Notch pathway

DSL ligands

Two families of DSL ligands can recognise the NOTCH receptors in mammals: DLL (delta-like ligands) and JAG (jagged), which correspond to the Serrate ligands of Drosophila. These ligands are type I transmembrane proteins (i.e., their N-terminal part is in the extracellular space).

DSL ligands have a short intracytoplasmic domain, a single transmembrane domain and a large characteristic extracellular domain, made of 6–14 tandem repeats of EGF-like domains, a DOS (delta and OSM-11-like) domain absent from DLL3 and DLL4 and a DSL domain. DLL3 and DLL4, devoid of a DOS domain, require a DLK (delta-like protein homologue) coligand, equipped with DOS and EGF-like domains but devoid of a DSL domain. The simultaneous presence of DOS and DSL domains appears necessary for the ligand–receptor interaction, the DOS domain being either directly brought by the ligand (for JAG1, JAG2 and DLL1) or by a coligand (for DLL3 and 4). The protein structure of ligands and coligands of the Notch pathway is presented on Fig. 8.1.

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Fig. 8.1. DSL ligands. DSL ligands contain a series of domains that are essential for their function: the DOS and DSL recognition domains and 6–14 EGF-like domains. The JAG ligands contain in addition a cysteine-rich domain. DLL3 and DLL4 have no DOS domain and depend upon the presence of the coligands DLK1 and DLK2, which harbour a DOS domain but no DSL domain

NOTCH Receptors

DSL ligands are recognised by NOTCH receptors; four NOTCH receptors exist in humans, which are also type I transmembrane proteins. Whereas the protein sequence in Drosophila is continuous and covers the extraand intracellular parts, NOTCH receptors are heterodimeric in mammals, the monomers being generated in the Golgi apparatus by post-translational cleavage of a precursor containing the whole sequence, followed by non-covalent reassociation of the two monomers. The cleavage is realised by furin, a member of the subtilisin-like proprotein convertase family at a site called S1, and the resulting monomers are called NECD (notch extracellular domain) and NTMICD (notch transmembrane and intracellular domain).

In their extracellular part, the NOTCH receptors consist of 29–36 tandem repeats of EGF-like motifs, which contain O-glycosylation sites for fucose or glucose residues. The EGF-like motifs are followed by a NRR (negative regulatory region) sequence consisting of three tandem repeats of LNR (lin12 and notch repeats) motifs and a heterodimerisation (HD) domain where the S1 cleavage site is located. After the transmembrane domain, the intracellular part contains a RAM (RBPJK [recombination signal binding protein for immunoglobulin kappa J region]–association module) motif, followed by nuclear localisation sequences (NLS), ankyrin repeats (ANK) and a transactivation domain harbouring PEST (proline–glutamic acid–serine–threonine-rich) domains. Figure 8.2 presents the general structure of the NOTCH receptors.

NOTCH receptor activation and signal transmission

Ligand–receptor binding occurs by homotypic interaction of the EGF-like domains and induces the proteolytic cleavage of the receptor by a metalloproteinase, ADAM10 (a disintegrin and metalloproteinase) or ADAM17 (also known as TACE, TNF-alpha converting enzyme), at the level of the site S2 located within the heterodimerisation domain, thus in the extracellular part of the receptor, 12 amino acid residues ahead of the transmembrane domain. This cleavage is possible thanks to a conformational change releasing the S2 site from the protection exerted by the LNR domains. The intermediate protein is called NEXT (notch extracellular truncation) and is the substrate of the proteolytic activity exerted by the γ-secretase complex, at two transmembrane sites called S3 and S4. After cleavage, the NOTCH intracellular domain (NICD) is able to migrate to the nucleus, thanks to the unmasking of the NLS. γ-secretase is a multimeric transmembrane complex harbouring presenilins 1 and 2 (genes PSEN1 and 2), nicastrin (gene NCSTN) and activation and stabilisation factors of these proteases.

In the nucleus, NICD interacts, thanks to its RAM domain, with a DNA-binding protein of the CSL (CBF [Core-binding factor-1] /su(H)/lag-1) family, which is RBPJK in humans. The ANK domain then recruits a coactivator called MAML (mastermind-like protein), which in turn recruits the MED8 (mediator of RNA polymerase II transcription, subunit 8) factor, allowing the transactivation of target genes. Figure 8.3 presents the steps of the post-translational maturation of the NOTCH receptors, interaction with DSL ligands, proteolysis, migration of the active part to the nucleus and interaction with activating and repressing transcriptional complexes.

