Dependence receptors: a new paradigm in cell signaling and cancer therapy
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Oncogene (2010), 1–18
& 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00
www.nature.com/onc
REVIEW
Dependence receptors: a new paradigm in cell signaling and cancer therapy
D Goldschneider and P Mehlen
Apoptosis, Cancer and Development Laboratory- Equipe labellisée ‘La Ligue’, CNRS UMR5238, Centre Léon Bérard, Université de
Lyon, Lyon, France
Dependence receptors (DRs) now form a family of more (Stupack et al., 2001). All of them are involved in both
than a dozen membrane receptors that are not linked by nervous system development and cancer progression.
their structure, but by common functional traits. The most
notable is their ability to trigger two opposite signaling
pathways: in the presence of ligand, these receptors Dependence receptors: a short history
activate classic signaling pathways implicated in cell
survival, migration and differentiation. In the absence of Neurotrophin receptor p75NTR was the first DR to be
ligand, they do not stay inactive, rather they elicit an described. P75NTR was discovered as one of two
apoptotic signal. Thus, cells expressing this kind of receptors able to bind nerve growth factor (Chao
receptor are dependent on the presence of ligand in the et al., 1986), the other being TrkA (Kaplan et al.,
extracellular environment to survive. This review will 1991). TrkA was rapidly shown to mediate the known
recapitulate the increasing data regarding the molecular responses to NGF, such as neurite outgrowth and
mechanisms associated with DRs, their potential implica- neuronal survival (Lee et al., 2001a; Lykissas et al.,
tion during development, as well as their deregulation 2007), whereas the precise biological role of p75NTR
during tumorigenesis and, finally, their emergence as new remained misunderstood. p75NTR was shown to collabo-
possible therapeutic targets for cancer treatment. rate with TrkA to form high-affinity sites for NGF
Oncogene advance online publication, 22 February 2010; binding (Hempstead et al., 1991). In addition, p75NTR
doi:10.1038/onc.2010.13 was shown to alter the ligand specificity of other Trk
receptors. For example, brain-derived neurotrophic
Keywords: dependence receptors; apoptosis; caspase;
factor, NT3 and NT4/5 can all bind TrkB in the absence
tumor progression; cancer therapy
of p75NTR, whereas only brain-derived neurotrophic
factor does so in the presence of p75NTR. In contrast,
coexpression of p75NTR with TrkC results in a relaxation
Membrane receptors are classically considered as inactive in its absolute specificity for NT3 (Hempstead, 2002). At
unless bound to their ligand. However, increasing observa- the time of its discovery, p75NTR was considered as a
tions demonstrate that some receptors, in addition to their unique type of protein but, subsequently, a large
‘positive’ signaling when their ligand is present, transduce a superfamily of tumor necrosis factor (TNF) receptors
‘negative’ signal that induces apoptosis in the absence of were found to share the overall structure of p75NTR
ligand (Figure 1). Cells expressing these receptors are thus (Liepinsh et al., 1997). Identification of this superfamily
dependent on the presence of ligand to survive. These helped elucidate some of the biological functions of
receptors are named ‘dependence receptors.’ To date, the p75NTR, including its link to cell death regulation. The
dependence receptor (DR) family is composed of more relationship between these TNF death receptors, which
than a dozen members including DCC (deleted in color- induce cell death on binding of proapoptotic ligand such
ectal carcinoma) (Mehlen et al., 1998), UNC5Hs (un- as TNF or FasL, and p75NTR, which binds NGF, a
coordinated 5 homologs), neogenin (Matsunaga et al., trophic factor known to induce cell survival, led DE
2004), p75NTR (p75 neurotrophin receptor) (Rabizadeh Bredesen and colleagues to propose that p75NTR induces
et al., 1993), RET (rearranged during transfection) cell death when unoccupied by NGF, whereas binding
(Bordeaux et al., 2000), TrkC (tyrosine kinase receptor C) of NGF blocks apoptosis (Rabizadeh et al., 1993)
(Tauszig-Delamasure et al., 2007), Ptc (patched) (Thibert (Figure 2 and Table 1). This finding suggested that
et al., 2003), EphA4 (ephrin type A receptor 4) (Furne p75NTR expression creates a state of cellular dependence
et al., 2009), ALK (anaplastic lymphoma kinase) (Mourali on NGF. Further studies with knockout mice confirmed
et al., 2006), MET (Tulasne et al., 2004) and some integrins this notion. First, p75NTR-deficient mice have an
increased number of cholinergic neurons, somal hyper-
Correspondence: Dr P Mehlen, Apoptosis, Cancer and Development trophy and hyperinnervation in some areas of the
Laboratory- Equipe labellisée ‘La Ligue’, CNRS UMR5238, Centre hippocampus (Yeo et al., 1997; Naumann et al., 2002).
Léon Bérard, Université de Lyon, 28 rue Laennec, Lyon, Rhone 69008, In addition, crossing NGF hemizygous mice, which
France.
E-mail: mehlen@lyon.fnclcc.fr display a reduction in cholinergic cell numbers, with
Received 1 October 2009; revised 2 January 2010; accepted 6 January p75NTR null mice showed that loss of p75NTR partially
2010 restores cholinergic cell numbers (Naumann et al.,A new paradigm in cell signaling and cancer therapy
D Goldschneider and P Mehlen
2
2002). However, the overall picture of p75NTR function binding rather than ligand withdrawal (Casaccia-Bon-
became more complicated when some studies showed nefil et al., 1996; Frade et al., 1996). Particularly, in
that p75NTR induced apoptosis in response to ligand addition to its ability to bind mature neurotrophins,
Table 1 DRs and their known ligands
Receptors Ligands
p75NTR NGF, proNGF, BDNF, NT-3, NT-4/5,
b-amyloid, prion
DCC netrin-1, netrin-4
Neogenin netrin-1, RGMa, RGMb, RGMc, netrin-3, netrin-4
Unc50 s netrin-1, netrin-4
RET GDNF, neurturin, artemin, persephin
Ptc Shh
TrkC NT-3
EphA4 ephrinB3, ephrinA1, ephrinA4
ALK pleiotrophin, midkin, jeb
Survival, MET HGF
Migration, APOPTOSIS AR androgens
Differentiation Integrin avb3 extracellular matrix
and a5b1
Figure 1 The DR model. DRs have two faces: in the presence of
their respective ligand, they transduce a positive signal of The above listed are the DRs and their known ligands. In case of
differentiation, migration or survival, whereas in the absence of multiple ligands, those which were shown to block apoptotic function
their ligand, they do not stay inactive but, rather, induce apoptosis. are underlined. ALK is a particular case: its putative ligands have not
Thus, cells expressing such receptors are dependent on ligand yet been tested for blocking apoptosis, only agonist antibodies have
availability for survival. been used.
Figure 2 Representation of the DR family. The functional domains present in extra and intracellular domains are represented. DRs
are not related to each other according to their structure, but according to their ability to induce apoptosis in the absence of ligand. All
of them are caspase substrates, except p75NTR and integrins. The position of caspase cleavage sites is indicated. Localization of ADD,
which has been more or less precisely determined depending on receptor, is indicated by double arrows.
