J. Endocrinol. Invest. 36: 781-787, 2013 DOI: 10.3275/9021

SHORT REVIEW

Multiple endocrine neoplasia syndromes associated with mutation of p27 M. Lee and N.S. Pellegata Institute of Pathology, Helmholtz Zentrum München-German Research Center for Environmental Health, Neuherberg, Germany

ABSTRACT. Multiple endocrine neoplasias (MEN) are autosomal dominant disorders characterized by the occurrence of tumors in at least two endocrine glands. Until recently, two MEN syndromes were known, i.e. the MEN type 1 (MEN1) and type 2 (MEN2), which are caused by germline mutations in the MEN1 and RET genes, respectively. These two syndromes are characterized by a different tumor spectrum. A few years ago we described a variant of the MEN syndromes, which spontaneously developed in a rat colony and was named MENX. Affected animals consistently develop multiple endocrine tumors, with a spectrum that shares features with both MEN1 and MEN2 human syndromes. Genetic studies identified a germline mutation in the Cdkn1b gene, encoding the p27 cell cycle inhibitor, as

the causative mutation for MENX. Capitalizing on these findings, germline mutations in the human homologue, CDKN1B, were searched for and identified in patients with multiple endocrine tumors. As a consequence of this discovery, a novel human MEN syndrome, named MEN4, was recognized, which is caused by heterozygous mutations in p27. These studies identified Cdkn1b/CDKN1B as a novel tumor susceptibility gene for multiple endocrine tumors in both rats and humans. Here we review the characteristics of the MENX and MEN4 syndromes and we briefly address the main function of p27 and how it is affected by MENX- or MEN4-associated mutations. (J. Endocrinol. Invest. 36: 781-787, 2013) ©2013, Editrice Kurtis

INTRODUCTION

tions identified in MEN1 and RET and their impact on the clinical management of the mutation-carriers have been reviewed in detail elsewhere (6, 7). Noteworthy, approximately 10% of patients presenting with a MEN1-like phenotype do not carry detectable MEN1 gene mutations (8). In recent years, a novel MEN syndrome has been discovered, which is caused by mutations in the CDKN1B gene encoding the p27 tumor suppressor and was named MEN4 (9). Studies on a rat strain predisposed to multiple endocrine tumors (named MENX) identified Cdkn1b as a novel susceptibility gene for such tumors, and provided a new gene to also analyze in patients with a MEN1-like phenotype and no mutations in the canonical susceptibility genes. These studies led to the discovery of the MEN4 syndrome. This review focuses on the rat and human MEN syndromes associated with germline mutation of p27.

Multiple endocrine neoplasia (MEN) syndromes are genetic disorders characterized by proliferative lesions (usually hyperplasia or adenoma) arising in at least two endocrine tissues (1). The predisposition to MEN syndromes is inherited as autosomal dominant trait with high penetrance. The endocrine tumors in MEN syndromes are histologically similar to their sporadic counterpart, but they are often multifocal and preceded by hyperplasia. MEN1, or Wermer syndrome, is characterized by the occurrence of pituitary, parathyroid, and pancreatic hyperplasia or neoplasia, sometimes accompanied by gastrointestinal endocrine tumors (2, 3). The MEN2 syndrome is further divided in MEN2A, which is characterized by parathyroid hyperplasia, pheochromocytomas, and C-cell hyperplasia or carcinoma, and MEN2B, where parathyroid hyperplasia is absent but cutaneous neuromas, ganglioneuromas of the intestine and marfanoid body habitus can occur (4, 5). A variant of MEN2A exists where patients have a strong predisposition to develop medullary thyroid cancer but not the other features of the syndrome, and it is termed familiar medullary thyroid cancer (4, 5). Loss-offunction mutations in the MEN1 gene cause the MEN1 syndrome, while activating mutations in the proto-oncogene RET cause MEN2. The characteristics of the muta-

