Aberrant methylation of the SPARC gene promoter and its clinical implication in gastric cancer.
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ABSTRACT:
SUBMITTER: Chen ZY
PROVIDER: S-EPMC4268651 | biostudies-other | 2014
REPOSITORIES: biostudies-other
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Project description:Epigenetic silencing of tumor suppressor genes is a new focus of investigation in the generation and proliferation of carcinomas. Secreted protein acidic and rich in cysteine (SPARC) is reportedly detrimental to the growth of ovarian cancer cells and has been shown to be epigenetically silenced in several cancers. We hypothesized that SPARC is downregulated in ovarian cancer through aberrant promoter hypermethylation. To that end, we analyzed SPARC expression in ovarian cancer cell lines and investigated the methylation status of the Sparc promoter using methylation-specific polymerase chain reaction. Our results show that SPARC mRNA expression is decreased in three (33%) and absent in four (44%) of the nine ovarian cancer cell lines studied, which correlated with hypermethylation of the Sparc promoter. Treatment with the demethylating agent 5-aza-2'-deoxycytidine rescued SPARC mRNA and protein expression. Addition of exogenous SPARC, as well as ectopic expression by an adenoviral vector, resulted in decreased proliferation of ovarian cancer cell lines. Investigation of primary tumors revealed that the Sparc promoter is methylated in 68% of primary ovarian tumors and that the levels of SPARC protein decrease as the disease progresses from low to high grade. Lastly, de novo methylation of Sparc promoter was shown to be mediated by DNA methyltransferase 3a. These results implicate Sparc promoter methylation as an important factor in the genesis and survival of ovarian carcinomas and provide new insights into the potential use of SPARC as a novel biomarker and/or treatment modality for this disease.
| S-EPMC2631137 | biostudies-literature
Project description:AimTo investigate the methylation status of secreted protein acidic and rich in cysteine (SPARC) in human hepatocellular carcinoma (HCC) and evaluate its clinical implication.MethodsThe methylation status of SPARC was analyzed in one HCC cell line (SMMC-7721) and 60 pairs of HCC and corresponding nontumorous tissues by methylation-specific polymerase chain reaction and bisulfite sequencing. The expression of SPARC mRNA and protein were examined by reverse transcription polymerase chain reaction and immunohistochemistry, respectively. The correlations between the methylation status and the gene expression, the clinicopathological parameters, as well as the prognosis after surgery were analyzed.ResultsIn the SMMC-7721 cell line, the loss of SPARC expression was correlated with the aberrant methylation and could be reactivated by the demethylating agent 5-aza-2'-deoxycytidine. Methylation frequency of SPARC in HCC was significantly higher than that in the corresponding nontumorous tissues (45/60 vs 7/60, P < 0.001), and it was correlated with the pathological classification (P = 0.019). The downregulation of the SPARC mRNA expression in HCC was correlated with the SPARC methylation (P = 0.040). The patients with methylated SPARC had a poorer overall survival than those without methylated SPARC (28.0 mo vs 41.0 mo, P = 0.043).ConclusionAberrant methylation is an important mechanism for SPARC inactivation in HCC and SPARC methylation may be a promising biomarker for the diagnosis and prognosis of HCC.
| S-EPMC3342602 | biostudies-literature
Project description:BackgroundColorectal cancer (CRC) multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect.Methodology/principal findingsWe examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals) and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2), RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008) and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047) as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006). Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17), SFRP1 (r = 0.83, 0.06), HPP1 (r = 0.64, p = 0.17), 3OST2 (r = 0.83, p = 0.06) and GATA4 (r = 0.6, p = 0.24). Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant difference in any evaluated gene.ConclusionsThese results provide a proof-of-concept that gene promoter methylation is associated with tumor multiplicity. This underlying epigenetic defect may have noteworthy implications in the prevention of patients with sporadic CRC.
| S-EPMC2808250 | biostudies-literature
Project description:SPARC (secreted protein acidic and rich in cysteine) is an extracellular Ca2+-binding matricellular glycoprotein associated with the regulation of cell adhesion and growth. We investigated loss of expression of SPARC gene and promoter methylation in lung cancers and correlated the data with clinicopathological features. We observed loss of SPARC expression in 12 of 20 (60%) lung cancer cell lines. Treatment of expression-negative cell lines with a demethylating agent restored expression in all cases. Methylation frequencies of SPARC gene were 55% in 20 lung cancer cell lines. Primary tumours had methylation at a rate of 69% (119 of 173), while nonmalignant lung tissues (n=60) had very low rates (3%). In lung adenocarcinomas, SPARC methylation correlated with a negative prognosis (P=0.0021; relative risk 4.65, 95% confidence interval 1.75-12.35, multivariate Cox's proportional-hazard model). Immunostaining revealed protein expression in bronchial epithelium (weak intensity) and in juxtatumoral stromal tissues (strong intensity) accompanied by frequent loss in cancer cells that correlated with the presence of methylation (P<0.001). Our findings are of biological interest and potentially of clinical importance in human lung cancers.
