Zinc finger transcription factors are involved broadly in development and tumorigenesis. Here, we report that the little studied zinc finger transcription factor ZNF382 functions as a tumor suppressor in multiple carcinomas. Although broadly expressed in normal tissues, ZNF382 expression was attenuated in multiple carcinoma cell lines due to promoter CpG methylation. ZNF382 was also frequently methylated in multiple primary tumors (nasopharyngeal, esophageal, colon, gastric, and breast). Ectopic expression of ZNF382 in silenced tumor cells significantly inhibited their clonogenicity and proliferation and induced apoptosis. We further found that ZNF382 inhibited NF-κB and AP-1 signaling and downregulated the expression of multiple oncogenes including MYC, MITF, HMGA2, and CDK6, as well as the NF-κB upstream factors STAT3, STAT5B, ID1, and IKBKE, most likely through heterochromatin silencing. ZNF382 could suppress tumorigenesis through heterochromatin-mediated silencing, as ZNF382 was colocalized and interacted with heterochromatin protein HP1 and further changed the chromatin modifications of ZNF382 target oncogenes. Our data show that ZNF382 is a functional tumor suppressor frequently methylated in multiple carcinomas. Cancer Res; 70(16); 6516–26. ©2010 AACR.

Cancer is caused by aberrant gene regulation, including inactivation of negative regulators of cell proliferation [including tumor suppressor genes (TSG)] and activation of positive regulators (such as oncogenes; ref. 1). TSGs function through the following mechanisms: protecting the genome from mutagenic events, fine tuning the cell cycle regulation, inducing apoptosis in cells that escape normal cell cycle control, and inhibiting cellular migration and metastasis (2). While genetic alteration is a hallmark of human cancers, aberrant epigenetic modification of tumor cells is also important in initiating carcinogenesis (3). Promoter CpG methylation causes the loss of TSG functions, which occurs frequently during tumor development and progression (4). Currently, it is well accepted that epigenetic alterations even precede genetic changes during tumorigenesis (4). Clinically, TSG methylation can be used as an epigenetic biomarker for tumor diagnosis and prognosis prediction.

Zinc finger protein (ZFP) is the largest family of transcriptional factors with their zinc fingers binding to promoters to activate or repress gene expression (5). ZFPs also interact with other proteins to function in various signaling pathways and sometimes even bind to dsRNA (6). About one third of ZFPs contain a Krüppel-associated box (KRAB) domain. KRAB-containing ZFPs (KRAB-ZFP) are involved in cell differentiation, proliferation, apoptosis, and neoplastic transformation (7). For instance, ATM and p53–associated KZNF protein (APAK) can interact with p53 to suppress p53-mediated apoptosis (8); ZNF23 has growth-inhibitory potential (9); and ZBRK1, a BRCA1-dependent transcriptional repressor, plays a critical role in the control of cell growth and survival (10).

KRAB-ZFPs are also involved in heterochromatin formation, which in turn leads to epigenetic silencing. KRAB-ZFPs bind to specific DNA sequences and recruit KRAB-associated protein 1(KAP1), which forms heterochromatin with HP1, SETDB1, and histone deacetylase (HDAC) inhibitor to silence target gene expression (7). The active form of KRAB-ZFPs is required to initiate heterochromatin formation; thus, irregular KRAB-ZFPs result in failed formation of heterochromatin and uncontrollable gene expression leading to tumorigenesis.

A large number of ZFPs are located in several gene clusters at the long arm of chromosome 19. Deletion of 19q is frequent in multiple cancers including cervical cancer, esophageal squamous cell carcinoma (ESCC), and nasopharyngeal carcinoma (NPC; refs. 1113). Here, we investigated the potential tumor suppressor function of ZNF382, a novel 19q13.12 gene that we identified previously (14). We found that ZNF382 was epigenetically inactivated in common carcinomas including NPC and ESCC. We further showed that ectopic expression of ZNF382 suppressed clonogenicity and proliferation and induced apoptosis in tumor cells lacking endogenous ZNF382 expression. ZNF382 also repressed NF-κB and AP-1 signaling and inhibited the expression of multiple oncogenes including the NF-κB upstream factors STAT3, STAT5B, ID1, and IKBKE.

Cell lines, tumors, and normal tissue samples

A series of tumor cell lines were used (15). Immortalized normal epithelial cell lines (NP69, NE1, NE3, NE083, Het-1A, HMEC, and HMEpC) were also included. HCT116 cell lines with genetic knockout (KO) of DNA methyltransferases (DNMT): HCT116 DNMT1−/− (1KO), HCT116 DNMT3B−/− (3BKO), and HCT116 DNMT1−/− DNMT3B−/− (DKO) cells (gifts of Bert Vogelstein, Johns Hopkins University, Baltimore, MD), were grown with either 0.4 mg/mL geneticin or 0.05 mg/mL hygromycin or both. These cell lines were obtained either from the American Type Culture Collection or from our collaborators with no authentication performed. Human normal adult and fetal tissue RNA samples were purchased commercially (Stratagene or Millipore Chemicon; ref. 16). DNA samples of normal nasopharyngeal and esophageal tissues from healthy individuals were extracted as described previously (16). Samples of Asian Chinese primary NPC, nude mouse–passaged NPC tumors originated from North Africans, paired Hong Kong Chinese esophageal carcinomas (T) and the matching surgical marginal normal tissues (N), and other carcinomas were used for methylation study (1518).

Semiquantitative reverse transcription-PCR and real-time PCR

The expression of ZNF382 in cell lines and tissues was examined by reverse transcription-PCR (RT-PCR), with GAPDH as an internal control. Primers used are listed in Supplementary Table S1. RT-PCR was performed for 32 cycles with hot-start Go-Taq (Promega; ref. 16). The signals were quantified by densitometry using the ImageJ software (http://rsbweb.nih.gov/ij/) developed by Wayne Rasband (Research Services Branch, National Institute of Mental Health, Bethesda, Maryland). Real-time PCR was performed according to the manufacturer's protocol (HT7900 system, Applied Biosystems), with the expression level of ZNF382 in normal tissues (larynx and esophagus) set as baseline. To screen for ZNF382 target genes, we examined a panel of oncogenes (primers listed in Supplementary Table S2). For chromatin immunoprecipitation (ChIP)-quantitative PCR, the primers for c-MYC and STAT3 promoters are listed in Supplementary Table S1.