A MULTIPLE ENDOCRINE NEOPLASIA IN THE RAT (MENX) About 10 years ago a rat cohort started to spontaneously develop multiple endocrine tumors with high penetrance. Upon careful histological examination, it was noted that affected rats present with tumors that overlap the spectrum of MEN1 and MEN2 human tumor syndromes. Specifically, they present with multifocal anterior pituitary adenoma and bilateral adrenal pheochromocytoma (frequency 100%), as well as extra-adrenal pheochromocytoma (paraganglioma), thyroid C-cell hyperplasia, parathyroid hyperplasia, and pancreatic islet cells hyperplasia (10). This multi-tumor syndrome was named MENX (10). Malignancies in MENX-affected rats show a clear progression with time: these animals develop adreno-

Key-words: Cdkn1b, MENX, MEN4, multiple endocrine neoplasias, p27. Correspondence: N.S. Pellegata, PhD, Institute of Pathology, Helmholtz Zentrum München-German Research Center for Environmental Health, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany E-mail: [email protected] Accepted May 31, 2013. First published online June 26, 2013.

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THE CDKN1B GENE AND ITS PROTEIN

medullary hyperplasia at 3-4 months of age, which progresses to tumors (pheochromocytoma) by 6-8 months of age. Multifocal adenohypophyseal neoplasms develop at about 4 months of age and merge into larger adenomas by 8-12 months of age. Additional phenotypic characteristics of the MENX-affected rats include: macroscopically visible bilateral juvenile cataracts (10), increased body size, and organomegaly, particularly of spleen and thymus, in mutants vs wild-type littermates (9). Whereas wild-type rats live approximately 24-30 months, the average life span of MENX-affected rats is 10±2 months (9). The cause of death of affected rats has not been formally established, although the high blood pressure associated with the pheochromocytomas might be the leading cause of morbidity. As opposed to the human MEN syndromes, the predisposition of MENX-affected rats to develop multiple endocrine tumors seems to be inherited as a recessive trait, so that homozygosity for the underlying genetic mutation is required to develop the disease phenotype (9). Linkage analysis followed by a positional cloning approach identified Cdkn1b as the gene responsible for the MENX syndrome. Indeed, all affected rats carry a homozygous tandem duplication of eight nucleotides (c.520-528dupTTCAGAC, RefSeq: NM_031762) in exon 2 of the gene (9). This mutation causes a frameshift and the mutant transcript encodes a protein which has a Cterminal sequence different from the wild-type p27 protein, referred to as p27fs177. The wild-type p27 (p27wt) protein is 198 amino acids long, while the mutated protein, due to the frameshift, is 221 amino acids long. This Cdkn1b mutation was identified in all MENX-affected rats tested (n>300), whereas it was never observed in unaffected littermates or in control wild-type rats of commercially available inbred strains. Following the identification of the causative genetic mutation, the expression of the Cdkn1b gene in MENX rats was explored in more detail. The analysis of various tissues of 2-month-old affected rats (before they develop malignancies) and wild-type age-matched littermates (controls) showed that the level of Cdkn1b mRNA is highly similar in both animal groups. Furthermore, correct splicing of the mutant mRNA was demonstrated in mutant rat tissues (9). When the expression of the p27 protein was assessed by western blotting, a faint band corresponding in size to the predicted p27fs177 mutant protein could be observed, but only in some rat tissues (i.e. thymus and thyroid). Immunohistochemical staining, performed with an antip27-specific antibody on a broader variety of tissues, showed extremely low (thyroid, pituitary, thymus, parathyroid, and brain) or absent (adrenals, lung, kidney, liver, and testis) p27 immunoreactivity in tissues of affected rats before they eventually developed neoplasias, whereas the same tissues obtained from wildtype rats showed strong nuclear positivity for p27 (9). Subsequent in vitro studies demonstrated that the MENXassociated mutation does not affect the transcription of Cdkn1b, but renders the encoded p27fs177 protein highly unstable (11). Upon transfection in various recipient cell lines, we found that p27fs177 is rapidly degraded, in part through the proteasome, since treatment with proteasome inhibitors rescues the level of the protein (11).

