MEN1 silencing triggers the dysregulation of mTORC1 and MYC pathways in ER+ breast cancer cells.

Menin, encoded by the MEN1 gene, has been identified as a critical factor regulating ESR1 transcription, playing an oncogenic role in ER+ breast cancer (BC) cells. Here, we further dissected the consequences of menin inactivation in ER+ BC cells by focusing on factors within two major pathways involved in BC, mTOR and MYC. MEN1 silencing in MCF7 and T-47D resulted in an increase in phosphor-p70S6K1, phosphor-p85S6K1 and phosphor-4EBP1 expression. The use of an AKT inhibitor inhibited the activation of S6K1 and S6RP triggered by MEN1 knockdown (KD). Moreover, MEN1 silencing in ER+ BC cells led to increased formation of the eIF4E and 4G complex. Clinical studies showed that patients with menin-low breast cancer receiving tamoxifen plus everolimus displayed a trend toward better overall survival. Importantly, MEN1 KD in MCF7 and T-47D cells led to reduced MYC expression. ChIP analysis demonstrated that menin bound not only to the MYC promoter but also to its 5' enhancer. Furthermore, E2-treated MEN1 KD MCF7 cells displayed a decrease in MYC activation, suggesting its role in estrogen-mediated MYC transcription. Finally, expression data mining in tumors revealed a correlation between the expression of MEN1 mRNA and that of several mTORC1 components and targets and a significant inverse correlation between MEN1 and two MYC inhibitory factors, MYCBP2 and MYCT1, in ER+ BC. The current work thus highlights altered mTORC1 and MYC pathways after menin inactivation in ER+ BC cells, providing insight into the crosstalk between menin, mTORC1 and MYC in ER+ BC.

Reduced menin expression leads to decreased ERα expression and is correlated with the occurrence of human luminal B-like and ER-negative breast cancer subtypes.

PURPOSE: Menin, encoded by the MEN1 gene, was recently reported to be involved in breast cancers, though the underlying mechanisms remain elusive. In the current study, we sought to further determine its role in mammary cells. METHODS: Menin expression in mammary lesions from mammary-specific Men1 mutant mice was detected using immunofluorescence staining. RT-qPCR and western blot were performed to determine the role of menin in ERα expression in human breast cancer cell lines. ChIP-qPCR and reporter gene assays were carried out to dissect the action of menin on the proximal ESR1 promoter. Menin expression in female patients with breast cancer was analyzed and its correlation with breast cancer subtypes was investigated. RESULTS: Immunofluorescence staining revealed that early mammary neoplasia in Men1 mutant mice displayed weak ERα expression. Furthermore, MEN1 silencing led to both reduced ESR1 mRNA and ERα protein expression in MCF7 and T47D cells. To further dissect the regulation of ESR1 transcription by menin, we examined whether and in which way menin could regulate the proximal ESR1 promoter, which has not been fully explored. Using ChIP analysis and reporter gene assays covering - 2500 bp to + 2000 bp of the TSS position, we showed that the activity of the proximal ESR1 promoter was markedly reduced upon menin downregulation independently of H3K4me3 status. Importantly, by analyzing the expression of menin in 354 human breast cancers, we found that a lower expression was associated with ER-negative breast cancer (P = 0.041). Moreover, among the 294 ER-positive breast cancer samples, reduced menin expression was not only associated with larger tumors (P = 0.01) and higher SBR grades (P = 0.005) but also with the luminal B-like breast cancer subtype (P = 0.006). Consistent with our clinical data, we demonstrated that GATA3 and FOXA1, co-factors in ESR1 regulation, interact physically with menin in MCF7 cells, and MEN1 knockdown led to altered protein expression of GATA3, the latter being a known marker of the luminal A subtype, in MCF7 cells. CONCLUSION: Taken together, our data provide clues to the important role of menin in ERα regulation and the formation of breast cancer subtypes.

MEN1 silencing aggravates tumorigenic potential of AR-independent prostate cancer cells through nuclear translocation and activation of JunD and ß-catenin.

