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...     

Of mice and MEN1: Insulinomas in a conditional mouse knockout

Patients with multiple endocrine neoplasia type 1 (MEN1) develop multiple endocrine tumors, primarily affecting the parathyroid, pituitary, and endocrine pancreas, due to the inactivation of the MEN1 gene. A conditional mouse model was developed to evaluate the loss of the mouse homolog, Men1, in the pancreatic beta cell. Men1 in these mice contains exons 3 to 8 flanked by loxP sites, such that, when the mice are crossed to transgenic mice expressing cre from the rat insulin promoter (RIP-cre), exons 3 to 8 are deleted in beta cells. By 60 weeks of age, >80% of mice homozygous for the floxed Men1 gene and expressing RIP-cre develop multiple pancreatic islet adenomas. The formation of adenomas results in elevated serum insulin levels and decreased blood glucose levels. The delay in tumor appearance, even with early loss of both copies of Men1, implies that additional somatic events are required for adenoma formation in beta cells. Comparative genomic hybridization of beta cell tumor DNA from these mice reveals duplication of chromosome 11, potentially revealing regions of interest with respect to tumorigenesis.     

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.     

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.     

Haploinsufficient and predominant expression of multiple endocrine neoplasia type 1 (MEN1)-related genes, MLL, p27 and p18 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.     

Interplay between Menin and K-Ras in Regulating Lung Adenocarcinoma

MEN1, which encodes the nuclear protein menin, acts as a tumor suppressor in lung cancer and is often inactivated in human primary lung adenocarcinoma. Here, we show that the inactivation of MEN1 is associated with increased DNA methylation at the MEN1 promoter by K-Ras. On one hand, the activated K-Ras up-regulates the expression of DNA methyltransferases and enhances the binding of DNA methyltransferase 1 to the MEN1 promoter, leading to increased DNA methylation at the MEN1 gene in lung cancer cells; on the other hand, menin reduces the level of active Ras-GTP at least partly by preventing GRB2 and SOS1 from binding to Ras, without affecting the expression of GRB2 and SOS1. In human lung adenocarcinoma samples, we further demonstrate that reduced menin expression is associated with the enhanced expression of Ras (p < 0.05). Finally, excision of the Men1 gene markedly accelerates the K-Ras^G12D-induced tumor formation in the Men1^f/f;K-Ras^G12D/+;Cre ER mouse model. Together, these findings uncover a previously unknown link between activated K-Ras and menin, an important interplay governing tumor activation and suppression in the development of lung cancer.     

In vitro hematopoietic differentiation of mouse embryonic stem cells requires the tumor suppressor menin and is mediated by Hoxa9

Inactivating mutations in the tumor suppressor gene MEN1 cause the inherited cancer syndrome multiple endocrine neoplasia type 1 (MEN1). The ubiquitously expressed MEN1 encoded protein, menin, interacts with MLL (mixed-lineage leukemia protein), and together they are essential components of a multiprotein complex with histone methyl transferase activity. MLL is also essential for hematopoiesis, and plays a critical role in leukemogenesis via epigenetic regulation of Hoxa9 expression that also requires menin. Therefore we chose to explore the role of menin in hematopoiesis. We generated Men1^−/− embryonic stem (ES) cell lines, and induced them to differentiate in vitro. While these cells were able to form embryoid bodies (EBs) expressing the early markers Flk-1 and c-Kit, their ability to further differentiate into hematopoietic colonies was compromised. The Men1^−/− ES cells show reduced expression of Hoxa9 that can be recovered by reexpression of Menin. We demonstrate that the block in differentiation of Men1^−/− ES cell lines can be rescued not only by the expression of menin but also that of Hoxa9. These results suggest that, similar to MLL, menin is required for hematopoiesis, and this requirement may be mediated through regulation of Hoxa9 expression.     

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.     

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.     

Conditional deletion of menin results in antral G cell hyperplasia and hypergastrinemia

Antral gastrin is the hormone known to stimulate acid secretion and proliferation of the gastric corpus epithelium. Patients with mutations in the multiple endocrine neoplasia type 1 (MEN1) locus, which encodes the protein menin, develop pituitary hyperplasia, insulinomas, and gastrinomas in the duodenum. We previously hypothesized that loss of menin leads to derepression of the gastrin gene and hypergastrinemia. Indeed, we show that menin represses JunD induction of gastrin in vitro. Therefore, we examined whether conditional deletion of Men1 (Villin-Cre and Lgr5-EGFP-IRES-CreERT2), with subsequent loss of menin from the gastrointestinal epithelium, increases gastrin expression. We found that epithelium-specific deletion of Men1 using Villin-Cre increased plasma gastrin, antral G cell numbers, and gastrin expression in the antrum, but not the duodenum. Moreover, the mice were hypochlorhydric by 12 mo of age, and gastric somatostatin mRNA levels were reduced. However, duodenal mRNA levels of the cyclin-dependent kinase inhibitor p27(Kip1) were decreased, and cell proliferation determined by Ki67 staining was increased. About 11% of the menin-deficient mice developed antral tumors that were negative for gastrin; however, gastrinomas were not observed, even at 12 mo of age. No gastrinomas were observed with conditional deletion of Men1 in the Lgr5 stem cells 5 mo after Cre induction. In summary, epithelium-specific deletion of the Men1 locus resulted in hypergastrinemia due to antral G cell hyperplasia and a hyperproliferative epithelium, but no gastrinomas. This result suggests that additional mutations in gene targets other than the Men1 locus are required to produce gastrin-secreting tumors.     

Molecular and genetic mechanisms of tumorigenesis in multiple endocrine neoplasia type-1

Multiple endocrine neoplasia type 1 (MEN1) is a rare but informative syndrome for endocrine tumorigenesis. Since its isolation, several groups have begun to determine the role of menin, the protein product of MEN1, in sporadic endocrine tumors as well as tumors of the MEN1 syndrome. Mutations of menin have been reported in more than 400 families and tumors, most of which are truncating mutations, thus supporting the function of menin as a tumor suppressor. The exact function of menin is unknown, but overexpression of menin inhibits proliferation of Ras-transformed NIH3T3 cells. Since menin interacts with proteins from both the TGF beta and AP-1 signaling pathways, perhaps its tumor suppressor function is related to these key cell growth pathways. In this review we will discuss the various clinical manifestations of MEN1 syndrome, potential mechanisms of MEN1 tumorigenesis, and mutations associated with MEN and sporadic endocrine tumors.     

The role of tumor suppressor menin in IL-6 regulation in mouse islet tumor cells

Menin is a gene product of multiple endocrine neoplasia type1 (Men1), an inherited familial cancer syndrome characterized by tumors of endocrine tissues. To gain insight about how menin performs an endocrine cell-specific tumor suppressor function, we investigated the possibility that menin was integrated in a cancer-associated inflammatory pathway in a cell type-specific manner. Here, we showed that the expression of IL-6, a proinflammatory cytokine, was specifically elevated in mouse islet tumor cells upon depletion of menin and Men^−/− MEF cells, but not in hepatocellular carcinoma cells. Histone H3 lysine (K) 9 methylation, but not H3 K27 or K4 methylation, was involved in menin-dependent IL-6 regulation. Menin occupied the IL-6 promoter and recruited SUV39H1 to induce H3 K9 methylation. Our findings provide a molecular insight that menin-dependent induction of H3 K9 methylation in the cancer-associated interleukin gene might be linked to preventing endocrine-specific tumorigenesis.