Functional characterization of a CDKN1B mutation in a Sardinian kindred with multiple endocrine neoplasia type 4

in Endocrine Connections
View More View Less
  • 1 Endocrine Unit 2, Endocrinology Unit, Institute of Pathology, Department of Surgical, Department of Clinical and Experimental Medicine, University Hospital of Pisa, University of Pisa, Via Paradisa 2, Pisa, Italy

Inactivating germline mutations of the CDKN1B gene encoding the nuclear cyclin-dependent kinase inhibitor P27kip1 protein have been reported in patients with multiple endocrine neoplasia type 4 (MEN4), a MEN1-like phenotype without MEN1 mutations. The aim of this study was to characterize in vitro the germline CDKN1B mutation c.374_375delCT (S125X) we detected in a patient with MEN4. The proband was affected by primary hyperparathyroidism due to multiglandular parathyroid involvement and gastro–entero–pancreatic tumors. We carried out subcellular localization experiments by transfection with plasmid vectors expressing the WT or mutant CDKN1B cDNA into the eukaryotic human cervix adenocarcinoma (HeLa) and GH3 cell lines. Results from western blotting studies indicated that fusion proteins were expressed at equal levels. The mutated protein was shorter compared with the WT protein and lacked the highly conserved C-terminal domain, which includes the bipartite nuclear localization signal at amino acids 152/153 and 166/168. In HeLa and GH3 cells, WT P27 localized in the nucleus, whereas the P27_S125X protein was retained in the cytoplasm, predicting the loss of tumor-suppressive function. The proband's tumoral parathyroid tissue did not show allelic loss, because both WT and mutant alleles were determined to be present by sequencing the somatic DNA. Immunohistochemistry revealed a complete loss of nuclear expression of P27 in a parathyroid adenoma, which had been removed by the second surgery in the patient. In conclusion, our results confirm the pathogenic role of the c.374_375delCT CDKN1B germline mutation in a patient with MEN4.

Abstract

Inactivating germline mutations of the CDKN1B gene encoding the nuclear cyclin-dependent kinase inhibitor P27kip1 protein have been reported in patients with multiple endocrine neoplasia type 4 (MEN4), a MEN1-like phenotype without MEN1 mutations. The aim of this study was to characterize in vitro the germline CDKN1B mutation c.374_375delCT (S125X) we detected in a patient with MEN4. The proband was affected by primary hyperparathyroidism due to multiglandular parathyroid involvement and gastro–entero–pancreatic tumors. We carried out subcellular localization experiments by transfection with plasmid vectors expressing the WT or mutant CDKN1B cDNA into the eukaryotic human cervix adenocarcinoma (HeLa) and GH3 cell lines. Results from western blotting studies indicated that fusion proteins were expressed at equal levels. The mutated protein was shorter compared with the WT protein and lacked the highly conserved C-terminal domain, which includes the bipartite nuclear localization signal at amino acids 152/153 and 166/168. In HeLa and GH3 cells, WT P27 localized in the nucleus, whereas the P27_S125X protein was retained in the cytoplasm, predicting the loss of tumor-suppressive function. The proband's tumoral parathyroid tissue did not show allelic loss, because both WT and mutant alleles were determined to be present by sequencing the somatic DNA. Immunohistochemistry revealed a complete loss of nuclear expression of P27 in a parathyroid adenoma, which had been removed by the second surgery in the patient. In conclusion, our results confirm the pathogenic role of the c.374_375delCT CDKN1B germline mutation in a patient with MEN4.

Introduction

Multiple endocrine neoplasia type 1 (MEN1, OMIM #131100) is a rare autosomal dominant endocrine disorder characterized by the occurrence of parathyroid adenoma/hyperplasia, duodeno–pancreatic neuroendocrine tumors (NETs), and anterior pituitary tumors in the same individual (1). A minority of affected patients may also develop a wide spectrum of endocrine and non-endocrine manifestations, such as adrenal cortical tumors, foregut carcinoid tumors, angiofibromas, collagenomas, and lipomas, thus contributing to the heterogeneity of the phenotypic presentation. The rare combinations of less common manifestations of MEN1 are known as ‘phenocopy variants’ (2).

Germline heterozygous loss-of-function mutations of the tumor suppressor MEN1 gene – the main molecular defect causing the MEN1 syndrome – have been detected in about 70–80% and 30% of patients with familial and sporadic MEN1 respectively (3). The percentage in familial MEN1 rises to 90% if a search for large germline deletions is performed (4, 5, 6). Thus, a definite proportion of familial and sporadic MEN1 patients do not carry MEN1 mutations, indicating that other tumor-susceptibility genes may be involved in the pathogenesis of this syndrome. Following the identification of a germline change in the cyclin-dependent kinase (CDK) inhibitor Cdkn1b gene as the causative mutation of a variant of both MEN1 and MEN2 human syndromes in a rat colony (MENX syndrome), mutations of the human homologue CDKN1B were searched for in individuals with the MEN1 clinical phenotype, but without MEN1 mutations (7). Nine different germline mutations in the coding, as well as in the 5′-UTR of the CDKN1B gene have been described in patients with familial or sporadic MEN1-like syndromes, but negative for MEN1 mutations (7, 8, 9, 10, 11, 12, 13). Results from in vitro studies have confirmed the pathogenic role of these mutations (14).

The cases with mutations of CDKN1B are now classified as having the MEN4 syndrome (OMIM #610755), although they do not have any peculiar phenotypic manifestations compared with MEN1-mutation-positive cases. However, given the small number of reported MEN4 patients, the clinical penetrance of the disease and the precise tumor spectrum of the syndrome are still to be defined.

