Notch Signaling Activation as a Hallmark for Triple-Negative Breast Cancer Subtype.

Triple-negative breast cancer (TNBC) is a subgroup of 15%-20% of diagnosed breast cancer patients. It is generally considered to be the most difficult breast cancer subtype to deal with, due to the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), which usually direct targeted therapies. In this scenario, the current treatments of TNBC-affected patients rely on tumor excision and conventional chemotherapy. As a result, the prognosis is overall poor. Thus, the identification and characterization of targets for novel therapies are urgently required. The Notch signaling pathway has emerged to act in the pathogenesis and tumor progression of TNBCs. Firstly, Notch receptors are associated with the regulation of tumor-initiating cells (TICs) behavior, as well as with the aetiology of TNBCs. Secondly, there is a strong evidence that Notch pathway is a relevant player in mammary cancer stem cells maintenance and expansion. Finally, Notch receptors expression and activation strongly correlate with the aggressive clinicopathological and biological phenotypes of breast cancer (e.g., invasiveness and chemoresistance), which are relevant characteristics of TNBC subtype. The purpose of this up-to-date review is to provide a detailed overview of the specific role of all four Notch receptors (Notch1, Notch2, Notch3, and Notch4) in TNBCs, thus identifying the Notch signaling pathway deregulation/activation as a pathognomonic feature of this breast cancer subtype. Furthermore, this review will also discuss recent information associated with different therapeutic options related to the four Notch receptors, which may be useful to evaluate prognostic or predictive indicators as well as to develop new therapies aimed at improving the clinical outcome of TNBC patients.

Deregulated miRNAs in bone health: Epigenetic roles in osteoporosis.

MicroRNA (miRNA) has shown to enhance or inhibit cell proliferation, differentiation and activity of different cell types in bone tissue. The discovery of miRNA actions and their targets has helped to identify them as novel regulations actors in bone. Various studies have shown that miRNA deregulation mediates the progression of bone-related pathologies, such as osteoporosis. The present review intends to give an exhaustive overview of miRNAs with experimentally validated targets involved in bone homeostasis and highlight their possible role in osteoporosis development. Moreover, the review analyzes miRNAs identified in clinical trials and involved in osteoporosis.

Engineered exosomes: A new promise for the management of musculoskeletal diseases.

BACKGROUND: Exosomes are nanovesicles actively secreted by potentially all cell types, including tumour cells, with the primary role of extracellular systemic communication mediators, both at autocrine and paracrine levels, at short and long distances. Recently, different studies have used exosomes as a delivery system for a plethora of different molecules, such as drugs, microRNAs and proteins. This has been made possible thanks to the simplicity in exosomes engineering, their great stability and versatility for applications in oncology as well as in regenerative medicine. SCOPE OF REVIEW: The aim of this review is to provide information on the state-of-the-art and possible applications of engineered exosomes, both for cargo and specific cell-targeting, in different pathologies related to the musculoskeletal system. MAJOR CONCLUSIONS: The use of exosomes as therapeutic agents is rapidly evolving, different studies explore drug delivery with exosomes using different molecules, showing an enormous potential in various research fields such as oncology and regenerative medicine. GENERAL SIGNIFICANCE: However, despite the significant progress made by the different studies carried out, currently, the use of exosomes is not a therapeutic reality for the considerable difficulties to overcome.

In Silico Shear and Intramural Stresses are Linked to Aortic Valve Morphology in Dilated Ascending Aorta.

