Netrin-1, a missing link between chronic inflammation and tumor progression.

Abstract

Netrin-1 discovered as a neuronal navigation cue, has been recently proposed to play a crucial role during colorectal tumorigenesis by regulating apoptosis. This survival activity is mediated via the inhibition of the so-called netrin-1 dependence receptors. The netrin-1 receptors, DCC (for Deleted in Colorectal Cancer) and UNC5H (UNC5 homologues), indeed belong to the functional family of dependence receptors that share the ability to induce apoptosis in the absence of their ligands and such a trait has been hypothesized to confer these receptors a tumor suppressor activity as their presence render cell survival dependent on ligand availability. As a consequence, human tumors show either a loss of dependence receptors or a gain of netrin-1, allowing tumors to escape this safeguard mechanism. We recently found that netrin-1 is a direct transcriptional target of the transcription factor NFκB, and that a fraction of colorectal tumors show a netrin-1 gain parallel to NFκB activation. Moreover, colorectal cancers from patients affected by inflammatory bowel diseases (IBD) show upregulation of netrin-1. Several evidences suggest a tight link between chronic inflammation and tumorigenesis, mainly through NFκB activation. We propose that induction of netrin-1 expression via NFκB in IBD patients could affect colorectal tumor promotion and progression and that inhibition of netrin-1 could be an innovative target for drug therapy in inflammation-driven colorectal cancers.

Full text

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www.landesbioscience.com Cell Cycle 1253
Cell Cycle 9:7, 1253-1262; April 1, 2010; © 2010 Landes Bioscience
extrA view
extrA view
Key words: chronic inflammation,
apoptosis, colorectal tumorigenesis, gene
expression, drug therapy, dependence
receptors
Submitted: 12/16/09
Accepted: 12/28/09
Previously published online:
www.landesbioscience.com/journals/cc/
article/11072
*Correspondence to: Patrick Mehlen;
Email: mehlen@lyon.fnclcc.fr
N
proposed to play a crucial role during
colorectal tumorigenesis by regulating
apoptosis. This survival activity is medi-
ated via the inhibition of the so-called
netrin-1 dependence receptors. The
netrin-1 receptors, DCC (for Deleted in
Colorectal Cancer) and UNC5H (UNC5
homologues), indeed belong to the func-
tional family of dependence receptors
that share the ability to induce apopto-
sis in the absence of their ligands and
such a trait has been hypothesized to
confer these receptors a tumor suppres-
sor activity as their presence render cell
survival dependent on ligand availability.
As a consequence, human tumors show
either a loss of dependence receptors or
a gain of netrin-1, allowing tumors to
escape this safeguard mechanism. We
recently found that netrin-1 is a direct
transcriptional target of the transcrip-
tion factor NFκB, and that a fraction of
colorectal tumors show a netrin-1 gain
parallel to NFκB activation. Moreover,
colorectal cancers from patients affected
by inflammatory bowel diseases (IBD)
show upregulation of netrin-1. Several
evidences suggest a tight link between
chronic inflammation and tumorigen-
esis, mainly through NFκB activation.
We propose that induction of netrin-1
expression via NFκB in IBD patients
could affect colorectal tumor promotion
and progression and that inhibition of
netrin-1 could be an innovative target
for drug therapy in inflammation-driven
colorectal cancers.
etrin-1, discovered as a neuronal
navigation cue, has been recently
Introduction
Netrin-1 is a laminin-related molecule
initially discovered as a diffusible mol-
ecule produced by a ventral structure
in the developing spinal cord, the floor
plate, which attracts commissural axons.1
Netrin-1 was thus shown to act as a
chemoattractive or chemorepulsive cue for
many migrating axons and neurons dur-
ing the development of nervous system.
This effect is regulated by the interac-
tion with its main receptors, DCC (for
Deleted in Colorectal Cancer)2-4 and
UNC5H (UNC5 homologue).5,6 DCC
is a type I transmembrane glycoprotein
protein of roughly 175–190 kDa with a
single membrane spanning domain. The
sequences present in DCC’s large extra-
cellular domain of roughly 1,100 amino
acids bear strong similarity to those found
in neural cell adhesion molecule (NCAM)
protein family members, and include four
immunoglobulin-like domains and six
fibronectin type III-like motifs (Fig. 1).