Methylation-specific PCR and bisulfite genomic sequencing

DNA bisulfite treatment, methylation-specific PCR (MSP), and bisulfite genomic sequencing (BGS) were performed as previously described (19). Several pairs of MSP primers targeting the methylated or unmethylated alleles of the promoter region were designed and tested, and only the optimal pairs with best amplification efficiency and specificity were used (ZNF382m3/ZNF382m5 and ZNF382u3/ZNF382u5; Supplementary Table S1). These primer pairs were also tested for not amplifying any unbisulfited DNA and thus were specific to bisulfite-converted DNA. MSP was performed for 35 cycles using AmpliTaq-Gold (Applied Biosystems; ref. 19). BGS primers were ZNF382BGS3 and ZNF382BGS2 (Supplementary Table S1). Amplified BGS products were TA-cloned, and five to six randomly chosen colonies were sequenced.

5-Aza-2′-deoxycytidine and trichostatin A treatment

Tumor cells (1 × 105/mL) were allowed to grow overnight. The culture medium was then replaced with fresh medium containing 5-aza-2′-deoxycytidine (Aza) at a final concentration of 5 to 10 μmol/L (Sigma-Aldrich; ref. 20). Cells were allowed to grow for 72 hours with change of Aza-containing medium every 24 hours; some were further treated with the HDAC inhibitor trichostatin A (TSA) for an additional 24 hours. Cells were then harvested for DNA and RNA extractions.

Stress treatment

Heat shock was performed as previously described (15). In brief, the cells were incubated at 42°C for 1 hour, followed by recovery in 5% CO2 incubator at 37°C for 2 hours. Then, the cells were harvested for RNA extraction.

Gene cloning and plasmid construction

The full-length open reading frame (ORF) of ZNF382 was cloned from adult testis cDNA library by nested-PCR. ZNF382CF2/ZNF382CR2 was used for the first-round PCR and ZNF382CF3/ZNF382CR3 was subsequently applied for the second-round PCR. All PCR reactions were performed with Pfu polymerase (Stratagene). The PCR product was then digested with BamHI and ligated into pcDNA3.1(+) with the sequence and orientation confirmed. The same ORF segment was also subcloned into pIRES-ZsGreen1 for flow cytometry analysis.

Subcellular localization

HCT116 or COS7 cells (5 × 104) were seeded on coverslips in a six-well plate. Cells were transfected with pcDNA3.1(+)-Flag-ZNF382 using Fugene 6 (Roche). In COS7 cells, ZNF382 was also cotransfected with RFP-HP1γ (21). Twenty-four hours after transfection, cells were fixed and stained with anti-Flag M2 monoclonal antibody (F3165, Sigma), then incubated with FITC-conjugated rabbit anti-mouse IgG F(ab)2 antibody (F0313, DAKO). Subsequently, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and imaged using an inverted fluorescence microscope (Olympus, Japan).

Colony formation assay

Monolayer culture was performed. HONE1, KYSE510, and HCT116 cells (1 × 105 per well) were seeded in a 12-well plate and transfected with pcDNA3.1(+)-Flag-ZNF382 plasmid or the control vector (0.8 μg each), using Fugene 6. Forty-eight hours after transfection, cells were collected and plated at appropriate density in a six-well plate and subjected to G418 (0.4 mg/mL) selection for 10 to 12 days, with selective medium refreshed every 3 days. Surviving colonies (>50 cells per colony) were counted after staining with Gentian Violet (ICM Pharma). Total RNA from the transfected cells was extracted, treated with DNase I, and analyzed by RT-PCR to confirm the ectopic expression of ZNF382. All the experiments were performed in triplicate wells three times.

Cell proliferation assay

Cell proliferation was measured by flow cytometry (BD Biosciences) with 7-hydroxy-9H(1,3-dichloro-9,9-dimethylacridin-2-one succinimidyl ester (DDAO-SE; Invitrogen) staining. The cells were transiently transfected with pIRES-ZsGreen1-ZNF382 or control vector. Twenty-four hours after transfection, 1 × 105 cells were replated in a 12-well plate. After another 24 hours, cells were washed with PBS and incubated with 5 μmol/L DDAO-SE (Molecular Probes) in PBS for 15 minutes at 37°C. The reaction was stopped by replacing the staining solution with fresh, prewarmed medium, after which the cells were incubated for an additional 30 minutes at 37°C. Cells were then harvested at the starting time point and at 48 hours, and the fluorescence intensity was measured by flow cytometry for green fluorescent protein (GFP)–positive cells. The experiment was repeated three times independently.

Apoptosis assay

Apoptosis was assessed using the Annexin V-phycoerythrin (PE) Apoptosis Detection Kit I (BD Biosciences) by flow cytometry. Cell apoptosis and viability were measured by Annexin V-PE and 7-amino-actinomycin (7-AAD) staining. Cell populations were counted as viable (Annexin V, 7-AAD), early apoptotic (Annexin V+, 7-AAD), necrotic (Annexin V, 7-AAD+), and late apoptotic cells (Annexin V+, 7-AAD+). HCT116 cells transfected with pIRES-ZsGreen1 or pIRES-ZsGreen1-ZNF382 were harvested at 48 hours after transfection. GFP-positive cells were sorted by flow cytometry and the apoptotic status was analyzed after staining with Annexin V-PE and 7-AAD. Both late and early apoptotic cells were counted together for relative apoptotic changes. All the experiments were performed three times.

Apoptosis was also determined by DAPI staining. Forty-eight hours after transfection of pIRES-ZsGreen1-ZNF382 or control vector, cells were fixed with pre-cold methanol for 10 minutes at −20°C. Subsequently, cell nuclei were stained with DAPI (0.5 μg/mL) for 10 minutes at room temperature and examined under a fluorescence microscope. Condensed or fragmented nuclei indicated apoptotic changes.

Coimmunoprecipitation and Western blot

For coimmunoprecipitation, 1 × 106 HEK293 cells were cotransfected with 2 μg of pcDNA3.1(+)-Flag-ZNF382 and pEGFP-C1-HP1β (mouse HP1β; a gift from Alain Verreault, Institute for Research in Immunology and Cancer, Montreal, Canada) using Fugene 6. Forty-eight hours after transfection, cells were lysed in 200 μL of lysis buffer [50 mmol/L NaCl, 20 mmol/L Tris (pH 7.6), 1% NP40, 1× protease inhibitor mixture] for 1 hour on ice. Cell lysates were centrifuged at 10,000 × g at 4°C for 10 minutes. A total of 200 μg of protein were used for coimmunoprecipitation reaction. First, total protein lysates were precleared by 20 μL of protein G-agarose with incubation at 4°C for 30 minutes. After a brief centrifugation, the supernatant was transferred to a new 1.5-mL tube. The immunoprecipitation reaction was performed with 2 μg of the indicated antibody and 50 μL of protein-G slurry for overnight incubation at 4°C. The precipitated materials were separated by SDS-PAGE, transferred onto a polyvinylidene difluoride (PVDF) membrane, and immunoblotted as described previously (18).

