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Recurrent alterations in promoter methylation of tumor suppressor genes (TSGs) and LINE1 (L1RE1) repeat elements were previously reported in pheochromocytoma and abdominal paraganglioma. This study was undertaken to explore CpG methylation abnormalities in an extended tumor panel and assess possible relationships between metastatic disease and mutation status. CpG methylation was quantified by bisulfite pyrosequencing for selected TSG promoters and LINE1 repeats. Methylation indices above normal reference were observed for DCR2 (TNFRSF10D), CDH1, P16 (CDKN2A), RARB, and RASSF1A. Z-scores for overall TSG, and individual TSG methylation levels, but not LINE1, were significantly correlated with metastatic disease, paraganglioma, disease predisposition, or outcome. Most strikingly, P16 hypermethylation was strongly associated with SDHB mutation as opposed to RET/MEN2, VHL/VHL, or NF1-related disease. Parallel analyses of constitutional, tumor, and metastasis DNA implicate an order of events where constitutional SDHB mutations are followed by TSG hypermethylation and 1p loss in primary tumors, later transferred to metastatic tissue. In the combined material, P16 hypermethylation was prevalent in SDHB-mutated samples and was associated with short disease-related survival. The findings verify the previously reported importance of P16 and other TSG hypermethylation in an independent tumor series. Furthermore, a constitutional SDHB mutation is proposed to predispose for an epigenetic tumor phenotype occurring before the emanation of clinically recognized malignancy.

Background

Epigenetic inactivation of tumor suppressor genes (TSG) by promoter CpG island hypermethylation is a hallmark of cancer. To assay its extent in human lymphoma, methylation of 24 TSG was analyzed in lymphoma-derived cell lines as well as in patient samples.

Methods

We screened for TSG methylation using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in 40 lymphoma-derived cell lines representing anaplastic large cell lymphoma, Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma and mantle cell lymphoma (MCL) as well as in 50 primary lymphoma samples. The methylation status of differentially methylated CD44 was verified by methylation-specific PCR and bisulfite sequencing. Gene expression of CD44 and its reactivation by DNA demethylation was determined by quantitative real-time PCR and on the protein level by flow cytometry. Induction of apoptosis by anti-CD44 antibody was analyzed by annexin-V/PI staining and flow cytometry.

Results

On average 8 ± 2.8 of 24 TSG were methylated per lymphoma cell line and 2.4 ± 2 of 24 TSG in primary lymphomas, whereas 0/24 TSG were methylated in tonsils and blood mononuclear cells from healthy donors. Notably, we identified that CD44 was hypermethylated and transcriptionally silenced in all BL and most FL and DLBCL cell lines, but was usually unmethylated and expressed in MCL cell lines. Concordant results were obtained from primary lymphoma material: CD44 was not methylated in MCL patients (0/11) whereas CD44 was frequently hypermethylated in BL patients (18/29). In cell lines with CD44 hypermethylation, expression was re-inducible at mRNA and protein levels by treatment with the DNA demethylating agent 5-Aza-2'-deoxycytidine, confirming epigenetic regulation of CD44. CD44 ligation assays with a monoclonal anti-CD44 antibody showed that CD44 can mediate apoptosis in CD44+ lymphoma cells. CD44 hypermethylated, CD44- lymphoma cell lines were consistently resistant towards anti-CD44 induced apoptosis.

Conclusion

Our data show that CD44 is epigenetically regulated in lymphoma and undergoes de novo methylation in distinct lymphoma subtypes like BL. Thus CD44 may be a promising new epigenetic marker for diagnosis and a potential therapeutic target for the treatment of specific lymphoma subtypes.

Aberrant methylation of CpG islands in promoter regions of tumor suppressor genes (TSG) has been demonstrated in epithelial origin tumors. However, the methylation profiling of tumor-related gene promoter regions in cutaneous melanoma tumors has not been reported. Seven known or candidate TSGs that are frequently hyper-methylated in carcinomas were assessed by methylation-specific polymerase chain reaction (MSP) in 15 melanoma cell lines and 130 cutaneous melanoma tumors. Four TSGs were frequently hypermethylated in 86 metastatic tumor specimens: retinoic acid receptor-β2 (RAR-β2) (70%), RAS association domain family protein 1A (RASSF1A) (57%), and O6-methylguanine DNA methy-latransferase (MGMT) (34%), and death-associated protein kinase (DAPK) (19%). Hypermethylation of MGMT, RASSF1A, and DAPK was significantly lower in primary melanomas (n = 20) compared to metastatic melanomas. However, hypermethylation of RAR-β2 was 70% in both primary and metastatic melanomas. Cell lines had hypermethylation profiles similar to those of metastatic melanomas. The analysis of these four markers of metastatic tumors demonstrated that 97% had ≥1 gene(s) and 59% had ≥2 genes hypermethylated. The methylation of genes was verified by bisulfite sequencing. The mRNA transcripts could be re-expressed in melanoma cell lines having hypermethylated genes following treatment with 5′-aza 2′-deoxycytidine (5Aza-dC). Analysis of melanoma patients’ plasma (preoperative blood; n = 31) demonstrated circulating hypermethylated MGMT, RAR-β2, and RASSF1A DNA for at least one of the markers in 29% of the patients. Our findings indicate that the incidence of TSG hypermethylation increases during tumor progression. Methylation of TSG may play a significant role in cutaneous melanoma progression.

