Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b).

Pancreatic islet ß-cells that lack the MEN1-encoded protein menin develop into tumors. Such tumors express the phosphorylated isoform of the ß-cell differentiation transcription factor HLXB9. It is not known how phospho-HLXB9 acts as an oncogenic factor in insulin-secreting ß-cell tumors (insulinomas). In this study we investigated the binding partners and target genes of phospho-HLXB9 in mouse insulinoma MIN6 ß-cells. Co-immunoprecipitation coupled with mass spectrometry showed a significant association of phospho-HLXB9 with the survival factor p54nrb/Nono (54-kDa nuclear RNA-binding protein, non-POU-domain-containing octamer). Endogenous phospho-HLXB9 co-localized with endogenous Nono in the nucleus. Overexpression of HLXB9 decreased the level of overexpressed Nono but not endogenous Nono. Anti-phospho-HLXB9 chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) identified the c-Met inhibitor, Cblb, as a direct phospho-HLXB9 target gene. Phospho-HLXB9 occupied the promoter of Cblb and reduced the expression of Cblb mRNA. Cblb overexpression or HLXB9 knockdown decreased c-Met protein and reduced cell migration. Also, increased phospho-HLXB9 coincided with reduced Cblb and increased c-Met in insulinomas of two mouse models of menin loss. These data provide mechanistic insights into the role of phospho-HLXB9 as a pro-oncogenic factor by interacting with a survival factor and by promoting the oncogenic c-Met pathway. These mechanisms have therapeutic implications for reducing ß-cell proliferation in insulinomas by inhibiting phospho-HLXB9 or its interaction with Nono and modulating the expression of its direct (Cblb) or indirect (c-Met) targets. Our data also implicate the use of pro-oncogenic activities of phospho-HLXB9 in ß-cell expansion strategies to alleviate ß-cell loss in diabetes.

Epigenetic regulation in the tumorigenesis of MEN1-associated endocrine cell types.

Epigenetic regulation is emerging as a key feature in the molecular characteristics of various human diseases. Epigenetic aberrations can occur from mutations in genes associated with epigenetic regulation, improper deposition, removal or reading of histone modifications, DNA methylation/demethylation and impaired non-coding RNA interactions in chromatin. Menin, the protein product of the gene causative for the multiple endocrine neoplasia type 1 (MEN1) syndrome, interacts with chromatin-associated protein complexes and also regulates some non-coding RNAs, thus participating in epigenetic control mechanisms. Germline inactivating mutations in the MEN1 gene that encodes menin predispose patients to develop endocrine tumors of the parathyroids, anterior pituitary and the duodenopancreatic neuroendocrine tissues. Therefore, functional loss of menin in the various MEN1-associated endocrine cell types can result in epigenetic changes that promote tumorigenesis. Because epigenetic changes are reversible, they can be targeted to develop therapeutics for restoring the tumor epigenome to the normal state. Irrespective of whether epigenetic alterations are the cause or consequence of the tumorigenesis process, targeting the endocrine tumor-associated epigenome offers opportunities for exploring therapeutic options. This review presents epigenetic control mechanisms relevant to the interactions and targets of menin, and the contribution of epigenetics in the tumorigenesis of endocrine cell types from menin loss.

Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells.

The long noncoding RNA (lncRNA) MEG3 is significantly downregulated in pancreatic neuroendocrine tumors (PNETs). MEG3 loss corresponds with aberrant upregulation of the oncogenic hepatocyte growth factor (HGF) receptor c-MET in PNETs. Meg3 overexpression in a mouse insulin-secreting PNET cell line, MIN6, downregulates c-Met expression. However, the molecular mechanism by which MEG3 regulates c-MET is not known. Using chromatin isolation by RNA purification and sequencing (ChIRP-Seq), we identified Meg3 binding to unique genomic regions in and around the c-Met gene. In the absence of Meg3, these c-Met regions displayed distinctive enhancer-signature histone modifications. Furthermore, Meg3 relied on functional enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), to inhibit c-Met expression. Another mechanism of lncRNA-mediated regulation of gene expression utilized triplex-forming GA-GT rich sequences. Transfection of such motifs from Meg3 RNA, termed triplex-forming oligonucleotides (TFOs), in MIN6 cells suppressed c-Met expression and enhanced cell proliferation, perhaps by modulating other targets. This study comprehensively establishes epigenetic mechanisms underlying Meg3 control of c-Met and the oncogenic consequences of Meg3 loss or c-Met gain. These findings have clinical relevance for targeting c-MET in PNETs. There is also the potential for pancreatic islet ß-cell expansion through c-MET regulation to ameliorate ß-cell loss in diabetes.