Functional interaction between tumor suppressor menin and activator of S-phase kinase

Multiple endocrine neoplasia type I (MEN1), a hereditary tumor syndrome, is characterized by the development of tumors in multiple endocrine organs. The gene mutated in MEN1 patients, Men1, encodes a tumor suppressor, menin. Overexpression of menin leads to inhibition of Ras-transformed cells. However, it is unclear whether menin is essential for repression of cell proliferation, and if it is, how it inhibits cell proliferation. Here, we show that targeted disruption of the Men1 gene leads to enhanced cell proliferation, whereas complementation of menin-null cells with menin reduces cell proliferation. Moreover, menin interacts with activator of S-phase kinase (ASK), a component of the Cdc7/ASK kinase complex that is crucial for cell proliferation, but does not appear to alter Cdc7 kinase activity in in vitro kinase assays. We identify the COOH terminus of menin as the domain that mediates the specific interaction with ASK. Notably, wild-type menin completely represses ASK-induced cell proliferation, although it does not obviously affect the steady-state cell cycle profile of ASK-infected cells. Interestingly, disease-related COOH-terminal menin mutants that do not interact with ASK completely fail to repress ASK-induced cell proliferation. Together, these findings demonstrate a functional link between menin and ASK in the regulation of cell proliferation.     

Stable overexpression of MEN1 suppresses tumorigenicity of RAS

Although there is indirect genetic evidence that MEN1, the gene for multiple endocrine neoplasia type 1, is a tumor suppressor gene, little is known about the MEN1-encoded protein, menin. Menin was stably overexpressed in a well-characterized murine tumor cell line, (valine-12)-RAS-transformed NIH3T3 cells. Menin overexpression reverted the morphology of the RAS-transformed NIH3T3 cells towards the more flattened and more spread, fibroblastic shape of wild type NIH3T3 cells. The proliferation rate of the RAS-transformed cells in 0.5% calf serum was also slower with menin overexpression. Menin overexpression reduced the RAS-induced clonogenicity in soft agar. Menin also reduced tumor growth after injection of cells in nude mice. In conclusion, stable overexpression of MEN1 suppressed partially the RAS-mediated tumor phenotype in vitro and in vivo. Overexpressed menin protein had biological effects, directly supporting MEN1 gene function as a tumor suppressor.     

Characterization of the MEN1 gene product, menin, by site-specific polyclonal antibodies.

The gene associated with multiple endocrine neoplasia type 1 (MEN 1), designated MEN1, has recently been identified. This gene shows no homology to other known genes, and its expression is not restricted to endocrine organs as estimated by northern blotting. Expression of the MEN1 gene product, menin, has been studied only in a few tissues. In this report, expression of menin in various cells and mouse tissues was studied using two polyclonal antibodies against menin. Expression of menin as a 76 kDa single protein was observed in all cell lines examined, regardless of origin. Two nuclear localization signals of the menin have been reported, but through the study of mutant menin in lymphocytes from subjects with MEN 1, impaired nuclear localization of the mutant menin was observed even though the mutant retained one of the two nuclear localization signals (NLSs). Menin was stable in vitro with a half-life of over 24 h at 37 degrees C. In the cell, the half-life of wild-type menin was about 10 h, while that of the mutant was about 2 h. The mutant rapidly disappeared from the nucleus.     

The tumor suppressor protein menin inhibits AKT activation by regulating its cellular localization

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder associated mainly with tumors of multiple endocrine organs. Mutations in the MEN1 gene that encodes for the menin protein are the predominant cause for hereditary MEN1 syndrome. Though menin is a tumor suppressor, its molecular mechanism of action has not been defined. Here, we report that menin interacts with AKT1 in vitro and in vivo. Menin downregulates the level of active AKT and its kinase activity. Through interaction with AKT1, menin suppresses both AKT1-induced proliferation and antiapoptosis in nonendocrine and endocrine cells. Confocal microscopy analysis revealed that menin regulates AKT1 in part by reducing the translocation of AKT1 from the cytoplasm to the plasma membrane during growth factor stimulation. Our findings may be generalizable to other cancers, insofar as we found that loss of menin expression was also associated with AKT activation in a mouse model of pancreatic islet adenoma. Together, our results suggest menin as an important novel negative regulator of AKT kinase activity.     

