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MEN1 is the main gene responsible for tumorigenesis of syndromic and sporadic primary hyperparathyroidism (PHPT). Germline mutations of the CDKN1B/p27 Kip gene have been associated with multiple endocrine tumors in rats and humans. To evaluate the involvement of the CDKN1B gene and its relationship with MEN1 in sporadic PHPT, we carried out sequencing and loss of heterozygosity analyses of the CDKN1B gene in 147 sporadic parathyroid adenomas. p27 immunohistochemistry and genetic screening of the MEN1 gene were performed in 50 cases. Three germline CDKN1B variants (c.-80C>T, c.-29_-26delAGAG, c.397C>A) were identified in 3/147 patients. Reduction of CDKN1B gene transcription rate was demonstrated in vitro for the novel c.-80C>T and the c.-29_-26delAGAG variants. Loss of p27 expression was detected in the tumor carrying the c.-29_-26delAGAG variant. Two tumors carrying the CDKN1B variants also harbored a MEN1 mutation. Fifty-four percent of 50 CDKN1B mutation-negative tumors had a reduction of p27 nuclear staining. Somatic MEN1 mutations, identified in 15/50 samples, significantly segregated in tumors negative for nuclear and cytoplasmic p27 staining. The germline nature of the CDKN1B mutations suggests that they might predispose to PHPT. The lack of somatic CDKN1B mutations in our samples points to a rare involvement in parathyroid adenomas, despite the frequent loss of nuclear p27 expression. MEN1 biallelic inactivation seems to be directly related to down-regulation of p27 expression through the inhibition of CDKN1B gene transcription.

Multiple endocrine neoplasia (MEN) syndromes are autosomal dominant diseases with high penetrance characterized by proliferative lesions (usually hyperplasia or adenoma) arising in at least two endocrine tissues. Four different MEN syndromes have been so far identified: MEN type 1 (MEN1), MEN2A (also referred to as MEN2), MEN2B (or MEN3) and MEN4, which have slightly varying tumor spectra and are caused by mutations in different genes. MEN1 associates with loss-of-function mutations in the MEN1 gene encoding the tumor suppressor menin. The MEN2A and MEN2B syndromes are due to activating mutations in the proto-oncogene RET (Rearranged in Transfection) and are characterized by different phenotypic features of the affected patients. MEN4 was the most recent addition to the family of the MEN syndromes. It was discovered less than 10 years ago thanks to studies of a rat strain that spontaneously develops multiple endocrine tumors (named MENX). These studies identified an inactivating mutation in the Cdkn1b gene, encoding the putative tumor suppressor p27, as the causative mutation of the rat syndrome. Subsequently, germline mutations in the human ortholog CDKN1B were also found in a subset of patients with a MEN-like phenotype and this led to the identification of MEN4. Small animal models have been instrumental in understanding important biochemical, physiological and pathological processes of cancer onset and spread in intact living organisms. Moreover, they have provided us with insight into gene function(s) and molecular mechanisms of disease progression. We here review the currently available animal models of MEN syndromes and their impact on the elucidation of the pathophysiology of these diseases, with a special focus on the rat MENX syndrome that we have been characterizing., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Genetic alterations frequently are involved in the development of a pituitary adenoma in young age. We here characterize the functional role of a deletion in CDKN1B 5'-UTR region (c.-29_-26delAGAG) identified in an acromegalic patient that developed a growth hormone in pituitary adenoma during childhood. Our results show that the identified novel heterozygous deletion in the CDKN1B 5'-UTR region associates with a reduction in CDKN1B mRNA levels, a predicted altered secondary mRNA structure, and a reduced CDKN1B 5'-UTR transcriptional activity in vitro. The patient displayed loss of heterozygosity in the same CDKN1B 5'-UTR region at tissue level and the 5'UTR region containing the deleted sequence encompasses a GRE. These findings indicate that the identification of functional alterations of newly discovered genetic derangements need to be fully characterized and always correlated with the clinical manifestations.

