[CANCER RESEARCH 63, 4836 – 4841, August 15, 2003]

Pancreatic ␤-Cell-specific Ablation of the Multiple Endocrine Neoplasia Type 1 (MEN1) Gene Causes Full Penetrance of Insulinoma Development in Mice1 Philippe Bertolino, Wei-Min Tong, Pedro Luis Herrera, Huguette Casse, Chang Xian Zhang,2 and Zhao-Qi Wang Genetic and Cancer Laboratory, CNRS, UMR5641, Faculty of Medicine University Lyon 1, 69373, Lyon, France [P. B., H. C., C. X. Z.]; IARC, 69008, Lyon, France [P. B., W-M. T., Z-Q. W.], and Department of Morphology, School of Medicine, University of Geneva, CH-1211 Geneva 4, Switzerland [P. L. H.]

ABSTRACT 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 ␤ 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 ␤-cell dedifferentiation, angiogenesis, and altered expression of both E-cadherin and ␤-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.

INTRODUCTION The MEN1 gene has been identified as the gene responsible for MEN13 (1, 2), a hereditary syndrome transmitted with an autosomal dominant trait (MEN1; Online Mendelian Inheritance in Man no. 131100). The disease is characterized by a predisposition to multiple endocrine tumors in the parathyroid, endocrine pancreas, and anterior pituitary, as well as adrenal cortical tumors, although less frequently, and foregut diffused endocrine carcinoids. Several nonendocrine tumors have also been seen in patients suffering from MEN1, such as lipoma and angioma (3). The germ line mutation of the MEN1 gene has been detected in ⬃85% of familial MEN1 cases (4 –5), and somatic mutations have also been found in several types of sporadic endocrine tumors, especially in sporadic parathyroid adenomas (6), gastrinomas, and insulinomas (7). Both germ line and sporadic mutations show a typical “loss of function” profile, with different types of mutation detected along the whole coding sequence (8). However, these observations do not establish any genotype-phenotype correlation. In addition, the loss of heterozygosity has been observed both in familial MEN1 tumor tissues and in their sporadic counterparts, suggesting the tumor suppressing nature of the MEN1 gene (9 –10). Although much effort has been made to address the function of the MEN1 gene in vivo and in tumorigenesis, little is known to date about Received 12/26/02; revised 3/21/03; accepted 6/5/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by the Association for International Cancer Research, United Kingdom; the Association pour la Recherche Contre le Cancer, France; the Programme Emmergence from the Re´gion Rhoˆne-Alpes; and La Ligue Contre le Cancer du Rhoˆne. P. B. is the recipient of a fellowship from the French Ministry of Education, and P. L. H. is funded by the Juvenile Diabetes Research Foundation and the Swiss National Science Foundation. 2 To whom requests for reprints should be addressed, at Laboratoire Ge´ne´tique, CNRS, UMR5641, Faculty of Medicine University Lyon 1, 8 avenue Rockefeller, 69373 Lyon Cedex, France. Phone: 33-4-78-77-72-13; Fax: 33-4-78-77-72-20; E-mail: zhang@rockefeller. univ-lyon1.fr. 3 The abbreviations used are: MEN1, multiple endocrine neoplasia type 1; Rip; rat insulin promoter; Cre, causes recombination.

the latter because of the unavailability of null mutant cells lines from MEN1 patients. Recently, Crabtree et al. (11) reported that heterozygous Men1 knockout mice develop multiple endocrine tumors. However, neither gastrinoma, the most frequently observed entero-pancreatic endocrine tumor in MEN1 patients, nor glucagonoma was reported in this model. It has been noticed that these mice also develop endocrine tumors mainly seen in other polyendocrinopathies, such as pheochromocytoma, with a substantial incidence (11). To study the role of the MEN1 gene in endocrine malignancy, we generated Men1 knockout mice using the conventional strategy. Whereas homozygous mutant (Men1T/T) mice died at E11.5–E13.5 with multiple developmental defects, including nonclosure of the neural tube, heart hypotrophy, and an altered organization of the epithelial and hematopoietic compartments in the liver, heterozygous Men1⫹/T mice appeared to be normal and did not show any phenotypic abnormalities at a young age (12). However, at ⬃12 months of age, Men1⫹/T mice started to develop the full range of major endocrine tumors seen in MEN1 patients, affecting the parathyroid, pancreatic islets, pituitary, and adrenal glands.4 The tumor spectrum is reminiscent of cases seen in MEN1 patients. Furthermore, these tumors were associated with a loss of heterozygosity of Men1, suggesting that the Men1 gene is completely inactivated during the development of these tumors. However, whether the complete loss of Men1 function is prerequisite and causal genetic event responsible for tumor development in MEN1 patients could not be addressed in the above models. In addition, because of the embryonic lethality of homozygous Men1 mutant mice in mid-gestation, the function of menin in the development of the endocrine organs remains elusive. To address these questions, mice carrying a floxed Men1 gene (13), generated by the conditional knockout technique, would be useful for disrupting both alleles of the gene within specifically targeted cells. We report here that specific somatic deletion of the Men1 gene in ␤ cells results in insulinoma formation with multiple progression features, providing, for the first time, the direct evidence that menin is a tumor suppressor in insulinoma genesis. MATERIALS AND METHODS Men1F/F Knockout Mice and Men1F/F-RipCreⴙ Conditional Knockout Mice. Mice carrying the floxed allele (Men1⫹/F) were generated by crossing the previously generated Men1⫹/T mice (see Ref. 12; Fig. 1) with SycP1Cre transgenic mice expressing the Cre recombinase in testis during spermatogenesis (14). Specifically, Men1⫹/F mice were obtained from offspring of male Men1⫹/T-SycP1Cre⫹ mice crossed with Men1⫹/T mice. They were then bred to homozygosity and termed Men1F/F. Men1F/F mice were then crossed with RipCre transgenic mice expressing Cre under the control of the rat insulin promoter (15), to generate the Men1⫹/F-RipCre⫹ mice. Histological and Immunohistochemical Analysis of Pancreatic Tissues. Pancreases were collected from mice at predetermined time points and fixed in 4% neutral-buffered formaldehyde for at least 24 h, followed by dehydration and paraffin embedding. Histopathological analysis was carried out on 3-␮m sections stained with H&E. Immunohistochemical staining was performed on serial sections essentially, as described previously (16), using antibodies against insulin (polyclonal, 1:400; DAKO, Carpinteria, CA), glucagon (poly-

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Unpublished observations.

ROLE OF THE Men1 GENE IN ␤-CELL TUMORIGENESIS

Fig. 1. Generation of the Men1 floxed allele and conditional disruption of the Men1 gene in pancreatic ␤ cells. A, structure and restriction map of the wild-type murine Men1 allele (⫹), targeting allele (T), floxed allele (F), and deleted allele (⌬). Exons are indicated by a black box with numbers; Neo and TK correspond to the neomycin and thymidine kinase genes. G, BglII; B, BamHI. Open arrows indicate the transgenic mice expressing Cre recombinase used to obtain the designated alleles. SycP1Cre mice were used to obtain mice carrying the floxed or deleted Men1 allele from the Men1⫹/T mutant mice. RipCre transgenic mice were crossed with Men1F/F mice to obtain mice carrying the deleted allele specifically in pancreatic ␤ cells. B, Southern blot analysis of tail DNA from generated mice. Tail DNA was digested by BamHI and BglII and hybridized with the pmM1–5⬘ probe. C, depletion of menin in pancreatic islets of Men1F/F-RipCre⫹ mice. Immunohistochemistry analysis using an antimenin antibody was performed in pancreas sections of Men1F/F-RipCre⫹ mice 2– 6 months of age and sections of Men1F/F-RipCre⫺ mice at 6 months of age. Note that the loss of menin staining in the nuclei was already found in many cells at 2 months and occurred in a majority of islet cells at 6 months in Men1F/F-RipCre⫹ mice. Original magnification, ⫻40.

