Gastroenterology

Gastroenterology

Volume 153, Issue 6, December 2017, Pages 1555-1567.e15
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
Gastrin Induces Nuclear Export and Proteasome Degradation of Menin in Enteric Glial Cells

https://doi.org/10.1053/j.gastro.2017.08.038Get rights and content

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.

Section snippets

Human Samples

De-identified surgical samples of human gastrinoma from 1996 to 2007 were obtained from the Department of Pathology at the University of Michigan, Center for Tumor Biology Barts Cancer Institute, Queen Mary University, London and the Royal Liverpool University Hospital, Liverpool, UK and are listed in Table 1. Sample access was approved by University of Michigan Institutional Review Board #HUM00115310.

Animals and Cell Culture

All animal experiments were approved by the University of Michigan's Committee on the Use and

Gastrin-expressing Cells Identified in Duodenal Lamina Propria

We previously showed that the OMS mice develop gastric carcinoids in the stomach corpus.20 Examination of the duodenums of these mice revealed numerous gastrin-positive cells in the lamina propria (LP-Gastrin+; Figure 1A and B). LP-Gastrin+ cells almost disappeared 4 months after withdrawal of omeprazole (OM), respectively (Figure 1C). H&E analysis revealed misshapen villi and dilated lamina propria in the OMS mice that returned to normal 4 months after withdrawing OM (Supplementary Figure 1A).

Discussion

Menin functions as a tumor suppressor in neuroendocrine tumors, including gastrinomas.36 Accordingly, we previously showed that modulating menin levels directly regulates gastrin gene expression through JUND in cell lines.17, 18 However, deleting Men1 from the intestinal epithelium does not induce gastrinomas, suggesting that modulating additional loci restricted to the spectrum of tissues developing neuroendocrine tumors contribute to the neoplastic transformation of gastrin-expressing

Acknowledgments

The authors thank Linda Samuelson for critically reviewing the manuscript, and support from R37 DK045729, Digestive Disease Center, UM Cancer Center Cores.

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    Conflicts of interest Drs Grembecka and Cierpicki receive research support from Kura Oncology. They are also receiving compensation as members of the scientific advisory board of Kura Oncology, and have an equity ownership in the company. The remaining authors disclose no conflicts.

    Funding R37 DK45729 (to J.L.M.) and Digestive Disease Center 5P30 DK034933; R01 CA160467 (to J.G.) and R01 CA200660 (to J.G.); R01 DK66604 (to M.J.L.); Molecular Core; UM Cancer Center P30 CA046592 Tissue and Transgenic Mouse Core.

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