BACKGROUND: Recent studies highlighted the increased frequency of AR-low or -negative prostate cancers (PCas) and the importance of AR-independent mechanisms in driving metastatic castration-resistant PCa (mCRPC) development and progression. Several previous studies have highlighted the involvement of the MEN1 gene in PCa. In the current study, we focused on its role specifically in AR-independent PCa cells. METHODS: Cell tumorigenic features were evaluated by proliferation assay, foci formation, colony formation in soft agar, wound healing assay and xenograft experiments in mice. Quantitative RT-PCR, Western blot and immunostaining were performed to determine the expression of different factors in human PCa lines. Different ChIP-qPCR-based assays were carried out to dissect the action of JunD and ß-catenin. RESULTS: We found that MEN1 silencing in AR-independent cell lines, DU145 and PC3, resulted in an increase in anchorage independence and cell migration, accompanied by sustained MYC expression. By searching for factors known to positively regulate MYC expression and play a relevant role in PCa development and progression, we uncovered that MEN1-KD triggered the nuclear translocation of JunD and ß-catenin. ChIP and 3C analyses further demonstrated that MEN1-KD led to, on the one hand, augmented binding of JunD to the MYC 5' enhancer and increased formation of loop structure, and on the other hand, increased binding of ß-catenin to the MYC promoter. Moreover, the expression of several molecular markers of EMT, including E-cadherin, BMI1, Twist1 and HIF-1α, was altered in MEN1-KD DU145 and PC3 cells. In addition, analyses using cultured cells and PC3-GFP xenografts in mice demonstrated that JunD and ß-catenin are necessary for the altered tumorigenic potential triggered by MEN1 inactivation in AR-independent PCa cells. Finally, we observed a significant negative clinical correlation between MEN1 and CTNNB1 mRNA expression in primary PCa and mCRPC datasets. CONCLUSIONS: Our current work highlights an unrecognized oncosuppressive role for menin specifically in AR-independent PCa cells, through the activation of JunD and ß-catenin pathways.

Men1 disruption in Nkx3.1-deficient mice results in ARlow/CD44+ microinvasive carcinoma development with the dysregulated AR pathway.

Dysregulated androgen receptor (AR) plays a crucial role in prostate cancer (PCa) development, though further factors involved in its regulation remain to be identified. Recently, paradoxical results were reported on the implication of the MEN1 gene in PCa. To dissect its role in prostate luminal cells, we generated a mouse model with inducible Men1 disruption in Nkx3.1-deficient mice in which mouse prostatic intraepithelial neoplasia (mPIN) occur. Prostate glands from mutant and control mice were analyzed pathologically and molecularly; cellular and molecular analyses were carried out in PCa cell lines after MEN1 knockdown (KD) by siRNA. Double-mutant mice developed accelerated mPIN and later displayed microinvasive adenocarcinoma. Markedly, early-stage lesions exhibited a decreased expression of AR and its target genes, accompanied by reduced CK18 and E-cadherin expression, suggesting a shift from a luminal to a dedifferentiated epithelial phenotype. Intriguingly, over 60% of menin-deficient cells expressed CD44 at a later stage. Furthermore, MEN1 KD led to the increase in CD44 expression in PC3 cells re-expressing AR. Menin bound to the proximal AR promoter and regulated AR transcription via the H3K4me3 histone mark. Interestingly, the cell proliferation of AR-dependent cells (LNCaP, 22Rv1, and VCaP), but not of AR-independent cells (DU145, PC3), responded strongly to MEN1 silencing. Finally, menin expression was found reduced in some human PCa. These findings highlight the regulation of the AR promoter by menin and the crosstalk between menin and the AR pathway. Our data could be useful for better understanding the increasingly reported AR-negative/NE-negative subtype of PCa and the mechanisms underlying its development.

Foxa2, a novel protein partner of the tumour suppressor menin, is deregulated in mouse and human MEN1 glucagonomas.