The CDKN1B gene encodes a nuclear protein named P27 (also known as KIP1), a member of the CDK inhibitors family. P27 regulates the transition of G1 phase to S phase by inhibiting the activity of CDKs and by promoting exit from the cell cycle. The subcellular localization of P27 appears to be central to regulation of its function. The antiproliferative role of P27 depends on its presence in the nucleus, the cellular compartment where it complexes with its target kinases (cyclin E-CDK2 and cyclin A-CDK2). Conversely, in the cytoplasm, despite being deprived of its tumor suppressor role, P27 drives pro-oncogenic functions, such as apoptosis and cell motility, and promotes cell proliferation by complexing to cyclin D/CDK4,6 (15, 16). P27 harbors a CDK-binding domain in the N-terminal half of the protein, which is necessary for the mediation of the CDK-inhibitory functions. Moreover, P27 contains a bipartite nuclear localization signal (NLS) at amino acids 152/153 and 166/168 in its C-terminal part. Although CDKN1B is considered to be a tumor suppressor gene, somatic loss-of-function mutations in this gene have rarely been detected in different cancers (17, 18, 19). Conversely, loss or decreased expression of the P27 protein has been reported in many human cancers, where it is often associated with a poor prognosis (20). Transcriptional, translational, and post-translational modifications of P27 (i.e., phosphorylation events), leading to mis-localization and/or sequestration of the P27 protein in the cytoplasm and subsequent degradation of the protein by proteasome-dependent mechanisms, account for the loss of its physiological role in the control of the cell cycle (21, 22). Nonetheless, biallelic inactivation of CDKN1B is a rare event, suggesting that haploinsufficiency may explain the tumorigenic progression (23, 24, 25, 26). The CDKN1B gene is therefore considered to be an atypical tumor suppressor gene.

We previously reported a novel loss-of-function mutation of the CDKN1B gene detected in a Sardinian kindred with MEN4 (27, 28). The index case was later seen elsewhere and the detailed description of the patient has been recently published (29). Herein, we extend the genetic analysis and describe the functional characterization of the mutation.

Materials and methods

Informed consent was obtained from the patient and one of the proband's three sons for all procedures used in this study. Our internal review board approved the study.

Tissue samples

Formalin-fixed paraffin embedded (FFPE) parathyroid tissue (superior right gland) removed at the second parathyroid surgery was retrieved from pathological archives.

Genetic studies

DNA was extracted from index patient's peripheral leukocytes with a Maxwell 16 Instrument according to the manufacturer's instructions (Promega Corp.). FFPE tissues were manually microdissected from two sections and samples were submitted to xylene deparaffination and then lysed and digested with proteinase K. DNA extraction was performed using the spin column procedure (QIAmp minikit; Qiagen). As no mutations in MEN1 were identified, the genetic analysis of the coding region and intron/exon boundaries of the CDKN1B (NM_004064.3) gene was carried out using the BigDye Sequencing Reaction Kit v.1.1. The reaction products were separated on an ABI 3130XL automatic sequencer (Applied Biosystems). To assess for loss-of-heterozigosity (LOH), the fragment of interest in the tumoral DNA was sequenced.

Immunohistochemistry

The immunohistochemical analysis was performed using the avidin–biotin–peroxidase complex method with the Ventana Benchmark immunostaining system (Ventana Medical System, Tucson, AZ, USA) following the manufacturer's instructions. A monoclonal anti-P27 antibody (clone SX53G, Ventana Medical System) was used to detect expression of P27. A negative control was included in the analysis by omitting the primary antibody. For the assessment of immunoreactivity, at least ten randomly selected fields under high-power magnification (×20) were chosen. In each experiment, adjacent normal parenchyma and/or infiltrating lymphocytes served as an internal positive control. Two specimens of normal parathyroid gland, inadvertently removed from normocalcemic subjects undergoing surgery for nontoxic multinodular goiter, were also used as controls. The cells were scored as positive if specific nuclear staining was detected, independently of the intensity of staining. Tumor staining was quantified according to the percentage of cells showing specific nuclear staining.

Transfection studies

The CDKN1B mutation identified in the proband by sequencing was introduced by site-directed mutagenesis (Quikchange II Site-Directed Mutagenesis Kit, Stratagene, La Jolla, CA, USA) into the full-length WT human CDKN1B cDNA cloned in a pEYFP (EYFP, enhanced yellow fluorescent protein) plasmid backbone. The mutant protein was expressed in human cervix adenocarcinoma (HeLa) and rat pituitary epithelial-like tumor (GH3) cell lines, maintained in DMEM media supplemented with 10% fetal bovine serum, 20 mM l-glutamine, 100 units/ml of penicillin G sodium, and 100 μg/ml streptomycin, or F-12K medium supplemented with 15% horse serum, 2.5% fetal bovine serum, 20 mM l-glutamine, 100 units/ml of penicillin G sodium, and 100 μg/ml streptomycin, respectively (Invitrogen). Transient transfections were carried out as reported previously (7). For western blotting and immunofluorescence analyses, the resulting vector expressed the mutant P27 protein as a fusion protein with the YFP tag at the N-terminus. Indirect immunofluorescence was performed using a monoclonal anti-P27 antibody (BD Biosciences, San Jose, CA, USA) as reported previously (7).

Results

Case report

The relevant clinical details of the index case are briefly reported. Primary hyperparathyroidism (PHPT) was firstly diagnosed in the index case, a 41-year-old woman (serum calcium 11.65 mg/dl (8.4–10.2 mg/dl) and parathyroid hormone (PTH) 189 pg/ml (10–65 pg/ml)). In the same year, she underwent inferior left and right parathyroidectomy (PTx). The two superior parathyroid glands appeared normal at neck exploration and both were biopsied. The histological examination of the inferior parathyroid glands showed an oxyphil chief cells adenoma. The biopsied superior parathyroid glands were histologically normal. Serum calcium levels normalized after surgery and no further biochemical testing was performed until the age of 48 years when a relapse of PHPT was diagnosed (serum calcium 11.3 mg/dl and PTH 201 pg/ml). Two years later, at the age of 50 years, the patient was referred to the Endocrine Unit of the University Hospital of Cagliari, where she was followed from February 2007 to October 2011. The clinical and biochemical evaluation confirmed the diagnosis of relapsing PHPT and the patient underwent a second neck exploration. The superior right parathyroid gland was removed and the histological examination showed a 20 mm oxyphil chief cells adenoma. A search for mutations of the MEN1 gene yielded negative results. The subsequent follow-up was notable for the evidence of gastro–entero–pancreatic NETs, which were initially treated with proton-pump inhibitors and somatostatin analogs, and then successfully by surgery. At follow-up evaluation in October 2011, a relapse of PHPT was evident. Total serum calcium and PTH were 10.6 mg/dl and 138 pg/ml respectively. A 99 m-Tc-Sestamibi scan showed an uptake in the left paratracheal region. Anterior pituitary function was normal and a pituitary MRI showed slight enlargement of the left side of the gland, in the absence of focal lesions. Surveillance was advised.

