Heterozygous Men1 mutant mice develop a range of endocrine tumors mimicking multiple endocrine neoplasia type 1

Multiple endocrine neoplasia type 1 (MEN1) is a hereditary syndrome characterized by the occurrence of multiple endocrine tumors of the parathyroid, pancreas, and anterior pituitary in patients. To study tumorigenesis related to the MEN1 syndrome, we have generated Men1 knockout mice using the gene targeting approach. Heterozygous Men1 mutant mice developed the same range of major endocrine tumors as is seen in MEN1 patients, affecting the parathyroid, pancreatic islets, pituitary and adrenal glands, as well as the thyroid, and exhibiting multistage tumor progression with metastatic potential. In particular, extrapancreatic gastrinoma, pancreatic glucagonoma, and mixed hormone-producing tumors in islets were observed. In addition, there was a high incidence of gonadal tumors of endocrine origin, i.e. Leydig cell tumors, and ovary sex-cord stromal cell tumors in heterozygous Men1 mutant mice. Hormonal disturbance, such as abnormal PTH and insulin levels, was also observed in these mice. These tumors were associated with loss of heterozygosity of the wild-type Men1 allele, suggesting that menin is involved in suppressing the development of these endocrine tumors. All of these features are reminiscent of MEN1 symptoms in humans and establish heterozygous Men1 mutant mice as a suitable model for this disease.     

MEN1胰岛瘤中细胞周期蛋白cyclin B2高表达的作用和机制

多发性内分泌肿瘤1-(multiple endocrine neoplasia type 1,MEN1)是一种常染色体显性遗传的肿瘤综合征,患者常表现出多发性的内分泌器官肿瘤,包括垂体瘤、甲状旁腺瘤和胰岛瘤.抑癌基因Men1的突变导致MENl的发生,其编码的蛋白为核蛋白menin.Menin可以抑制包括胰岛β细胞在内的...     

p18Ink4c, but not p27Kip1, collaborates with Men1 to suppress neuroendocrine organ tumors

Mutant mice lacking both cyclin-dependent kinase (CDK) inhibitors p18(Ink4c) and p27(Kip1) develop a tumor spectrum reminiscent of human multiple endocrine neoplasia (MEN) syndromes. To determine how p18 and p27 genetically interact with Men1, the tumor suppressor gene mutated in familial MEN1, we characterized p18-Men1 and p27-Men1 double mutant mice. Compared with their corresponding single mutant littermates, the p18(-/-); Men1(+/-) mice develop tumors at an accelerated rate and with an increased incidence in the pituitary, thyroid, parathyroid, and pancreas. In the pituitary and pancreatic islets, phosphorylation of the retinoblastoma (Rb) protein at both CDK2 and CDK4/6 sites was increased in p18(-/-) and Men1(+/-) cells and was further increased in p18(-/-); Men1(+/-) cells. The remaining wild-type Men1 allele was lost in most tumors from Men1(+/-) mice but was retained in most tumors from p18(-/-); Men1(+/-) mice. Combined mutations of p27(-/-) and Men1(+/-), in contrast, did not exhibit noticeable synergistic stimulation of Rb kinase activity, cell proliferation, and tumor growth. These results demonstrate that functional collaboration exists between p18 and Men1 and suggest that Men1 may regulate additional factor(s) that interact with p18 and p27 differently.     

Menin induces apoptosis in murine embryonic fibroblasts

Multiple endocrine neoplasia type I (MEN1) is a hereditary tumor syndrome characterized by multiple endocrine and occasionally non-endocrine tumors. The tumor suppressor gene Men1, which is frequently mutated in MEN1 patients, encodes the nuclear protein menin. Although many tumor suppressor genes are involved in the regulation of apoptosis, it is unclear whether menin facilitates apoptosis. Here we show that ectopic overexpression of menin via adenoviruses induces apoptosis in murine embryonic fibroblasts. The induction of apoptosis depends on Bax and Bak, two proapoptotic proteins. Moreover, loss of menin expression compromises apoptosis induced by UV irradiation and tumor necrosis factor-alpha (TNF-alpha), whereas complementation of menin-null cells with menin restores sensitivity to UV- and TNF-alpha-induced apoptosis. Interestingly, loss of menin reduces the expression of procaspase 8, a critical protease that is essential for apoptosis induced by death-related receptors, whereas complementation of the menin-null cells up-regulates the expression of procaspase 8. Furthermore, complementation of menin-null cells with menin increases the activation of caspase 8 in response to TNF-alpha treatment. These results suggest a proapoptotic function for menin that may be important in suppressing the development of MEN1.     

