Carcinogenesis vol.30 no.3 pp.
377386, 2009
doi:10.1093/carcin/bgp014
Advance Access publication January 9, 2009
REVIEW
The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the
tumour microenvironment
Alexander Greenhough, Helena J.M.Smartt, Amy
E.Moore, Heather R.Roberts, Ann C.Williams, Christos
Paraskeva
and Abderrahmane Kaidi1
Cancer Research UK Colorectal Tumour Biology Group, Department of
Cellular and Molecular Medicine, University of Bristol, University Walk,
Clifton, Bristol BS8 1TD, UK and 1The Wellcome Trust/Cancer Research UK
Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge
CB2 1QN, UK


To whom correspondence should be addressed. Tel: 44 117 331 2072;
Fax: 44 117 928 7896;
Email: c.paraskeva@bristol.ac.uk
It is widely accepted that alterations to cyclooxygenase-2 (COX-2)
expression and the abundance of its enzymatic product prosta-
glandin E2 (PGE2) have key roles in influencing the development
of colorectal cancer. Deregulation of the COX-2/PGE2 pathway
appears to affect colorectal tumorigenesis via a number of distinct
mechanisms: promoting tumour maintenance and progression,
encouraging metastatic spread, and perhaps even participating
in tumour initiation. Here, we review the role of COX-2/PGE2
signalling in colorectal tumorigenesis and highlight its ability to
influence the hallmarks of cancerattributes defined by Hana-
han and Weinberg as being requisite for tumorigenesis. In addi-
tion, we consider components of the COXprostaglandin pathway
emerging as important regulators of tumorigenesis; namely, the
prostanoid (EP) receptors, 15-hydroxyprostaglandin dehydroge-
nase and the prostaglandin transporter. Finally, based on recent
findings, we propose a model for the cellular adaptation to the
hypoxic tumour microenvironment that encompasses the inter-
play between COX-2, hypoxia-inducible factor 1 and dynamic
switches in b-catenin function that fine-tune signalling networks
to meet the ever-changing demands of a tumour.
A plethora of data indicates that human cancer is a multistage genetic
and epigenetic disease. Like all cancers, colorectal cancers are
thought to originate from a single replication-competent cell (stem
cell or proliferative progenitor cell) (1). In colorectal tumours, the
initiating event is thought to be the acquisition of a genetic alteration
that deregulates the gatekeeping pathwaythe adenomatous poly-
posis coli (APC)/b-catenin pathway (also known as the WNT-signal-
ling pathway)with tumour progression occurring through clonal
selection via the protracted acquisition of multiple genetic lesions
(1,2). These alterations, while having the potential to produce a wide
array of different cancer cell genotypes, have been proposed to affect
six principal aspects of cell physiology that are obligate for cancer
development. These are termed the hallmarks of cancer (3): ac-
quired capabilities that represent breaches to the normal regulatory
mechanisms controlling cell survival, proliferation, migration, inva-
sion and the interactions with neighbouring cells and stroma.
Abbreviations: APC, adenomatous polyposis coli; COX, cyclooxygenase;
EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated pro-
tein kinase; GSK3b, glycogen synthase kinase-3 beta; HIF-1, hypoxia-inducible
factor-1; MAPK, mitogen-activated protein kinase; NSAID, non-steroidal anti-
inflammatory drug; PGE2, prostaglandin E2; PGES, prostaglandin E synthase;
PGF2a, prostaglandin F2a; PGT, prostaglandin transporter; PI3K, phosphatidyli-
nositol-3-OH kinase; PPAR, peroxisome proliferator-activated receptor;
15-PGDH, 15-hydroxyprostaglandin dehydrogenase; TCF, T-cell factor; TGFb,
transforming growth factor-beta; VEGF, vascular endothelial growth factor.
The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
The genetic changes that occur in colorectal cancer appear to more
or less follow the histological progression from small, pre-malignant
adenomas, to advanced metastatic tumours, with both early and late
stages of the disease associated with the number of sequentially ob-
tained genetic alterations (reviewed in ref. 4). This implies that each
successive genetic hit confers an advantageous characteristic upon
the expanding tumour mass. While it may be true that numerous
different genes can become altered during the development of a given
tumour, it has also recently been proposed that all cancers arise and
are maintained by the deregulation of a relatively small number of
signalling pathways (1,5). This is an attractive premise from a thera-
peutic standpoint, given that there are far fewer cancer-related path-
ways than cancer-related genes (1).
