Abstract
Since the discovery of the multiple endocrine neoplasia type 1 (MEN1) gene in 1997, elucidation of the molecular function of its protein product, menin, has been a challenge. Biochemical, proteomics, genetics and genomics approaches have identified various potential roles, which converge on gene expression regulation. The most consistent findings show that menin connects transcription factors and chromatin-modifying enzymes, in particular, the histone H3K4 methyltransferase complexes MLL1 and MLL2. Chromatin immunoprecipitation combined with next-generation sequencing has enabled studying genome-wide dynamics of chromatin binding by menin. We propose that menin regulates cell type-specific transcriptional programs by linking chromatin regulatory complexes to specific transcription factors. In this fashion, the MEN1 gene is a tumor suppressor gene in the endocrine tissues that are affected in MEN1. Recent studies have hinted at possibilities to pharmacologically restore the epigenetic changes caused by loss of menin function as therapeutic strategies for MEN1, for example, by inhibition of histone demethylases. The current lack of appropriate cellular model systems for MEN1-associated tumors is a limitation for compound testing, which needs to be addressed in the near future. In this review, we look back at the past twenty years of research on menin and the mechanism of disease of MEN1. In addition, we discuss how the current understanding of the molecular function of menin offers future directions to develop novel treatments for MEN1-associated endocrine tumors.
Introduction
In the 1997 paper reporting the positional cloning of the multiple endocrine neoplasia type 1 (MEN1) gene, the authors proposed the name menin for its protein product. Chandrasekharappa and coworkers noted that analysis of the 610 amino acid sequence provided few clues to menin’s function and that there were no apparent molecular similarities to previously known proteins (Chandrasekharappa et al. 1997). The MEN1 gene is expressed very widely and menin was found to reside primarily in the cell nucleus (Guru et al. 1998).
MEN1 patients are predisposed to develop parathyroid adenomas, neuroendocrine tumors (NETs) of the duodenum and the pancreas and pituitary adenomas, which often manifest at a young age. In addition, adrenal adenomas, neuroendocrine tumors of the stomach, thymus or lungs, lipomas, leiomyomas and meningiomas can be observed (Thakker et al. 2012). Recently, an increased risk to develop breast cancer was added to the MEN1 spectrum (Dreijerink et al. 2014). Metastatic pancreatic neuroendocrine tumors (pNETs) are the most frequent MEN1-related cause of death in MEN1 patients (Conemans et al. 2017). MEN1 patients and MEN1 gene mutation carriers are monitored at regular intervals in order to detect and treat manifestations in a timely manner.
The first genetics studies showed that MEN1 germ line mutations found in MEN1 families were mostly scattered through the coding exons and had clearly inactivating effects on menin (Chandrasekharappa et al. 1997, Lemmens et al. 1997). The identification of inactivating mutations, together with the previously reported loss of heterozygosity (LOH) at the MEN1 locus at chromosome 11q13 in MEN1-related insulinoma demonstrated that the MEN1 gene acts as a classic tumor suppressor gene in MEN1 (Larsson et al. 1988). This was confirmed in mouse models: homozygous deletion of the Men1 gene is embryonic lethal at the mid-gestation stage. However, mice carrying heterozygous Men1 germ line mutations develop phenotypes that show close resemblance to MEN1 manifestations in humans as well as loss of the wild-type allele in the tumor tissues (reviewed in Agarwal 2014). In addition to inherited tumors in MEN1 patients, the MEN1 gene is also commonly mutated in sporadic parathyroid adenomas and pNETs (Heppner et al. 1997, Jiao et al. 2011, Scarpa et al. 2017).
The majority of MEN1 germ line mutations are nonsense and frameshift mutations causing premature stop codons (Lemos & Thakker 2008). Expression of truncated menin proteins is probably suppressed by nonsense-mediated mRNA decay (Zetoune et al. 2008). In addition, the majority of missense MEN1 mutations lead to unstable proteins, which are prone to ubiquitin-dependent proteasomal degradation (Yaguchi et al. 2004). Proteasomal inhibition or silencing of expression of the responsible carboxy-terminus of Hsc70-interacting protein (CHIP) E3 ubiquitin ligase was demonstrated to increase expression of menin mutant proteins (Canaff et al. 2012). The solution of the crystal structure of menin has enabled three-dimensional mapping of MEN1 mutations (Huang et al. 2012). Although there have been reports about selected missense mutations and intron mutations occurring in familial isolated hyperparathyroidism or in families with relatively mild phenotypes, there is no clear genotype–phenotype correlation in MEN1 (reviewed by Lips et al. 2012). The penetrance of MEN1 manifestations is highly variable, even within MEN1 families carrying the same gene mutation.
