Multiple Endocrine Neoplasia Syndromes from Genetic and Epigenetic Perspectives.

Multiple endocrine neoplasia (MEN) syndromes are infrequent inherited disorders in which more than one endocrine glands develop noncancerous (benign) or cancerous (malignant) tumors or grow excessively without forming tumors. There are 3 famous and well-known forms of MEN syndromes (MEN 1, MEN 2A, and MEN 2B) and a newly documented one (MEN4). These syndromes are infrequent and occurred in all ages and both men and women. Usually, germ line mutations that can be resulted in neoplastic transformation of anterior pituitary, parathyroid glands, and pancreatic islets in addition to gastrointestinal tract can be an indicator for MEN1. The medullary thyroid cancer (MTC) in association with pheochromocytoma and/or multiple lesions of parathyroid glands with hyperparathyroidism can be pointer of MEN2 which can be subgrouped into the MEN 2A, MEN 2B, and familial MTC syndromes. There are no distinct biochemical markers that allow identification of familial versus nonfamilial forms of the tumors, but familial MTC usually happens at a younger age than sporadic MTC. The MEN1 gene (menin protein) is in charge of MEN 1 disease, CDNK1B for MEN 4, and RET proto-oncogene for MEN 2. The focus over the molecular targets can bring some hope for both diagnosis and management of MEN syndromes. In the current review, we look at this disease and responsible genes and their cell signaling pathway involved.

Introduction

Multiple endocrine neoplasia (MEN) disorders are infrequent and hereditary diseases that can be developed into the number of endocrine glands and result in tumor formation or grow exceptionally no tumor creation.[1-4] The MENs are run in families because they are the exact consequence of genetic mutations and their symptoms are completely dissimilar dependent on the involving glands.[5-7] The profiling and screening of candidate genes can be informative to detect disease people who have or are susceptible to MEN syndromes.[8-12] The MEN syndromes occur in 3 forms which are types 1, 2A, and 2B and through specific novel variants such as MEN 4.[13] These main types have some similarity and are atypically outsized glands with additional hormone production.[14] The genetic alterations (mutation) are mainly responsible for MEN syndrome formation and the main candidate gene accountable for type 1 disease is well known, whereas in people with types 2A and 2B, defects in some genes can be responsible for these 2 types of disease formation. People with MEN type 1 usually develop tumors of 2 or more often in the parathyroid gland, pancreas, pituitary gland, and less often in thyroid gland and adrenal glands.[15-18] More often than not, people with MEN type 1 will develop the parathyroid-related benign tumors with excessive parathyroid hormone production.[19] This extra parathyroid hormone generally increases the calcium levels in the blood or even occasionally triggering formation of kidney stones. In 30% to 80% of people with MEN 1 hormone-producing cells (islet cells) of the pancreas, tumors can be seen (Table 1).

Table 1.

The major conditions and features of MEN (multiple endocrine neoplasia).

MEN type	Gene	Conditions (features)
MEN 1 (Wermer)	MEN 1 (menin)	Hyperparathyroidism (95%)Pancreas tumors (30%-80%)Pituitary gland tumors (30%-42%)Rarely (facial angiofibromas, collagenomas, lipomas, meningiomas, ependymomas)
MEN2A (Sipple)	RET (specially codon 634)	Thyroid gland tumors (specifically medullary carcinoma) (95%)Pheochromocytoma (tumor of the adrenal glands) (40%-50%)Hyperparathyroidism (10%-20%)
MEN2b (multiple mucosal neuroma syndrome)	RET (specially codon 918)	Neuromas (99%)Physical characteristics similar to those in people with Marfan syndrome (99%)Thyroid gland tumors (specifically medullary carcinoma) (95%)
MEN4	CDNK1B	Parathyroid and anterior pituitary tumors (possibly associated with adrenal, renal, and reproductive organ tumors)

MEN Type 1

Multiple endocrine neoplasia 1 (MEN 1, OMIM no. 131100), famous as Wermer disease, is a familial disease and autosomal dominant cancer connected with the endocrine (hormone-producing) glands neoplasia.[20-22] The maximum joint tumors with MEN1 are the parathyroid glands, islet cells of the pancreas, and pituitary glands.[13,23] Additional rare endocrine tumors realized in MEN1 contain adrenal cortical tumors, carcinoid tumors and infrequent pheochromocytomas, and some parts of the digestive tract. Moreover, there are some nonendocrine tumors in MEN1 such as facial angiofibromas, collagenomas (flesh-colored tumors on the skin), lipomas, leiomyomas, meningiomas, and ependymomas.[13]