In order to identify novel candidate tumor suppressor genes (TSGs) implicated in renal cell carcinoma (RCC), we performed genome-wide methylation profiling of RCC using the HumanMethylation27 BeadChips to assess methylation at >14,000 genes. Two hundred and twenty hypermethylated probes representing 205 loci/genes were identified in genomic CpG islands. A subset of TSGs investigated in detail exhibited frequent tumor methylation, promoter methylation associated transcriptional silencing and reactivation after demethylation in RCC cell lines and downregulation of expression in tumor tissue (e.g., SLC34A2 specifically methylated in 63% of RCC, OVOL1 in 40%, DLEC1 in 20%, TMPRSS2 in 26%, SST in 31% and BMP4 in 35%). As OVOL1, a putative regulator of c-Myc transcription, and SST (somatostatin) had not previously been linked to cancer and RCC, respectively, we (1) investigated their potential relevance to tumor growth by RNAi knockdown and found significantly increased anchorage-independent growth and (2) demonstrated that OVOL1 knockdown increased c-Myc mRNA levels.

Tumor Suppressor genes (TSGs) often locate at chromosomal regions with frequent deletions in tumors. Loss of 16q23 occurs frequently in multiple tumors, indicating the presence of critical TSGs at this locus, such as the well-studied WWOX. Herein we found that ADAMTS18, located next to WWOX, was significantly downregulated in multiple carcinoma cell lines. No deletion of ADAMTS18 was detected with multiplex differential DNA-PCR or high resolution 1-Mb array-based CGH analysis. Instead, methylation of the ADAMTS18 promoter CpG Island was frequently detected with methylation-specific PCR and bisulfite genome sequencing in multiple carcinoma cell lines and primary carcinomas, but not in any non-tumor cell line and normal epithelial tissue. Both pharmacological and genetic demethylation dramatically induced ADAMTS18 expression, indicating that CpG methylation directly contributes to the tumor-specific silencing of ADAMTS18. Ectopic ADAMTS18 expression leads to significant inhibition of both anchorage-dependent and -independent growth of carcinoma cells lacking the expression. Thus, through functional epigenetics, we identified ADAMTS18 as a novel functional tumor suppressor, being frequently inactivated epigenetically in multiple carcinomas.

Tumour suppressor genes (TSGs) were frequently inactivated through promoter hypermethylation in gastric carcinoma as well as pre-malignant gastric lesions, suggesting that promoter hypermethylation can be used as a marker to define novel TSGs and also biomarkers for early detection of gastric cancer. In an effort to search for such genes aberrantly methylated in gastric cancer development, fibulin 1 (FBLN1) was found as a candidate TSG epigenetically downregulated in gastric cancer. FBLN1 expression was downregulated in all of gastric cancer cell lines used (100%, 7 out of 7) and the primary gastric carcinoma tissues (84%, 86 out of 102) and significantly restored after pharmacological demethylation. Hypermethylation of the FBLN1 promoter was frequently (71%, 5 out of 7) detected in gastric cancer cell lines and primary gastric carcinoma tissues. Ectopic expression of FBLN1 led to the growth inhibition of gastric cancer cells through the induction of apoptosis. In summary, FBLN1 was identified as a novel candidate TSG epigenetically downregulated in gastric cancer.

Colorectal cancers (CRC)-and probably all cancers-are caused by alterations in genes. This includes activation of oncogenes and inactivation of tumor suppressor genes (TSGs). There are many ways to achieve these alterations. Oncogenes are frequently activated by point mutation, gene amplification, or changes in the promoter (typically caused by chromosomal rearrangements). TSGs are typically inactivated by mutation, deletion, or promoter methylation, which silences gene expression. About 15% of CRC is associated with loss of the DNA mismatch repair system, and the resulting CRCs have a unique phenotype that is called microsatellite instability, or MSI. This paper reviews the types of genetic alterations that can be found in CRCs and hepatocellular carcinoma (HCC), and focuses upon the epigenetic alterations that result in promoter methylation and the CpG island methylator phenotype (CIMP). The challenge facing CRC research and clinical care at this time is to deal with the heterogeneity and complexity of these genetic and epigenetic alterations, and to use this information to direct rational prevention and treatment strategies.