OBJECTIVE/BACKGROUND: The development of ascending aortic dilatation in patients with bicuspid aortic valve (BAV) is highly variable, and this makes surgical decision strategies particularly challenging. The purpose of this study was to identify new predictors, other than the well established aortic size, that may help to stratify the risk of aortic dilatation in BAV patients. METHODS: Using fluid-structure interaction analysis, both haemodynamic and structural parameters exerted on the ascending aortic wall of patients with either BAV (n = 21) or tricuspid aortic valve (TAV; n = 13) with comparable age and aortic diameter (42.7 ± 5.3 mm for BAV and 45.4 ± 10.0 mm for TAV) were compared. BAV phenotypes were stratified according to the leaflet fusion pattern and aortic shape. RESULTS: Systolic wall shear stress (WSS) of BAV patients was higher than TAV patients at the sinotubular junction (6.8 ± 3.3 N/m2 for BAV and 3.9 ± 1.3 N/m2 for TAV; p = .006) and mid-ascending aorta (9.8 ± 3.3 N/m2 for BAV and 7.1 ± 2.3 N/m2 for TAV; p = .040). A statistically significant difference in BAV versus TAV was also observed for the intramural stress along the ascending aorta (e.g., 2.54 × 105 ± 0.32 × 105 N/m2 for BAV and 2.04 × 105 ± 0.34 × 105 N/m2 for TAV; p < .001) and pressure index (0.329 ± 0.107 for BAV and 0.223 ± 0.139 for TAV; p = .030). Differences in the BAV phenotypes (i.e., BAV type 1 vs. BAV type 2) and aortopathy (i.e., isolated tubular vs. aortic root dilatations) were associated with asymmetric WSS distributions in the right anterior aortic wall and right posterior aortic wall, respectively. CONCLUSION: These findings suggest that valve mediated haemodynamic and structural parameters may be used to identify which regions of aortic wall are at greater stress and enable the development of a personalised approach for the diagnosis and management of aortic dilatation beyond traditional guidelines.

Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression.

Deregulated Notch signaling is associated with T-cell Acute Lymphoblastic Leukemia (T-ALL) development and progression. Increasing evidence reveals that Notch pathway has an important role in the invasion ability of tumor cells, including leukemia, although the underlying molecular mechanisms remain mostly unclear. Here, we show that Notch3 is a novel target protein of the prolyl-isomerase Pin1, which is able to regulate Notch3 protein processing and to stabilize the cleaved product, leading to the increased expression of the intracellular domain (N3IC), finally enhancing Notch3-dependent invasiveness properties. We demonstrate that the combined inhibition of Notch3 and Pin1 in the Notch3-overexpressing human leukemic TALL-1 cells reduces their high invasive potential, by decreasing the expression of the matrix metalloprotease MMP9. Consistently, Pin1 depletion in a mouse model of Notch3-induced T-ALL, by reducing N3IC expression and signaling, impairs the expansion/invasiveness of CD4(+)CD8(+) DP cells in peripheral lymphoid and non-lymphoid organs. Notably, in in silico gene expression analysis of human T-ALL samples we observed a significant correlation between Pin1 and Notch3 expression levels, which may further suggest a key role of the newly identified Notch3-Pin1 axis in T-ALL aggressiveness and progression. Thus, combined suppression of Pin1 and Notch3 proteins may be exploited as an additional target therapy for T-ALL.

Notch and NF-kB signaling pathways regulate miR-223/FBXW7 axis in T-cell acute lymphoblastic leukemia.

Notch signaling deregulation is linked to the onset of several tumors including T-cell acute lymphoblastic leukemia (T-ALL). Deregulated microRNA (miRNA) expression is also associated with several cancers, including leukemias. However, the transcriptional regulators of miRNAs, as well as the relationships between Notch signaling and miRNA deregulation, are poorly understood. To identify miRNAs regulated by Notch pathway, we performed microarray-based miRNA profiling of several Notch-expressing T-ALL models. Among seven miRNAs, consistently regulated by overexpressing or silencing Notch3, we focused our attention on miR-223, whose putative promoter analysis revealed a conserved RBPjk binding site, which was nested to an NF-kB consensus. Luciferase and chromatin immunoprecipitation assays on the promoter region of miR-223 show that both Notch and NF-kB are novel coregulatory signals of miR-223 expression, being able to activate cooperatively the transcriptional activity of miR-223 promoter. Notably, the Notch-mediated activation of miR-223 represses the tumor suppressor FBXW7 in T-ALL cell lines. Moreover, we observed the inverse correlation of miR-223 and FBXW7 expression in a panel of T-ALL patient-derived xenografts. Finally, we show that miR-223 inhibition prevents T-ALL resistance to γ-secretase inhibitor (GSI) treatment, suggesting that miR-223 could be involved in GSI sensitivity and its inhibition may be exploited in target therapy protocols.

The molecular basis of notch signaling regulation: a complex simplicity.