In April 2012, the patient was referred to the University Hospital of Florence where in November 2012 the left superior parathyroid gland was removed and a parathyroid fragment implanted in the non-dominant forearm (29).

Genetic analyses

The search for mutations in MEN1 in the entire coding region and splice sites, previously performed, gave negative results. This prompted us to search for mutations of the CDKN1B gene and a novel germline heterozygous deletion was found in exon 1 of the CDKN1B gene, c.374_375delCT (according to the latest Human Genome Variation Society nomenclature –http://www.hgvs.org/mutnomen; the nucleotide numbering reflects coding DNA, with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence). The two-nucleotide deletion causes a frameshift in the coding sequence, leading to a substitution of a serine (TCT) with a stop codon (TGA) and the production of a truncated P27 protein (S125X), consisting of 124 rather than 198 amino acids of the WT protein. The P27_S125X protein lacks the C-terminal domain, which contains the NLS required to enter the nucleus where the protein exerts its CDK-inhibitory function.

The proband's tumoral parathyroid tissue did not show allelic loss, because both WT and mutant alleles were demonstrated to be present by sequencing the somatic DNA. Genetic testing for the CDKN1B mutation was advised to all first-degree relatives, but only one of the three proband's sons agreed to be investigated. The results of the genetic test were negative.

Immunohistochemistry

A complete loss of nuclear P27 expression was observed in the parathyroid tumor of the proband. The adjacent normal parathyroid tissue showed strong nuclear staining (the percentage of positive cells ranging between 50% and 100%) similar to that observed in normal parathyroid tissue. Representative images are shown in Fig. 1.

Figure 1
Figure 1

Immunohistochemical staining for P27. (A) Normal parathyroid gland from a control individual. The parathyroid cells show a diffuse nuclear immunoreactivity (×20). (B) Superior right parathyroid adenoma removed at second parathyroidectomy from the MEN4 proband scored as negative (×20). (C) Image obtained of a different area of the same section as (B) showing a rim of normal parathyroid tissue (upper part of the image) with a diffuse nuclear staining adjacent to the parathyroid adenoma (lower part) (×20).

Citation: Endocrine Connections 4, 1; 10.1530/EC-14-0116

Transfection studies

To determine the effect of mutation of human P27 on protein localization, we generated WT P27 and P27_S125X YFP-tagged proteins. The results of the western blotting analysis indicated that the fusion proteins were expressed at equal levels (Fig. 2A). As expected, the P27_S125X protein was smaller as compared with the WT P27 fusion protein. This phenomenon was reproducibly observed in HeLa and GH3 cell lines and for independent DNA clones of the same construct. We determined the cellular localization of the fusion proteins by using an anti-YFP and indirect immunofluorescence. In both cell lines, P27_WT localized in the nucleus, whereas the P27_S125X protein was retained in the cytoplasm (Fig. 2B).

Figure 2
Figure 2

Expression and subcellular localization of exogenous proteins in HeLa and GH3 cells transfected with the indicated constructs. GH3 cells do not express endogenous P27. (A) Expression of proteins in transfected cells. 50 μg total protein obtained from cell lysates were separated by electrophoresis, blotted, and probed with YFP tagged-antibodies. Results from immunoblotting indicate that the fusion proteins are equally expressed. To control for equal loading of lysates, the membrane was probed with the anti-tubulin MAB. As expected, the P27_S125X protein, lacking 73 amino acids, is approximately 10 kDa smaller as compared with the P27_WT fusion protein. (B) Subcellular localization of YFP-tagged P27 proteins. Both HeLa (upper panels) and GH3 (lower panels) cells were fixed 24 h after transfection and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The cells were examined for direct YFP fluorescence. P27_S125X localized mainly in the cytoplasm, whereas P27_WT was in the nucleus.

Citation: Endocrine Connections 4, 1; 10.1530/EC-14-0116

Discussion

The medical history of the patient described herein (multiglandular PHPT and multiple gastro–entero–pancreatic tumors) was consistent with the diagnosis of MEN1 syndrome, but unexpectedly, the genetic testing of the MEN1 gene gave a negative result. The search for mutations of other genes involved in parathyroid tumorigenesis allowed the identification of a c.374_375delCT germline mutation in the CDKN1B gene, leading to the diagnosis of MEN4. The age at diagnosis of PHPT in our patient (41 years), which was similar to those previously reported for other MEN4 cases carrying nonsense or frameshift mutations of CDKN1B (W76X and K25fs), was older than the mean age of diagnosis of PHPT in patients with MEN1 syndrome (25 years) (30), but younger than that of MEN4 patients harboring missense (56 years) and 5′UTR mutations in CDKN1B (67 years) (31). No other phenotypic differences were observed between patients with MEN4 carrying the c.374_375delCT truncating mutation or other mutations of CDKN1B.

Herein we report the functional characterization of the c.374_375delCT CDKN1B germline mutation. HeLa and GH3 cells transfected with the CDKN1B WT or mutant cDNA efficiently translated the constructs and the fusion proteins were expressed at equal levels, as demonstrated by western blotting analysis. The mutated protein was shorter (lacking the last 74 amino acids) compared with the WT protein and lacked the highly conserved C-terminal domain, which includes the bipartite NLS (amino acids 152/153 and 166/168). This protein abnormality would be predicted to result in its retention in the cytoplasm and loss of tumor-suppressive function. In addition, the lack of the C-terminal domain would also be predicted to result in the loss of the binding domains of for some cytoplasmic interacting partners of P27 involved in the regulation of cellular functions independent of progression of the cell cycle, such as differentiation and migration (Fig. 3). As a matter of fact, the results of in vitro studies in HeLa cell lines indicated that the P27_S125X protein was localized in the cytoplasm, confirming that the mutant protein had lost its ability to transfer into the nucleus. This in vitro phenotype is similar to that of cells transfected with sequence encoding the W76X nonsense variant found in the first MEN4 patient, affected by PHPT and acromegaly (7). Notably, both the S125X and the W76X mutations have previously been detected in a somatic setting in a small intestine NET (SI-NET) case and an adult T-cell leukemia/lymphoma, respectively, strengthening support for their role in tumorigenesis (17, 19).