The human CDKN1B gene (like rat Cdkn1b) has two coding exons resulting in a 2.4 Kb long coding region. CDKN1B is located on chromosome 12p13, a chromosomal region known to undergo hemizygous loss in hematological malignancies (12). CDKN1B encodes the p27 protein, a cyclin-dependent kinase (CDK) inhibitor, whose main function is to control the progression from G1 to S phase (13). p27 binds to, and inhibits, cyclinE/CDK2 and cyclinA/CDK2 complexes in response to either mitogenic or anti-mitogenic stimuli. When the CDK2 kinase is bound by p27 it cannot phosphorylate its main targets, i.e. the retinoblastoma (pRb) family of proteins. If pRb is not phosphorylated, it cannot release E2F transcription factors, and this prevents the transcription of genes responsible for the progression from the G1 to the S phase. Thus, binding of p27 to cyclinE,A/CDK2 arrests cell cycle progression in the G1 phase (13). p27 is also required for the cytoplasmic assembly and nuclear import of cyclinD/CDK complexes, molecules that promote cell cycle progression (14). Because p27 is a critical regulator of cell cycle progression, its activity is tightly regulated at different levels: transcriptional, translational, and post-translational. Although transcriptional regulation of Cdkn1b (15, 16) and control of mRNA translation (17) have been reported, the best known mechanism regulating the function of p27 acts at the post-translational level and modulates the amount of protein through ubiquitin-mediated proteasomal degradation (18). In growth-arrested cells, high levels of nuclear p27 bind to cyclin/CDK complexes, thereby blocking cell cycle progression (13). Upon mitogenic stimulation, the cells need to rapidly eliminate p27 so that they can re-enter the cell cycle, and they do so through proteasome-mediated degradation. Two pathways responsible for p27 degradation have been described: the first involves the ubiquitylation-promoting complex KPC1/KPC2 and occurs in the cytoplasm (19), while the second pathway takes place in the nucleus and is mediated by the ubiquitin ligase SKP2 (20). In early G1, p27 is phosphorylated at the Ser10 residue and this promotes the export of the protein to the cytoplasm where it is then degraded through the KPC1 pathway (21). In late G1, p27 is phosphorylated at the Thr187 residue by cyclinE,A/ CDK2 complexes and this creates a recognition site for the SKP2 ubiquitin ligase which induces p27 poly-ubiquitylation and subsequent degradation by the proteasome (20). Given the pivotal role of p27 in regulating cell cycle progression, it is not surprising that the amount of p27 is reduced/lost in many cancers, including those of the colon, breast, prostate, and stomach (22). p27 down-regulation is usually associated with poor prognosis (22). The observation that often the reduction in p27 protein levels is not associated to a decrease in CDKN1B mRNA expression led to the speculation that the mechanism regulating p27 down-regulation might be enhanced proteolysis. Experimental evidence showed this to be the case in aggressive colorectal carcinoma samples, where the loss/reduction of p27 expression is caused by accelerated proteasome-dependent degradation of the protein (23), likely due to increased expression of the SKP2 ubiq-

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THE DISCOVERY OF GERMLINE CDKN1B MUTATIONS IN HUMAN PATIENTS

uitin ligase (24). Subsequently, additional tumor cell types were shown to employ this molecular mechanism to reduce the amount of p27. In addition to the amount of protein, the non-covalent sequestration of p27 in the cytoplasm can also modulate the protein’s activity. Indeed, when p27 is mislocalized to the cytoplasm through phosphorylation or protein binding (25), it can no longer bind and inhibit Cyclin/CDK complexes and may acquire additional functions that are not completely understood. Mislocalization of p27 in the cytoplasm through phosphorylation of Thr157 by protein kinase b (AKT) has frequently been observed in carcinoma of the breast, and is associated with unfavorable prognosis (26). Studies on mouse models expressing a mutant p27 that cannot bind Cyclin/CDK complexes suggested that cytoplasmic p27 has a pro-oncogenic role in some cell types (27).