Foxa2, known as one of the pioneer factors, plays a crucial role in islet development and endocrine functions. Its expression and biological functions are regulated by various factors, including, in particular, insulin and glucagon. However, its expression and biological role in adult pancreatic α-cells remain elusive. In the current study, we showed that Foxa2 was overexpressed in islets from α-cell-specific Men1 mutant mice, at both the transcriptional level and the protein level. More importantly, immunostaining analyses showed its prominent nuclear accumulation, specifically in α-cells, at a very early stage after Men1 disruption. Similar nuclear FOXA2 expression was also detected in a substantial proportion (12/19) of human multiple endocrine neoplasia type 1 (MEN1) glucagonomas. Interestingly, our data revealed an interaction between Foxa2 and menin encoded by the Men1 gene. Furthermore, using several approaches, we demonstrated the relevance of this interaction in the regulation of two tested Foxa2 target genes, including the autoregulation of the Foxa2 promoter by Foxa2 itself. The current study establishes menin, a novel protein partner of Foxa2, as a regulator of Foxa2, the biological functions of which extend beyond the pancreatic endocrine cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Islet Cells Serve as Cells of Origin of Pancreatic Gastrin-Positive Endocrine Tumors.

The cells of origin of pancreatic gastrinomas remain an enigma, since no gastrin-expressing cells are found in the normal adult pancreas. It was proposed that the cellular origin of pancreatic gastrinomas may come from either the pancreatic cells themselves or gastrin-expressing cells which have migrated from the duodenum. In the current study, we further characterized previously described transient pancreatic gastrin-expressing cells using cell lineage tracing in a pan-pancreatic progenitor and a pancreatic endocrine progenitor model. We provide evidence showing that pancreatic gastrin-expressing cells, found from embryonic day 12.5 until postnatal day 7, are derived from pancreatic Ptf1a(+) and neurogenin 3-expressing (Ngn3(+)) progenitors. Importantly, the majority of them coexpress glucagon, with 4% coexpressing insulin, indicating that they are a temporary subpopulation of both alpha and beta cells. Interestingly, Men1 disruption in both Ngn3 progenitors and beta and alpha cells resulted in the development of pancreatic gastrin-expressing tumors, suggesting that the latter developed from islet cells. Finally, we detected gastrin expression using three human cohorts with pancreatic endocrine tumors (pNETs) that have not been diagnosed as gastrinomas (in 9/34 pNETs from 6/14 patients with multiple endocrine neoplasia type 1, in 5/35 sporadic nonfunctioning pNETs, and in 2/20 sporadic insulinomas), consistent with observations made in mouse models. Our work provides insight into the histogenesis of pancreatic gastrin-expressing tumors.

Transdifferentiation of pancreatic α-cells into insulin-secreting cells: From experimental models to underlying mechanisms.

Pancreatic insulin-secreting ß-cells are essential regulators of glucose metabolism. New strategies are currently being investigated to create insulin-producing ß cells to replace deficient ß cells, including the differentiation of either stem or progenitor cells, and the newly uncovered transdifferentiation of mature non-ß islet cell types. However, in order to correctly drive any cell to adopt a new ß-cell fate, a better understanding of the in vivo mechanisms involved in the plasticity and biology of islet cells is urgently required. Here, we review the recent studies reporting the phenomenon of transdifferentiation of α cells into ß cells by focusing on the major candidates and contexts revealed to be involved in adult ß-cell regeneration through this process. The possible underlying mechanisms of transdifferentiation and the interactions between several key factors involved in the process are also addressed. We propose that it is of importance to further study the molecular and cellular mechanisms underlying α- to ß-cell transdifferentiation, in order to make ß-cell regeneration from α cells a relevant and realizable strategy for developing cell-replacement therapy.

p.Ala541Thr variant of MEN1 gene: a non deleterious polymorphism or a pathogenic mutation?