Figure 3
Figure 3

Schematic representation of WT and truncated P27_S125X protein. The binding domains to major interacting partners of P27 are represented as boxes filled with different patterns. Figures indicate the positions of the first and the last amino acid of each domain. The truncated P27_S125X protein lacks the C-terminal half of the protein, thereby loosing the nuclear localization signal, and the binding sites for RhoA, Stathmin, and Rac-dependent cell migration (also known as the ‘scatter domain’) and part of the binding site of p38Jab1.

Citation: Endocrine Connections 4, 1; 10.1530/EC-14-0116

Results from immunohistochemical studies were indicative of a complete loss of nuclear expression of P27 in the patient's parathyroid adenoma. No staining was evident in the cytoplasm. Conversely, a strong P27 staining was retained in a rim of normal parathyroid tissue surrounding the adenoma. A reduction in P27 protein in a different parathyroid adenoma obtained from the same patient at a later parathyroid surgery was also observed (29). The same authors found significant overexpression of CDKN1B mRNA compared with CDKN1B-non-mutated parathyroid tumors and normal parathyroid. The cDNA sequencing of the patient's tumoral mRNA revealed the presence of the WT, but not the mutated mRNA. No explanation is given for the lack of transcription of the mutated allele. The author's conclusion was that ‘in this case, the downregulation of the P27 protein could be at a post-transcriptional and/or post-translational level’. Only one study has previously evaluated the sequence of mRNA in a renal angiomyolipoma and normal renal tissue from a patient with MEN4 harboring the W76X mutation (7). The authors found that the WT and the W76X mutated alleles were equally transcribed in both tissues, indicating that both alleles were translated. This is in keeping with the results of our in vitro studies, which indicated that the P27_S125X mutant construct was fully translated into a truncated protein.

The presence of a hemizygous deletion of the CDKN1B gene in human hematopoietic malignancies, ovarian and prostate cancers, associated with a reduced expression of P27 is indicative of a CDKN1B haploinsufficient behavior in those tumors (25). Animal models also provide direct evidence of the role of P27 haploinsufficiency in the development of cancer and may explain the later onset of tumors in hemizygous compared with homozygous deficient mice (23, 32).

Haploinsufficiency of CDKN1B has not been clearly demonstrated in MEN4-associated tumors. Results of LOH studies, using either microsatellites flanking CDKN1B or sequencing the tumoral DNA, revealed allelic loss only in two out of five MEN4-associated tumors (a bronchial carcinoid and a small-cell neuroendocrine cervical carcinoma) (8, 10), but not in a parathyroid adenoma coexistent in the patient with bronchial carcinoid (10). The complete loss of P27 protein according to immunohistochemistry in these LOH-positive tumors indicates that CDKN1B behaves as a classical tumor suppressor gene. Conversely, the reduced expression of P27 protein in two of the three remaining LOH-negative tumors is indicative of a haploinsufficient behavior. Finally, the lack of P27 expression in the third LOH-negative case (W76X) indicates that a second somatic hit, other than LOH, inactivates the WT allele.

Both pathogenetic mechanisms (haploinsuffiency or tumor-suppressor behavior) appear to be operating in asynchronous parathyroid adenomas of the patient described herein. Indeed, our finding of a complete loss of P27 protein, in the absence of allelic loss, is indicative of an alternative somatic hit (genetic or epigenetic) at the CDKN1B locus. Conversely, the reduction in the level of P27 protein observed in a previous study (29) in the absence of allelic loss, is indicative of haploinsufficiency. This observation is in keeping with the finding of different somatic events in a patient with recurrent PHPT harboring a germline CDC73 mutation (33). The positive P27 staining in the rim of normal tissue surrounding the parathyroid adenoma in our patient provides strong support for the occurrence of a second somatic hit at the CDKN1B locus.

In conclusion, our results reveal the pathogenic role of the c.374_375delCT CDKN1B germline mutation in a patient with MEN4. The absence of allelic loss and complete lack of nuclear P27 expression in the parathyroid adenoma indicate that a second somatic hit, other than LOH, may inactivate the WT allele.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

The work was in part supported by Krebshilfe grant to N S Pellegata (grant number 107973) and Ministero dell'Istruzione, dell'Università e della Ricerca to C Marcocci (grant number 20094T89BR).

References

  • 1

    Marcocci C, Cetani F. Clinical practice. Primary hyperparathyroidism. New England Journal of Medicine 2011 365 23892397. (doi:10.1056/NEJMcp1106636).

    • Search Google Scholar
    • Export Citation
  • 2

    Turner JJ, Christie PT, Pearce SH, Turnpenny PD, Thakker RV. Diagnostic challenges due to phenocopies: lessons from multiple endocrine neoplasia type1 (MEN1). Human Mutation 2010 31 E1089E1101. (doi:10.1002/humu.21170).

    • Search Google Scholar
    • Export Citation
  • 3

    Lemos MC, Thakker RV. Multiple endocrine neoplasia type 1 (MEN1): analysis of 1336 mutations reported in the first decade following identification of the gene. Human Mutation 2008 29 2232. (doi:10.1002/humu.20605).

    • Search Google Scholar
    • Export Citation
  • 4

    Cavaco BM, Domingues R, Bacelar MC, Cardoso H, Barros L, Gomes L, Ruas MM, Agapito A, Garrao A, Pannett AA et al. . Mutational analysis of Portuguese families with multiple endocrine neoplasia type 1 reveals large germline deletions. Clinical Endocrinology 2002 56 465473. (doi:10.1046/j.1365-2265.2002.01505.x).

