The Stress-Responsive Gene GADD45G Is a Functional Tumor

Human Cancer Biology

The Stress-Responsive Gene GADD45G Is a Functional Tumor Suppressor, with Its Response to Environmental Stresses Frequently Disrupted Epigenetically in MultipleTumors Jianming Ying,1, 2 Gopesh Srivastava,3 Wen-Son Hsieh,1, 4 Zifen Gao,5 Paul Murray,6 Shuen-Kuei Liao,7 Richard Ambinder,4 and Qian Tao1,2,4

Abstract The CpG island of GADD45G was identified as a target sequence during the identification of hypermethylated genes using methylation-sensitive representational difference analysis combined with 5-aza-2V-deoxycytidine demethylation. Located at the commonly deleted region 9q22, GADD45G is a member of the DNA damage-inducible gene family. In response to stress shock, GADD45Ginhibits cell growth and induces apoptosis. Same as other GADD45 members, GADD45G is ubiquitously expressed in all normal adult and fetal tissues. However, its transcrip- tional silencing or down-regulation and promoter hypermethylation were frequently detected in tumor cell lines, including 11of 13 (85%) non-Hodgkin’s lymphoma, 3 of 6 (50%) Hodgkin’s lymphoma, 8 of 11 (73%) nasopharyngeal carcinoma, 2 of 4 (50%) cervical carcinoma, 5 of 17 (29%) esophageal carcinoma, and 2 of 5 (40%) lung carcinoma and other cell lines but not in any immortalized normal epithelial cell line, normal tissue, or peripheral blood mononuclear cells. The silencing of GADD45G could be reversed by 5-aza-2V-deoxycytidine or genetic double knockout of DNMT1 and DNMT3B, indicating a direct epigenetic mechanism. Aberrant methylation was further frequently detected in primary lymphomas although less frequently in primary carcinomas. Only one single sequence change in the coding region was detected in 1of 25 cell lines examined, indicating that genetic inactivation of GADD45G is very rare. GADD45G could be induced by heat shock or UV irradiation in unmethylated cell lines; however, this stress response was abolished when its promoter becomes hypermethylated. Ectopic expression of GADD45G strongly suppressed tumor cell growth and colony formation in silenced cell lines. These results show that GADD45G can act as a functional new-age tumor suppressor but being frequently inactivated epigenetically in multiple tumors.

Epigenetic inactivation of tumor suppressor genes (TSG) is transcription silencing (1). Increasing number of TSGs has frequently associated with tumor pathogenesis (1). The major been documented with epigenetic inactivation in tumors, such mechanism of this epigenetic inactivation is through hyper- as p16INK4a,hMLH1,VHL,BRCA1,andRASSF1A (2). methylation of promoter CpG islands, which leads to the Furthermore, promoter hypermethylation can be used as a binding of transcription repressors, compressed chromatin, and biological marker for the identification of novel candidate TSGs and tumor diagnosis (3). Various methylation-based strategies, including methylation-sensitive representational 1 2 Authors’Affiliations: Johns Hopkins Singapore, Singapore; Cancer Epigenetics difference analysis (MS-RDA; ref. 4), restriction landmark Laboratory, Department of Clinical Oncology, Sir YK Pao Cancer Center, Chinese genome scanning (5), arbitrarily primed PCR (6), and CpG University of Hong Kong; 3Department of Pathology, University of Hong Kong, Hong Kong; 4Sydney Kimmel Comprehensive Cancer Center, Johns Hopkins island microarray (7), have been developed and proven to be School of Medicine, Baltimore, Maryland; 5Department of Pathology, Peking useful for identifying hypermethylated sequences. MS-RDA University Health Science Center, Beijing, China; 6Cancer Research UK Institute has been successfully used to identify silenced genes in for Cancer Studies, University of Birmingham, Birmingham, United Kingdom; tumors, imprinted regions on mouse chromosome 2, and and 7Graduate Institute of Clinical Medical Sciences, Chang Gung University, Tai w a n , C h in a differentially methylated regions after viral infections Received 2/4/05; revised 4/4/05; accepted 4/13/05. (reviewed in ref. 4). Grant support: A*STAR, Singapore, Chinese University of Hong Kong, and GADD45 is a family of proteins involved in DNA damage Lymphoma Specialized Programs of Research Excellence grant P50CA96888 response and cell growth arrest. GADD45A was initially (R. Ambinder). identified as a gene rapidly induced by DNA-damaging agents, 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 such as methylmethane sulfonate, UV radiation, hydroxyurea, with 18 U.S.C. Section 1734 solely to indicate this fact. and ionizing radiation (8). It is a classic TP53-regulated Requests for reprints: Qian Tao, Department of Clinical Oncology, Sir YK Pao gene, and GADD45A-null mice exhibit a phenotype similar to Cancer Center, Prince of Wales Hospital, Chinese University of Hong Kong, TP53-null mice (9). GADD45B (MyD118) was firstly identi- Room 315, Hong Kong. Phone: 852-2632-1340; Fax: 852-2648-8842; E-mail: [email protected]. fied as a myeloid differentiation responsive gene, activated in F 2005 American Association for Cancer Research. M1 myeloid leukemia cells by interleukin-6 after induction of doi:10.1158/1078-0432.CCR-05-0267 terminal differentiation (10). GADD45G (GRP17/CR6) was

