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A novel germline CDKN1B mutation causing multiple endocrine tumors: clinical, genetic and functional characterization - PubMed

Case Reports

. 2010 Nov;31(11):E1825-35.

doi: 10.1002/humu.21354.

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Free PMC article

Case Reports

A novel germline CDKN1B mutation causing multiple endocrine tumors: clinical, genetic and functional characterization

Sara Molatore et al. Hum Mutat. 2010 Nov.

Free PMC article

Abstract

Multiple endocrine neoplasia (MEN) syndromes are characterized by tumors involving two or more endocrine glands. Two MEN syndromes have long been known: MEN1 and MEN2,caused by germline mutations in MEN1 or RET, respectively. Recently, mutations in CDKN1B,encoding the cyclin-dependent kinase (Cdk) inhibitor p27, were identified in patients having a MEN1-like phenotype but no MEN1 gene mutations. Currently, the molecular mechanisms mediating the role of p27 in tumor predisposition are ill defined. We here report a novel germline missense variant in CDKN1B (c.678C>T, p.P69L) found in a patient with multiple endocrine tumors. We previously reported a nonsense p27 mutation (c.692G>A, p.W76X) in two patients with MEN1-like phenotype. Functional assays were used to characterize p27P69L and p27W76X in vitro. We show that p27P69L is expressed at reduced level and is impaired in both binding toCdk2 and inhibiting cell growth. p27W76X, which is mislocalized to the cytoplasm, can no longer efficiently bind Cyclins-Cdks, nor inhibit cell growth or induce apoptosis. In the patient’s tumor tissues, p27P69L associates with reduced/absent p27 expression and in one tumor with loss-of heterozygosity.Our results extend previous findings of CDKN1B mutations in patients with MEN1-related states and support the hypothesis of a tumor suppressor role for p27 in neuroendocrine cells.

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Figures

Figure 1
Figure 1

Characterization of the P69L and W76X mutations in p27. A, Sequence chromatograms showing the novel nucleotide change identified in patient FL compared to a control normal individual. B, Expression of the p27W76X and p27P69L mutant proteins 24h after transfection of MCF7 cells. Western blotting (WB) was performed with a monoclonal anti-p27 antibody. C, Localization of the p27 proteins. Asynchronously growing p27-negative MEFs were transiently transfected with the different YFP fusion constructs. Cells were fixed 24h later and nuclei were counterstained with DAPI. Cells were examined for direct YFP fluorescence. D, p27P69L does not associate with Cdk2. Lysates obtained upon transfection of HeLa with p27wt or p27P69L were incubated with purified GST-Cdk2 protein. Following GST pull-down, 50 µgr of the original lysate (Input) and the entire pull-down (GST-Cdk2) were analyzed by Western blotting (WB) with antibodies against p27 or Cdk2.

Figure 2
Figure 2

Degradation kinetics of the p27W76X and p27P69L mutant proteins and pathways of degradation. A, p27P69L is slightly more unstable than p27wt. Asynchronously growing MCF7 cells were transiently transfected with the different constructs and 24h later treated with CHX for the indicated times. Western blotting (WB) was performed using an anti-p27 antibody. The transfected (Tx) exogenous p27 proteins and the endogenous (Endo) p27 are indicated on the left. To control for equal loading the membranes were probed with anti-α-tubulin antibody. B, Degradation of p27W76X and p27P69L is in part mediated by Skp2 and KPC1. MCF7 cells were transfected with siRNA against Skp2 or KPC1 together with expression plasmids for YFP-p27wt, -p27W76X and -p27P69L and analyzed 48h later by western blotting for p27. Transfected (Tx) exogenous p27 proteins and the endogenous (Endo) p27 are indicated on the left. C, Scrambled siRNA (Scr. siRNA) has no effect on the expression of p27 proteins. Only the transfected proteins are shown.

Figure 3
Figure 3

Effect of the p27 mutations on protein binding, cell growth and apoptosis. A, Asynchronously growing HeLa cells were transfected with p27W76X and collected 24h later. Five hundred microgr of the lysate were immunoprecipitated (IP) o.n. with the anti-YFP antibody. Fifty microgr of the original lysate (Input) and the entire IP (IP YFP) were analyzed by Western blotting (WB) with antibodies against the indicated proteins. B, p27-negative GH3 cells were transfected with the indicated expression plasmids and then selected in G418-containing media for 3 weeks. Colonies were stained and counted. The values represent the mean of 2 independent experiments (each consisting of 6 separate plates) ±SD. C, Growth curves of representative GH3-derived clones expressing p27W76X. At day 1 and day 9 cells were lysed and total proteins were analyzed by Western blotting (WB). Values are the mean of 2 independent experiments ±SD. D, p27wt, p27W76X and p27P69L fusion proteins were transiently transfected into HeLa cells and 48h later stained with DAPI. Cells positive for YFP (=transfected) and showing chromatin condensation (=apoptotic) were counted and expressed as the percentage of the YFP-positive cells without features of apoptosis (Y axis). The values represent the average of 6 independently-transfected plates ±SD.

Figure 4
Figure 4

Expression of p27P69L and p27W76X in patient tissues. A-F, Immunohistochemical staining with an anti-p27 specific antibody. A-B, Staining of the normal kidney tissue from the W76X mutation-positive patient who developed renal angiomyolipoma (A), and from a normal control individual (B). The patient with the germline p27 mutation shows cytoplasmic staining in the renal tubules, whereas the normal individual only shows nuclear p27 immunoreactivity in some tubules. C-D, Staining of the parathyroid tumor from the P69L mutation-positive patient (C) and of a similar tumor from a sporadic mutation-negative patient (D). The p27 immunoreactivity of the tumor cells in the mutation-positive patient is dramatically reduced compared to the interspersed endothelial cells (indicated by arrows in the Inset). E-F, Staining of one bronchial carcinoid from the P69L mutation-positive patient (E) and of a similar tumor from a sporadic patient without mutation in p27 (F). There is virtually lack of p27 immunoreactivity of the tumor cells of the mutation-positive patient compared to the interspersed endothelial cells (indicated by an arrow in the Inset). Bar, 50 µM. Original magnification: A-F, 200X; Insets, 400X. G, LOH analysis of the tissues of the P69L mutation-positive patient for 3 micro satellite markers adjacent to the p27 locus. PT, parathyroid tumor; Carc N, normal tissue adjacent to the bronchial carcinoid; Carc T, bronchial carcinoid (tumor tissue).

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