The Biology Behind

GADD45 Deregulation in : Frequently Methylated Tumor Suppressors and Potential TherapeuticTargets 55Commentary onYing et al., p. 6442 Luiz F. Zerbini and Towia A. Libermann

In this issue of Clinical Cancer Research, Ying et al. (1) have (NF-nB) in human cancer. We reported that the transcription investigated the hypothesis that GADD45c, a member of the factor NF-nB, which is deregulated and activated in many tumors family originally identified as a growth arrest– and and a critical regulator of survival, mediates repres- DNA damage–inducible , could serve as a functional sion of GADD45a and GADD45c , and NF-nB- , and moreover, as a therapeutic target. mediated repression of these two GADD45 is essential for DNA is an important regulator of gene escape from programmed cell death (3, 4). Together, these transcription, and its role in carcinogenesis has been a topic findings show the importance of GADD45g for the development of considerable interest in the last few years. Epigenetic and progression of various cancer types pointing out different alterations, such as abnormal DNA methylation patterns, are mechanisms to inactivate GADD45g functions in cancer. associated with many human tumor types and are now recognized as one of the most common molecular alterations ,DNA Methylation,Prognostic,and in human cancer and a cause of oncogenesis (Fig. 1; ref. 2). Cancer Among epigenetic modifications, hypermethylation, which represses transcription of the regions of tumor Methylation of cytosines is an evolutionary conserved suppressor genes leading to gene silencing, has been most mechanism of gene regulation that plays a critical role during extensively studied. New techniques have been developed to normal development and genetic defects associated with perform -wide screening for alterations in DNA methylation result in multiple developmental diseases. Because methylation patterns not only to identify tumor suppressor cancer cells many times express various features of undifferen- genes but also to find patterns that can be used in diagnosis and tiated or dedifferentiated cells, it is not surprising that prognosis and may influence future treatment regimens (2). methylation of genes is frequently observed in cancer, although The studies of Ying et al. provide strong evidence that a loss of global methylation is observed concomitant with GADD45g is frequently epigenetically inactivated in various methylationofspecificCpGislands.Avastamountof types of and tumor cell lines and remarkable findings information has been gained about aberrant methylation emerge: first, the authors identified CpG islands in the patterns in human cancers. Tumor-specific methylation GADD45c gene as tumor-specific, rarely mutated, but com- changes in different genes have been identified and the monly hypermethylated target sequences; second, GADD45g potential clinical applications of these data had an effect on expression as well as stress-induced activation is reduced or cancer diagnosis, prognosis, and therapeutics. Consequently, silenced due to its promoter hypermethylation. Using deme- several DNA methylase inhibitors such as 5-azacytidine, thylating agents, they showed that methylation is directly decitabine, and MG98 are currently in clinical trials for various responsible for silencing of the GADD45g promoter, a process types of cancer. For example, a phase III clinical trial of 5- that is pharmacologically reversible. Furthermore, ectopic over- azacitidine in myelodysplastic syndrome patients resulted in expression of GADD45g results in reduced tumor growth and significantly improved response rate, quality of life, and colony formation supporting the notion that GADD45c is a enhanced overall survival (5). tumor suppressor gene. What is unexpected and thus far unexplained is the tumor- These data confirm and extend previous data about reduced specific methylation of specific subsets of genes, in particular, expression of GADD45g in several other types of cancer. tumor suppressor genes such as GADD45c in the current study. Furthermore, we (3) recently focused on a similar question The precise mechanisms are not known, although some increase n with regard to GADD45g regulation by nuclear factor- B in DNMT1 (2, 6) expression has been observed in some cancers. Epigenetics can be described as a stable alteration in gene expression potential that takes place during development and Authors’ Affiliation: Beth Israel Deaconess Medical Center Genomics Center cell proliferation, without any change in gene sequence. DNA and Dana-Farber/Harvard Cancer Center Cancer Proteomics Core, Beth Israel methylation is one of the most common epigenetic events in the Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts mammalian genome. Although heritable, this process is Received 7/7/05; accepted 7/7/05. Grant support: NIHgrants 1RO1CA85467 and P50 CA105009. reversible, making it an interesting therapeutic target. Because Requests for reprints: Towia A. Libermann, Beth Israel Deaconess Medical cancer is a result of aberrant gene expression, it is no surprise Center Genomics Center and New England Baptist Bone and Joint Institute, Beth that methylation plays a crucial role. Israel Deaconess Medical Center and Harvard Medical School, Harvard Institutes DNA methylation is a chemical modification, resulting in the of Medicine, 4 Blackfan Circle, Boston, MA 02115. Phone: 617-667-3393; Fax: 617-975-5299; E-mail: [email protected]. addition of a methyl group at the carbon 5 position of the F 2005 American Association for Cancer Research. cytosine ring. Although most cytosine methylation occurs in doi:10.1158/1078-0432.CCR-05-1475 the sequence context 5VCpG3V, it can also engage CpA and CpT

