Menin is a ubiquitously expressed 67-kDa protein that is predominantly localized in the nucleus due to the presence of two nuclear localization signals (NLS), NLS1 (amino acid residues 479–497) and NLS2 (amino acid residues 588–608) [58]; a third accessory NLS, NLSa (amino acid residues 546–572), has also been reported [59]. Menin is highly conserved in animal species but with no known homologs in yeast and nematodes. The crystal structure of Nematostella menin (the starlet sea anemone) and human menin has been deciphered, and they are very similar [60, 61]. The structure resembles a curved left hand, with a deep pocket formed by the thumb and the palm [60, 61]. Menin is an α-helical protein with 4 domains: a long β-hairpin N-terminal domain, a transglutaminase-like domain that forms the thumb, a helical palm domain that contains three tetratricopeptide motifs, followed by a C-terminal fingers domain [60, 61]. The deep cavity in the palm may act as a binding site for interacting proteins [60, 61]. Both structures lack some parts of menin that have been predicted as disordered regions – in Nematostella menin, an unstructured loop (amino acid residues 426–442) and the C-terminus (amino acid residues 487–539), and in human menin, an internal unstructured loop (amino acid residues 460–519) [60, 61]. Therefore, the crystal structure of the entire menin protein in its free form or together with an interacting partner remains to be determined.

Menin does not show homology to any known proteins and it does not possess any enzymatic activity. Therefore to elucidate the function of menin, interacting proteins have been sought, and approximately 40 different proteins have been shown to partner with menin – transcription factors, chromatin modification factors, transcription initiation and elongation factors, cytoskeletal proteins associated with cell division/adhesion/motility, DNA repair proteins, an mRNA biogenesis factor, and signaling mediators in the cytoplasm (Fig. 3.2) [37, 62–64]. These interactions predict that menin may participate in a variety of functions in different cell types. A majority of the interactions are with proteins involved in transcriptional regulation; however, menin by itself does not possess a DNA-binding domain to directly regulate transcription [60, 61]. Protein and DNA interaction studies have revealed some menin target genes (cell cycle inhibitors p18 and p27, the long noncoding RNA MEG3, IGFBP2, and the HOX genes) that are regulated by menin from interactions with the mixed lineage leukemia (MLL) protein in a protein complex that is responsible for a specific chromatin mark of gene activation, histone H3 lysine 4 trimethylation (H3K4me3) [65–73]. Loss of menin results in loss of H3K4me3 at specific genes and, at the same genes, gain of H3K27me3 that is an epigenetic mark of gene repression [71]. Phosphorylation at six different amino acid residues of menin has been reported – Ser394, Thr397, Thr399, Ser487, Ser543, and Ser583 – constitutively (Ser543) or in response to DNA damage (Ser394 and Ser487) or upon loss of phosphorylation at Ser394 (Thr397 and Thr399) [74]. Menin is reported to undergo another posttranslational modification, SUMOylation at Lys591 [75]. The impact of phosphorylation or SUMOylation on the crystal structure of menin or in protein–protein interactions is not known. Most of the protein interaction studies have been conducted in cell lines unrelated to NETs or other tumors of MEN1 [76]. In order to confirm the MEN1-related relevance of the functions of menin through its interacting proteins, there is a need to develop cell lines from tissues (normal and tumor) affected in MEN1. Such studies may help to define the biological functions of menin that are critical to prevent the development of MEN1-related tumors.