Objective: To date, no evidence of robust genotype-phenotype correlation or disease modifiers for multiple endocrine neoplasia type 1 (MEN1) syndrome has been described, leaving the highly variable clinical presentation of patients unaccounted for., Design: As the CDKN1B (p27) gene causes MEN4 syndrome and it is transcriptionally regulated by the product of the MEN1 gene (menin), we sought to analyze whether p27 influences the phenotype of MEN1-mutated patients. The cohort consisted of 100 patients carrying germline MEN1 gene mutations and 855 population-matched control individuals., Methods: Genotyping of the coding p27 c.326T>G (V109G) variant was performed by sequencing and restriction site digestion, and the genotypes were associated with clinical parameters by calculating odds ratios (ORs) and their 95% CIs using logistic regression., Results: There were significant differences in p27 V109G allele frequencies between controls and MEN1-mutated patients (OR=2.55, P=0.019, CI=1.013-5.76). Among patients who are ≥30 years old carrying truncating MEN1 mutations, the T allele was strongly associated with susceptibility to tumors in multiple glands (three to four glands affected vs one to two glands affected; OR=18.33; P=0.002, CI=2.88-16.41). This finding remained significant after the Bonferroni's multiple testing correction, indicating a robust association. No correlations were observed with the development of MEN1-related tumors such as hyperparathyroidism, pituitary adenomas, and enteropancreatic and adrenocortical tumors., Conclusions: Our study suggests that the p27 tumor suppressor gene acts as a disease modifier for the MEN1 syndrome associated with MEN1 germline mutations. If confirmed in independent patient cohorts, this finding could facilitate the management of this clinically complex disease., (© 2014 European Society of Endocrinology.)

Germline mutations in p27(kip1) are associated with increased susceptibility to multiple endocrine neoplasias (MEN) both in rats and humans; however, the potential role of common polymorphisms of this gene in endocrine tumor susceptibility and tumorigenesis remains mostly unrecognized. To assess the risk associated with polymorphism rs2066827 (p27-V109G), we genotyped a large cohort of Brazilian patients with sporadic endocrine tumors (pituitary adenomas, n=252; pheochromocytomas, n=125; medullary thyroid carcinoma, n=51; and parathyroid adenomas, n=19) and 885 population-matched healthy controls and determined the odds ratios and 95% CIs. Significant associations were found for the group of patients with pituitary adenomas (P=0.01), particularly for those with ACTH-secreting pituitary adenomas (P=0.005). In contrast, no association was found with GH-secreting pituitary tumors alone or with the sporadic counterpart of MEN2-component neoplasias. Our in vitro analyses revealed increased colony formation and cell growth rate for an AtT20 corticotropin mouse cell line overexpressing the p27-V109G variant compared with cells transfected with the WT p27. However, the genotypic effects in genetic and in vitro approaches were divergent. In accordance with our genetic data showing specificity for ACTH-secreting pituitary tissues, the overexpression of p27-V109G in a GH3 somatotropin rat cell line resulted in no difference compared with the WT. Pituitary tumors are one of the major clinical components of syndromes associated with the p27 pathogenic mutations MENX and MEN4. Our genetic and in vitro data indicate that the common polymorphism rs2066827 may play a role in corticotropinoma susceptibility and tumorigenesis through a molecular mechanism not fully understood thus far.

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.

Aims: The aim of this study is to evaluate the pathological features, serum hormone levels and ex vivo cultures of pituitary adenomas that occur in rats affected by MENX syndrome. MENX is multiple endocrine neoplasia syndrome caused by a germline mutation in the cell cycle inhibitor p27. Characterization of MENX adenomas is a prerequisite to exploit this animal model for molecular and translational studies of pituitary adenomas., Methods: We investigated MENX pituitary adenomas with immunohistochemistry, double immunofluorescence, electron microscopy, reverse transcription polymerase chain reaction (RT-PCR), measurement of serum hormone levels and ex vivo cultures., Results: Adenomas in MENX rats belong to the gonadotroph lineage. They start from 4 months of age as multiple neoplastic nodules and progress to become large lesions that efface the gland. Adenomas are composed of chromophobic cells predominantly expressing the glycoprotein alpha-subunit (αGSU). They show mitotic activity and high Ki67 labelling. A few neoplastic cells co-express gonadotropins and the transcription factor steroidogenic factor 1, together with growth hormone or prolactin and Pit-1, suggesting that they are not fully committed to one cell lineage. Ex vivo cultures show features similar to the primary tumour., Conclusions: Our results suggest that p27 function is critical to regulate gonadotroph cells growth. The MENX syndrome represents a unique model to elucidate the physiological and molecular mechanisms mediating the pathogenesis of gonadotroph adenomas., (© 2012 The Authors. Neuropathology and Applied Neurobiology © 2012 British Neuropathological Society.)