OncogeneA new paradigm in cell signaling and cancer therapy
D Goldschneider and P Mehlen
3
other cell-death-inducing ligands have been proposed embryogenesis. In vertebrates, besides netrin-1, four
for p75NTR, such as pro-NGF (Lee et al., 2001b), other netrins have been described: netrin-2/3/2like,
b-amyloid (Yaar et al., 2002) and prion (Della-Bianca netrin-4/b, netrin-G1 and netrin-G2 (Puschel, 1999;
et al., 2001) peptides. The decision between ligand- Mehlen and Mazelin, 2003). Netrins are structurally
induced apoptosis and ligand-inhibited apoptosis related to the short arm of laminin (g for netrin 1–3 and
mediated by p75NTR likely depends on cell type and b for netrin-4, G1 and G2). Netrins 1–4 are secreted,
development stage (Barrett and Bartlett, 1994). Because whereas netrin-G1 and G2 are membrane anchored by
the idea of a receptor triggering apoptosis when means of a glycophosphatidylinol tail. Secreted netrins
unbound to its ligand contradicted the dogma regarding exert their biological functions by binding to receptors
receptor signaling and the trophic theory, considering such as DCC, UNC5, neogenin and DSCAM, whereas
p75NTR as a classic death receptor had more success than netrin Gs do not interact with these receptors (Rajase-
considering it as a DR. Consequently, DR p75NTR has kharan and Kennedy, 2009). Interestingly, netrin-5 has
been rather forgotten, although it was the first to be recently appeared in databases, which seems to be
described and no definitive evidence has demonstrated related to the netrin 1–3 group according to its sequence.
that it is not a DR. Netrin-1 is the most studied member of the netrin family
The concept reemerged with DCC. DCC was dis- and to date it seems to be the main ligand for DCC, as
covered in 1990 as a putative cell-surface receptor well as for UNC5 receptors (see below), although a
encoded by a gene frequently deleted through allelic recent report mentioned that netrin-4 could interact with
loss in colorectal carcinoma (Fearon et al., 1990). DCC and UNC5H1 (Qin et al., 2007).
Observation that DCC expression is reduced or lost in The UNC5 receptor was initially identified in
colorectal cancer led to the proposal that DCC Caenorhabditis elegans as an axonal guidance trans-
expression represented a constraint for disease progres- membrane receptor (Leung-Hagesteijn et al., 1992) and,
sion and is therefore a tumor suppressor. This hypo- on the basis of a genetic screen, was predicted to interact
thesis was supported by the fact that DCC expression is with UNC6 (the C. elegans netrin-1 ortholog) (Hedge-
lost or reduced in various cancers (Mehlen and Fearon, cock et al., 1990). Four homologs of UNC5 have been
2004) and that its loss of expression is associated with described in mammals (UNC5H1, 2, 3 and 4 in rodents
poor prognosis (Shibata et al., 1996; Sun et al., 1999). In and UNC5A, B, C and D in humans) (Leonardo et al.,
addition, restoration of DCC expression can suppress 1997). Although DCC alone is implicated in the
tumorigenic property in vitro and in nude mice chemoattractive effect of netrin-1, it has been proposed
(Klingelhutz et al., 1993; Velcich et al., 1999). The that UNC5, associated with DCCs through their
DCC extracellular domain shares structural features intracellular domains, is responsible for the repulsive
with certain types of cell-adhesion molecules, such as effect of netrin-1 (Hong et al., 1999). Besides this role,
NCAM (Cho et al., 1994) (Figure 2 and Table 1), but its UNC5 receptors are now known to have critical roles in
intracellular domain shows little similarity with known other cellular processes, such as neuronal migration
proteins; hence in spite of the above-mentioned studies (Mehlen and Furne, 2005) and embryonic angiogenesis
on cancers, little was known about the precise biological (Lu et al., 2004). Interestingly, UNC5 proteins contain a
role of DCC. It was rediscovered as the receptor for death domain related to the death domain of the TNF
netrin-1 (Keino-Masu et al., 1996), a diffusible molecule receptor superfamily in their cytoplasmic part. As
originally identified as a chemoattractant for commis- UNC5 receptors were netrin-1 receptors and contained
sural axons in the vertebral spinal cord (Serafini et al., a death domain, it was suggested that they were possible
1994). The classic view for netrin-1 is that a gradient of DRs (Figure 2 and Table 1). Along this line, UNC5
this cue diffuses from a ventral spinal cord structure, the receptors are able to induce apoptosis in the absence of
floor plate, and orients the growth of commissural axons netrin-1, whereas addition of ligand efficiently blocked
as they extend circumferentially toward the ventral this effect (Llambi et al., 2001; Wang et al., 2008).
midline of the embryonic nervous system. The key role A DCC homolog was discovered and called neogenin
of DCC and netrin-1 in mediating axon outgrowth and (Vielmetter et al., 1994; Meyerhardt et al., 1997).
pathfinding is supported by a large number of studies, Although there is only one member of the DCC receptor
particularly the analysis of DCC and netrin-1 knockout family in C. elegans and Drosophila (UNC40 and
mice, which display similar defects in the central nervous frazzled, respectively), vertebrates have evolved two
system (Serafini et al., 1996; Fazeli et al., 1997). Such a closely related orthologs, DCC and neogenin. Owing to
dual role for a receptor, implicated during development its identity of sequence with DCCs, especially in their
and functioning as a putative tumor suppressor, seems ectodomain, neogenin was initially considered to be a
to be a common trait for DRs. DCC was thus proposed netrin-1 receptor as well. However, more recently, the
as a DR when it was shown that its expression in various propensity of netrin-1 to function as a ligand for
cancer cell lines that lacked endogenous DCC expres- neogenin was challenged when neogenin seemed to have
sion induced cell death and that addition of netrin-1 much higher affinity for RGM (repulsive guidance
blocked apoptosis (Mehlen et al., 1998; Chen et al., molecule), another guidance molecule, and to mediate
1999). its repulsive effect (Rajagopalan et al., 2004). RGM was
Netrin-1 is in fact the founding member of a family of first identified as a repulsive, membrane-bound cue
extracellular proteins found throughout the animal responsible for the mapping of temporal retinal axons to
kingdom and that direct cell and axon migration during the posterior region of the chick tectum (Monnier et al.,
OncogeneA new paradigm in cell signaling and cancer therapy
D Goldschneider and P Mehlen
4
2002). In mammals, three RGMs exist: RGMa, the These ‘non-liganded integrins,’ which are either un-
closest ortholog of chick RGM, RGMb/DRAGON and ligated or occupied with a soluble antagonist, not only
RGMc/hemojuvelin. RGMa and b are both expressed in disrupt survival signaling but also actively induce
the central nervous system and follow a complementary apoptosis, hence supporting the view of integrins as
expression pattern (Niederkofler et al., 2004), whereas DRs (Stupack et al., 2001) (Figure 2 and Table 1).