In this paper, we review the literature addressing the molecular function of menin and the pathogenesis of MEN1. We highlight a number of mechanisms that have been validated in a translational fashion in MEN1 model systems. Recent studies have indicated potential therapeutic interventions for MEN1. Strategies aimed to restore the function of menin could result in mechanism-based therapies for MEN1 and MEN1-associated tumors, in particular pNETs.
Menin is involved in transcriptional regulation
After the discovery of the MEN1 gene, many research groups set out to identify menin-interacting proteins in order to understand the pathogenesis of MEN1 (reviewed in Matkar et al. 2013). The vast majority of menin-interacting partners include proteins involved in transcriptional regulation and chromatin modification. Transcription of genes into messenger RNA is carried out by the RNA polymerase II (pol II) enzyme together with the basal transcription machinery, which direct initiation of mRNA synthesis at transcription start sites (TSS) of gene promoters (Fig. 1). Gene-specific transcription factors coregulate transcription by binding to specific DNA sequences that may either be located close to gene promoters but also at distant regulatory elements termed enhancers. Although enhancers may be far away from TSS from a linear perspective, these sites can be looped to TSS to enable interactions of DNA-bound proteins and thus affect transcription initiation. Gene-specific transcription factors are often expressed in certain tissues, which results in tissue-specific enhancer function. Prominent enhancer clusters are sometimes referred to as super-enhancers (Whyte et al. 2013). Chromatin accessibility and modification constitute additional layers of transcriptional regulation. DNA is wrapped around histone proteins into chromatin. The functional unit of chromatin is called a nucleosome and consists of a histone octamer (each containing two sets of the core histones H2A, H2B, H3 and H4) and ~150 base pairs of DNA. Post-translational modifications of histones such as acetylation and methylation strongly correlate with the functional properties of the genome. Histone H3 lysine 4 trimethylation (H3K4me3) marks active gene promoters and histone H3 lysine 36 (H3K36) methylation accumulates toward the 3′-ends of actively transcribed genes. In contrast, histone H3 lysine 9 trimethylation (H3K9me3), histone H3 lysine 27 trimethylation (H3K27me3) and loss of histone acetylation are associated with transcriptional repression. As a rule, histone marks are dynamic modifications that can be written, read and erased by dedicated proteins. The combinatorial interplay of the basal transcription factors, pol II recruitment, enhancer-binding transcription factors and chromatin structure and modifications determines the mRNA expression signatures of cells and tissues. Disruption of mRNA expression, for example, by mutations in genes encoding transcriptional regulators, is a major contributor for oncogenic transformation of cells into tumor cells (reviewed by Bradner et al. 2017). Below and in Fig. 1, we give an overview of the published menin interactions with transcription factors and chromatin regulatory proteins.
The MEN1 gene product menin connects transcription factors to histone-modifying protein complexes. Menin can interact with several classes of transcription factors listed in the left panel. Transcription factors bind directly or indirectly in the case of β-catenin to specific DNA sequences at sites termed enhancers. Enhancers may be located far away from transcription start sites (TSS), however, through DNA looping enhancer-bound proteins can interact directly with the TSS. Menin links transcription factors to chromatin modifying protein complexes, such as the MLL1 and MLL2-containing complexes that have H3K4me3 methyltransferase activity to activate transcription of target genes as shown in the middle panel. Other menin chromatin modifying interactors include HDACs, SIRT1, PRMT5 and SUV39H1. Histone deacetylation, H4R3me2s and H3K9me3 are associated with repression of transcription. HDAC, histone deacetylase; MEN1, Multiple endocrine neoplasia type 1; MLL, mixed-lineage leukemia; PRMT, protein arginine N-methyltransferase; SIRT, sirtuin; SUV39H1, suppressor of variegation 3–9 Homolog 1.
