC, esophageal and colon carci- cycles with hot-start, using AmpliTaq Gold DNA Polymerase noma cell lines. We also noticed that normal IRF8 (Applied Biosystems, Foster City, CA, USA) as previously expression is maintained in all the immortalized reported. Glyceraldehyde 3-phosphate dehydrogenase epithelial cell lines, comparable to normal tissues, (GAPDH) was used as a control (Tao et al., 2002). RT–PCR indicating that IRF8 methylation is not an early event primers were designed to span introns to prevent amplification of genomic DNA. Primer sequences are provided in Supple- during carcinogenesis and not methylated during mentary Table 1. immortalization. In summary, we found that IRF8 is frequently Western blotting analysis inactivated by promoter methylation in multiple tumors Western blotting analysis was performed as previously including NPC, esophageal, cervical, breast, lung and described (Ying et al., 2008). Antibody against IRF8 colorectal carcinomas, which disrupts its response to (a´ -ICSBP/IRF8, C-19) was obtained from Santa Cruz IFN-g-induced activation. We further demonstrated Biotechnology (Santa Cruz, CA, USA), and antibody against that IRF8 could act as a functional TSG in multiple tublin was purchased from Neomarkers (Fremont, CA, USA).

Oncogene Methylation of IRF8 in multiple carcinomas KY Lee et al 5275 Bisulfite treatment and promoter methylation analysis performed in triplicate wells for three times. Data were Bisulfite modification of DNA, MSP and BGS were carried presented as relative colony formation ability±s.d. Statistical out as previously described (Tao et al., 2002; Ying et al., 2006). analysis was carried out by Student’s t-test, a Po0.05 was Both MSP and BGS were performed for 40 cycles using considered as statistically significant difference. AmpliTaq Gold DNA Polymerase with hot-start. MSP primers were tested for not amplifying any unbisulfited DNA. For BGS, the PCR products were cloned into pCR4- Acknowledgements TOPO (Invitrogen, Carlsbad, CA, USA). Four to six colonies were randomly chosen and sequenced. Primer sequences are This project was supported by a Michael and Betty Kadoorie shown in Supplementary Table 1. Cancer Genetics Research Program (MBKCGRP) Grant to QT and a Hong Kong RGC Central Allocation Grant (CA06/ Colony formation assay 07.SC03, QT). We thank Drs Bert Vogelstein, George Tsao Mammalian expression vector pcDNA3.1( þ )-IRF8 encoding (Dolly Huang), Sun Young Rha and Kaitai Yao for some cell the full-length open reading frame of human IRF8 gene was lines, DSMZ (German Collection of Microorganisms and Cell constructed and used (Hashmueli et al., 2003). For colony Cultures) for the KYSE cell lines [Shimada et al., Cancer 69: formation assay using monolayer culture, cells (2 Â 105 per 277–284 (1992)], Dr C Langford at the Wellcome Trust Sanger well) were plated in a 12-well plate and transfected with Institute, Cambridge, UK for aCGH slides, and Tzer-Jing expression plasmids pcDNA3.1( þ )-IRF8 or the empty vector Seng (Johns Hopkins Singapore) for her valuable help in pcDNA3.1( þ ) (0.5 mg each), using Fugene6.0 (Roche, Basel, aCGH analysis. Switzerland). Cells were collected and plated in a six-well plate 48 h post-transfection, and selected for 10–14 days with G418 (0.4 mg/ml). Surviving colonies (X50 cells per colony) were Note added in proof: counted after staining with Gentian Violet (ICM Pharma, During the final preparation of this paper, Yang et al. Singapore). Total RNA from the transfected cells was reported IRF8 methylation in a single colon cell line (SW620), extracted, treated with DNase I and analysed by RT–PCR to which is related to apoptotic resistance and tumor metastasis confirm the expression of IRF8. All the experiments were (Yang et al., Cancer Res. 2007; 67: 3301–3309).

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene