P27kip1 controls H-Ras/MAPK activation and cell cycle entry via modulation of MT stability

The cyclin-dependent kinase (CDK) inhibitor p27(kip1) is a critical regulator of the G1/S-phase transition of the cell cycle and also regulates microtubule (MT) stability. This latter function is exerted by modulating the activity of stathmin, an MT-destabilizing protein, and by direct binding to MTs. We recently demonstrated that increased proliferation in p27(kip1)-null mice is reverted by concomitant deletion of stathmin in p27(kip1)/stathmin double-KO mice, suggesting that a CDK-independent function of p27(kip1) contributes to the control of cell proliferation. Whether the regulation of MT stability by p27(kip1) impinges on signaling pathway activation and contributes to the decision to enter the cell cycle is largely unknown. Here, we report that faster cell cycle entry of p27(kip1)-null cells was impaired by the concomitant deletion of stathmin. Using gene expression profiling coupled with bioinformatic analyses, we show that p27(kip1) and stathmin conjunctly control activation of the MAPK pathway. From a molecular point of view, we observed that p27(kip1), by controlling MT stability, impinges on H-Ras trafficking and ubiquitination levels, eventually restraining its full activation. Our study identifies a regulatory axis controlling the G1/S-phase transition, relying on the regulation of MT stability by p27(kip1) and finely controlling the spatiotemporal activation of the Ras-MAPK signaling pathway

SRSF2 mutations in epithelial ovarian cancer

Resistance to platinum chemotherapy regimens represents a major obstacle in the successful treatment of epithelial ovarian cancer (EOC) patients. Among the molecular mechanism responsible for resistance to platinum, alternative splicing, which is induced upon platinum treatment, can control apoptosis by regulating the expression of apoptotic protein variants with opposite functions. Alterations in alternative splicing are found in tumors and can hinder apoptotic response. In the present study we sequenced SRSF2, a splicing factor that regulates Caspase-8 and Caspase-9 variants, in search of mutations that could possibly explain alternative mechanisms of platinum resistant in EOC

Downregulation of miR-223 Expression Is an Early Event during Mammary Transformation and Confers Resistance to CDK4/6 Inhibitors in Luminal Breast Cancer

miR-223 is an anti-inflammatory miRNA that in cancer acts either as an oncosuppressor or oncopromoter, in a context-dependent manner. In breast cancer, we demonstrated that it dampens the activation of the EGF pathway. However, little is known on the role of miR-223 during breast cancer onset and progression. miR-223 expression was decreased in breast cancer of luminal and HER2 subtypes and inversely correlated with patients' prognosis. In normal luminal mammary epithelial cells, miR-223 acted cell autonomously in the control of their growth and morphology in three-dimensional context. In the MMTV-Δ16HER2 transgenic mouse model, oncogene transformation resulted in a timely abrogation of miR-223 expression, likely due to activation of E2F1, a known repressor of miR-223 transcription. Accordingly, treatment with CDK4/6 inhibitors, which eventually results in restraining E2F1 activity, restored miR-223 expression and miR-223 ablation induced luminal breast cancer resistance to CDK4/6 inhibition, both in vitro and in vivo. Notably, miR-223 expression was lost in microdissected ductal carcinoma in situ (DCIS) from patients with luminal and HER2-positive breast cancer. Altogether, these results identify downmodulation of miR-223 as an early step in luminal breast cancer onset and suggest that it could be used to identify aggressive DCIS and predict the response to targeted therapy. SIGNIFICANCE: miR-223 may represent a predictive biomarker of response to CDK4/6 inhibitors and its loss could identify DCIS lesions that are likely to progress into invasive breast cancer

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

p27Kip1-stathmin interaction influences sarcoma cell migration and invasion

Emerging evidences suggest that cyclin-dependent kinase inhibitors (CKIs) can regulate cellular functions other than cell cycle progression, such as differentiation and migration. Here, we report that cytoplasmic expression of p27kip1 affects microtubule (MT) stability following cell adhesion on extracellular matrix (ECM) constituents. This p27kip1 activity is due to its ability to bind and impair the function of the MT-destabilizing protein stathmin. Accordingly, upregulation of p27kip1 or downregulation of stathmin expression results in the inhibition of mesenchymal cell motility. Moreover, high stathmin and low cytoplasmic p27kip1 expression correlate with the metastatic phenotype of human sarcomas in vivo. This study provides a functional link between proliferation and invasion of tumor cells based on diverse activities of p27kip1 in different subcellular compartments

Proteomic and Postproteomic Characterization of Keratan Sulfate-Glycanated Isoforms of Thyroglobulin and Transferrin Uniquely Elaborated by Papillary Thyroid Carcinomas.