Citation: Endocrine-Related Cancer 24, 10; 10.1530/ERC-17-0281
Menin binds to transcription factors
JunD
The first menin-interacting protein that was identified is JunD by Agarwal and coworkers. It was found in a yeast-two hybrid screen experiment (Agarwal et al. 1999). JunD is a member of the activator protein 1 transcription (AP-1) factor family. AP-1 transcription factors act in a gene-specific manner and are downstream in the signaling cascade elicited by the RAS oncogene. The transcriptional activity of JunD is repressed by menin in reporter assays, putatively due to recruitment of histone deacetylase activity (Agarwal et al. 1999, Gobl et al. 1999, Kim et al. 2003). Menin interferes with Jun kinase (JNK)-mediated phosphorylation of JunD and also c-Jun, potentially interfering with RAS signaling (Gallo et al. 2002). In 2012, menin was co-crystallized with a JunD peptide, which allowed detailed mapping of the interaction surface (Huang et al. 2012). Several independent research groups have confirmed the stable nature of the menin-JunD interaction. However, the pathogenic relevance for MEN1 remains elusive to date.
SMAD transcription factors
Transforming growth factor beta (TGF-β) and bone morphogenic protein (BMP) signaling is transduced to gene transcription via SMA and MAD (SMAD) gene-specific transcription factors. Genomic SMAD-binding sites can be both at promoter-proximal and enhancer elements. Menin has been reported to interact with and co-activate SMAD1, SMAD3 and SMAD5 (Sowa et al. 2003, 2004). In this fashion, menin can suppress prolactin and parathyroid hormone expression in murine cells, which is in line with the loss of function of menin in pituitary and parathyroid adenomas (Hendy et al. 2005). The molecular mechanism of SMAD co-activation by menin is presently unknown.
Forkhead transcription factors
Forkhead box proteins are gene-specific transcription factors that bind to DNA sequences and are downstream effectors of several signaling pathways. Menin has been reported to interact with several FOX family members including FOXN1/checkpoint suppressor 1 (CHES1) and FOXO1 (Busygina et al. 2006, Wuescher et al. 2011). In sporadic breast cancer cells, menin is enriched at enhancers that contain FOXA1-binding motifs (Dreijerink et al. 2017). In mouse embryonic stem cell (mESC)-derived embryoid bodies, lower Foxa1 and Foxa2 mRNA levels were observed in the absence of Men1 gene expression (Zhang et al. 2011). In addition, menin was found to be able to interact with Foxa2 and to regulate Foxa2 expression in mouse pancreatic cell lines indicating a potential role in MEN1-associated pNET tumorigenesis (Bonnavion et al. 2017).
MYC
MYC (c-Myc) is a transcription factor that binds to E-boxes in the DNA to activate transcription of genes involved in tumorigenesis. Increased MYC expression and activity is correlated with cell proliferation rates, and this is observed in many cancer types. Both activating and repressive effects of menin on MYC action have been reported. Menin has been suggested to interact with MYC and the SKI-interacting protein (SKIP) coactivator at the HIV-1 promoter (Bres et al. 2009). In HEK293 embryonic kidney cells, menin and the mixed-lineage leukemia (MLL) complex were found to interact with the transcription factor FUSE-binding protein 1 (FBP1/FUSBP1) to stimulate MYC expression (Zaman et al. 2014). It was recently reported that in fibrosarcoma cells, menin can also interact directly with MYC and regulate MYC target genes in an H3K4me3-independent manner (Wu et al. 2017). More in line with its tumor suppressive function, loss of menin was shown to result in the upregulation of the c-Myc mRNA in mouse-derived pancreatic cells, possibly via increased Hedgehog signaling pathway activity (Gurung et al. 2013a).
Nuclear receptors
Based on the observation that the amino acid sequence of menin contains a putative nuclear receptor interaction motif, menin was identified as a coactivator of nuclear receptor (NR)-mediated transcription. Menin is able to interact with the estrogen receptor alpha (ERα) ligand-binding domain in an estradiol (E2)-dependent fashion (Dreijerink et al. 2006). This interaction is important for transcription of ERα target genes through recruitment of H3K4me3 methyltransferase activity. Menin also regulates transcription of the ESR1 gene, which encodes ERα in breast cancer and pituitary cells (Dreijerink et al. 2017). Subsequent work showed that menin is a generic co-regulator for nuclear receptor-mediated transcription as it interacts with many other NR family members including the vitamin D receptor (VDR), the retinoid X receptor (RXR), the peroxisome proliferator-activated receptors (PPAR) alpha and gamma, the liver X receptor (LXR) alpha and the androgen receptor (AR) (Dreijerink et al. 2006, 2009a, Cheng et al. 2011, 2015, Malik et al. 2015). The clinical relevance of the menin–NR interaction was investigated in normal parathyroid and MEN1 patient-derived parathyroid adenoma tissues. In MEN1 tumors, lower levels of VDR target gene messenger RNAs are in line with the loss of the coactivator function of menin (Dreijerink et al. 2009b).