RGMc is mainly expressed in striated muscles and liver Beside these receptors, some classical tyrosine kinase
(Schmidtmer and Engelkamp, 2004). Neogenin is a DR receptors have also emerged as DRs. RET was the first
in that it can induce apoptosis when overexpressed in one (Figure 2 and Table 1). RET is the signaling
chick neural tube, and its ligand, RGM, but not netrin- component of a multisubunit complex that functions as
1, counteracts this process (Matsunaga et al., 2004) a receptor for glial cell line-derived neurotrophic factor
(Figure 2 and Table 1). It should be noted that the (Jing et al., 1996), neurturin, artemin and persephin
question of neogenin’s ligand remains tricky: despite its (Kotzbauer et al., 1996), four homologous neurotrophic
higher affinity for RGM, neogenin seems to be able to factors related to the transforming growth factor-b
mediate netrin-1 signaling in axon attraction (Wilson family. The receptor complex also includes (GPI)-
and Key, 2006) or cell adhesion (Srinivasan et al., 2003). anchored proteins GFRa1, 2, 3 and 4 that are required
Moreover, neogenin has also been proposed to mediate for RET dimerization and dictate ligand selectivity
netrin-3 signaling during myotube formation (Kang (Baloh et al., 2000; Scott and Ibanez, 2001). After
et al., 2004), as well as netrin-4 signaling in angiogenesis interaction with its ligands, RET undergoes autopho-
(Lejmi et al., 2008). sphorylation and then interacts with multiple effectors
Patched (Ptc) is a 12-transmembrane receptor that is such as phospholipase C, Shc, enigma, Grb2, Grb7/
part of the complex responsible for sonic hedgehog Grb10, Src kinase and Ras-GAP (Santoro et al., 1994;
(Shh) morphogen signaling (Figure 2 and Table 1). Shh Arighi et al., 1997; Lorenzo et al., 1997). Gain-
binds Ptc and thus abolishes the Ptc-repressing effect on of-function mutations of the RET gene have been
smoothened (Smo). Smo is a seven-transmembrane associated with multiple endocrine neoplasia type 2
receptor that activates downstream Gli transcription (MEN 2), an autosomal dominant inherited cancer
factors (Murone et al., 1999). Shh is a glycoprotein syndrome (Mulligan et al., 1993), whereas loss-of-
secreted by the notochord and floor plate during function mutations of RET have been associated with
development, after a ventro-dorsal concentration gra- Hirschsprung disease (aganglionosis, HSCR), a frequent
dient in the ventral neural tube. This gradient deter- congenital intestinal malformation (1 in 5000 live births)
mines the induction and specification of ventral neurons characterized by the absence of neural crest-derived
in the vertebrate neural tube (Jessell, 2000). In addition parasympathetic neurons of the hindgut (Edery et al.,
to its morphogen activity, Shh was also shown to be a 1994; Romeo et al., 1994). In vitro, MEN 2-associated
survival factor: indeed, Le Douarin and colleagues mutations lead to ligand-independent constitutive acti-
discovered that experimental withdrawal of Shh in vation of RET kinase activity either through covalent
chick embryos by partial destruction of the notochord dimerization of the receptor (MEN 2A) (Santoro et al.,
leads to massive cell death in the developing neural tube 1995) or through direct structural changes in its kinase
(Charrier et al., 1999, 2001). It was subsequently demon- domains (MEN 2B) (Songyang et al., 1995). In contrast,
strated that Shh functions as a survival factor by the mechanisms leading to the absence of intramural
inhibiting the apoptotic function of Ptc (Thibert et al., ganglion cells of the hindgut observed in HSCR remain
2003). Thus, Ptc is a DR and can signal independently incompletely understood. The observation that RET is
of Smo. involved in both cancer progression and nervous system
Integrins are the main receptors that mediate cellular development, similar to previously identified DRs, led to
interactions with extracellular matrix ligands such as the question as to whether it could also be one of them.
laminins, collagens and fibronectins (Hood and Cher- It was then shown that, in different settings, expression
esh, 2002). They are heterodimeric (ab) type I trans- of RET induced apoptosis in the absence, but not in the
membrane receptors, and provide a connection between presence, of glial cell line-derived neurotrophic factor
the matrix and the cytoskeleton. Integrins have tradi- (Bordeaux et al., 2000; Canibano et al., 2007).
tionally been considered as prosurvival receptors, on the Trk receptors (TrkA, B and C) are the main
basis of the concept of ‘anchorage dependence’ (Stupack neurotrophin receptors. TrkA is the receptor for NGF,
and Cheresh, 2002). Integrin-mediated adhesion sup- TrkB is the receptor for brain-derived neurotrophic
ports the formation of cytoskeletal and contractile factor and NT4/5, whereas TrkC is the receptor for NT3
elements, promotes cellular resistance to exogenous (Kaplan and Miller, 2000). The classic neurotrophic
apoptotic stimuli and facilitates signaling by trophic theory proposes that neuronal survival depends on the
factor receptors. Most cells require integrin-mediated presence of trophic factors such as neurotrophins (Levi-
adhesion to respond to trophic factors. This has led to Montalcini and Angeletti, 1963; Huang and Reichardt,
the proposal that controlling cell adhesion and geome- 2001), and that cell death, which occurs when these
try, thereby permitting responsiveness to survival factors become limited, is strictly due to loss of survival
factors, may be the critical function of integrins in signals. On neurotrophin binding, Trk receptors activate
maintaining cell viability. However, expression of PI3K/Akt and Ras/MAP kinase pathways that are
certain b3 or b1 integrins can also induce apoptosis, if thought to inhibit the naturally occurring death pro-
immobilized substrates are not available as ligands. gram in neurons (Kaplan and Miller, 2000). However, in
OncogeneA new paradigm in cell signaling and cancer therapy
D Goldschneider and P Mehlen
5
light of what was known about DRs, it was tempting to To close this list, it should be mentioned that a
hypothesize that neurotrophin binding served also to nontransmembrane receptor, androgen receptor (AR),
block an active apoptotic signal from Trks. Interest- has also been proposed to be a DR (Ellerby et al., 1999).