    • Search Google Scholar
    • Export Citation
  • 5

    Fukuuchi A, Nagamura Y, Yaguchi H, Ohkura N, Obara T, Tsukada T. A whole MEN1 gene deletion flanked by Alu repeats in a family with multiple endocrine neoplasia type 1. Japanese Journal of Clinical Oncology 2006 36 739744. (doi:10.1093/jjco/hyl089).

    • Search Google Scholar
    • Export Citation
  • 6

    Tham E, Grandell U, Lindgren E, Toss G, Skogseid B, Nordenskjold M. Clinical testing for mutations in the MEN1 gene in Sweden: a report on 200 unrelated cases. Journal of Clinical Endocrinology and Metabolism 2007 92 33893395. (doi:10.1210/jc.2007-0476).

    • Search Google Scholar
    • Export Citation
  • 7

    Pellegata NS, Quintanilla-Martinez L, Siggelkow H, Samson E, Bink K, Hofler H, Fend F, Graw J, Atkinson MJ. Germ-line mutations in p27Kip1 cause a multiple endocrine neoplasia syndrome in rats and humans. PNAS 2006 103 1555815563. (doi:10.1073/pnas.0603877103).

    • Search Google Scholar
    • Export Citation
  • 8

    Georgitsi M, Raitila A, Karhu A, van der Luijt RB, Aalfs CM, Sane T, Vierimaa O, Makinen MJ, Tuppurainen K, Paschke R et al. . Germline CDKN1B/p27Kip1 mutation in multiple endocrine neoplasia. Journal of Clinical Endocrinology and Metabolism 2007 92 33213325. (doi:10.1210/jc.2006-2843).

    • Search Google Scholar
    • Export Citation
  • 9

    Agarwal SK, Mateo CM, Marx SJ. Rare germline mutations in cyclin-dependent kinase inhibitor genes in multiple endocrine neoplasia type 1 and related states. Journal of Clinical Endocrinology and Metabolism 2009 94 18261834. (doi:10.1210/jc.2008-2083).

    • Search Google Scholar
    • Export Citation
  • 10

    Molatore S, Marinoni I, Lee M, Pulz E, Ambrosio MR, degli Uberti EC, Zatelli MC, Pellegata NS. A novel germline CDKN1B mutation causing multiple endocrine tumors: clinical, genetic and functional characterization. Human Mutation 2010 31 E1825E1835. (doi:10.1002/humu.21354).

    • Search Google Scholar
    • Export Citation
  • 11

    Malanga D, De Gisi S, Riccardi M, Scrima M, De Marco C, Robledo M, Viglietto G. Functional characterization of a rare germline mutation in the gene encoding the cyclin-dependent kinase inhibitor p27Kip1 (CDKN1B) in a Spanish patient with multiple endocrine neoplasia-like phenotype. European Journal of Endocrinology 2012 166 551560. (doi:10.1530/EJE-11-0929).

    • Search Google Scholar
    • Export Citation
  • 12

    Belar O, De La Hoz C, Perez-Nanclares G, Castano L, Gaztambide S, Spanish MENG . Novel mutations in MEN1, CDKN1B and AIP genes in patients with multiple endocrine neoplasia type 1 syndrome in Spain. Clinical Endocrinology 2012 76 719724. (doi:10.1111/j.1365-2265.2011.04269.x).

    • Search Google Scholar
    • Export Citation
  • 13

    Occhi G, Regazzo D, Trivellin G, Boaretto F, Ciato D, Bobisse S, Ferasin S, Cetani F, Pardi E, Korbonits M et al. . A novel mutation in the upstream open reading frame of the CDKN1B gene causes a MEN4 phenotype. PLoS Genetics 2013 9 e1003350. (doi:10.1371/journal.pgen.1003350).

    • Search Google Scholar
    • Export Citation
  • 14

    Lee M, Pellegata NS. Multiple endocrine neoplasia type 4. Frontiers of Hormone Research 2013 41 6378. (doi:10.1159/000345670).

  • 15

    Sicinski P, Zacharek S, Kim C. Duality of p27Kip1 function in tumorigenesis. Genes and Development 2007 21 17031706. (doi:10.1101/gad.1583207).

    • Search Google Scholar
    • Export Citation
  • 16

    Larrea MD, Wander SA, Slingerland JM. p27 as Jekyll and Hyde: regulation of cell cycle and cell motility. Cell Cycle 2009 8 34553461. (doi:10.4161/cc.8.21.9789).

    • Search Google Scholar
    • Export Citation
  • 17

    Morosetti R, Kawamata N, Gombart AF, Miller CW, Hatta Y, Hirama T, Said JW, Tomonaga M, Koeffler HP. Alterations of the p27KIP1 gene in non-Hodgkin's lymphomas and adult T-cell leukemia/lymphoma. Blood 1995 86 19241930.

    • Search Google Scholar
    • Export Citation
  • 18

    Stephens PJ, Tarpey PS, Davies H, Van Loo P, Greenman C, Wedge DC, Nik-Zainal S, Martin S, Varela I, Bignell GR et al. . The landscape of cancer genes and mutational processes in breast cancer. Nature 2012 486 400404. (doi:10.1038/nature11017).

    • Search Google Scholar
    • Export Citation
  • 19

    Francis JM, Kiezun A, Ramos AH, Serra S, Pedamallu CS, Qian ZR, Banck MS, Kanwar R, Kulkarni AA, Karpathakis A et al. . Somatic mutation of CDKN1B in small intestine neuroendocrine tumors. Nature Genetics 2013 45 14831486. (doi:10.1038/ng.2821).

    • Search Google Scholar
    • Export Citation
  • 20

    Chu IM, Hengst L, Slingerland JM. The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nature Reviews. Cancer 2008 8 253267. (doi:10.1038/nrc2347).