Beta-CATENIN
The wingless-related integration site (Wnt)/β-catenin signaling route has important roles not only in development, but also in cancer biology. Menin has been reported to interact with the major effector of this pathway: β-catenin. In addition, menin was found to interact with one of β-catenin’s associated transcription factors, T-cell factor 3 (TCF3) and to have a role in the stimulation of Wnt signaling in murine islet cells (Chen et al. 2008). Conversely, in mouse insulinoma cells lacking Men1, nuclear staining of β-catenin was present, a sign of active Wnt signaling in these tissues (Cao et al. 2009). Inhibition of Wnt signaling resulted in reduced proliferation of Men1-deficient rodent beta-cells. Accordingly, combining a beta cell-specific mouse knockout model of the Men1 gene with knockout of the Ctnnb1 gene (encoding β-catenin) resulted in lower tumor numbers and sizes in mice defective in Men1 and Ctnnb1 vs Men1 alone (Jiang et al. 2014). These results suggest that activated Wnt signaling after loss of MEN1 gene expression could be a targetable mechanism in MEN1-related tumors.
Menin interacts with chromatin regulatory proteins
MLL1/MLL2
In 2004, using a combined approach of immunoprecipitation and mass spectrometry in 293T cells, Hughes and coworkers identified menin as part of a histone methyltransferase (HMT) complex that contained the trithorax family member mixed-lineage leukemia protein 2 (MLL2; also termed lysine methyltransferase 2B (KMT2B)) (Hughes et al. 2004). The complex was found to methylate histone H3K4 and a direct interaction between menin and pol II was discovered. Yokoyama and coworkers used biochemical purification to identify a similar menin-containing complex that contained MLL1 (KMT2A) but not MLL2 in K562 erythroleukemia cells (Yokoyama et al. 2004). MLL1 and MLL2 are proteins that contain su(var)3–9, enhancer-of-zeste and trithorax (SET) domains and are part of a family of H3K4 methylation writers that are derived from the yeast Set1 protein (reviewed in Smith et al. 2011). In humans, six distinct Set1-like complexes exist, each assembled around one of the SET1/MLL proteins (Fig. 2). Using quantitative mass spectrometry, Van Nuland and coworkers determined the composition and stoichiometry of the human SET1/MLL complexes in HeLa cervical carcinoma cells and found that menin is exclusively present in the MLL1/MLL2 complexes in substoichiometric quantities but not in the other SET1/MLL complexes (van Nuland et al. 2013).
Composition of human SET1/MLL complexes in Hela cells based on immunoprecipitation experiments combined with mass spectrometry (adapted from (van Nuland et al. 2013)). The SET1–2 (also termed KMT2F-G) and MLL1–4 (KMT2A-D) proteins are the catalytic subunits of six distinct human Set1-like complexes. These complexes contain four common subunits: ASH2L, DPY30, RBBP5 and WDR5, but also complex-specific components. Menin is exclusively found in MLL1- and MLL2-containing complexes. In MLL2 complexes in particular, menin has also been co-precipitated together with PSIP1. ASH2L, Absent, small, or homeotic-like; BOD, Bcl-2-related ovarian death gene; CFP1, CXXC finger protein 1; HCF, host cell factor; KDM, lysine demethylase; KMT, lysine methyltransferase; MLL, mixed-lineage leukemia; NCOA, nuclear receptor coactivator; OGT, O-Linked N-Acetylglucosamine transferase; PA1, PTIP-associated protein; PSIP1, PC4- and SFRS1-interacting protein; PTIP, Pax-interacting protein; RBBP, RB-binding protein; SET, suvar enhancer-of-zeste, trithorax; WDR, WD40 repeat.