The DCC cytoplasmic domain of roughly
325 amino acids shows little similarity to
proteins with well-established functions.
UNC5H receptors regroup actually a fam-
ily of 4 receptors: UNC5H1, UNC5H2,
UNC5H3 and UNC5H4 also called
UNC5A-B-C-D in human. These recep-
tors are type I transmembrane receptors
composed of two immunoglobulin-like
(Ig-like) domains, two thrombospondin
type II domain, a transmembrane domain,
a Zu-5 domain (homologous to Zo-1 pro-
tein) and a Death Domain (Fig. 1). Both
DCC and UNC5H have been shown to
transduce navigation signals to neurons or
axons in response to netrin-1.
Netrin-1, a missing link between chronic inflammation and tumor
progression
Andrea Paradisi and Patrick Mehlen*
Apoptosis, Cancer and Development Laboratory—Equipe labellisée ‘La Ligue’; CNRS UMR5238; Université de Lyon; Centre Léon Bérard, Lyon

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1254 Cell Cycle volume 9 issue 7
a result of netrin-1-dependent cell death
inhibition in the intestinal epithelium.7 In
the same line, a netrin-1 gain has recently
been described in several human can-
cers, such as colorectal cancer,22,23 meta-
static breast cancers,24 lung cancer,25 and
neuroblastoma26 and this netrin-1 over-
expression has been shown as a selective
advantage for tumor progression.23-26 The
netrin-1/dependence receptors pair repre-
sents therefore a relevant mechanism to
control tumor development and cellular
escape. While several evidences show that,
at genetic or epigenetic level, the down-
regulation of dependence receptor expres-
sion occurs through loss of heterozygosity
or promoter methylation,17,20,27 the mecha-
nism allowing netrin-1 upregulation is
not completely understood. Recently, we
found that netrin-1 is a transcriptional
target of NFκB,22 a master transcription
factor that is well known to link cell sur-
vival and tumor development. Here we
describe the regulation of the netrin-1
gene expression by NFκB, and more spe-
cifically during inflammation-driven col-
orectal tumorigenesis.
is that the expression of such a receptor rep-
resents a protective mechanism that limits
tumor development through apoptosis
induction of tumor cells that would grow
or migrate beyond the regions of ligand
availability. Consequently, cell transfor-
mation toward malignant/metastatic phe-
notype is associated with the constitutive
inhibition of the death signal induced by
these receptors and this can be achieved by
at least three mechanisms: either the loss
of dependence receptor expression, the
loss/mutation of the death signaling above
these receptors or the autocrine expression
of the ligand. This has been demonstrated
per se for netrin-1 dependence receptors.
Indeed, DCC expression is lost in a large
fraction of human cancers,17,18 as well as
UNC5H genes, hence suggesting that the
loss of these receptors represents a selec-
tive advantage for tumor development.19-21
Along this line, UNC5H3 mutant mice
show increased intestinal tumor progres-
sion.20 Moreover, in mice the targeted
overexpression of netrin-1 throughout
the digestive tract increases initiation and
promotion of intestinal tumorigenesis, as
However, in the last past years netrin-1
has been proposed as a completely dif-
ferent molecule regulating cell survival
and modulating tumorigenesis.7 Indeed,
netrin-1 receptors DCC and UNC5H
belong to a new family of cellular receptor,
the so-called “dependence receptors”.8,9
Such receptors, which include also the low-
affinity neurotrophin receptor p75NTR,10
RET,11 TrKC,12 Patched13 and neogenin14
initiate two completely opposite signal-
ing pathways: when ligand is available,
these receptors transduce a positive signal
leading to cellular proliferation, differen-
tiation, migration or survival. However, in
the absence of their ligand, these receptors
are not inactive, like “classical” receptors,
but rather induce a “negative signaling”
that triggers caspase-dependent apoptotic
cell death (Fig. 1).15 Then, the expression
of the dependence receptors within a cell
renders this cell dependent on the presence
of the ligand for its survival in the cellular
environment (reviewed in refs. 15 and 16).