The molecular genetics of inherited phaeochromocytoma have received considerable attention, but the somatic genetic and epigenetic events that characterise tumourigenesis in sporadic phaeochromocytomas are less well defined. Previously, we found considerable overlap between patterns of promoter region tumour suppressor gene (TSG) hypermethylation in two neural crest tumours, neuroblastoma and phaeochromocytoma. In order to identify candidate biomarkers and epigenetically inactivated TSGs in phaeochromocytoma and neuroblastoma, we characterised changes in gene expression in three neuroblastoma cell lines after treatment with the demethylating agent 5-azacytidine. Promoter region methylation status was then determined for 28 genes that demonstrated increased expression after demethylation. Three genes HSP47, homeobox A9 (HOXA9) and opioid binding protein (OPCML) were methylated in >10% of phaeochromocytomas (52, 17 and 12% respectively). Two of the genes, epithelial membrane protein 3 (EMP3) and HSP47, demonstrated significantly more frequent methylation in neuroblastoma than phaeochromocytoma. These findings extend epigenotype of phaeochromocytoma and identify candidate genes implicated in sporadic phaeochromocytoma tumourigenesis.

Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis. By integrating gene expression and methylation array analysis we identified novel candidate genes frequently methylated in melanoma. We validated the methylation status of the most promising genes using highly sensitive Sequenom Epityper assays in a large panel of melanoma cell lines and resected melanomas, and compared the findings with those from cultured melanocytes. We found transcript levels of UCHL1, COL1A2, THBS1 and TNFRSF10D were inversely correlated with promoter methylation. For THBS1 and UCHL1 the effect of this methylation on expression was confirmed at the protein level. Identification of these candidate TSGs and future research designed to understand how their silencing is related to melanoma development will increase our understanding of the etiology of this cancer and may provide tools for its early diagnosis.

Promoter region hypermethylation and transcriptional silencing is a frequent cause of tumour suppressor gene (TSG) inactivation in many human cancers. Previously, to identify candidate epigenetically inactivated TSGs in renal cell carcinoma (RCC), we monitored changes in gene expression in four RCC cell lines after treatment with the demethylating agent 5-azacytidine. This enabled us to identify HAI-2/SPINT2 as a novel epigenetically inactivated candidate RCC TSG. To identify further candidate TSGs, we undertook bioinformatic and molecular genetic evaluation of a further 60 genes differentially expressed after demethylation. In addition to HAI-2/SPINT2, four genes (PLAU, CDH1, IGFB3 and MT1G) had previously been shown to undergo promoter methylation in RCC. After bioinformatic prioritisation, expression and/or methylation analysis of RCC cell lines±primary tumours was performed for 34 genes. KRT19 and CXCL16 were methylated in RCC cell lines and primary RCC; however, 22 genes were differentially expressed after demethylation but did not show primary tumour-specific methylation (methylated in normal tissue (n=1); methylated only in RCC cell lines (n=9) and not methylated in RCC cell lines (n=12)). Re-expression of CXCL16 reduced growth of an RCC cell line in vitro. In a summary, a functional epigenomic analysis of four RCC cell lines using microarrays representing 11 000 human genes yielded both known and novel candidate TSGs epigenetically inactivated in RCC, suggesting that this is valid strategy for the identification of novel TSGs and biomarkers.

The class III histone deactylase (HDAC), SIRT1, has cancer relevance because it regulates lifespan in multiple organisms, down-regulates p53 function through deacetylation, and is linked to polycomb gene silencing in Drosophila. However, it has not been reported to mediate heterochromatin formation or heritable silencing for endogenous mammalian genes. Herein, we show that SIRT1 localizes to promoters of several aberrantly silenced tumor suppressor genes (TSGs) in which 5′ CpG islands are densely hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed. Heretofore, only type I and II HDACs, through deactylation of lysines 9 and 14 of histone H3 (H3-K9 and H3-K14, respectively), had been tied to the above TSG silencing. However, inhibition of these enzymes alone fails to re-activate the genes unless DNA methylation is first inhibited. In contrast, inhibition of SIRT1 by pharmacologic, dominant negative, and siRNA (small interfering RNA)–mediated inhibition in breast and colon cancer cells causes increased H4-K16 and H3-K9 acetylation at endogenous promoters and gene re-expression despite full retention of promoter DNA hypermethylation. Furthermore, SIRT1 inhibition affects key phenotypic aspects of cancer cells. We thus have identified a new component of epigenetic TSG silencing that may potentially link some epigenetic changes associated with aging with those found in cancer, and provide new directions for therapeutically targeting these important genes for re-expression.