clonal, 1:500; NovoCastra, United Kingdom), menin (polyclonal, 1:200; Santa-Cruz Biotechnology, CA), E-cadherin (monoclonal, 1:400; Zymed Laboratories, San Francisco, CA), and ␤-catenin (Monoclonal, 1:4000; Transduction Laboratories, Lexington, KY). Immunohistochemical staining using antibody against CD31 (monoclonal, 1/200; BD Bioscience, CA) was performed with paraformaldehyde-fixed sections. Isolation of Mouse Pancreatic Islets and PCR Analysis. Pancreatic islets were isolated from mice at 2–15 months of age according to the protocol described previously (17). Briefly, pancreases were digested with collagenase and dissociated vigorously by mechanical pipetting. Islets were “hand-picked” from dark field dishes under a dissecting microscope and pooled for further analysis. DNA was extracted and amplified using the following 3 primers: 2f0, 5⬘-CTTACCTCTTCTCATGTCTG; 3f1, 5⬘-GGATTCTGCCCCAGGC; and 3r1, 5⬘-CACCTCCATCTTACGGTCG. Each PCR reaction was carried out in a 25-␮l reaction mixture containing 1 ␮l of template DNA, 1 ␮M of each primer, 1 mM of each deoxynucleoside triphosphate, and 1 unit of Tag polymerase. The reaction mixture was denatured for 5 min at 94°C and incubated for 35 cycles (denaturing at 94°C for 30 s, annealing at 56°C for 45 s, and extending at 72°C for 105 s). Quantification of Blood Glucose and Serum Insulin Level. All measurements were carried out on animals of 2–15 months of age. Mice were fasted 4 h before blood and serum collection. For glucose measurement, blood samples were collected from the tail veins, and glucose concentration was determined using a Glucotrend 2 kit (Roche). For insulin serum level quantification, blood was collected from the retro-orbital plexus, serum was obtained after clotting, and separation was obtained by centrifugation. Quantification

was performed with a solid-phase, two-site ELISA immunoassay specific for mouse insulin (Ultrasensitive mouse insulin ELISA; Mercodia). Assays for each serum were performed in duplicate and repeated twice.

RESULTS Generation of Mice with Tissue-specific Deletion of the Men1 Gene in Pancreatic ␤ Cells. First we generated a mouse strain (12) containing the targeted allele (T) by injection of Men1⫹/T ES cells into mouse blastocysts (Fig. 1A). The generation of the mice carrying the conditional allele in which the third exon of the Men1 gene was flanked by two lox-P sites [designated as floxed (F) allele; see Fig. 1A] was achieved by crossing Men1⫹/T mice with SycP1Cre transgenic mice that express the Cre recombinase in the testis during an early stage of meiosis (14). This approach allowed the generation of mice carrying either the floxed (F) or deleted (⌬) Men1 allele (Fig. 1A), which were identified by Southern blot analysis (Fig. 1B). Intercrossing of Men1⫹/F mice and breeding of Men1⫹/F mice with Men1⫹/D mice gave rise to viable Men1F/F or Men1F/D mice. Western blot analysis of Men1F/F and Men1F/D embryos revealed normal menin levels that were comparable with those in wild-type mice (data not shown). These data suggest that the floxed Men1 allele was functional. To specifically delete the Men1 gene in pancreatic ␤-islet cells,

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Men1F/F mice were crossed with mice expressing Cre recombinase driven by the rat insulin promoter (RipCre; see Ref. 15). The excision of the floxed Men1 allele was determined by PCR analysis in physically isolated pancreatic islets of Men1F/F-RipCre⫹ mice, whereas the same excision was not detected from other tissues examined, including brain, spleen, liver, lung, kidney, testis/ovary, and skin in these mice (data not shown). To further verify the inactivation of menin protein in ␤ cells attributable to the specific disruption of the gene, we analyzed menin expression in Men1F/F-RipCre⫹ islets by immunohistochemical staining. Indeed, loss of menin expression was observed in a substantial number of cells in the islet of 2-month-old Men1F/FRipCre⫹ mice, and the proportion of the menin-negative cells increased remarkably 4 – 6 months (Fig. 1C). To the contrary, all of the pancreatic islet cells expressed menin in Men1F/F-RipCre⫺ (Fig. 1C) or Men1⫹/⫹-RipCre⫹ mice (data not shown). ␤-Cell-specific Deletion of the Men1 Gene Induces Insulinomas. To analyze the consequences of menin deletion in normal and tumor development, we monitored Men1F/F-RipCre⫹ mice for 15 months. The RipCre transgene used in this study expresses specifically in ␤ cells and induces excision of the genomic sequence flanked by two loxP sites as early as embryonic day 11 (18). To better visualize the progression of islet tumors, we isolated islets from pancreases of Men1F/F-RipCre⫹ mice. It appeared that islets exhibited enlargement at as early as 2 months of age, which progressed with aging (Fig. 2A). Consistent with this finding, histological examination revealed that

