ECE2022 Eposter Presentations Calcium and Bone (114 abstracts)
Aberrant promoter methylation, expression and function of RASSF1A gene in a series of Italian parathyroid tumors
Chiara Verdelli 1 , Fabrizio Federico Pio 2 , Giulia Stefania Tavanti 1,3 , Paola Maroni 4 , Annamaria Morotti 5 , Vito Guarnieri 6 , Elena Pardi 7 , Filomena Cetani 8 , Alfredo Scillitani 9 , Riccardo Maggiore 10 , Valentina Vaira 11 , Lucia Anna Muscarella 2 & Sabrina Corbetta 12
1Laboratory of Experimental Endocrinology, IRCCS Istituto Ortopedico Galeazzi, Milano, Italy; 2Laboratory of Oncology, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy; 3, Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milano, Italy; 4Laboratory of experimental Biochemistry & Molecular Biology, IRCCS Istituto Ortopedico Galeazzi, Milano, Italy; 5Department of Pathophysiology and Transplantation, University of Milan, Milan, Milan, Italy; 6Division of Medical Genetics, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy; 7Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy; 8Endocrine Unit, University Hospital of Pisa, Pisa, Italy; 9Endocrinology Unit, IRCCS Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy; 10Endocrine Surgery, IRCCS Ospedale San Raffaele, Milan, Italy; 11Division of Pathology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Department of Pathophysiology and Organ Transplantation, University of Milan, Milan, Italy; 12Endocrinology and Diabetology Service, IRCCS Istituto Ortopedico Galeazzi, Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milano, Italy.
Aberrant epigenetic features occurring in parathyroid tumors involve DNA methylation, histone methylation, and non-coding RNAs. RASSF1A and APC were frequently downregulated in human cancers. Here, we investigated RASSF1A and APC methylation status in a series of parathyroid tumors from Italian patients with primary hyperparathyroidism (PAds, n=80), confirming RASSF1A and APC promoter methylation as a hallmark of sporadic parathyroid adenomas. Morever, we extended the analysis in parathyroid carcinomas (PCas, n=9), which displayed RASSF1A promoter methylation, while APC promoter was methylated only in 2 samples. In PAds, RASSF1A promoter methylation levels positively correlated with the methylation levels of APC promoter, suggesting a common methylation process for both genes. We focused the attention on the oncosuppressor RASSF1A. Our results showed that RASSF1A transcripts were significantly reduced in PAds (n=35) when compared with normal parathyroid glands (PaNs; n=3), though RASSF1A mRNA levels and levels of RASSF1A promoter methylation did not correlate. At protein level, RASSF1A was detectable by immunohistochemistry in the cytoplasm of cells in PaNs (n=3) and in the rim of PaN surrounding parathyroid adenomas, while cells in PAds (n=11) showed weakly positive citoplasmic staining. PCas (n=6) were definitely negative both at cytoplasmic and nucelar levels. Furthermore, we investigated 2 potential epigenetic modifiers involved in RASSF1A promoter methylation: the methylatransferase DNMT1 and the antisense lncRNA RASSF1-AS1. DNMT1 methylates both RASSF1A and APC gene promoters. We found that DNMT activity, investigated in PAds nuclear extracts (n=16), was inversely correlated with RASSF1A protein levels (r2=0.400, P=0.009), supporting the involvement of deregulated DNMT activity in the aberrant RASSF1A promoter methylation. RASSF1-AS1 (alias ANRASSF1) was implicated in a locus-specific mechanism for the RASSF1A epigenetic repression, mediated by Polycomb Repressive Complex 2 (PRC2), reinforcing RASSF1A long-term epigenetic silencing. In PAds, ANRASSF1 levels positively correlated with expression levels of RASSF1A (r=0.788, P=0.0001). Similarly, RASSF1A promoter methylation negatively correlated with ANRASSF1A mRNA levels (r=−0.366, P=0.031). These findings exclude ANRASSF1 in the methylation process of the RASSF1A promoter in PAds. Finally, using HEK293A cells transiently transfected with human CASR as experimental model (CASR-HEK293A), we investigated the effects of RASSF1A gene silencing on pERK/ERK levels stimulated by the CASR agonist R568. Efficient RASSF1A silencing increased basal pERK/ERK levels and blunted the pERK/ERK increase induced by CASR activation, suggesting that loss of RASSF1A may contribute to the parathyroid cell desensitization towards extracellular calcium concentrations observed in parathyroid tumors.
In conclusion, RASSF1A and APC promoter methylation is a hallmark of parathyroid adenomas.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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