Methylation-associated down-regulation of RASSF1A and up-regulation of RASSF1Cin pancreatic endocrine tumors

Abstract

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.

RASSF tumor suppressor gene family: Biological functions and regulation

“…Pyrosequencing revealed high variability of a distribution and level of RASSF1A promoter methylation within and among pancreatic cancer samples [129]. The same variability was shown ten years earlier in breast cancer using an oligonucleotide-based microarray technique [130].…”

Genetic and epigenetic drivers of neuroendocrine tumours (NET)

“…Several studies indicated RASSF1A as one of the most hypermethylated genes in PanNET and more frequently in metastatic tumours compared to nonmetastatic ones. Interestingly, RASSF1A promoter was found methylated in normal pancreas as well, although to a lower level (Dammann et al 2003, Malpeli et al 2011. We found VHL promoter (VHL-associated gene) hyper-methylation in a subset of PanNET in correlation with intra-tumoural hypoxia and poor prognosis (Schmitt et al 2009).…”

Neuroendocrine tumours: cracking the epigenetic code

“…Separate studies have identified RASSF1 promoter methylation in 80-83% of pNETs making it the most frequently altered gene in sporadic pNET identified to date. In some cases, methylation is also identified in matched normal pancreas, however at a lower level of expression (Dammann et al 2003a, Malpeli et al 2011. RASSF1 methylation has been noted to be more frequent in metastatic tumours (100%) than non-metastatic (71%) (Malpeli et al 2011).…”

“…In contrast to RASSF1A, the RASSF1C isoform expression is 11.4 times higher in pNET than in normal tissue (PZ0.001) (Malpeli et al 2011). RASSF1C has been shown to have a role inhibiting b-catenin degradation and given the above findings may also have a pathogenic role in pNET development (Estrabaud et al 2007).…”

“…In some cases, methylation is also identified in matched normal pancreas, however at a lower level of expression (Dammann et al 2003a, Malpeli et al 2011. RASSF1 methylation has been noted to be more frequent in metastatic tumours (100%) than non-metastatic (71%) (Malpeli et al 2011). Methylation of the RASSF1 promoter occurs less frequently in pancreatic ductal adenocarcinoma (Dammann et al 2003a).…”