Following the identification of the genetic mutation causing the MENX syndrome in rats, we investigated whether mutations in the human homologue CDKN1B could explain some of the MEN1-like cases without mutations in MEN1. We screened for the presence of CDKN1B germline mutations several patients fulfilling these criteria and we identified a germline heterozygous TGG>TAG (c.227G>A; RefSeq: AY890407) nonsense mutation at codon 76 (p.Trp76stop, W76X) in a female proband with GH-secreting pituitary adenoma (causing acromegaly; age 30 yr) and primary hyperparathyroidism (age 46 yr) (9). Analysis of the proband’s relatives confirmed that in her family, the p27W76X mutation segregates with the predisposition to tumors belonging to the MEN1 tumor spectrum. Indeed, one of the proband’s sisters, also a carrier of the germline c.227G>A mutation, developed renal angiomyolipoma (age 55 yr), a tumor type that has been previously associated with the MEN1 syndrome (9). Subsequently, other groups identified germline CDKN1B mutations in patients with features suggestive of MEN1. More recently, also patients with predisposition only to pituitary adenomas and patients with a sporadic presentation of parathyroid adenomas were found to carry CDKN1B gene mutations (see below). To date a total of 13 germline base substitutions in CDKN1B have been identified in patients with various combinations of endocrine tumors, some having a MEN1-like phenotype (MEN4 syndrome) and others having sporadic presentation of the disease or only pituitary adenomas (Table 1 and Fig. 1). Most of the identified changes in CDKN1B are missense mutations, and therefore their role as “pathogenic” substitutions is difficult to determine. However, since these changes were not found in control unaffected individuals, and in some cases alter the protein’s activity, we consider them as potentially pathogenic and hereafter we refer to them as “mutations”. The germline changes with no detectable molecular phenotype, and/or also occurring in control individuals, are considered genetic variants of unknown significance and are here only briefly mentioned. All the identified changes in CDKN1B occur in heterozygosity, thereby suggesting a dominant inheritance of the tumor predisposition.

CDKN1B SEQUENCE POLYMORPHISMS AND SOMATIC CHANGES IN HUMAN PATIENTS Several single nucleotide polymorphisms (SNP) have been identified in the human CDKN1B gene, including three which are potentially functional: −838C>A (rs36228499), −79C>T (rs34330), and 326T>G (V109G, rs2066827). Epidemiological studies have identified a significant association between coding SNP rs2066827 (V109G) and cancers of the prostate and breast, as well as oral squamous cell carcinoma (28-30), but the results of different studies were not always consistent and thus the role of this polymorphism in cancer risk remains controversial (31). Whether this amino acid change causes a change in p27 functions is currently unknown. Recently, SNP −79C>T (rs34330) was associated with the risk of developing papillary thyroid carcinoma (follicular variant) (32). The authors went on to demonstrate that the variant allele is transcribed with reduced efficiency in vitro and, consequently, there is a lower amount of p27 protein in the cells. Whereas the down-regulation of p27 expression is among the most frequent alterations in human cancers, somatic CDKN1B mutations are extremely rare and to date only a handful has been reported in the literature. A nonsense mutation has been identified in an adult T-cell leukemia/lymphoma (p27W76X) (33), another nonsense mutation was found in a breast cancer sample (p27Q104X) (34) and a missense change in an unclassified myeloproliferative disorder (p27I119T) (35). More recently, a missense change causing an amino acid substitution (p27P133T) and a 25 nucleotide deletion starting at codon 25 (c.582del25) were identified in sporadic parathyroid adenomas (36). The c.582del25 mutation deletes part of the splice donor site in intron 2 and causes a frameshift, as demonstrated by the presence of abnormally spliced transcripts in the peripheral blood of the mutation-carrier patient (36). Interestingly, some of these somatic changes were also identified in the germline of individuals presenting with neuroendocrine tumors and are described here below. The finding that the same mutation arose independently in somatic and germline cells could indicate that these variants have a pro-oncogenic effect.

