Characterization of a naturally-occurring p27 mutation predisposing to multiple endocrine tumors

<p>Abstract</p> <p>Background</p> <p>p27Kip1 (p27) is an important negative regulator of the cell cycle and a putative tumor suppressor. The finding that a spontaneous germline frameshift mutation in <it>Cdkn1b </it>(encoding p27) causes the MENX multiple endocrine neoplasia syndrome in the rat provided the first evidence that <it>Cdkn1b </it>is a tumor susceptibility gene for endocrine tumors. Noteworthy, germline p27 mutations were also identified in human patients presenting with endocrine tumors. At present, it is not clear which features of p27 are crucial for this tissue-specific tumor predisposition in both rats and humans. It was shown that the MENX-associated <it>Cdkn1b </it>mutation causes reduced expression of the encoded protein, but the molecular mechanisms are unknown. To better understand the role of p27 in tumor predisposition and to characterize the MENX animal model at the molecular level, a prerequisite for future preclinical studies, we set out to assess the functional properties of the MENX-associated p27 mutant protein (named p27fs177) <it>in vitro </it>and <it>in vivo</it>.</p> <p>Results</p> <p><it>In vitro</it>, p27fs177 retains some properties of the wild-type p27 (p27wt) protein: it localizes to the nucleus; it interacts with cyclin-dependent kinases and, to lower extent, with cyclins. In contrast to p27wt, p27fs177 is highly unstable and rapidly degraded in every phase of the cell-cycle, including quiescence. It is in part degraded by Skp2-dependent proteasomal proteolysis, similarly to p27wt. Photobleaching studies showed reduced motility of p27fs177 in the nucleus compared to p27wt, suggesting that in this compartment p27fs177 is part of a multi-protein complex, likely together with the degradation machinery. Studies of primary rat newborn fibroblasts (RNF) established from normal and MENX-affected littermates confirmed the rapid degradation of p27fs177 <it>in vivo </it>which can be rescued by Bortezomib (proteasome inhibitor drug). Overexpression of the negative regulators microRNA-221/222 plays no role in regulating the amount of p27fs177 in RNFs and rat tissues.</p> <p>Conclusion</p> <p>Our findings show that reduced p27 levels, not newly acquired properties, trigger tumor formation in rats, similarly to what has been observed in mice. The molecular characteristics of p27fs177 establish MENX as a useful preclinical model to evaluate compounds that inhibit p27 degradation for their efficacy against endocrine tumors.</p

Functional Imaging of Pheochromocytoma with 68Ga-DOTATOC and 68C-HED in a Genetically Defined Rat Model of Multiple Endocrine Neoplasia

Rats affected by the MENX multitumor syndrome develop pheochromocytoma (100%). Pheochromocytomas are uncommon tumors and animal models are scarce, hence the interest in MENX rats to identify and preclinically evaluate novel targeted therapies. A prerequisite for such studies is a sensitive and noninvasive detection of MENXassociated pheochromocytoma. We performed positron emission tomography (PET) to determine whether rat pheochromocytomas are detected by tracers used in clinical practice, such as 68Ga-DOTATOC (somatostatin analogue) or 11C-Hydroxyephedrine (HED), a norepinephrine analogue. We analyzed four affected and three unaffected rats. The PET scan findings were correlated to histopathology and immunophenotype of the tumors, their proliferative index, and the expression of genes coding for somatostatin receptors or the norepinephrine transporter. We observed that mean 68Ga-DOTATOC standard uptake value (SUV) in adrenals of affected animals was 23.3 ± 3.9, significantly higher than in control rats (15.4 ± 7.9; P = .03). The increase in mean tumor-to-liver ratio of 11C-HED in the MENX-affected animals (1.6 ± 0.5) compared to controls (0.7 ± 0.1) was even more significant (P = .0016). In a unique animal model, functional imaging depicting two pathways important in pheochromocytoma biology discriminated affected animals from controls, thus providing the basis for future preclinical work with MENX rats