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Fig. 1. A model for the molecular mechanisms by which DNA methylation may result in transcriptional silencing of tumor suppressor genes in cancer. Most CpGs inthe genome are methylated in the normal cellular state, whereas CpGs in CpG islands are normally unmethylated, regardless of the transcription state of the gene. During tumorigenesis, CpG islands in the promoter regions of tumor suppressor genes become hypermethylated, which spread through the promoter and silence the gene. dinucleotides (6). Clusters of CpG sites are occasionally present colorectal, , and (9). Therefore, the findings in the genome, designated as CpG islands, ranging from 0.5 to by Ying et al. (1) about the frequent methylation of the 5 kb and occurring on average every 100 kb, with distinctive GADD45c gene in a variety of cancers is an important first properties. These regions are unmethylated, GC rich (60-70%), step to determine the relevance of this methylation event for with no suppression of the frequency of the dinucleotide CpG cancer prognosis. (7). Approximately half of all the genes in humans have CpG islands (8), and many of them are present in promoter regions GADD45 Family critical for gene regulation, although CpG islands are also located at various positions throughout genes (9), such as in The GADD45 gene family encodes three structurally highly exons and introns, or further downstream (10). Cancer-specific related growth arrest– and DNA damage–inducible , gene primarily affect CpG islands in promoter GADD45a, GADD45h, and GADD45g (Fig. 2; ref. 13). They regions of tumor suppressor genes and other, presumably contain only one discernible homology domain to other critical genes, leading to silencing of multiple genes in any proteins (i.e., to ribosomal proteins). Although the role of given cancer cell. These methylations are gene and cancer GADD45 in growth arrest and is not entirely clear, specific, resulting in cancer-specific methylation patterns that several, sometimes conflicting aspects of GADD45 function not only correlate with the cancer type but may also correlate have been revealed. GADD45 proteins are primarily nuclear with stage, outcome, and response to therapy. Therefore, proteins that interact with various –related proteins identification of cancer-specific gene methylation such as for such as the G2 cell cycle–specific , Cdc2/Cdk1, prolifer- GADD45c is an important field of cancer research and has ating cell nuclear antigen, and the cell cycle kinase inhibitor resulted in the identification of novel tumor suppressor genes (waf1) (13). and diagnostic and prognostic methylation patterns. GADD45 proteins play a role in the G2-M checkpoint in The procedures and techniques to investigate the epigenetic response to DNA damage. GADD45a activates -dependent process are diverse, complex, and often conflicting to some G2-M arrest and inhibits cdc2 kinase that can be blocked by degree. Different methodologies are employed to detect overexpression of providing a link between p53- methylation at CpG sites in specific target areas of these dependent cell cycle checkpoint and DNA repair (13). genes. Each approach may use one or the other of two Interestingly, unlike GADD45a, neither GADD45h nor principal techniques. Recent advances in the techniques for GADD45g seem to inhibit Cdc2 kinase or induce G2-M arrest. detection of methylation include powerful tools such as GADD45a-null mice have a phenotype similar to p53-deficient sodium bisulfite conversion, cDNA microarray, restriction mice, including genomic instability and increased radiation genomic scanning, and CpG island microarray (11). The carcinogenesis, suggesting that GADD45a and p53 are involved sensitivity and specificity of DNA methylation markers in in similar cellular pathways (13). Indeed GADD45a but not cancer diagnosis depends on several factors, including the type GADD45h and GADD45g is a direct downstream target of p53. of cancer and the gene to be studied and the technique The role of GADD45 genes in cell cycle arrest has been involved (12). well established, whereas the role GADD45 genes play in Because DNA methylation is closely related to the develop- apoptosis remains unclear. Overexpression of GADD45 in ment of cancer, it would be interesting to know whether its normal human fibroblasts causes G2-M arrest but not presence or absence affects the prognosis as well. This would apoptosis. Nevertheless, genotoxic stress or Brca1-induced help in modifying initial treatment options, monitoring apoptosis seem to involve GADD45a-mediated activation of patient response to therapy, and predicting survival. Recently, the stress-responsive c-jun NH2-terminal kinase and/or p38 several studies have shown methylation of certain genes to be mitogen-activated kinase (13, 14). NF-nB-induced cell closely related to the prognosis of cancer including for survival has been proposed to be mediated by induction of