For Western blot analysis, 48 hours after transfection, cells were harvested and lysed in lysis buffer [10 mmol/L Tris-HCl (pH 7.4), 1% SDS, 10% glycerol, 5 mmol/L MgCl2, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium orthovandate, 5 μg/mL leupeptin, and 21 μg/mL aprotinin]. A total of 30 μg of protein lysates were separated by SDS-PAGE and transferred onto a PVDF membrane. The dilution of primary antibodies was according to the company's recommendation. Proteins were visualized using the enhanced chemiluminescence detection system.

Chromatin immunoprecipitation

ChIP assays were performed according to the protocol of the kit manufacturer (Upstate). Specific H3K4me3 antibody was used (ab8580, Abcam). The same amount of nonspecific IgG was used as antibody control. Both input DNA and immunoprecipitated DNA were purified with Qiagen DNA mini kit. All samples were analyzed by real-time PCR. The fold enrichment of target sequence was determined using the following formula: fold enrichment = 2(ΔCT of input − ΔCT of IP'ed DNA). The experiment was repeated three times independently.

Luciferase reporter assay

To screen for signaling pathways modulated by ZNF382, several signaling pathway luciferase reporters were evaluated in ZNF382-transfected HCT116 and HEK293 cells, including NF-κB-luc, p53-luc, AP-1-luc, SRE-luc (Stratagene), and TOPFLASH [kindly provided by Prof. Christof Niehrs, German Cancer Research Center (DKFZ), Heidelberg, Germany]. Luciferase assay was done as previously described (22). All the experiments were performed in triplicates.

Statistical analysis

Data were presented as mean ± SD. Statistical analysis was carried out with Student's t test. P < 0.05 was considered as statistically significant difference.

Expression profiling of ZNF382 in normal tissues and tumor cell lines

We previously identified a novel KRAB-ZFP, ZNF382, at 19q13.12 (14). By semiquantitative RT-PCR, which is more sensitive than the previous Northern blot, we found that ZNF382 was expressed in all normal adult tissues and fetal tissues examined, as well as in normal peripheral blood mononuclear cells, but with varying expression levels (Fig. 1A). In contrast, ZNF382 expression was frequently silenced or reduced in multiple carcinoma cell lines of nasopharyngeal, lung, esophageal, colon, stomach, breast, and cervical cancers, but infrequently in liver and prostate cancers (Fig. 1B). This expression pattern was further confirmed by real-time PCR in some representative cell lines (Fig. 1C). ZNF382 was also highly expressed in seven immortalized normal epithelial cell lines examined (NP69, NE1, NE3, NE083, Het-1A, HMEC, and HMEpC). These results suggest that ZNF382 is a broadly expressed gene but is frequently disrupted in multiple carcinoma cell lines.

Figure 1.

A, top, the ZNF382 CGI. Transcription start site is indicated by a curved arrow. The CGI, MSP, and BGS regions analyzed are indicated. Bottom, expression profile of ZNF382 in human normal adult and fetal tissues by semiquantitative RT-PCR with GAPDH as a control. B, representative analyses of ZNF382 expression and promoter methylation in tumor cell lines and immortalized epithelial cells. M, methylated; U, unmethylated. C, real-time PCR analysis of ZNF382 expression in several representative cell lines. The expression level of ZNF382 in NPC cell lines was normalized to that in normal larynx, whereas the expression in esophageal carcinoma cell lines was compared with that in normal esophagus. D, high-resolution methylation analysis of the ZNF382 promoter by BGS in tumor cell lines and immortalized normal cell line NP69.

Figure 1.

A, top, the ZNF382 CGI. Transcription start site is indicated by a curved arrow. The CGI, MSP, and BGS regions analyzed are indicated. Bottom, expression profile of ZNF382 in human normal adult and fetal tissues by semiquantitative RT-PCR with GAPDH as a control. B, representative analyses of ZNF382 expression and promoter methylation in tumor cell lines and immortalized epithelial cells. M, methylated; U, unmethylated. C, real-time PCR analysis of ZNF382 expression in several representative cell lines. The expression level of ZNF382 in NPC cell lines was normalized to that in normal larynx, whereas the expression in esophageal carcinoma cell lines was compared with that in normal esophagus. D, high-resolution methylation analysis of the ZNF382 promoter by BGS in tumor cell lines and immortalized normal cell line NP69.

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Frequent inactivation of ZNF382 by promoter CpG methylation

Sequence analysis revealed that the ZNF382 promoter contains a CpG island (CGI; Fig. 1A), indicating CpG methylation may be a major mechanism silencing its expression in cancer cells (20). MSP analysis showed that the ZNF382 promoter was methylated in 6 of 6 NPC, 1 of 1 hypopharyngeal, 3 of 5 (60%) of lung, 16 of 18 (89%) esophageal, 4 of 4 colon, 15 of 15 (100%) gastric, 4 of 13 (30%) hepatocellular, 5 of 10 (50%) breast, and 3 of 4 cervical cancer cell lines and with no methylation detected in 3 prostate cancer cell lines (Fig. 1B). In contrast, obvious ZNF382 methylation was not detected in 7 immortalized normal epithelial cell lines (Fig. 1B), suggesting that the methylation of ZNF382 is tumor specific. A good correlation between downregulation and methylation of ZNF382 was observed in these carcinoma cell lines. We further examined ZNF382 methylation in detail by high-resolution BGS analysis. The results confirmed those of the MSP analysis (Fig. 1D).

Pharmacologic and genetic demethylation restores ZNF382 expression

To determine whether CpG methylation directly mediates ZNF382 downregulation, several carcinoma cell lines, C666-1, HK1, HNE1, HONE1, HCT116, EC109, KYSE510, KYSE520, MB231, and MB435, were treated with the DNA methyltransferase inhibitor Aza alone or combined with the HDAC inhibitor TSA. The treatment restored ZNF382 expression, accompanied by a decrease of methylated promoter alleles and an increase of unmethylated alleles (Fig. 2A). ZNF382 expression could also be activated by genetic demethylation through double KO of both DNMT1 and DNMT3B (DKO cell line), but not through single KO of DNMT1 or DNMT3B (1KO or 3BKO cell line), in a colorectal cancer cell line model (Fig. 2B), indicating that the maintenance of ZNF382 methylation was mediated by DNMT1 and DNMT3B together, similar to the other TSGs we have examined (15, 16). Detailed BGS analysis confirmed the demethylation of ZNF382 (Fig. 2C). These results indicate that promoter methylation directly mediates the transcriptional silencing of ZNF382.

Figure 2.

