Debora Bencivenga

P27^Kip1 is a key cell cycle gatekeeper governing the timing of Cyclin-dependent kinase (CDK) activation/inactivation and, consequently, cell proliferation. Structurally, the protein is largely unfolded, a feature that strongly increases its plasticity and interactors and enhances the number of regulated cellular processes. p27^Kip1, like other intrinsically unstructured proteins, is post-translationally modified on several residues. These modifications affect its cellular localization and address p27^Kip1 for specific interactions/functions. Several germline or somatic CDKN1B (the p27^Kip1 encoding gene) mutations have been demonstrated to be associated with multiple endocrine neoplasia type 4 (MEN4), hairy cell leukemia, small-intestine neuroendocrine tumors, and breast and prostate cancers. Here, we analyzed the effect of four CDKN1B missense and nonsense mutations found in patients affected by MEN4 or cancers, namely, c.349C>T, p.P117S; c.397C>A, p.P133T; c.487C>T, p.Q163*; and c.511G>T, p.E171*. By transfecting breast cancer cell lines, we observed increased growth and cell motility for all the investigated mutants compared to wild-type p27^Kip1 transfected cells. Furthermore, we discovered that the mutant forms exhibited altered phosphorylation on key residues and different localization or degradation mechanisms in comparison to the wild-type protein and suggested a possible region as crucial for the lysosome-dependent degradation of the protein. Finally, the loss of p27^Kip1 ability in blocking cell proliferation was in part explained through the different binding efficiency that mutant p27^Kip1 forms exhibited with Cyclin/Cyclin-dependent Kinase complexes (or proteins involved indirectly in that binding) with respect to the WT.

P57^Kip2 is a member of the cyclin-dependent kinase (CDK) Interacting Protein/Kinase Inhibitory Protein (CIP/Kip) family that also includes p21^Cip1/WAF1 and p27^Kip1. Different from its siblings, few data are available about the p57^Kip2 protein, especially in humans. Structurally, p57^Kip2 is an intrinsically unstructured protein, a characteristic that confers functional flexibility with multiple transient interactions influencing the metabolism and roles of the protein. Being an IUP, its localization, stability, and binding to functional partners might be strongly modulated by post-translational modifications, especially phosphorylation. In this work, we investigated by two-dimensional analysis the phosphorylation pattern of p57^Kip2 in different cellular models, revealing how the human protein appears to be extensively phosphorylated, compared to p21^Cip1/WAF1 and p27^Kip1. We further observed clear differences in the phosphoisoforms distributed in the cytosolic and nuclear compartments in asynchronous and synchronized cells. Particularly, the unmodified form is detectable only in the nucleus, while the more acidic forms are present in the cytoplasm. Most importantly, we found that the phosphorylation state of p57^Kip2 influences the binding with some p57^Kip2 partners, such as CDKs, LIMK1 and CRM1. Thus, it is necessary to completely identify the phosphorylated residues of the protein to fully unravel the roles of this CIP/Kip protein, which are still partially identified.

Multiple endocrine neoplasia (MEN) syndromes are part of a spectrum of clinically well-defined tumor syndromes ultimately characterized by histologically similar tumors arising in patients and families with mutations in one of the following four genes: MEN1, RET, CDKN1B, and MAX. The high level of genetic and phenotypic heterogeneity has been linked to phenocopies and modifying genes, as well as unknown mechanisms that might be investigated in the future based on preclinical and translational considerations. MEN1, also known as Wermer’s syndrome (OMIM *131100), is an autosomal dominant syndrome codifying for the most frequent MEN syndrome showing high penetrance due to mutations in the MEN1 gene; nevertheless, clinical manifestations vary among patients in terms of tumor localization, age of onset, and clinical aggressiveness/severity, even within the same families. This has been linked to the effect of modifying genes, as described in the review. MEN 2-2b-4 and 5 also show remarkable clinical heterogeneity. The traditional view of genetically predisposing monogenic or multifactorial disorders is no longer valid, and mandates a change in scientific focus. Phenotypes are indeed rarely consistent across genetic backgrounds and environments. In the future, understanding factors and genetic variants that control cellular functions and the expression of disease genes should provide insights into fundamental disease processes, providing implications for counseling and therapeutic and prophylactic possibilities.

This PRISMA-ScR driven scoping review aims to evaluate the influence of magnetic field stimulation on dental implant osseointegration. Seven databases were screened adopting ad-hoc strings. All clinical and preclinical studies analyzing the effects of magnetic fields on dental implant osseointegration were included. From 3124 initial items, on the basis of the eligibility criteria, 33 articles, regarding both Pulsed ElectroMagnetic Fields (PEMF) and Static magnetic Fields from permanent Magnets (SFM) were finally included and critically analyzed. In vitro studies showed a positive effect of PEMF, but contrasting effects of SFM on bone cell proliferation, whereas cell adhesion and osteogenic differentiation were induced by both types of stimulation. In vivo studies showed an increased bone-to-implant contact rate in different animal models and clinical studies revealed positive effects on implant stability, under magnetic stimulation. In conclusion, although positive effects of magnetic exposure on osteogenesis activity and osseointegration emerged, this scoping review highlighted the need for further preclinical and clinical studies. More standardized designs, accurate choice of stimulation parameters, adequate methods of evaluation of the outcomes, greater sample size and longer follow-ups are needed to clearly assess the effect of magnetic fields on dental implant osseointegration.