The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27(KIP1), an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27(KIP1) expression and/or localization are often observed in tumor cells, further confirming its central role in regulating the cell cycle. Recently, germline mutations in CDKN1B have been associated with the inherited multiple endocrine neoplasia syndrome type 4, an autosomal dominant syndrome characterized by varying combinations of tumors affecting at least two endocrine organs. In this study we identified a 4-bp deletion in a highly conserved regulatory upstream ORF (uORF) in the 5'UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm. This deletion causes the shift of the uORF termination codon with the consequent lengthening of the uORF-encoded peptide and the drastic shortening of the intercistronic space. Our data on the immunohistochemical analysis of the patient's pancreatic lesion, functional studies based on dual-luciferase assays, site-directed mutagenesis, and on polysome profiling show a negative influence of this deletion on the translation reinitiation at the CDKN1B starting site, with a consequent reduction in p27(KIP1) expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27(KIP1) activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27(KIP1) activity can also be modulated by an uORF and mutations affecting uORF could change p27(KIP1) expression. This study adds the CDKN1B gene to the short list of genes for which mutations that either create, delete, or severely modify their regulatory uORFs have been associated with human diseases., Competing Interests: The authors have declared that no competing interests exist.

A few years ago a novel multiple endocrine neoplasia syndrome, named multiple endocrine neoplasia type 4 (MEN4), was discovered thanks to studies conducted on a MEN syndrome in the rat (named MENX). The rat and the human syndromes are both caused by germline mutations in the Cdkn1b/CDKN1B gene, respectively. This gene encodes p27Kip1, a putative tumor suppressor which binds to and inhibits cyclin/cyclin-dependent kinase complexes, thereby preventing cell cycle progression. MEN4 patients carry heterozygous mutations at various residues of p27Kip1 and present with endocrine lesions mainly belonging to a MEN1-like spectrum: their most common phenotypic features are parathyroid and pituitary adenomas. Recently, germline mutations in p27kip1 were also identified in patients with a sporadic parathyroid disease presentation. In vitro functional analysis of several CDKN1B sequence changes identified in MEN4 patients detected impaired activity of the encoded p27Kip1 variant proteins (e.g. reduced expression, mislocalization or poor binding to interaction partners), thereby highlighting the characteristics of the protein which are critical for tumor suppression. Although the number of MEN4 patients is low, the discovery of this syndrome has demonstrated a novel role for CDKN1B as a tumor susceptibility gene for neuroendocrine tumors. Here, we review the clinical characteristics of the MEN4 syndrome and the molecular phenotype of the associated p27Kip1 mutations., (Copyright © 2013 S. Karger AG, Basel.)

Familial isolated pituitary adenoma (FIPA) occurs in families and is unrelated to multiple endocrine neoplasia type 1 and Carney complex. Mutations in AIP account only for 15-25% of FIPA families. CDKN1B mutations cause MEN4 in which affected patients can suffer from pituitary adenomas. With this study, we wanted to assess whether mutations in CDKN1B occur among a large cohort of AIP mutation-negative FIPA kindreds. Eighty-eight AIP mutation-negative FIPA families were studied and 124 affected subjects underwent sequencing of CDKN1B. Functional analysis of putative CDKN1B mutations was performed using in silico and in vitro approaches. Germline CDKN1B analysis revealed two nucleotide changes: c.286A>C (p.K96Q) and c.356T>C (p.I119T). In vitro, the K96Q change decreased p27 affinity for Grb2 but did not segregate with pituitary adenoma in the FIPA kindred. The I119T substitution occurred in a female patient with acromegaly. p27(I119T) shows an abnormal migration pattern by SDS-PAGE. Three variants (p.S56T, p.T142T, and c.605+36C>T) are likely nonpathogenic because In vitro effects were not seen. In conclusion, two patients had germline sequence changes in CDKN1B, which led to functional alterations in the encoded p27 proteins in vitro. Such rare CDKN1B variants may contribute to the development of pituitary adenomas, but their low incidence and lack of clear segregation with affected patients make CDKN1B sequencing unlikely to be of use in routine genetic investigation of FIPA kindreds. However, further characterization of the role of CDKN1B in pituitary tumorigenesis in these and other cases could help clarify the clinicopathological profile of MEN4.

Multiple endocrine neoplasias are autosomal dominant disorders characterized by the occurrence of tumors in at least two endocrine glands. Two MEN syndromes have long been known and are well characterized: the MEN type 1 (MEN1) and type 2 (MEN2). These syndromes are caused by germline mutations in the MEN1 and RET genes, respectively, and have a different tumor spectrum. Recently, a variant of the MEN syndromes arose spontaneously 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, heterozygous 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 mutations in p27. Altogether, 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 they are affected by MENX/4-associated mutations.

Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling cascade occurs in a variety of human malignancies, where it sustains tumor cell proliferation and survival. Pharmacologic blockade of this pathway exerts antineoplastic activity by triggering apoptosis and/or cell-cycle arrest. Pituitary adenomas show activation of the PI3K/AKT/mTOR pathway, but only a fraction of them respond in vitro to the antiproliferative action of rapamycin and RAD001 (mTOR inhibitors), possibly because of the described negative feedback loop on AKT which reactivates the signaling cascade. Rats affected by the multiple endocrine neoplasia-like syndrome (MENX) develop pituitary adenomas showing increased activated AKT. In this study, we comparatively investigated the antitumor potential of the novel dual PI3K/mTOR inhibitor NVP-BEZ235 and the single mTOR inhibitor RAD001 on rat pituitary adenoma cells in primary culture. NVP-BEZ235 inhibits the PI3K pathway both upstream and downstream of AKT, thereby preventing the negative feedback loop. NVP-BEZ235 was more effective than RAD001 in reducing cell viability of pituitary adenomas. Consistently, NVP-BEZ235 treatment decreased Akt and S6 phosphorylation and triggered apoptosis. Because MENX is caused by a germline loss-of-function mutation in the cell-cycle inhibitor p27Kip1, we investigated the relationship between this defect and response to NVP-BEZ235 treatment. The levels of p27Kip1 positively correlate with the response to NVP-BEZ235 treatment. Combined treatment with NVP-BEZ235 and the proteasome inhibitor bortezomib, which increases p27Kip1 amount, shows synergistic antiproliferative effects on pituitary adenoma cells. Our data suggest that NVP-BEZ235 may represent an effective therapeutic modality for pituitary adenomas and that p27Kip1 levels represent a potential predictor of response to dual PI3K/mTOR inhibition., (©2011 AACR)

Context: Typical nonfamilial (sporadic) parathyroid adenomas are common endocrine tumors for which no predisposing germline DNA variants and only a few clonally altered genes that drive parathyroid tumorigenesis have been identified. CDKN1B, encoding cyclin-dependent kinase inhibitor p27(kip1), has recently been implicated in a multiple endocrine tumor phenotype in rats and, rarely, in a human familial MEN1 (multiple endocrine neoplasia type 1)-like disorder., Objective: We sought to determine whether mutation of CDKN1B might contribute to the development of common sporadic parathyroid adenomas., Patients and Design: We sequenced the CDKN1B gene in 86 parathyroid adenomas from patients with typical, sporadic presentations of primary hyperparathyroidism. Identified alterations were categorized as somatic or germline, and their functional consequences were examined., Results: CDKN1B sequence abnormalities were identified in four parathyroid adenomas. Acquired biallelic alteration of CDKN1B, resulting from somatic mutation plus loss of heterozygosity, was detected in one tumor. Germline origin was documented in two cases despite nonfamilial presentations. None of the observed alterations were found in 240 CDKN1B alleles from normal individuals, nor among more than 2,000 previously reported alleles. Most identified variants reduced p27(kip1) protein levels or altered in vitro stability., Conclusions: In typical, sporadic parathyroid adenomas, CDKN1B mutation can be somatic and clonal, indicative of a directly conferred selective advantage in parathyroid tumorigenesis. Additionally, the identification of germline CDKN1B variants in patients with sporadic presentations provides evidence for CDKN1B as a susceptibility gene in the development of typical parathyroid adenomas.

Multiple endocrine neoplasias (MEN) are autosomal dominant disorders characterized by the occurrence of tumors in at least two endocrine glands. Two types of MEN syndromes have long been known: MEN type 1 (MEN1) and MEN type 2 (MEN2), associated with a different spectrum of affected organs. MEN1 and MEN2 are caused by germline mutations in the MEN1 tumor suppressor gene and the RET proto-oncogene, respectively. Lately, a new type of MEN was identified (named MEN4) which is due to mutations in the CDKN1B gene, encoding for p27kip1 (p27), a cyclin-dependent kinase (Cdk) inhibitor that regulates the transition of cells from G1 to S phase. p27 is a non-canonical tumor suppressor since it is usually not somatically mutated in human cancers but it is often downregulated by post-translational mechanisms. The discovery of MEN4 has defined a new role for CDKN1B as a tumor susceptibility gene for multiple endocrine tumors. To date, six germline CDKN1B mutations have been found in patients with a MEN1-like phenotype but negative for MEN1 mutations. Due to the limited number of patients so far identified, the phenotypic features of MEN4 are not clearly defined. Here, we review the clinical and molecular characteristics of the MEN4 syndrome and summarize the main functions of p27 to better comprehend how their alteration can predispose to neuroendocrine tumors., (Copyright © 2010 S. Karger AG, Basel.)