Men1 is the most often happening form of MENs and it is more often than not associated with primary hyperparathyroidism (distinguished by the presence of parathyroid adenomas or hyperplasia), which happens in about 90% of patients.[24] The treatment strategy typically can be surgery and drug that increases dopamine activity prescription, an effective prolactin-inhibiting factor in patients with prolactin-secreting pituitary tumors.[25] Moreover, surgery or a proton pump inhibitor (a drug that blocks gastric acid secretion) could be used for patients with gastrinomas to reduce levels of gastric acid and peptic ulcers. There are some suggestions over the comparison of the conservational management and surgery. For example, MEN1 patient’s nonfunctioning pancreatic neuroendocrine tumors with size more than 2 cm would be preferred to manage through observant waiting, while the surgery in nonfunctioning pancreatic neuroendocrine tumors with size more than 3 cm should be taken into consideration.[26,27]

MEN1 disease is a consequence of the MEN1 gene mutation whose genetic locus is chromosome 11q13 (Figure 1).[28,29] The role of MEN1 gene is a tumor suppressor confirmed by microsatellite analysis in cancerous tissues of MEN1 patients.[30-33]

Figure 1.

MEN1 gene in genomic location: bands according to ensemble (http://www.genecards.org/cgi-bin/carddisp.pl?gene=MEN1).

Germ line mutation in the MEN1 gene resulting in loss of heterozygosity (LOH) at both alleles of MEN1 in the endocrine tumor and can be extent throughout the coding region of the gene.[34,35] The protein product of MEN1 comprises 610 residues and is completely consensus from Drosophila melanogaster to humans,[36] contrary to yeast or Caenorhabditis elegans, signifying its new evolutionarily origin.[36] Despite the fact that during mouse embryogenesis, MEN1 gene is ubiquitously expressed in countless tissues and organs during mouse embryonic development,[37-39] its role is completely restricted and tissue specific in the way that even exhibiting contrasting function between different organs.[38] In endocrine organs, MEN1 suppresses tumorigenesis in some organs, such as lung, prostate, and breast, and it makes worse diabetes in mouse models.[40-46] Interestingly, there are some reports over the role of MEN1 function of further organs such as liver and bone.[47-50] In mouse mesenchymal and osteoblastic cells, it is related to β-catenin, cell-cell adhesion, and gene transcription factor, which is essential for osteoblast differentiation.[49] Also, MEN1 protein (menin) is considered to maintain bone morphogenetic protein 2 (BMP-2), TGF-β super family of proteins, and Runt-related transcription factor 2 (Runx2), resulting in mesenchymal cells to osteoblasts differentiation.[51] Overexpression of MEN1 repressed the ALP activity induced by JunD. Actually, it has been recommended that menin destroys the maturation of osteoblast, through stopping the differentiation of JunD.[52,53] Menin can be activated in fibrinogenes via TGF-beta.[54] It has been shown that menin inhibits gene transcription through different chromatin-modifying enzymes or posttranscriptionally acting. What is more is G2-M phase transition stopping through cyclin B2 expression.[55,56] It also plays its role through JunD-mediated gene transcription and other mechanisms.[55,57-60] More than that menin in a straight line interacts with the p65 subunit of NF-κB to repress NF-κB–dependent transcription.[61,62] There are some suggesting interactions with the PTN gene as a pro-proliferative receptor in lung cancer cells for the inhibition of complex 2 (PRC2) attachment to the PTN gene promoter in addition to enhancing the suppressive chromatin spot H3K27me3.[41]

Menin is able to induce posttranscriptional modification through increasing the microRNA expression like microRNA-26a (miR-26a) which is crucial for osteoblastic differentiation.[63] More than nuclear localization of menin, it is present in cytoplasm or even extracellular spaces suggesting that it has additional role in control of multiple signaling pathways, ranging from Ras to Akt to Hedgehog signaling (Figure 2). It was shown that with supporting of the transforming growth factor type β signaling pathway, cell proliferation inhibition removed.[65] For Wnt signaling and glyco-kipoprotein emission the trascriptional co-activator btea-caten controls homeostasis in embryonic and adult development.[66] Menin holds back extracellular regulated protein kinase-1/2 (ERK-1/2) mitogen-activated protein kinase pathway which is a downstream target in Ras pathway.[67-69] The correlation of menin with reduced activity of protein kinase Akt1 in cultured cells and mouse pancreatic denoted that translocation of Akt1 to the cell membrane is inhibited by menin.[70] Further studies had shown that the transcription factor FOXO1 in the cytoplasm of hepatocytes correlated with menin; however, it is uncertain how menin cooperates with FOXO1 and what are its biological outcomes.[71] There are some indicators of menin role in taking on PRMT5 to the promoter of the Gas1 gene, a fundamental part for binding of Sonic hedgehog (Shh) ligand, to activate the Hedgehog signaling pathway.[62]

Figure 2.

