Aberrant methylation and chromosomal alterations involving RASSF1 locus in pancreatic neoplasms

RASSF1 is a tumour suppressor gene, whose promoter hypermethylation has been suggested as a major event in the pathogenesis of different tumors, including pancreatic endocrine tumor (PETs) and the more aggressive pancreatic ductal adenocarcinoma (PDAC). Furthermore, allelic losses at 3p21.3, which includes the locus of RASSF1, have been recently related to PET. Despite this, previous studies did not give a direct proof that RASSF1 reduced expression was related to its promoter methylation. In this study we conducted an exhaustive analysis of RASSF1 status at both genetic and epigenetic level in order to better define its role as a putative marker of pancreatic cancers as well as clearly depict RASSF1-associated transcription regulatory event in these malignancies. Specifically, we analyzed both in PET and PDAC: (i) the methylation status of RASSF1 by methylation-specific PCR and sequencing (ii) the expression of RASSF1 variants by quantitative RT-PCR and immunofluorescence (iii) RASSF1 DNA copy number by fluorescence in situ hybridization (FISH) in a larger series of tumor specimens. Our analysis dealing with PET indicated the existence in the RASSF1 locus of a methylation-associated relay circuitry down-regulating RASSF1A and boosting RASSF1C, thus highlighting methylation as a fine-tuning process of RASSF1 gene activity in PET. Looking at copy number status, RASSF1 locus alterations were found in 25% of PET cases, with losses predominant over gains (19% vs. 6%). Interestingly, the higher proportion of monosomic cells in PET was positively correlated with an advanced stage of disease. Otherwise, the lower RASSF1A expression found in PDAC with respect to normal pancreas was not associated with either inhibitory or tuning effect of methylation on RASSF1A transcription. Surprisingly, FISH analysis despite the high frequency of 3p loss among PDAC cases (52% of losses vs 20% of gains), highlighted the excess of CEP3 copy number as the best prognostic factor affecting survival of PDAC patients. We can conclude that the RASSF1 locus suffers of changes at epigenetic and transcriptional level during tumorigenesis but methylation cannot be considered a decisive marker lesion for PET and PDAC. On the other hand, down-regulation of RASSF1A and alterations in copy number of chromosome 3p may represent relevant events for the pathogenesis and progression of both types of tumors.