Mammalian CDC14 phosphatases control exit from stemness in pluripotent cells.

Maintenance of stemness is tightly linked to cell cycle regulation through protein phosphorylation by cyclin-dependent kinases (CDKs). However, how this process is reversed during differentiation is unknown. We report here that exit from stemness and differentiation of pluripotent cells along the neural lineage are controlled by CDC14, a CDK-counteracting phosphatase whose function in mammals remains obscure. Lack of the two CDC14 family members, CDC14A and CDC14B, results in deficient development of the neural system in the mouse and impairs neural differentiation from embryonic stem cells (ESCs). Mechanistically, CDC14 directly dephosphorylates specific proline-directed Ser/Thr residues of undifferentiated embryonic transcription Factor 1 (UTF1) during the exit from stemness, triggering its proteasome-dependent degradation. Multiomic single-cell analysis of transcription and chromatin accessibility in differentiating ESCs suggests that increased UTF1 levels in the absence of CDC14 prevent the proper firing of bivalent promoters required for differentiation. CDC14 phosphatases are dispensable for mitotic exit, suggesting that CDC14 phosphatases have evolved to control stemness rather than cell cycle exit and establish the CDK-CDC14 axis as a critical molecular switch for linking cell cycle regulation and self-renewal.

Acellular human corneal matrix sheets seeded with human adipose-derived mesenchymal stem cells integrate functionally in an experimental animal model.

PURPOSE: To evaluate the in vivo biocompatibility of grafts composed of sheets of decellularized human corneal stroma with or without the recellularization of human adipose derived adult stem cells (h-ADASC) into the rabbit cornea. METHODS: Sheets of human corneal stroma of 90 µm thickness were decellularized, and their lack of cytotoxicity was assayed. The recellularization was achieved by the injection of 2 × 10(5) labeled h-ADASC in the graft followed by five days of cell culture. The grafts were implanted in vivo into a stromal pocket at 50% depth. After a triple-masked three-month follow-up, the animals were euthanized and the biointegration of the graft, the viability of the stem cells and the expression of keratocan (human keratocyte-specific protein) were assessed. RESULTS: The decellularized stromal sheets showed an intact extracellular matrix with a decellularization rate of 92.8% and an excellent recellularization capacity in vitro with h-ADASC. A complete and stable graft transparency was observed during the full follow-up, with absence of any clinical sign of rejection. The postmortem analysis demonstrated the survival of the transplanted human stem cells inside the graft and their differentiation into functional keratocytes, as assessed by the expression of human keratocan. CONCLUSIONS: We report a new model of lamellar keratoplasty that requires only a simple and safe procedure of liposuction and a donor allogeneic cornea to provide an optically transparent autologous stromal graft with excellent biocompatibility and integration into the host tissue in a rabbit model.

Circulating immune biomarkers in peripheral blood correlate with clinical outcomes in advanced breast cancer.

Identification of the different elements intervening at the tumor microenvironment seems key to explain clinical evolution in several tumor types. In this study, a set of immune biomarkers (myeloid derived suppressor cells, regulatory T cells, and OX40 + and PD-1 + T lymphocytes counts) in peripheral blood of patients diagnosed with advanced breast cancer were analyzed along of first line antineoplastic therapy. Subsequently, a comparison between groups with clinical benefit versus progression of disease and with a healthy women cohort was executed. Results reflected that patients showed higher basal levels of myeloid derived suppressor cells (35.43, IR = 180.73 vs 17.53, IR = 16.96 cells/µl; p = 0.001) and regulatory T cells (32.05, IR = 29.84 vs 22.61, IR = 13.57 cells/µl; p = 0.001) in comparison with healthy women. Furthermore, an increase in the number of activated T lymphocytes (expressing OX40), a decrease of immune inhibitory cells (MDSCs and Tregs) and inhibited T lymphocytes (expressing PD-1) were observed along the treatment in patients with clinical benefit (p ≤ 0.001). The opposite trend was observed in the case of disease progression. These findings suggest that some critical immune elements can be easily detected and measured in peripheral blood, which open a new opportunity for translational research, as they seem to be correlated with clinical evolution, at least in ABC.