CONTEXT: Multiple Endocrine Neoplasia Type 1 (MEN1) is an autosomal dominant inherited syndrome, related to mutations in the MEN1 gene. Controversial data suggest that the nonsynonymous p.Ala541Thr variant, usually considered as a non-pathogenic polymorphism, may be associated with an increased risk of MEN1-related lesions in carriers. OBJECTIVE: The aim of this study was to evaluate the pathogenic influence of the p.Ala541Thr variant on clinical and functional outcomes. PATIENTS AND METHODS: We analysed a series of 55 index patients carrying the p.Ala541Thr variant. Their clinical profile was compared to that of 117 MEN1 patients. The biological impact of the p.Ala541Thr variant on cell growth was additionally investigated on menin-deficient Leydig cell tumour (LCT)10 cells generated from Men1+/Men1- heterozygous knock-out mice, and compared with wild type (WT). RESULTS: The mean age at first appearance of endocrine lesions was similar in both p.Ala541Thr carriers and MEN1 patients, but no p.Ala541Thr patient had more than one cardinal MEN1 lesion at initial diagnosis. A second MEN1 lesion was diagnosed in 13% of MEN1 patients and in 7% of p.Ala541Thr carriers in the year following preliminary diagnosis. Functional studies on LCT10 cells showed that overexpression of the p.Ala541Thr variant did not inhibit cell growth, which is in direct contrast to results obtained from investigation of WT menin protein. CONCLUSION: Taken together, these data raise the question of a potential pathogenicity of the p.Ala541Thr missense variant of menin that commonly occurs within the general population. Additional studies are required to investigate whether it may be involved in a low-penetrance MEN1 phenotype.

Both PAX4 and MAFA are expressed in a substantial proportion of normal human pancreatic alpha cells and deregulated in patients with type 2 diabetes.

Pax4 and MafA (v-maf musculoaponeurotic fibrosarcoma oncogene homolog A) are two transcription factors crucial for normal functions of islet beta cells in the mouse. Intriguingly, recent studies indicate the existence of notable difference between human and rodent islet in terms of gene expression and functions. To better understand the biological role of human PAX4 and MAFA, we investigated their expression in normal and diseased human islets, using validated antibodies. PAX4 was detected in 43.0±5.0% and 39.1±4.0% of normal human alpha and beta cells respectively. We found that MAFA, detected in 88.3±6.3% insulin(+)cells as in the mouse, turned out to be also expressed in 61.2±6.4% of human glucagons(+) cells with less intensity than in insulin(+) cells, whereas MAFB expression was found not only in the majority of glucagon(+) cells (67.2±7.6%), but also in 53.6±10.5% of human insulin(+) cells. Interestingly, MAFA nuclear expression in both alpha and beta cells, and the percentage of alpha cells expressing PAX4 were found altered in a substantial proportion of patients with type 2 diabetes. Both MAFA and PAX4 display, therefore, a distinct expression pattern in human islet cells, suggesting more potential plasticity of human islets as compared with rodent islets.

High incidence of mammary intraepithelial neoplasia development in Men1-disrupted murine mammary glands.

Mutations of the MEN1 tumour suppressor gene predispose patients to the development of multiple endocrine neoplasia type 1 (MEN1) syndrome, which is characterized by multiple endocrine tumours, including prolactinomas. The recent findings of the interaction between menin, encoded by the MEN1 gene, and the oestrogen receptor, as well as the observation of rare cases of mammary carcinomas in our heterozygous Men1 mutant mice, led us to investigate a putative tumour suppressor function of the Men1 gene in mouse mammary cells by disrupting the gene in luminal epithelial cells. A significantly higher incidence of mammary intraepithelial neoplasia (MIN) was observed in mutant WapCre-Men1(F/F) mice (51.5%) than in WapCre-Men1(+/+) (0%) or Men1(F/F) (7.1%) control mice. The majority of MIN observed in the mutant mice displayed complete menin inactivation. Because of the leakage of WapCre transgene expression, prolactinomas were observed in 83.3% of mutant mice, leading to premature death. As there was no correlation between MIN development and elevated serum prolactin levels, and phospho-STAT5 expression was decreased in mammary lesions, the increased incidence of MIN lesions was most likely due to Men1 disruption rather than to prolactinoma development. Interestingly, in MIN lesions, we found a decrease in membrane-associated E-cadherin and beta-catenin expression, the latter of which is a menin partner. Finally, reduced menin expression was found in a large proportion of two independent cohorts of patients with breast carcinomas. Taken together, the current work indicates a role of Men1 inactivation in the development of mammary pre-cancerous lesions in mice and a potential role in human mammary cancer.