Citation: Endocrine-Related Cancer 24, 10; 10.1530/ERC-17-0281
In mouse embryonic fibroblasts (MEFs), the menin–MLL1 complex was found to be present at the Hoxc8 locus, both at the TSS and at an upstream enhancer site (Hughes et al. 2004). H3K4me3 is associated with active promoters and menin was found to be an activator of Hoxc8 transcription. Further evidence of menin-dependent H3K4me3 at target genes was provided by experiments using E2-induced transcription in MCF-7 breast cancer cells. After silencing of MEN1 expression, E2-induced H3K4me3 was significantly lower on the TSS of the TFF1 ERα target gene (Dreijerink et al. 2006). Genome-wide analysis of menin-binding sites supported colocalization of menin, MLL1 and H3K4me3 in a variety of cell types (Scacheri et al. 2006). The menin–MLL1 complex can be tethered to chromatin together with the lens epithelium-derived growth factor (LEDGF)/PC4 and SFRS1-interacting protein (PSIP1), a H3K36-binding protein (Yokoyama & Cleary 2008). In HeLa cells, PSIP1 was found to be mostly present in menin–MLL2 complexes (van Nuland et al. 2013).
The crystallization of menin both from Nematostella vectensis and subsequently a human protein construct has enabled the three-dimensional analysis of the menin–MLL interaction (Murai et al. 2011, Huang et al. 2012). The interaction surfaces of menin both with MLL1 and LEDGF and JunD were mapped to a central cavity of menin. However, these interactions cannot occur simultaneously, supporting differential roles for menin either as an interactor of JunD or as a component of MLL1/MLL2 complexes. Strong genetic evidence for the contribution of loss of H3K4me3 to MEN1 tumorigenesis was provided in a study by Lin and coworkers who showed that additional genetic ablation of the retinoblastoma binding protein 2 (Rbp2; also known as Jarid1a or lysine demethylase 5a (Kdm5a)) H3K4 demethylase in Men1-deficient mice decreased pNET formation and prolonged survival (Lin et al. 2011).
HDAC/SIRT
The silencing by menin of JunD-mediated transcription has been attributed to repressive chromatin modifications. Gobl and coworkers showed that repression of JunD-dependent transcription could be reversed using the histone deacetylase (HDAC) inhibitor trichostatin A (Gobl et al. 1999). Kim and coworkers found an association with SIN3A–HDAC complexes (Kim et al. 2003), which could not be confirmed by quantitative proteomic analysis of HeLa cells (van Nuland et al. 2013). In a more recent study, an interaction between menin and the histone deacetylase sirtuin 1 (SIRT1) was reported. Menin was shown to be required for the recruitment of SIRT to the Cd36 gene promoter in mouse hepatocytes (Cao et al. 2013).
PRMT5
Work from the Hua group described menin as a repressor of Hedgehog signaling by histone H4 arginine 3 (R3) symmetric dimethylation (H4R3me2s). H4R3me2s is a repressive transcription mark. The protein arginine N-methyltransferase PRMT5 was identified as a menin interactor by mass spectrometry of purified flag-tagged menin-bound proteins in 293T cells (Gurung et al. 2013b). The Hedgehog signaling gene transcription factor GLI1 gene was found to be a target gene of the menin–PRMT5 mechanism. Administration of a Hedgehog signaling inhibitor resulted in reduced proliferation of insulinoma cells in a beta cell-specific MEN1 mouse model.
SUV39H1
In a Men1-knockout MEF cell line, global reduction of histone H3 lysine 9 trimethylation (H3K9me3) was observed. A candidate approach of overexpressing menin and several H3K9 methyltransferases in 293T cells resulted in the identification of the suppressor of variegation 3–9 Homolog 1 (SUV39H1) protein as a menin-interacting protein (Yang et al. 2013). In addition to MEN1 gene mutations, sporadic pNETs often harbor loss-of-function mutations in the death domain-associated protein (DAXX) and alpha-thalassemia X-linked mental retardation protein (ATRX) tumor suppressor genes (Jiao et al. 2011). DAXX and ATRX are both involved in telomere lengthening and histone H3.3 exchange. In a recent report, Feng and coworkers reported a direct interaction between menin and DAXX after ectopic overexpression in 293T cells. In murine insulin-producing cells, Menin and DAXX enhanced H3K9me3 at the promoter of the membrane metallo-endopeptidase (Mme) gene coinciding with SUV39H1 recruitment at the same sites (Feng et al. 2017).