An international call for a new grading system for cerebral and cerebellar cavernomas.

Surgical indications for cerebral cavernous malformations remain significantly center- and surgeon-dependent. Available grading systems are potentially limited, as they do not include epileptological and radiological data. A novel grading system is proposed for supratentorial and cerebellar cavernomas: it considers neuroradiological features (bleeding, increase in size), neurological status (focal deficits and seizures), location of the lesion and age of the patient. The score ranges from -1 to 10; furthermore, surgery should be considered when a score of 4 or higher is present. Based on neuroradiological characteristics, 0 points are assigned if the CCM is stable in size at different neuroradiological controls, 1 point if there is an increase in volume during follow-up, 2 points if intra- or extra-lesional bleeding <1 cm is present and 3 points if the CCM produced a hematoma >1 cm. Regarding focal neurological deficits, 0 points are assigned if absent and 2 points if present. For seizures, 0 points are assigned if absent, 1 point if present, but controlled by medications, and 2 points if drug resistant. We considered the site of the CCM, and in case of deep-seated lesions in a critical area (basal ganglia, thalamus) 1 point (-1) is subtracted, while for subcortical or deep cerebellar lesions 0 points are assigned, for CCMs in a cortical critical area 1 point is assigned and in case of lesions in cortical not in critical area or superficial cerebellar area, 2 points are assigned. As far as age is concerned, 0 points are assigned for patients older than 50 years and 1 point for patients younger than 50. In conclusion, a novel grading for surgical decision making in cerebral cavernomas, based on the experience of selected neurosurgeons, basic scientists, and patients, is suggested with the aim of further improving and standardizing the treatment of CCMs. The aim of this paper was also to call for both retrospective and prospective multicenter studies with the aim of testing the efficacy of the grading system in different centers.

Derived Neutrophil-to-Lymphocyte Ratio Predicts Pathological Complete Response to Neoadjuvant Chemotherapy in Breast Cancer.

BACKGROUND: Derived neutrophil-to-lymphocyte ratio (dNLR) is a biomarker associated with clinical outcome in breast cancer (BC). We analyzed the association of dNLR with pathological complete response (pCR) in triple-negative BC (TNBC) patients receiving neoadjuvant chemotherapy (CT). METHODS: This is a retrospective analysis of two randomized studies involving early stage/locally advanced TNBC patients receiving anthracycline/taxane-based CT+/-carboplatin (GEICAM/2006-03) or nab-paclitaxel/paclitaxel followed by anthracycline regimen (ETNA). dNLR was calculated as the ratio of neutrophils to the difference between total leukocytes and neutrophils in peripheral blood before CT (baseline) and at the end of treatment (EOT). Logistic regression analyses were used to explore dNLR association with pCR. RESULTS: In total, 308 TNBC patients were analyzed, 216 from ETNA and 92 from GEICAM/2006-03. Baseline median dNLR was 1.61 (interquartile range (IQR): 1.25-2.04) and at EOT 1.53 (IQR: 0.96-2.22). Baseline dNLR showed positive correlation with increased tumor size (p-value = 1e-04). High baseline dNLR, as continuous variable or using median cutoff, was associated with lower likelihood of pCR in univariate analysis. High EOT dNLR as continuous variable or using quartiles was also associated with lower pCR rate in uni- and multivariate analyses. CONCLUSIONS: High baseline and EOT dNLR correlates with lower benefit from neoadjuvant CT in TNBC.

Pembrolizumab Plus Gemcitabine in the Subset of Triple-Negative Advanced Breast Cancer Patients in the GEICAM/2015-04 (PANGEA-Breast) Study.