The Notch receptors have attracted considerable attention for their ability to control cellular functions that regulate embryo development and tissue homeostasis. Notch receptors act by controlling the expression of a specific set of target genes. If Notch signaling system can be so simple, and yet so complex in its pleiotropic effects, then a sophisticated network of regulatory mechanisms is required to maintain the control over the initiation, activity and termination of this signaling pathway. A multitude of regulatory mechanisms has been discovered that controls the interaction of Notch receptors with their ligands, the assembling of a Notch transcriptional activation complex and the termination of Notch signals. The intracellular and extracellular domains of the Notch receptors are synthesized as single proteins, pairing with each other during their trafficking through the exocytotic route. The mechanisms operating in the phase preceding the generation of the heterodimeric signal-competent Notch receptors can be as elaborate and physiologically important as those operating downstream of Notch receptor activation. These regulatory mechanisms, which are essential to understand the role of Notch signaling in human physiology and pathology are reviewed here.

PCAF ubiquitin ligase activity inhibits Hedgehog/Gli1 signaling in p53-dependent response to genotoxic stress.

The Hedgehog (Hh) signaling regulates tissue development, and its aberrant activation is a leading cause of malignancies, including medulloblastoma (Mb). Hh-dependent tumorigenesis often occurs in synergy with other mechanisms, such as loss of p53, the master regulator of the DNA damage response. To date, little is known about mechanisms connecting DNA-damaging events to morphogen-dependent processes. Here, we show that genotoxic stress triggers a cascade of signals, culminating with inhibition of the activity of Gli1, the final transcriptional effector of Hh signaling. This inhibition is dependent on the p53-mediated elevation of the acetyltransferase p300/CBP-associated factor (PCAF). Notably, we identify PCAF as a novel E3 ubiquitin ligase of Gli1. Indeed PCAF, but not a mutant with a deletion of its ubiquitination domain, represses Hh signaling in response to DNA damage by promoting Gli1 ubiquitination and its proteasome-dependent degradation. Restoring Gli1 levels rescues the growth arrest and apoptosis effect triggered by genotoxic drugs. Consistently, DNA-damaging agents fail to inhibit Gli1 activity in the absence of either p53 or PCAF. Finally, Mb samples from p53-null mice display low levels of PCAF and upregulation of Gli1 in vivo, suggesting PCAF as potential therapeutic target in Hh-dependent tumors. Together, our data define a mechanism of inactivation of a morphogenic signaling in response to genotoxic stress and unveil a p53/PCAF/Gli1 circuitry centered on PCAF that limits Gli1-enhanced mitogenic and prosurvival response.

Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia.

Notch3 and pTalpha signaling events are essential for T-cell leukemogenesis and characterize murine and human T-cell acute lymphoblastic leukemia. Genetic ablation of pTalpha expression in Notch3 transgenic mice abrogates tumor development, indicating that pTalpha signaling is crucial to the Notch3-mediated leukemogenesis. Here we report a novel direct interaction between Notch3 and pTalpha. This interaction leads to the recruitment and persistence of the E3 ligase protein c-Cbl to the lipid rafts in Notch3-IC transgenic thymocytes. Conversely, deletion of pTalpha in Notch3 transgenic mice leads to cytoplasmic retention of c-Cbl that targets Notch3 protein to the proteasomal-degradative pathway. It appears that protein kinase C theta (PKCtheta), by regulating tyrosine and serine phosphorylation of Cbl, is able to control its function. We report here that the increased Notch3-IC degradation correlates with higher levels of c-Cbl tyrosine phosphorylation in Notch3-IC/pTalpha(-/-) double-mutant thymocytes, which also display a decreased PKCtheta activity. Our data indicate that pTalpha/pre-T-cell receptor is able to regulate the different subcellular localization of c-Cbl and, by regulating PKCtheta activity, is also able to influence its ubiquitin ligase activity upon Notch3 protein.

'Hypersynchronisation' by tissue velocity imaging in patients with cardiac amyloidosis.

