Aberrant methylation of SPARC in human lung cancers.
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ABSTRACT:
SUBMITTER: Suzuki M
PROVIDER: S-EPMC2361924 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
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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.
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Project description:MicroRNAs (miRs) contribute to cancer development and progression by acting as oncogenes and tumor suppressor genes. miR-9 family members (miR-9s), including miR-9-1, 9-2 and 9-3, have been shown to be oncogenically involved through the downregulation of E-cadherin expression, which promotes the epithelial-mesenchymal transition. Tumor suppressive roles of miR-9s have also been reported to silence miR-9 through methylation, which is associated with an shortened overall survival (OS) period in several types of cancer. In this study, the impact of miR-9s methylation on non-small cell lung cancers (NSCLC) was investigated. In total, 293 resected NSCLC samples were examined and the miR-9s methylation status was determined using a combined bisulfite restriction analysis. miR-9 expression was analyzed by in situ hybridization. Methylation of miR-9-1, 9-2 and 9-3 was present in 20 (7%), 33 (11%) and 34 (12%) of the cases, respectively. Methylation of any miR-9s (miR-9s methylation) was observed in 76 of the cases (26%), and miR-9 expression was silenced in cases with miR-9s methylation. Logistic regression analysis demonstrated that male gender [odds ratio (OR), 2.0; 95% confidence interval (95% CI), 1.1-3.6; P=0.01] and pathologically negative lymph node metastasis (OR, 4.8; 95% CI, 1.4-17.2; P=0.002) were independent relative factors for miR-9s methylation. Additionally, miR-9s methylation [hazard ratio (HR), 4.2; 95% CI, 1.2-27.0; P=0.026] and early pathological stage (HR, 8.3; 95% CI, 2.1-28.6; P=0.004) were found to be independent predictive factors for prolonged OS time by the Cox proportional hazard test. miR-9s methylation which induces expression silencing is common in NSCLC cases without lymph nodal metastasis, suggesting that miR-9s are oncogenically involved in NSCLC carcinogenesis through the promotion of tumor metastasis.
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Project description:Secreted protein acidic and rich in cysteine (SPARC) gene has been shown to be epigenetically silenced in several cancers. We investigated the loss of expression and promoter methylation of this tumor suppressor gene in gastric cancers and correlated the data with clinicopathological features. We observed the loss of SPARC mRNA and SPARC protein expression in 7 of 10 (70%) gastric cancer cell lines. Upon treatment of expression-negative cell lines with a demethylating agent, expression of mRNA and protein was restored in all cells. Methylation rate of SPARC gene was 80% in ten gastric cancer cell lines and 74% (163 of 220) in primary tumors, while it was 5% in normal gastric mucosa (n = 40). In intestinal gastric cancer, SPARC methylation correlated with a negative prognosis (P < 0.001; relative risk 2.754, 95% confidence interval 1.780-4.261). Immunostaining revealed that SPARC protein was overexpressed in stromal fibroblasts adjacent to neoplastic epithelium but rarely expressed in the primary gastric cancer cells. These results implicate SPARC promoter methylation as an important factor in the tumorigenesis of gastric carcinomas and provide new insights into the potential use of SPARC as a novel biomarker and the potential clinical importance in human gastric cancers.
| S-EPMC4268651 | biostudies-other
Project description:BackgroundCancer is a broad group of genetic diseases which account for millions of deaths worldwide each year. Cancers are classified by various clinical, pathological and molecular methods, but even within a well-characterized disease, there is a significant inter-patient variability in survival, response to treatment, and other parameters. Especially in molecular level, tumours of the same category can appear significantly dissimilar due to complex combinations of genetic aberrations leading to a similar malignancy. We extended the current classification methods by studying tumour heterogeneity at pathway level.MethodsWe computed the rate of alterations in 1994 pathways and 2210 tumours consisting of eight different cancers. Using gene set enrichment analysis, each sample was computed a pathway aberration profile that reflected its molecular state. The profiles were analysed together to infer the characteristic aberration rates for each pathway within each cancer. Subgroups of tumours defined by similar pathway aberrations were identified using clustering analyses. The pathway aberration and gene expression profiles of the subgroups were consecutively compared across all eight cancer types to search for similar tumours crossing the standard classification.ResultsWe identified pathways and processes that were common to all cancers as well as traits that are unique to a cancer type or closely related cancers. Studying the gene expression patterns within the pathway context suggested potential alteration mechanisms. Clustering analysis revealed five clinically relevant subgroups of tumours in four cancers that exhibited significant differences in survival compared to others. The cross-cancer analysis of the subgroups resulted in the identification of tumours that shared potentially significant alterations.ConclusionsThis study represents the first effort to extend the molecular characterizations towards pathway level descriptions across the family of cancers. In addition to providing a proof-of-concept for single sample pathway aberration analysis in this context, we present a comprehensive pathway aberration dataset that can be used to study pathway aberration patterns within or across cancers. Significant similarities between subgroups of different cancers on pathway and gene expression levels provide interesting hypotheses for understanding variable drug response, or transferring treatments across diseases by identifying common druggable pathways or genes, for example.