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GADD45h expression and down-regulation of c-jun-NH2- GADD45 gene has been observed thus far. GADD45a kinase activity (15, 16). A yeast two-hybrid screen revealed expression is also reduced in ovarian cancer, although the direct interaction of all three GADD45 family members with precise mechanism has not been elucidated. The GADD45a the upstream kinase MTK1/mitogen-activated protein kinase promoter is methylated in the majority of breast cancers kinase kinase 4 that activates both p38 and c-jun-NH2-kinase resulting in reduced expression when compared with normal in response to environmental stresses. All three GADD45 breast (18). It is thus interesting to notice that in members activate MTK1 kinase activity leading to p38/c-jun- the current publication no differences in GADD45g methyl- NH2-kinase activation and apoptosis. Recent findings estab- ation were observed in breast cancer cell lines suggesting lished the GADD45 family as a critical mediator of apoptosis specific methylation of the GADD45a gene in breast cancer. in cancer cells (3). NF-nB-mediated cell survival in cancer In pituitary adenomas, silencing of the GADD45c gene in cells is absolutely dependent on two GADD45 family 67% of patients is primarily associated with methylation of members, GADD45a and GADD45g (Fig. 3). NF-nB-mediated the GADD45c gene and reversal of this epigenetic change repression of GADD45a and g is sufficient for cancer cell results in re-expression (19). GADD45g is also down- survival and repression of the GADD45a and GADD45c genes regulated in anaplastic thyroid cancer and in 65% of is for a large part the result of NF-nB-mediated up-regulation hepatocellular carcinomas due to hypermethylation of the of c-myc, another oncogene frequently overexpressed in a GADD45g promoter. Interestingly, the GADD45b gene is variety of cancers. Whether NF-nB-mediated repression of methylated and silenced in as well, GADD45a and GADD45c gene expression in cancer may indicating a strong linkage between at least two GADD45 involve also changes in methylation is not known. Inhibition genes and cancer. Our observation that activated NF-nB of NF-nB in cancer cells results in GADD45a- and GADD45g- leads to repression of GADD45a and GADD45g expression dependent induction of apoptosis and inhibition of tumor in various types of cancer together with the frequent growth (3) correlating well with the findings of Ying et al. (1) constitutive activation of NF-nB in cancers, furthermore, that ectopic expression of GADD45g in cancer cells inhibits suggests that there are at least two mechanisms whereby tumor growth. GADD45 genes become repressed in cancer. Thus, repression GADD45a and GADD45g induction has been shown to be of GADD45 gene expression in various types of cancer via methylation or NF-nB activation seems a critical step in essential for c-jun-NH2-kinase activation and apoptosis in cancer cells, highlighting the notion that GADD45a and oncogenesis and highlights the role of the GADD45 gene GADD45g repression via methylation or other mechanisms family in regulating DNA damage, cell cycle, and cell n plays an unambiguous and universal role in the ability of survival. Because activation of NF- B is a critical step for certain types of cancer to escape from programmed cell death many cells to escape programmed cell death and this is (3). Changes in GADD45a and GADD45g expression are dependent on GADD45a and GADD45g repression, methyl- likely to play a role in the proapoptotic functions of various ation of the GADD45c gene as reported by Ying et al. may anticancer drugs as well. Whether these drugs act via result in a similar phenotype (i.e., helping cancer cells to inhibition of NF-nB or changes in methylation is not clear. survive and become resistant to stress and DNA damage). Although members of the GADD45 family seem infre- The current publication by Ying et al. (1) analyzes the quently mutated in cancer as far as currently known, reduced methylation status of two regions in the GADD45g promoter expression of the three GADD45 family members due to in a total of 75 cell lines as well as primary tissues and promoter methylation has been frequently observed in tumors. They show that promoter hypermethylation is several types of human cancer. In resectable invasive frequently detected in tumors cell lines, including 85% of pancreatic ductal carcinomas, GADD45a is frequently mutat- non-Hodgkin, 50% of Hodgkin lymphoma, 73% of naso- ed, and mutation combined with the p53 status correlates pharyngeal, 50% of cervical, 29% of esophageal, and 40% of with survival of invasive pancreatic ductal carcinoma patients lung carcinoma but not in immortalized normal epithelial (17). This is the only type of cancer where mutation of a cell lines, normal tissues, or peripheral blood mononuclear