OBJECTIVE: It is unknown if some patients with cardiac amyloidosis (CA) have mechanical dyssynchrony, as has been demonstrated in patients with ischaemic and dilated cardiomyopathies. The aim of this study was to assess mechanical dyssynchrony in patients with CA using tissue velocity imaging (TVI) and to define its usefulness for risk stratification. DESIGN AND PATIENTS: We included 121 patients with primary amyloidosis and 37 age-matched and sex-matched controls. Patients were divided into two groups: 60 with advanced-CA and 61 with no-advanced-CA, according to left ventricular (LV) wall thickness and diastolic dysfunction. Dyssynchrony assessment included: (1) atrioventricular dyssynchrony (dys), (2) interventricular dys, (3) intraventricular dys assessed longitudinally, using the standard deviation of time to systolic peak velocity (Ts-SD) of the 12 basal and mid level LV segments, and (4) intraventricular dys assessed radially, using the difference in radial Ts between mid anteroseptal and mid posterior segments. OUTCOME: Primary end-point was all-cause death. During a median follow-up of 13 months there were 35 events among patients. RESULTS: Contrary to the hypothesis, the intraventricular dys indices in advanced-CA patients were reduced compared to either the no-advanced-CA group or to controls (Ts-SD: 12.1 (9.0); 35.1 (18.6); 24.5 (14.1), respectively, p<0.001). This reduction was primarily the result of decreased ejection time (ET). Moreover, ET was the most significant predictor of survival (HR = 0.98, p<0.001). CONCLUSIONS: The regional timing of systolic motion measured by TVI was abnormally synchronised in the patients with advanced-CA. ET reduction plays a prominent part in this process and should be considered an essential parameter for assessment of patients with cardiac amyloidosis.

Notch3: from subtle structural differences to functional diversity.

The Notch3 gene was identified, at the beginning of 90s, as the third mammalian Notch and was initially reported as being expressed in proliferating neuroepithelium. Since then, increasing evidence has demonstrated a number of structural and functional differences between Notch3 and both Notch1 and Notch2, which exhibit the highest structural similarity among the four mammalian Notch receptors. Possibly due to its more restricted tissue distribution, targeted deletion of murine Notch3 does not lead to embryonic lethality as is observed with targeted deletion of Notch1 and Notch2. However, genetic mutation, amplification and deregulated expression of Notch3 have been correlated with the disruption of cell differentiation in transgenic mice and to development of diseases in mice and humans. This review discusses the possible relationships between the structural differences and the nonredundant roles that Notch3 plays in the pathogenesis of the human disease cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy and in the regulation of murine T-cell differentiation and leukemogenesis.

Lack of amplification phenomena of ribosomal RNA genes in sea urchin.

Careful quantitative analyses by spot hybridization to homologous probes demonstrate that no rDNA amplification occurs during Paracentrotus lividus oogenesis. The same approach was used to measure the copy number of the genes involved in ribosome biogenesis. Surprisingly, differently from the organisms in which the lack of rDNA amplification phenomena was observed, a very low number of constitutive rDNA repeats was found in this organism.

Constitutive activation of NF-kappaB and T-cell leukemia/lymphoma in Notch3 transgenic mice.

The multiplicity of Notch receptors raises the question of the contribution of specific isoforms to T-cell development. Notch3 is expressed in CD4(-)8(-) thymocytes and is down-regulated across the CD4(-)8(-) to CD4(+)8(+) transition, controlled by pre-T-cell receptor signaling. To determine the effects of Notch3 on thymocyte development, transgenic mice were generated, expressing lck promoter-driven intracellular Notch3. Thymuses of young transgenics showed an increased number of thymocytes, particularly late CD4(-)8(-) cells, a failure to down-regulate CD25 in post-CD4(-)8(-) subsets and sustained activity of NF-kappaB. Subsequently, aggressive multicentric T-cell lymphomas developed with high penetrance. Tumors sustained characteristics of immature thymocytes, including expression of CD25, pTalpha and activated NF-kappaB via IKKalpha-dependent degradation of IkappaBalpha and enhancement of NF-kappaB-dependent anti-apoptotic and proliferative pathways. Together, these data identify activated Notch3 as a link between signals leading to NF-kappaB activation and T-cell tumorigenesis. The phenotypes of pre-malignant thymocytes and of lymphomas indicate a novel and particular role for Notch3 in co-ordinating growth and differentiation of thymocytes, across the pre-T/T cell transition, consistent with the normal expression pattern of Notch3.

The thymus at the crossroad of neuroimmune interactions.