Clin Cancer Res 2005;11(18) September 15, 2005 6442 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Epigenetic Silencing of GADD45G in Multiple Tumors identified as an interleukin-2-induced immediate-early gene KYSE70, KYSE140, KYSE150, KYSE180, KYSE270, KYSE410, KYSE450, (11–13). These family proteins share 55% to 58% amino acid KYSE510, and KYSE520), colorectal (HCT116, HT-29, HCT15, SW48, and LoVo), cervical (HeLa, CaSki, C33A, and SiHa), lung (A549, homology and mediate the activation of the p38/c-Jun NH2- terminal kinase pathway via MTK1/MEKK4 in response to H1299, H2126, H1395, and H292), hepatocellular (HepG2, huH1, huH4, huH6, and huH7), gastric (KatoIII), and laryngeal carcinoma various environmental stresses (11). They can also inhibit (HEp-2), non-Hodgkin’s lymphoma (BJAB, CA46, Rael, Namalwa, Raji, cell proliferation at different stages, including G1-S and G2-M AG876, OCI-Ly1, Ly3, Ly7, Ly8, Ly13.2, Ly17, and Ly18), Hodgkin’s checkpoints, and induce cell apoptosis (11, 13–15). GADD45B lymphoma (L428, L540, L591, HD-LM2, HD-MY-Z, and KM-H2), and is down-regulated in hepatocellular carcinoma through methy- leukemia (HL-60, THP-1, and K562). Four immortalized normal lation (16), and GADD45G is also down-regulated in anaplastic epithelial cell lines (NP69, NE1, NE3, and RHEK-1; refs. 19, 20) with thyroid cancer and pituitary adenoma (17, 18). many features of normal epithelial cells were also used. HCT116 To identify aberrantly methylated genes, we used a novel DNMT1À/À (1KO), HCT116 DNMT3BÀ/À (3BKO), and HCT116 / / approach by combining MS-RDA with demethylation treatment DNMT1À ÀDNMT3BÀ À (DKO) cells (gifts of Bert Vogelstein, Johns in cell lines of nasopharyngeal carcinoma, a prevalent tumor in Hopkins, Baltimore, MD) were grown with either 0.4 mg/mL geneticin our locality of Southern China and Southeast Asia. We or 0.05 mg/mL hygromycin (21). DNA and RNA of primary carcinomas and lymphomas have been described (19, 22–25). Cell identified GADD45G as a target gene for epigenetic inactivation lines were treated with 5-aza-2V-deoxycytidine (Sigma, St. Louis, MO) as in nasopharyngeal carcinoma as well as multiple other types of previously (19). For the treatment combining 5-aza-2V-deoxycytidine carcinomas and lymphomas. We further showed that exoge- and trichostatin A (Cayman Chemical Co., Ann Arbor, MI), cells were nous expression of GADD45G strongly suppressed tumor cell treated with 5-aza-2V-deoxycytidine for 3 days and subsequently with growth and colony formation, indicating that GADD45G can trichostatin A (100 ng/mL) for 24 hours. act as a functional TSG. Stress treatments. Heat shock was done as previously (19), except for an incubation at 42jC for 1 hour with recovery at 37jC for Materials and Methods 2 hours. For UV treatment, medium was removed and the flask was turned upside down to face the light source in a UV cross-linker 500 Cell lines and primary tumors. In total, 75 cell lines of carcinomas (Amersham Biosciences, Piscataway, NJ). Cells were irradiated for a and lymphomas were used, including nasopharyngeal (C666-1, CNE-1, dose of 70 J/m2. After irradiation, fresh medium was added, and the CNE-2, HK1, NPC-BM1, HNE1, HNE2, HNE3, HONE-1, 5-8F, and cells were recovered at 37jC for 1 hour and then harvested. HKM1), breast (MCF-7, T47D, ZR-75-1, and MDA-MB231), esophageal Methylation-sensitive representational difference analysis. MS-RDA (EC1, EC18, EC109, HKESC-1, HKESC-2, HKESC-3, SLMT-1, KYSE30, was done as previously (26). We used the genomic DNA of a

