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.

C/EBP beta is considered a key element of interleukin‐6 (IL‐6) signalling as well as an important transcriptional regulator of the IL‐6 gene itself. We describe here how mice lacking C/EBP beta develop a pathology similar to mice overexpressing IL‐6 and nearly identical to multicentric Castleman's disease in human patients, with marked splenomegaly, peripheral lymphadenopathy and enhanced haemopoiesis. Humoral, innate and cellular immunity are also profoundly distorted, as shown by the defective activation of splenic macrophages, the strong impairement of IL‐12 production, the increased susceptibility to Candida albicans infection and the altered T‐helper function. Our data show that C/EBP beta is crucial for the correct functional regulation and homeostatic control of haemopoietic and lymphoid compartments.

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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.

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.

Thymic-derived dysregulated tolerance has been suggested to occur in type 1 diabetes via impaired generation of CD4+CD25+ T regulatory cells, leading to autoimmune β cell destruction. In this study, we demonstrate that Notch3 expression is a characteristic feature of CD4+CD25+ cells. Furthermore, streptozotocin-induced autoimmune diabetes fails to develop in transgenic mice carrying the constitutively active intracellular domain of Notch3 in thymocytes and T cells. The failure to develop the disease is associated with an increase of CD4+CD25+ T regulatory cells, accumulating in lymphoid organs, in pancreas infiltrates and paralleled by increased expression of IL-4 and IL-10. Accordingly, CD4+ T cells from Notch3-transgenic mice inhibit the development of hyperglycemia and insulitis when injected into streptozotocin-treated wild-type mice and display in vitro suppressive activity. These observations, therefore, suggest that Notch3-mediated events regulate the expansion and function of T regulatory cells, leading to protection from experimental autoimmune diabetes and identify the Notch pathway as a potential target for therapeutic intervention in type 1 diabetes.

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.

Constitutive activation of the transmembrane receptor, Notch3, and loss of function of the hematopoietic transcription repressor, Ikaros (IK), play direct roles in T‐cell differentiation and leukemogenesis that are dependent on pre‐T‐cell receptor (pre‐TCR) signaling. We demonstrate the occurrence of crosstalk between Notch3 and IK that results in transcriptional regulation of the gene encoding the pTα chain of the pre‐TCR. We also show that, in the presence of the pre‐TCR, constitutive activation of Notch3 in thymocytes causes increased expression of dominantnegative non‐DNA‐binding IK isoforms, which are able to restrain the IK inhibition of Notch3's transcriptional activation of pTα. This effect appears to be mediated by Notch3's pre‐TCR‐dependent upregulation of the RNA‐binding protein, HuD. Notch3 signaling thus appears to play a critical role in the diminished IK activity described in several lymphoid leukemias. By exerting transcription‐activating and transcription‐repressing effects on the pTα promoter, Notch3 and IK cooperate in the fine‐tuning of pre‐TCR expression and function, which has important implications for the regulation of thymocyte differentiation and proliferation.