DNA Methylation Pattern in Somatotroph Pituitary Neuroendocrine Tumors

Abstract

<b><i>Introduction:</i></b> Growth hormone secretion by sporadic somatotroph neuroendocrine pituitary tumors (PitNETs) is a major cause of acromegaly. These tumors are relatively heterogenous in terms of histopathological and molecular features. Our previous transcriptomic profiling of somatotroph tumors revealed three distinct molecular subtypes. This study aimed to investigate the difference in DNA methylation patterns in subtypes of somatotroph PitNETs and its role in distinctive gene expression. <b><i>Methods:</i></b> Genome-wide DNA methylation was investigated in 48 somatotroph PitNETs with EPIC microarrays. Gene expression was assessed with RNAseq. Bisulfite pyrosequencing and qRT-PCR were used for verifying the results of DNA methylation and gene expression. <b><i>Results:</i></b> Clustering tumor samples based on methylation data reflected the transcriptome-related classification. Subtype 1 tumors are densely granulated without <i>GNAS</i> mutation, characterized by high expression of <i>NR5A1</i> (SF-1) and <i>GIPR</i>. The expression of both genes is correlated with specific methylation of the gene body and promoter. This subtype has a lower methylation level of 5′ gene regions and CpG islands than the remaining tumors. Subtype 2 PitNETs are densely granulated and frequently <i>GNAS</i>-mutated, while those in subtype 3 are mainly sparsely granulated. Methylation/expression analysis indicates that ∼50% genes located in differentially methylated regions are those differentially expressed between tumor subtypes. Correlation analysis revealed DNA methylation-controlled genes, including <i>CDKN1B</i>, <i>CCND2</i>, <i>EBF3</i>, <i>CDH4</i>, <i>CDH12</i>, <i>MGMT</i>, <i>STAT5A</i>, <i>PLXND1</i>, <i>PTPRE</i>, and <i>MMP16</i>, and genes encoding ion channels and semaphorins. <b><i>Conclusion:</i></b> DNA methylation profiling confirmed the existence of three molecular subtypes of somatotroph PitNETs. High expression of <i>NR5A1</i> and <i>GIPR</i> in subtype 1 tumors is correlated with specific methylation of both genes.