Ta b l e 1. Sequences of primers used in this study

PCR Primers Size (bp) Annealing Cycles temperature (°C) RT-PCR GADD45A F: 5V-TGAGTGAGTGCAGAAAGCAG 18 1 55 35, 37 R: 5V-TTTGCTGAGCACTTCCTCCA GADD45B F: 5V-AACATGACGCTGGAAGAGCT 247 55 35, 37 R: 5V-AGAAGGACTGGATGAGCGTG GADD45G F: 5V-AACTAGCTGCTGGTTGATCG 178 55 35, 37 R1: 5V-CGTTCAAGACTTTGGCTGAC R2 (for direct sequencing and cloning): 539 55 32 5V-ACCACGTCGATCAGACCAAG GAPDH 33: 5V-GATGACCTTGCCCACAGCCT 304 60 25 55: 5V-ATCTCTGCCCCCTCTGCTGA MSP for the GADD45B promoter GADD45B m3: 5V-GAAAGTTCGGGTCGTTTCGC 134 60 40 m4: 5V-GAAAACCGAATAAATAACCGCG u3: 5V-TTTGAAAGTTTGGGTTGTTTTGT 139 58 40 u4: 5V-ACAAAAACCAAATAAATAACCACA MSP for the GADD45G promoter Region1(topstrand) m1:5V-ACGTGGTTTTTTGGTACGAGTC 160 64 40 m2: 5V-GCCCACCACCAACGAATACG u1: 5V-ATGTGGTTTTTTGGTATGAGTT 160 58 40 u2: 5V-ACCCACCACCAACAAATACA Region 2 (bottom strand) bm1: 5V-CGGAATTGTGTTTTGGTCGC 185 58 40 bm2: 5V-ACCAACCTATATAAAAACGCG bu1: 5V-TTTGGAATTGTGTTTTGGTTGT 188 58 40 bu2: 5V-CACCAACCTATATAAAAACACA BGS for the GADD45G promoter BGS1: 5V-GTAGATTTGAGGTATTGTTATTT 355 56 40 BGS2: 5V-CCTAAAACCCACCTAACTATA

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Fig. 1. Schematic structure of the GADD45G CpG island. A, locations of the hypermethylated fragment identified by MS-RDA, the core promoter, exons 1to 3, and part of exon 4 (filled rectangles). The transcription start site mapped by 5V-Rapid Amplification of cDNA Ends is indicated by a bent arrow. Six heat shock factors within the core promoter are also shown. Two MSP regions and the BGS region analyzed are indicated. B, expression profiling of GADD45A, GADD45B,andGADD45G in human normal adult and fetal tissues by semiquantitative RT-PCR, with GAPDH as a control. S.M., skeleton muscle; B.M., bone marrow. C, representative expression of GADD45A, GADD45B,andGADD45G by semiquantitative RT-PCR (top three) and methylation status of the GADD45B and GADD45G promoters by MSP (bottom four) in various tumor cell lines. M, methylated; U, unmethylated. D, analysis of GADD45G cDNA sequence variations by direct sequencing. +, with sequence variation; À,novariation.

