Constitutional activation of IL-6-mediated JAK/STAT pathway through hypermethylation of SOCS-1 in human gastric cancer cell line

The interleukin-mediated Janus kinase (JAK)/STAT pathway plays a crucial role in carcinogenesis. Recently, increased STAT3 activity was found in hepatocellular carcinoma and multiple myeloma in which there was silencing of SOCS-1 (suppressor of cytokine signalling-1) by gene promoter hypermethylation. We investigated the expression level of interleukin-6 (IL-6) and SOCS-1 in gastric cancer cell lines. Expression of SOCS-1 correlated with IL-6 level in most of the cell lines, except for AGS cells in which SOCS-1 was absent despite a high level of IL-6 production. Methylation analysis by methylation-specific polymerase chain reaction and bisulphite sequencing revealed that CpG island of SOCS-1 was densely methylated in AGS cells. Demethylation treatment by 5'aza-deoxycytidine restored SOCS-1 expression and also suppressed constitutive STAT3 phosphorylation in AGS cells. Moreover, methylation of SOCS-1 was detected in 27.5% (11 of 40) of primary gastric tumours samples, 10% (one of 10) of adjacent noncancer tissues but not in any (zero of nine) normal gastric mucosa. Methylation of SOCS-1 also correlated with the loss of mRNA expression in some primary gastric cancers. In conclusion, this is the first report to demonstrate that hypermethylation of SOCS-1 led to gene silencing in gastric cancer cell line and primary tumour samples. Downregulation of SOCS-1 cooperates with IL-6 in the activation of JAK/STAT pathway in gastric cancer.     

Decreased expression of GRIM-19 by DNA hypermethylation promotes aerobic glycolysis and cell proliferation in head and neck squamous cell carcinoma

To identify novel tumor suppressor genes that are down-regulated by promoter hypermethylation in head and neck squamous cell carcinoma (HNSCC), genome-wide methylation profiling was performed using a methylated DNA immunoprecipitation (MeDIP) array in HNSCC and normal mucosa tissue samples. Promoter hypermethylation of the candidate gene, gene associated with retinoid-interferon induced mortality-19 (GRIM-19), was confirmed in HNSCC cell lines. Multivariate regression analysis determined that GRIM-19 hypermethylation was an independent significant factor for HNSCC diagnosis (OR:125.562; P < 0.001). HNSCC patients with lower ratio of GRIM-19/ACTB hypermethylation had increased overall and disease free survival. Furthermore, the optimal cutoff provided 90% sensitivity and 77% specificity of GRIM-19 hypermethylation as a diagnostic marker for HNSCC. Ectopic expression of GRIM-19 in HNSCC cells led to increased oxygen consumption, reduced glycolysis and decreased cell proliferation. HNSCC cells ectopically expressing GRIM-19 displayed increased p53 activity as well as decreased Stat3 and HIF-1α activities. Moreover, GRIM-19 knockdown not only resulted in decreased oxygen consumption and increased aerobic glycolysis but also promoted cell proliferation and tumorigenic capacity in HNSCC cells. Our data indicate that decreased GRIM-19 expression due to promoter hypermethylation may be important in head and neck carcinogenesis by promoting cell proliferation and regulating metabolic activity.     

Frequent 3p allele loss and epigenetic inactivation of the RASSF1A tumour suppressor gene from region 3p21.3 in head and neck squamous cell carcinoma

Studies of allelic imbalance and suppression of tumourigenicity have consistently suggested that the short arm of chromosome three (3p) harbours tumour suppressor genes (TSGs) whose inactivation leads to the development of various types of neoplasia including head and neck squamous cell carcinoma (HNSCC). Previously, we defined a critical minimal region of 120kb at 3p21.3 that contains overlapping homozygous deletions in lung and breast tumour lines and isolated eight genes from the minimal region. Mutation analysis in a large panel of lung and breast cancers revealed only rare mutations, but the majority of lung tumour lines showed loss of expression for one of the eight genes (RASSF1A) due to hypermethylation of a CpG island in the promoter region of RASSF1A. We found RASSF1A to be methylated in the majority of lung tumours, but to a lesser extent in breast and ovarian tumours. In order to define the role of 3p TSGs, in particular RASSF1A in HNSCC, we (a) analysed 43 primary HNSCC for allelic loss in regions proposed to contain 3p TSGs (3p25-26, 3p24, 3p21-22, 3p14 and 3p12), (b) analysed 24 HNSCC for evidence of RASSF1A methylation and (c) undertook mutation analysis of RASSF1A in HNSCC. We found that 81% of HNSCC showed allele loss at one or more 3p markers, 66% demonstrated loss for 3p21.3 markers and 56% showed allelic losses at 3p12 loci. Thus, 3p loss is common in HNSCC and extensive 3p loss occurs even in early stage tumours. RASSF1A promoter region hypermethylation was found in 17% (4/24) of the sporadic HNSCC, but RASSF1A mutations were not identified. Furthermore, we found RASSF1A methylation to be significantly higher in poorly differentiated then in moderate to well differentiated HNSCC (P=0.0048). Three of the four tumours showing RASSF1A methylation also underwent 3p21.3 allelic loss, hence RASSF1A behaves as a classical TSG (two hits, methylation and loss). One tumour with RASSF1A methylation had retention of markers at 3p providing further evidence of specific inactivation of RASSF1A as a critical step in some HNSCC. Although the frequency of 3p21.3 allele loss was substantially higher than that of RASSF1A methylation this does not necessarily suggest that other genes from 3p21.3 are also implicated in HNSCC, as 3p21.3 LOH was invariably found with LOH at other 3p loci. Thus, the presence of 3p21.3 allele loss without RASSF1A methylation might reflect a propensity for 3p21.3 loss to occur as a secondary consequence of large 3p deletions targeted at other 3p TSG regions. Furthermore, in the presence of homozygous inactivation of other 3p TSGs, RASSF1A haploinsufficiency might be sufficient to promote tumourigenesis in many HNSCC.     