Restoration of ZNF382 by demethylation. A, pharmacologic demethylation with Aza alone or Aza combined with TSA (A + T) activates ZNF382 expression in tumor cell lines, accompanied by demethylation of the promoter. B, genetic demethylation through double KO of DNMT1 and DNMT3B in HCT116 cells restores ZNF382 expression, but not with single KO of DNMT1 (1KO) or DNMT3B (3BKO). C, detailed BGS analysis confirms the demethylation of ZNF382 promoter in HCT116-DKO cell line. D, epigenetic silencing of ZNF382 results in the disruption of its response to heat shock stress. Top, locations of heat shock factor (HSF) binding sites and the ZNF382 promoter CGI are indicated; bottom, ZNF382 expression is upregulated in weakly unmethylated (HK1 and CNE1) or hemi-unmethylated (CNE2) cell lines but not in the fully methylated cell line (HCT116) upon heat shock treatment, and pharmacologic demethylation of HCT116 is able to restore this heat shock response of ZNF382.

Figure 2.

Restoration of ZNF382 by demethylation. A, pharmacologic demethylation with Aza alone or Aza combined with TSA (A + T) activates ZNF382 expression in tumor cell lines, accompanied by demethylation of the promoter. B, genetic demethylation through double KO of DNMT1 and DNMT3B in HCT116 cells restores ZNF382 expression, but not with single KO of DNMT1 (1KO) or DNMT3B (3BKO). C, detailed BGS analysis confirms the demethylation of ZNF382 promoter in HCT116-DKO cell line. D, epigenetic silencing of ZNF382 results in the disruption of its response to heat shock stress. Top, locations of heat shock factor (HSF) binding sites and the ZNF382 promoter CGI are indicated; bottom, ZNF382 expression is upregulated in weakly unmethylated (HK1 and CNE1) or hemi-unmethylated (CNE2) cell lines but not in the fully methylated cell line (HCT116) upon heat shock treatment, and pharmacologic demethylation of HCT116 is able to restore this heat shock response of ZNF382.

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Promoter methylation disrupts the stress response of ZNF382

Three heat shock factor binding sites were predicted in the ZNF382 promoter [one localizes within the CGI whereas the other two are adjacent to the CGI (MatInspector; http://www.genomatix.de)], indicating that ZNF382 might be a stress-responsive gene (Fig. 2D). We thus examined the response of ZNF382 to heat shock stimulation in several cell lines. ZNF382 expression was significantly increased on heat shock treatment in HK1, CNE1 (weakly unmethylated), and CNE2 (hemi-unmethylated) cell lines. This response was, however, abolished in completely methylated HCT116 cells. In contrast, heat shock–activated ZNF382 expression was rescued in pharmacologically demethylated HCT116 cells (Fig. 2D). Taken together, these results show that ZNF382 is indeed stress responsive, and promoter methylation disrupts its cellular protective response to environmental stresses.

Frequent ZNF382 methylation in primary tumors

We further examined ZNF382 methylation in multiple primary tumors and the corresponding normal tissues. ZNF382 was not methylated in normal nasopharyngeal (0 of 10) and esophageal epithelial tissues (0 of 7), but was frequently methylated in 88.9% (48 of 54) of Asian Chinese primary NPC, 2 of 3 nude mice-passaged undifferentiated NPC tumors from North Africans (C15, C17, and C18), and 71.4% (20 of 28) of esophageal, 72.7% (8 of 11) of colon, 63.6% (7 of 11) of gastric, and 18.2% (2 of 11) of breast tumors (Fig. 3A and B). Aberrant methylation was also detected in 10.7% (3 of 28) paired surgical marginal esophageal tissues from esophageal carcinoma patients, which could be due to the presence of premalignant lesions or infiltrating tumor cells. The transcript level of ZNF382 was reduced in 19 of 21 methylated primary tumors but not in unmethylated primary tumors as examined by semiquantitative RT-PCR (Fig. 3C), indicating a good correlation between ZNF382 promoter methylation and its transcriptional silencing in primary tumors. It should be noted that primary tumors, if not microdissected, would also have background expression of ZNF382 transcripts due to infiltrating normal cells (17), which could explain the situation of the two not downregulated, methylated cases (#62 and #73). Nevertheless, these results show that ZNF382 methylation is a common event in multiple tumorigenesis.

Figure 3.

Representative analyses of ZNF382 methylation in normal tissues and primary tumors by MSP. A, normal epithelial tissues. B, primary NPC, esophageal carcinomas (T), and their surgical marginal normal tissues (N) and other carcinomas. C, semiquantitative RT-PCR analysis of ZNF382 expression in primary NPC relative to GAPDH expression levels. The expression levels of ZNF382 in NPC tumors were normalized to that in the normal control cell line NP69.

Figure 3.

Representative analyses of ZNF382 methylation in normal tissues and primary tumors by MSP. A, normal epithelial tissues. B, primary NPC, esophageal carcinomas (T), and their surgical marginal normal tissues (N) and other carcinomas. C, semiquantitative RT-PCR analysis of ZNF382 expression in primary NPC relative to GAPDH expression levels. The expression levels of ZNF382 in NPC tumors were normalized to that in the normal control cell line NP69.

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ZNF382 is a nuclear protein inhibiting the NF-κB and AP-1 signaling pathways

KS1, the rat homologue of human ZNF382, is a nuclear protein that functions as a transcription repressor (23, 24). We examined the subcellular localization of ZNF382. Indirect immunofluorescent staining showed that Flag-tagged ZNF382 was also a nuclear protein (Fig. 4A). Furthermore, by screening several signaling pathways with luciferase reporter assays, we found that ZNF382 significantly suppressed NF-κB and AP-1 reporter activities in HCT116 tumor cells; however, in an immortalized normal epithelial cell line, HEK293, it only significantly inhibited AP-1 activity (Fig. 4B). We further confirmed the inhibitory effect of ZNF382 on NF-κB activity in two other tumor cell lines, KYSE150 and HNE1 (Fig. 4C). Both NF-κB and AP-1 signaling are important in cell proliferation, survival, apoptosis, and malignant transformation (25, 26). Thus, ZNF382 could induce apoptosis and inhibit cell proliferation by suppressing both the NF-κB and the AP-1 signaling pathways.

Figure 4.

A, nuclear localization of ZNF382 by indirect immunofluorescence in HCT116 and COS7 cell lines. Left, FITC green fluorescence of Flag-tagged ZNF382; middle, DAPI-stained cell nuclei; right, merged images. B, the effects of ZNF382 on several signaling pathways in HCT116 and HEK293 cells were assessed by dual-luciferase reporter assays. *, P < 0.05; **, P < 0.01. C, ZNF382 significantly inhibits NF-κB activity in KYSE150 and HNE1 cell lines. *, P < 0.05; **, P < 0.01.