It is interesting to note that most of the
known dependence receptors are candidate
tumor suppressors. The overall hypothesis
Figure 1. Netrin-1 receptors and the dependence receptor notion. Netrin-1 transduces a “positive” signal when bound to its receptors DCC and
UNC5H. the different receptor domains are indicated. Netrin-1 binds DCC on the 4th and 5th fibronectin-like domains, and UNC5H on the two ig-like
domains. in the presence of netrin-1, DCC and UNC5H dimerize and activate several pathways involved in cell survival, proliferation and axon guid-
ance. in the absence of netrin-1, both receptors monomerize and induce “negative” signaling pathways that trigger caspase-dependent apoptosis. ig,
immunoglobulin; DCC, Deleted in Colorectal Cancer.

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role of TLR signaling in maintaining
the intestinal homeostasis is one of the
emerging themes in innate immunity in
the gut.31 In normal settings, TLR recep-
tors protect the intestinal epithelial bar-
rier and confer commensal tolerance. In
abnormal settings, aberrant TLR signal-
ing may stimulate different inflammatory
responses leading to acute and chronic
intestinal inflammation.42 Along this
line, although TLR receptors are weakly
expressed and detected in primary IECs,
thus minimizing recognition of luminal
bacteria in the healthy intestine,43,44 TLR4
is increased in primary IECs during active
UC.45 It has also been shown that changes
in DC function may also contribute to the
pathogenesis of IBD.46 With respect to
NODs, cytoplasmatic proteins that were
among the first NLRs reported, a strong
association of Nod2 gene mutations and
Crohn’s disease was reported47,48 even if
the mechanisms by which Nod2 muta-
tions confer an increased susceptibility
to Crohn’s disease are unknown. It has
been shown that patients carrying Nod2
polymorphisms have increased levels of
antibodies against luminal microbes,49,50
suggesting that defective handling of the
luminal flora plays a role in IBD.
NFκB as a Link between
Inflammation and Cancer
One of the key players in inflammatory
process is nuclear factor κB (NFκB) tran-
scription factor family, composed by a
number of closely related protein dimers
that bind a common sequence motif. So
far, five distinct NFκB subunits have been
identified in mammalian cells: NFκB1
(p50/p105), NFκB2 (p52/p100), RelA
(p65), RelB and c-Rel.51,52 The different
heterodimers bind to specific promoter
and initiate transcription of a wide range
of genes involved in inflammation, cell
death, survival and tissue repair.53 In rest-
ing cells, NFκB dimers are sequestered in
the cytoplasm by members of the inhibi-
tor of κB (IκB) family, structurally related
proteins, among which the isoform IκBα
is the most abundant.54 The classical and
the alternative pathways represent the two
distinct known NFκB activation path-
ways.55 The former is triggered mainly
by bacterial and viral infection, through
contains a very large number of commen-
sal bacteria. As the intestinal mucosa holds
the highest number of immune cells in the
body,39 it is not surprising that the gut
constitutes the largest lymphoid organ in
the body.38 Indeed, the intestinal immune
system has to prevent infection while
avoiding the development of destructive
inflammatory responses to the normal
microbiota. This requirement is possible
as a result of the intestinal epithelium cel-
lular composition, constituted primarily
by intestinal epithelial cells (IECs), which
form an impermeable barrier between the
body and the luminal contents. Besides
their role in importing luminal nutrients,
IECs play an active role in immunity by
producing antimicrobial peptides and
pro-inflammatory cytokines following
activation of PRRs by microbes.40 The
importance of the IEC dual function
against pathogens (i.e., direct antimicro-
bial function and a sentinel function to
alert the immune system) could be appreci-
ated by several mouse models showing that
disrupted signaling into IECs increases
susceptibility to intestinal inflammation.40
In addition to the immune function pro-
vided by IECs, populations of immune
cells can be found within the intestinal
tissue, such as dendritic cells (DCs) that
sample the gut luminal contents,41 and
intraepithelial lymphocytes (IEL)38 resid-
ing in the lamina propria, a layer of con-
nective tissue supporting the epithelium.