The numerous relationships existing between the nervous and the immune systems suggest that the neural networks present in the intrathymic microenvironment may influence T-cell development. We previously reported that thymic neural-crest-derived stromal cells are involved in a neural differentiation pathway and are able to produce neurotrophic factors and neurokines that are in turn able to increase and/or modulate thymic-stromal cell neuronal phenotype. We also showed that EGF promotes a neural phenotype in thymic epithelial cells by enhancing the expression of neuronal-specific markers, neurotransmitters, and neuropoietic cytokines, such as IL-6 and CNTF. More recently we showed that the effect of EGF in directing thymic epithelial cells toward a neural-oriented cell fate is mediated by modulating the expression of genes directly involved in neurotypic differentiation (i.e., thrombospondin-1). EGF-induced regulation of stromal cells may also affect T-cell differentiation, as we observed that an EGF-pretreatment reduces the ability of thymic epithelial cells to sustain thymocyte differentiation in vitro. Finally, we demonstrated that a complex network involving the neurotrophin BDNF and its specific receptors may have a role in sustaining thymocyte precursor survival and supporting the thymocyte differentiation process. All together, our results suggest that the thymus may be the site of integration of different neuroimmune networks that are potentially involved in the regulation of thymocyte survival and/or differentiation.

Modulation of gene expression following long-term synaptic depression in the striatum.

A number of behavioural and cellular studies have suggested that activity-dependent synaptic plasticity associated with learning and memory may lead to the expression of various genes whose protein products can play a critical role in memory acquisition and consolidation. Long-term potentiation (LTP) and long-term depression (LTD) represent two forms of synaptic plasticity which have been widely studied by electrophysiological techniques. However, the molecular mechanisms at target gene involved in the generation of long term depression remain to be determined. To elucidate the molecular mechanism underlying activity dependent synaptic remodeling in striatal long term depression, we used the mRNA differential display technology to isolate genes that are induced or modulated by high frequency stimulation of the corticostriatal pathway in a rat brain slice preparation. We have differentially displayed, by means of reverse transcriptase-polymerase chain reaction, mRNA species isolated from striatal slices in which long term depression was induced by tetanic stimuli as well as from slices stimulated at low frequency. We then compared radio-labeled RT-PCR banding patterns to isolate cDNAs that are differentially expressed. Three independent cDNAs were isolated and identified whose mRNA level were enhanced by tetanic stimulation inducing long term depression. We provide evidence that two of these genes encode proteins involved in synaptic vesicle trafficking (dynamin I and amphiphysin II). Moreover, expression of tissue plasminogen activator (t-PA) gene was also increased following striatal long term depression. Our data suggest that a complex pattern of genes acting at presynaptic level and extracellularly may be involved in LTD-associated synaptic remodeling.

Expression pattern of notch1, 2 and 3 and Jagged1 and 2 in lymphoid and stromal thymus components: distinct ligand-receptor interactions in intrathymic T cell development.

The suggested role of Notch1 or its mutants in thymocyte differentiation and T cell tumorigenesis raises the question of how the different members of the Notch family influence distinct steps in T cell development and the role played by Notch ligands in the thymus. We report here that different Notch receptor-ligand partnerships may occur inside the thymus, as we observed differential expression of Notch1, 2 and 3 receptors, their ligands Jagged1 and 2, and downstream intracellular effectors hairy and Enhancer of Split homolog 1 (HES-1) and hairy and Enhancer of Split homolog 5 (HES-5), depending on ontogenetic stage and thymic cell populations. Indeed, while Jagged2 is expressed in both stromal cells and thymocytes, Jagged1 expression is restricted to stromal cells. Moreover, a differential distribution of Notch3, with respect to Notch1, was observed in distinct age-related thymocyte subsets. Finally, Notch3 was preferentially up-regulated in thymocytes, following the induction of their differentiation by interaction with thymic epithelial cells expressing the cognate Jagged1 and 2 ligands, suggesting that, besides Notch1, Notch3 may also be involved in distinct steps of thymocyte development. Our results suggest that the Notch signaling pathway is involved in a complex interplay of T cell developmental stages, as a consequence of the heterogeneity and specific expression of members of the Notch receptor family and their cognate ligands, in distinct thymic cell compartments.