Promoter methylation of MGMT, MLH1 and RASSF1A tumor suppressor genes in head and neck squamous cell carcinoma: pharmacological genome demethylation reduces proliferation of head and neck squamous carcinoma cells

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.     

Hypermethylation-associated inactivation of the SOCS-1 gene, a JAK/STAT inhibitor, in human pancreatic cancers

BACKGROUND: SOCS-1, a JAK-binding protein (SSI-1/SOCS-1/JAB), regulates the JAK/STAT signal transduction pathway that relays signals from various cytokines in the extracellular matrix into the cell. Inactivation of the SOCS-1 gene by methylation has been previously described in hepatocellular carcinomas and multiple myeloma. The purpose of the present work was to analyze the expression of the SOCS-1 gene and identify inactivation of this gene by methylation in pancreatic cancers. METHODS: 20 samples were analyzed. We identified the expression of SOCS-1 gene using RT-PCR and the mechanism of inactivation in this gene by methylation assay. RESULTS: We documented marked suppression of SOCS-1 mRNA and reduction of SOCS-1 protein in 7 of 14 primary pancreatic cancers examined; moreover, CpG-rich regions upstream of the SOCS-1 gene were hypermethylated in 8 of the 14 tumors. CONCLUSIONS: The results suggested that this gene is silenced in a substantial portion of pancreatic cancers through mechanisms that cause methylation in the promoter region.     

PTEN,DMBT1, and p16 alterations in diffusely infiltrating astrocytomas

Losses at chromosomes 10q and 9p are genetic events implicated in the genesis of diffusely infiltrating astrocytomas. We studied the role of PTEN (10q23.3), DMBT1 (10q25.3-26.1) and p16 (9p21) tumor suppressor genes in a series of 50 low- and high-grade astrocytomas. A genetic screening for point mutations, homozygous deletions and promoter hypermethylation was done by PCR-SSCP, direct sequencing, differential PCR, and methylation specific PCR. PTEN was mutated in 23% and homozygously deleted in 9% GBMs, but not in grade II and III astrocytomas. Homozygous deletions of DMBT1 were found in 5% GBMs and 13% low-grade astrocytomas, but not in anaplastic astrocytomas. p16 suffered homozygous deletion in 27% GBMs and 13% low-grade astrocytomas, though no p16 point mutations were found in any of the 50 astrocytomas. p16 promoter hypermethylation was found in 9% GBMs and 6% low-grade astrocytomas. Our results might indicate that PTEN alterations are late genetic events in the progression of low to high-grade astrocytomas, versus DMBT1 and p16 alterations that also occur in low-grade astrocytomas, and might be considered as early genetic events. The majority of samples did not present more than one alteration. Taken together, these data suggest that there are multiple independent mechanisms to control cell growth and prevent GBM tumorigenesis, and that aberrations in several distinct pathways or at several sites within a pathway are needed to overcome these control mechanisms.     

O6-methylguanine-DNA methyltransferase gene: epigenetic silencing and prognostic value in head and neck squamous cell carcinoma.

BACKGROUND: Alkylating N-nitroso compounds can interact directly with DNA, forming O(6)-alkylguanine, a DNA adduct proved to be mutagenic and carcinogenic if not sufficiently repaired. A specific DNA repair enzyme, O(6)-methylguanine-DNA methyltransferase (MGMT), can remove the alkyl group from the O(6)-position of the guanine, thereby preventing its mutagenic and carcinogenic effects. Inactivation of the MGMT gene in association with promoter hypermethylation results in persistence of O(6)-alkylguanine in DNA, leading to G:C to A:T transition mutation and these G:C to A:T transition mutations can inactivate p53 tumor suppressor gene or activate ras proto-oncogene. METHODS: We analyzed MGMT promoter hypermethylation and protein expression patterns in 94 cases of primary head and neck squamous cell carcinoma (HNSCC) by methylation-specific PCR (MSP) and immunohistochemical staining. The results were then correlated with clinical follow-up data. RESULTS: MGMT promoter hypermethylation was present in 17 of 94 patients (18.1%) and apparent loss of protein expression was seen in 19 of 93 HNSCC patients (20.4%). The presence of MGMT promoter hypermethylation was significantly correlated with loss of MGMT protein expression in HNSCC. Both MGMT promoter hypermethylation and loss of protein expression were significantly correlated to increased tumor recurrences and decreased patient survival, independent of other risk factors, such as tumor site, tumor size, nodal status, age, and chemoradiation therapy. CONCLUSIONS: MGMT promoter hypermethylation and apparent loss of protein expression are reliable and independent prognostic factors in HNSCC. The above study may also provide guideline or basis for applying alkylating antitumor agents to patients with HNSCC that display MGMT promoter hypermethylation and/or loss of MGMT protein expression.     