nasopharyngeal carcinoma cell line CNE-1 (the Driver) and CNE-1 Analyses of tumor suppressor functions. The full-length cDNA of treated with 50 Amol/L 5-aza-2V-deoxycytidine (the Tester) and did two GADD45G was PCR cloned from human trachea RNA (BD Clontech, cycles of competitive hybridization. PCR products from the second Palo Alto, CA), sequence verified, and then subcloned into both hybridization were cloned into the pCR2.1-Topo vector (Invitrogen, pcDNA3.1(+) and pcDNA3.1(À) vectors (Invitrogen) to generate sense Carlsbad, CA), sequenced, and analyzed using the BLASTN algorithm plasmid pcDNA3.1(+)GADD45G and antisense plasmid pcDNA3.1(À) (National Center for Biotechnology Information). GADD45G. For colony formation assay using monolayer culture, cells 5V-Rapid Amplification of cDNA Ends. We determined the GADD45G (2 Â 105 per well) were plated in a 12-well plate and transfected with transcription start site using 5V-Rapid Amplification of cDNA Ends 2 Ag sense, antisense, or vector plasmid using LipofectAMINE 2000 version 2.0 (Invitrogen). Briefly, the first-strand cDNA was synthesized (Invitrogen). Cells were stripped off, plated in a six-well plate 48 from placenta RNA using primer GADD45GR2 (Table 1). Homopol- hours post-transfection, and selected for 2 to 3 weeks with 0.4 to 0.5 ymeric tails were then added to the 3V ends with terminal deoxy- mg/mL G418. Surviving colonies (z50 cells per colony) were counted nucleotidyl transferase. PCR was done using Abridged Anchor Primer after staining with Giemsa. For colony formation assay using soft agar and a second gene-specific primer GADD45GR1 (Table 1). The Rapid culture, cells were transfected as above and suspended in RPMI 1640 Amplification of cDNA Ends product was enriched by reamplifying containing 0.35% agar, 10% fetal bovine serum, and G418 and with the Abridged Universal Amplification Primer and GADD45GR1, then layered on RPMI 1640 containing 0.5% agar, 10% fetal bovine cloned, and sequenced. serum, and G418 in a six-well plate 48 hours post-transfection. Semiquantitative reverse transcription-PCR analysis. Reverse tran- Colonies were counted and photographed 16 days post-transfection. scription-PCR (RT-PCR) was done as previously (22, 27). Primers are Total RNA from transfected cells was extracted, treated with DNase I, shown in Table 1. and analyzed by RT-PCR to confirm the ectopic expression of Bisulfite treatment and promoter methylation analysis. Bisulfate GADD45G. All experiments were done in triplicate wells and repeated modification of DNA, methylation-specific PCR (MSP), and bisulfite thrice. genomic sequencing (BGS) were carried out as previously (27, 28). Cell proliferation assay. CA46 cells (1 Â 106 per well in 12-well GADD45B promoter and two regions of the GADD45G promoter were plate) were transfected with empty vector or pcDNA3.1(+)GADD45G analyzed by MSP (Table 1). MSP primers were tested previously for not and plated in a 12-well plate (7.5 Â 104 per well) 12 hours post- amplifying any unbisulfited DNA. transfection. Cells were counted at indicated time points using a