The NOTCH receptor, which must be cleaved and whose intracellular part constitutes in fact the second messenger, can be used only once: ligand and receptor availability at the surface of the two cells exchanging a message is, therefore, a decisive factor for signalling. Synthesis and degradation of the ligands and receptors are the key elements of this regulation. Ubiquitinylation plays a crucial role for ligand and receptor endocytosis and turnover (see Annex C).

Textbook of Cell Signalling in Cancer_ An Educational Approach-Springer International Publishing (2015) 8.2

Fig. 8.2. NOTCH receptors. The four NOTCH heterodimeric receptors are built on the same model and associate an extracellular domain (NECD) and a transmembrane and intracellular domain (NTMICD), tied together via a non-covalent interaction at the level of a heterodimerisation domain HD; the two monomers result from the cleavage of a unique precursor protein, which is cleaved within the HD domain in the Golgi apparatus by furin. NECD contains 29–36 EGF-like domains and three LNR domains involved in the negative regulation of the Notch signalling pathway by protecting the S2 cleavage site of the HD domain. NTMICD contains a RAM domain, involved in the interaction with DNA-binding proteins, together with nuclear localisation sequences NLS, ankyrin repeats ANK involved in the recruitment of the transcriptional activator MAML and a transactivation domain containing PEST motifs

The heterodimerisation HD domain, containing the successive proteolytic cleavage sites, is certainly the most sensitive domain of the NOTCH receptors. Glycosylation of the EGF-like motifs of the NOTCH receptors also represents an important feature for the regulation of this signalling pathway. Finally, numerous crosstalks with other signalling pathways have been described at the level of the transcription of target genes: for instance, the JAG1 ligand is the product of a gene activated by β -catenin (Chap. 7).

Textbook of Cell Signalling in Cancer_ An Educational Approach-Springer International Publishing (2015) 8.3

Fig. 8.3. Notch signalling pathway. (A) After synthesis, the NOTCH receptor is fucosylated and glycosylated on serine and threonine residues of EGF-like domains in the endoplasmic reticulum. (B) In the Golgi apparatus, it is cleaved by furin at the S1 site of the HD domain, but the two cleavage products, NECD and NTMICD, remain associated by non-covalent bonds localised at the level of the HD domain and thus form a heterodimer. (C) Once in the plasma membrane, the NOTCH receptor can be recognised by the DSL ligands of a neighbour cell by homotypic interaction involving the EGF-like domains. (D) This recognition induces the unmasking of the cleavage site S2 of the HD domain of NTMICD by an ADAM proteinase, allowing then a new cleavage at the S3 and S4 transmembrane sites by the γ-secretase proteolytic complex. (E) The truncated receptor, NICD, can then migrate to the nucleus thanks to its nuclear localisation sites (NLS). (F) NICD is recognised in the nucleus, at the level of its RAM domain, by a CSL protein, which binds it to DNA sequences. (F) This allows the recruitment of a MAML protein, which binds to the ANK domains of NICD, so that transcription of target genes can occur. (G) NICD is then destroyed in the proteasome after ubiquitinylation, thanks to the PEST domains localised on its C-terminal part. (H) Similarly, the DSL ligands are digested in the proteasome after ubiquitinylation

Oncogenic alterations

The Notch pathway plays a crucial role in the multiplication of stem cells in intestinal villi and breast cancers; its inhibition allows the reorientation of these cells toward differentiation. Among the NOTCH target genes are SNAI1 (SNAIL) and SNAI2 (SLUG), which are transcriptional repressors of E-cadherin, an epithelial adhesion protein whose expression must be inhibited to allow cell migration (Chap. 7). The Notch pathway is thus responsible for the crucial steps of the epithelial-to-mesenchymal transition (EMT). The Notch pathway is also involved in an important step of angiogenesis: the formation of vascular ramifications. The endothelial tip cells synthesise, upon VEGF impulse, a DSL ligand able to activate the proliferation of another type of endothelial cell, the stalk cells, which constitute the new vessel branches and their anastomoses.