    • Search Google Scholar
    • Export Citation
  • 21

    Viglietto G, Motti ML, Fusco A. Understanding p27kip1 deregulation in cancer: down-regulation or mislocalization. Cell Cycle 2002 1 394400. (doi:10.4161/cc.1.6.263).

    • Search Google Scholar
    • Export Citation
  • 22

    Vervoorts J, Luscher B. Post-translational regulation of the tumor suppressor p27KIP1. Cellular and Molecular Life Sciences 2008 65 32553264. (doi:10.1007/s00018-008-8296-7).

    • Search Google Scholar
    • Export Citation
  • 23

    Fero ML, Randel E, Gurley KE, Roberts JM, Kemp CJ. The murine gene p27Kip1 is haplo-insufficient for tumour suppression. Nature 1998 396 177180. (doi:10.1038/24179).

    • Search Google Scholar
    • Export Citation
  • 24

    Philipp-Staheli J, Payne SR, Kemp CJ. p27Kip1: regulation and function of a haploinsufficient tumor suppressor and its misregulation in cancer. Experimental Cell Research 2001 264 148168. (doi:10.1006/excr.2000.5143).

    • Search Google Scholar
    • Export Citation
  • 25

    Le Toriellec E, Despouy G, Pierron G, Gaye N, Joiner M, Bellanger D, Vincent-Salomon A, Stern MH. Haploinsufficiency of CDKN1B contributes to leukemogenesis in T-cell prolymphocytic leukemia. Blood 2008 111 23212328. (doi:10.1182/blood-2007-06-095570).

    • Search Google Scholar
    • Export Citation
  • 26

    Taguchi R, Yamada M, Horiguchi K, Tomaru T, Ozawa A, Shibusawa N, Hashimoto K, Okada S, Satoh T, Mori M. Haploinsufficient and predominant expression of multiple endocrine neoplasia type 1 (MEN1)-related genes, MLL, p27Kip1 and p18Ink4C in endocrine organs. Biochemical and Biophysical Research Communications 2011 415 378383. (doi:10.1016/j.bbrc.2011.10.077).

    • Search Google Scholar
    • Export Citation
  • 27

    Cetani F, Pardi E, Borsari S, Saponaro F, Banti C, Vignali E, Cianferotti l, Di Rosa G, Mastinu M, Mariotti S et al. Genetic analyses of CDKN1B and AIP genes in familial primary hyperparathyroidism. Journal of Bone and Mineral Research 2012 27 Suppl.1 Annual Meeting of the American Society for Bone and Mineral Research S159

  • 28

    Pardi E, Borsari S, Saponaro F, Banti C, Pellegata N, Lee M, Vignali E, Meola A, Mastinu M, Mariotti S et al. . Genetic analysis of CDKN1B gene in familial primary hyperparathyroidism. Endocrine Abstracts 2013 32 OC2.5. (doi:10.1530/endoabs.32.OC2.5).

    • Search Google Scholar
    • Export Citation
  • 29

    Tonelli F, Giudici F, Giusti F, Marini F, Cianferotti L, Nesi G, Brandi ML. A heterozygous frameshift mutation in exon 1 of CDKN1B gene in a patient affected by MEN4 syndrome. European Journal of Endocrinology 2014 171 K7K17. (doi:10.1530/EJE-14-0080).

    • Search Google Scholar
    • Export Citation
  • 30

    Marx SJ. Hyperparathyroid and hypoparathyroid disorders. New England Journal of Medicine 2000 343 18631875. (doi:10.1056/NEJM200012213432508).

    • Search Google Scholar
    • Export Citation
  • 31

    Lee M, Pellegata NS. Multiple endocrine neoplasia syndromes associated with mutation of p27. Journal of Endocrinological Investigation 2013 36 781787. (doi:10.3275/9021).

    • Search Google Scholar
    • Export Citation
  • 32

    Cook WD, McCaw BJ. Accommodating haploinsufficient tumor suppressor genes in Knudson's model. Oncogene 2000 19 34343438. (doi:10.1038/sj.onc.1203653).

    • Search Google Scholar
    • Export Citation
  • 33

    Cetani F, Pardi E, Ambrogini E, Viacava P, Borsari S, Lemmi M, Cianferotti L, Miccoli P, Pinchera A, Arnold A et al. . Different somatic alterations of the HRPT2 gene in a patient with recurrent sporadic primary hyperparathyroidism carrying an HPRT2 germline mutation. Endocrine-Related Cancer 2007 14 493499. (doi:10.1677/ERC-06-0092).

    • Search Google Scholar
    • Export Citation

K Benfini is now at Section of Endocrinology, Department of Medical Sciences, University of Ferrara, Ferrara, Italy

 

     European Society of Endocrinology

     Society for Endocrinology

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 1029 190 53
PDF Downloads 476 120 22
  • View in gallery

    Immunohistochemical staining for P27. (A) Normal parathyroid gland from a control individual. The parathyroid cells show a diffuse nuclear immunoreactivity (×20). (B) Superior right parathyroid adenoma removed at second parathyroidectomy from the MEN4 proband scored as negative (×20). (C) Image obtained of a different area of the same section as (B) showing a rim of normal parathyroid tissue (upper part of the image) with a diffuse nuclear staining adjacent to the parathyroid adenoma (lower part) (×20).

  • View in gallery

    Expression and subcellular localization of exogenous proteins in HeLa and GH3 cells transfected with the indicated constructs. GH3 cells do not express endogenous P27. (A) Expression of proteins in transfected cells. 50 μg total protein obtained from cell lysates were separated by electrophoresis, blotted, and probed with YFP tagged-antibodies. Results from immunoblotting indicate that the fusion proteins are equally expressed. To control for equal loading of lysates, the membrane was probed with the anti-tubulin MAB. As expected, the P27_S125X protein, lacking 73 amino acids, is approximately 10 kDa smaller as compared with the P27_WT fusion protein. (B) Subcellular localization of YFP-tagged P27 proteins. Both HeLa (upper panels) and GH3 (lower panels) cells were fixed 24 h after transfection and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The cells were examined for direct YFP fluorescence. P27_S125X localized mainly in the cytoplasm, whereas P27_WT was in the nucleus.

