Demethylation status of somatic DNA extracted from pituitary neuroendocrine tumors indicates proliferative behavior

Abstract

Background: Cytosine intermediaries 5-methylcytosine (5mC) and 5-hydroxymethylcytosine(5hmC), epigenetic hallmarks, have never been investigated in pituitary neuroendocrine tumors(PitNET).Objective: To examine methylation-demethylation status of global deoxyribonucleic acid (DNA)in PitNET tissues and to assess its correlation with clinical and biological parameters.Materials and Methods: Altogether, 57 PitNET and 25 corresponding plasma samples werecollected. 5mC and 5hmC were investigated using liquid chromatography-tandem massspectrometry. Expression of DNA methyltransferase 1 (DNMT1); tet methylcytosine dioxygenase1 through 3 (TET1-3); and ubiquitin-like, containing PHD and RING finger domains 1 and2 (UHRF1-2) were measured by reverse transcription-polymerase chain reaction. Levels of5hmC and UHRF1-2 were explored by immunohistochemistry. Effect of demethylating agentdecitabine was tested on pituitary cell lines.Results: 5hmC/5mC ratio was higher in less differentiated PitNET samples. A negativecorrelation between Ki-67 proliferation index and 5hmC, 5hmC to 5mC ratio were revealed.Higher 5mC was observed in SF-1 + gonadotroph adenomas with a higher Ki-67 index.Expressions of TET2 and TET3 were significantly higher in adenomas with higher proliferationrate. UHRF1 showed gradually increased expression in higher proliferative adenoma samples,and a significant positive correlation was detected between UHRF2 expression and 5hmC level.Decitabine treatment significantly decreased 5mC and increased 5hmC levels in both cell lines,accompanied with decreased cell viability and proliferation.Conclusion: The demethylation process negatively correlated with proliferation rate andthe ratio of 5hmC to 5mC was higher in less differentiated adenomas. Therefore, epigenetic markers can be potential biomarkers for PitNET behavior. Altering the epigenome in adenomacells by decitabine decreased proliferation, suggesting that this treatment might be a novelmedical treatment for PitNET.