Lessons from the menin cistrome
An experimental method named chromatin immunoprecipitation (ChIP) allows for the detection of DNA protein interactions after formaldehyde crosslinking. This enables mapping of DNA-binding motifs of transcriptional regulators or the presence of histone modifications on the DNA. This mapping can be carried out by testing candidate sites using targeted PCR, as has been reported for several of the target genes mentioned above. However, by combining ChIP with high-throughput analyses of bound DNA, such as a DNA chip (ChIP on a chip) or more recently next-generation sequencing (ChIP-seq) the presence of a given transcriptional regulator or a histone modification of interest can be studied on a genome-wide scale in an unbiased fashion. The combined set of genomic binding elements for a transcriptional regulator has been termed the cistrome (Lupien & Brown 2009). The first study addressing the menin cistrome by Agarwal & Jothi demonstrated binding of menin mostly at promoters and genes. However, intergenic loci comprised 33% of binding sites in HeLa S3 cervical carcinoma cells (Agarwal & Jothi 2012). Subsequent ChIP-chip studies of HeLa S3, HepG2 and pancreatic islet cells showed that menin, MLL1 and the SET1/MLL complex subunit RB-binding protein 5 (Rbbp5) localized to the promoters of thousands of human genes but not always simultaneously. The data suggested that menin is present at sites of active transcription as menin occupancy generally correlated with high gene expression. Loss of menin did not result in significant changes in most transcript levels, except for the Motor Neuron And Pancreas Homeobox 1 (MNX1)/Homeobox Protein HB9 (HLXB9) gene (Scacheri et al. 2006). In a comparative study of mESCs and pancreatic islet-like endocrine cells, average H3K4me3 levels at TSS did not differ between wild-type and Men1-knockout cells. However, at a selected number of loci H3K4me3 was lower in the absence of Men1, in particular, H3K4me3 levels at the long non-coding RNA Meg3 locus and several Hox genes were decreased in islet cells (Agarwal & Jothi 2012). In a study in mouse pancreatic tissues, menin-dependent H3K4me3 loss was accompanied by gain of the repressive mark H3K27me3 at menin target loci (Lin et al. 2015).
While the MEN1 gene functions as a tumor suppressor gene in MEN1, it has an oncogenic role in sporadic breast cancer cells. A recent integrative analysis of the menin cistrome, H3K4me3 and gene expression in the well-established MCF-7 ER+ breast cancer cell line was carried out to further investigate this oncogenic mechanism of menin-MLL1/MLL2 target gene regulation. A menin–MLL1/MLL2 target gene was defined as a gene that has menin-dependent H3K4me3 and mRNA levels. In accordance with previous studies, only a limited number of genes fulfilled these requirements. Menin–MLL1/MLL2 target genes not only had menin bound at their TSS, but also showed an enrichment of menin binding to distal enhancer sites that showed looping to the target gene TSS (Dreijerink et al. 2017). From these observations, it can be concluded that not only the presence of menin at TSS, but rather the combination of the presence of menin at TSS and enhancer sites, determines target gene specificity of menin–MLL1/MLL2 complexes. In the case of MCF-7 breast cancer cells, menin was present at breast cancer-specific FOXA1- and GATA3-bound enhancers. There was a large degree of overlap between menin-bound enhancers and known super-enhancers. Several master transcription factors for breast cancer were among the menin–MLL1/MLL2 target genes in MCF-7 cells. In fact, the ESR1 gene, encoding ERα, turned out to be a major target gene. Menin also co-activates the ERα protein at the protein level and simultaneous regulation of the ESR1 gene could be an auto-regulatory mechanism to control the core transcriptional regulatory circuitry in mammary cells. Similar regulatory circuitries could exist in MEN1-affected tissues, e.g. involving FOXA2 and MNX1/HLXB9 in pancreatic neuroendocrine cells. An obvious next step, which is unfortunately hampered by the current lack of appropriate model systems, would be to take a similar integrative ChIP-seq and RNA-seq approach in MEN1 tumor cells to identify the relevant transcription factors and target genes that are responsible for MEN1 tumorigenesis.