Figure 4.

A, nuclear localization of ZNF382 by indirect immunofluorescence in HCT116 and COS7 cell lines. Left, FITC green fluorescence of Flag-tagged ZNF382; middle, DAPI-stained cell nuclei; right, merged images. B, the effects of ZNF382 on several signaling pathways in HCT116 and HEK293 cells were assessed by dual-luciferase reporter assays. *, P < 0.05; **, P < 0.01. C, ZNF382 significantly inhibits NF-κB activity in KYSE150 and HNE1 cell lines. *, P < 0.05; **, P < 0.01.

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ZNF382 inhibits clonogenicity and proliferation and induces apoptosis of tumor cells

To assess the functions of ZNF382 in tumor cells, Flag-ZNF382–expressing plasmid was transfected into HONE1, KYSE510, and HCT116 cells with completely methylated/silenced ZNF382. Ectopic expression of ZNF382 dramatically reduced the colony formation efficiency of these cells (down to 10.2–42.9%; P < 0.01; Fig. 5A). The ZNF382 inhibition of cell proliferation was further determined by flow cytometry with DDAO-SE staining. Forty-eight hours after transfection, the fluorescence intensity in ZNF382-transfected HCT116 cells was higher than that in control vector–transfected cells after GFP sorting, which provided direct evidence that ZNF382 could inhibit tumor cell proliferation (Fig. 5B).

Figure 5.

ZNF382 is a functional TSG. A, left, representative colony formation assay with monolayer culture. Right, ectopic ZNF382 expression in tumor cells was confirmed by RT-PCR. Bottom, quantitative analysis of colony formation ability in ZNF382-transfected cells. The number of G418-resistant colonies (>50 cells) in each vector-transfected cells was set to 100. **, P < 0.01. B, ZNF382 inhibits cell proliferation in HCT116 cells by DDAO-SE assay. The fluorescence intensities of ZNF382- and control vector–transfected HCT116 cells at different time points were indicated by different colors. C, left, relative proportions of apoptotic cells in ZNF382- and control vector–transfected HCT116 cells as analyzed by flow cytometry after staining with Annexin V and 7-AAD. Right, Western blot of apoptotic indicator, cleaved PARP, in ZNF382-transfected HCT116 and HNE1 cells. D, ZNF382 induced the apoptosis-associated cell nuclear changes. Cells were stained with DAPI and photographed under a fluorescence microscope 48 h after transfection. Red arrows, transfection-positive cells.

Figure 5.

ZNF382 is a functional TSG. A, left, representative colony formation assay with monolayer culture. Right, ectopic ZNF382 expression in tumor cells was confirmed by RT-PCR. Bottom, quantitative analysis of colony formation ability in ZNF382-transfected cells. The number of G418-resistant colonies (>50 cells) in each vector-transfected cells was set to 100. **, P < 0.01. B, ZNF382 inhibits cell proliferation in HCT116 cells by DDAO-SE assay. The fluorescence intensities of ZNF382- and control vector–transfected HCT116 cells at different time points were indicated by different colors. C, left, relative proportions of apoptotic cells in ZNF382- and control vector–transfected HCT116 cells as analyzed by flow cytometry after staining with Annexin V and 7-AAD. Right, Western blot of apoptotic indicator, cleaved PARP, in ZNF382-transfected HCT116 and HNE1 cells. D, ZNF382 induced the apoptosis-associated cell nuclear changes. Cells were stained with DAPI and photographed under a fluorescence microscope 48 h after transfection. Red arrows, transfection-positive cells.

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To explore the mechanism of tumor suppression by ZNF382, we performed an apoptosis assay using flow cytometry with Annexin V-PE and 7-AAD double staining. pIRES-ZsGreen1, which could translate the gene of interest and ZsGreen1 from a single bicistronic mRNA, was used to sort transfected positive cells. We determined the percentage of Annexin V(+/−) and 7-AAD(+/−) cells for GFP-positive cells. In HCT116 cells, ectopic ZNF382 expression resulted in a significant increase of apoptotic cells as compared with control (Fig. 5C). Apoptotic induction was further confirmed by DAPI staining at the individual-cell level. pIRES-ZsGreen1-ZNF382–transfected cells (GFP positive) showed obvious condensed or fragmented nuclei, a remarkable cell apoptotic feature, which was not observed in vector control–transfected cells (Fig. 5D). Evidence of apoptosis was also detected by Western blot. Ectopic expression of ZNF382 could significantly increase the cleavage of poly(ADP-ribose) polymerase (PARP) in HCT116 and HNE1 cells as compared with control (Fig. 5C). Taken together, these results support that ZNF382 functions as a tumor suppressor by inducing apoptosis and inhibiting the proliferation of tumor cells.

ZNF382 represses the expression of multiple oncogenes including NF-κB pathway upstream effectors through heterochromatin silencing

Because ZNF382 (KS1) is a repressor, we screened for its potential target genes by examining the expression changes of 34 major oncogenes that have been reported to be involved in proliferation/transformation/apoptosis inhibition and signaling pathways, after ectopic ZNF382 expression in HCT116. Real-time PCR analysis revealed that, as compared with vector control–transfected cells, ZNF382 expression significantly downregulated the expression of multiple oncogenes at the mRNA level, including STAT3, MYC, ID1, IKBKE, STAT5B, MITF, HMGA2, and CDK6, whereas the expression of other oncogenes such as ID2 and CCND1 was not affected (Fig. 6A). Downregulation of STAT3 and STAT5B was further confirmed by Western blot (Fig. 6A). Some of these oncogenes were known to be NF-κB pathway upstream effectors, such as STAT3, STAT5B, IKBKE, and ID1 (2730). Thus, ZNF382 could inhibit the NF-κB pathway by suppressing its upstream effectors.

Figure 6.

A, left, suppression of multiple oncogenes by ZNF382 expression as evaluated by real-time PCR in HCT116 cells. All the downregulated genes are shown. *, P < 0.05; **, P < 0.01. Right, ectopic ZNF382 expression downregulated the protein levels of STAT3 and STAT5B in HCT116 cells. B, left, colocalization of RFP-HP1γ and Flag-ZNF382 (detected by FITC-conjugated antibody) in COS7 cells. Right, coimmunoprecipitation showed the physical interaction between ZNF382 and HP1β. C, ZNF382 decreases H3K4me3 levels on the promoters of STAT3 and c-MYC by ChIP assay. **, P < 0.01. D, a possible mechanism model of tumor suppression by ZNF382. ZNF382 suppresses the expression of MYC, CDK6, and MITF, which controls apoptosis and proliferation. ZNF382 also suppresses the expression of STAT3, STAT5B, ID1, and IKBKE and further inhibits NF-κB and AP-1 signaling.