ingly, when Trk receptors were evaluated as possible AR is a member of the nuclear receptor superfamily of
DRs, TrkC was the only one the enforced expression of ligand-activated transcription factors. Binding of andro-
which induced cell death in the absence of ligand gens such as testosterone by the AR leads to nuclear
(Tauszig-Delamasure et al., 2007). Thus, TrkC is a DR, translocation and transcriptional activity. Gene regula-
whereas TrkA and B are not, suggesting that even a tion by the AR affects widespread processes such as
closely related receptor can acquire a different activity male gonadal development, cell survival and muscular
with regard to cell survival and apoptosis (Figure 2 and development, among many others (Lee and Chang,
Table 1). 2003). AR displays a profile similar to that of membrane
Anaplastic lymphoma kinase (ALK) tyrosine kinase is DRs; it is a caspase substrate and its expression induces
a member of the insulin receptor superfamily (Figure 2 apoptosis in the absence of ligand, whereas the addition
and Table 1). It was initially identified as part of the of ligand inhibits receptor-induced cell death (Ellerby
oncogenic nucleophosmin–ALK fusion protein resulting et al., 1999). Mutations in the AR are associated with
from the t(2;5) translocation that is frequently asso- both prostate cancer and neurodegeneration. Neurode-
ciated with anaplastic large-cell lymphoma (Morris generation-associated mutants give rise to Kennedy’s
et al., 1994). Nucleophosmin allows dimerization of disease, a syndrome associated with the degeneration of
the fusion protein, causing constitutive activation of motor neurons in the brainstem and spinal cord,
ALK kinase and downstream activation of phospholi- resulting in weakness and muscular atrophy. Interest-
pase C-g, PI3K, STATs and pp60c-src (Allouche, 2007). ingly, mutations associated with neurodegeneration
The native full-length ALK receptor is mainly expressed consist in expansion (430) of polyglutamine tracts
in discrete regions of the developing central and present in the N-terminal part of the receptor, whereas
peripheral nervous system (Iwahara et al., 1997). shortened tracts (p22) are associated with a greater
Mourali et al. (2006) forced ALK expression in cells of risk of developing prostate cancer (Nelson and Witte,
lymphoid and neuronal origin to investigate wild-type 2002; Clark et al., 2003). AR, similar to p75NTR, has been
ALK functions. They observed that ALK enhanced or quite forgotten as a DR. Paradoxically, the word
triggered apoptosis in these cells and that treatment with ‘dependence’ is frequently associated with AR in the
agonist antibodies mimicking ALK ligand prevented cell field of prostate cancer. Indeed, studies have led to the
death induction (Mourali et al., 2006). concept that prostate secretory epithelial cells require
Eph receptors constitute the largest family of tyrosine testosterone for survival, and the withdrawal of
kinase receptors and bind ligands called ephrins testosterone leads to apoptosis in these cells (Craft and
(Figure 2 and Table 1). Eph receptors regulate a diverse Sawyers, 1998). Thus, neoplastic prostate epithelial cells
array of cellular processes during development, such as are often treated by hormone deprivation because it
axon guidance, angiogenesis, or cell migration and leads to apoptosis as a result of their dependence on
positioning (Pasquale, 2005). More recently, some testosterone. It is quite regrettable that no link has ever
ephrin and Eph receptors have been found to affect cell been made between the dependency phenomenon of
death in neurogenic regions. Activation of EphA7 by prostate cancer cells and the fact that AR could behave
overexpressed ephrinA5 in the embryonic cortex re- as a DR.
sulted in neural progenitor apoptosis (Depaepe et al.,
2005). On the other hand, lack of ephrinB3 is associated
with apoptosis in the subventricular zone of adult mice DRs are caspase substrates
(Ricard et al., 2006), suggesting that its receptor could
function as a DR. EphA4 has been shown to function as The molecular hallmark of programmed cell death
a DR, the apoptotic activity of which is impaired by its (apoptosis) is the activation of caspases (Thornberry
ligand ephrinB3 (Furne et al., 2009). and Lazebnik, 1998). During apoptosis, caspases, which
Another tyrosine kinase receptor should probably be form a family of cysteine-dependent aspartate-directed
added to the list: MET, the receptor for hepatocyte proteases, can cleave a wide range of substrates, thereby
growth factor (Figure 2 and Table 1). MET is well inactivating survival and activating proapoptotic me-
known for its essential role in normal development and chanisms. Except for the fact that they induce apoptosis
cell survival. Interestingly, it was reported that MET in the absence of their ligand, the other most common
was cleaved under stress conditions by caspase, thereby characteristic of DR is that they are cleaved by caspases.
generating an apoptotic fragment (Tulasne et al., 2004). Receptor cleavage is important for apoptotic function,
As we will see below, caspase cleavage is one of the most as mutation of the cleavage site abolishes cell death
common traits for apoptotic signaling of DRs. More- induction. DCC, neogenin, Ptc, ALK, EphA4 are
over, hepatocyte growth factor inhibits this caspase cleaved once, roughly in the middle of their intracellular
cleavage and concomitantly apoptosis. Therefore, domain (Mehlen et al., 1998; Thibert et al., 2003;
although it is not clear whether MET is able to initiate Matsunaga et al., 2004; Mourali et al., 2006; Furne
apoptosis by itself in the absence of ligand, as other et al., 2009). The cleavage site of UNC5 receptors is very
DRs do, this receptor presents some striking similarities close to the plasma membrane (Llambi et al., 2001),
with DRs. whereas RET, Trkc and MET have two cleavage sites
OncogeneA new paradigm in cell signaling and cancer therapy
D Goldschneider and P Mehlen
6
Table 2 Conservation of caspase cleavage sites of DR among species
Homo Pan Bos Canis Mus Rattus Gallus Xenopus Xenopus Danio
sapiens troglodytes taurus familiaris musculus norvegicus gallus tropicalis laevis rerio
DCC LSVD LSVD NA LSVD LSVD LSVD NA NA LTVD No
UNC5B DITD DITD DITD DITD DITD DITD DITD NA DITD EITD
Neogenin CCTD CCTD CCTD CCTD CCTD CCTD PCAD? GPED? NA CTTD
Ptc PETD PETD PETD PETD PETD PETD NEDD? HEND? HEND? No
RET VSVD VSVD VSVD VSVD VPVD VSVD VSVD NA MSVD VAID
DYLD DYLD DYLD DYLD DYLD DYLD DYLD DYLD DYLD
TrkC SSLD SSLD SSLD SSLD SSLD SSLD SSLD NA NA NA
ILVD ILVD ILVD ILVD ILVD ILVD ILVD
EphA4 LEDD LEDD LEDD LEDD LEDD LEDD LEDD LEDD LEDD LEED
ALK DELD DELD DELD DELD DELD DELD DELD DEMD NA DELD
MET ESVD ESVD ESVD ESVD ESVD ESVD ESVD ESVD ESVD ESVD
DNAD DNAD No DNID DNID DNID DNTD No No SNLD?
Caspase cleavage sites of DR have been mapped by experiment on human or rodent proteins. This table lists these sites for various DRs and the
amino acids found at the same position in sequence from other species. Caspase cleavage sites are well conserved in mammals and variably
conserved in other vertebrates: for example, cleavage site of EphA4 is better conserved than those of neogenin or patched. In some cases, sequence
is not perfectly conserved but only a slight variation is observed between species (amino acid is shown in bold). In other cases an aspartate residue is
found at the correct position, but the surrounding residues are not conserved (shown in italic). No: means that no asparte residue has been found by
sequence alignment. NA: means not analyzed or not found in databases. Alignments have been performed using sequence from Ensembl or NCBI
databases.
(Bordeaux et al., 2000; Foveau et al., 2007; Tauszig- cleavage inhibition. In addition, ligand binding has been
Delamasure et al., 2007). The cases of p75NTR and proposed to have other structural effects, such as
integrins are less clear, as there is no solid evidence that receptor multimerization. In fact, with the exception of
caspase cleavage is needed for their proapoptotic effect. Ptc and integrins, most DRs display homo-multimeriza-
Interestingly, caspase cleavage sites of DRs seem to be tion properties in the presence of their respective ligand
conserved in mammals, variably in other vertebrates but (Wang et al., 2000; Manie et al., 2001; Stein et al., 2001;
never in orthologs in lower organisms, such as nematode Mille et al., 2009a). Initially described as being
or Drosophila (Table 2) (Mehlen and Thibert, 2004). important for positive signaling, receptor multimeriza-
These findings may suggest that the appearance as a tion also seems to have a role in blocking apoptosis
caspase substrate, and therefore the mediation of the induction. P75NTR exerts its proapoptotic effect as a
dependence state, is a relatively late event in the monomer, whereas multimerization abrogates this effect
evolution of these proteins. This may make sense, given (Rabizadeh et al., 2000). In the same way, results
the greater plasticity of the mammalian nervous system obtained with DCC and UNC5H2 showed that these
compared with those of invertebrates and the necessity receptors trigger cell death when their ligand-induced
for more complex and higher lifespan mammals to multimerization is hindered (Mille et al., 2009a). Netrin-
develop antitumor mechanisms. 1 has also been proposed to suppress the apoptotic
function of UNC5H2 by inducing interaction of the
receptor with the GTPase PIKE-L (Tang et al., 2008).
