REVIEWS
THE ROLE OF NOTCH IN TUMORIGENESIS: ONCOGENE OR TUMOUR SUPPRESSOR? Freddy Radtke* and Kenneth Raj ‡ Notch signalling participates in the development of multicellular organisms by maintaining the self-renewal potential of some tissues and inducing the differentiation of others. Involvement of Notch in cancer was first highlighted in human T-cell leukaemia, fuelling the notion that aberrant Notch signalling promotes tumorigenesis. However, there is mounting evidence that Notch signalling is not exclusively oncogenic. It can instead function as a tumour suppressor. HAPLOINSUFFICIENCY
A situation in which a loss-offunction phenotype is produced by mutation of one allele of a gene in a diploid cell, even though the other allele is wild type.
*Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Switzerland. ‡ Swiss Institute of Experimental Cancer Research (ISREC), Chemin des Boveresses 155, 1066 Epalinges, Switzerland. Correspondence to F.R. e-mail:
[email protected] doi:10.1038/nrc1186
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In 1917, Thomas Hunt Morgan and colleagues described a strain of Drosophila with notches at the end of their wing blades1 (FIG. 1a). This curious trait was attributed to a partial loss of function (HAPLOINSUFFICIENCY) of what would later be identified as the Notch gene. Notch, which was cloned in the mid-1980s by the groups of ArtavanisTsakonas2 and Young 3, encodes a receptor with a single transmembrane domain. Although synthesized as a single precursor protein, Notch is cleaved in two during its transport to the cell surface and, as a consequence, exists as a heterodimeric receptor4,5. The extracellular part of Notch possesses many epidermal-growth-factor (EGF)like repeats followed by three cysteine-rich Notch/Lin12 repeats (LN). The amino-terminal EGF-like repeats participate in ligand binding, whereas the LN repeats prevent signalling in the absence of ligand. The cytoplasmic extension of Notch conveys the signal to the nucleus; it contains a RAM domain, six ankyrin (also known as CDC10) repeats, two nuclear-localization signals, a transcription transactivation domain (TAD) and a PEST sequence. Although only a single Notch protein and two ligands (Delta (Dl) and Serrate (Ser)) are present in Drosophila, mammals such as mice and humans possess four Notch proteins (NOTCH1–4)6–10 and five ligands, named Delta-like-1,-3 and -4 (DLL1, DLL3 and DLL4; REFS 11–13) and Jagged 1 and Jagged 2 (JAG1 and JAG2) — which are Ser-like ligands (REFS 14,15; FIG. 1b,c). Although, overall, the structures of the four Notch receptors are very similar, they show differences in the extracellular and cytoplasmic parts. The NOTCH1 and NOTCH2
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receptors contain 36 EGF repeats in their ectodomain, whereas NOTCH3 harbours 34 and NOTCH4 only 29 repeats. Additional differences are found within the cytoplasmic domain; specifically, NOTCH1 contains a strong TAD, and NOTCH2 a weak TAD, but no TAD is present in NOTCH3 and NOTCH4. The main structural differences between the ligand family members are the number and spacing of EGF-like repeats in the extracellular domain and the presence of a cysteine-rich domain — which is located downstream of the EGF-like repeats — in Ser, JAG1 and JAG2 (FIG. 1c). Notch signalling is initiated by a receptor–ligand interaction between two neighbouring cells, which leads to two successive proteolytic cleavages that liberate the cytoplasmic portion of Notch (Notch-IC) from the membrane. Notch-IC enters the nucleus and binds to the transcription factor CSL. In the absence of Notch signalling, CSL binds to the promoters of its target genes and recruits corepressors and histone deacetylases, which inhibit transcription16–18. When Notch-IC is present, however, it competes with the inhibitory proteins to bind to CSL. It then recruits co-activators, including Mastermind, and histone acetyltransferases, which convert CSL from a transcriptional repressor to a transcriptional activator19–22 (FIG. 2). Although only a few transcription targets of Notch signalling have been identified so far, it is already clear that these targets vary in both their nature and their effects on the cell. One of the best characterized Notch targets is the HES (hairy/enhancer of split) family of
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REVIEWS
Summary • Signalling between Notch receptors and ligands influences many differentiation processes and cell-fate decisions during embryonic and postnatal development. • Stem-cell maintenance, binary cell-fate decisions and induction of differentiation are three main functions of Notch signalling in self-renewing tissues. • Notch can function as an oncogene.Aberrant expression of the dominant active cytoplasmic domain of Notch receptors in haematopoietic cells because of chromosomal translocation or viral integrations causes T-cell leukaemias in mice and humans. • Notch needs to cooperate with oncoproteins that can override the G1–S checkpoint in order to cause cancer. • Notch receptors and ligands are re-expressed in certain human carcinomas, which is compatible with the ability of Notch to maintain stem cells or precursor cells in an undifferentiated state. • Recent data show that Notch1 can also function as a tumour suppressor in mouse skin by inducing Waf1 and repressing Shh and Wnt signalling. • Notch has two faces; one that promotes and the other that suppresses tumorigenesis. Which of the two faces is shown is dependent on the cellular context and the crosstalk with other signal-transduction pathways.