Figure 6.

A, left, suppression of multiple oncogenes by ZNF382 expression as evaluated by real-time PCR in HCT116 cells. All the downregulated genes are shown. *, P < 0.05; **, P < 0.01. Right, ectopic ZNF382 expression downregulated the protein levels of STAT3 and STAT5B in HCT116 cells. B, left, colocalization of RFP-HP1γ and Flag-ZNF382 (detected by FITC-conjugated antibody) in COS7 cells. Right, coimmunoprecipitation showed the physical interaction between ZNF382 and HP1β. C, ZNF382 decreases H3K4me3 levels on the promoters of STAT3 and c-MYC by ChIP assay. **, P < 0.01. D, a possible mechanism model of tumor suppression by ZNF382. ZNF382 suppresses the expression of MYC, CDK6, and MITF, which controls apoptosis and proliferation. ZNF382 also suppresses the expression of STAT3, STAT5B, ID1, and IKBKE and further inhibits NF-κB and AP-1 signaling.

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To explore the possible mechanism of transcriptional repression mediated by ZNF382, we checked the involvement of heterochromatin silencing, as previous study revealed that KS1 is located in the nucleus and interacts with KAP1 (23, 24) whereas KAP1 is known to form a complex with heterochromatin proteins (HP) to exert gene silencing (31). Indirect immunofluorescence staining for Flag-tagged ZNF382 in HCT116 and COS7 cells clearly showed that ZNF382 is a nuclear protein (Fig. 4A) and also colocalizes with HP1 (Fig. 6B). The interaction between ZNF382 and HP1β was further confirmed by coimmunoprecipitation assay (Fig. 6B). It is well known that H3K4me3 (trimethylation of histone H3 at the lysine 4 residue) is a marker of transcriptionally active chromatin, whereas H3K9me3 modification is a marker of inactive chromatin (32). Our ChIP assays further showed that ectopic expression of ZNF382 could significantly decrease the levels of H3K4me3 on the promoters of its target oncogenes, STAT3 and c-MYC (Fig. 6C). Taken together, our results suggest that ZNF382 is functionally involved in heterochromatin-mediated gene silencing, by the suppression of multiple oncogenes.

In this report, we found that ZNF382, a KRAB-ZFP we identified previously, is frequently downregulated by promoter CpG methylation in multiple tumors. We further found that ectopic expression of ZNF382 in silenced carcinoma cells dramatically inhibits their colony formation by inhibiting cell proliferation and inducing apoptosis. We also showed that ZNF382 inhibits NF-κB and AP-1 signaling and suppresses the expression of multiple oncogenes likely through heterochromatin silencing. Thus, ZNF382 functions as a broad tumor suppressor in human cancers.

KRAB-ZFPs play important roles in various epigenetic regulations. During early mouse development, KRAB domain triggers de novo promoter methylation (33). Zfp57 maintains both maternal and paternal imprints in mice (34). The KRAB domain binds to KAP1, which further recruits HP1, SETDB1 (H3K9 methyltransferase), and HDAC complexes, whereas the zinc finger domain specifically binds to nucleation sites, forming heterochromatin for gene silencing (35). Defects in chromatin-related activities, such as chromatin assembly and remodeling, may trigger tumorigenesis (36). The disruption of DNA binding proteins (KRAB-ZFPs) may thus cause failure in heterochromatin formation and contribute to tumorigenesis. We found that ZNF382 colocalizes and interacts with heterochromatin protein HP1, together with the previous report of the colocalization of the ZNF382 rat homologene KS1 with KAP1 (24), indicating that ZNF382 is indeed involved in heterochromatin formation and silencing. Thus, ZNF382 might directly bind to target gene promoters as a repressor (24) or form heterochromatin on target gene promoters together with KAP1 and HP1, leading to transcriptional repression of target genes. The epigenetic disruption of ZNF382 would cause the aberrant expression of normally repressed genes mediated by heterochromatin.

Indeed, we found that ZNF382 could significantly repress the RNA levels of multiple oncogenes involved in neoplastic transformation, apoptosis, cell cycle, and proliferation, including MYC, STAT3, ID1, IKBKE, STAT5B, MITF, HMGA2, and CDK6. We further verified that the downregulation of STAT3 and MYC by ZNF382 was mainly mediated through heterochromatin silencing. Among these targets, MYC, MITF, and CDK6 are involved in cell cycle regulation and apoptosis (3739). STAT3, MITF, and HMGA2 are involved in neoplastic transformation (4042). Some targets are NF-κB upstream regulatory factors, such as STAT3, STAT5B, ID1, and IKBKE. STAT3 and STAT5B are required for maintaining NF-κB activity (27, 28). ID1 promotes cell survival by regulating NF-κB activity in prostate and breast cancers (30, 43); IKBKE is an oncogene controlling the activity of NF-κB in cell proliferation and malignant transformation (29). In agreement, ZNF382 inhibits the oncogenic NF-κB signaling pathway as well as the AP-1 signaling pathway.

NF-κB is a dimeric transcriptional factor, involved in the regulation of cell proliferation, apoptosis, angiogenesis, and cell invasion (25). NF-κB is highly activated in many cancers, accompanied by the upregulation of its downstream oncoproteins. Thus, the NF-κB pathway is considered as a cell survival and antiapoptotic signaling pathway (25). Some TSGs exert their tumor-suppressive functions by inhibiting the NF-κB pathway, such as CYLD and CHFR (44, 45). NF-κB also cross-talks with the AP-1 signaling pathway (46). AP-1 is another dimeric factor regulating multiple cellular processes including proliferation, apoptosis, and differentiation. Activated AP-1 not only induces apoptosis for certain tumors or specific stages of tumorigenesis but also promotes cell survival for other tumor types (26). Several TSGs can inhibit the proliferation of colon cancer by inhibiting AP-1 activity, such as PDCD4 and HINT1 (47, 48). KS1, the rat homologue of human ZNF382, antagonizes Ras-, Gα12-, or Gα13-induced neoplastic transformation, but does not induce apoptosis (23). Since NF-κB and AP-1 are both downstream targets of Ras, Gα12 and Gα13 (25, 26, 49, 50), ZNF382 could antagonize Ras oncogene–induced transformation by inhibiting downstream events such as NF-κB and AP-1 signaling.