Characterisation of prostate cancer lesions in heterozygous Men1 mutant mice.

BACKGROUND: Mutations of the MEN1 gene predispose to multiple endocrine neoplasia type 1 (MEN1) syndrome. Our group and others have shown that Men1 disruption in mice recapitulates MEN1 pathology. Intriguingly, rare lesions in hormone-dependent tissues, such as prostate and mammary glands, were also observed in the Men1 mutant mice. METHODS: To study the occurrence of prostate lesions, we followed a male mouse cohort of 47 Men1+/- mice and 23 age-matched control littermates, starting at 18 months of age, and analysed the prostate glands from the cohort. RESULTS: Six Men1+/- mice (12.8%) developed prostate cancer, including two adenocarcinomas and four in situ carcinomas, while none of the control mice developed cancerous lesions. The expression of menin encoded by the Men1 gene was found to be drastically reduced in all carcinomas, and partial LOH of the wild-type Men1 allele was detected in three of the five analysed lesions. Using immunostaining for the androgen receptor and p63, a basal epithelial cell marker, we demonstrated that the menin-negative prostate cancer cells did not display p63 expression and that the androgen receptor was expressed but more heterogeneous in these lesions. Furthermore, our data showed that the expression of the cyclin-dependent kinase inhibitor CDKN1B (p27), a Men1 target gene known to be inactivated during prostate cell tumorigenesis, was notably decreased in the prostate cancers that developed in the mutant mice. CONCLUSION: Our work suggests the possible involvement of Men1 inactivation in the tumorigenesis of the prostate gland.

Alpha cell-specific Men1 ablation triggers the transdifferentiation of glucagon-expressing cells and insulinoma development.

BACKGROUND & AIMS: The tumor suppressor menin is recognized as a key regulator of pancreatic islet development, proliferation, and beta-cell function, whereas its role in alpha cells remains poorly understood. The purpose of the current study was to address this issue in relation to islet tumor histogenesis. METHODS: We generated alpha cell-specific Men1 mutant mice with Cre/loxP technology and carried out analyses of pancreatic lesions developed in the mutant mice during aging. RESULTS: We showed that, despite the alpha-cell specificity of the GluCre transgene, both glucagonomas and a large amount of insulinomas developed in mutant mice older than 6 months, accompanied by mixed islet tumors. Interestingly, the cells sharing characteristics of both alpha and beta cells were identified shortly after the appearance of menin-deficient alpha cells but well before the tumor onset. Using a genetic cell lineage tracing analysis, we demonstrated that insulinoma cells were directly derived from transdifferentiating glucagon-expressing cells. Furthermore, our data indicated that the expression of Pdx1, MafA, Pax4, and Ngn3 did not seem to be required for the initiation of this transdifferentiation. CONCLUSIONS: Our work shows cell transdifferentiation as a novel mechanism involved in islet tumor development and provides evidence showing that menin regulates the plasticity of differentiated pancreatic alpha cells in vivo, shedding new light on the mechanisms of islet tumorigenesis.

GC-MS analysis of liposoluble constituents from the stems of Cynomorium songaricum.

In order to evaluate the differences and similarities between the liposoluble constituents in Cynomorium songaricum populations, stem liposoluble constituents in five populations of C. songaricum collected from three different geographic regions and four different hosts were obtained by solvent extraction and analyzed by GC-MS. Cluster analysis of the percentage composition of 80 compounds showed differences in chemical composition which were related to the geographic origin rather than the host. Hexadecanoic acid was the most abundant compound in the essential oils of C. songaricum from hosts Nitraria sibirica and Nitraria tanguticum. Whereas (Z)-9-octadecenoic acid was accumulated in the oils of C. songaricum from Zygophyllum xanthoxylum and Peganum harmala. Four of the five populations had characteristic components, which were specific to each population.