  • View in gallery

    Schematic representation of WT and truncated P27_S125X protein. The binding domains to major interacting partners of P27 are represented as boxes filled with different patterns. Figures indicate the positions of the first and the last amino acid of each domain. The truncated P27_S125X protein lacks the C-terminal half of the protein, thereby loosing the nuclear localization signal, and the binding sites for RhoA, Stathmin, and Rac-dependent cell migration (also known as the ‘scatter domain’) and part of the binding site of p38Jab1.

  • 1

    Marcocci C, Cetani F. Clinical practice. Primary hyperparathyroidism. New England Journal of Medicine 2011 365 23892397. (doi:10.1056/NEJMcp1106636).

    • Search Google Scholar
    • Export Citation
  • 2

    Turner JJ, Christie PT, Pearce SH, Turnpenny PD, Thakker RV. Diagnostic challenges due to phenocopies: lessons from multiple endocrine neoplasia type1 (MEN1). Human Mutation 2010 31 E1089E1101. (doi:10.1002/humu.21170).

    • Search Google Scholar
    • Export Citation
  • 3

    Lemos MC, Thakker RV. Multiple endocrine neoplasia type 1 (MEN1): analysis of 1336 mutations reported in the first decade following identification of the gene. Human Mutation 2008 29 2232. (doi:10.1002/humu.20605).

    • Search Google Scholar
    • Export Citation
  • 4

    Cavaco BM, Domingues R, Bacelar MC, Cardoso H, Barros L, Gomes L, Ruas MM, Agapito A, Garrao A, Pannett AA et al. . Mutational analysis of Portuguese families with multiple endocrine neoplasia type 1 reveals large germline deletions. Clinical Endocrinology 2002 56 465473. (doi:10.1046/j.1365-2265.2002.01505.x).

    • Search Google Scholar
    • Export Citation
  • 5

    Fukuuchi A, Nagamura Y, Yaguchi H, Ohkura N, Obara T, Tsukada T. A whole MEN1 gene deletion flanked by Alu repeats in a family with multiple endocrine neoplasia type 1. Japanese Journal of Clinical Oncology 2006 36 739744. (doi:10.1093/jjco/hyl089).

    • Search Google Scholar
    • Export Citation
  • 6

    Tham E, Grandell U, Lindgren E, Toss G, Skogseid B, Nordenskjold M. Clinical testing for mutations in the MEN1 gene in Sweden: a report on 200 unrelated cases. Journal of Clinical Endocrinology and Metabolism 2007 92 33893395. (doi:10.1210/jc.2007-0476).

    • Search Google Scholar
    • Export Citation
  • 7

    Pellegata NS, Quintanilla-Martinez L, Siggelkow H, Samson E, Bink K, Hofler H, Fend F, Graw J, Atkinson MJ. Germ-line mutations in p27Kip1 cause a multiple endocrine neoplasia syndrome in rats and humans. PNAS 2006 103 1555815563. (doi:10.1073/pnas.0603877103).

    • Search Google Scholar
    • Export Citation
  • 8

    Georgitsi M, Raitila A, Karhu A, van der Luijt RB, Aalfs CM, Sane T, Vierimaa O, Makinen MJ, Tuppurainen K, Paschke R et al. . Germline CDKN1B/p27Kip1 mutation in multiple endocrine neoplasia. Journal of Clinical Endocrinology and Metabolism 2007 92 33213325. (doi:10.1210/jc.2006-2843).

    • Search Google Scholar
    • Export Citation
  • 9

    Agarwal SK, Mateo CM, Marx SJ. Rare germline mutations in cyclin-dependent kinase inhibitor genes in multiple endocrine neoplasia type 1 and related states. Journal of Clinical Endocrinology and Metabolism 2009 94 18261834. (doi:10.1210/jc.2008-2083).

    • Search Google Scholar
    • Export Citation
  • 10

    Molatore S, Marinoni I, Lee M, Pulz E, Ambrosio MR, degli Uberti EC, Zatelli MC, Pellegata NS. A novel germline CDKN1B mutation causing multiple endocrine tumors: clinical, genetic and functional characterization. Human Mutation 2010 31 E1825E1835. (doi:10.1002/humu.21354).

    • Search Google Scholar
    • Export Citation
  • 11

    Malanga D, De Gisi S, Riccardi M, Scrima M, De Marco C, Robledo M, Viglietto G. Functional characterization of a rare germline mutation in the gene encoding the cyclin-dependent kinase inhibitor p27Kip1 (CDKN1B) in a Spanish patient with multiple endocrine neoplasia-like phenotype. European Journal of Endocrinology 2012 166 551560. (doi:10.1530/EJE-11-0929).

    • Search Google Scholar
    • Export Citation
  • 12

    Belar O, De La Hoz C, Perez-Nanclares G, Castano L, Gaztambide S, Spanish MENG . Novel mutations in MEN1, CDKN1B and AIP genes in patients with multiple endocrine neoplasia type 1 syndrome in Spain. Clinical Endocrinology 2012 76 719724. (doi:10.1111/j.1365-2265.2011.04269.x).

    • Search Google Scholar
    • Export Citation
  • 13

    Occhi G, Regazzo D, Trivellin G, Boaretto F, Ciato D, Bobisse S, Ferasin S, Cetani F, Pardi E, Korbonits M et al. . A novel mutation in the upstream open reading frame of the CDKN1B gene causes a MEN4 phenotype. PLoS Genetics 2013 9 e1003350. (doi:10.1371/journal.pgen.1003350).

    • Search Google Scholar
    • Export Citation
  • 14

    Lee M, Pellegata NS. Multiple endocrine neoplasia type 4. Frontiers of Hormone Research 2013 41 6378. (doi:10.1159/000345670).

  • 15

    Sicinski P, Zacharek S, Kim C. Duality of p27Kip1 function in tumorigenesis. Genes and Development 2007 21 17031706. (doi:10.1101/gad.1583207).