the islet hyperplasia appeared to be more evident at 4 months of age, and the majority of islets were hyperplastic or dysplastic at 6 months in Men1F/F-RipCre⫹ mice. Strikingly, insulinomas were found in 5 of 12 (41.5%) Men1F/F-RipCre⫹ mice at 6 months of age, whereas multiple islet tumors were commonly seen in these mice after 6 months of age (Table 1; see also Fig. 2A). Increased frequency of tumors was associated with the aging of Men1F/F-RipCre⫹ mice, and reached 100% at 10 months of age. We also noticed that all Men1F/F-RipCre⫹ mice examined after 10 months of age developed advanced insulinomas (carcinomas), often forming several macroscopic tumor masses (Fig. 2A). Of the 38 Men1F/F-RipCre⫺ or Men1⫹/⫹-RipCre⫹ islets analyzed, there were no obvious abnormalities. Although the inactivation of menin was readily detectable in many ␤ cells at 2 months of age (see Fig. 1C), there is a comparable intensity of insulin immunoreactivity, and proportion of insulin/glucagon positive (␤/␣) cells between islets from Men1F/F-RipCre⫹ and Men1F/F-RipCre⫺ mice (Fig. 2B), suggesting a dispensable role of menin in islet development. However, insulin immunoreactivity was reduced, although clearly positive, in a proportion of cells in hyperplastic/dysplastic islets and strongly decreased in more advanced insulinomas from Men1F/F-RipCre⫹ mice at 8 months compared with those in age-matched Men1F/F-RipCre⫺ controls (Fig. 2B). The progressive loss of insulin-positive staining in these tumors suggests a dedifferentiation process during insulinoma development, a phenom-

Fig. 2. Multiple insulinomas in Men1F/F-RipCre⫹ mice. A, macroscopic view of physically isolated islets from Men1F/F-RipCre⫹ mice between 2 and 15 months of age shows islet tumor development and progression. Note that the angiogenesis appears in insulinomas of 6-month-old Men1F/F-RipCre⫹ mice and becomes extensive in those that are 10 months old. Bars correspond to 1 mm. B, histological analysis performed on Men1F/F-RipCre⫹ mice 2–10 months of age shows the tumor progression associated with marked angiogenesis from the age of 6 months (arrows). Massive vascularization is evident in the entirety of the islet in 10-month-old mice. Insulin and glucagon stainings confirm that these tumors are insulinomas. Note that the number of insulin positive islet cells is reduced slightly at 4 months and extensively by the age of 10 months. No obvious abnormalities are found in the Men1F/F-RipCre⫺ mice. Original magnification, ⫻40.

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Table 1 Tumor development in ␤-cell-specific Men1 knockout mice Men1F/F-RipCre⫹ and Men1F/F-RipCre⫺ mice were monitored for tumor development and examined by histology at 2, 4, 6, 8 and 10 months respectively. Age

Islet phenotypea

Men1F/F-RipCre⫹

2 months

Normal Hyperplasia

8/11 (72.7%) 3/11 (27.3%)

4 months

Normal Hyper-/Dysplasia

1/8 (12.5%) 7/8 (87.5%)

6 months

Normal Hyper-/Dysplasia Insulinoma

12/12 (100%) 5/12 (41.5%)

Normal Hyper-/Dysplasia Insulinoma

16/16 (100%) 14/16 (87.5%)

Normal Hyper-/Dysplasia Insulinoma

11/11 (100%) 11/11 (100%)

8 months

10 months

b

Men1F/F-RipCre⫺ 6/6 (100%) 6/6 (100%) 10/10 (100%)

9/9 (100%)

7/7 (100%)

a

Histological spectra of islets. Number of mice of each histological category over the total number of the mice examined. The percentage of mice in each histological category is shown in parentheses. Note that the same pancreas may contain both hyper-/dysplastic islets and insulinomas. b

enon observed previously in a transgenic model of insulinoma (19). Although glucagon-positive ␣ cells were present in normal and hyperplastic/dysplastic islets, as well as in early insulinomas from Men1F/F-RipCre⫹ mice, ␣ cells were rarely seen in advanced insulinomas (carcinomas), possibly because of the disorganized architecture of the pancreatic islet (Fig. 2B). Tumor Progression Features of Men1-deficient Insulinomas. Tumor growth depends largely on angiogenesis, the formation of new blood vessels (20). To investigate whether angiogenesis is switched on in Men1-deficient insulinoma formation, we examined histological sections and physically isolated islets. Macroscopic analysis showed that angiogenesis was evident in isolated islets starting from the age of 6 months by blood islands consequent to microhemorrhaging (Fig. 2A). Histological analysis revealed that vasculature could be seen in

