Gastrin Induces Nuclear Export and Proteasome Degradation of Menin in Enteric Glial Cells.

BACKGROUND & AIMS: The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner's glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. METHODS: Primary enteric glial cultures were generated from the VillinCre:Men1FL/FL:Sst-/- mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. RESULTS: Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas. CONCLUSIONS: MEN1-associated gastrinomas, which develop in the submucosa, might arise from enteric glial cells through hormone-dependent PKA signaling. This pathway disrupts nuclear menin function, leading to hypergastrinemia and associated sequelae.

Pathophysiology of Gastric NETs: Role of Gastrin and Menin.

PURPOSE OF REVIEW: Neuroendocrine tumors (NETs) were initially identified as a separate entity in the early 1900s as a unique malignancy that secretes bioactive amines. GI-NETs are the most frequent type and represent a unique subset of NETs, because at least 75% of these tumors represent gastrin stimulation of the enterochromaffin-like cell located in the body of the stomach. The purpose of this review is to understand the specific role of gastrin in the generation of Gastric NETs (G-NETs). RECENT FINDINGS: We review here the origin of enterochromaffin cells gut and the role of hypergastrinemia in gastric enteroendocrine tumorigenesis. We describe generation of the first genetically engineered mouse model of gastrin-driven G-NETs that mimics the human phenotype. The common mechanism observed in both the hypergastrinemic mouse model and human carcinoids is translocation of the cyclin-dependent inhibitor p27kip to the cytoplasm and its subsequent degradation by the proteasome. Therapies that block degradation of p27kip, the CCKBR2 gastrin receptor, or gastrin peptide are likely to facilitate treatment.

Deletion of Men1 and somatostatin induces hypergastrinemia and gastric carcinoids.

BACKGROUND: Gastric carcinoids are slow growing neuroendocrine tumours arising from enterochromaffin-like (ECL) cells in the corpus of stomach. Although most of these tumours arise in the setting of gastric atrophy and hypergastrinemia, it is not understood what genetic background predisposes development of these ECL derived tumours. Moreover, diffuse microcarcinoids in the mucosa can lead to a field effect and limit successful endoscopic removal. OBJECTIVE: To define the genetic background that creates a permissive environment for gastric carcinoids using transgenic mouse lines. DESIGN: The multiple endocrine neoplasia 1 gene locus (Men1) was deleted using Cre recombinase expressed from the Villin promoter (Villin-Cre) and was placed on a somatostatin null genetic background. These transgenic mice received omeprazole-laced chow for 6 months. The direct effect of gastrin and the gastrin receptor antagonist YM022 on expression and phosphorylation of the cyclin inhibitor p27Kip1 was tested on the human human gastric adenocarcinoma cell line stably expressing CCKBR (AGSE) and mouse small intestinal neuroendocrine carcinoma (STC)-1 cell lines. RESULTS: The combination of conditional Men1 deletion in the absence of somatostatin led to the development of gastric carcinoids within 2 years. Suppression of acid secretion by omeprazole accelerated the timeline of carcinoid development to 6 months in the absence of significant parietal cell atrophy. Carcinoids were associated with hypergastrinemia, and correlated with increased Cckbr expression and nuclear export of p27Kip1 both in vivo and in gastrin-treated cell lines. Loss of p27Kip1 was also observed in human gastric carcinoids arising in the setting of atrophic gastritis. CONCLUSIONS: Gastric carcinoids require threshold levels of hypergastrinemia, which modulates p27Kip1 cellular location and stability.

Transcription Factor ZBP-89 Drives a Feedforward Loop of ß-Catenin Expression in Colorectal Cancer.

In colorectal cancer, APC-mediated induction of unregulated cell growth involves posttranslational mechanisms that prevent proteasomal degradation of proto-oncogene ß-catenin (CTNNB1) and its eventual translocation to the nucleus. However, about 10% of colorectal tumors also exhibit increased CTNNB1 mRNA. Here, we show in colorectal cancer that increased expression of ZNF148, the gene coding for transcription factor ZBP-89, correlated with reduced patient survival. Tissue arrays showed that ZBP-89 protein was overexpressed in the early stages of colorectal cancer. Conditional deletion of Zfp148 in a mouse model of Apc-mediated intestinal polyps demonstrated that ZBP-89 was required for polyp formation due to induction of Ctnnb1 gene expression. Chromatin immunoprecipitation (ChIP) and EMSA identified a ZBP-89-binding site in the proximal promoter of CTNNB1 Reciprocally, siRNA-mediated reduction of CTNNB1 expression also decreased ZBP-89 protein. ChIP identified TCF DNA binding sites in the ZNF148 promoter through which Wnt signaling regulates ZNF148 gene expression. Suppression of either ZNF148 or CTNNB1 reduced colony formation in WNT-dependent, but not WNT-independent cell lines. Therefore, the increase in intracellular ß-catenin protein initiated by APC mutations is sustained by ZBP-89-mediated feedforward induction of CTNNB1 mRNA. Cancer Res; 76(23); 6877-87. ©2016 AACR.