CDKN1B MUTATIONS IN PATIENTS WITH THE MEN4 SYNDROME Nine germline base substitutions in CDKN1B have been so far identified in association with a MEN1-like phenotype. We here discuss these changes, their effect on the protein function and the phenotype of the mutation-positive patients, presenting them in the order in which they occur along the gene sequence from the 5’ UTR to the 3’ end of CDKN1B (Table 1 and Fig. 1). A new mutation was recently discovered in a highly conserved Open Reading Frame (ORF) located in the 5’ UTR region of CDKN1B (c.-456-453delCCTT) in an Italian patient presenting with acromegaly and well-differentiated non-functioning pancreatic endocrine tumor (37). This deletion abolishes the termination codon of the ORF,

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Fig. 1 - Schematic structure of the p27 protein, with the upstream 5’ UTR of the CDKN1B gene. In the black box is indicated the protein sequence, with the various protein binding domains. The identified germline mutations are indicated by arrows. Upstream of the CDKN1B gene there is an open reading frame (ORF). CAP indicates the translation initiation site. CycA: Cyclin A; Cdk2: cyclin-dependent kinase 2; Grb2: growth factor receptor-bound protein 2; Jab1: c-jun activation domain binding protein 1; RhoA: Ras homolog gene family, member A; NLS: nuclear localization signal.

tumor (age 69) and primary hyperparathyroidism (age 74) (38); a mutation at the –7 position in the Kozak sequence (ATG-7G>C) in a patient with a parathyroid tumor, bilateral adrenal masses and uterine fibroids (39). In peripheral blood leukocytes of the patient with the c.-32/-29del mutation, the amount of CDKN1B transcript was significantly reduced compare to a normal healthy control (38). The ATG-7(g>c) nucleotide substitution reduces the translation efficiency of the variant allele, which in turn causes a reduced amount of the encoded p27 protein when compared with the wild-type CDKN1B allele (39). The remaining 6 mutations so far identified in CDKN1B occur in the coding sequence of the gene. A 19-bp duplication (c.59-77dup19) in exon 1, which causes a frameshift

thereby lengthening its coding sequence and shortening the space between the ORF and the beginning of the CDKN1B gene. This change does not affect the rate of CDKN1B transcription but affects the translation re-initiation at the CDKN1B transcript by altering the intercistronic distance between ORF and gene. The causative role of this mutation in reducing p27 levels, as confirmed by in vitro assays, is in agreement with the decrease in p27 expression observed in the tumor tissues of the mutation-carrier patient (37). Additional mutations discovered in the 5’ UTR region of the CDKN1B gene are: a heterozygous GAGA deletion at the -29 position (c.-32/-29del), which was identified in a 69-yr old Spanish female patient with gastric carcinoid

Table 1 - Summary of the CDKN1B germline mutations identified to date, their phenotypic features and molecular characteristics. CDKN1B mutation

Patient phenotype

Familial

In vitro phenotype

Acromegaly, non-functioning pancreatic endocrine neoplasm

Not specified

Reduced p27 protein level

Ref.

In 5’ UTRa -456_-453delCCTT

37

ATG-32_-29

PHPTb, gastric carcinoid tumor

NO

Reduced mRNA and protein level

38

ATG-7G>C

PHPT, bilateral adrenal mass non-functioning, uterine fibroid

Yes (MEN1-like)

Reduced p27 protein level

39

PHPT PHPT (Hepatic metastasis), Zollinger-Ellison syndrome, gastrinoma PHPT, non-functioning pituitary microadenoma, bronchial carcinoids, papillary thyroid carcinoma, multiple lung metastases PHPT (2 parathyroid tumors), Zollinger-Ellison syndrome, mass in duodenum and tail of pancreas Prolactinoma, breast cancer Acromegaly PHPT (1 parathyroid tumor)

NO Not specified

Reduced p27 protein stability N.d.c

36 41

Yes (MEN1-like)