Pheochromocytomas are neoplasias of neural crest origin that arise from the chromaffin cells of the adrenal medulla. Pheochromocytomas arise with complete penetrance in rats homozygous for a germ-line frameshift mutation of Cdkn1b, encoding the cell cycle inhibitor p27KIP1 (MENX syndrome). We performed a genome-wide scan for allelic imbalance comparing 20 rat pheochromocytoma DNAs with normal rat DNA to better understand the pathobiology of the tumors and to correlate the findings with human pheochromocytoma. We identified allelic imbalance (AI) at candidate regions on rat chromosomes 8 and 19. Interestingly, the regions often lost in rat tumors are syntenic to regions involved in human pheochromocytomas. Fluorescence in situ hybridization analysis further validated the AI data. Sdhd and Rassf1a were analyzed in detail as they map to regions of AI on chromosome 8 and their homologues are implicated in human pheochromocytoma: we found no genetic mutations nor decreased expression. We also analyzed additional candidate genes, that is, rat homologues of genes predisposing to human pheochromocytoma and known tumor-suppressor genes, but we found no AI. In contrast, we observed frequent overexpression of Cdkn2a and Cdkn2c, encoding the cell cycle inhibitors p16INK4a and p18INK4c, respectively. The relative small number of allelic changes we found in rat pheochromocytoma might be related to their nonmalignant status and losses at chromosomes 8 and 19 are events that precede malignancy. Because of the high concordance of affected loci between rat and human tumors, studies of the MENX-associated pheochromocytomas should facilitate the identification of novel candidate genes implicated in their human counterpart.

In the past 3 years new insight into the etiopathogenesis of hereditary endocrine tumors has emerged from studies conducted on MENX, a rat multiple endocrine neoplasia (MEN) syndrome. MENX spontaneously developed in a rat colony and was discovered by serendipity when these animals underwent complete necropsy, as they were found to consistently develop multiple endocrine tumors with a spectrum similar to both MEN type 1 (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 the MENX syndrome. Capitalizing on these findings, we and others identified heterozygous germline mutations in the human homologue, CDKN1B, in patients with multiple endocrine tumors. As a consequence of these observations a novel human MEN syndrome, named MEN4, was recognized which is caused by mutations in p27. Altogether these studies identified Cdkn1b/CDKN1B as a novel tumor susceptibility gene for multiple endocrine tumors in both rats and humans. In this chapter we present the MENX syndrome and its phenotype, and we compare it to the human MEN syndromes; we discuss the current state of knowledge regarding the genes associated to inherited MEN, with a particular focus on CDKN1B; we present recent clinical and basic findings about the MEN4 syndrome and the functional characterization of the CDKN1B mutations identified. These findings are placed in the broader context of how p27 dysregulation might affect neuroendocrine cell function and trigger tumorigenesis., (Copyright 2010 Elsevier B.V. All rights reserved.)

MENX is a recessive multiple endocrine neoplasia-like syndrome in the rat. The tumor spectrum in MENX overlaps those of human multiple endocrine neoplasia (MEN) types 1 and 2. We mapped the MenX locus to the distal part of rat chromosome 4, excluding the homologs of the genes responsible for the MEN syndromes (RET and MEN1) and syndromes with an endocrine tumor component (VHL and NF1). We report the fine mapping of the disease locus and the identification of a homozygous frameshift mutation in Cdkn1b, encoding the cyclin-dependent kinase inhibitor p27(Kip1). As a consequence of the mutation, MENX-affected rats show dramatic reduction in p27(Kip1) protein. We have identified a germ-line nonsense mutation in the human CDKN1B gene in a MEN1 mutation-negative patient presenting with pituitary and parathyroid tumors. Expanded pedigree analysis shows that the mutation is associated with the development of an MEN1-like phenotype in multiple generations. Our findings demonstrate that germ-line mutations in p27(Kip1) can predispose to the development of multiple endocrine tumors in both rats and humans.