This interaction triggers the activation of PI3 kinase
Role of ligand binding signaling and consequently the inhibition of UNC5H2
proapoptotic function. Furthermore, recent data from
Another common feature of DRs is the inhibition of cristallography evidenced that the UNC5H2 intra-
apoptosis induction on ligand binding. The main cellular domain adopts an autoinhibited closed con-
hypothesis with regard to the role of ligand binding is formation. In this conformation, ZU5 and death
inhibition of the caspase cleavage. This hypothesis has domains bind to each other and are thus unable to
been partly confirmed for some DRs, UNC5H2 induce cell death. Netrin-1 is unable to prevent
(Tanikawa et al., 2003), TrkC (Tauszig-Delamasure apoptosis induced by the UNC5H2 mutant that has a
et al., 2007), EphA4 (Furne et al., 2009) and MET constitutive open conformation, which leads the authors
(Tulasne et al., 2004; Foveau et al., 2007). For other to suggest that netrin-1 somehow stabilizes the auto-
receptors, the effect of ligand on caspase cleavage inhibited conformation of UNC5H2 (Wang et al., 2009).
cannot be assessed because cleavage products have It must be noted that, for receptors accepting more
relatively short half-lives and are thus hardly detectable than one ligand, the antiapoptotic effect has not been
in vivo. On the other hand, ectopic expression of a demonstrated for all ligands. For example, only glial cell
truncated receptor, mimicking the caspase cleaved line-derived neurotrophic factor was shown to block
receptor, leads to apoptosis induction even in the RET-induced apoptosis (Bordeaux et al., 2000), no data
presence of ligand (Mehlen et al., 1998; Thibert et al., are available for neurturin, artemin and persephin,
2003; Matsunaga et al., 2004). This is an indirect although neurturin, similar to glial cell line-derived
argument in favor of a role for the ligand in caspase neurotrophic factor, was recently proposed to be a
OncogeneA new paradigm in cell signaling and cancer therapy
D Goldschneider and P Mehlen
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survival factor for parasympathetic neurons (Peterziel netrin-1, DCC recruits and activates caspase 9, thereby
et al., 2007). In the case of neogenin, the ligand allowing caspase 3 activation, but this process does not
antiapoptotic effect was reported only for chick RGM/ require cytochrome c release and subsequent formation
RGMa (Matsunaga et al., 2004) but not for other of an apoptosome (cytochrome c/apaf-1/caspase 9)
members of the RGM family or for netrin-1/3/4. EphA4- complex, as is the case in the classic mitochondrial
associated cell death induction is hindered by ephrinB3, pathway (Forcet et al., 2001). DCC does not interact
but not by its other known ligands, ephrinA1 and directly with caspase 9, hence it may recruit one or more
ephrinA4 (Furne et al., 2009). With regard to ALK, only adaptor proteins (Figure 3). One of them could be
ligand-mimicking antibodies have been used because the DIP13a (DCC-interacting protein 13-a), a protein
identity of the receptor’s physiological ligand is still a identified as an interactor of DCC ADD, and shown
matter of debate. Pleiotrophin and midkine, two heparin- to be important for DCC-induced cell death (Liu et al.,
binding growth factors, have been proposed to function 2002). However, the precise role of DIP13a in DCC-
as ALK ligand but these proteins fail to make a general triggered apoptosis remains quite obscure, as it does not
consensus. This controversy is further supported by the seem to mediate interaction of DCC with caspase 9 and
genetic identification in Drosophila melanogaster of an further studies performed on DIP13a, also known as
ALK ligand, jelly belly (jeb), which has no similarities APPL1 (adaptor protein containing PH domain, PTB
with pleiotrophin or midkine (Englund et al., 2003; Lee domain and leucine zipper motif 1), have not provided
et al., 2003; Loren et al., 2003). clear evidence for a role of this protein in apoptosis
Finally, in the particular case of integrins, the ligand induction. In addition, palmitoylation and lipid raft
must be an immobilized substrate ligand to block localization were reported to be a prerequisite for DCC
apoptosis as, contrary to other DRs, soluble ligand proapoptotic activity, both in vitro and in primary
is not sufficient to counteract cell death induction commissural neurons (Furne et al., 2006). Lipid rafts are
(Stupack, 2005). ordered membrane microdomains enriched in sphingo-
lipids and cholesterol, and are proposed to have an
important role in cell signaling, in particular through the
organization of surface receptors, signaling enzymes and
Proapoptotic signaling by DRs adaptor molecules into complexes at specific sites in the
membrane (Simons and Toomre, 2000; Hueber, 2003). It
DRs share the property of being caspase amplifiers; was then shown that DCC presence in lipid rafts is
indeed most of them fail to induce apoptosis in the required to allow caspase-9/DCC interaction, suggesting
presence of general caspase inhibitors such as zVADfmk that this caspase-activating complex occurs in and takes
(Mehlen and Thibert, 2004). The way that leads to advantage of lipid rafts.
caspase activation and amplification has begun to be The discovery of a caspase-activating complex re-
decrypted specifically for some of them. First, all DRs cruited to a DR was recently made for Patched. Ptc was
contain, in their intracellular part, a domain required for indeed found to interact through its ADD, and only in
apoptosis induction. This domain, the ADD (addiction/ the absence of its ligand Shh, with DRAL/FHL2 (Mille
dependence domain) (Bredesen et al., 1998), is required et al., 2009b). DRAL was already known to promote
and often sufficient for cell death induction. Caspase apoptosis through an unknown mechanism in a wide
cleavage, except for p75NTR and integrins, is thought to be variety of cells when overexpressed (Scholl et al., 2000)
responsible for unmasking the ADD. In most cases, and to interact with TUCAN, a CARD-containing
ADD is borne by the remaining membrane-anchored adaptor protein for caspases 1 and 9 (Stilo et al., 2002).
fragment. In the case of UNC5H, RET, TrkC and MET Mille and colleagues showed that the Ptc–DRAL
receptors, however, it is the cytosolic generated fragment association serves as a platform for recruiting TUCAN
that is proapoptotic. ADDs are usually unique regions (and/or NALP1, a protein closely related to TUCAN)
that are not structurally related to known protein and caspase 9 (Figure 4). This then allows caspase 9
functional domains. Two notable exceptions are p75NTR recruitment to Ptc and caspase 9 activation. The
and UNC5H receptors: in those two cases, two regions complex involving DRAL, TUCAN and caspase 9 was
corresponding to known functional domains are respon- named dependosome, by analogy to other known
sible for apoptosis induction. The first region is their caspase-activating complexes such as the apoptosome
death domain, which is structurally related to the death (comprising Apaf-1, cytochrome c and caspase 9), DISC
domain of receptors of the TNF receptor superfamily (comprising Fas, FADD and caspase 8), the PIDDo-
(Hofmann and Tschopp, 1995; Bredesen et al., 1998; some (comprising PIDD, RAID and caspase 2) and the
Llambi et al., 2001). The second region is the chopper inflammosome (comprising NALPs, ASC, caspases 1
domain for p75NTR (Coulson et al., 2000) and the ZU5 and 5). Additional studies are required to determine
domain for UNC5H (Williams et al., 2003). whether this dependosome is a common platform for
After ADD release/exposition, caspase amplification other DRs.
seems to be more or less direct, depending on receptors. In any case, if such a dependosome existed and were
In some cases, ADD recruits caspase-activating com- common to DRs, the initiator recruited caspase could
plexes that are different from those implicated in death not always be caspase 9. Indeed, Stupack et al. (2001)
receptors and in intrinsic mitochondrial classical elegantly showed that integrins, as DRs, trigger
apoptotic pathways. For example, in the absence of apoptosis through recruitment of caspase 8.