Tumour suppressor menin is essential for development of the pancreatic endocrine cells.

Mutations of the multiple endocrine neoplasia type 1 (MEN1) gene predispose patients to MEN1 that affects mainly endocrine tissues, suggesting important physiological functions of the gene in adult endocrine cells. Homozygous disruption of Men1 in mice causes embryonic lethality, whereas the eventual involvement of the gene in embryonic development of the endocrine cells remains unknown. Here, we show that homozygous Men1 knockout mice demonstrate a reduced number of glucagon-positive cells in the E12.5 pancreatic bud associated with apoptosis, whereas the exocrine pancreas development in these mice is not affected. Our data suggest that menin is involved in the survival of the early pancreatic endocrine cells during the first developmental transition. Furthermore, chimerism assay revealed that menin has an autonomous and specific effect on the development of islet cells. In addition, using pancreatic bud culture mimicking the differentiation of alpha- and beta-cells during the second transition, we show that loss of menin leads to the failure of endocrine cell development, altered pancreatic structure and a markedly decreased number of cells expressing neurogenin 3, indicating that menin is also required at this stage of the endocrine pancreas development. Taken together, our results suggest that menin plays an indispensable role in the development of the pancreatic endocrine cells.

MEN1 mutation analysis in Chinese patients with multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN1) is an inherited tumour syndrome characterized by the development of tumours of the parathyroid, anterior pituitary and pancreatic islets, etc. Heterozygous germ line mutations of MEN1 gene are responsible for the onset of MEN1. We investigated the probands and 31 family members from eight unrelated Chinese families associated with MEN1 and identified four novel mutations, namely 373_374ins18, 822delT, 259delT and 1092delC, as well as three previously reported mutations, such as 357_360delCTGT, 427_428delTA and R108X (CGA>TGA) of MEN1 gene. Furthermore, we detected a loss of heterozygosity (LOH) at chromosome 11q in the removed tumours, including gastrinoma, insulinoma and parathyroid adenoma from two probands of MEN1 families. RT-PCR and direct sequencing showed that mutant MEN1 transcripts remained in the MEN1-associated endocrine tumours, whereas normal menin proteins could not be detected in those tumours by either immunohistochemistry or immunoblotting. In conclusion, MEN1 heterozygous mutations are associated with LOH and menin absence, which are present in MEN1-associated endocrine tumours.

Upregulation of the tumor suppressor gene menin in hepatocellular carcinomas and its significance in fibrogenesis.

The molecular mechanisms underlying the progression of cirrhosis toward hepatocellular carcinoma were investigated by a combination of DNA microarray analysis and literature data mining. By using a microarray screening of suppression subtractive hybridization cDNA libraries, we first analyzed genes differentially expressed in tumor and nontumor livers with cirrhosis from 15 patients with hepatocellular carcinomas. Seventy-four genes were similarly recovered in tumor (57.8% of differentially expressed genes) and adjacent nontumor tissues (64% of differentially expressed genes) compared with histologically normal livers. Gene ontology analyses revealed that downregulated genes (n = 35) were mostly associated with hepatic functions. Upregulated genes (n = 39) included both known genes associated with extracellular matrix remodeling, cell communication, metabolism, and post-transcriptional regulation gene (e.g., ZFP36L1), as well as the tumor suppressor gene menin (multiple endocrine neoplasia type 1; MEN1). MEN1 was further identified as an important node of a regulatory network graph that integrated array data with array-independent literature mining. Upregulation of MEN1 in tumor was confirmed in an independent set of samples and associated with tumor size (P = .016). In the underlying liver with cirrhosis, increased steady-state MEN1 mRNA levels were correlated with those of collagen alpha2(I) mRNA (P < .01). In addition, MEN1 expression was associated with hepatic stellate cell activation during fibrogenesis and involved in transforming growth factor beta (TGF-beta)-dependent collagen alpha2(I) regulation. In conclusion, menin is a key regulator of gene networks that are activated in fibrogenesis associated with hepatocellular carcinoma through the modulation of TGF-beta response.