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Project description:BackgroundADAMs (a disintegrin and metalloproteinase) have long been associated with tumor progression. Recent findings indicate that members of the closely related ADAMTS (ADAMs with thrombospondin motifs) family are also critically involved in carcinogenesis. Gene silencing through DNA methylation at CpG loci around e.g. transcription start or enhancer sites is a major mechanism in cancer development. Here, we aimed at identifying genes of the ADAM and ADAMTS family showing altered DNA methylation in the development or colorectal cancer (CRC) and other epithelial tumors.MethodsWe investigated potential changes of DNA methylation affecting ADAM and ADAMTS genes in 117 CRC, 40 lung cancer (LC) and 15 oral squamous-cell carcinoma (SCC) samples. Tumor tissue was analyzed in comparison to adjacent non-malignant tissue of the same patients. The methylation status of 1145 CpGs in 51 ADAM and ADAMTS genes was measured with the HumanMethylation450 BeadChip Array. ADAMTS16 protein expression was analyzed in CRC samples by immunohistochemistry.ResultsIn CRC, we identified 72 CpGs in 18 genes which were significantly affected by hyper- or hypomethylation in the tumor tissue compared to the adjacent non-malignant tissue. While notable/frequent alterations in methylation patterns within ADAM genes were not observed, conspicuous changes were found in ADAMTS16 and ADAMTS2. To figure out whether these differences would be CRC specific, additional LC and SCC tissue samples were analyzed. Overall, 78 differentially methylated CpGs were found in LC and 29 in SCC. Strikingly, 8 CpGs located in the ADAMTS16 gene were commonly differentially methylated in all three cancer entities. Six CpGs in the promoter region were hypermethylated, whereas 2 CpGs in the gene body were hypomethylated indicative of gene silencing. In line with these findings, ADAMTS16 protein was strongly expressed in globlet cells and colonocytes in control tissue but not in CRC samples. Functional in vitro studies using the colorectal carcinoma cell line HT29 revealed that ADAMTS16 expression restrained tumor cell proliferation.ConclusionsWe identified ADAMTS16 as novel gene with cancer-specific promoter hypermethylation in CRC, LC and SCC patients implicating ADAMTS16 as potential biomarker for these tumors. Moreover, our results provide evidence that ADAMTS16 may have tumor suppressor properties.
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Project description:The p16INK4a (p16) tumor suppressor gene is frequently inactivated by homozygous deletion or methylation of the 5' CpG island in cell lines derived from human non-small-cell lung cancers. However, the frequency of dysfunction in primary tumors appears to be significantly lower than that in cell lines. This discordance could result from the occurrence or selection of p16 dysfunction during cell culture. Alternatively, techniques commonly used to examine tumors for genetic and epigenetic alterations may not be sensitive enough to detect all dysfunctions within the heterogeneous cell population present in primary tumors. If p16 inactivation plays a central role in development of non-small-cell lung cancer, then the frequency of gene inactivation in primary tumors should parallel that observed in cell lines. The present investigation addressed this issue in primary rat lung tumors and corresponding derived cell lines. A further goal was to determine whether the aberrant p16 gene methylation seen in human tumors is a conserved event in this animal model. The rat p16 gene was cloned and sequenced, and the predicted amino acid sequence of its product found to be 62% homologous to the amino acid sequence of the human analog. Homozygous deletion accounted for loss of p16 expression in 8 of 20 cell lines, while methylation of the CpG island extending throughout exon 1 was observed in 9 of 20 cell lines. 2-Deoxy-5-azacytidine treatment of cell lines with aberrant methylation restored gene expression. The methylated phenotype seen in cell lines showed an absolute correlation with detection of methylation in primary tumors. Aberrant methylation was also detected in four of eight primary tumors in which the derived cell line contained a deletion in p16. These results substantiate the primary tumor as the origin for dysfunction of the p16 gene and implicate CpG island methylation as the major mechanism for inactivating this gene in the rat lung tumors examined. Furthermore, rat lung cancer appears to be an excellent model in which to investigate the mechanisms of de novo gene methylation and the role of p16 dysfunction in the progression of neoplasia.