OncogeneA new paradigm in cell signaling and cancer therapy
D Goldschneider and P Mehlen
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Netrin-1
α
DIP13α
X
CAS
Cleavage 9
PAS
E
Caspase 3
Caspase 3
activation
Amplification of
receptor cleavage
Apoptosis
Figure 3 Model of cell death induction by DCC. In the presence of netrin-1, DCC is dimerized and interacts with procaspase 3.
Following ligand withdrawal, DCC becomes a monomer and is cleaved, possibly by bound caspase 3 or another activated protease.
Cleavage leads to ADD exposure and to its direct interaction with apoptotic partners such as DIP13a, the role of which remains
unclear, or to indirect interaction with caspase 9. Consequently, these interactions lead to caspase 9 activation, which in turn activates
caspase 3.
Shh
Shh
Cleavage
Caspase 3
activation
Amplification of
receptor cleavage
Apoptosis
Figure 4 Model of cell death induction by Ptc. Following ligand withdrawal, Ptc is cleaved by caspase (or by another activated
protease), thus allowing exposure of its ADD. Ptc recruits DRAL, which in turns recruits TUCAN (or NALP1) and caspase 9.
Formation of this complex leads to caspase 9 activation and consequently to caspase 3 activation.
The formation of a caspase-activating complex does fact, apoptotic pathways downstream of UNC5H3 and
not seem to be the only mechanism used by DRs to 4 have not yet been documented, and functional data are
trigger apoptosis. As an example, the mechanism of available only for UNC5H1 and 2. UNC5H1 induces
UNC5H-induced apoptosis has been documented. apoptosis by interacting with NRAGE (neurotrophin
Despite their structural homology, members of the receptor-interacting MAGE homolog) through its ZU5
UNC5H family seem to mediate their apoptotic signal domain (Williams et al., 2003). NRAGE possibly
by interacting preferentially with distinct partners. In transduces UNC5H1-induced apoptosis through at least
OncogeneA new paradigm in cell signaling and cancer therapy
D Goldschneider and P Mehlen
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Unc5H2
Netrin-1
Cleavage
ZU5 ZU5 ZU5
DD DD P 5
ZU
DAPK
DD
Amplification of
Closed DA
receptor cleavage
conformation PK
DAPK
P
Caspase 3
activation
Apoptosis
Figure 5 Model of cell death induction by UNC5H2/UNC5B. In the presence of netrin-1, UNC5H2 and UNC5B are dimerized, and
their intracellular domain adopts a close conformation in which ZU5 and death domains interact with each other. DAPK interacts
with the closed intracellular domain, but is in an inactive autophosphorylated state. Following ligand withdrawal, UNC5H2 receptor
becomes a monomer, whereas the intracellular domain undergoes both caspase (or another protease) cleavage and opening/
dissociation of ZU5 and DD in parallel. Relation and chronology between cleavage and opening remain unclear, but these
modifications should allow interaction between the DD of UNC5H and DAPK, thus leading to DAPK activation and initiation of an
apoptotic program. Precisely how activated DAPK induces apoptosis remains to be determined.
two pathways: one implicating the degradation of the UNC5H2 through their non-‘death domain’-interacting
caspase inhibitor XIAP, and the other implicating regions, whereas, in the absence of netrin-1, DAPK
activation of the proapoptotic JNK signaling pathway. interacts with the death domain of UNC5H2,
NRAGE can also interact with UNC5H2 and 3, but which allows DAPK activation (Figure 5). This hypoth-
with a much weaker binding affinity. On the other hand, esis strongly fits with the more recent study by
UNC5H2 triggers apoptosis mainly by recruiting the Wang et al. (2009), arguing that UNC5H2 can adopt a
serine/threonine death-associated protein kinase closed conformation, preventing association of the
(DAPK) (Llambi et al., 2005). UNC5H2 and DAPK death domain with other proteins. Downstream
bind each other in part through their respective death effectors of DAPK in UNC5H2-mediated cell death
domain, but not only so, as deletion of these domains is remain to be identified, but DAPK is already known to
not sufficient to abrogate their association. Surprisingly, trigger cell death through p53-dependent and -indepen-
although UNC5H2-mediated induction of DAPK ac- dent mechanisms. Moreover, phosphorylation of the
tivity is observed only in the absence of netrin-1 and myosin light chain by DAPK leads to membrane
requires UNC5H2 caspase cleavage, DAPK seems to blebbing, a hallmark of programmed cell death
interact constitutively with UNC5H2, that is, not only (Gozuacik and Kimchi, 2006). Interestingly, it was
in the absence of netrin-1. Indeed, DAPK is known to recently suggested that UNC5H2 was not the only DR
be capable of autophosphorylation, which inhibits to trigger apoptosis through DAPK, as neogenin was
its activity by inducing a conformational change also shown to interact with DAPK and to require
(Shohat et al., 2001). On this basis, and according to DAPK for apoptosis induction (Fujita et al., 2008), in
their observations, Llambi and colleagues proposed the same way that DCC, UNC5H1, 2 and 3 also require
that, in the presence of netrin-1, DAPK is in an inactive lipid raft association to induce cell death (Maisse et al.,
autophosphorylated state, and it interacts with 2008).