Synopsis

The propensity for cancer to arise and progress is influenced not only by gene mutations (genetic abnormalities), but also by defects in gene expression programs that are inherited from one dividing cell to another. This change in the inheritance of gene expression patterns not associated with changes in the primary DNA sequence is referred to as an epigenetic abnormality. In virtually every form of cancer, tumor suppressor genes (TSGs) and candidate TSGs are epigenetically altered such that the ability of these genes to become activated and lead to production of the corresponding proteins is lost. This so-called gene “silencing” is often linked with abnormal accumulation of methyl groups to DNA (DNA methylation) in a region of the gene that controls its expression. The SIRT1 protein is an enzyme that can remove acetyl groups attached to specific amino acids in a number of different protein targets and thereby regulate gene silencing in yeast. However, in mammalian cells this has not been demonstrated. Here, the authors show SIRT1 is involved in epigenetic silencing of DNA-hypermethylated TSGs in cancer cells. Inhibition of SIRT1 by multiple approaches leads to TSG re-expression and a block in tumor-causing networks of cell signaling that are activated by loss of the TSGs in a wide range of cancers. This finding has important ramifications for the biology of cancer in terms of what maintains abnormal gene silencing. Furthermore, the authors propose that their observations may have potential clinical relevance in suggesting new means for restoring expression of abnormally silenced genes in cancer.

Promoter hypermethylation of tumor suppressor genes (TSGs) is a common feature of primary cancer cells. However, to date the somatic epigenetic events that occur in head and neck squamous cell carcinoma (HNSCC) tumorigenesis have not been well-defined. In the present study, we analyzed the promoter methylation status of the genes mutL homolog 1 (MLH1), Ras-association domain family member 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in 23 HNSCC samples, three control tissues and one HNSCC cell line (UM-SCC 33) using methylation-specific PCR (MSP). The expression of the three proteins was quantified by semi-quantitative immunohistochemical analysis. The cell line was treated with the demethylating agent 5-azacytidine (5-Aza) and the methylation status after 5-Aza treatment was analyzed by MSP and DNA sequencing. Proliferation was determined by Alamar blue staining. We found that the MGMT promoter in 57% of the analyzed primary tumor samples and in the cell line was hypermethylated. The MLH promoter was found to be methylated in one out of 23 (4%) tumor samples while in the examined cell line the MLH promoter was unmethylated. The RASSF1A promoter showed methylation in 13% of the tumor samples and in the cell line. MGMT expression in the group of tumor samples with a hypermethylated promoter was statistically significantly lower compared to the group of tumors with no measured hypermethylation of the MGMT promoter. After treatment of the cell line with the demethylating agent 5-Aza no demethylation of the methylated MGMT and RASSF1A genes were determined by MSP. DNA sequencing verified the MSP results, however, increased numbers of unmethylated CpG islands in the promoter region of MGMT and RASSF1A were observed. Proliferation was significantly (p<0.05) reduced after treatment with 5-Aza. In summary, we have shown promoter hypermethylation of the tumor suppressor genes MGMT and RASSF1A in HNSCC, suggesting that this epigenetic inactivation of TSGs may play a role in the development of HNSCC. 5-Aza application resulted in partial demethylation of the MGMT and RASSF1A TSGs and reduced proliferation of the tumor cells suggesting further evaluation of 5-Aza for HNSCC treatment.

Background

Tumor suppressor gene (TSG) RASSF1A and candidate TSG BLU are two tandem head-to-tail genes located at 3p21.3. We hypothesized that there may be a concordance on their gene expression and promoter methylation status. If not, then there may be an insulator located between RASSF1A and BLU genes that provides a barrier activity.