Menin in numerous cell signaling pathways.[64]

It can be said that menin mediated inhibition of cell proliferation inhibition through interaction with (a) histone-modifying enzymes (MLL, EZH2, and HDACs); (b) the relations with several transcription factors, such as JunD, nuclear factor κB (NF-κB), peroxisome proliferator-activated receptors (PPARγ), and vitamin D receptor (VDR), to stimulate or repress gene transcription; (c) cell proliferation arresting by means of transforming growth factor β1 (TGF-β) signaling and Wnt/β-catenin pathways; (d) the destruction of pro-proliferative factors such as insulinlike growth factors I and II (Igf-I and Igf-II) and parathyroid hormone-related protein (PTHrP) involved in endocrine tumors; and (e) the direct effect on cell cycle progression.[13,57,72]

More often than not, in 5% to 10% of MEN1 patients, no mutations of the MEN1 gene can be detected that could harbor mutations involving other genes[34,73] such as the CDNK1B gene (12p13.1-p12) responsible for coding 196 amino acid cyclin-dependent kinase inhibitor (CK1) p27kip1 mutation, highlighting as a responsible gene of recessive MEN-like syndrome referred to as MEN4.[74] The exact MEN4 tumorigenesis molecular mechanisms are not clear yet, although it is supposed that the mutated allele possibly will be responsible for the reduction of p27 protein localized in the nucleus and, thus, competent to exert its role of negative regulator of cell cycle progression and cell growth. Now, 9 dissimilar CDKN1B pathogenic modifications have been recognized but no precise medical features is available to discriminate MEN4 from MEN1.

In addition to genetic alterations, there are some epigenetic mechanisms that act as a regulatory element through changing the gene expression patterns without altering the sequence of the genome.[75] Between epigenetic mechanisms and DNA methylation, the important ones that happen in around 3% of cytosines of cytosine-guanine dinucleotide (CpG) islands are present in the genome.[76] Menin can interact with lysine methyltransferase 2D (MLL2) and regulate its histone methyl transferase activity.[77] It was shown that insulinlike growth factor 2 (Igf2) was overexpressed in Men1 mutant mice as a result of hypermethylation of the intragenic differentially methylated regulatory regions (DMR2) of the Igf2 gene.[78] Promoter hypermethylation was shown in one of the cell cycle regulator genes (RB1, P14ARF, P15 (INK4b) (CDKN2B), P16 (CDKN2A), P21 (CDKN1A), P27, and P73(TP73)) in pituitary tumors, that is, one of the MEN1-associated tumors.[79] Moreover, there are some evidence of promoter hypermethylation in the retinoblastoma 1, P14 (ARF), P16, P73, metalloproteinase inhibitor 3 (TIMP3), O-6-methylguanine DNA methyltransferase (MGMT), DAPK (DAPK1), THBS1, and CASP8 genes.[80-83] Also, the fibroblast growth factor receptor (FGFR2), a member of the FGF family with a critical role in pituitary development, decreased in human pituitary tumors as a result of gene promoter methylation. The genotype/phenotype association in Korean MEN1 patients suggested some altered DNA methylations to track the main reason of tumorigenesis.[84]

MEN Type 2

Multiple endocrine neoplasia type 2 (MEN2) is a hereditary disease resulting in additional abnormal activation of one or more of the endocrine glands which could be resulting in a wide range of tumor formations, including adrenal (about half the time), parathyroid (20% of the time), and thyroid (almost all of the time).[85] MEN2 is triggered by malfunction of the RET (REarranged during Transfection) gene.[86,87] MEN 2 is classified to 3 different subtypes: MEN 2A, MEN 2B, and FMTC (familial medullary thyroid carcinoma) with the possibility of medullary carcinoma of the thyroid (MTC) development. In fact, both MEN 2A and MEN 2B have a bigger hazard for pheochromocytoma, whereas MEN 2A has a higher possibility for parathyroid adenoma or hyperplasia.[88] MTC classically happens in early childhood in MEN 2B, first years of adulthood in MEN 2A, and middle age in FMTC.[89,90] Although MEN 2 was detected the first time at the University Hospital of Freiburg, Germany, in the 19th century, the connection of an MTC and a pheochromocytoma was initially explained in 1961.[91-93] Between different MEN 2 subtypes, MEN 2A is the mainly frequent form which develops MTC in the form of multifocal, bilateral, and nearly linked to the C-cell hyperplasia.[13,94,95] Half of MEN 2A patients are at the risk of pheochromocytoma involving both adrenal glands[96-98] and one-fourth of MEN 2A patients are able to grow multiple adenomatous parathyroid glands with hyperparathyroidism.[99] Moreover, co-occurrence of Hirschsprung’s disease (HSCR) and coetaneous lichen amyloidosis was shown.[100-102] MEN 2 is the source of numerous tumors in one patient, although not essentially at the equal time.[87]