The PANGEA-Breast trial evaluated a new chemo-immunotherapeutic combination that would synergistically induce long-term clinical benefit in HER2-negative advanced breast cancer patients. Treatment consisted of 21-day cycles of 200 mg of pembrolizumab (day 1) plus gemcitabine (days 1 and 8). The primary objective was the objective response rate (ORR). The tumor infiltrating lymphocytes (TILs) density and PD-L1 expression in tumor, and the myeloid-derived suppressor cells (MDSCs) level in peripheral blood, were analyzed to explore associations with treatment efficacy. Considering a two-stage Simon's design, the study recruitment was stopped after its first stage as statistical assumptions were not met. A subset of 21 triple-negative breast cancer (TNBC) patients was enrolled. Their median age was 49 years; 15 patients had visceral involvement, and 16 had ≤3 metastatic locations. Treatment discontinuation due to progressive disease (PD) was reported in 16 patients. ORR was 15% (95% CI 3.2-37.9). Four patients were on treatment >6 months before PD. Grade ≥3 treatment-related adverse events were observed in 8 patients, where neutropenia was the most common. No association was found between TILs density, PD-L1 expression or MDSCs levels and treatment efficacy. ORR in TNBC patients also did not meet the assumptions, but 20% were on treatment >6 months.

Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage.

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A and contribute to delaying cell-cycle progression, thereby preventing genomic instability. Here we report that beta-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of beta-TrCP1 and beta-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that beta-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of beta-TrCP expression results in radioresistant DNA synthesis in response to DNA damage--a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that beta-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation.

Cdc25A phosphatase: combinatorial phosphorylation, ubiquitylation and proteolysis.

In eukaryotic cells, control mechanisms of cell-cycle progression have evolved to accurately monitor the integrity of genetic information to be transferred to the progeny. Cdc25A phosphatase is an essential activator of cell-cycle progression and is targeted by checkpoint signals. Ubiquitylation regulates Cdc25A activity through fine tuning of its protein levels. Two different ubiquitin ligases (APC/C and SCF complex) are involved in Cdc25A turnover. While APC/C is involved in regulating Cdc25A at the exit of mitosis, SCF regulates the abundance of Cdc25A in S phase and G2. In response to DNA damage or to stalled replication, the activation of the ATM and ATR protein kinases leads to Chk1 and Chk2 activation and to Cdc25A hyperphosphorylation. These events stimulate SCF-mediated ubiquitylation of Cdc25A and its proteolysis. This contributes to delaying cell-cycle progression, thereby preventing genomic instability. Based on recent findings, we discuss the role of Cdc25A ubiquitylation and degradation in cell-cycle progression and in response to DNA damage. Moreover, we discuss the role of phosphorylation at multiple sites in triggering ubiquitylation signals.

Hierarchical order of phosphorylation events commits Cdc25A to betaTrCP-dependent degradation.

We have recently demonstrated that regulation of Cdc25A protein abundance during S phase and in response to DNA damage is mediated by SCF(betaTrCP) activity. Based on sequence homology of known betaTrCP substrates, we found that Cdc25A contains a conserved motif (DSG), phosphorylation of which is required for interaction with betaTrCP.1 Here, we show that phosphorylation at Ser 82 within the DSG motif anchors Cdc25A to betaTrCP and that Chk1-dependent phosphorylation at Ser 76 affects this interaction as well as betaTrCP-dependent degradation. We propose that a hierarchical order of phosphorylation events commits Cdc25A to betaTrCP-dependent degradation. According to our model, phosphorylation at Ser 76 is a "priming" step required for Ser 82 phosphorylation, which in turn allows recruitment of Cdc25A by betaTrCP and subsequent betaTrCP-dependent degradation.

Biointegration of corneal macroporous membranes based on poly(ethyl acrylate) copolymers in an experimental animal model.