Methodology/Principal Findings

We first identified potential transcriptionally important CpG sites using the methylation-specific oligonucleotide array in relation to mRNA expression of RASSF1A and BLU genes in primary lung tumors. We demonstrated that E2F1 bound to the potential transcriptionally important CpG sites in RASSF1A gene of a normal lung cell line expressing RASSF1A transcripts, whereas loss of E2F1 binding to RASSF1A in A549 cancer cell line was the result of DNA methylation. Both RASSF1A and BLU genes had their own potential transcriptionally important CpG regions. However, there was no correlation of methylation status between RASSF1A and BLU. Using gel shift assay and chromatin immunoprecipitation-PCR (ChIP-PCR), we found that CCCTC-binding factor (CTCF) bound to insulator sequences located between these two genes. Bisulfite sequencing and ChIP-PCR revealed distinct methylation and chromatin boundaries separated by the CTCF binding domains in normal cells, whereas such distinct epigenetic domains were not observed in cancer cells. Note that demethylation reagent and histone deacetylase inhibitor treatments led to CTCF binding and recovery of barrier effect for RASSF1A and BLU genes in cancer cells.

Conclusions/Significance

Our study dissects the potential transcriptionally important CpG sites for RASSF1A and BLU genes at the sequence level and demonstrates that CTCF binding to the insulator of BLU gene provides a barrier activity within separate epigenetic domains of the juxtaposed BLU and RASSF1A loci in the 3p21.3 gene cluster region.

Malignant pleural mesothelioma (MPM) is a rapidly fatal tumor with increasing incidence worldwide responsible for many thousands of deaths annually. Although there is a clear link between exposure to asbestos and mesothelioma, and asbestos is known to be both clastogenic and cytotoxic to mesothelial cells, the mechanisms of causation of MPM remain largely unknown. However, there is a rapidly emerging literature that describes inactivation of a diverse array of tumor suppressor genes (TSGs) via promoter DNA CpG methylation in MPM, although the etiology of these alterations remains unclear. We studied the relationships among promoter methylation silencing, asbestos exposure, patient demographics and tumor histology using a directed approach; examining six cell cycle control pathway TSGs in an incident case series of 70 MPMs. Promoter hypermethylation of APC, CCND2, CDKN2A, CDKN2B, HPPBP1 and RASSF1 were assessed. We observed significantly higher lung asbestos body burden if any of these cell cycle genes were methylated (P < 0.02), and there was a significant trend of increasing asbestos body counts as the number of methylated cell cycle pathway genes increased from 0 to 1 to >1 (P < 0.005). This trend of increasing asbestos body count and increasing number of methylated cell cycle pathway genes remained significant (P < 0.05) after controlling for age, gender and tumor histology. These data suggest a novel tumorigenic mechanism of action of asbestos and may contribute to the understanding of precisely how asbestos exposure influences the etiology and clinical course of malignant mesothelioma.

Promoter hypermethylation mediated by DNA methyltransferases (DNMTs) is the main reason for epigenetic inactivation of tumor suppressor genes (TSGs). Previous studies showed that DNMT1 and DNMT3B play an important role in CpG island methylation in tumorigenesis. Little is known about the role of DNMT3A in this process, especially in hepatocellular carcinoma (HCC). In the present study, increased DNMT3A expression in 3 out of 6 HCC cell lines and 16/25 (64%) HCC tissues implied that DNMT3A is involved in hepatocellular carcinogenesis. Depletion of DNMT3A in HCC cell line SMMC-7721 inhibited cell proliferation and decreased the colony formation (about 65%). Microarray data revealed that 153 genes were upregulated in DNMT3A knockdown cells and that almost 71% (109/153) of them contain CpG islands in their 5′ region. 13 of them including PTEN, a crucial tumor suppressor gene in HCC, are genes involved in cell cycle and cell proliferation. Demethylation of PTEN promoter was observed in DNMT3A-depleted cells implying that DNMT3A silenced PTEN via DNA methylation. These results provide insights into the mechanisms of DNMT3A to regulate TSGs by an epigenetic approach in HCC.

Background

Inactivaion of tumor suppressor genes (TSGs) by promoter CpG methylation frequently occurs in tumorigenesis, even in the early stages, contributing to the initiation and progression of human cancers. Deleted in lung and esophageal cancer 1 (DLEC1), located at the 3p22-21.3 TSG cluster, has been identified frequently silenced by promoter CpG methylation in multiple carcinomas, however, no study has been performed for lymphomas yet.

Methods

We examined the expression of DLEC1 by semi-quantitative reverse transcription (RT)-PCR, and evaluated the promoter methylation of DLEC1 by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS) in common lymphoma cell lines and tumors.