islets from the Men1F/F-RipCre⫹ mice at as early as 4 months of age (Fig. 2B). Strikingly, extensive vascularization was observed in insulinomas from most of the mice at 6 – 8 months of age and also in advanced insulinomas at 10 months (arrows in Fig. 2B). Moreover, an increased blood-vessel density was further confirmed by CD31 immunostaining in these islet tumors (Fig. 3A). Down-regulation or loss of E-cadherin has been shown to play a role in the transition from ␤-cell adenoma to islet carcinoma in the Rip1-Tag2 mouse model (21). To further characterize Men1-deficient insulinoma progression, we examined E-cadherin and ␤-catenin expressions at different stages of these Men1-deficient islets. Immunohistological analysis revealed that although the membrane expression of E-cadherin was on approximately the same level in early insulinomas (Fig. 3A, middle panel) as in normal islets (Fig. 3A, upper panel), it was decreased in advanced insulinomas (Fig. 3A, lower panel). We next analyzed the ␤-catenin expression in Men1-deficient islets and found that although its membrane expression was slightly reduced in early insulinomas (Fig. 3A, middle panel), ␤-catenin expression was further down-regulated in advanced islet carcinomas and was also detected in the cytoplasmic compartment (Fig. 3A, lower panel). This translocation of the ␤-catenin expression pattern was observed in Men1F/F-RipCre⫹ mice after 8 months of age. These data indicate that alteration of E-cadherin and ␤-catenin expression represents an important feature of insulinoma progression in the Men1 somatic-deleted mouse model. Inactivation of the Men1 Gene in ␤ Cells Causes Hormonal Disturbance. Hormonal dysregulation is often associated with insulinomas in MEN1 patients (3). To verify whether these mice also exhibit this physiological abnormality, we measured blood glucose and insulin levels. Although the blood glucose level remained normal until the age of 8 months, it decreased thereafter when compared with that of the control animals (Men1F/F-RipCre⫺) and continued to decrease with age (Fig. 3B). We also quantified the serum insulin

Fig. 3. Altered expression of angiogenic and cell adhesion molecules in insulinoma development and hormone disturbance in Men1F/F-RipCre⫹ mice. A, immunohistochemical staining of pancreas sections from Men1F/F-RipCre⫹ and Men1F/F-RipCre⫺ mice with antibodies against CD31, E-cadherin (E-Cad), and ␤-catenin (␤-Cat) corresponding to a higher magnification of the black squared region indicated in H&E staining sections. An increased CD31 immunostaining during progression of angiogenesis is seen in tumors. The reduction of E-cadherin membrane staining is found in the most advanced tumors (carcinoma, lower panel). ␤-catenin membrane staining in islets (Isl) versus in the exocrine compartment (Ex) is already decreased in adenomic islets (middle panel) in comparison with normal islets (upper panel). Note that loss of membrane-bound ␤-catenin and strong cytoplasmic ␤-catenin staining are seen in advanced insulinomas. Blood glucose (B) and serum insulin (C) analyses show the progressive decrease of mean blood glucose and increase of the serum insulin level during tumor progression in Men1F/F-RipCre⫹ mice compared with Men1F/F-RipCre⫺ mice. The number of mice analyzed for each time point and each genotype is given along the bars or curves. Measurement was performed in duplicate and repeated twice.