The interplay between IECs and inflam-
matory cells is likely to be a crucial process
during inflammation-associated tumor
development. This is particularly true for
inflammatory bowel diseases (IBD), such
as ulcerative colitis (UC) and Crohn’s
disease, that are characterized by chronic
intestinal inflammation induced by dereg-
ulated response to the intestinal flora.38
IBD patients show a major risk of colorec-
tal cancer and, even if the mechanisms
that link these chronic inflammatory state
and tumorigenesis are partially unknown,
it has been suggested that pathogenic
microbes initiate human IBD. Since IBD
is associated with inappropriate inflamma-
tion in the presence of normal commensal
bacteria, several groups have considered
the possible involvement of TLRs and
NODs, the two major forms of innate
immune system sensors. The important
Intestinal Inflammation and
Colorectal Cancer
Inflammation is a fundamental response to
the loss of cellular and tissue homeostasis
due to stress, injury and infection.28,29 An
inflammatory site is characterized by local
expression of pro-inflammatory cytok-
ines, chemokines and adhesion molecules,
which regulate the coordinated recruit-
ment of leukocytes to the site of infec-
tion or injury.30 The first line of defense
against pathogens and tissue damage is
the innate immune system.31 Initiation of
the innate response for microbial infec-
tion is triggered by pathogen recognition
receptors (PRRs), such as the membrane-
bound Toll-like receptors (TLRs) and the
cytoplasmatic receptors that belong to the
nucleotide binding and oligomerization
domain (NOD)-like receptor (NLR) fam-
ily.32,33 A normal inflammatory response
has to be self-limited and involves the
downregulation of
gene expression and increased expression
of anti-inflammatory genes. The molecu-
lar mechanisms that regulate the resolu-
tion of inflammation are not completely
understood, but it has been shown that
persistent inflammation increases the risk
of cancer development.30 During the 19th
century, the German pathologist Rudolf
Virchow first noted leukocytes in cancer
tissue and first reported a possible link
between inflammation and cancer.34 This
association has been confirmed by a sub-
stantial number of epidemiologic, gene
association and molecular studies, lead-
ing to the elucidation of the precise role of
inflammation in cancer. Indeed, it appears
that chronic inflammation stimulates
tumor development and plays a critical
role in initiating, sustaining and promot-
ing tumor growth.35,36 Consistent with
this hypothesis, many of the receptors
and cytokines involved in innate immune
responses, such as TNFα, IL-1, IL-6 and
IL-8, have also been linked to tumor pro-
gression processes.
Among the different organ systems
affected by chronic inflammation, the
gastrointestinal tract represents an impor-
tant entry site for pathogens, since it is
the largest surface of contact between the
body and the external environment.37,38
Furthermore, the gastrointestinal lumen
pro-inflammatory

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1256 Cell Cycle volume 9 issue 7
angiogenesis and metastasis—such as
MMPs, adhesion molecules and vascu-
lar endothelial growth factor (VEGF).81
Therefore, it was reasonable hypothesis to
predict that NFκB activation in chronic
inflammation could also promote tumor
development.82 Cancer is the result of
uncontrolled cellular growth that occurs
together with the invasion of surrounding
tissue and the spread of malignant cells.52
Tumorigenesis can be divided into three
phases: initiation, promotion and progres-
sion.83 Initiation involves genomic altera-
tions (by chemical or physical agents),
leading to the activation of oncogenes
and/or the inactivation of tumor-suppres-
sor genes. Promotion is characterized by
clonal expansion of transformed cells, due
to increased proliferation and/or reduced
cell death. Progression is characterized by
an increase in size tumor, invasion and
metastasis. It is now well accepted that
inflammation and NFκB could affect
each of these three stages, holding pro-
tumorigenic effects. In chronic inflamma-
tory diseases, such as IBD, it is thought
that pro-inflammatory factors cause accu-
mulating DNA damage in quiescent pre-
malignant cells, e.g., IECs in the case of
intestinal chronic inflammation, thereby
pushing these cells over the threshold
to become malignant. In the same time,
