Gene expression profiling in insulinomas of Men1 beta-cell mutant mice reveals early genetic and epigenetic events involved in pancreatic beta-cell tumorigenesis.

ABSTRACT

Mutations of the MEN1 gene lead to the occurrence of multiple endocrine neoplasia type 1 (MEN1). To gain insights into the mechanisms of the tumorigenesis related to MEN1 inactivation, we have used mice in which the Men1 gene was specifically disrupted in pancreatic beta-cells. In these mice, we observed full penetrance of insulinoma with defined histological characteristics of tumorigenesis. To identify the genetic factors taking part in the tumour development, we performed gene expression profiling analysis of these insulinomas at different stages. Here, we show that in late stage insulinomas, 56 genes are up-regulated and 194 are down-regulated more than fourfold compared with normal pancreatic islets. Clustering analysis reveals the deregulation of Hox gene family and the genes involved in cell proliferation and cell cycle control. The altered expression of Igf2, Igfbp3 and Igfbp6 as well as cyclin A2, B2 and D2 are confirmed by quantitative RT-PCR, with the overexpression of all the three cyclins found in early stage insulinomas. Moreover, an increased proportion of cyclin A2- and D2-expressing cells and the overexpression of insulin-like growth factor 2 (IGF2) protein are detected in mouse Men1 insulinomas by immunostaining. Interestingly, the analysis of DNA methylation patterns by quantitative serial pyrosequencing reveals that four specific CpGs in the intragenic differentially methylated region 2 (DMR2) region of the Igf2 gene known to augment transcription through methylation are significantly hypermethylated in insulinomas of Men1 beta-cell mutant mice at 6 and 10 months of age, even before IGF2 overexpression can be detected. Thus, our data indicate the involvement of both genetic and epigenetic mechanisms in early tumorigenesis of beta-cells related to MEN1 inactivation.