Results

Here we report that DLEC1 is readily expressed in normal lymphoid tissues including lymph nodes and PBMCs, but reduced or silenced in 70% (16/23) of non-Hodgkin and Hodgkin lymphoma cell lines, including 2/6 diffuse large B-cell (DLBCL), 1/2 peripheral T cell lymphomas, 5/5 Burkitt, 6/7 Hodgkin and 2/3 nasal killer (NK)/T-cell lymphoma cell lines. Promoter CpG methylation was frequently detected in 80% (20/25) of lymphoma cell lines and correlated with DLEC1 downregulation/silencing. Pharmacologic demethylation reversed DLEC1 expression in lymphoma cell lines along with concomitant promoter demethylation. DLEC1 methylation was also frequently detected in 32 out of 58 (55%) different types of lymphoma tissues, but not in normal lymph nodes. Furthermore, DLEC1 was specifically methylated in the sera of 3/13 (23%) Hodgkin lymphoma patients.

Conclusions

Thus, methylation-mediated silencing of DLEC1 plays an important role in multiple lymphomagenesis, and may serve as a non-invasive tumor marker for lymphoma diagnosis.

Promoter CpG methylation of tumour suppressor genes (TSGs) is an epigenetic biomarker for TSG identification and molecular diagnosis. We screened genome wide for novel methylated genes through methylation subtraction of a genetic demethylation model of colon cancer (double knockout of DNMT1 and DNMT3B in HCT116) and identified DLEC1 (Deleted in lung and oesophageal cancer 1), a major 3p22.3 TSG, as one of the methylated targets. We further found that DLEC1 was downregulated or silenced in most colorectal and gastric cell lines due to promoter methylation, whereas broadly expressed in normal tissues including colon and stomach, and unmethylated in expressing cell lines and immortalised normal colon epithelial cells. DLEC1 expression was reactivated through pharmacologic or genetic demethylation, indicating a DNMT1/DNMT3B-mediated methylation silencing. Aberrant methylation was further detected in primary colorectal (10 out of 34, 29%) and gastric tumours (30 out of 89, 34%), but seldom in paired normal colon (0 out of 17) and gastric (1 out of 20, 5%) samples. No correlation between DLEC1 methylation and clinical parameters of gastric cancers was found. Ectopic expression of DLEC1 in silenced HCT116 and MKN45 cells strongly inhibited their clonogenicity. Thus, DLEC1 is a functional tumour suppressor, being frequently silenced by epigenetic mechanism in gastrointestinal tumours.

Background

Both gastric and colorectal cancers (CRC) are the most frequently occurring malignancies worldwide with the overall survival of these patients remains unsatisfied. Identification of tumor suppressor genes (TSG) silenced by promoter CpG methylation uncovers mechanisms of tumorigenesis and identifies new epigenetic biomarkers for early cancer detection and prognosis assessment. Cystathionine-beta-synthase (CBS) functions in the folate metabolism pathway, which is intricately linked to methylation of genomic DNA. Dysregulation of DNA methylation contributes substantially to cancer development.

Methodology/Principal Findings

To identify potential TSGs silenced by aberrant promoter methylation in CRC, we analyzed tumor and adjacent tissues from CRC cases using the Illumina Human Methylation45 BeadChip. We identified hypermethylation of the CBS gene in CRC samples, compared to adjacent tissues. Methylation and decreased mRNA expression of CBS were detected in most CRC cell lines by methylation-specific PCR and semiquantitative RT-PCR, as well as in gastric cancer. Treatment with 5-aza-2'-deoxycytidine and/or trichostatin A reversed methylation and restored CBS mRNA expression indicating a direct effect. Aberrant methylation was further detected in 31% of primary CRCs (29 of 96) and 55% of gastric tumors (11 of 20). In contrast, methylation was seldom found in normal tissues adjacent to the tumor. CBS methylation was associated with KRAS mutations in primary CRCs (P = 0.04, by χ2-test). However, no association was found between CBS methylation or KRAS mutations with cancer relapse/metastasis in Stage II CRC patients.

Conclusion

A novel finding from this study is that the folate metabolism enzyme CBS mRNA levels are frequently downregulated through CpG methylation of the CBS gene in gastric cancer and CRC, suggesting that CBS functions as a tumor suppressor gene. These findings warrant further study of CBS as an epigenetic biomarker for molecular diagnosis of gastrointestinal cancers.

Background

Alterations in DNA methylation in cancer include global hypomethylation and gene-specific hypermethylation. It is not clear whether these two epigenetic errors are mechanistically linked or occur independently. This study was performed to determine the relationship between DNA hypomethylation, hypermethylation and microsatellite instability in cancer.

