Menin Promotes the Wnt Signaling Pathway in Pancreatic Endocrine Cells Gao Chen, Jingbo A, Min Wang, Steven Farley, Lung-Yi Lee, Lung-Ching Lee, and Mark P. Sawicki David Geffen School of Medicine at University of California at Los Angeles and the Greater Los Angeles VA Medical Center, Los Angeles, California Abstract Introduction Menin is a tumor suppressor protein mutated in Multiple endocrine neoplasia type 1 (MEN1) is an patients with multiple endocrine neoplasia type 1. We autosomal dominant disease characterized by the development show that menin is essential for canonical Wnt/B- of parathyroid hyperplasia, pancreatic islet cell tumors, and catenin signaling in cultured rodent islet tumor cells. anterior pituitary endocrine tumors (1). Besides these classic In these cells, overexpression of menin significantly tumors, several other endocrine and nonendocrine tumors may enhances TCF gene assay reporter activity in response develop, including lipomas, dermatofibromas, collagenomas, to B-catenin activation. Contrastingly, inhibition of ependymomas, meningiomas, schwannomas, carcinoids, adre- menin expression with Men1 siRNA decreases TCF nal cortical tumors, and thyroid tumors. The MEN1 gene is reporter gene activity. Likewise, multiple endocrine highly conserved and encodes for a 67-kDa nuclear protein, neoplasia type 1 disease associated missense called menin, which has been implicated in DNA metabolism mutations of menin abrogate the ability to increase TCF and transcription regulation (2). MEN1 patients have trunca- reporter gene activity. We show that menin physically tion mutations and missense mutations that are predicted to interacts with proteins involved in the canonical Wnt inactivate menin function consistent with a tumor suppressor À/À signaling pathway, including B-catenin, TCF3 (TCFL1), protein (3). Homozygous knockout mice (Men1 )die and weakly with TCF4 (TCFL2). Menin overexpression in utero (E11.5-13.5) with multiple developmental defects, increases expression of the Wnt/B-catenin downstream suggesting that menin that has an essential role in early target gene Axin2, which is associated with increased development (4). Similar to MEN1 patients, heterozygous À/+ H3K4 trimethylation of the Axin2 gene promoter. knockout mice (Men1 ) develop normally, but the adult mice Moreover, inhibition of menin expression by siRNA eventually grow endocrine tumors (5). abrogates H3K4 trimethylation and Axin2 gene To determine how the loss of menin promotes endocrine expression. Based on these studies, we hypothesized tumor development, most investigations have focused on the that Wnt signaling could inhibit islet cell proliferation role of menin in transcription regulation. Menin interacts with because loss of menin function is thought to increase several transcription factors including members of the activator endocrine tumor cell proliferation. TGP61 rodent protein-1 (JunD), transforming growth factor-h/bone morpho- islet tumor cells treated with a glycogen synthase genetic protein (RUNX2, SMAD1, SMAD3, and SMAD5), and kinase 3B inhibitor that increases Wnt pathway nuclear factor-nB (p65, p50, and p52) signal transduction signaling had decreased cell proliferation compared pathways (6-11). More recently, menin was identified in MLL with vehicle-treated cells. Collectively, these and MLL2 histone methyltransferase complexes and found data suggest that menin has an essential role in essential for the histone methyltransferase activity (12-16). Wnt/B-catenin signaling through a mechanism that Pancreas development, in particular islets, is mediated by eventually affects histone trimethylation of the expression of a specific program of growth factors including the downstream target gene Axin2, and activation of Wnt proteins (17-24). In general, Wnt proteins regulate Wnt/B-catenin signaling inhibits islet tumor cell development through actions on cell proliferation, differentia- proliferation. (Mol Cancer Res 2008;6(12):1894–907) tion, cell fate decisions, apoptosis, axial polarity, axonal guidance, and adhesion (25). In the canonical Wnt pathway, the extracellular Wnt ligand (20 different genes in mammalian cells) binds the cell surface receptor frizzled (10 FZD genes in mammalian cells) and the coreceptor LRP5/6 (low density Received 12/14/07; revised 8/4/08; accepted 8/25/08. lipoprotein receptor–related proteins 5 and 6). This initiates a Grant support: NIH grant R01CA095148 and VA Merit grants (M.P. Sawicki). cascade of intracellular mediators that results in h-catenin 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 stabilization and subsequent h-catenin nuclear localization to accordance with 18 U.S.C. Section 1734 solely to indicate this fact. regulate