Failure to bind to CDK2, impaired growth suppression

42

Suspected MEN1

Failure to bind to Grb2

39

Yes (FIPAd-like) Yes (FIPA-like) NO

Failure to bind to Grb2 Abnormal migration by SDS-PAGE No phenotype identified

43 43 36

In the coding sequence Missense G9R A55T P69L

P95S

K96Q I119T P133T Nonsense W76X

PHPT, Acromegaly

Yes (MEN1-like)

Cytoplasmic localization

9

Frameshift K25fs Stop>Q

PHPT, Cushing’s disease PHPT (3 parathyroid tumors)

NO Yes (MEN1-like)

N.d. Reduced p27 protein level

40 39

auntranslated

region; bprimary hyperparathyroidism; cnot determined; dfamilial isolated pituitary adenoma.

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variant encodes a protein expressed at lower level than p27wt upon ectopic overexpression in cultured cells, probably because it is rapidly degraded by the proteasome (39).

after codon 25 (p.K25fs), was identified in a Dutch patient diagnosed with ACTH-secreting pituitary adenoma (Cushing’s disease; age 46 yr), small-cell neuroendocrine cervical carcinoma (age 45 yr), and hyperparathyroidism (age 47 yr). Due to this frameshift, the variant transcript is predicted to encode a p27 protein with an amino acid sequence different from p27wt starting from codon 25, and significantly shorter than p27wt. The cervical carcinoma of the patient showed loss of the wild-type CDKN1B allele and no p27 protein expression (40). Recently, a cohort of familial and sporadic Spanish patients with a MEN1 phenotype was screened for mutations in MEN1, CDKN1B, and AIP, and a novel missense mutation in CDKN1B (c.163G>A; p. A55T) was found in a lady presenting with Zollinger-Ellison syndrome with gastrinoma and hepatic metastases (age 42 yr), and primary hyperparathyroidism (age 51 yr). The potential effect of this change on p27 activity has not yet been assessed (41). In an Italian patient with suspected MEN1 a missense mutation at codon 69 of p27, which results in a proline to threonine amino acid substitution (c.678C>T, p.P69L), was found. This patient was diagnosed with primary hyperparathyroidism caused by a parathyroid adenoma (age 67 yr), a papillary thyroid carcinoma (age 64 yr) and a non-functioning pituitary microadenoma (age 79 yr) (42). The P69L change substitutes one of the proline residues mediating the interaction between p27wt and the CDK2 protein and consequently we could demonstrate that the p27P69L mutant protein binds CDK2 with much lower affinity than p27wt in vitro. p27P69L is also expressed at reduced level both in transfected cells and in the tumor tissues of the mutation-carrier individual, likely because it is less stable than p27wt. Moreover, p27P69L is impaired at inhibiting cell growth (42). The heterozygous TGG>TAG (c.227G>A) nonsense mutation at codon 76, which determines the premature truncation of the protein (p.W76X), was detected in a 48-yrold Caucasian female presenting with GH-secreting pituitary tumor (acromegaly; age 30 yr) and primary hyperparathyroidism (age 46 yr). In the family of the index case, this mutation is inherited together with the predisposition to MEN1-like tumors, since a mutation-carrier sister of the proband presented with renal angiomyolipoma (age 55 yr) (9). The p27W76X peptide no longer possesses the nuclear localization signal of the full-length protein and it is therefore mislocalized to the cytoplasm in vitro and in the tissues of a mutation-positive patient. As a consequence of its cytoplasmic localization, p27W76X has lost the ability to inhibit the growth of GH3 pituitary adenoma cells in vitro (42). CDKN1B sequencing identified a CCC>TCC missense mutation at codon 95 (c. p.P95S) in an individual affected by primary hyperparathyroidism and displaying masses in both duodenum and pancreas (39). In another patient with a family history of primary hyperparathyroidism a change in CDKN1B was found which changes the stop codon to glutamine (TAA>CAA; stop>Q), thereby generating a protein 60 amino acids longer than p27wt (39). The p27P95S variant changes one of the residues mediating the binding of p27 to the Grb2 adaptor protein and as a consequence the mutant p27 protein does not bind Grb2 as efficiently as p27wt in vitro. The p27 stop->Q