Early Rehabilitation Reduces Time to Decannulation in Patients With Severe Acquired Brain Injury: A Retrospective Study

Purpose: Early decannulation is considered a main rehabilitative goal in tracheostomized patients. Our aim is to evaluate whether a very early rehabilitation protocol helps to reduce the tracheostomy duration in patients affected by an Acquired Brain Injury (ABI).Methods: Data about consecutive tracheostomized patients admitted in our Neuro-Rehabilitation Unit (NRU) were retrospectively collected. We defined two groups: Early Rehabilitation Group patients came from our ICU, where they started the rehabilitative treatment; Delayed Rehabilitation Group patients arrived from external ICUs and started rehabilitation in our NRU. Primary outcome was the time from tracheostomy to decannulation. Secondary outcomes were: ICU length of stay, time from NRU admission to decannulation, Glasgow Coma Scale, Coma Recovery Scale revised and Levels of Cognitive Functioning scores at NRU discharge and the re-cannulation rate.Results: We enrolled 66 patients, 40 in the Early Rehabilitation Group and 26 in the Delayed Rehabilitation Group. 70% of patients for each group could be decannulated (p = 0.73) and were analyzed. Only one patient was re-cannulated. Early Rehabilitation Group showed a shorter tracheostomy duration (61.0 vs. 94.5 days, p = 0.013), a higher probability of occurrence of decannulation (p = 0.008) and a lower ICU length of stay (30.0 vs. 52.0 days, p = 0.001). The time to decannulation in NRU was similar between groups (30.0 vs. 45.50 days, p = 0.14). All the scale scores had a significant improvement in both groups (p &lt; 0.0001 all).Conclusions: The present study shows that an early neuro-rehabilitation protocol helps to reduce the time to decannulation in tracheostomized patients affected by ABI

A Novel Mutation in the Upstream Open Reading Frame of the CDKN1B Gene Causes a MEN4 Phenotype

PubMed ID: 23555276This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Role of T198 Modification in the Regulation of p27Kip1 Protein Stability and Function

The tumor suppressor gene p27Kip1 plays a fundamental role in human cancer progression. Its expression and/or functions are altered in almost all the different tumor histotype analyzed so far. Recently, it has been demonstrated that the tumor suppression function of p27 resides not only in the ability to inhibit Cyclins/CDKs complexes through its N-terminal domain but also in the capacity to modulate cell motility through its C-terminal portion. Particular interest has been raised by the last amino-acid, (Threonine 198) in the regulation of both protein stability and cell motility

Somatic Mutation and Germline Sequence Abnormalities in CDKN1B, Encoding p27Kip1, in Sporadic Parathyroid Adenomas

DNA sequence abnormalities have identified the CDKN1B gene as a novel contributor, and potential susceptibility gene, in the development of typical sporadic parathyroid adenomas

Functional analysis and case-control study of -160C/A polymorphism in the E-cadherin gene promoter: association with cancer risk

Background: A C→A polymorphism within the CDH1 (E-cadherin) promoter seems to be associated with a reduced efficiency of gene transcription in vitro. Due to the crucial role of E-cadherin in epithelia, tissue-specific effect of C→A change on CDH1 transcription was tested and a case-control study was performed on patients affected with epithelial tumors. Patients and Methods: The –178/+93 CDH1 region containing either C or A nucleotide was inserted upstream of the Luciferase reporter gene in the pGL-2 vector, and the construct activity was assessed by transient transfection assay in HeLa and HCT116 cells. Results: A significantly lower activity for pGL-2A was found compared to pGL-2C, both in HeLa (54% decrease) and in HCT116 (67% decrease) cells. Genotyping of 246 controls and 505 patients affected with breast, gastric, colorectal, cervical and endometrial cancers demonstrated an association between the A allele and an increased risk of colorectal, gastric and endometrial tumors (1.66-, 1.81- and 2.35-fold, respectively). Conclusion: Our data support the notion that the A allele may act as a low-penetrance cancer susceptibility gene