title: Methylation-associated down-regulation of RASSF1A and up-regulation of RASSF1C in pancreatic endocrine tumors.
abstract: BACKGROUND
  RASSF1A 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.
  
  
  METHODS
  We 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.
  
  
  RESULTS
  MSP 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.
  
  
  CONCLUSIONS
  RASSF1A 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.
authors.affiliation: Department of Pathology and Diagnostics, University of Verona, Verona, Italy. giorgio.malpeli@univr.it
authors.forename: Giorgio
authors.initials: G
authors.lastname: Malpeli
authors.forename: Eliana
authors.initials: E
authors.lastname: Amato
authors.forename: Mario
authors.initials: M
authors.lastname: Dandrea
authors.forename: Caterina
authors.initials: C
authors.lastname: Fumagalli
authors.forename: Valentina
authors.initials: V
authors.lastname: Debattisti
authors.forename: Letizia
authors.initials: L
authors.lastname: Boninsegna
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pelosi
authors.forename: Massimo
authors.initials: M
authors.lastname: Falconi
authors.forename: Aldo
authors.initials: A
authors.lastname: Scarpa
mesh_terms: D000328:Adult; D000368:Aged; D001374:Azacitidine / Q000031:analogs & derivatives; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D000077209:Decitabine; D015536:Down-Regulation; D005091:Exons; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016014:Linear Models; D008297:Male; D008875:Middle Aged; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D018709:Statistics, Nonparametric; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D020033:Protein Isoforms; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; D000077209:Decitabine; D001374:Azacitidine