FUCOSE RESIDUES
Sugar residues that are attached to certain EGF repeats of Notch receptors. In the presence of the corresponding saccharides, Fringe proteins can elongate these sugar chains. BINARY CELL-FATE DECISION
The situation in which a precursor cell has to choose between two different cell fates.
NATURE REVIEWS | C ANCER
transcription factors23,24, which negatively modulate the expression of genes such as the achaete-scute family that induce neuronal differentiation. Notch1-IC also directly stimulates expression of the cell-cycle regulator Waf1 (also known as Cip1 and p21) in primary mouse keratinocytes 25.The increase in Waf1 concentration causes proliferating keratinocytes to withdraw from the cell cycle and helps to initiate terminal differentiation. These two examples illustrate that Notch signalling activates a diverse repertoire of genes, the products of which can activate or inhibit many different cellular functions. Notch signalling can be modulated at several levels. Fringe proteins — which are glycosyl transferases — initiate elongation of O-linked FUCOSE RESIDUES on certain EGF-like repeats of Notch receptors. This modification prevents activation of Notch signalling by Jagged protein, but not by Delta-like ligands (for reviews, see REFS 26,36). Although the Jagged and Delta-like ligands possess several similar features at the protein level, the effects they elicit can be different. In vitro, a DLL1 signal via Notch drives haematopoietic precursor cells to differentiate into the T-cell lineage, whereas a JAG1 signal prevents differentiation of bone-marrow-progenitor cells28–30. Whether or not these differences are caused by modifiers such as the fringe proteins awaits further investigations. Readers who are interested in modifiers of Notch signalling are referred to reviews addressing this subject26,31. The range of proteins that can modulate Notch activity, and the crosstalk between the Notch pathway and other signal-transduction pathways, make the outcome of Notch activation difficult to predict. No single property of Notch signalling is consistently manifested each time this pathway is activated. Instead, the consequence of Notch signalling is greatly, if not entirely, dependent on the cell type. In addition to the classical signalling pathway with Notch-IC and CSL, mounting
evidence points to the existence of a CSL-independent Notch-signalling pathway, which awaits further characterization32,33 (FIG. 2). Notch function
Notch signalling impinges on a wide variety of cellular processes, including the maintenance of stem cells, specification of cell fate, differentiation, proliferation and apoptosis. At the moment, three functions of Notch are thought to be important in the context of the role of Notch in cancer. Maintenance of an undifferentiated state. Notch signalling in the vertebrate nervous system is usually thought to influence the balance between the progenitor cell pool and its differentiating progeny34,35 (FIG. 3a). Gain-of-function studies in the chicken and frog using a dominant active Notch-IC show that forced Notch signalling prevents progenitors from undergoing neurogenesis, whereas blocking this pathway leads to excessive neurogenesis and depletion of the progenitor pool36,37. Similarly, exposure of haematopoietic stem cells to JAG1 increases the proportion of stem cells as opposed to differentiating cells. Therefore, Notch signalling induces these cells to retain a stem-cell-like character 28,38. Participation in cell-fate decisions. In addition to preserving some cells (such as stem cells) in their native state, Notch signalling also participates in BINARY CELLFATE DECISIONS. This is most apparent during the development, in Drosophila, of neuronal-precursor cells of the sensory organs, which originate from a group of equipotent cells that have the capacity to develop into either neuronal-precursor cells or epidermal cells (FIG. 3b). Initially, the precursor cells express Notch and its ligand, but the concentrations of these proteins start to differ between neighbouring cells — the mechanisms behind these changes in concentration are unknown. Small differences in receptor and/or ligand concentrations are amplified over time, leading to cells that exclusively express either Notch or its ligand. The cell that receives Notch signals is inhibited from pursuing the path that leads to neuronal development and adopts an epidermal-cell fate, whereas cells that exclusively express ligands are driven into the neuronal-cell fate39. Notch signalling can also occur between two developmentally distinct cells — referred to as inductive cell-fate determination40. In this case, Notch and its ligands are expressed exclusively on two different cell types. The cell expressing the receptor, and therefore the recipient of the Notch signal, is induced to differentiate into a particular cell lineage. For example, a bipotential mouse neural-crest stem cell can be induced by Notch to adopt a glial-cell fate as opposed to a neuronal one by Notch ligands expressed on neuroblasts41. Mouse thymic epithelial cells expressing ligands for Notch1 induce early lymphocyte precursors to adopt the T-cell fate as soon as they enter the thymus, whereas in the absence of Notch1 signalling these precursors adopt the B-cell fate as the default pathway (reviewed in REF. 42).