Menin-dependent gene expression signatures
The fate and function of a cell are determined by its gene expression signature. As menin is a transcriptional regulator, MEN1-related tumorigenesis is likely to be the result of aberrant tumor suppressive gene expression due to the loss of menin. Restoration of expression of menin target genes in MEN1-affected tissues could therefore have therapeutic consequences.
Among the first gene sets described to be regulated by menin were the cyclin-dependent kinase-inhibiting (CDKI) genes CDKN2C and CDKN1B encoding the p18INK4c and p27KIP1 proteins, respectively. CDKIs are targets of cyclin-dependent kinases and have important roles in cell cycle control (Milne et al. 2005). Genetic knockout mouse models of the Cdkn2c and Cdkn1b genes develop a mix of MEN1 and multiple endocrine neoplasia type 2 (MEN2)-type endocrine tumors, underscoring the potential importance of these genes for endocrine tumorigenesis (Franklin et al. 1998). Moreover, multiple endocrine neoplasia type 4 (MEN4) is a rare inherited condition characterized by pituitary and parathyroid adenomas and caused by germ line mutations in the CDKN1B gene (Molatore et al. 2010). Using a candidate approach, menin and MLL1 were found to be present at the Cdkn2c and Cdkn1b promoters in MEFs and later also in mouse pancreatic tumors (Karnik et al. 2005, Milne et al. 2005). Activation of these genes could be an important tumor suppressive function of menin. CDK inhibition could be explored as a therapeutic strategy for MEN1 tumors. However, a recent integrative genomics study in Men1-depleted pancreatic islets from 2-month-old mice, the CDK genes were not among the menin–MLL1/MLL2 target genes (Lin et al. 2015).
An experimental approach using H3K4me3 ChIP-seq and microarrays in menin-null vs wild-type mESCs identified the long non-coding RNA Meg3 as a potential menin–MLL1/MLL2 target. A similar analysis in pancreatic islet-like endocrine cells revealed several Hox genes, which are the classic MLL target genes, as menin–MLL1/MLL2 target genes (Agarwal & Jothi 2012). Loss of MEG3 has been reported in sporadic pituitary adenomas (Zhang et al. 2003). In a follow-up study in mouse pancreatic islet cells, overexpression of Meg3 coincided with downregulation of the c-Met oncogene. Compared with normal islets, human MEN1-associated PNETs expressed lower levels of MEG3 and higher levels of c-MET, suggesting that c-Met could be an interesting future treatment target in MEN1-related pNETs (Modali et al. 2015).
Genome-wide analysis of menin-dependent H3K4me3 combined with gene expression data revealed that the MNX1/HLXB9 gene is a gene that is activated by the menin–MLL1/MLL2 complex (Scacheri et al. 2006). The MNX1/HLXB9 protein is a transcription factor that is important for pancreas and beta cell development. In addition, MNX1/HLXB9 was found to interact with menin in overexpression experiments in MIN6 mouse insulin-producing cells and to induce apoptosis and reduced proliferation in this cell model (Shi et al. 2013). In a broader sense, differential Men1-dependent expression of pancreatic transcription factors, such as MNX1/HLXB9, Foxa2, MafA, MafB and Pdx1 has been described in several reports (Fontaniere et al. 2006, Scacheri et al. 2006, Zhang et al. 2011, Lu et al. 2012, Hamze et al. 2013). In fact, the Men1 gene appears to be essential for normal development of the endocrine but not of the exocrine pancreas in mice. In ex vivo culturing experiments of the pancreatic bud, in the absence of menin, the number of cells expressing the pancreatic endocrine precursor transcription factor Neurog3 was markedly reduced (Fontaniere et al. 2008). Beta cell-specific Men1 knockout has also been shown to be able to initiate the development of trans-differentiated glucagon-expressing tumors, further highlighting the critical role of menin in endocrine pancreatic development (Li et al. 2015).
In tissues that are not affected in MEN1, menin was found not to be involved in anti-proliferative gene expression and to have different functions. For example, menin regulates a gene signature in pro-B cells that affects B cell differentiation but not proliferation (Li et al. 2013). Moreover, in contrast to its tumor-suppressive role in MEN1-related tumors, in sporadic breast cancer cells, menin regulates a growth promoting gene expression signature (Dreijerink et al. 2017). These observations should be kept in mind when considering approaches aimed at restoring the loss of menin function.