Indicators of MEN2 can be a lump in the front of the neck and sweating, irregular heartbeat, and headaches.[103] MEN2 can be identified with several blood and urine tests, a biopsy or computed tomographic scan, magnetic resonance imaging, or ultrasound scans. The adrenal tumors are identified pheochromocytoma, and the thyroid tumors are described as medullary thyroid carcinoma (MTC) which inherited in dominant autosomal pattern.[22,104-108] In fact, parafollicular C cells of the thyroid gland and calcitonin-secreting cells can give rise to MTC which is approximately the first appearance of MEN2 all times in very young children.[109] It can be led to hyperplasia of the C cells and make the patients candidate for total thyroidectomy (TT), whereas patients with hyperplasia progressed to carcinoma are not treatable through this operation.[110] In addition, genetic testing is another accessible tool, so checking the mutation of some candidate genes involving RET exons 10, 11, 13, 14, 15, and 16 can be informative to determine that many MEN2 carriers undergo TT ahead of showing MTC symptoms.[22] The MTCs are likely to metastasize to central and lateral, cervical, and adjacent lymph nodes or farther in lung, liver, or bone.[22] The aggressiveness of MTC linked to the MEN2 is dependent to the exact mutated RET codon (Table 2),[112,113] so finding the genotype-phenotype correlation has been specified recently.[114,115]

Table 2.

Risk for aggressive MTC based on genotype and recommended interventions.

ATA[a] risk level	Level D (highest risk)	Level C	Level B	Level A
Pathogenic variants[b,c]	p.Ala883Phep.Met918Thrp.Val804Met+p.Glu805Lys[d] p.Val804Met+p.Tyr806Cys[d] p.Val804Met+p.Ser904Cys[d]	p.Cys634Arg/Gly/Phe/Ser/Trp/Tyr	p.Cys609Phe/Arg/Gly/Ser/Tyrp.Cys611Arg/Gly/Phe/Ser/Trp/Tyrp.Cys618Arg/Gly/Phe/Ser/Tyrp.Cys620Arg/Gly/Phe/Ser/Trp/Tyrp.Cys630Arg/Phe/Ser/Tyrp.Asp631Tyrp.633/9 bp dupp.634/12 bp dupp.Val804Met+p.Val778Ile[d]	p.Arg321Glyp.531/9 bp dupp.532 dupp.Cys515Serp.Gly533Cysp.Arg600Glnp.Lys603Glup.Tyr606Cysp.635/insert ELCR; p.Thr636Prop.Lys666Glup.Glu768Aspp.Asn777Serp.Leu790Phep.Val804Leu/Metp.Gly819Lysp.Arg833Cysp.Arg844Glnp.Arg866Trpp.Ser891Alap.Arg912Pro
Age of prophylactic surgery	As soon as possible in first year of life	<5 y	Consider <5 y; may delay if criteria met[e]	May delay beyond age 5 y if criteria met[e]

Adapted from American Thyroid Association Guidelines Task Force (2009).[111]

ATA, American Thyroid Association.

p.Ser649Leu and p.Tyr791Phe have been removed from this list as they were reclassified as benign variants.[116]

Pathogenic variant designations have not been edited by Gene Reviews staff and may not be standard nomenclature.

Pathogenic variants in cis configuration on one allele.

Criteria: typical annual basal and or stimulated serum calcitonin; normal yearly neck ultrasound examination; family history of fewer aggressive MTC.

RET proto-oncogene chromosomal locus is 10q11.21 and encodes a transmembrane receptor and member of the tyrosine protein kinase family protein with 1114 amino acids and 124 319 Da weight (Figures 3 and 4).

Figure 3.