Characterization of the MEN1 Gene Product, Menin, by Site-specific Polyclonal Antibodies

The gene associated with multiple endocrine neoplasia type 1 (MEN 1), designated MEN1 , has recently been identified. This gene shows no homology to other known genes, and its expression is not restricted to endocrine organs as estimated by northern blotting. Expression of the MEN1 gene product, menin, has been studied only in a few tissues. In this report, expression of menin in various cells and mouse tissues was studied using two polyclonal antibodies against menin. Expression of menin as a 76 kDa single protein was observed in all cell lines examined, regardless of origin. Two nuclear localization signals of the menin have been reported, but through the study of mutant menin in lymphocytes from subjects with MEN 1, impaired nuclear localization of the mutant menin was observed even though the mutant retained one of the two nuclear localization signals (NLSs). Menin was stable in vitro with a half-life of over 24 h at 37°C. In the cell, the half-life of wild-type menin was about 10 h, while that of the mutant was about 2 h. The mutant rapidly disappeared from the nucleus.     

Mutation of tumor suppressor gene Men1 acutely enhances proliferation of pancreatic islet cells

Multiple endocrine neoplasia type 1 (MEN1), an inherited tumor syndrome affecting endocrine organs including pancreatic islets, results from mutation of the tumor suppressor gene Men1 that encodes protein menin. Although menin is known to be involved in regulating cell proliferation in vitro, it is not clear how menin regulates cell cycle and whether mutation of Men1 acutely promotes pancreatic islet cell proliferation in vivo. Here we show that excision of the floxed Men1 in mouse embryonic fibroblasts (MEF) accelerates G(0)/G(1) to S phase entry. This accelerated S-phase entry is accompanied by increased cyclin-dependent kinase 2 (CDK2) activity as well as decreased expression of CDK inhibitors p18(Ink4c) and p27(Kip1). Moreover, Men1 excision results in decreased expression of p18(Ink4c) and p27(Kip1) in the pancreas. Furthermore, complementation of menin-null cells with wild-type menin represses S-phase entry. To extend the role of menin in repressing cell cycle in cultured cells to in vivo pancreatic islets, we generated a system in which floxed Men1 alleles can be excised in a temporally controllable manner. As early as 7 days following Men1 excision, pancreatic islet cells display increased proliferation, leading to detectable enlargement of pancreatic islets 14 days after Men1 excision. These observations are consistent with the notion that an acute effect of Men1 mutation is accelerated S-phase entry and enhanced cell proliferation in pancreatic islets. Together, these results suggest a molecular mechanism whereby menin suppresses MEN1 tumorigenesis at least partly through repression of G(0)/G(1) to S transition.     

Menin associates with FANCD2, a protein involved in repair of DNA damage

Multiple endocrine neoplasia type I (MEN1) is an inherited tumor syndrome characterized by tumors in multiple endocrine organs including the parathyroids, pancreatic islets, and the pituitary. The gene mutated in MEN1 patients, Men1, encodes a protein of 610 amino acid residues, menin, and mutations in the Men1 gene lead to the MEN1 syndrome. Although the chromosomal instability in the peripheral lymphocytes from the MEN1 patients has been reported previously, it is not clear whether menin is involved in repair of DNA damage. Here we show that menin specifically interacts with FANCD2, a protein encoded by a gene involved in DNA repair and mutated in patients with an inherited cancer-prone syndrome, Fanconi anemia. The interaction between menin and FANCD2 is enhanced by gamma-irradiation. Moreover, loss of menin expression in mouse embryonic fibroblasts leads to increased sensitivity to DNA damage. Furthermore, menin is localized to chromatin and nuclear matrix, and the association with nuclear matrix is enhanced by gamma-irradiation. Together, these results suggest that menin plays a critical role in repair of DNA damage in concert with FANCD2.     