Relevance of menin research for MEN1-related tumors and potential therapeutic opportunities
The current view of the molecular function of menin is that menin links gene-specific transcription factors to chromatin modification in a cell-specific context. Menin is able to bind to MYC, nuclear receptors and β-catenin as well as AP-1, SMAD and Forkhead transcription factors. Menin is a stable component of the MLL1 and MLL2 histone methyltransferase complexes and possibly also interacts with histone deacetylases, PRMT5 and SUV39H1. In MEN1-associated tissues, loss of menin leads to the disruption of anti-proliferative gene expression programs and to the development of endocrine tumors. Restoration of the epigenetic perturbations or correction of the function of aberrantly expressed genes in the absence of menin holds promise for molecular mechanism-based means to treat or prevent MEN1-related tumors. The most compelling evidence supporting this strategy was reported by Lin and coworkers in 2011 and 2015 (Lin et al. 2011, 2015). In mice with beta cell-specific menin depletion, combined genetic knockout of the Rbp2/Jarid1a/Kdm5a gene, resulted in reduced tumor formation and increased survival. Rbp2 is a histone demethylase for H3K4me2/3. At the TSS of the insulin-like growth factor 2 mRNA-binding protein 2 (Igf2bp2) gene, the menin-dependent absence of H3K4me3 in Men1 fl/fl, RIP-Cre mice could be reversed by simultaneously knocking out Rbp2 (Lin et al. 2015).
These observations indicate that compensation of the loss of H3K4me3 mark by the loss of menin at certain target genes may restore the function of menin in pancreatic tumors. Members of the JARID/lysine demethylase 5 (KDM5) family have H3K4me3 demethylase acitivity. The enzymatic activity of the KDM5A-D proteins is dependent on iron and alpha-ketoglutarate (αKG). KDM inhibitors that occupy the αKG-binding site are a promising new class of compounds that is currently under development and could be used to rescue the loss of H3K4me3 in MEN1 tumors (Horton et al. 2016, Vinogradova et al. 2016). Conversely, as the loss of H3K4me3 at menin target genes was found to coincide with gain of H3K27me3, compounds inhibiting H3K27 methyltransferase activity could have similar effects on menin target genes affected by the loss of menin (Lin et al. 2015).
A recent candidate compound screen in neuroendocrine tumor cell lines and in a pancreatic beta cell-specific Men1 knockout mouse model by Lines and coworkers identified JQ1 as a potential treatment for neuroendocrine tumors (Lines et al. 2017). Of note, no H3K4 demethylase inhibitors were tested in this study. JQ1 is a compound that targets the bromodomain and extraterminal domain (BET) protein family members that can bind active histone acetyl marks as well as other acetylated proteins through their bromodomains. The BET family includes the bromodomain-containing BRD2, BRD3, BRD4 and BRDT proteins. BET inhibition has been shown to be particularly effective in cancers that express high levels of the oncogenic transcription factor MYC by inhibiting MYC expression (reviewed by Shu & Polyak 2017). JQ1 reduced the proliferation rate and increased apoptosis in a MEN1 pNET mouse model. However, a MYC-dependent mechanism could not be confirmed in the NET cell lines (Lines et al. 2017).
Apart from mouse models, there are no established experimental model systems based on human cells for MEN1-associated tumors. A large portion of the basic research on menin has been performed in cell types that are not affected in MEN1 (reviewed in Pieterman et al. 2014). The widely used neuroendocrine cell lines BON-1 and QGP-1 do not harbor MEN1 gene mutations (Boora et al. 2015). There is an urgent need for cell lines derived from MEN1-associated tumors. The MEN1 mouse models that have been developed mimic MEN1 to a large extent except for the fact that MEN1 mice do not develop non-functioning pNETs, which constitute the most important threat to the life expectancy of MEN1 patients. This poses an important problem for MEN1 research. In order to be able to properly test and validate future epigenetic treatments for MEN1-related tumors, novel preclinical models will have to be developed. Xenograft models, in which human tumor material is implanted and propagated in mice, may hold promise for the future. In addition, the establishment of three-dimensional tumor cell organoid cultures could enable large-scale experiments and drug screens aimed at restoring transcriptional regulation in MEN1-associated tumors (van de Wetering et al. 2015).
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
M B is supported by the National Cancer Institute, grant number: P01 CA080111.
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