It is now definitively established that a large part of the human genome is transcribed. However, only a scarce percentage of the transcriptome (about 1.2%) consists of RNAs that are translated into proteins, while the large majority of transcripts include a variety of RNA families with different dimensions and functions. Within this heterogeneous RNA world, a significant fraction consists of sequences with a length of more than 200 bases that form the so-called long non-coding RNA family. The functions of long non-coding RNAs range from the regulation of gene transcription to the changes in DNA topology and nucleosome modification and structural organization, to paraspeckle formation and cellular organelles maturation. This review is focused on the role of long non-coding RNAs as regulators of cyclin-dependent kinase inhibitors’ (CDKIs) levels and activities. Cyclin-dependent kinases are enzymes necessary for the tuned progression of the cell division cycle. The control of their activity takes place at various levels. Among these, interaction with CDKIs is a vital mechanism. Through CDKI modulation, long non-coding RNAs implement control over cellular physiology and are associated with numerous pathologies. However, although there are robust data in the literature, the role of long non-coding RNAs in the modulation of CDKIs appears to still be underestimated, as well as their importance in cell proliferation control.

The Cyclin-dependent kinase (CDK) regulator p27^Kip1 is a gatekeeper of G1/S transition. It also regulates G2/M progression and cytokinesis completion, via CDK-dependent or -independent mechanisms. Recently, other important p27^Kip1 functions have been described, including the regulation of cell motility and migration, the control of cell differentiation program and the activation of apoptosis/autophagy. Several factors modulate p27^Kip1 activities, including its level, cellular localization and post-translational modifications. As a matter of fact, the protein is phosphorylated, ubiquitinated, SUMOylated, O-linked N-acetylglicosylated and acetylated on different residues. p27^Kip1 belongs to the family of the intrinsically unstructured proteins and thus it is endowed with a large flexibility and numerous interactors, only partially identified. In this review, we look at p27^Kip1 properties and ascribe part of its heterogeneous functions to the ability to act as an anchor or scaffold capable to participate in the construction of different platforms for modulating cell response to extracellular signals and allowing adaptation to environmental changes.

P57^Kip2 protein is a member of the CIP/Kip family, mainly localized in the nucleus where it exerts its Cyclin/CDKs inhibitory function. In addition, the protein plays key roles in embryogenesis, differentiation, and carcinogenesis depending on its cellular localization and interactors. Mutations of CDKN1C, the gene encoding human p57^Kip2, result in the development of different genetic diseases, including Beckwith–Wiedemann, IMAGe and Silver–Russell syndromes. We investigated a specific Beckwith–Wiedemann associated CDKN1C change (c.946 C>T) that results in the substitution of the C-terminal amino acid (arginine 316) with a tryptophan (R316W-p57^Kip2). We found a clear redistribution of R316W-p57^Kip2, in that while the wild-type p57^Kip2 mostly occurs in the nucleus, the mutant form is also distributed in the cytoplasm. Transfection of two expression constructs encoding the p57^Kip2 N- and C-terminal domain, respectively, allows the mapping of the nuclear localization signal(s) (NLSs) between residues 220–316. Moreover, by removing the basic RKRLR sequence at the protein C-terminus (from 312 to 316 residue), p57^Kip2 was confined in the cytosol, implying that this sequence is absolutely required for nuclear entry. In conclusion, we identified an unreported p57^Kip2 NLS and suggest that its absence or mutation might be of relevance in CDKN1C-associated human diseases determining significant changes of p57^Kip2 localization/regulatory roles.

CDKN1B haploinsufficiency promotes the development of several human cancers. The gene encodes p27^Kip1, a protein playing pivotal roles in the control of growth, differentiation, cytoskeleton dynamics, and cytokinesis. CDKN1B haploinsufficiency has been associated with chromosomal or gene aberrations. However, very few data exist on the mechanisms by which CDKN1B missense mutations facilitate carcinogenesis. Here, we report a functional study on a cancer‐associated germinal p27^Kip1 variant, namely glycine9‐>arginine‐p27^Kip1 (G9R‐p27^Kip1) identified in a parathyroid adenoma. We unexpectedly found that G9R‐p27^Kip1 lacks the major tumor suppressor activities of p27^Kip1 including its antiproliferative and pro‐apoptotic functions. In addition, G9R‐p27^Kip1 transfection in cell lines induces the formation of more numerous and larger spheres when compared to wild‐type p27^Kip1‐transfected cells. We demonstrated that the mutation creates a consensus sequence for basophilic kinases causing a massive phosphorylation of G9R‐p27^Kip1 on S12, a residue normally never found modified in p27^Kip1. The novel S12 phosphorylation appears responsible for the loss of function of G9R‐p27^Kip1 since S12AG9R‐p27^Kip1 recovers most of the p27^Kip1 tumor suppressor activities. In addition, the expression of the phosphomimetic S12D‐p27^Kip1 recapitulates G9R‐p27^Kip1 properties. Mechanistically, S12 phosphorylation enhances the nuclear localization of the mutant protein and also reduces its cyclin‐dependent kinase (CDK)2/CDK1 inhibition activity. To our knowledge, this is the first reported case of quantitative phosphorylation of a p27^Kip1 variant on a physiologically unmodified residue associated with the loss of several tumor suppressor activities. In addition, our findings demonstrate that haploinsufficiency might be due to unpredictable post‐translational modifications due to generation of novel consensus sequences by cancer‐associated missense mutations.