OncogeneA new paradigm in cell signaling and cancer therapy
D Goldschneider and P Mehlen
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The fact that DRs are at the same time caspase TrkC-induced apoptosis in the classic neurotrophin
substrates and caspase activators/amplifiers points out a theory. The classic dogma suggests that each neuron is
paradox. How can a receptor that requires caspase moving toward death unless a survival/neurotrophin
cleavage to be a proapoptotic molecule participate in signal is provided. The integration of the DR notion
apoptosis induction? One possibility could be the within this neurotrophin theory would then be that each
initiation of DR cleavage by a noncaspase protease, neuron is actively pushed toward apoptosis by a DR,
which would be sufficient to generate a caspase such as TrkC, in the context of ligand limitation,
amplification loop. Another view could be that caspases whereas ligand presence not only activates survival
are never completely inactive, even in nonapoptotic signals but also blocks the active process of cell death.
cells, and that this residual caspase activity is sufficient The idea of TrkC being a DR is particularly attractive
to detect receptors disengaged from their ligand. while analyzing data from knockout mice for neuro-
Interestingly, caspase 3 was observed to interact with trophins and their respective receptors. Indeed, inactiva-
DCC, downstream of its cleavage site, only in non- tion of TrkA or NGF in mice results in the same amount
apoptotic conditions, that is, in the presence of netrin-1, of sensory neuron loss at birth (that is, nociceptive
or when the DCC caspase cleavage site is mutated neurons) (Crowley et al., 1994). Similarly, inactivation
(Forcet et al., 2001). It is tempting to speculate that, of either TrkB or brain-derived neurotrophic factor
when dimerized in the presence of its ligand, DCC results in an equivalent loss of mechanoceptive neurons
adopts a conformation that prevents its cleavage by (Ernfors et al., 1994; Minichiello et al., 1995). On the
caspases, whereas it can be efficiently cleaved as a other hand, neonates invalidated for TrkC present a loss
monomer in the absence of ligand. This event results in of 30% DRG neurons, whereas NT-3/ neonates have
unmasking its ADD, thus allowing capase 9 activation lost 70% of them (Tessarollo et al., 1994, 1997). This is
and caspase 3 amplification. in agreement with the view that TrkC, contrary to TrkA
and B, can trigger, when deprived of ligand, an active
apoptotic signaling. A confirmation that TrkC apopto-
tic signaling controls the fate of sensory neurons is
Role of DRs during embryonic development provided by an experience in which microinjection in
sensory neurons of a mutated intracellular domain of
Before being identified as DRs, all members of this TrkC, known to interfere with TrkC apoptotic function,
functional family were already known to be implicated dramatically enhanced survival of NT-3-deprived neu-
in nervous system development. Implications of their rons (Tauszig-Delamasure et al., 2007).
positive signaling in the presence of their ligand are The ligand/DR pair ephrinB3/EphA4 was also
largely documented, whereas a role for negative recently demonstrated, by studying knockout mice, to
proapoptotic signaling long remained essentially spec- have an important role in regulating cell number in an
ulative. However, several lines of evidence now accu- adult mouse subventricular zone through apoptosis
mulate to further support the view that DRs participate modulation. Indeed, extinction of eprhinB3 results in
in nervous system development through their proapop- increased apoptosis in subventricular zone, whereas the
totic function as well. Because of this ability to trigger absence of EphA4 results in an excessive number of
apoptosis in settings of ligand absence or limitation, neuroblasts in this zone (Furne et al., 2009). In addition,
they were hypothesized to control cell numbers in some infusion of soluble ephrinB3 into the lateral ventricle
specific areas of the developing brain and to dictate reduced cell death. Thus, it is tempting to speculate that
adequate territories of neuron migration and axon EphA4, as a DR, is important in regulating the fate of
projection by eliminating those localized out of regions neuronal stem cells during brain development.
of ligand availability. For example, netrin-1 receptors do An elegant study from Palmer and colleagues
not only mediate the chemotropic effect of netrin-1 in evidenced a role for the ALK/jeb pair in the Drosophi-
the developing nervous system but also seem to regulate la-developing visual system. ALK is expressed and
the survival of olivary neurons, as these cells, known to required in target neurons in the optic lobe, whereas
express DCC and UNC5H receptors, display increased jeb is primarily generated by photoreceptor axons and
apoptosis in netrin-1/ mice (Llambi et al., 2001). functions in the eye to control target selection of specific
Moreover, netrin-1 functions as a survival factor for photoreceptor cell axons. Interestingly, the level of
spinal cord commissural neurons, which was shown in neuronal cell death (measured by active caspase 3 level)
both primary neuron cultures and animal models (Furne in the ALK expressing optic lobe medulla increases in
et al., 2008). mutants lacking an expression of jeb. Moreover,
Similarly, it was shown that Shh, the ligand of DR Ptc, caspase-dependent neuronal apoptosis dramatically
is not only a morphogen but also a survival factor decreases in mutants overexpressing jeb (Bazigou
(Charrier et al., 1999, 2001). Interestingly, it was et al., 2007). These results suggest that ALK could have
demonstrated that Shh functions as a survival factor by a role in the physiological negative selection of neurons
inhibiting the apoptotic function of Ptc and that this shaping the optic lobe in the Drosophila nervous system
proapoptotic function is crucial for adequate neural tube by favoring apoptosis in the absence of the ALK ligand.
development (Thibert et al., 2003; Mille et al., 2009b). Interestingly, even though the data reported so far on
The recent observation that neurotrophin receptor the role of DRs during development seem to be linked to
TrkC is a DR also led to questioning the implication of nervous system development, it has recently been
OncogeneA new paradigm in cell signaling and cancer therapy
D Goldschneider and P Mehlen
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unbound receptor
apoptotic cancer cell
bound receptor cancer cell
normal cell
Figure 6 DRs and tumor suppression. Normal cells located in their normal environment express DRs that are bound to ligands
supplied by local source. In contrast, in this limited and constant ligand concentration environment, highly proliferative tumor cells
display some unbound DRs and thus undergo apoptosis. In the case of metastatic tumor cells escaping from the primary site, migration
away from locally supplied ligands leads to apoptosis because of unbound receptors. DRs could thus counteract tumor growth and/or
invasiveness. Loss of expression of these receptors or overexpression of ligands would represent a selective advantage for cancer cells.
proposed that these DRs may also be involved outside on chromosome 18q, DCC is submitted to loss of
the developing nervous system. Along this line, netrin-1 heterozygosity in over 70% of colorectal cancers
was recently shown to control the survival of endothelial (Fearon et al., 1990). DCC is also submitted to loss of
cells and to promote angiogenesis, at least in part, by heterozygosity and/or to decreased expression in various
blocking apoptosis triggered by its unbound UNC5H2 other cancers including gastric, prostate, endometrial,
receptor (Castets et al., 2009). The DR activity of ovarian, esophageal, breast and testicular cancer, as well
UNC5H2 can indeed conciliate conflicting results as neuroblastoma and hematological malignancies
regarding the implication of netrin-1 in angiogenesis (Mehlen and Fearon, 2004). Loss of DCC expression
(Lu et al., 2004; Wilson et al., 2006). is often associated with poor prognosis and advanced
cancer or metastasis (Shibata et al., 1996; Saito et al.,
1999), suggesting a role of DCC loss in cancer
progression rather than in cancer initiation. Moreover,
DRs are altered during tumor progression restoration of DCC expression can suppress tumorigenic
growth properties in vitro or in nude mice (Klingelhutz
In addition to a role during embryonic development, the et al., 1993; Velcich et al., 1999; Kato et al., 2000;
DR model also predicts a role for such receptors as Rodrigues et al., 2007). On the other hand, the fact that
tumor suppressors, because of their ability to promote only 10–15% of colon cancers carry mutations in DCC
cell death when disengaged from their ligand. A tumor and the lack of a tumor predisposing effect of DCC
cell submitted to an abnormal environment (highly inactivation in mouse models (Fazeli et al., 1997) led
proliferative cells in an environment with limited and some investigators to conclude that DCC had little or no
constant ligand concentration or metastatic cells migrat- biological role in colon cancer, and that its inactivation
ing to sites in which ligand is absent) would display was an epiphenomenon. However, such a categoric
unbound DRs and thus undergo apoptosis (Figure 6). judgment was partly due to the lack of understanding of
This mechanism would represent an alternative safe- the biological roles of DCC, which has since been
guard mechanism to limit tumor progression. It is then compensated by its characterization as a DR (Grady,
expected that in aggressive tumors, tumor cells have to 2007).
