Notch signaling pathway | EPFL Graph Search

The Notch signaling pathway is a highly conserved cell signaling system present in most animals. Mammals possess four different notch receptors, referred to as NOTCH1, NOTCH2, NOTCH3, and NOTCH4. The notch receptor is a single-pass transmembrane receptor protein. It is a hetero-oligomer composed of a large extracellular portion, which associates in a calcium-dependent, non-covalent interaction with a smaller piece of the notch protein composed of a short extracellular region, a single transmembrane-pass, and a small intracellular region. Notch signaling promotes proliferative signaling during neurogenesis, and its activity is inhibited by Numb to promote neural differentiation. It plays a major role in the regulation of embryonic development. Notch signaling is dysregulated in many cancers, and faulty notch signaling is implicated in many diseases, including T-cell acute lymphoblastic leukemia (T-ALL), cerebral autosomal-dominant arteriopathy with sub-cortical infarcts and leukoe

The Notch signaling pathway

Karine Schouwey

In animals, pigment cells are essential for coloration, as seen in feathers, fur, skin and eyes. In mammals, neural crest-derived melanocytes constitute the major population of pigment cells that is located mainly in the skin epidermis, as well as in hair follicles. A minor population of pigment cells originates from the optic cup and forms the retinal pigment epithelium (RPE), a cell monolayer lying between the neural retina and the choroid. In many cell types, the Notch signaling pathway regulates diverse biological processes such as cell fate decision, differentiation, stem cell maintenance or apoptosis. In my thesis project, the role of this pathway was investigated using Notch1, Notch2 and RBP-Jκ conditional knockout mice. Disruption of the Notch pathway by inactivation of Notch1 and Notch2, or RBP-Jκ in the melanocyte lineage using Tyr::Cre transgenic mice resulted in a gene dosage-dependent precocious hair graying, with the most pronounced effect seen in mice lacking both Notch1 and Notch2 in melanocytes. Histologically, this coat color dilution was explained by the progressive elimination of melanocytes and melanocyte stem cells, identified in skin or embryos by X-Gal staining (Dct::LacZ reporter mouse line) and immunofluorescence analyses. As in neural crest-derived melanocytes, members of the Notch signaling pathway are normally expressed in pigment cells of the RPE. The RPE-restricted deletion of RBP-Jκ using Tyrp1::Cre transgenic mice resulted in a microphthalmic eye phenotype and thus indicated a role of the Notch pathway in the development of the RPE. Further analyses demonstrated that misregulation of Notch signaling in RPE induced a reduction in the number of these pigment cells. In addition to these knockout studies, I have generated transgenic mouse lines that express the constitutively activated intracellular part of Notch (NotchIC) in melanocytes (Dct::NotchIC) and RPE cells (Tyrp1::NotchIC). Since these mice rescue the phenotypes observed in the conditional Notch receptor knockouts, they are instrumental to assess the implication of active Notch signaling in pigment cell development and tumorigenesis. Moreover, it might be determined whether constitutive activation of Notch can prevent physiological hair graying and loss of stem cells. In summary, my results underline the importance of fine-tuned Notch signaling for proper hair pigmentation and, more generally, for pigment cell development.

EPFL2009

Identification and functional characterization of protein kinases that modulate Notch signaling under physiological conditions and in cancer