In conclusion, we found that ZNF382 is a functional TSG, inducing apoptosis, inhibiting cell proliferation, and suppressing multiple oncogenes. The antitumorigenic effect of ZNF382 may act through suppressing both the NF-κB and the AP-1 signaling pathways (Fig. 6D), whereas epigenetic silencing of ZNF382 would activate these cancer signaling pathways during tumorigenesis. In addition, the high frequency and tumor-specific methylation of ZNF382 in NPC and esophageal carcinomas indicate that it could be used as a potential epigenetic biomarker for their molecular diagnosis.

No potential conflicts of interest were disclosed.

We thank Bert Vogelstein, George Tsao, Michael Obster (Dolly Huang), Kai-Tai Yao, and Gui-yuan Li for some of the cell lines; DSMZ (German Collection of Microorganisms and Cell Cultures) for the KYSE cell lines (Shimada et al., Cancer 69:277-284;1992); Jianming Ying for normal nasopharyngeal DNA samples; and Ying Ying for critical reading of the manuscript.

Grant Support: Hong Kong RGC grant 474407, Chinese University of Hong Kong Scheme C, and a Chinese University of Hong Kong direct grant.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1
Coleman
WB
,
Tsongalis
GJ
. 
Molecular mechanisms of human carcinogenesis
.
EXS
2006
;
96
:
321
49
.
2
Hayslip
J
,
Montero
A
. 
Tumor suppressor gene methylation in follicular lymphoma: a comprehensive review
.
Mol Cancer
2006
;
5
:
44
.
3
Herman
JG
. 
Hypermethylation of tumor suppressor genes in cancer
.
Semin Cancer Biol
1999
;
9
:
359
67
.
4
Jones
PA
,
Baylin
SB
. 
The fundamental role of epigenetic events in cancer
.
Nat Rev Genet
2002
;
3
:
415
28
.
5
Cowger
JJ
,
Zhao
Q
,
Isovic
M
,
Torchia
J
. 
Biochemical characterization of the zinc-finger protein 217 transcriptional repressor complex: identification of a ZNF217 consensus recognition sequence
.
Oncogene
2007
;
26
:
3378
86
.
6
Yang
M
,
Wu
S
,
Su
X
,
May
WS
. 
JAZ mediates G1 cell-cycle arrest and apoptosis by positively regulating p53 transcriptional activity
.
Blood
2006
;
108
:
4136
45
.
7
Urrutia
R
. 
KRAB-containing zinc-finger repressor proteins
.
Genome Biol
2003
;
4
:
231
.
8
Tian
C
,
Xing
G
,
Xie
P
, et al
. 
KRAB-type zinc-finger protein Apak specifically regulates p53-dependent apoptosis
.
Nat Cell Biol
2009
;
11
:
580
91
.
9
Huang
C
,
Jia
Y
,
Yang
S
, et al
. 
Characterization of ZNF23, a KRAB-containing protein that is downregulated in human cancers and inhibits cell cycle progression
.
Exp Cell Res
2007
;
313
:
254
63
.
10
Tan
W
,
Zheng
L
,
Lee
WH
,
Boyer
TG
. 
Functional dissection of transcription factor ZBRK1 reveals zinc fingers with dual roles in DNA-binding and BRCA1-dependent transcriptional repression
.
J Biol Chem
2004
;
279
:
6576
87
.
11
Tsuda
H
,
Takarabe
T
,
Okada
S
, et al
. 
Different pattern of loss of heterozygosity among endocervical-type adenocarcinoma, endometrioid-type adenocarcinoma and adenoma malignum of the uterine cervix
.
Int J Cancer
2002
;
98
:
713
7
.
12
Du
PL
,
Dietzsch
E
,
Van
GM
, et al
. 
Mapping of novel regions of DNA gain and loss by comparative genomic hybridization in esophageal carcinoma in the Black and Colored populations of South Africa
.
Cancer Res
1999
;
59
:
1877
83
.
13
Shao
JY
,
Huang
XM
,
Yu
XJ
, et al
. 
Loss of heterozygosity and its correlation with clinical outcome and Epstein-Barr virus infection in nasopharyngeal carcinoma
.
Anticancer Res
2001
;
21
:
3021
9
.
14
Luo
K
,
Yuan
W
,
Zhu
C
, et al
. 
Expression of a novel Krupple-like zinc-finger gene, ZNF382, in human heart
.
Biochem Biophys Res Commun
2002
;
299
:
606
12
.
15
Cui
Y
,
Ying
Y
,
van
HA
, et al
. 
OPCML is a broad tumor suppressor for multiple carcinomas and lymphomas with frequently epigenetic inactivation
.
PLoS ONE
2008
;
3
:
e2990
.
16
Ying
J
,
Li
H
,
Seng
TJ
, et al
. 
Functional epigenetics identifies a protocadherin PCDH10 as a candidate tumor suppressor for nasopharyngeal, esophageal and multiple other carcinomas with frequent methylation
.
Oncogene
2006
;
25
:
1070
80
.
17
Liu
XQ
,
Chen
HK
,
Zhang
XS
, et al
. 
Alterations of BLU, a candidate tumor suppressor gene on chromosome 3p21.3, in human nasopharyngeal carcinoma
.
Int J Cancer
2003
;
106
:
60
5
.
18
Wang
Y
,
Li
J
,
Cui
Y
, et al
. 
CMTM3, located at the critical tumor suppressor locus 16q22.1, is silenced by CpG methylation in carcinomas and inhibits tumor cell growth through inducing apoptosis
.
Cancer Res
2009
;
69
:
5194
201
.
19
Tao
Q
,
Huang
H
,
Geiman
TM
, et al
. 
Defective de novo methylation of viral and cellular DNA sequences in ICF syndrome cells
.
Hum Mol Genet
2002
;
11
:
2091
102
.
20
Qiu
GH
,
Tan
LK
,
Loh
KS
, et al
. 
The candidate tumor suppressor gene BLU, located at the commonly deleted region 3p21.3, is an E2F-regulated, stress-responsive gene and inactivated by both epigenetic and genetic mechanisms in nasopharyngeal carcinoma
.
Oncogene
2004
;
23
:
4793
806
.
21
Fukagawa
T
,
Nogami
M
,
Yoshikawa
M
, et al
. 
Dicer is essential for formation of the heterochromatin structure in vertebrate cells
.
Nat Cell Biol
2004
;
6
:
784
91
.
22
Ying
J
,
Li
H
,
Yu
J
, et al
. 