CDKN1B GERMLINE MUTATIONS IN ENDOCRINE DISEASE In addition to patients with a MEN1-like phenotype, also patients with the predisposition to only pituitary adenomas or with a sporadic presentation of parathyroid adenomas may occasionally bear mutations in CDKN1B (Table 1 and Fig. 1). Sequence analysis of 124 affected subjects belonging to familial pituitary adenoma (FIPA) families and having no mutations in the AIP gene identified two point mutations in two patients: c.286A>C (p.K96Q) and c.356T>C (p.I119T). The K96Q mutation occurs in the proline rich domain (amino acids 90-96) of p27 which mediates the binding to the adaptor molecule Grb2, and indeed the variant p27K96Q protein cannot bind Grb2 in vitro (43). The p27I119T protein displays an abnormal migration pattern by polyacrylamide gel electrophoresis, in that it migrates slower than the wild-type protein (43). The unique migration pattern of p27I119T, suggestive of noncanonical post-translational modifications, is not due to increased phosphorylation at the newly-created threonine residue. Since glycosylation occurs at serine, threonine or aspartic acid residues, the migration shift associated with the 119T residue could be caused by glycosylation of the protein, thereby conferring greater stability. In agreement with this finding, p27I119T is more stable than wild-type p27 in vitro. In this FIPA patient’s cohort, additional variants of p27 were identified, namely: p.S56T, p.T142T, c.605+36C>T. Since these substitutions were occasionally observed in control unaffected individuals and were not predicted, by in silico modeling, to affect p27 function, are likely to be non-pathogenic (43). In addition to patients selected because of a familial presentation of neuroendocrine disease, also patients presenting with single gland parathyroid adenomas, presumed to be sporadic, were found to carry germline p27 changes. One patient, a 68-yr-old man, with a typical, single-gland, mildly symptomatic tumor presentation, had a heterozygous germline single nucleotide change at base 25 in CDKN1B exon 1 (c.25G>A, p.G9R). The germline DNA of another patient, a 53-yr-old woman presenting with fatigue, slightly elevated serum calcium and PTH levels, showed a heterozygous single nucleotide substitution (c.397C>A, p. P133T) (36). In vitro studies showed that the p27G9R variant associates with reduced protein stability, which in turn translates into a very low level of expression of p27 in the tumor tissue of the mutation-carrier individual. No evidence that the p27P133T variant affects p27 function could be gathered by various in vitro assays and therefore it is to be considered a variant of unknown pathogenic significance (36). CONCLUSIONS Studies on the MENX rat syndrome led to the identification of a novel MEN syndrome in patients, which is

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caused by germline mutations in CDKN1B, encoding the cell cycle inhibitor p27. This syndrome, named MEN4, has a tumor spectrum similar to MEN1, although CDKN1B mutations were found to occur also in patients presenting with only pituitary adenomas or single-gland parathyroid adenomas. The phenotypical and molecular features of MENX/MEN4 here summarized, together with studies of engineered mouse models with defective or mutant p27, confirm a critical role for p27 in regulating the proliferation of neuroendocrine cells. We speculate that in these cells other cyclin-CDK inhibitors cannot efficiently compensate for the lack or reduction of functional p27, and this ultimately leads to uncontrolled cell division. Cells and tissues less sensitive to reduced p27 activity most likely rely on other molecules to regulate cell cycle progression. This might explain the tissue-specific tumor spectrum of the MEN4 syndrome. Our understanding of the functions of p27 involved in neuroendocrine tumor predisposition will improve with the identification and characterization of new germline CDKN1B mutations. This knowledge may ultimately help in devising more effective targeted therapeutic strategies for mutation-positive patients.

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ACKNOWLEDGMENTS

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We thank the members of the laboratory for stimulating discussions. The work of the authors is supported by the Deutsche Forschungsgemeinschaft (SFB 824) and by the Deutsche Krebshilfe (grant 109223).

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