NFκB activation that could be due to
PRR engagement, carcinogens or follow
oncogene activation, prevents apoptosis
of pre-malignant cells, while in inflam-
matory cells, such as macrophages, DCs
and neutrophils, recruited to the tumor
microenvironment, triggers the produc-
tion of cytokines and growth factors, as
well as angiogenic factors. These proteins
allow tumor cells to proliferate, invade
and eventually metastasize. This suggests
that NFκB-activated inflammatory cells
are important mediators of tumor promo-
tion and progression.34,84
A definitive evidence for the role of
NFκB in bridging inflammation and can-
cer was provided by the seminal work by
Karin and colleagues. They have shown,
using a mice model of CAC (colitis-asso-
ciated cancer), that the IKKβ-dependent
NFκB activation pathway represents a
critical molecular link between inflam-
mation and cancer.85 Mice were injected
with the pro-carcinogen azoxymethane
of pathogens, mainly through PPR recep-
tors, such as TLRs. Ligand binding to
these receptors induces the recruitment or
the activation of adapter proteins through
the Toll/IL-1 Receptor (TIR) domain,
leading to the activation of IKK complex,
degradation of IκBα and liberation of
NFκB heterodimers.70 Moreover, NFκB
activation could also be achieved by cyto-
plasmatic PRRs, such as NOD receptors,
which are able to survey the cytosol for
the presence of bacterial peptiodoglycan
and then detect intracellular pathogens.33
Activation of NFκB triggers intestinal
inflammation responses in either IECs
or intestinal hematopoietic cells, such as
macrophages or DCs,70,71 and the conse-
quent upregulation of adhesion molecules
and chemokines leads to the recruitment
and activation of effector cells—i.e., neu-
trophils and then later macrophages and
other leukocytes. NFκB is also respon-
sible for the production of antimicrobial
effector molecules, such as α-defensins,
into the intestinal lumen,72 and as such
play an important role for the survival
of recruited neutrophils in an inflamma-
tory environment.73 Finally, NFκB also
regulates the expression of matrix metal-
loproteinases (MMPs), that are crucial
mediators of local inflammation and leu-
kocyte recruitment.74,75
Due to its central role in inflamma-
tion responses, it is not surprising if
NFκB has been rapidly considered as a
bridge between chronic inflammation
and cancer.56,76 Activated NFκB is indeed
detected in colonic mucosa of patients
suffering from IBD, as compared to
healthy subjects.77-79 Similarly, high levels
of nuclear p65 and c-Rel accumulation
has been observed in inflamed tissues
of Crohn’s disease patients, suggesting
enhanced NFκB transcriptional activity
in these patients.80 Activation of NFκB in
IBD could result of an aberrant PRR sig-
naling, that seems to be occur in this dis-
ease, and this could contribute to tumor
formation in patients affected by IBD.
Indeed, NFκB has been linked with cellu-
lar transformation and it is constitutively
active in most tumor cells. In tumor cells,
it regulates the expression of a large frac-
tion of anti-apoptotic genes, but also of
genes involved in tumoral proliferation—
such as c-myc and cyclin D1,—invasion,
pro-inflammatory cytokines, all of which
activate the IκB kinase (IKK) complex.
This complex is composed by two cata-
lytic subunits, IKKα and IKKβ, and a
regulatory subunit, IKKγ (also known
as NEMO).52,56 Activated IKK complex
phosphorylates NFκB-bound IκB proteins
and targets them for polyubiquitination
and degradation.57 IκB-free-NFκB dim-
ers can thus translocate to the nucleus and
mediate transcription of several hundred
target genes.58 The activation of NFκB
dimers is fast, an essential feature to allow
host response to pathogens. However, the
ability to switch-off NFκB signaling is
equally important. The principal mecha-
nism used to attenuate NFκB activation
entails the NFκB-mediated upregulation
of IκB proteins. Degraded IκB proteins
are replaced by NFκB-dependent de novo
synthesis of these inhibitors, generating a
negative feedback loop for NFκB activity.
The alternative NFκB activation pathway,
triggered by certain members of the TNF
family,55,58 is independent of IKKβ and
IKKγ and involves the upstream kinase
NFκB-inducing kinase (NIK), which
activates IKKα. The alternative pathway
results in specific activation of p52-RelB
heterodimers, through specific IKKα
phosphorylation of p52-p100 (NFκB2).