target gene transcription (26). In the absence of Wnt Note: Supplementary data for this article are available at Molecular Cancer ligand, h-catenin is phosphorylated in a cytoplasmic complex Research Online (http://mcr.aacrjournals.org/). Requests for reprints: Mark P. Sawicki, David Geffen School of Medicine, CHS with casein kinase 1, glycogen synthase kinase 3h (GSK3h), 72-215, Los Angeles, CA 90095-6904. Phone: 310-268-3298; Fax: 310-268- axin 1, and the tumor suppressor adenomatous polyposis coli. 3026. E-mail: [email protected] h Copyright D 2008 American Association for Cancer Research. Phosphorylated -catenin is ubiquitylated and targeted for doi:10.1158/1541-7786.MCR-07-2206 degradation by the proteasome. Wnt binds the Fzd receptor and 1894 Mol Cancer Res 2008;6(12). December 2008 Downloaded from mcr.aacrjournals.org on September 23, 2021. © 2008 American Association for Cancer Research. Menin Is Essential for Wnt Signaling 1895 stops phosphorylation of h-catenin. The hypophosphorylated (dephosphorylated) h-catenin is not altered by menin over- h-catenin translocates to the nucleus where it associates with expression. the T-cell–specific transcription factor/lymphoid enhancer binding factor 1 (TCF/LEF) family transcription factors and Menin Is Essential for b-Catenin Activation of a TCF regulates Wnt responsive genes expression. Responsive Reporter Components of the Wnt pathway regulate pancreas deve- To determine whether menin is involved with Wnt/h-catenin lopment, although the mechanism is not fully understood. signaling, we studied mouse TGP-61 islet tumor cells trans- Numerous members of the Wnt pathway are expressed during fected with a TCF responsive luciferase reporter vector the development of the mouse pancreas and in the adult organ (Super8XTOPflash, M50) containing eight TCF/LEF binding (27). In particular, activated (dephosphorylated) h-catenin and sites or a control vector (Super8XFOPflash, M51) that has the parahox protein Pdx-1, which signals pancreas lineage mutant TCF/LEF binding sites (32). As expected, over- development, are both expressed in the pancreas epithelium expression of either wild-type h-catenin or the constitutively during development (20). Pdx-1 promoter directing Wnt activated phosphorylation mutant h-catenin (S37A; ref. 33) misexpression in a transgenic mouse model results in pancreas increased TCF reporter gene assay activity (Fig. 2A). Menin agenesis (28). A h-catenin conditional knockout mouse, alone did not activate the reporter gene, but cotransfection of however, loses normal pancreatic acinar tissue development, menin and either h-catenin or activated h-catenin (S37A) but islet development is preserved (22). Whereas it is clear that strongly stimulated the reporter gene activity severalfold above Wnt signaling and h-catenin are important for pancreas that seen with either h-catenin or h-catenin (S37A) alone. One development, its role in pancreatic endocrine tumor develop- possible explanation for the increased reporter activity with ment is unknown. menin overexpression could be altered h-catenin expression, Based on the role of menin in multiple cell signaling but Western blot analysis shows similar expression of h-catenin pathways and the broad role of Wnt signaling in tumor when menin is co-overexpressed with h-catenin or h-catenin development, we hypothesized that menin may be important for (S37A; Fig. 2A, right). To determine whether menin was Wnt/h-catenin signaling in endocrine tumors. In this report, we affecting the reporter promoter through a mechanism not investigate the role of menin in Wnt/h-catenin signaling in a involving the TCF response elements, similar experiments were mouse islet cell line. These data suggest menin is important for done comparing the response of the control reporter gene, regulation of Wnt signaling in the endocrine pancreas through a Super8XFOPflash (plasmid M51 labeled ‘‘F’’ in Fig. 2B), mechanism that involves histone H3K4 trimethylation. which has mutant TCF binding sites, with the response from the nonmutated reporter Super8XTOPflash (plasmid M50 labeled Results ‘‘T’’ in Fig. 2B). Menin and h-catenin did not activate the Expression of Endogenous b-Catenin, TCF3, and TCF4 in negative control reporter gene Super8XFOPflash that has Endocrine and Nonendocrine Cell Lines and the Effect of mutant TCF binding sites (Fig. 2B). Expression of the Menin Overexpression overexpressed proteins is shown in Fig. 2B (right), indicating Canonical Wnt pathway protein expression in endocrine that the transfections of multiple plasmids did not significantly tumor cell lines has not been previously reported. Therefore, we interfere with each other. We then wondered whether Wnt/h- first determined