| S-EPMC2361924 | biostudies-literature
Project description:BackgroundThe CBFA2T3 locus located on the human chromosome region 16q24.3 is frequently deleted in breast tumors. CBFA2T3 gene expression levels are aberrant in breast tumor cell lines and the CBFA2T3B isoform is a potential tumor suppressor gene. In the absence of identified mutations to further support a role for this gene in tumorigenesis, we explored whether the CBFA2T3B promoter region is aberrantly methylated and whether this correlates with expression.ResultsAberrant hypo and hypermethylation of the CBFA2T3B promoter was detected in breast tumor cell lines and primary breast tumor samples relative to methylation index interquartile ranges in normal breast counterpart and normal whole blood samples. A statistically significant inverse correlation between aberrant CBFA2T3B promoter methylation and gene expression was established.ConclusionCBFA2T3B is a potential breast tumor suppressor gene affected by aberrant promoter methylation and gene expression. The methylation levels were quantitated using a second-round real-time methylation-specific PCR assay. The detection of both hypo and hypermethylation is a technicality regarding the methylation methodology.
| S-EPMC516017 | biostudies-literature
Project description:ABCG2 is a ubiquitous ATP-binding cassette transmembrane protein that is important in clinical drug resistance. Little is known about the mechanism(s) regulating the expression of ABCG2. We hypothesized that DNA methylation could play a role in the epigenetic regulation of ABCG2 gene expression. The promoter methylation status of three renal carcinoma cell lines was assessed with restriction enzyme digestion-coupled PCR and bisulfite genomic sequencing. Both UOK121 and UOK143, with known methylation of the VHL promoter, showed induction of ABCG2 expression after 5-aza-2'-deoxycytidine (5-aza-dC) treatment, suggesting that aberrant methylation of the ABCG2 gene was associated with gene silencing. In vitro methylation of the ABCG2 promoter-driven luciferase reporter vector resulted in a significant inhibition of transcription. Our data suggested that the ABCG2 gene is regulated coordinately at both histone and DNA levels. A chromatin immunoprecipitation assay demonstrated that the methylated promoter in UOK121 and UOK143, but not the unmethylated one in UOK181, is associated with the methyl CpG binding domain proteins (MBDs), MBD2 and MeCP2. Histone deacetylase 1 and a corepressor, mSin3A, were identified binding to the promoter region containing the CpG island, thereby suppressing ABCG2 transcription. Reactivation of ABCG2 was achieved by treatment with 5-aza-dC, a demethylating agent, concomitant with the release of MBDs from the promoter. Furthermore, the association of methylated lysine 9 on histone H3, a hallmark of promoter methylation, with the promoter was reduced following 5-aza-dC treatment. These data suggest that DNA methylation-dependent formation of a repressor complex in the CpG island contributes to inactivation of ABCG2.
| S-EPMC1636795 | biostudies-literature
Project description:BackgroundYAP, a potent oncogene and major downstream effector of the mammalian Hippo tumor suppressor pathway can act as either oncogene or tumor suppressor gene based on the type of tissue involved. Despite various studies, the role and mechanism through which YAP mediates its tumor suppressor or oncogenic effects are not yet fully understood. Therefore in the present study we aimed to investigate YAP at DNA, mRNA and protein level and also attempted to correlate our molecular findings with various clinicopathological variables of the patients.MethodsThe study comprised of a total 137 genetically unrelated women with sporadic breast cancer cases and normal adjacent tissues not infiltrated with tumor. Mutation of YAP gene was analyzed by automated DNA sequencing. YAP promoter methylation was studied using MS-PCR. Expression at mRNA and protein level was studied using qPCR and IHC respectively.ResultsIn our study YAP mRNA expression was found to be 8.65 ± 6.17 fold downregulated in 67.15% cases. The expression of YAP when analyzed at the protein level by IHC was found to be absent in 78.83% cases. Results from MS-PCR analysis showed that YAP promoter methylation plays an important role in declining the expression of YAP protein. The absence of YAP protein coincided with 86.60% methylated cases thereby showing a very strong correlation (p = 0.001). We also investigated YAP mutation at the major check point sites in the Hippo pathway and observed no mutation. A significant association was observed on correlating mRNA expression with clinical stages (p = 0.038) and protein expression with ER status (p = 0.018) among Indian breast cancer patients.ConclusionThe expression of YAP was found to be downregulated in response to aberrant promoter methylation. The downregulation of YAP are consistent with previous studies suggesting it to have a tumor suppressive role in breast cancer. We did not observe any mutation at the major check point sites in the Hippo pathway.