Analysis of p27(Kip1) expression in insulinomas developed in pancreatic beta-cell specific Men1 mutant mice.

Multiple Endocrine Neoplasia type 1 (MEN1) is a hereditary disease characterised by the occurrence of multiple endocrine tumours. The biological functions of the responsible gene, MEN1, and its encoded protein, menin, remain so far largely elusive. The recent generation of Men1 mutant mice by our group and other laboratories provides powerful tools allowing for the identification of cellular and molecular events that occur after gene disruption. Interestingly, it has been recently reported that p27(Kip1) expression is regulated by menin and that decreased p27(Kip1) expression can be found in MEN1 insulinomas and parathyroid adenomas. In order to address whether and when p27(Kip1) expression alters during insulinoma development in pancreatic beta-cell-specific Men1 mutant mice, we analysed p27(Kip1) expression in islet lesions from mutant mice at different ages. Our data revealed that p27(Kip1) protein expression was reduced in 40 out of 52 (77%) insulinomas analysed, whereas the remaining 12 insulinomas (23%) did not show altered p27(Kip1) expression. No difference between the insulinomas with and without decreased p27(Kip1) expression could be observed in terms of histological features or menin inactivation. Furthermore, our analysis on hyperplastic and dysplastic islets developed in young mutant mice showed the lack of detectable alteration in p27(Kip1) expression, despite evident loss of menin expression in a substantial proportion of islet cells. Our work confirms the altered p27(Kip1) expression reported in tumours from MEN1 patients, whereas it suggests that other molecular events may also participate in the tumorigenesis process initiated by the Men1 gene inactivation.

Reduction of menin expression enhances cell proliferation and is tumorigenic in intestinal epithelial cells.

Menin, the product of the tumor suppressor gene MEN1, is widely expressed in mammalian endocrine and non-endocrine tissues, including intestine. Its known abundant expression in several types of cells with high proliferative capacity led us to investigate the physiological function of the protein menin in intestinal epithelium, one of the most rapidly growing epithelia. Here we showed that the Men1 gene is mainly expressed in the crypt compartment of the proximal small intestine and that its expression was increased during fasting in vivo, both suggesting a role of menin in the control of cell growth. Indeed, specific reduction of menin expression by transfected antisense cDNA in the rat duodenal crypt-like cell line, IEC-17, increased cell proliferation. The latter is correlated to a loss of cell-cycle arrest in G(1) phase by resting cells and an overexpression of cyclin D1 and cyclin-dependent kinase (Cdk)-4. Furthermore, these cells lost the inhibition of proliferation induced by transforming growth factor-beta1, associated with a decrease of transforming growth factor-beta type II receptor expression. As a result of deregulated proliferation, antisense menin transfected IEC-17 cells became tumorigenic as shown in vitro as well as in vivo in immunosuppressed animals. These results indicate that menin contributes to proliferation control in intestinal epithelial cells. The present study reveals an unknown physiological function for menin in intestine that may be important in the regulation of epithelial homeostasis.

Pancreatic beta-cell-specific ablation of the multiple endocrine neoplasia type 1 (MEN1) gene causes full penetrance of insulinoma development in mice.

The function of the predisposition gene to multiple endocrine neoplasia type 1 (MEN1) syndrome remains largely unknown. Previous studies demonstrated that null mutation of the Men1 gene caused mid-gestation lethality in mice, whereas heterozygous Men1 knockout mice developed multiple endocrine tumors late in life. To seek direct evidence on the causal role of menin in suppressing tumor development, we generated mice in which the Men1 gene was disrupted specifically in pancreatic beta cells. These mice began to develop hyperplastic islets at as early as 2 months of age and insulinomas at 6 months of age. The islet lesions exhibited features of multistage tumor progression, including beta-cell dedifferentiation, angiogenesis, and altered expression of both E-cadherin and beta-catenin. Additionally, disturbance of blood insulin and glucose levels correlated with tumor development, mimicking human MEN1 symptoms. Our data indicate that this strain of mice provides a powerful tool for the study of the mechanisms of tumorigenesis related to MEN1 disease.