Brn-4 transcription factor expression targeted to the early developing mouse pancreas induces ectopic glucagon gene expression in insulin-producing beta cells

The endocrine pancreas is comprised of beta and alpha cells producing the glucostatic hormones insulin and glucagon, respectively, and arises during development by the differentiation of stem/progenitor cells in the foregut programmed by the beta cell lineage-specific homeodomain protein Idx-1. Brain-4 (Brn-4) is expressed in the pancreatic anlaga of the mouse foregut at e10 in the alpha cells and transactivates glucagon gene expression. We expressed Brn-4 in pancreatic precursors or beta cell lineage in transgenic mice by placing it under either Idx-1 or insulin promoter (rat insulin II promoter) control, respectively. Idx-1 expression occurs at developmental day e8.5, and insulin expression occurs at e9.5, respectively. Misexpression of Brn-4 by the Idx-1 promoter results in ectopic expression of the proglucagon gene in insulin-expressing pancreatic beta cells, whereas misexpression by rat insulin II promoter did not. The early developmental expression of Brn-4 appears to be a dominant regulator of the glucagon expressing alpha cell lineage, even in the context of the beta cell lineage.     

Beta cells within single human islets originate from multiple progenitors

Background

In both humans and rodents, glucose homeostasis is controlled by micro-organs called islets of Langerhans composed of beta cells, associated with other endocrine cell types. Most of our understanding of islet cell differentiation and morphogenesis is derived from rodent developmental studies. However, little is known about human islet formation. The lack of adequate experimental models has restricted the study of human pancreatic development to the histological analysis of different stages of pancreatic development. Our objective was to develop a new experimental model to (i) transfer genes into developing human pancreatic cells and (ii) validate gene transfer by defining the clonality of developing human islets.

Methods and Findings

In this study, a unique model was developed combining ex vivo organogenesis from human fetal pancreatic tissue and cell type-specific lentivirus-mediated gene transfer. Human pancreatic progenitors were transduced with lentiviruses expressing GFP under the control of an insulin promoter and grafted to severe combined immunodeficient mice, allowing human beta cell differentiation and islet morphogenesis. By performing gene transfer at low multiplicity of infection, we created a chimeric graft with a subpopulation of human beta cells expressing GFP and found both GFP-positive and GFP-negative beta cells within single islets.

Conclusion

The detection of both labeled and unlabeled beta cells in single islets demonstrates that beta cells present in a human islet are derived from multiple progenitors thus providing the first dynamic analysis of human islet formation during development. This human transgenic-like tool can be widely used to elucidate dynamic genetic processes in human tissue formation.     

Genetic ablation of the tumor suppressor menin causes lethality at mid-gestation with defects in multiple organs

Patients suffering from multiple endocrine neoplasia type 1 (MEN1) are predisposed to multiple endocrine tumors. The MEN1 gene product, menin, is expressed in many embryonic, as well as adult tissues, and interacts with several proteins in vitro and in vivo. However, the biological function of menin remains largely unknown. Here we show that disruption of the Men1 gene in mice causes embryonic lethality at E11.5-E13.5. The Men1 null mutant embryos appeared smaller in size, frequently with body haemorrhages and oedemas, and a substantial proportion of them showed disclosure of the neural tube. Histological analysis revealed an abnormal development of the nervous system and heart hypotrophy in some Men1 null embryos. Furthermore, Men1 null livers generally displayed an altered organization of the epithelial and hematopoietic compartments associated with enhanced apoptosis. Chimerism analysis of embryos generated by injection of Men1 null ES cells, showed that cells lacking menin do not seem to have a general cell-autonomous defect. However, primary Men1 null embryonic fibroblasts entered senescence earlier than their wild-type counterparts. Despite normal proliferation ability, Men1 null ES cells exhibited a deficiency to form embryoid bodies, suggesting an impaired differentiation capacity in these cells. The present study demonstrates that menin plays an important role in the embryonic development of multiple organs in addition to its proposed role in tumor suppression.     

