RASSF1 è un gene soppressore tumorale, la cui ipermetilazione del promotore è stata associata alla patogenesi di diversi tipi di tumore, tra cui i tumori endocrini del pancreas (PET) e il più aggressivo adenocarcinoma pancreatico (PDAC). La perdita allelica della regione 3p21.3, in cui è compreso il locus di RASSF1, è inoltre un evento che è stato recentemente associato all’insorgenza dei PET. Tuttavia, studi precedenti non hanno fornito un prova sperimentale diretta dell’effettiva riduzione dell’espressione di RASSF1A, in relazione alla metilazione. In questo studio è stata condotta un’analisi approfondita dello stato di RASSF1 a livello genetico ed epigenetico al fine di definirne il ruolo come putativo marcatore per la diagnosi precoce di cancro del pancreas; inoltre, scopo di questo lavoro è stato anche quello di cercare di delineare chiaramente l’evento regolatorio associato alla trascrizione di RASSF1 in questi tipi di neoplasie. In particolare, abbiamo determinato in PET e PDAC: (i) lo stato di metilazione di RASSF1 utilizzando la PCR metilazione-specifica ed il sequenziamento del DNA (ii) l’espressione delle varianti di mRNA di RASSF1 tramite RT-PCR e immunofluorescenza (iii) il numero di copie del gene RASSF1 mediante l’impiego dell’ibridazione fluorescente in situ (FISH) su una larga casistica di tumori endocrini e metastasi. La nostra analisi sui PET ha indicato l’esistenza di un circuito regolatorio associato alla metilazione che comporta la downregolazione di RASSF1A e l’incremento di RASSF1C, evidenziando quindi la metilazione come un processo che regola finemente l’attività del gene RASSF1 nei PET. Per quanto riguarda il numero di copie del gene d’interesse, sono state trovate alterazioni del locus di RASSF1 nel 25% dei casi con perdite predominanti rispetto a guadagni (19% vs 6%). In particolare, la più alta proporzione di cellule monosomiche nei PET correlava con uno stadio più avanzato della malattia. Diversamente, nei PDAC la ridotta espressione di RASSF1A nei PET rispetto al tessuto normale non era invece associata ad alcun effetto inibitorio o regolatorio della metilazione sulla trascrizione di RASSF1A. Nonostante l’elevata frequenza di perdita del 3p riscontrata nei casi di PDAC a disposizione (52% di perdite e 20% di guadagni), l’analisi FISH ha evidenziato che il numero in eccesso di copie del centromero del cromosoma 3 risulta il miglior fattore prognostico per la sopravvivenza dei pazienti affetti da PDAC. In conclusione quindi il locus di RASSF1 è caratterizzato da cambiamenti a livello epigenetico e trascrizionale durante la tumorigenesi, ma la metilazione non può essere considerata un marker decisivo per PET e PDAC. D’altra parte, la downregolazione di RASSF1A e le alterazioni nel numero di copie del cromosoma 3p potrebbero rappresentare eventi rilevanti per la patogenesi e la progressione di entrambi i tipi di neoplasie pancreatiche.
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.
Aberrant methylation and chromosomal alterations involving RASSF1 locus in pancreatic neoplasms
AMATO, Eliana
2010-01-01
Abstract
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.File | Dimensione | Formato | |
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