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      Methylation-associated down-regulation of RASSF1A and up-regulation of RASSF1C in pancreatic endocrine tumors

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          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.

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          Most cited references30

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          Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3.

          Allelic loss at the short arm of chromosome 3 is one of the most common and earliest events in the pathogenesis of lung cancer, and is observed in more than 90% of small-cell lung cancers (SCLCs) and in 50-80% of non-small-cell lung cancers (NSCLCs). Frequent and early loss of heterozygosity and the presence of homozygous deletions suggested a critical role of the region 3p21.3 in tumorigenesis and a region of common homozygous deletion in 3p21.3 was narrowed to 120 kb (ref. 5). Several putative tumour-suppressor genes located at 3p21 have been characterized, but none of these genes appear to be altered in lung cancer. Here we describe the cloning and characterization of a human RAS effector homologue (RASSF1) located in the 120-kb region of minimal homozygous deletion. We identified three transcripts, A, B and C, derived from alternative splicing and promoter usage. The major transcripts A and C were expressed in all normal tissues. Transcript A was missing in all SCLC cell lines analysed and in several other cancer cell lines. Loss of expression was correlated with methylation of the CpG-island promoter sequence of RASSF1A. The promoter was highly methylated in 24 of 60 (40%) primary lung tumours, and 4 of 41 tumours analysed carried missense mutations. Re-expression of transcript A in lung carcinoma cells reduced colony formation, suppressed anchorage-independent growth and inhibited tumour formation in nude mice. These characteristics indicate a potential role for RASSF1A as a lung tumour suppressor gene.
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            Wnt signaling regulates pancreatic beta cell proliferation.

            There is widespread interest in defining factors and mechanisms that stimulate proliferation of pancreatic islet cells. Wnt signaling is an important regulator of organ growth and cell fates, and genes encoding Wnt-signaling factors are expressed in the pancreas. However, it is unclear whether Wnt signaling regulates pancreatic islet proliferation and differentiation. Here we provide evidence that Wnt signaling stimulates islet beta cell proliferation. The addition of purified Wnt3a protein to cultured beta cells or islets promoted expression of Pitx2, a direct target of Wnt signaling, and Cyclin D2, an essential regulator of beta cell cycle progression, and led to increased beta cell proliferation in vitro. Conditional pancreatic beta cell expression of activated beta-catenin, a crucial Wnt signal transduction protein, produced similar phenotypes in vivo, leading to beta cell expansion, increased insulin production and serum levels, and enhanced glucose handling. Conditional beta cell expression of Axin, a potent negative regulator of Wnt signaling, led to reduced Pitx2 and Cyclin D2 expression by beta cells, resulting in reduced neonatal beta cell expansion and mass and impaired glucose tolerance. Thus, Wnt signaling is both necessary and sufficient for islet beta cell proliferation, and our study provides previously unrecognized evidence of a mechanism governing endocrine pancreas growth and function.
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              Role of the Ras-association domain family 1 tumor suppressor gene in human cancers.

              In recent years, the list of tumor suppressor genes (or candidate TSG) that are inactivated frequently by epigenetic events rather than classic mutation/deletion events has been growing. Unlike mutational inactivation, methylation is reversible and demethylating agents and inhibitors of histone deacetylases are being used in clinical trails. Highly sensitive and quantitative assays have been developed to assess methylation in tumor samples, early lesions, and bodily fluids. Hence, gene silencing by promoter hypermethylation has potential clinical benefits in early cancer diagnosis, prognosis, treatment, and prevention. The hunt for a TSG located at 3p21.3 resulted in the identification of the RAS-association domain family 1, isoform A gene (RASSF1A). RASSF1A falls into the category of genes frequently inactivated by methylation rather than mutational events. This gene is silenced and frequently inactivated by promoter region hypermethylation in many adult and childhood cancers, including lung, breast, kidney, gastric, bladder, neuroblastoma, medulloblastoma, gliomas and it has homology to a mammalian Ras effector (i.e., Nore1). RASSF1A inhibits tumor growth in both in vitro and in vivo systems, further supporting its role as a TSG. We and others identified the gene in 2000, but already there are over a 150 publications demonstrating RASSF1A methylation in a large number of human cancers. Many laboratories including ours are actively investigating the biology of this novel protein family. Thus far, it has been shown to play important roles in cell cycle regulation, apoptosis, and microtubule stability. This review summarizes our current knowledge on genetic, epigenetic, and functional analysis of RASSF1A tumor suppressor gene and its homologues.
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                Author and article information

                Journal
                BMC Cancer
                BMC Cancer
                BioMed Central
                1471-2407
                2011
                12 August 2011
                : 11
                : 351
                Affiliations
                [1 ]Department of Pathology and Diagnostics, University of Verona, Verona, Italy
                [2 ]ARC-NET Center for Applied Research on Cancer, the Hospital and University of Verona, Verona, Italy
                [3 ]Department of Pathology and Laboratory Medicine, National Cancer Institute and University of Milan, Milan, Italy
                [4 ]Department of Surgery and Oncology, the Hospital and University of Verona, Verona, Italy
                Article
                1471-2407-11-351
                10.1186/1471-2407-11-351
                3170651
                21838870
                c864240c-3d79-489f-bd13-1744849936cb
                Copyright ©2011 Malpeli et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 31 January 2011
                : 12 August 2011
                Categories
                Research Article

                Oncology & Radiotherapy
                Oncology & Radiotherapy

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