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Project description:Epigenetic changes through altered DNA methylation have been implicated in critical aspects of tumor progression, and have been extensively studied in a variety of cancer types. In contrast, our current knowledge of the aberrant genomic DNA methylation in tumor-associated fibroblasts (TAFs) or other stromal cells that act as critical coconspirators of tumor progression is very scarce. To address this gap of knowledge, we conducted genome-wide DNA methylation profiling on lung TAFs and paired control fibroblasts (CFs) from non-small cell lung cancer patients using the HumanMethylation450 microarray. We found widespread DNA hypomethylation concomitant with focal gain of DNA methylation in TAFs compared to CFs. The aberrant DNA methylation landscape of TAFs had a global impact on gene expression and a selective impact on the TGF-? pathway. The latter included promoter hypermethylation-associated SMAD3 silencing, which was associated with hyperresponsiveness to exogenous TGF-?1 in terms of contractility and extracellular matrix deposition. In turn, activation of CFs with exogenous TGF-?1 partially mimicked the epigenetic alterations observed in TAFs, suggesting that TGF-?1 may be necessary but not sufficient to elicit such alterations. Moreover, integrated pathway-enrichment analyses of the DNA methylation alterations revealed that a fraction of TAFs may be bone marrow-derived fibrocytes. Finally, survival analyses using DNA methylation and gene expression datasets identified aberrant DNA methylation on the EDARADD promoter sequence as a prognostic factor in non-small cell lung cancer patients. Our findings shed light on the unique origin and molecular alterations underlying the aberrant phenotype of lung TAFs, and identify a stromal biomarker with potential clinical relevance.
| S-EPMC4662832 | biostudies-other
Project description:We identify a gene, SLC5A8, and show it is a candidate tumor suppressor gene whose silencing by aberrant methylation is a common and early event in human colon neoplasia. Aberrant DNA methylation has been implicated as a component of an epigenetic mechanism that silences genes in human cancers. Using restriction landmark genome scanning, we performed a global search to identify genes that would be aberrantly methylated at high frequency in human colon cancer. From among 1,231 genomic NotI sites assayed, site 3D41 was identified as methylated in 11 of 12 colon cancers profiled. Site 3D41 mapped to exon 1 of SLC5A8, a transcript that we assembled. In normal colon mucosa we found that SLC5A8 exon 1 is unmethylated and SLC5A8 transcript is expressed. In contrast, SLC5A8 exon 1 proved to be aberrantly methylated in 59% of primary colon cancers and 52% of colon cancer cell lines. SLC5A8 exon 1 methylated cells were uniformly silenced for SLC5A8 expression, but reactivated expression on treatment with a demethylating drug, 5-azacytidine. Transfection of SLC5A8 suppressed colony growth in each of three SLC5A8-deficient cell lines, but showed no suppressive effect in any of three SLC5A8-proficient cell lines. SLC5A8 exon 1 methylation is an early event, detectable in colon adenomas, and in even earlier microscopic colonic aberrant crypt foci. Structural homology and functional testing demonstrated that SLC5A8 is a member of the family of sodium solute symporters, which are now added as a class of candidate colon cancer suppressor genes.
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Project description:Lung adenocarcinoma (LUAD) is a common diagnosed disease with high-mortality rate, and its prognostic implications are under discovered. DNA methylation aberrations are not only an important event for dysregulation of gene expression during tumorigenesis but also a revolution in epigenetics by identifying key prognostic biomarkers for multiple cancers. In this study, we analyzed methylation status of 485 578 CpG sites and RNA-seq transcriptomes of 20 532 genes for 1095 LUAD samples in TCGA database. The association between DNA methylation and the prognostic value of the corresponding gene expression was identified as well. In total, ten aberrantly methylated and dysregulated genes (AURKA, BLK, CNTN2, HMGA1, PTTG1, TNS4, DAPK2, MFSD2A, THSD1, and WNT7A) were highlighted which were significantly correlated with overall survival of 492 LUAD patients, which were all reported as tumor-associated genes in other various cancers and worthy of further investigated and might be used as therapeutic targets for LUAD. Together, methylation aberrances regulate gene expression level during tumorigenesis and influence prognosis of LUAD patients. Integrating knowledge of epigenetics and expression of genes can be useful for an in-depth understanding of cancer mechanism and for the eventual purpose of precisely prognostic and therapeutic target verification.
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