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Project description:Gastric cancer is one of the most prevalent malignant tumors worldwide. Immunological checkpoint inhibitors of the programmed death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) signaling pathway are effective in the treatment of various malignant tumor types, but the potential of such immunotherapeutic techniques for the treatment of gastric cancer is yet to be elucidated. The purpose of the present study was to investigate the methylation of the PD-L1 gene promoter and its clinical significance in advanced gastric cancer, as this may suggest the use of PD-L1 promoter methylation as a novel biomarker for gastric cancer progression. In a total of 70 samples, the methylation rate of the PD-L1 gene promoter region was significantly higher in gastric cancer tissues compared with adjacent tissues. A high level of PD-L1 promoter methylation was associated with lymph node staging, and resulted in poorer prognoses in patients with advanced gastric cancer. A total of 26 patients exhibited highly methylated PD-L1; in this group, the median progression-free survival time of patients receiving platinum/fluorouracil chemotherapy was 4.2 months longer than those receiving paclitaxel/fluorouracil chemotherapy, and the risk of disease progression in patients receiving paclitaxel/fluorouracil chemotherapy was 5.009 times higher compared with patients who received platinum/fluorouracil chemotherapy. Additionally, PD-L1 promoter methylation was significantly correlated with PD-L1 expression, and the progression of advanced gastric cancer. In conclusion, high methylation levels of the PD-L1 promoter region may be a faciliatory mechanism enabling gastric cancer tumorigenesis, and may also represent an independent prognostic factor for chemotherapeutic efficacy in patients with advanced gastric cancer.
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Project description:The aims of our study were to elucidate the role of methylation of a large panel of genes during multistage pathogenesis of bladder cancer and to correlate our findings with patient age and other clinicopathologic features.We studied the methylation status of 21 genes by quantitative methylation-specific PCR in an evaluation set of 25 tumor and 5 normal samples. Based on methylation frequency in tumors and normals in gene evaluation set, we selected 7 candidate genes and tested an independent set of 93 tumors and 26 normals. The presence or absence of methylation was evaluated for an association with cancer using cross-tabulations and chi(2) or Fisher's exact tests as appropriate. All statistical tests were two-sided.Most primary tumors (89 of 93, 96%) had methylation of one or more genes of independent set; 53 (57%) CCNA1, 29 (31%) MINT1, 36 (39%) CRBP, 53 (57%) CCND2, 66 (71%) PGP9.5, 60 (65%) CALCA, and 78 (84%) AIM1. Normal uroepithelium samples from 26 controls revealed no methylation of the CCNA1 and MINT1 genes, whereas methylation of CRBP, CCND2, PGP9.5, and CALCA was detected at low levels. All the 7 genes in independent set were tightly correlated with each other and 3 of these genes showed increased methylation frequencies in bladder cancer with increasing age. PGP9.5 and AIM1 methylation correlated with primary tumor invasion.Our results indicate that the methylation profile of novel genes in bladder cancers correlates with clinicopathologic features of poor prognosis and is an age-related phenomenon.
| S-EPMC2778751 | biostudies-literature
Project description:Glioma includes astrocytoma, oligodendroglioma, ependymoma and glioblastoma. We previously reported the epigenetic silencing of paternally expressed gene 3 (PEG3) in glioma cell lines. In this study, we investigated methylation of an exonic CpG island in the promoter region and the expression of PEG3 gene in 20 glioma and 5 non-tumor tissue samples. We found wide variations in the methylation level. Hypomethylaiton and hypermethylation was found in 3 and 4 glioma tissue samples, respectively. Monoallelic expression, which is an evidence of an imprinted gene, was maintained in eight out of nine informative cases which have T/C polymorphisms in PEG3. The lower gene expression, which suggested epigenetic silencing of PEG3, was confirmed statistically in glioblastoma using quantitative reverse-transcription polymerase chain reaction. Interestingly, we found higher expression of PEG3 in two out of three oligodendrogliomas. A negative correlation between the methylation level and gene expression was shown by regression analysis. These results suggest that the abnormal regulation of PEG3 is associated with several glioma subtypes and that it plays an important role in tumorigenesis.
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