Clin Cancer Res 2005;11(18) September 15, 2005 6444 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Epigenetic Silencing of GADD45G in Multiple Tumors hemocytometer based on trypan blue exclusion. The experiment was non-Hodgkin’s lymphoma, 50% (3 of 6) Hodgkin’s lympho- done in triplicate wells and repeated thrice. ma, 73% (8 of 11) nasopharyngeal carcinoma, 29% (5 of 17) Screening for GADD45G mutations. Single-stranded cDNA was esophageal carcinoma, 50% (2 of 4) cervical carcinoma, and synthesized with SuperScript II reverse transcriptase (Invitrogen) using 40% (2 of 5) lung carcinoma and other tumor cell lines. All the total RNA from cell lines or cell lines treated with 5-aza-2V- cell lines with hypermethylation had either reduced or silenced deoxycytidine. RT-PCR was used to amplify the full-length coding sequence with Accume Pfx DNA polymerase (Invitrogen) and primers expression depending on the extent of methylation level. No GADD45GF and GADD45GR2. PCR products were electrophoresed, methylation was found in the four immortalized normal purified, and sequenced in both directions. epithelial cell lines, which had virtually normal phenotypes Statistical analysis. Statistical significance of the assays was analyzed and expressed this gene (Fig. 2A). In contrast, no methylation by t test. P < 0.05 was taken as statistically significant. of the GADD45B promoter was detected, which correlated with its broad expression in cell lines (Fig. 1C). Results and Discussion To further examine GADD45G methylation in more detail, we analyzed 12 cell lines, 2 normal tissues, and 5 tumors using Identification of the CpG island of GADD45G as a hyper- the high-resolution BGS analysis (Fig. 2B). We analyzed 33 methylated target. By combining MS-RDA with pharmacologic CpG sites spanning the core promoter and part of exon 1 in a demethylation, DNA fragments hypermethylated in CNE-1 but 355-bp region. The BGS results strongly correlated with the demethylated after 5-aza-2V-deoxycytidine treatment were iden- MSP analysis. Methylated CpG sites were not found or only tified, cloned, and analyzed by bioinformatics. All but 2 of the 31 unique fragments identified have typical CpG islands.8 Among them, a 296-bp fragment located at 9q22, a locus frequently deleted in multiple tumors (29), contains part of the Ta b l e 2 . Frequencies of GADD45G methylation in cell exon 3, exon 4, and intron 3 of the GADD45G gene (ref. 11; lines and primary tumors Fig. 1A). Frequent epigenetic silencing of GADD45G but not GADD45B Samples Promoter in various cell lines. We then examined the expression of methylation (%) GADD45G, together with other family members GADD45A Tu m o r c e l l l i n e s and GADD45B, in a series of cell lines to determine whether Non-Hodgkin’s lymphoma 11/13 (85) there are widespread epigenetic inactivation of these genes in Hodgkin’s lymphoma 3/6 (50) tumors. We analyzed 11 nasopharyngeal carcinoma and 56 Leukemia 1/3 (33) other cell lines using semiquantitative RT-PCR. We found that Nasopharyngeal carcinoma 8/11 (73) GADD45G expression was remarkably reduced or silenced in Cervical carcinoma 2/4 (50) 8 of 11 nasopharyngeal carcinoma, 11 of 13 non-Hodgkin’s Lung carcinoma 2/5 (40) lymphoma, 3 of 6 Hodgkin’s lymphoma, and 13 of 37 other Esophageal carcinoma 5/17 (29) cell lines. In contrast, the expression levels of GADD45A and Hepatocellular carcinoma 1/5 (20) GADD45B remained high in virtually all cell lines, with Colorectal carcinoma 1/5 (20) GADD45B the highest (Fig. 1C). Furthermore, along with Laryngeal carcinoma 1/1 GADD45A and GADD45B, GADD45G was readily detected in Breast carcinoma 0/4 all30normaladultandfetaltissues,includingnormal Gastric carcinoma 0/1 peripheral blood mononuclear cells (Fig. 1B). Primary lymphomas Before we could assess the epigenetic alterations of Endemic Burkitt’s lymphoma 7/8 (88) GADD45G, we determined the accurate location of the Diffuse large B-cell lymphoma 5/13 (38) promoter and the transcription start site using 5V-Rapid Follicular lymphoma 1/6 (16) Amplification of cDNA Ends. We obtained a strong PCR band Post-transplant lymphoma 4/13(33) of f260 nucleotides (data not shown). Sequence analysis of Nasal NK/Tcell lymphoma 5/8 (63) the product (AY845250) showed that the identified transcrip- Hodgkin’s lymphoma 10/29 (34) tion start site matched exactly the published cDNA 5Vsequence Other types of lymphoma 0/11 (NM_006705) in National Center for Biotechnology Informa- Primary carcinomas tion database. The GADD45G promoter is located within a Nasopharyngeal carcinoma 6/38 (16) typical CpG island (19), containing the core promoter, exons 1 Esophageal carcinoma 3/27 (11) to 3, and part of exon 4 (Fig. 1A). CpG islands are frequently Breast carcinoma 0/20 silenced by methylation in tumors as an alternative epigenetic Gastric carcinoma 2/19 (11) mechanism to inactivate TSG functions (1). We suspected that Hepatocellular carcinoma 0/6 the silencing of GADD45G might also be mediated through Immortalized normal epithelial cell lines epigenetic regulation. Therefore, the methylation status of two RHEK-1,NE1,NE3,NP69 0/4 regions in the GADD45G promoter was analyzed by MSP in a Normal tissues total of 75 cell lines (Fig. 1C; Table 2). The MSP results of Peripheral blood mononuclear cell 0/12 region 2 were identical to that of region 1 (data not shown). In Lymph node 0/3 total, GADD45G methylation was detected in 85% (11 of 13) Nasopharynx 0/10 Breast tissue 0/7 Esophageal epithelium 0/7 8 J. Ying and Q. Tao, in preparation.