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level by ELISA. Although the insulin level in Men1F/F-RipCre⫹ and control mice was equal at the age of 2 months, it was superior in Men1F/F-RipCre⫹ mice compared with age-matched controls starting at the age of 4 months (Fig. 3B), with an ⬃1.25-fold increase. Such an increase continued with age (Fig. 3B): 1.43 fold at 6 months, 1.7 fold at 8 months, 2.2 fold at 10 months, and 14 fold by the age of 15 months. We noted that the increased insulin level apparently paralleled the development of insulinomas, but correlated inversely with blood glucose levels in Men1F/F-RipCre⫹ mice (see Fig. 2). DISCUSSION The previous study by Crabtree et al. (11), as well as the observations from our laboratory (12), demonstrated that whereas homozygous Men1 mutant mice die at mid-gestation, older heterozygous Men1 knockout mice develop multiple endocrine tumors, mainly in the pancreas and parathyroid, pituitary, and adrenal glands, with insulinomas being the most common lesions (11).4 Tumor spectra, and the loss of the wild-type Men1 allele in tumors of heterozygous Men1⫹/T mice, are consistent with what happens in human MEN1 patients. In the present study, we established a direct cause/effect relationship between menin inactivation and endocrine tumor development by generating a Men1 knockout mouse model with the Men1 gene specifically disrupted in ␤ cells, one of the major endocrine cell types affected in MEN1 disease. We noticed that the latency period is substantially reduced compared with that observed in Men1⫹/T (11),4 as well as Men1F/T mice (data not shown). These data indicate that the complete loss of menin function is a limiting step in inducing endocrine cell transformation. Despite these findings, the insulinomas in our somatic deletion of Men1 model appeared at 6 – 8 months of age, suggesting that once menin is completely inactivated, other genetic events may be necessary to fully drive ␤ cells into neoplasm. One of the advantages of this conditional knockout approach is the ability to monitor the entire process of tumorigenesis, including hyperplasia, dysplasia, and adenoma/carcinoma formation. Indeed, we were able to follow development of islet lesions, as well as angiogenesis and progressive change of adhesion molecules, during tumor progression, both of which play an important role in insulinoma progression (20, 21). In fact, early insulinomas in our Men1 somatic deletion mice already looked angiogenic, whereas extensive vascularization was seen in all of the advanced insulinomas without exception. Meanwhile, we found that the membrane expression of both Ecadherin and ␤-catenin was down-regulated in advanced insulinomas. More interestingly, the reduction of ␤-catenin membrane expression started even in early insulinomas, whereas the intracellular expression of ␤-catenin was seen in advanced insulinomas. These features suggest that the altered expression of ␤-catenin is an important event in insulinoma progression. It is known that ␤-catenin is not only important for the establishment and maintenance of cell-to-cell adhesion, but also crucial for the regulation of gene expression through Wnt signaling. However, we did not find any significant alteration of c-Myc and cyclin-D1 expression in these islet tumor cells (data not shown), both genes being regulated by the Wnt pathway (22, 23) and playing an important role in homeostasis of ␤ cells (19). It is interesting to note that transgenic mice expressing SV40 T antigen under control of the rat insulin promoter developed hyperplastic and tumor lesions in islet ␤ cells beginning at 9 –10 weeks after birth (24). In addition, ectopic coexpression of c-Myc and Bcl-XL in transgenic mice caused an immediate and uniform malignant tumor progression within 7 days of c-Myc activation, along with the suppression of c-Myc-induced apoptosis (19). Extensive studies on these mouse insulinoma models have identified the genetic and cellular

events involved in islet-tumor development, including those related to oncogene activation, cell-cycle control, apoptosis, and angiogenesis in islet tumorigenesis (25). Although there are some common features between the above-mentioned insulinoma models and our own, such as dedifferentiation of ␤ cells, angiogenic islets, and multistage tumorigenesis, our insulinoma model differs from the above-mentioned models by a relatively long latency period. It seems that the molecular events causing tumor development in our model may not be the same. Nevertheless, our results, together with findings from a previous report (11), demonstrate that menin is a key molecule to suppress tumor development. Menin is known to interact with several partners, such as JunD, Smad3, nm23, pem, and the major components of nuclear factor ␬B family p65, p52, and p50 (26 –30). The possible involvement of the interaction between menin and other partners in the cellular functions that may be important in islet tumorigenesis requires further investigation. Because of early embryonic lethality in Men1 null mice (11, 12), the role of menin in the development of endocrine tissues could not be addressed. The fact that the entire islet architecture appears intact in Men1F/F-RipCre⫹ mice at 2 months when somatic inactivation of Men1 in mice has occurred indicates that the development of islets is not affected in the absence of menin. These findings, together with multiple defects in null Men1 mouse embryos, suggest that menin may play a distinct role in different cell types and in normal differentiation versus tumorigenesis. Given the unique and cell-type-specific disruption of the Men1 gene in ␤-islet cells and the availability of the conditional Men1 mouse model carrying the floxed Men1 allele, these mice provide a powerful tool to dissect genetic and molecular events causing not only insulinomas in the present study, but also tumors from other tissues, including the parathyroids and the pituitary, affected in MEN1 disease. Finally, these mice, which could have the Men1 gene inactivated in specific tissues or at specific stages, would allow further assessment of menin and its interacting partners in various biological processes, which, ultimately, would advance the understanding of the mechanism underlying tumorigenesis related to MEN1 disease. ACKNOWLEDGMENTS We thank Franc¸ ois Cuzin (Centre De Biochimie, Universite´ De Nice, Nice, France) for providing SycP1Cre mice; Dominique Galendo and Marie-Pierre Cros (IARC, Lyon, France) for the maintenance of the mouse colonies; and Nicole Lyandrat, Sandra Roche, and Christine Carreira (IARC, Lyon, France) for histological assistance.

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