Sequence analysis of the MEN I gene in two patients with multiple cutaneous lipomas and endocrine tumors.

The discovery of mutations of the menin gene in a few multiple endocrine neoplasma type 1 (MEN I)-associated lipomas and loss of heterozygosity (LOH) on chromosome 11q13 in some sporadic lipomas has stimulated the hypothesis that lipomas may belong to the group of sporadic tumors caused by defects of the gene responsible for MEN I. Since it is unclear if the above hypothesis applies to all patients with lipoma or just to specific subsets, we searched to enlarge the database on this topic. For this purpose, we identified two patients with multiple cutaneous lipomas. One had an additional pituitary adenoma and familial presentation of multiple lipomas, the other had recurrent goiter in the setting of a family history of adenomatous goiter. Deoxyribonucleic acid (DNA) was analyzed by complete direct DNA sequencing of all coding exons and splice junctions of the MEN I gene. No mutation was identified in the coding exons of the menin gene. In contrast to former data on sporadic lipomas, these data are the first to render evidence that mutations of the MEN I gene may not be responsible for the formation of multiple lipomas, even if they appear in the context of other endocrine tumors.     

Haploinsufficient and predominant expression of multiple endocrine neoplasia type 1 (MEN1)-related genes, MLL, p27Kip1 and p18Ink4C in endocrine organs

Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominantly inherited syndrome characterized by parathyroid, gastro-entero-pancreatic and anterior pituitary tumors. Although the tissue selectivity of tumors in specific endocrine organs is the very essence of MEN1, the mechanisms underlying the tissue-selectivity of tumors remain unknown. The product of the Men1 gene, menin, and mixed lineage leukemia (MLL) have been found to cooperatively regulate p27(Kip1)/CDKN1B (p27) and p18(Ink4C)/CDKN2C (p18) genes. However, there are no reports on the tissue distribution of these MEN1-related genes. We investigated the expression of these genes in the endocrine and non-endocrine organs of wild-type, Men1 knockout and MLL knockout mice. Men1 mRNA was expressed at a similar level in endocrine and non-endocrine organs. However, MLL, p27 and p18 mRNAs were predominantly expressed in the endocrine organs. Notably, p27 and MLL mRNAs were expressed in the pituitary gland at levels approximately 12- and 17-fold higher than those in the liver. The heterozygotes of Men1 knockout mice the levels of MLL, p27 and p18 mRNAs did not differ from those in the wild-type mice. In contrast, heterozygotes of MLL knockout mice showed significant reductions in p27 mRNA as well as protein levels in the pituitary and p27 and p18 in the pancreatic islets, but not in the liver. This study demonstrated for the first time the predominant expression MEN1-related genes, particularly MLL and p27, in the endocrine organs, and a tissue-specific haploinsuffiency of MLL, but not menin, may lead to a decrease in levels of p27 and p18 mRNAs in endocrine organs. These findings may provide basic information for understanding the mechanisms of tissue selectivity of the tumorigenesis in patients with MEN1.     

Genome-wide characterization of menin-dependent H3K4me3 reveals a specific role for menin in the regulation of genes implicated in MEN1-like tumors

Inactivating mutations in the MEN1 gene predisposing to the multiple endocrine neoplasia type 1 (MEN1) syndrome can also cause sporadic pancreatic endocrine tumors. MEN1 encodes menin, a subunit of MLL1/MLL2-containing histone methyltransferase complexes that trimethylate histone H3 at lysine 4 (H3K4me3). The importance of menin-dependent H3K4me3 in normal and transformed pancreatic endocrine cells is unclear. To study the role of menin-dependent H3K4me3, we performed in vitro differentiation of wild-type as well as menin-null mouse embryonic stem cells (mESCs) into pancreatic islet-like endocrine cells (PILECs). Gene expression analysis and genome-wide H3K4me3 ChIP-Seq profiling in wild-type and menin-null mESCs and PILECs revealed menin-dependent H3K4me3 at the imprinted Dlk1-Meg3 locus in mESCs, and all four Hox loci in differentiated PILECs. Specific and significant loss of H3K4me3 and gene expression was observed for genes within the imprinted Dlk1-Meg3 locus in menin-null mESCs and the Hox loci in menin-null PILECs. Given that the reduced expression of genes within the DLK1-MEG3 locus and the HOX loci is associated with MEN1-like sporadic tumors, our data suggests a possible role for menin-dependent H3K4me3 at these genes in the initiation and progression of sporadic pancreatic endocrine tumors. Furthermore, our investigation also demonstrates that menin-null mESCs can be differentiated in vitro into islet-like endocrine cells, underscoring the utility of menin-null mESC-derived specialized cell types for genome-wide high-throughput studies.     