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Fig. 2. Analyses of the methylation status of GADD45G in primary tumors. A, representative MSP analysis of GADD45G in primary tumors, normal tissues, and immortalized normal cell lines. PCR bands marked with Mindicate methylated promoter, whereas PCR bands marked with U indicate unmethylated promoter. B, high-resolution mapping of the methylation status of every CpG site in the GADD45G promoter by BGS in normal tissues, tumor cell lines, primary tumors, and cell lines treated with 5-aza-2V- deoxycytidine. A 355-bp region spanning the core promoter with 33 CpG sites was analyzed.Transcription start site is labeled as a curved arrow. Each CpG site is shown at the top row as an individual number (#). Percentage methylation was determined as percentage of methylated cytosines from 8 to 10 sequenced colonies. On the right side is the MSP results and GADD45G expression levels. M, methylated; U, unmethylated; (M), weakly methylated; (U), weakly unmethylated; NA, not available; LN, lymph node; GC, gastric carcinoma; BL, Burkitt’s lymphoma; DLBCL, diffuse large B-cell lymphoma; PTLD, post-transplant lymphoma.

scattered in cell lines expressing GADD45G. In contrast, GADD45G could also be activated to similar extent as the densely methylated CpG sites were detected in all silenced 5-aza-2V-deoxycytidine–treated in HCT116, which has a minor- cell lines (Fig. 2B). Our data indicate that epigenetic silencing ity of methylated alleles, by genetic biallelic disruption of both of GADD45G is involved in the pathogenesis of a few DNMT1 and DNMT3B (DKO) but not DNMT1 or DNMT3B tumors. alone (ref. 21; data not shown). In contrast, neither GADD45A Activation of GADD45G expression by pharmacologic and nor GADD45B expression was affected in the DKO cell genetic demethylation. To test whether methylation is directly line (data not shown). These results indicate that the responsible for silencing GADD45G, we treated five cell lines maintenance of GADD45G promoter methylation is mediated (Rael, Namalwa, Raji, L428, and EC109) with hypermethylated by DNMT1 and DNMT3B together, like other known typical and silenced promoter with the demethylating agent 5-aza-2V- TSGs (30).9 deoxycytidine. 5-Aza-2V-deoxycytidine restored GADD45G GADD45G methylation is tumor specific. We further inves- expression in all of them although with different efficiency tigated the presence of GADD45G methylation in a large (Fig. 3A). No significant increase of either GADD45A or collection of primary tumors, including various lymphomas GADD45B expression was detected (data not shown). Con- and carcinomas and normal tissues (Fig. 2A and B; Table 2). comitantly, after 5-aza-2V-deoxycytidine treatment, unmethy- GADD45G methylation was detected in multiple tumor types lated GADD45G alleles were increased as determined by both with different frequencies, more frequently in lymphomas than MSP and BGS (Figs. 2B and 3A). The Rael cell line showed carcinomas and in carcinoma cell lines than primary carcinomas marginal restoration of GADD45G after 5-aza-2V-deoxycytidine (73% versus 16% in nasopharyngeal carcinoma and 29% versus treatment. However, further treatment with 5-aza-2V-deoxycyti- 11% in esophageal carcinoma; Table 2), indicating that some cell dine combined with histone deacetylase inhibitor, trichostatin A, resulted in synergistic activation of GADD45G to greater levels (data not shown), suggesting that histone deacetylation is also involved in repressing GADD45G in this cell line. 9 G.H. Qiu and Q.Tao, submitted for publication.