Germline homozygous loss of the Men1 gene (Men1−/−) in mice results in death in utero between embryonic days 10.5 and 14.5, but germline heterozygous loss of the Men1 gene (Men1+/−) does not cause embryonic lethality [77–81]. After 9–16 months, Men1(+/−) mice develop MEN1-related endocrine tumors: parathyroid tumors, pancreatic islet tumors (mainly insulinoma), and anterior pituitary tumors (mainly prolactinoma in female) [77, 79–81]. Gastrinomas are seen in only one of the four different mouse models of MEN1 [79]. Foregut carcinoids are not seen in the mouse models of MEN1. Other tumors observed in the Men1(+/−) mouse model of MEN1 are adrenal cortical tumors, gonadal tumors (Leydig cells in male or ovarian stroma in female), and bilateral pheochromocytoma [82, 83]. Similar to MEN1-related human tumors, the tumors in Men1(+/−) mice develop after loss of the wild-type Men1 allele (LOH). Given that menin may regulate the expression of cell cycle genes, mouse models with loss of tumor suppressor genes known to regulate the cell cycle (Rb or p53) or cell cycle regulator genes (Cdk2, Cdk4, p18, or p27) have been generated in the Men1(+/−) background to study the impact on the development of MEN1-related NETs, PNETs in particular (Table 3.1). Combined loss of Men1(+/−) with Rb or p53 did not show any significant effect [84–86]. Combined loss of Men1(+/−) with p18 but not p27 accelerated the rate of insulinoma formation and with an increased tumor incidence [87]. Mice with combined loss of Men1(+/−) with Cdk4 but not Cdk2 did not develop insulinoma [88]. These mouse models show that p18 inactivation and Cdk4 activation may be critical for PNET formation upon menin loss.

Mice with tissue-specific homozygous loss of Men1 in the parathyroids, pancreas, or pancreatic islet β-cells are viable and develop tumors in the specific tissue at an earlier age than in the Men1(+/−) mice [89–93]. The exception is a mouse model with liver-specific homozygous Men1 knockout that lacks tumors in the liver, a tissue not associated with tumors in MEN1 patients, underscoring tissue-specific actions of menin in MEN1-related target tissues [94]. The tissue-specific action of menin as a tumor suppressor in the pancreatic β-cells is highlighted by the observation that homozygous loss of Men1 in β-cells (Men1(f/f); RIP-Cre) or in the whole pancreas (Men1(f/f); PDX1-Cre), both show only β-cell tumors (insulinomas) [90–93]. Another interesting observation in mouse models is that mice with pancreatic α-cell-specific loss of Men1 develop insulinomas rather than the expected glucagonomas due to possible transdifferentiation of α-cells into β-cells, or perhaps paracrine signals that induce β-cell proliferation [95, 96]. The molecular mechanisms underlying β-cell-specific tumorigenesis from menin loss remain to be determined. Perhaps menin has tissue-specific interactions or regulates tissue-specific factors as indicated in a few studies that have implicated β-cell-specific differentiation factors MafA or Hlxb9/Mnx1 [97–100]. Effect of menin loss together with target genes associated with β-cell proliferation and function has been studied in mouse models (Table 3.1). Combined genetic manipulation of candidate genes in mice with β-cell-specific Men1 knockout (Men1(f/f); RIP-Cre) shows that expression of activated K-RAS(G12D) enhances rather than inhibits β-cell proliferation, β-catenin loss can suppress islet growth and tumorigenesis, histone demethylase Rbp2 loss decreases islet tumor formation and prolongs survival, and ActivinB loss prolongs survival after 10 months of age [101–104]. Further studies of these mouse models may help to understand β-cell-specific disease-associated pathways and to develop potential therapies for MEN1-related PNETs.

Mouse models of Men1 loss have served as preclinical models to study the potential of various treatment options in PNETs (insulinoma) and pituitary tumors (prolactinoma) – an angiogenesis inhibitor (anti-VEGF-A monoclonal antibody, mAb G6-31), a small molecular tyrosine kinase inhibitor of all VEGF receptors (sunitinib), a somatostatin analog (pasireotide/SOM230), and menin replacement therapy [105–107]. Similar preclinical investigations may be possible from the results described above about studies in mouse models with combined loss of Men1 and genetic manipulation of candidate genes such as inhibitors of CDK, β-catenin, ActivinB, or histone demethylation.