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REVIEWS a Normal wing
'Notched' wing
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dNotch
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Ser DSL
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Figure 1 | Notch receptor and ligands. a | Wing blade of a wild-type Drosophila melanogaster (left), and of a mutant with a partial loss of the Notch gene (right). The notches, which are absent in the wild type, but clearly visible at the border of the wing blade, have given the name to the implicated gene (photographs kindly provided by Mark Fortini). b | Structure of Notch proteins and their ligands. Drosophila has one Notch receptor (dNotch) and vertebrates have four (NOTCH1–4), which are presented on the cell surface as heterodimers. The ectodomain of Notch receptors contains epidermal-growth-factor (EGF)-like repeats and a cysteine-rich Notch/Lin12 domain (LN); this is followed by a transmembrane domain, the RAM domain and six ankyrin repeats (ANK, also known as CDC10 repeats), two nuclear-localization signals (NLSs), followed by the transactivation domain (TAD) and a PEST sequence. NOTCH1 contains a strong and NOTCH2 a weak transactivation domain in the cytoplasmic part of the receptor. c | Two transmembrane-bound ligands for Notch have been identified in Drosophila, named Delta (Dl) and Serrate (Ser). The vertebrates possess three Delta homologues, called Delta-like (DLL)-1, -3 and -4, and two Serrate homologues, Jagged 1 (JAG1) and Jagged 2 (JAG2). The ligands harbour an amino-terminal structure called DSL (Delta, Serrate and LAG-2), which is common to all family members, followed by EGF-like repeats. Serrate, Jagged1 and Jagged2 harbour a cysteine-rich domain (CR) following the EGF-like repeats.
Induction of terminal differentiation. Instead of influencing the choice between two possible cell fates, Notch signalling between developmentally related cell types can induce or enhance terminal differentiation. In human skin, DLL1-induced Notch signalling initiates a terminal-differentiation programme 43 (FIG. 3c). In the adult mouse skin and keratinocytes, where Dll1 is not expressed, this differentiation programme is triggered by Jaggedmediated Notch signalling, which induces early differentiation markers and cell-cycle arrest by upregulating Waf1(REF. 25).
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Notch as an oncogene
T-cell leukaemia. The oncogenic role of Notch was first identified in human T-cell neoplasia. Some of these cancer cells possess a specific chromosomal translocation that attaches a portion of chromosome 7 to chromosome 9, which contains the T-cellreceptor-β (TCRβ) gene. The fusion of these two loci — t(7;9)(q34;q34.3) — was first identified by Jeff Sklar and co-workers in a T-cell acute lymphoblastic leukaemia (T-ALL)44. As the gene at the chromosome 7 locus that is fused to the TCRβ promoter/enhancer is very similar to Drosophila Notch, it was named
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Jagged
Notch heterodimer
TACE
γ-Secretase (PS) Cytoplasm
CSL independent?
Nucleus Golgi
Notch precursor CoR NIC CoA CSL dependent CoR CSL
CSL
Target genes
Figure 2 | Notch signalling. Notch proteins are synthesized as precursor proteins that are processed by a furin-like convertase in the Golgi before being transported to the cell surface, where they reside as heterodimers. Interaction of Notch receptors with their ligands, such as Delta-like or Jagged, leads to a cascade of proteolytic cleavages. The first cleavage is mediated by TACE (tumour-necrosis factor-α-convering enzyme/metalloproteinase), followed by a second cleavage mediated by the γ-secretase activity of presenilins (PS), which liberates the cytoplasmic domain —Notch intracellular domain (NIC) — of the Notch receptors. The liberated NIC enters the nucleus and binds to the transcription factor CSL, which displaces co-repressors (CoR) and recruits co-activators (CoA), leading to transcriptional activation of downstream target genes. This pathway is known as the CSL-dependent pathway. Genetic evidence points to the existence of a CSL-independent pathway that is poorly characterized at present.
