Menin determines K-RAS proliferative outputs in endocrine cells

Abstract

R e s e a R c h a R t i c l e4 0 9 4 jci.org Volume 124 Number 9 September 2014 het; Figure 1B). We discovered that pancreata from E13.5 Kras-het embryos contained more endocrine cells, as determined by combined glucagon and insulin staining, than did WT pancreata, while overall pancreatic size remained unchanged (Figure 1, C-E, and Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI69004DS1). By P5, Kras-het animals had a relative expansion of both the glucagon-expressing α cell and insulin-expressing β cell populations without any increase in the exocrine pancreas (Figure 1, F-H, and Supplemental Figure 2). Further, Kras-het adults had improved glucose tolerance compared with that of WT controls (Supplemental Figure 3), consistent with an increase in functional β cell capacity. Next, we sought to determine the source of additional endocrine cells in Kras-het animals. The fetal pancreas generates endocrine cells by neogenesis, followed by rapid expansion of those cells by proliferation during perinatal and postnatal growth (Figure 2A and ref. 13). Using expression of the transcription factor NEUROG3 to identify the transient precursor cells generated susceptible to MEN1 gene mutation, menin normally prevents the MAPK effector pathway from driving proliferation, while leaving inhibitory effector pathways such as RASSF1A intact. In this model, loss of menin causes proliferation in susceptible cells due to removal of the blockage of MAPK-driven proliferation downstream of K-RAS, while loss of K-RAS signaling increases proliferation by decreasing unopposed RASSF1A activity. Our data explain the absence of activating KRAS mutations and the high frequency of MEN1 and RASSF1A inactivation in pancreatic endocrine tumors. Our study also suggests potential antiproliferative strategies for treating these tumors and proproliferative therapies for diseases that result from a deficiency of endocrine cell types, such as β cells, in both type 1 and type 2 diabetes. ResultsTo test K-RAS function in pancreatic endocrine cell growth, we used mice carrying the Kras G12D allele, which is a null allele in the absence of Cre recombinase (12), in place of 1 WT allele (Kras- G12D-knock-in allele, which contains the G12D mutation in exon 2 and an upstream LoxP-Stop-LoxP sequence (12), to an active allele expressing constitutively active K-RAS G12D after Cre-mediated recombination. (C and D) Projection images of the dorsal pancreas were collected by whole-mount confocal microscopy from E13.5 mouse embryos of the indicated genotypes and immunostained for E-cadherin (blue) and glucagon and insulin (green) (n = 6 Kras WT, n = 6 Kras-het). (E) Quantification of immunostained areas in the epithelial pancreas (E-cadherin) and endocrine pancreas (glucagon and insulin) relative to WT embryos. (F-H) Pancreatic sections from P5 WT (F) and Kras-het (G) neonates were immunostained for glucagon (red) and insulin (green), with quantification of the relative insulin and glucagon areas and pancreatic mass (H...

Ras signaling through RASSF proteins

“…Moreover, RASSF1A promoter methylation correlates with the worst prognosis in Ras driven tumors [20], and suppression of RASSF1A by shRNA promotes metastasis in mutant Ras cell lines [24]. In transgenic systems, RASSF1A loss enhances K-Ras induced proliferation [25]. …”

The future: genetics advances in MEN1 therapeutic approaches and management strategies

“…These mouse models have shown that the expression of activated K-RAS(p.G12D) enhances rather than inhibits β-cell proliferation, β-catenin loss can suppress insulinoma tumorigenesis, histone demethylase retinoblastoma binding protein-2 (Rbp2) loss decreases insulinoma formation and prolongs survival, ActivinB loss prolongs survival, and MLL1/KMT2A loss leads to earlier onset of tumor formation (4 months vs. 6 months) and shortened lifespan by promoting tumor progression and angiogenesis (Chamberlain, et al 2014; Jiang, et al 2014; Lin, et al 2011; Lin, et al 2016; Ripoche, et al 2015). Such studies have dual benefits as they help to understand tumorigenesis and to reveal β-cell proliferation mechanisms for developing β-cell replacement strategies to help diabetic patients who suffer from functional β-cell loss (Garcia-Ocana and Stewart 2014).…”

Glucose Induces Mouse β-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor

“…Inhibition of MTOR with rapamycin reduces β-cell proliferation (15,29–33), but genetic manipulation of MTOR leads to less clear results, with some studies suggesting that MTOR drives β-cell proliferation (15,34–37) and others not (38–41). ERK, activated by glucose in β-cells (12), is proproliferative in other cell types but may play a paradoxical antiproliferative role in β-cells (42,43). …”