RET gene in genomic location: bands according to Ensembl, locations according to GeneLoc (and/or Entrez Gene and/or Ensembl if different) taken from http://www.genecards.org/cgi-bin/carddisp.pl?gene=RET.

Figure 4.

Activating mutations in multiple endocrine neoplasia over the RET proto-oncogene.[117,118]

The Ret gene established is expressed in definite line of cells which are originated from neural crest-like C cells of thyroid glands, migratory neural crest cells, the cells of dorsal-ventral axis of the neural tube, autonomic ganglion cells, and cells of enteric nervous system.[119-121]

The expression of RET gene stays controlled through DNA-binding factors to adjust transcription-like growth response protein-1 (EGR-1), transcription factor Spi-1/PU, and Sp3 transcription factor more than enhancer elements.[122-125] The alternative splicing of 3′ exonic regions of RET transcript is leading to 3 protein isoforms: RET9, RET43, and RET51 with different carboxyl terminus.[126-128] RET includes a big extracellular domain, same as other receptor tyrosine kinases that are another cell surface receptors and reveals multiple cadherin-like domains more than calcium-binding site.[129] This extracellular region is made of 4 cadherin-like repeats, important for stabilizing RET dimers, plus a membrane-proximal cysteine-rich module, in support of protein conformation and ligand binding.[129-132] After translation of RET transcripts, protein undergoes glycosylation as a posttranslational modification and an immature 155 KDa is produced which is additional processed and a mature plasma membrane is formed.[133,134] RET is involving in different cell signaling pathways and its role is completed through attachment of proteins of the glial cell line–derived neurotrophic factor (GDNF) family ligands (GFLs).[135] However, this attachment is mediated through an additional coreceptor: which is one of the 4 GDNF family receptor-α (GFRα) family members.[136] An additional new cell surface protein called Ret ligand 2 (RETL2) acts as a go-between GDNF-dependent ret signaling.[136] The extracellular domain of the RET has a kind of mutation which results in nonsynonymous substitution of amino acids (missense mutation), and this mutation frequently occurred in exons 10 and 11 and very uncommon mutations in exons 13, 14, and 15 which change cysteine codons.[136,137] The RET dimerization to form the active protein is influenced by replacing cysteine amino acids by other amino acid residues resulting in oncogenic potency of these RET mutations.[138] In MEN 2A, mainly RET mutations of codon 634 resulting to replace Cys634 by arginine (RET Cys634Arg) leads to parathyroid disease.[139] It has been shown that this RET mutation also could be presented in Hirschsprung’s disease connected to the MEN 2A,[140] and renal malformations in RETMEN2b transgenic mice linked to MEN 2A activating mutations.[141] The behavior of RET Cys634Arg mutation presents an extra aggressive MEN2A phenotype than Cys634Tyr mutation.[142] Opposite to some mutations of FMTC and MEN 2A, MEN 2B patients have mutation of codon 918 in exon 16 during which threonine (ACG) amino acid takes the place of methionine (ATG).[90,143] Despite the fact that RET hotspots are known, some recent studies have suggested that the whole coding region of the RET gene is supposed to be sequenced because a comprehensive analysis of the RET gene can reveal multiple germ line mutations in MEN 2.[144,145] The individualized prophylactic TT for MEN 2–related MTC was suggested according to prognostic incorporated testing of RET mutations and pre-serum calcitonin (Ct) levels in a group of Chinese.[146]

In addition to genetic mutations related to RET, there are some epigenetic profiles such as hypermethylation of CpG island promoters which is associated with transcriptional inactivation of tumor suppressor genes in different tumor formations.[147] For example, combined methylation of RASSF1A and p16 was established in MEN2-related pheochromocytomas.[148] In fact, inactivation of RASSF1A (RASSF1) through promoter hypermethylation can happen in thyroid cancer development and RASSF1 is tumor suppressor that is critical for phosphatidylinositol 3-kinase (PI3K)/Akt[88] pathway.[149-151]

Conclusions

MEN syndromes are a collection of autosomal dominant disease including MEN 1 (Wermer syndrome), MEN 2 (multiple endocrine adenomatosis), MEN 2A (Sipple syndrome), MEN 2B (mucosal neuroma syndrome), and MEN4. The information of MEN’s genetic alterations and the connection among genotype and phenotype could be beneficial for MEN disease management. The most important responsible genetic mutations are MEN1 gene in MEN1 (menin), RET gene mutation codon 634 in MEN2A, and RET gene mutation codon 918 in MEN2B, and CDNK1B in MEN4.