Clin Cancer Res 2005;11(18) September 15, 2005 6446 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Epigenetic Silencing of GADD45G in Multiple Tumors lines may have acquired GADD45G methylation during the whether its stress response would be affected by promoter establishment or maintenance process. Similar phenomenon methylation. Stress treatments (heat shock and UV irradiation) has been reported for some TSGs in other tumors (31, 32). None of normal and tumor cell lines with an unmethylated or only of the 39 normal tissues (12 peripheral blood mononuclear cells, partially methylated promoter resulted in increased GADD45G 3 lymph nodes, 10 nasopharynx, 7 normal breast tissues, and expression. However, this response was decreased or abolished 7 normal esophageal epithelia) had methylation as assessed by in cell lines with methylated promoter and was inversely either BGS or MSP. Therefore, hypermethylation of the correlated with the promoter methylation levels (Fig. 3C). GADD45G promoter is tumor specific. Under our conditions of stress treatment (heat shock at Genetic inactivation of GADD45G is very rare. Because TSGs 42jC for 1 hour with 2-hour recovery and 70 J/m2 UVC can also be inactivated genetically by various mutations, treatment with 1-hour recovery), we only observed marginal including deletions, and GADD45G is located in a frequently up-regulation of GADD45A but not GADD45B RNA. It has deleted locus, we tested this possibility for GADD45G and did been shown that different GADD45 genes respond to stress not detect its deletion in any cell line. We further used direct stimuli differently (i.e., GADD45A is inducible by p53, whereas sequencing to screen for any possible point mutation in 25 cell GADD45B and GADD45G are not, and interleukin-12 can lines, including expressing cell lines and silenced cell lines induce GADD45B but not GADD45G; ref. 15). Interestingly, treated with 5-aza-2V-deoxycytidine. Only a single sequence in treated normal (NE3) and some tumor cell lines (EC109 change G344A (G112E) in exon 3 was identified in one cell line and Rael), two new isoforms of GADD45B were induced after AG876 (Figs. 1D and 3B), in which the promoter was also heat shock (Fig. 3C). These isoforms have extended exon 1 of methylated. This sequence substitution might be a rare different lengths, resulting in the translation of a shorter mutation or a polymorphism. Therefore, the disruption of isoform (15 amino acids less) from an alternate ATG code GADD45G functions in tumor cell lines is solely through more downstream (accession nos. AY615270 and AY615271). epigenetic rather than genetic mechanism. The functions of these new GADD45B isoforms need further Promoter hypermethylation disrupts the stress response of study. GADD45G. Because GADD45 family proteins are involved GADD45G is a functional tumor suppressor. GADD45G is in cellular responses to environmental stresses, we inspected associated with proliferating cell nuclear antigen and the the GADD45G promoter for potential stress-responsive ele- cyclin-dependent kinase inhibitor p21WAF1/CIP1 and is involved ments. Multiple heat shock factor binding sites (6 in the core in the negative control of cell growth (13, 15). GADD45G is promoter, with a total of 26 sites in a region from À1,500 also associated with MTK1/MEKK4, which in turn activates the to +150) are present in the promoter (Fig. 1A), indicating that p38/c-Jun NH2-terminal kinase pathway leading to apoptosis, it is likely stress responsive. It has been reported that this in response to environmental stresses (11). Transient transfec- promoter is inducible by g-ray, H2O2, and UV irradiation (11). tion of GADD45 members could induce apoptosis in tumor We further tested whether it is responsive to heat shock and cells (11, 13, 15). The frequent epigenetic silencing of

Fig. 3. A, pharmacologic demethylation and induction of GADD45G in methylated and silenced tumor cell lines by 5-aza-2V- deoxycytidine. +, 5-aza-2V-deoxycytidine treated; À, untreated. B, only a single sequence change G443A (G112E) in the coding region of GADD45G was identified in 1 cell line, AG876, of 25 tumor cell lines analyzed. C, up-regulation of GADD45G, in response to stress treatment, is abolished in tumor cell lines with methylated promoter. Subconfluent normal (NE3) and tumor cell lines (CNE-2, HK1, EC109, and Rael) were exposed to 42jC heat shock or UV irradiation and then analyzed for GADD45A, GADD45B,andGADD45G expression. GADD45G methylation status in each cell line is shown (bottom), with percentage of methylated alleles indicated. The two novel heat shock ^ induced isoforms of GADD45B are indicated by asterisks. +, stress treated; À, mock control.

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Fig. 4. Tumor suppressor function analyses of GADD45G in multiple tumor cell lines. A, representative inhibition of colony formation in monolayer culture by GADD45G. HepG2 cells were transfected with pcDNA3.1(+)GADD45G, antisense plasmid (AS), or control vector and selected with G418 for 3 weeks. Quantitative analyses of colony numbers in several cell lines are shown on the right .The numbers of G418-resistant colonies in each control vector-transfected cell line were set to 100%. Columns, mean of at least three separate experiments; bars, SE. B, representative inhibition of colony formation by GADD45G in soft agar culture. HepG2 cells were transfected with pcDNA3.1(+)GADD45G or empty vector and selected in soft agar with G418 for16 days. Quantitative analyses of colony numbers after transfection and G418 selection in CNE-1and HepG2 are shown on the right. In each case, the numbers of G418-resistant colonies in vector-transfected cell lines were set to 100%. Columns, mean of at least three separate experiments; bars, SE. C, growth curves of CA46 cells after transfection with pcDNA3.1(+)GADD45G or control vector. At each indicated time point after transfection, cell numbers were counted and plotted. Points, mean of triplicate experiments; bars, SE. ***, P < 0.001.