Currently available keratoprosthesis models (nonbiological corneal substitutes) have a less than 75% graft survival rate at 2 years. We aimed at developing a model for keratoprosthesis based on the use of poly(ethyl acrylate) (PEA)-based copolymers, extracellular matrix-protein coating and colonization with adipose-derived mesenchymal stem cells. Human adipose tissue derived mesenchymal stem cells (h-ADASC) colonization efficiency of seven PEA-based copolymers in combination with four extracellular matrix coatings were evaluated in vitro. Then, macroporous membranes composed of the optimal PEA subtypes and coating proteins were implanted inside rabbit cornea. After a 3-month follow-up, the animals were euthanized, and the clinical and histological biointegration of the implanted material were assessed. h-ADASC adhered and survived when cultured in all PEA-based macroporous membranes. The addition of high hydrophilicity to PEA membranes decreased h-ADASC colonization in vitro. PEA-based copolymer containing 10% hydroxyethyl acrylate (PEA-HEA10) or 10% acrylic acid (PEA-AAc10) monomeric units showed the best cellular colonization rates. Collagen plus keratan sulfate-coated polymers demonstrated enhanced cellular colonization respect to fibronectin, collagen, or uncoated PEAs. In vivo implantation of membranes resulted in an extrusion rate of 72% for PEA, 50% for PEA-AAc10, but remarkably of 0% for PEA-HEA10. h-ADASC survival was demonstrated in all the membranes after 3 months follow-up. A slight reduction in the extrusion rate of h-ADASC colonized materials was observed. No significant differences between the groups with and without h-ADASC were detected respect to transparency or neovascularization. We propose PEA with low hydroxylation as a scaffold for the anchoring ring of future keratoprosthesis.

The Cdc14B phosphatase displays oncogenic activity mediated by the Ras-Mek signaling pathway.

Cdc14 is a dual-specific phosphatase with relevant functions during mitotic exit in yeast. The relevance of vertebrate Cdc14 phosphatases is not well understood due to the presence of two paralogs, Cdc14A and Cdc14B, and their dispensability for cell cycle progression. Here, we report that overexpression of mammalian Cdc14B, but not Cdc14A, leads to dramatic changes in morphology and malignant transformation of normal murine fibroblasts. Cdc14B disrupts the cytoskeletal F-actin organization with loss of actin stress fibers and vinculin adhesions in a phosphatase-dependent manner. These morphological changes are associated to cellular transformation, as Cdc14B-overexpressing cells display anchorage-independent growth and are able to form tumors in vivo. These alterations are similar to those induced by Ras oncogenes ,and both Cdc14B and H-RasV12 lead to similar changes in the transcriptional profile of transformed cells. Pharmacologic inhibition of the Ras-Mek pathway rescues these defects. These data suggest that Cdc14B, but not Cdc14A, is one of the few phosphatases that display oncogenic activity in mammals and point to the Ras-MAP kinase pathway as a major effector pathway during oncogenic transformation by Cdc14B.

Cdc14b regulates mammalian RNA polymerase II and represses cell cycle transcription.

Cdc14 is an essential phosphatase in yeast but its role in the mammalian cell cycle remains obscure. We report here that Cdc14b-knockout cells display unscheduled induction of multiple cell cycle regulators resulting in early entry into DNA replication and mitosis from quiescence. Cdc14b dephosphorylates Ser5 at the C-terminal domain (CTD) of RNA polymerase II, a major substrate of cyclin-dependent kinases. Lack of Cdc14b results in increased CTD-Ser5 phosphorylation, epigenetic modifications that mark active chromatin, and transcriptional induction of cell cycle regulators. These data suggest a function for mammalian Cdc14 phosphatases in the control of transcription during the cell cycle.

Karyotype instability and anchorage-independent growth in telomerase-immortalized fibroblasts from two centenarian individuals.

Several reports have shown that the ectopic expression of the human telomerase catalytic subunit gene (hTERT) leads to an indefinite extension of the life span of human fibroblasts cultured in vitro without the appearance of cancer-associated changes. We infected two fibroblast strains derived from centenarian individuals with an hTERT containing retrovirus and isolated transduced massive populations (cen2tel and cen3tel). In both populations, hTERT expression reconstituted telomerase activity and extended the life span. In cen2tel, a net telomere lengthening was observed while, in cen3tel, telomeres stabilized at a length lower than that detected in senescent parental cells. Interestingly, both cen2tel and cen3tel cells developed chromosome anomalies, numerical first and structural thereafter. Moreover, cen3tel cells acquired the ability to grow in the absence of solid support, a typical feature of transformed cells. The results we present here highlight an unexpected possible outcome of cellular immortalization driven by telomerase reactivation, and indicate that, in some cases, an artificial extension of cellular replicative capacity can increase the probability of occurrence of genomic alterations, which can lead to cellular transformation.