    • Search Google Scholar
    • Export Citation
  • 16

    Larrea MD, Wander SA, Slingerland JM. p27 as Jekyll and Hyde: regulation of cell cycle and cell motility. Cell Cycle 2009 8 34553461. (doi:10.4161/cc.8.21.9789).

    • Search Google Scholar
    • Export Citation
  • 17

    Morosetti R, Kawamata N, Gombart AF, Miller CW, Hatta Y, Hirama T, Said JW, Tomonaga M, Koeffler HP. Alterations of the p27KIP1 gene in non-Hodgkin's lymphomas and adult T-cell leukemia/lymphoma. Blood 1995 86 19241930.

    • Search Google Scholar
    • Export Citation
  • 18

    Stephens PJ, Tarpey PS, Davies H, Van Loo P, Greenman C, Wedge DC, Nik-Zainal S, Martin S, Varela I, Bignell GR et al. . The landscape of cancer genes and mutational processes in breast cancer. Nature 2012 486 400404. (doi:10.1038/nature11017).

    • Search Google Scholar
    • Export Citation
  • 19

    Francis JM, Kiezun A, Ramos AH, Serra S, Pedamallu CS, Qian ZR, Banck MS, Kanwar R, Kulkarni AA, Karpathakis A et al. . Somatic mutation of CDKN1B in small intestine neuroendocrine tumors. Nature Genetics 2013 45 14831486. (doi:10.1038/ng.2821).

    • Search Google Scholar
    • Export Citation
  • 20

    Chu IM, Hengst L, Slingerland JM. The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nature Reviews. Cancer 2008 8 253267. (doi:10.1038/nrc2347).

    • Search Google Scholar
    • Export Citation
  • 21

    Viglietto G, Motti ML, Fusco A. Understanding p27kip1 deregulation in cancer: down-regulation or mislocalization. Cell Cycle 2002 1 394400. (doi:10.4161/cc.1.6.263).

    • Search Google Scholar
    • Export Citation
  • 22

    Vervoorts J, Luscher B. Post-translational regulation of the tumor suppressor p27KIP1. Cellular and Molecular Life Sciences 2008 65 32553264. (doi:10.1007/s00018-008-8296-7).

    • Search Google Scholar
    • Export Citation
  • 23

    Fero ML, Randel E, Gurley KE, Roberts JM, Kemp CJ. The murine gene p27Kip1 is haplo-insufficient for tumour suppression. Nature 1998 396 177180. (doi:10.1038/24179).

    • Search Google Scholar
    • Export Citation
  • 24

    Philipp-Staheli J, Payne SR, Kemp CJ. p27Kip1: regulation and function of a haploinsufficient tumor suppressor and its misregulation in cancer. Experimental Cell Research 2001 264 148168. (doi:10.1006/excr.2000.5143).

    • Search Google Scholar
    • Export Citation
  • 25

    Le Toriellec E, Despouy G, Pierron G, Gaye N, Joiner M, Bellanger D, Vincent-Salomon A, Stern MH. Haploinsufficiency of CDKN1B contributes to leukemogenesis in T-cell prolymphocytic leukemia. Blood 2008 111 23212328. (doi:10.1182/blood-2007-06-095570).

    • Search Google Scholar
    • Export Citation
  • 26

    Taguchi R, Yamada M, Horiguchi K, Tomaru T, Ozawa A, Shibusawa N, Hashimoto K, Okada S, Satoh T, Mori M. Haploinsufficient and predominant expression of multiple endocrine neoplasia type 1 (MEN1)-related genes, MLL, p27Kip1 and p18Ink4C in endocrine organs. Biochemical and Biophysical Research Communications 2011 415 378383. (doi:10.1016/j.bbrc.2011.10.077).

    • Search Google Scholar
    • Export Citation
  • 27

    Cetani F, Pardi E, Borsari S, Saponaro F, Banti C, Vignali E, Cianferotti l, Di Rosa G, Mastinu M, Mariotti S et al. Genetic analyses of CDKN1B and AIP genes in familial primary hyperparathyroidism. Journal of Bone and Mineral Research 2012 27 Suppl.1 Annual Meeting of the American Society for Bone and Mineral Research S159

  • 28

    Pardi E, Borsari S, Saponaro F, Banti C, Pellegata N, Lee M, Vignali E, Meola A, Mastinu M, Mariotti S et al. . Genetic analysis of CDKN1B gene in familial primary hyperparathyroidism. Endocrine Abstracts 2013 32 OC2.5. (doi:10.1530/endoabs.32.OC2.5).

    • Search Google Scholar
    • Export Citation
  • 29

    Tonelli F, Giudici F, Giusti F, Marini F, Cianferotti L, Nesi G, Brandi ML. A heterozygous frameshift mutation in exon 1 of CDKN1B gene in a patient affected by MEN4 syndrome. European Journal of Endocrinology 2014 171 K7K17. (doi:10.1530/EJE-14-0080).

    • Search Google Scholar
    • Export Citation
  • 30

    Marx SJ. Hyperparathyroid and hypoparathyroid disorders. New England Journal of Medicine 2000 343 18631875. (doi:10.1056/NEJM200012213432508).

    • Search Google Scholar
    • Export Citation
  • 31

    Lee M, Pellegata NS. Multiple endocrine neoplasia syndromes associated with mutation of p27. Journal of Endocrinological Investigation 2013 36 781787. (doi:10.3275/9021).

    • Search Google Scholar
    • Export Citation
  • 32

    Cook WD, McCaw BJ. Accommodating haploinsufficient tumor suppressor genes in Knudson's model. Oncogene 2000 19 34343438. (doi:10.1038/sj.onc.1203653).

    • Search Google Scholar
    • Export Citation
  • 33

    Cetani F, Pardi E, Ambrogini E, Viacava P, Borsari S, Lemmi M, Cianferotti L, Miccoli P, Pinchera A, Arnold A et al. . Different somatic alterations of the HRPT2 gene in a patient with recurrent sporadic primary hyperparathyroidism carrying an HPRT2 germline mutation. Endocrine-Related Cancer 2007 14 493499. (doi:10.1677/ERC-06-0092).

    • Search Google Scholar
    • Export Citation