Methodology/Principal Findings

We examined 61 cancer cell lines and 60 colorectal carcinomas and their adjacent tissues using LINE-1 bisulfite-PCR as a surrogate for global demethylation. Colorectal carcinomas with sporadic microsatellite instability (MSI), most of which are due to a CpG island methylation phenotype (CIMP) and associated MLH1 promoter methylation, showed in average no difference in LINE-1 methylation between normal adjacent and cancer tissues. Interestingly, some tumor samples in this group showed increase in LINE-1 methylation. In contrast, MSI-showed a significant decrease in LINE-1 methylation between normal adjacent and cancer tissues (P<0.001). Microarray analysis of repetitive element methylation confirmed this observation and showed a high degree of variability in hypomethylation between samples. Additionally, unsupervised hierarchical clustering identified a group of highly hypomethylated tumors, composed mostly of tumors without microsatellite instability. We extended LINE-1 analysis to cancer cell lines from different tissues and found that 50/61 were hypomethylated compared to peripheral blood lymphocytes and normal colon mucosa. Interestingly, these cancer cell lines also exhibited a large variation in demethylation, which was tissue-specific and thus unlikely to be resultant from a stochastic process.

Conclusion/Significance

Global hypomethylation is partially reversed in cancers with microsatellite instability and also shows high variability in cancer, which may reflect alternative progression pathways in cancer.

A cyclin-dependent kinase inhibitor CDKN2A (p16/Ink4a) is a tumor suppressor and upregulated in cellular senescence. CDKN2A promoter methylation and gene silencing are associated with the CpG island methylator phenotype (CIMP) in colon cancer. However, prognostic significance of CDKN2A methylation or loss of CDKN2A (p16) expression independent of CIMP status remains uncertain. Utilizing a database of 902 colorectal cancers in two independent cohort studies (the Nurses' Health Study and the Health Professionals Follow-up Study), we quantified CDKN2A promoter methylation and detected hypermethylation in 269 tumors (30%). By immunohistochemistry, we detected loss of CDKN2A (p16) expression in 25% (200/804) of tumors. We analyzed for LINE-1 hypomethylation and hypermethylation at 7 CIMP-specific CpG islands (CACNA1G, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1); microsatellite instability (MSI); KRAS, BRAF and PIK3CA mutations; and expression of TP53 (p53), CTNNB1 (β-catenin), CDKN1A (p21), CDKN1B (p27), CCND1 (cyclin D1), FASN (fatty acid synthase), and PTGS2 (cyclooxygenase-2). CDKN2A promoter methylation and loss of CDKN2A (p16) were associated with shorter overall survival in univariate Cox regression analysis [hazard ratio (HR), 1.36; 95% CI, 1.10-1.66; p=0.0036 for CDKN2A methylation; HR, 1.30; 95% CI, 1.03-1.63; p=0.026 for CDKN2A (p16) loss] but not in multivariate analysis that adjusted for clinical and tumor variables including CIMP, MSI, and LINE-1 methylation. Neither CDKN2A promoter methylation nor loss of CDKN2A (p16) was associated with colorectal cancer-specific mortality in univariate or multivariate analysis. Despite its well-established role in carcinogenesis, CDKN2A (p16) promoter methylation or loss of expression in colorectal cancer is not independently associated with patient prognosis.

Background

So far, investigators have found numerous tumor suppressor genes (TSGs) and oncogenes (OCGs) that control cell proliferation and apoptosis during cancer development. Furthermore, TSGs and OCGs may act as modulators of transcription factors (TFs) to influence gene regulation. A comprehensive investigation of TSGs, OCGs, TFs, and their joint target genes at the network level may provide a deeper understanding of the post-translational modulation of TSGs and OCGs to TF gene regulation.

Methodology/Principal Findings

In this study, we developed a novel computational framework for identifying target genes of TSGs and OCGs using TFs as bridges through the integration of protein-protein interactions and gene expression data. We applied this pipeline to ovarian cancer and constructed a three-layer regulatory network. In the network, the top layer was comprised of modulators (TSGs and OCGs), the middle layer included TFs, and the bottom layer contained target genes. Based on regulatory relationships in the network, we compiled TSG and OCG profiles and performed clustering analyses. Interestingly, we found TSGs and OCGs formed two distinct branches. The genes in the TSG branch were significantly enriched in DNA damage and repair, regulating macromolecule metabolism, cell cycle and apoptosis, while the genes in the OCG branch were significantly enriched in the ErbB signaling pathway. Remarkably, their specific targets showed a reversed functional enrichment in terms of apoptosis and the ErbB signaling pathway: the target genes regulated by OCGs only were enriched in anti-apoptosis and the target genes regulated by TSGs only were enriched in the ErbB signaling pathway.