Fig. 2. Alignmentof theaminoacidsequencesof thehumanGADD45 genes.

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Fig. 3. Methylation and NF-nB-mediated cell survival in cancer cells by inactivating GADD45a and GADD45g expression. Methylation and NF-nB-mediated repression of GADD45a and GADD45g may be sufficient for cancer cell survival leading to mitogen-activated protein kinase kinase kinase 4 (MEKK4)/MTK1repression and escape from programmed cell death. cells. To gain more insight into the GADD45g methylation, they must be confirmed. Analysis of gene expression levels in a range also did high-resolution bisulfite genomic sequencing. They of tissues is sometimes helpful to evaluate its consequential found that densely methylated CpG sites were detected in all expression level (24) Thereafter, the connecting role of silenced cell lines, indicating that epigenetic silencing of methylation in gene silencing is analyzed by treating cells with GADD45g could be involved in the pathogenesis of tumors. a demethylating agent and observing the resulting up-regulation of gene expression. Many genes have been shown to be silenced in various cancers through MS-RDA32 (24) or MCA-RDA (24) Importance of Methylation for Gene Expression combined with 5-azadeoxycytidine treatment (25, 26). The A hypermethylation of CpG island in cancer is strongly ultimate central step is functional analysis of the candidate associated with transcriptional silencing of distinct subsets of tumor suppressor genes. The presence of mutations in these genes (2). Increased methylation in the promoter region of a genes in some tumor samples, if even only a few, is strong gene leads to reduced expression, whereas methylation in the evidence for a tumor suppressor gene. transcribed region has variable effects on gene expression In conclusion, the identification of CpG hypermethylation (20). Several mechanisms have been proposed to explain the events as tumor biomarkers can provide useful information for transcriptional repression by DNA methylation. The first early diagnosis, cancer risk assessment, and prognosis (2). mechanism involves direct interference with the binding of Methylated CpG islands are associated with virtually every type specific transcription factors to their recognition sites in their of tumor (2), may be grouped into tumor-specific marker panels respective promoters (21). The second mode of repression (2), and can be detected with a high degree of sensitivity (2). The involves a direct binding of specific transcriptional repressors mapping of hundreds to thousands of CpG island–associated to methylated DNA (22). The reduction in affinity of certain tumor suppressors and the discovery of the repressive properties factors, however, is often insufficient to account for the in- of methylation have had a great effect on disease prevention and activity of promoters in vivo (2). treatment (2). Differences in methylation patterns among Methylation of non-core regions within a promoter CpG tumors seem associated with patient outcome or other clinical island does not block gene transcription and is frequently responses and can be used as markers to classify tumors in observed in cancer and even in normal cells (23). When a core association with more effective treatments for patients. Most region is methylated in a cell, it is almost always associated importantly, the results provided by Ying et al. (1) establish the with that of non-core regions (23, 24). Genome-wide screening GADD45c gene as a major target for methylation in various of methylated regions in tumor DNA has led to the types of cancer and further support the notion that modulation identification of several tumor suppressor genes (25). of GADD45 gene family expression and function may be critical Numerous steps are necessary to identify methylation steps in cancer development and progression. These results also associated with a tumor suppressor gene. Initially, methylated imply that therapeutic strategies targeting the epigenetic core regions associated with specific gene silencing must be repression of the tumor suppressor GADD45g have the identified. Subsequently, expression of a gene in normal cells potential to result in strong anticancer effects.

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Luiz F. Zerbini and Towia A. Libermann

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