turndown this DR pathway. Consistent with this view, a In the same way, the UNC5H receptor family is
loss of receptor expression would then represent a downregulated in human cancers, including colorectal,
selective advantage for tumor cells and seem to be a breast, ovary, uterus, stomach, lung or kidney cancers
primary method to overcome this safeguard mechanism. (Thiebault et al., 2003). Recently, two studies have
Since its discovery in the 1990s, DCC has been focused on UNC5H3/UNC5C alteration in colorectal
suspected to be a tumor suppressor gene, even though carcinoma. UNC5C is indeed the most downregulated
no definitive evidence has been proposed so far. Located member of the UNC5H family (74–77% of cases,
OncogeneA new paradigm in cell signaling and cancer therapy
D Goldschneider and P Mehlen
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whereas UNC5H1/UNC5A and H2/B show a reduced expression by small interfering RNA or netrin-1
expression in 48 and 27% of the cases, respectively). titration by decoy soluble receptor ectodomain causes
This loss of expression observed in human primary apoptosis and prevents metastasis formation both in a
tumors, as well as in cell lines, is essentially due to syngenic mouse model and in a xenograft model
promoter methylation (Bernet et al., 2007; Shin et al., (Fitamant et al., 2008). In the same way, high levels of
2007). Furthermore, Bernet and colleagues took advan- netrin-1 were detected in almost 50% of non-small-cell
tage of a natural UNC5H3 loss-of-function occurring in lung cancer and in a large fraction of aggressive
mice (rcm, rostral cerebellar malformation) to demon- neuroblastoma. As in the breast cancer study, strategies
strate that UNC5H3 loss of function is associated with disrupting the netrin-1 autocrine loop led to apoptosis
tumor progression: mice that carry the APC1638N germ- induction and tumor growth inhibition in xenografted
line mutation, known to predispose mice to the models (Delloye-Bourgeois et al., 2009a, b). In these
development of low-grade adenoma (Sieber et al., three cases, apoptosis resulting from netrin-1 inhibition
2000), and are heterozygous or homozygous for mutant seems to be mediated by UNC5H receptors, rather than
UNC5H3, develop adenomas that progress to adeno- by DCC. Interestingly, in the case of aggressive
carcinoma at a higher frequency than what is seen in neuroblastomas, netrin-1 expression levels were found
APC1638N mice. Interestingly, loss of UNC5H3 function to have prognosis significance. Aggressive stage 4
in mice also correlates with apoptosis reduction in mice metastatic neuroblastoma is divided into three groups:
tumors. Another clue in favor of a role of the UNC5H stage 4 in children aged more than 1 year has the worse
family in cancer is that UNC5B and D are p53 target prognosis, whereas stage 4S and stage 4 in children less
genes, able to mediate part of p53 proapoptotic activity than 1-year old generally have a more favorable
(Tanikawa et al., 2003; Wang et al., 2008). prognosis, even though many infants succumb to
Patched is also a known tumor suppressor (Stone disease. High levels of netrin-1 were shown to correlate
et al., 1996). Inactive mutations of Ptc, as well as loss of with adverse outcome of stage 4S and stage 4 (o1 year).
expression, are found in basal cell carcinoma and A study by Link et al. (2007), reporting that netrin-1
medulloblastoma (Wicking and McGlinn, 2001). Ptc expression has significant impact on the overall survival
expression inhibits the hallmarks of cell transformation of patients with poorly differentiated pancreas tumors,
in vitro (Koike et al., 2002) and, interestingly, Ptc also completes this description of netrin-1 upregulation in
inhibits growth in soft agar of transformed cells. This is human neoplasias. Although the mechanism for auto-
linked to its proapoptotic function, as growth inhibition crine production of netrin-1 remains to be determined, it
does not occur in the presence of Shh, or of a general could be at least in part a result of nuclear factor-kB
caspase inhibitor, or when Ptc is mutated on its caspase activation, as netrin-1 is a direct target gene of this
cleavage site (Thibert et al., 2003). However, there is no transcription factor (Paradisi et al., 2008). Moreover,
strong in vivo evidence so far that Ptc functions as a according to the well-admitted link between inflamma-
tumor suppressor because it triggers apoptosis. tion and colorectal cancer predisposition, it has been
There is a wide spectrum of data supporting the role suggested that nuclear factor-kB activation resulting
of most DRs as tumor suppressors. As an example, from inflammatory stimulus could lead to local netrin-1
EphA4 is downregulated in invasive forms of breast production, and thus to tumor promotion by apoptosis
cancers (Fox and Kandpal, 2004), in liver and kidney inhibition. Along this line, colorectal tumor formation
cancers (Hafner et al., 2004) and in metastatic melano- in an animal model for chronic inflammation was
mas (Easty et al., 1997), whereas a progressive decrease inhibited by treatment with netrin-1 titrating agents
in p75NTR expression is described in prostate cancers (Paradisi et al., 2009).
(Pflug and Djakiew, 1998). TrkC is associated with good Finally, a third possible way for tumor cells to escape
prognosis in several cancers (Yamashiro et al., 1997). the proapoptotic activity of DRs would be to inactivate
However, the tumor suppressive functions of these their downstream signaling pathways. Notably, three
receptors are yet to be demonstrated per se. effectors of DRs display functional inactivation in
Whereas DR loss during tumorigenesis occurs in a human cancers: DAPK, DRAL and caspase 8, which
wide fraction of cancers, another selective advantage for transduce UNC5H2-, Ptc- and integrin-mediated apop-
tumor cells would be to constitutively overexpress tosis, respectively. DAPK loss of expression, essentially
ligand. There is now accumulating evidence with regard through promoter methylation, has now been described
to netrin-1 to support this idea. Indeed, forced expres- in a wide variety of cancers, including lymphoma,
sion of netrin-1 in the digestive tract of transgenic mice leukemia, brain tumors, bladder, breast, renal, cervix,
has been associated with decreased apoptosis in the prostate and colorectal carcinomas (Kissil et al., 1997;
intestine, development of advanced adenomas and Raveh and Kimchi, 2001; Gozuacik and Kimchi, 2006).
tumor progression to adenomacarcinoma in a setting Moreover, DAPK loss of expression is associated with a
of adenoma predisposition (Mazelin et al., 2004). More more malignant tumor phenotype and increased meta-
recently, high levels of netrin-1 were detected in a large static capacity. DAPK is absent in highly metastatic
panel of human cancers from distinct organs, and variants of mouse lung cancer cell lines, and is present in
netrin-1 overexpression was correlated with a blocking the low metastatic variants of those same cell lines
of the proapoptotic functions of netrin-1 receptors. (Inbal et al., 1997). In lung and head and neck cancers,
First, in breast cancer, netrin-1 was shown to be a DAPK promoter methylation was associated with
marker of metastatic disease: decrease in netrin-1 aggressive disease and poor survival (Sanchez-Cespedes
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