Conclusions/Significance

This study provides the first comprehensive investigation of the interplay of TSGs and OCGs in a regulatory network modulated by TFs. Our application in ovarian cancer revealed distinct regulatory patterns of TSGs and OCGs, suggesting a competitive regulatory mechanism acting upon apoptosis and the ErbB signaling pathway through their specific target genes.

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.

NOL7 is a putative tumor suppressor gene (TSG) localized to 6p23, a region with frequent loss of heterozygosity (LOH) in a number of cancers, including cervical cancer (CC). We have previously demonstrated that reintroduction of NOL7 into CC cells alters the angiogenic phenotype and suppressed tumor growth in vivo by 95%. Therefore, to understand its mechanism of inactivation in CC, we investigated the genetic and epigenetic regulation of NOL7. NOL7 mRNA and protein levels were assessed in thirteen CC cell lines and twenty-three consecutive CC specimens by RTQ-PCR, western blotting, and IHC. Methylation of the NOL7 promoter was analyzed by bisulfite sequencing and mutations were identified through direct sequencing. A CpG island with multiple CpG dinucleotides spanned the 5′UTR and first exon of NOL7. However, bisulfite sequencing failed to identify persistent sites of methylation. Mutational sequencing revealed that 40% of the CC specimens and 31% of the CC cell lines harbored somatic mutations that may affect the in vivo function of NOL7. Endogenous NOL7 mRNA and protein expression in CC cell lines was significantly decreased in 46% of the CC cell lines. Finally, immunohistochemistry demonstrated strong NOL7 nucleolar staining in normal tissues that decreased with histologic progression towards CC. NOL7 is inactivated in CC in accordance with Knudson's two-hit hypothesis through LOH and mutation. Together with evidence of its in vivo tumor suppression, these data support the hypothesis that NOL7 is the legitimate TSG located on 6p23.

Background:

The CpG island methylator phenotype (CIMP) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, associated with microsatellite instability-high (MSI-high) and BRAF mutations. 18q loss of heterozygosity (LOH) commonly present in colorectal cancer with chromosomal instability (CIN) is associated with global hypomethylation in tumor cell. A recent study has shown an inverse correlation between CIN and CIMP (determined by MINTs, p16, p14 and MLH1 methylation) in colorectal cancer. However, no study has examined 18q LOH in relation to CIMP-high, CIMP-low (less extensive promoter methylation) and CIMP-0 (CIMP-negative), determined by quantitative DNA methylation analysis.

Methods:

Utilizing MethyLight technology (real-time PCR), we quantified DNA methylation in 8 CIMP-specific promoters {CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1} in 758 non-MSI-high colorectal cancers obtained from two large prospective cohorts. Using four 18q microsatellite markers (D18S55, D18S56, D18S67 and D18S487) and stringent criteria for 18q LOH, we selected 374 tumors (236 LOH-positive tumors with ≥ 2 markers showing LOH; and 138 LOH-negative tumors with ≥ 3 informative markers and no LOH).

Results:

CIMP-0 (0/8 methylated promoters) was significantly more common in 18q LOH-positive tumors (59% = 139/236, p = 0.002) than 18q LOH-negative tumors (44% = 61/138), while CIMP-low/high (1/8–8/8 methylated promoters) was significantly more common (56%) in 18q LOH-negative tumors than 18q LOH-positive tumors (41%). These relations persisted after stratification by sex, location, or the status of MSI, p53 expression (by immunohistochemistry), or KRAS/BRAF mutation.

Conclusion:

18q LOH is correlated positively with CIMP-0 and inversely with CIMP-low and CIMP-high. Our findings provide supporting evidence for relationship between CIMP-0 and 18q LOH as well as a molecular difference between CIMP-0 and CIMP-low in colorectal cancer.

Promoter hypermethylation and heterochromatinization is a frequent event leading to gene inactivation and tumorigenesis. At the molecular level, inactivation of tumor suppressor genes in cancer has many similarities to the inactive X chromosome in female cells and is defined and maintained by DNA methylation and characteristic histone modifications. In addition, the inactive-X is marked by the histone macroH2A, a variant of H2A with a large non-histone region of unknown function. Studying tumor suppressor genes (TSGs) silenced in cancer cell lines, we find that when active, these promoters are associated with H2A.Z but become enriched for macroH2A1 once silenced. Knockdown of macroH2A1 was not sufficient for reactivation of silenced genes. However, when combined with DNA demethylation, macroH2A1 deficiency significantly enhanced reactivation of the tumor suppressor genes p16, MLH1 and Timp3 and inhibited cell proliferation. Our findings link macroH2A1 to heterochromatin of epigenetically silenced cancer genes and indicate synergism between macroH2A1 and DNA methylation in maintenance of the silenced state.