Loss of heterozygosity on chromosome 11 in sporadic gastrinomas

Summary Gastrinomas are pancreatic endocrine neoplasms that arise either sporadically or are inherited as part of the multiple endocrine neoplasia type I syndrome (MEN I). Loss of heterozygosity (LOH) in the region flanking the MEN I gene at chromosome 11q13 has been documented in a few sporadic and familial pancreatic endocrine tumors, but not previously in sporadic gastrinomas. It has therefore been suggested that gastrinomas develop by a mechanism different from other tumors associated with the MENI syndsrome. We report LOH on chromosome 11 in 5 of 11 sporadic gastrinomas. Four of these tumors have LOH for markers flanking the MEN I region. Molecular evaluation of segments of chromosomes 3, 13, and 17 known to contain cloned or putative tumor suppressor genes fail to show LOH except at one locus in one tumor. These data suggest that a tumor suppressor DNA segment exists at 11q13 that may be involved in the development of sporadic gastrinomas.     

Differentially expressed cDNAs in PLCbeta3-induced tumor suppression in a human endocrine pancreatic tumor cell line: activation of the human mismatch repair protein 3 gene

Phospholipase Cbeta3 (PLCB3) is located to chromosome 11q13 in the vicinity of the multiple endocrine neoplasia type1 (MEN1) gene and shows loss of expression in some neuroendocrine tumors. Transfection of PLCB3 to neuroendocrine cell lines induces growth suppression and phenotypic alterations, but the mechanisms remain unclear. To investigate the underlying events behind this tumor suppression, we performed an RT-Differential cDNA Display of total RNA from BON-1 (human endocrine pancreatic tumor cell line) transfected with PLCB3 and compared to wild type and BON-1 transfected with vector without insert. PLCB3 transfection resulted in increased expression of 4 genes and decreased of 2. The two inhibited were homologous to S100A3 and Chromogranin A. One of the four activated cDNAs could be identified as human mismatch repair protein 3 mRNA (hMSH3), and another was homologous to TIS/MA-3 mRNA (mouse topoisomerase suppressor inhibited gene/mouse apoptosis gene-3). Differential expression of these genes may contribute to the PLCB3-induced tumor suppression of neuroendocrine tumor cell lines.     

Molecular alterations during insulinoma tumorigenesis

Insulinomas are the most common functioning endocrine pancreatic tumors (EPTs). They present with clinical symptoms as a consequence of hypoglycemia induced by inappropriate insulin secretion. The etiology of these tumors is poorly understood. Some tumors may harbor MEN1 gene mutations, the susceptibility gene of the multiple endocrine neoplasia type I syndrome, but most cases show wildtype MEN1. Currently, no reliable clinical tests are available to differentiate benign from malignant tumors. Approximately 30% of the tumors are unresectable, and they often show different growth rates, which hampers treatment. Therefore, a better understanding of the molecular processes underlying the development and progression of insulinomas is required to improve diagnosis, prognosis and therapy. Here we summarize the progress that has been made in insulinoma research in the past decade. We describe the clinical detection, classification and treatment of these tumors, and review the multiplicity of molecular and genetic studies that investigated tumor development and progression using either primary tumors, transgenic mouse models or tumor-derived cell lines. The identification of many interactors of the MEN1 gene product menin, as well as recurrent chromosomal abnormalities that pinpoint candidate genes of interest will likely result in a better understanding of the molecular pathways involved in insulinoma tumorigenesis. In addition, these studies will pave the way for the identification of novel targets for therapeutical intervention and more reliable markers for clinical diagnosis and prognosis.