Dataset Information

RASSF1A Site-Specific Methylation Hotspots in Cancer and Correlation with RASSF1C and MOAP-1.

ABSTRACT: Epigenetic silencing of RASSF1A is frequently observed in numerous cancers and has been previously reported. The promoter region of RASSF1A is predicted to have 75 CpG sites, and very few studies demonstrate how the methylation of these sites affects expression. In addition, the expression relationship between RASSF1A and its downstream target, modulator of apoptosis 1 (MOAP-1), is poorly understood. In this study, we have explored the mRNA expression of RASSF1A, MOAP-1 and the well-characterized splice variant of RASSF1, RASSF1C, in cancer cell lines and primary tumors. We confirmed that the RASSF1A promoter is robustly methylated within a 32-CpG region in solid tumors and results in lower mRNA expression. The MOAP-1 promoter contains ~110 CpG sites, but was not found to be methylated in cancer cell lines when 19 predicted CpG sites were explored. Interestingly, MOAP-1 mRNA expression positively correlated with RASSF1A expression in numerous cancers, whereas RASSF1C expression remained the same or was increased in cell lines or tissues with epigenetic loss of RASSF1A. We speculate that MOAP-1 and RASSF1A may be more intimately connected than originally thought, and the expression of both are warranted in experimental designs exploring the biology of the RASSF1A/MOAP-1 molecular pathway.

SUBMITTER: Volodko N

PROVIDER: S-EPMC4931620 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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RASSF1A Site-Specific Methylation Hotspots in Cancer and Correlation with RASSF1C and MOAP-1.

Volodko Natalia N Salla Mohamed M Zare Alaa A Abulghasem El-Arbi el-A Vincent Krista K Benesch Matthew G K MG McMullen Todd P W TP Bathe Oliver F OF Postovit Lynne L Baksh Shairaz S

Cancers 20160610 6

Epigenetic silencing of RASSF1A is frequently observed in numerous cancers and has been previously reported. The promoter region of RASSF1A is predicted to have 75 CpG sites, and very few studies demonstrate how the methylation of these sites affects expression. In addition, the expression relationship between RASSF1A and its downstream target, modulator of apoptosis 1 (MOAP-1), is poorly understood. In this study, we have explored the mRNA expression of RASSF1A, MOAP-1 and the well-characterize ...[more]

PMID: 27294960

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Project description:BackgroundRASSF1A gene silencing by DNA methylation has been suggested as a major event in pancreatic endocrine tumor (PET) but RASSF1A expression has never been studied. The RASSF1 locus contains two CpG islands (A and C) and generates seven transcripts (RASSF1A-RASSF1G) by differential promoter usage and alternative splicing.MethodsWe studied 20 primary PETs, their matched normal pancreas and three PET cell lines for the (i) methylation status of the RASSF1 CpG islands using methylation-specific PCR and pyrosequencing and (ii) expression of RASSF1 isoforms by quantitative RT-PCR in 13 cases. CpG island A methylation was evaluated by methylation-specific PCR (MSP) and by quantitative methylation-specific PCR (qMSP); pyrosequencing was applied to quantify the methylation of 51 CpGs also encompassing those explored by MSP and qMSP approaches.ResultsMSP detected methylation in 16/20 (80%) PETs and 13/20 (65%) normal pancreas. At qMSP, 11/20 PETs (55%) and 9/20 (45%) normals were methylated in at least 20% of RASSF1A alleles.Pyrosequencing showed variable distribution and levels of methylation within and among samples, with PETs having average methylation higher than normals in 15/20 (75%) cases (P = 0.01). The evaluation of mRNA expression of RASSF1 variants showed that: i) RASSF1A was always expressed in PET and normal tissues, but it was, on average, expressed 6.8 times less in PET (P = 0.003); ii) RASSF1A methylation inversely correlated with its expression; iii) RASSF1 isoforms were rarely found, except for RASSF1B that was always expressed and RASSF1C whose expression was 11.4 times higher in PET than in normal tissue (P = 0.001). A correlation between RASSF1A expression and gene methylation was found in two of the three PET cell lines, which also showed a significant increase in RASSF1A expression upon demethylating treatment.ConclusionsRASSF1A gene methylation in PET is higher than normal pancreas in no more than 75% of cases and as such it cannot be considered a marker for this neoplasm. RASSF1A is always expressed in PET and normal pancreas and its levels are inversely correlated with gene methylation. Isoform RASSF1C is overexpressed in PET and the recent demonstration of its involvement in the regulation of the Wnt pathway points to a potential pathogenetic role in tumor development.

Dataset Information

RASSF1A Site-Specific Methylation Hotspots in Cancer and Correlation with RASSF1C and MOAP-1.

Publications

RASSF1A Site-Specific Methylation Hotspots in Cancer and Correlation with RASSF1C and MOAP-1.

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