Previous studies have suggested that surface components of papillary thyroid carcinoma (PTC) cells may be aberrantly glycanated, but the precise nature of these molecules has not been unveiled nor documented to be of clinical relevance. A monoclonal antibody was raised against a unique keratan sulfate (KS) determinant and used to differentially screen benign and malignant thyroid tissue for the expression of components carrying these moieties. In a total of 349 cases of benign and malignant thyroid lesions, 100% of the 115 PTC cases examined (including various histological subtypes) were found to contain KS-bearing molecules, whereas these were virtually absent from benign tissues and other thyroid tumors, with the exception of 21% of the follicular carcinoma cases analyzed. A composite immunoaffinity chromatography, immunochemistry, and mass spectrometric approach revealed that the PTC-specific KS-bearing macromolecules were unique glycoforms of thyroglobulin and transferrin. Combined, reciprocal immunoprecipitation and Western blotting further indicated that the former glycoform predominated and that most of the transferrin produced by PTC was glycanated with KS moieties. Fluorescent keratanase II-based fingerprinting of the KS moieties bound to these isoforms further demonstrated several PTC-specific peculiarities: 1) that a considerable portion of the moieties was covalently attached via a novel core protein linkage structure; 2) they had an unusual extended average length; 3) an unusual relative ratio of highly sulfated disaccharides terminating with (2-3)-linked N-acetylneuraminic acid capping residues; and 4) a novel unidentified oligosaccharide moiety at the nonreducing terminus. Comparative analysis of the relative distribution of transferrin in benign versus PTC tissues highlighted a marked malignancy-associated abundance of the molecule, with a >75% frequency in expression in PTC. These findings demonstrate that PTC cells synthesize unique post-translationally modified thyroglobulin and transferrin variants in situ that may be directly exploitable for diagnosis, through histological and noninvasive cytological procedures; for devising novel strategies for antibody-guided imaging of this tumor in vivo; and for postsurgery follow-up of PTC patients

Loss of p27_kip1 increases genomic instability and induces radio-resistance in luminal breast cancer cells

AbstractGenomic instability represents a typical feature of aggressive cancers. Normal cells have evolved intricate responses to preserve genomic integrity in response to stress, such as DNA damage induced by γ-irradiation. Cyclin-dependent kinases (CDKs) take crucial part to these safeguard mechanisms, but involvement of CDK-inhibitors, such as p27Kip1, is less clear. We generated immortalized fibroblasts from p27kip1 knock-out (KO) mouse embryos and re-expressed p27kip1 WT, or its mutant forms, to identify the function of different domains. We γ-irradiated fibroblasts and observed that loss of p27Kip1 was associated to accumulation of residual DNA damage, increased number of mitotic aberration and, eventually, to survival advantage. Nuclear localization and cyclin/CDK-binding of p27Kip1 were critical to mediate proper response to DNA damage. In human luminal breast cancer (LBC) p27kip1 is frequently down-modulated and CDKN1B, p27Kip1 gene, sporadically mutated. We recapitulated results obtained in mouse fibroblasts in a LBC cell line genetically manipulated to be KO for CDKN1B gene. Following γ-irradiation, we confirmed that p27kip1 expression was necessary to preserve genomic integrity and to recognize and clear-out aberrant cells. Our study provides important insights into mechanisms underlying radio-resistance and unveils the possibility for novel treatment options exploiting DNA repair defects in LBC.</jats:p

Stathmin Activity Influences Sarcoma Cell Shape, Motility, and Metastatic Potential

The balanced activity of microtubule-stabilizing and -destabilizing proteins determines the extent of microtubule dynamics, which is implicated in many cellular processes, including adhesion, migration, and morphology. Among the destabilizing proteins, stathmin is overexpressed in different human malignancies and has been recently linked to the regulation of cell motility. The observation that stathmin was overexpressed in human recurrent and metastatic sarcomas prompted us to investigate stathmin contribution to tumor local invasiveness and distant dissemination. We found that stathmin stimulated cell motility in and through the extracellular matrix (ECM) in vitro and increased the metastatic potential of sarcoma cells in vivo. On contact with the ECM, stathmin was negatively regulated by phosphorylation. Accordingly, a less phosphorylable stathmin point mutant impaired ECM-induced microtubule stabilization and conferred a higher invasive potential, inducing a rounded cell shape coupled with amoeboid-like motility in three-dimensional matrices. Our results indicate that stathmin plays a significant role in tumor metastasis formation, a finding that could lead to exploitation of stathmin as a target of new antimetastatic drugs