WNT5A exhibits tumor-suppressive activity through antagonizing the Wnt/β-catenin signaling, and is frequently methylated in colorectal cancer
.
Clin Cancer Res
2008
;
14
:
55
61
.
23
Gebelein
B
,
Fernandez-Zapico
M
,
Imoto
M
,
Urrutia
R
. 
KRAB-independent suppression of neoplastic cell growth by the novel zinc finger transcription factor KS1
.
J Clin Invest
1998
;
102
:
1911
9
.
24
Gebelein
B
,
Urrutia
R
. 
Sequence-specific transcriptional repression by KS1, a multiple-zinc-finger-Kruppel-associated box protein
.
Mol Cell Biol
2001
;
21
:
928
39
.
25
Basseres
DS
,
Baldwin
AS
. 
Nuclear factor-κB and inhibitor of κB kinase pathways in oncogenic initiation and progression
.
Oncogene
2006
;
25
:
6817
30
.
26
Eferl
R
,
Wagner
EF
. 
AP-1: a double-edged sword in tumorigenesis
.
Nat Rev Cancer
2003
;
3
:
859
68
.
27
Lee
H
,
Herrmann
A
,
Deng
JH
, et al
. 
Persistently activated Stat3 maintains constitutive NF-κB activity in tumors
.
Cancer Cell
2009
;
15
:
283
93
.
28
Han
X
,
Ren
X
,
Jurickova
I
, et al
. 
Regulation of intestinal barrier function by signal transducer and activator of transcription 5b
.
Gut
2009
;
58
:
49
58
.
29
Boehm
JS
,
Zhao
JJ
,
Yao
J
, et al
. 
Integrative genomic approaches identify IKBKE as a breast cancer oncogene
.
Cell
2007
;
129
:
1065
79
.
30
Ling
MT
,
Wang
X
,
Ouyang
XS
,
Xu
K
,
Tsao
SW
,
Wong
YC
. 
Id-1 expression promotes cell survival through activation of NF-κB signalling pathway in prostate cancer cells
.
Oncogene
2003
;
22
:
4498
508
.
31
Ryan
RF
,
Schultz
DC
,
Ayyanathan
K
, et al
. 
KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Kruppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing
.
Mol Cell Biol
1999
;
19
:
4366
78
.
32
Best
JD
,
Carey
N
. 
Epigenetic opportunities and challenges in cancer
.
Drug Discov Today
2010
;
15
:
65
70
.
33
Wiznerowicz
M
,
Jakobsson
J
,
Szulc
J
, et al
. 
The Kruppel-associated box repressor domain can trigger de novo promoter methylation during mouse early embryogenesis
.
J Biol Chem
2007
;
282
:
34535
41
.
34
Li
X
,
Ito
M
,
Zhou
F
, et al
. 
A maternal-zygotic effect gene, Zfp57, maintains both maternal and paternal imprints
.
Dev Cell
2008
;
15
:
547
57
.
35
Schultz
DC
,
Ayyanathan
K
,
Negorev
D
,
Maul
GG
,
Rauscher
FJ
 III
. 
SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins
.
Genes Dev
2002
;
16
:
919
32
.
36
Wynter
Coral
,
Alfonzo
Marcelo
,
Lippo de Becemberg
Itala
. 
Tumorigenesis is a disease of heterochromatin assembly and disruption of the epigenetic mark
. In:
Wong
David K.
, editor.
Tumorigenesis Research Advances
.
Nova Science publishers
; 
2007
, p.
201
226
.
37
Meyer
N
,
Penn
LZ
. 
Reflecting on 25 years with MYC
.
Nat Rev Cancer
2008
;
8
:
976
90
.
38
Dynek
JN
,
Chan
SM
,
Liu
J
,
Zha
J
,
Fairbrother
WJ
,
Vucic
D
. 
Microphthalmia-associated transcription factor is a critical transcriptional regulator of melanoma inhibitor of apoptosis in melanomas
.
Cancer Res
2008
;
68
:
3124
32
.
39
Steinman
RA
. 
Cell cycle regulators and hematopoiesis
.
Oncogene
2002
;
21
:
3403
13
.
40
Joo
A
,
Aburatani
H
,
Morii
E
,
Iba
H
,
Yoshimura
A
. 
STAT3 and MITF cooperatively induce cellular transformation through upregulation of c-fos expression
.
Oncogene
2004
;
23
:
726
34
.
41
Berlingieri
MT
,
Pierantoni
GM
,
Giancotti
V
,
Santoro
M
,
Fusco
A
. 
Thyroid cell transformation requires the expression of the HMGA1 proteins
.
Oncogene
2002
;
21
:
2971
80
.
42
Di
CF
,
Hillion
J
,
Hristov
A
, et al
. 
HMGA2 participates in transformation in human lung cancer
.
Mol Cancer Res
2008
;
6
:
743
50
.
43
Kim
H
,
Chung
H
,
Kim
HJ
, et al
. 
Id-1 regulates Bcl-2 and Bax expression through p53 and NF-κB in MCF-7 breast cancer cells
.
Breast Cancer Res Treat
2008
;
112
:
287
96
.
44
Hellerbrand
C
,
Bumes
E
,
Bataille
F
,
Dietmaier
W
,
Massoumi
R
,
Bosserhoff
AK
. 
Reduced expression of CYLD in human colon and hepatocellular carcinomas
.
Carcinogenesis
2007
;
28
:
21
7
.
45
Kashima
L
,
Toyota
M
,
Mita
H
, et al
. 
CHFR, a potential tumor suppressor, downregulates interleukin-8 through the inhibition of NF-κB
.
Oncogene
2009
;
28
:
2643
53
.
46
Fujioka
S
,
Niu
J
,
Schmidt
C
, et al
. 
NF-κB and AP-1 connection: mechanism of NF-κB-dependent regulation of AP-1 activity
.
Mol Cell Biol
2004
;
24
:
7806
19
.
47
Wang
Q
,
Sun
Z
,
Yang
HS
. 
Downregulation of tumor suppressor Pdcd4 promotes invasion and activates both β-catenin/Tcf and AP-1-dependent transcription in colon carcinoma cells
.
Oncogene
2008
;
27
:
1527
35
.
48
Wang
L
,
Zhang
Y
,
Li
H
,
Xu
Z
,
Santella
RM
,
Weinstein
IB
. 
Hint1 inhibits growth and activator protein-1 activity in human colon cancer cells
.
Cancer Res
2007
;
67
:
4700
8
.
49
Ki
SH
,
Choi
MJ
,
Lee
CH
,
Kim
SG
. 
Gα12 specifically regulates COX-2 induction by sphingosine 1-phosphate. Role for JNK-dependent ubiquitination and degradation of IκBα
.
J Biol Chem
2007
;
282
:
1938
47
.
50
Parnell
SC
,
Magenheimer
BS
,
Maser
RL
,
Zien
CA
,
Frischauf
AM
,
Calvet
JP
. 
Polycystin-1 activation of c-Jun N-terminal kinase and AP-1 is mediated by heterotrimeric G proteins
.
J Biol Chem
2002
;
277
:
19566
72
.

Supplementary data