The Notch pathway appears as an essential oncogenic pathway, and its role in oncogenesis is certainly far from being completely understood. Gain-of-function alterations in the Notch signalling pathway have been initially observed in T-cell lymphomas and lymphoblastic leukaemias. Thereafter, such alterations have been identified in other malignancies, especially colon and prostate cancers. The role of the Notch pathway in the fate of stem cells is certainly a key factor for the interest of this pathway in oncology.

A rare t(7;9) translocation in T-cell lymphoblastic leukaemias allowed the identification, as early as 1991, of the oncogenic role of a truncated, constitutively active NOTCH receptor. Later, numerous activating mutations have been identified, especially at the level of the HD and PEST domains. Mutations in the HD domain induce weakening of the interaction between NECD and NTMICD: this facilitates the subsequent cleavage at the S2 and S3–S4 sites for spontaneous release of NICD, without DSL ligand interaction. Furthermore, mutations and deletions of the C-terminal region may remove the NICD binding sites for its E3 ubiquitin ligase and stabilise the active form of the receptor at the level of its nuclear site of action.

Pharmacological targets

Pharmacological interventions on the successive steps of the NOTCH receptor activation aim at the inhibition of this signalling pathway in stem cell reproduction, oncogenesis and angiogenesis. Potential targets are found at the level of ligand– receptor interactions, receptor glycosylation, initial cleavage generating NECD and NTMICD, subsequent ADAMand γ-secretase-mediated cleavages, receptor or ligand ubiquitinylation, NICD interaction with nuclear proteins, etc.

Up to now, the essential target for pharmacological development has been the transmembrane cleavage mediated by the γ-secretase complex. γ-secretase inhibitors had been designed and developed for the treatment of Alzheimer disease, such as semagacestat and avagacestat, because the proteolytic activities generating the β -amyloid peptides are also catalysed by γ-secretase. Several clinical trials with original γ-secretase inhibitors have been undertaken in oncology. Another approach concerns the development of monoclonal antibodies targeting the NRR domain of the NOTCH receptors and the DLL ligand more specifically involved in angiogenesis. Demcizumab (anti-DLL4), OMP-52M51 (anti-NOTCH1) and OMP-59R5 (anti-NOTCH2) have entered clinical trials.

Further reading

Al-Hussaini H, Subramanyam D, Reedijk M, Sridhar SS. Notch signaling pathway as a therapeutic target in breast cancer. Mol Cancer Ther. 2011;10:9–15.

Aster JC, Blacklow SC. Targeting the Notch pathway: twists and turns on the road to rational therapeutics. J Clin Oncol. 2012;30:2418–20.

Brou C. Intracellular trafficking of Notch receptors and ligands. Exp Cell Res. 2009;315:1549–55.

Fortini ME. Notch signaling: the core pathway and its posttranslational regulation. Dev Cell. 2009;16:633–47.

Guruharsha KG, Kankel MW, Artavanis-Tsakonas S. The Notch signalling system: recent insights into the complexity of a conserved pathway. Nat Rev Genet. 2012;13:654–66.

Koch U, Radtke F. Notch and cancer: a double-edged sword. Cell Mol Life Sci. 2007;64:2746–62.

Kopan R, Ilagan MX. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell. 2009;137:216–33.

Nefedova Y, Gabrilovich D. Mechanisms and clinical prospects of Notch inhibitors in the therapy of hematological malignancies. Drug Resist Updat. 2008;11:210–8.

Qiao L, Wong BC. Role of Notch signaling in colorectal cancer. Carcinogenesis. 2009;30:1979–86. Ranganathan P, Weaver KL, Capobianco AJ. Notch signalling in solid tumours: a little bit of everything but not all the time. Nat Rev Cancer. 2011;11:338–51.

Rizzo P, Osipo C, Foreman K, Golde T, Osborne B, Miele L. Rational targeting of Notch signaling in cancer. Oncogene. 2008;27:5124–31.

Shao H, Huang Q, Liu ZJ. Targeting Notch signaling for cancer therapeutic intervention. Adv Pharmacol. 2012;65:191–234.

Tien AC, Rajan A, Bellen HJ. A Notch updated. J Cell Biol. 2009;184:621–9.

Wang Z, Li Y, Banerjee S, Sarkar FH. Emerging role of Notch in stem cells and cancer. Cancer Lett. 2009;279:8–12.

Yan M, Plowman GD. Delta-like 4/Notch signaling and its therapeutic implications. Clin Cancer Res. 2007;13:7243–6.