Deleted in colorectal cancer is a putative conditional tumor-suppressor gene inactivated by promoter hypermethylation in head and neck squamous cell carcinoma

Deleted in colorectal cancer (DCC) is a candidate tumor-suppressor gene located at chromosome 18q21. However, DCC gene was found to have few somatic mutations and the heterozygous mice (DCC(+/-)) showed a similar frequency of tumor formation compared with the wild-type mice (DCC(+/+)). Recently, DCC came back to the spotlight as a better understating of its function and relationship with its ligand (netrin-1) had shown that DCC may act as a conditional tumor-suppressor gene. We evaluated hypermethylation as a mechanism for DCC inactivation in head and neck squamous cell carcinoma (HNSCC). DCC promoter region hypermethylation was found in 75% of primary HNSCC. There was a significant correlation between DCC promoter region hypermethylation and DCC expression (assessed by immunohistochemistry; P = 0.021). DCC nonexpressing HNSCC cell lines JHU-O12 and JHU-O19 with baseline hypermethylation of the DCC promoter were treated with 5-aza-2'-deoxycytidine (a demethylating agent) and reexpression of DCC was noted. Transfection of DCC into DCC-negative HNSCC cell lines resulted in complete abrogation of growth in all cell lines, whereas additional cotransfection of netrin-1 resulted in rescue of DCC-mediated growth inhibition. These results suggest that DCC is a putative conditional tumor-suppressor gene that is epigenetically inactivated by promoter hypermethylation in a majority of HNSCC.     

Frequent hypermethylation of RASSF1A in early flat-type colorectal tumors

Flat colorectal tumors, characterized by high-grade dysplasia from early small flat mucosal lesions, exhibit a relatively aggressive clinical behavior and are known for their infrequent K-ras mutations. In this study, we investigated the methylation status of the RASSF1A promoter in association with 3p LOH and K-ras mutations in 48 flat colorectal tumors (39 early carcinomas and nine intramucosal high-grade dysplasias). RASSF1A hypermethylation was detected in 39 of 48 (81.3%) tumors and RASSF1A methylation was also detected in 19 of 39 (49%) normal colonic mucosal tissues. 3p21.3 LOH was detected in 20 of 42 (47.6%) cases, but RASSF1 methylation was detected in cases with LOH (14 cases) and retention of 3p21.3 (20 cases). K-ras mutations were detected in seven of 48 (14.6%) tumors and the concordant occurrence of K-ras mutation and RASSF1A methylation was detected in three of 48 cases (6.3%). Overall, there was a statistically significant mutually exclusive relationship between K-ras mutations and RASSF1A methylation. In conclusion, promoter hypermethylation of RASSF1A is a frequent event and may start early in the background normal mucosa in this tumor type. An alternative cascade of abnormalities in RAS transduction pathways may be responsible for the flat morphology and aggressive nature of flat colorectal neoplasms.     

Combined hypermethylation and chromosome loss associated with inactivation of SSI-1/SOCS-1/JAB gene in human hepatocellular carcinomas

We previously demonstrated using restriction landmark genomic scanning-based 2-dimensional genome electrophoresis method decreased results of 16 primary hepatocellular carcinomas (HCCs) revealed reduction of intensity of 60 NotI-landmark spots, and increase in five spots that were frequently observed in HCCs. Most frequently decreased spot (14/16 HCCs) was identified to it corresponds to a gene encoding SSI-1, a JAK-binding protein (SSI-1/SOCS-1/JAB) that regulated the JAK/STAT signal transduction pathway. This signaling pathway is important for relaying signals from various cytokines outside the cell to the inside. Expression level of SOCS-1 messenger RNA was markedly suppressed in 50% of HCCs (4/8). Loss of heterozygosity at the SSI-1 gene, was found in all cases with aberrant expression. Methylation analysis of the CpG-rich regions of SSI-1 gene revealed hypermethylation of these regions. In an additional series of methylation analysis using 30 HCCs, 16 (53%) showed hypermethylation of the gene. These results indicate that the SSI-1 gene is silenced in a substantial portion of HCC though the combined mechanisms of methylation of either 5' or exon CpG rich regions and by a chromosomal loss of the remaining allele.