Chhavi Jain

The Notch signaling pathway is a key regulator of cell fate decisions in embryonic development and in adult tissue homeostasis. Mounting evidence suggests that Notch signaling is frequently deregulated in human neoplasms, where depending upon the cellular context it can function both as an oncogene or a tumor suppressor. A plethora of studies shed light on how Notch crosstalks with signaling pathways downstream to manifest either its oncogenic or tumor suppressive activity. However, regulatory networks upstream of the Notch signaling pathway are still poorly understood. Since protein kinases are well-known regulators of a majority of cell signaling pathways, the aim of the current study was to identify novel positive and negative regulatory kinases that modulate Notch signaling. Using a HeLa cell based co-culture assay to read Notch-dependent luciferase activity, a small interference RNA (siRNA) library against the human kinase genome was previously tested in a high-throughput manner. Validation of top candidate kinases from the screening using both siRNA as well as pharmacological inhibitors led to further elimination of false positives and identification of Protein Kinase B (AKT2) and Calmodulin Kinase II (CaMKII) as key positive while Janus kinases (JAKs) as key negative regulators of the Notch signaling pathway. In the first part of the study, we analyzed the positive regulation of AKT2 and CaMKII kinases on Notch signaling in the T-cell acute lymphoblastic leukemia (T-ALL) cells where Notch is oncogenic. It was shown that pharmacological inhibition of either AKT2 or CaMKII kinase in T-ALL cell lines in vitro abrogated the expression of active Notch1 intracellular domain (N1-ICD) as well as of Notch target genes. Moreover, inhibition of these kinases blocked proliferation of T-ALL cells. In the second part of the study, we analyzed the negative regulation of JAK kinases on Notch signaling using a distinct physiological context where Notch is tumor suppressive. It was shown that pharmacological inhibition of JAK kinases led to an induction of Notch receptor transcription and of Notch target genes in the human primary epidermal keratinocytes (HPEK) as well as in the skin squamous cell carcinoma (SCC) cell lines. In addition, the pharmacological inhibition of JAK kinases induced a block in proliferation, cell cycle arrest and differentiation in HPEKs and skin SCCs by increased expression of early differentiation markers. Suppression of Notch signaling in primary keratinocytes counteracted the differentiation-inducing effect of JAK inhibition. Since JAK inhibition affected Notch transcription, global gene expression profiling using RNA sequencing was done to identify transcription factors involved in an indirect regulation of JAK kinases on the Notch cascade. EGR1 and EGR2 belonging to the early growth response family of transcription factors were significantly modulated downstream of JAK inhibition. In vivo, topical inhibition of JAK kinases provided marked resistance against chemically induced cutaneous carcinogenesis.Taken together, our data reveals a key role of AKT2 and CaMKII kinases in positive regulation while of JAK kinases in the negative regulation of Notch signaling, of potential relevance for combinatory approaches for cancer therapy. Pharmacological inhibitors against these kinases could be tested for their ability to modulate Notch signaling and may prove highly beneficial in the therapy of Notch-driven cancers.

EPFL2016

The Cell Differentiation Status Influences the Outcome of Notch-Induced Malignancy

Caroline Poisson

The Notch signaling cascade is a well conserved pathway that regulates many aspects of embryonic development and plays a crucial role in the differentiation process and tissue homeostasis in multiple organs of adult species. Several human pathological disorders, including cancer, arise as a consequence of the deregulation of the Notch signaling pathway. Notch deregulation, up to date, has been best characterized in acute T-cell lymphoblastic leukemia (T-ALL). Indeed, in T-ALL pediatric patients activating Notch1 mutations have been identified in more than 50% of the cases. Increased knowledge of the mechanisms underlying T-ALL development and maintenance is clearly needed as one out of 5 patients with T-ALL has a very poor prognosis. Moreover, the high toxicity of chemotherapeutic regimens has been associated with elevated risks of developing severe secondary health problems. In the present study, we characterized the role of Notch in T-ALL induction and maintenance. Using transgenic mouse models, we could demonstrate that the oncogenic potential of Notch is dependent on RBP-Jκ transcriptional activity. Indeed, the canonical Notch signaling pathway, mediated by the RBP-Jκ transcription factor, is essential for both Notch-mediated T-ALL induction and maintenance, as well as for the induction of a Notch-mediated lymphoproliferative disorder. Moreover, constitutive overexpression of Notch1 at discrete developmental stages within the hematopoietic system, led us to identify a correlation between the differentiation status of a cell, within the hierarchy of the hematopoietic tree, and its sensitivity to Notch1-induced transformation. We demonstrated that forced Notch1 expression in hematopoietic stem cells was not sufficient to induce T-ALL. Nevertheless, aberrant Notch1 expression in the bone marrow resulted in the development of a lethal lymphoproliferative disorder. Overexpression of Notch1 in thmus at the DN2 to DN3 stage of T-cell delvelopment generated an aggressive transplantable T-ALL with important metastatic potential. This suggested that progenitors already committed to the T-cell lineage are highly susceptible to Notch-mediated transformation, which is relevant to the human disease. Nevertheless, forced Notch1 expression after the β-selection checkpoint in the thymys, was no longer sufficient to induce transformation of late DN3 to DN4 thymocytes. We therefore established a murine genetic system that allowed us to dissect the mechanisms of aberrant Notch1 signaling in a lymphoproliferative disorder and a true T-ALL model. We were able to assess the genetic signature that is involved in the transplantability and metastatic potential of leukemic cells and generated a list of candidate genes implicated in these processes. In addition, we correlated genes identified in our murine models with genes associated as well in human T-ALL profiles to identify genes implicated in T-ALL pathogenesis in both mouse and human.

EPFL2011