A cancer-associated CDKN1B mutation induces p27 phosphorylation on a novel residue: a new mechanism for tumor suppressor loss-of-function

CDKN1B haploinsufficiency promotes the development of several human cancers. The gene encodes p27Kip1 , 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 p27Kip1 variant, namely glycine9->arginine-p27Kip1 (G9R-p27Kip1 ) identified in a parathyroid adenoma. We unexpectedly found that G9R-p27Kip1 lacks the major tumor suppressor activities of p27Kip1 including its antiproliferative and pro-apoptotic functions. In addition, G9R-p27Kip1 transfection in cell lines induces the formation of more numerous and larger spheres when compared to wild type p27Kip1 -transfected cells. We demonstrated that the mutation creates a consensus sequence for basophilic kinases causing a massive phosphorylation of G9R-p27Kip1 on S12, a residue normally never found modified in p27Kip1 . The novel S12 phosphorylation appears responsible for the loss of function of G9R-p27Kip1 since S12AG9R-p27Kip1 recovers most of the p27Kip1 tumor suppressor activities. In addition, the expression of the phosphomimetic S12D-p27Kip1 recapitulates G9R-p27Kip1 properties. Mechanistically, S12 phosphorylation enhances the nuclear localization of the mutant protein, and also reduces its CDK2/CDK1 inhibition activity. To our knowledge, this is the first reported case of quantitative phosphorylation of a p27Kip1 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

CDKN1B gene alterations and human cancers: mechanicistic investigations on G9R missense mutation

p27Kip1 (p27) was first discovered as a key regulator of cell proliferation, modulating Cyclin-dependent Kinases activity (1). Nearly two decades have elapsed since the discovery of p27 roles in the regulation of cytoskeletal dynamics and cellular plasticity, stem-cell proliferation and differentiation. This versatility has been attributed to its intrinsically unstructured nature that allows p27 to bind and modulate different proteins. Depending on the cell-specific context and its cellular compartimentalization, p27 acts both as a tumor-suppressor or tumor-promoter, and several post-synthetic modifications (mainly phosphorylations) control its commitment (1). Recently, mutations of CDKN1B (p27 encoding gene) have been found with a statistical significance in human cancers: along with frameshifts, some missense changes were associated to Multiple Endocrine Neoplasia, neuroendocrine tumours and other cancers (2). This study focuses on a germline mutation detected in sporadic parathyroid adenoma, Glycine9→Arginine (3). Glycine in position 9 is highly conserved in p27 across species and its substitution has been proposed to negatively affect the phosphorylation of adjacent Serine10 residue, the most abundant phosphorylation site of the protein. Therefore, we examined the phosphorylation pattern of this mutant protein by 2D-immunoblotting. Unexpectedly, Serine10 is still highly phosphorylated, at least as in the wild-type p27. Furthermore, a new phosphorylation site is probably generated by the missense mutation. Studies are in progress to identify the novel phosphorylation residue as well the protein kinase responsible for its modification. Functionally, despite its nuclear localization, G9Rp27 might contribute to enhance cell growth, motility and invasion; it also protects cells from apoptosis. Our studies indicate that G9Rp27 oncogenic activities depend on its phosphorylation pattern. In conclusion, this investigation confirms the importance of post-translational modifications in addressing the function of intrinsically unstructured proteins

High Dosage Lithium Treatment Induces DNA Damage and p57Kip2 Decrease

Lithium salt is the first-line therapeutic option for bipolar disorder and has been proposed as a potential antitumoral drug. The effects of LiCl treatment were investigated in SH-SY5Y, a human neuroblastoma cell line and an in vitro model of dopaminergic neuronal differentiation. LiCl, at the dosage used in psychiatric treatment, does not affect cell proliferation, while at higher doses it delays the SH-SY5Y cell division cycle and for prolonged usage reduces cell viability. Moreover, the ion treatment affects DNA integrity as demonstrated by accumulation of p53 and γH2AX (the phosphorylated form of H2AX histone), two important markers of genome damage. p57Kip2, a CIP/Kip protein, is required for proper neuronal maturation and represents a main factor of response to stress including genotoxicity. We evaluated the effect of lithium on p57Kip2 levels. Unexpectedly, we found that lithium downregulates the level of p57Kip2 in a dose-dependent manner, mainly acting at the transcriptional level. A number of different approaches, mostly based on p57Kip2 content handling, confirmed that the CKI/Kip reduction plays a key role in the DNA damage activated by lithium and suggests the unanticipated view that p57Kip2 might be involved in DNA double-strand break responses. In conclusion, our study identified novel roles for p57Kip2 in the molecular mechanism of lithium at high concentration and, more in general, in the process of DNA repair