MYELOID ORIGIN
Cells of myeloid origin include macrophages, granulocytes, megakaryocytes, erythroblasts and myeloid-dentritic cells, whereas cells of lymphoid origin include T cells, B cells, naturalkiller cells and lymphoid dendritic cells.
NATURE REVIEWS | C ANCER
TAN1 — for ‘translocation-associated Notch homologue’ 45 — and subsequently became known as human NOTCH1. Importantly, the t(7;9) translocation does not juxtapose the entire human NOTCH1 gene to the TCRβ locus, but just the carboxy-terminal region from within the EGF-repeat 34 of NOTCH1. This leads to expression of a truncated NOTCH1 protein that corresponds to NOTCH1-IC. As all T-ALLs with t(7;9) translocations show this feature, it was proposed that deregulated expression of the cytoplasmic part of the NOTCH1 protein causes T-ALL in humans (FIG. 4a).
This interpretation is supported by the generation of mouse models for T-ALL. Mice reconstituted with haematopoietic progenitor cells expressing the human NOTCH1-IC (TAN1) proteins develop T-cell leukaemia. During this process, they generate immature doublepositive (DP) T cells in the bone marrow 46 with simultaneous inhibition of B-cell development, indicating that NOTCH1 signalling drives haematopoietic progenitor cells into the T-cell lineage47. Indeed, loss-of-function experiments in which Notch1 in bone-marrow progenitors was inactivated, show that Notch1 is essential for normal T-cell lineage commitment48.Aberrant Notch1-IC expression in bone-marrow progenitors leads to immature DP T cells that are developmentally blocked at this stage49. These DP T cells were initially of polyclonal origin and not cycling. With increasing time, however, these mice develop highly aggressive monoclonal T-cell tumours, which indicates that additional mutations cooperate with Notch1-IC to transform non-cycling cells into aggressive, rapidly cycling tumour cells. The ankyrin repeats plus the TAD of Notch1 were sufficient for this cooperation50. Experimentally, Notch1 can collaborate with c-Myc51, E2A–PBX152 and Ikaros53 to induce T-ALL. Despite this, the complete in vivo molecular mechanism by which Notch1-IC transforms haematopoietic progenitor cells is unclear at present. Overexpression of Notch-ICs in haematopoietic cells gives rise exclusively to T-cell neoplasia. No tumours of MYELOID ORIGIN have been described so far, indicating that Notch1-IC cooperates with T-cell-specific signals to exert its oncogenic potential. Indeed, Allman et al.54 showed that mice transplanted with Notch1-IC-expressing bone-marrow-progenitor cells from either Rag2 –/– or Slp76 –/– mice — both of which lack pre-T-cell-receptor (preTCR) signalling — failed to develop T-cell leukaemia. Introduction of a TCRβ transgene into Rag2 –/– mice — to re-activate pre-TCR signalling — restored the oncogenic function of Notch1-IC, confirming that Notch1-IC-mediated transformation is dependent on a second T-cell-specific signal that is mediated by the pre-TCR54. Similar results were obtained with transgenic mice that express either Notch1-IC or Notch3-IC in the thymus55–57. So far, it is not clear whether this pre-TCR-mediated signal renders cells more permissive for acquisition of secondary mutations, or whether preTCR-induced signals actively synergize with Notch-ICs in tumour induction. T-cell neoplasia can also be caused by proviral integration into Notch genes, which leads to aberrant Notch signalling. Proviral integration of the Moloney murine leukaemia virus (MuLV) (FIG. 4b) or feline leukaemia virus into Notch1 or Notch2, respectively, causes T-cell leukaemia51,58. In both cases, the cytoplasmic domain of the Notch proteins is expressed under the influence of the viral promoter. Consistent with these findings, forced expression of the Notch ligand Dll4 also results in the development of T-cell leukaemia in one model59 and T-cell lymphoproliferative disease in another 60. Finally, Notch activity participates not only in the initiation of cancers, but also in their maintenance, as shown by Weng et al. for Notch-transformed T-ALL61.
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a Stem-cell
b Binary cell-fate decisions Lateral signalling
maintenance
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