This protein binds RelB through its IκB-
like domain and retains itself and RelB
into the cytoplasm.59 Activation of IKKα
results in the degradation of p100 and
nuclear entry of p52-RelB dimers. The
target genes of these two pathways are
different and therefore mediate different
functions.55
The implication of the classical IKKβ/
NFκB activation pathway in acute inflam-
mation and cell survival is well estab-
lished:60,61 NFκB activates the production
of key pro-inflammatory cytokines and
enzymes, including TNFα, IL-1β, IL-6
and COX-2,62 as well as the expression of
anti-apoptotic genes,63,64 such as Bcl-XL,65
FLICE-like inhibitory protein (FLIP)66,67
and members of the inhibitor of apoptosis
(IAP) family.68,69 These findings support
the view that NFκB has a dual function: it
mediates cell survival and at the same time
protects the local environment from infec-
tion and injury. As discussed above, to
activate an appropriate immune response,
the host must first recognize the presence

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www.landesbioscience.com Cell Cycle 1257
as mature macrophages, DCs and neu-
trophils. The role of NFκB in these cells
has been decrypted by ablating IKKβ
specifically in myeloid cells, which do not
undergo malignant transformation. This
deletion causes a decrease not only in the
tumor number, but also in the tumor size
probably as a consequence of decreased
proliferation in transformed IECs which
require growth factors produced by myel-
oid cells.85 These findings allow to propose
that IKKβ-dependent NFκB activation
affects CAC development through two
distinct cell type-specific mechanisms
(Fig. 2): in enterocytes, NFκB activates
anti-apoptotic genes, preventing the elim-
ination of pre-neoplastic cells, while in
myeloid cells it promotes the production
of cytokines and growth factors for the
pre-malignant enterocytes.52
shown by enterocyte-specific ablation of
IKKβ, leading to decreased NFκB activ-
ity in IECs. This selective inactivation
resulted in a 80% decrease in CAC tumor
incidence, without affecting the size and
composition of tumors or the induction of
oncogenic mutations, indicating that the
IKKβ-dependent NFκB activation path-
way in IECs operates during early tumor
promotion.85 Deletion of IKKβ in entero-
cytes does not affect DSS-induced inflam-
mation, indicating that the pro-tumoral
effect of NFκB in these cells is not due to
the activation of pro-inflammatory cytok-
ines, but rather to its ability to suppress
apoptotic cell death in pre-malignant pre-
cursors.56,85 However, another important
factor in inflammation-promoted colon
cancer is the production of pro-inflam-
matory mediators by myeloid cells, such
(AOM), which undergoes metabolic acti-
vation in IECs, leading to mutational acti-
vation of the β-catenin pathway. AOM
causes cancer with low incidence, but this
can be augmented by oral administra-
tion of dextran-sulfate sodium salt (DSS),
which induces acute colitis characterized
by bloody diarrhea, ulceration and infiltra-
tions with granulocytes.86,87 It is believed
that DSS is directly toxic to gut epithelial
cells of the basal crypts and therefore affects
the integrity of the mucosal barrier.87 This
causes the exposition of lamina propria’s
macrophages to normal enteric bacteria
and consequently the activation of NFκB
in these cells through TLR signaling,
leading to the production of pro-inflam-
matory cytokines that in turn activate
NFκB in IECs (Fig. 2). The importance
of NFκB signaling in this model has been
Figure 2. the central role of NFκB during inflammation-associated colorectal tumorigenesis. intestinal exposition to AOM or other pro-carcinogenic
initiates malignancy transformation in intestinal epithelial cells. DSS and other damaging agents affect the integrity of the mucosal barrier, causing
the exposition of inflammatory cells, residing in the lamina propria, to the enteric bacteria. Following tLr receptors activation and nuclear transloca-
tion of NFκB through iKKβ, activated NFκB in inflammatory cells induces the expression of several secreted pro-inflammatory cytokines, such as tNFα
and iL-1β, which enhance the growth and the survival of carcinoma cells. Activation of NFκB in the latter results in increased expression of survival
genes. As we have shown, NFκB-dependent upregulation of netrin-1 in malignant cells is fundamental to tumor promotion and progression. AOM,
azoxymethane; DSS, dextran-sulfate sodium salt; tNFα, tumor necrosis factor α; iL-1β, interleukin-1β; tNFr, tNFα receptor; iL1r, iL-1β receptor.