Reconstituted expression of menin in Men1-deficient mouse Leydig tumour cells induces cell cycle arrest and apoptosis

Multiple endocrine neoplasia type 1 (MEN1) is a hereditary syndrome caused by the inactivation of the responsible gene, MEN1. To date, the lack of MEN1-deficient cell lines derived directly from MEN1 tumours has hampered the detailed study of the MEN1 gene. We have established several stable Men1-deficient Leydig cell tumour (LCT) lines derived from a Leydig cell tumour developed in a male heterozygous Men1 mutant mouse. Our data show that these cell lines maintain the basic characteristics of Leydig cells in terms of both androgen synthesis and gene expression. Interestingly, reconstituted menin expression in one of Men1-deficient LCT cell lines resulted in cell growth inhibition, suggesting that the function of cell growth suppression of the menin pathway, apart from menin itself, is essentially preserved in these cells. Furthermore, we show that menin re-expression in these Men1-deficient cells leads to a block in the transition from G0/G1 to S phase of the cell cycle and an increase in apoptosis, accompanied by a marked increase of p18INK4C and p27Kip1 expression. The current study therefore highlights the importance of menin expression in cell cycle and cell survival control in endocrine cells, and may provide insights into the mechanisms of tumour suppression by menin in related endocrine tumours.     

Menin interacting proteins as clues toward the understanding of multiple endocrine neoplasia type 1

Multiple endocrine neoplasia type 1 (MEN1) is a familial cancer syndrome characterized mostly by tumors of the parathyroids, pancreas and anterior pituitary. The gene responsible, MEN1, encodes Menin, a 610 aminoacid nuclear protein with no sequence homology to other proteins. Although a mouse knock-out model is available, the function of Menin is still elusive. Proteins of known function are shown to interact with Menin: JunD, nuclear factor-KappaB, Smad3, Pem, Nm23H1, glial fibrillary acidic protein, Vimentin, and probably P53. Their partnership with Menin may correspond to a regulation of their activity, but their relevance to the various traits of MEN1 pathogenicity is not established. This raises fundamental issues on the regulation pathways implicated in this complex endocrine disease.     

Broad tumor spectrum in a mouse model of multiple endocrine neoplasia type 1

Multiple endocrine neoplasia type 1 (MEN1) is an inherited cancer predisposition syndrome typified by development of tumors in parathyroid, pituitary and endocrine pancreas, as well as less common sites including both endocrine and nonendocrine organs. Deletion or mutation of the tumor suppressor gene MEN1 on chromosome 11 has been identified in many cases of MEN1 as well as in sporadic tumors. The molecular biology of menin, the protein encoded by MEN1, remains poorly understood. Here we describe a mouse model of MEN1 in which tumors were seen in pancreatic islets, pituitary, thyroid and parathyroid, adrenal glands, testes and ovaries. The observed tumor spectrum therefore includes types commonly seen in MEN1 patients and additional types. Pancreatic pathology was most common, evident in over 80% of animals, while other tumor types developed with lower frequency and generally later onset. Tumors of multiple endocrine organs were observed frequently, but progression to carcinoma and metastasis were not evident. Tumors in all sites showed loss of heterozygosity at the Men1 locus, though the frequency in testicular tumors was only 36%, indicating that a different molecular mechanism of tumorigenesis occurs in those Leydig tumors that do not show loss of the normal Men1 allele. Menin expression was below the level of detection in ovary, thyroid and testis, but loss of nuclear menin immunoreactivity was observed uniformly in all pancreatic islet adenomas and in some hyperplastic islet cells, suggesting that complete loss of Men1 is a critical point in islet tumor progression in this model.