GADD45G in tumors and cell lines but not normal tissues suppressed tumor cell growth and colony formation of other suggests that it might be a functional tumor suppressor. To tumor cell lines, including nasopharyngeal, hepatocellular, test this hypothesis, we cloned the full-length GADD45G cDNA esophageal, and laryngeal carcinoma and lymphoma cell lines, into expression vectors and transfected it into several carci- further indicating that GADD45G can function as a TSG in noma and lymphoma cell lines, which had either complete multiple tumors. methylation and silencing (HepG2, EC109, HEp-2, and CA46) In summary, we identified the GADD45G CpG island as a or only weak expression with both methylated and unmethy- tumor-specific, hypermethylated target sequence. We also lated alleles (CNE-1 and HK1). The colony formation found that GADD45G expression is frequently reduced or efficiencies of each transfected cell line were evaluated by silenced in multiple tumors, including lymphomas and monolayer and soft-agar culture with G418 selection. Ectopic carcinomas, like other class II TSGs (33). This silencing is expression of GADD45G dramatically reduced the colony due to the hypermethylation of its promoter, which further formation efficiencies of these cell lines in both monolayer impairs its response to environmental stresses, but genetic cultures (down to 9-20% of controls in methylated cell lines inactivation of GADD45G is rare. We further showed that and 19-41% in hemimethylated cell lines; Fig. 4A) and soft- GADD45G can act as a functional TSG in multiple tumor agar assays (down to 8% of controls in CNE-1 and HepG2; cells. During the preparation of this article, Bahar et al. also Fig. 4B), suggesting that GADD45G can function as a bona fide reported the epigenetic down-regulation of GADD45G in TSG. Interestingly, HK1 has the relatively highest endoge- pituitary adenoma (34). This frequent inactivation of nous expression and lowest methylation level. The inhibition GADD45G in various tumors is consistent with its proposed of colony formation by ectopic GADD45G expression in HK1 role as a negative regulator of cell proliferation (13, 15) and also was also the least effective. Furthermore, cell proliferation shows the importance of GADD45G in preventing the assay was done for CA46 (Fig. 4C). Cells transfected with development of multiple tumors. As promoter hypermethyla- pcDNA3.1(+)GADD45G grew significantly slower than the tion is pharmacologically reversible using demethylating agents, mock-transfected cells (P < 0.001), indicating that GADD45G and GADD45G is rarely mutated in tumors, it is therefore a dramatically inhibits not only tumor cell colony formation but likely target for new epigenetic anticancer therapeutics. also their proliferation. Previously, it has been shown that GenBank accession numbers. The sequences of the 5V-Rapid ectopic GADD45G expression in lung carcinoma, pituitary Amplification of cDNA Ends product of GADD45G (accession tumor, and anaplastic thyroid carcinoma–derived cell lines no. AY845250) and heat shock–induced GADD45B isoforms resulted in a substantial inhibition of tumor cell growth (accession nos. AY615270 and AY615271) have been deposited (17, 18). As shown in this study, GADD45G also strongly to Genbank.

Clin Cancer Res 2005;11(18) September 15, 2005 6448 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Epigenetic Silencing of GADD45G in Multiple Tumors

Note Added in Proof Acknowledgments

During the final preparation of this article, Zerbini et al. reported that nuclear We thank Drs. Bert Vogelstein, Soh-ha Chan, Riccardo Dalla-Favera, Meehard factor-nB ^ mediated repression of GADD45A and GADD45G is essential for can- Herlyn, Dolly Huang, Katai Yao, Ya Cao, Thomas Putti, Guiyuan Li, Yixin Zeng, cercellsurvival(ZerbiniLF,etal.ProcNatlAcadSciUSA2004;101:13618^23), Sen-TienTsai, Sai WahTsao, Johng S. Rhim, Malini Olivo, Goh Boon Cher, and Lee thus pointing out another mechanism to inactive GADD45G functions in tumors. Soo Chin for some cell lines and samples.

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www.aacrjournals.org 6449 Clin Cancer Res 2005;11(18) September 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. The Stress-Responsive Gene GADD45G Is a Functional Tumor Suppressor, with Its Response to Environmental Stresses Frequently Disrupted Epigenetically in Multiple Tumors

Jianming Ying, Gopesh Srivastava, Wen-Son Hsieh, et al.

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