While perturbation of the WNT-signalling pathway is believed to
account for the initiation of colorectal tumours (6), the aberrant ex-
pression of cyclooxygenase-2 (COX-2) that occurs in the majority of
colorectal tumours is thought to play a crucial role during colorectal
cancer development (7). Deregulation of COX-2 expression leads to
an increased abundance of its principal metabolic product, prostaglan-
din E2 (PGE2), the pleiotropic effects of which appear to affect most,
if not all, of the hallmarks of cancer. This ability of the COX-2/PGE2
pathway to affect multiple aspects of cell physiology required for
tumour development and maintenance may also offer an explanation
for the effectiveness of COX inhibitors and non-steroidal anti-
inflammatory drugs (NSAIDs) at reducing both the incidence and
progression of intestinal tumours in animal models of cancer, and
more importantly in human cancer patients (reviewed in ref. 8).
In this review, we discuss the impact of the COX-2/PGE2 pathway
on colorectal cancer development and highlight its influence on each
of the hallmarks of cancer. Furthermore, we consider the emerging
regulators of COXprostaglandin signalling and novel roles for
COX-2/PGE2 signalling in the tumour microenvironment.
Cyclooxygenases and prostaglandins
NSAIDs such as aspirin have been used traditionally as analgesics and
anti-inflammatories for centuries and are some of the worlds most
widely used drugs. The seminal work by Sir John Vane delineated the
mechanism by which aspirin exerts its anti-inflammatory, analgesic
and antipyretic actions. In the early 1970s, the enzyme prostaglandin
G/H synthase, now more often referred to as COX, was elucidated as
the target for inhibition by aspirin and other NSAIDs (9). Since this
discovery, there has been a great deal of interest in identifying the
components of the COX pathway as well as its functions in both
physiological and pathological conditions. COX enzymes play key
roles in the biosynthesis of prostaglandins from arachidonic acid fol-
lowing its release from the plasma membrane by the action of phos-
pholipase-A2 (reviewed in ref. 10). In the initial cyclooxygenase
reaction, the COX enzymes catalyse the formation of the unstable
intermediate prostaglandin G2 from arachidonic acid, which is then
converted into prostaglandin H2 by the peroxidase activity of COX
(11). Prostaglandin H2 is the precursor for several structurally related
prostaglandins, which are formed by the action of specialized prosta-
glandin synthases (reviewed in ref. 12). The prostaglandins synthe-
sized by this pathway include the aforementioned PGE2, as well as
prostaglandin D2, prostaglandin F2a (PGF2a), prostaglandin I2 (also
known as prostacyclin) and thromboxane-A2. The actions of these
prostanoid ligands are mediated by their engagement of specific
cell-surface G-protein-coupled receptors designated EP14 for
PGE2 and prostaglandin F2a receptor, thromboxane A2 receptor,
prostaglandin I2 receptor and thromboxane A2 receptor for PGF2a,
prostaglandin D2, prostaglandin I2 and thromboxane-A2, respectively.
377
A.Greenhough et al.
These prostaglandins are important for a large number of normal
physiological processes in a broad range of tissues. These include
the modulation of immune responses, protection of the gastrointes-
tinal mucosa, maintenance of renal homeostasis and the regulation
of blood clotting. Furthermore, prostaglandins also function in path-
ological conditions where they can promote inflammation, swelling,
pain and fever (reviewed in ref. 10).
Later work on the biology of COX enzymes led to the distinction
between two COX enzymes: COX-1 (13) and COX-2 (14). A third
form (COX-3) has also been reported (15), although recent studies
indicate that this represents a splice variant of COX-1 that encodes
a truncated protein lacking enzymatic activity (16). In humans, COX-
1 is found constitutively expressed in a wide range of tissues including
the kidney, lung, stomach, small intestine and colon. Thus, COX-1 is
considered a housekeeping enzyme responsible for maintaining basal
prostaglandin levels important for tissue homeostasis. In contrast,
most tissues do not normally express COX-2 constitutively, notable
exceptions including the central nervous system (17), kidneys (18)
and seminal vesicles (19). However, the stimulation of COX-2 expres-
sion in Src-transformed fibroblasts (14), endothelial cells and mono-
cytes treated with the tumour promoter tetradecanoyl-phorbol-acetate
(20) or lipopolysaccharide (21) led to the notion that COX-2 is an
inducible enzyme that produces prostaglandins during inflammatory
and tumorigenic settings. Because of this, there has been a fervent
interest in studying the biology of COX-2 in relation to tumorigenesis,
particularly with regard to colorectal tumorigenesis where its actions
appear to have a major impact on tumour development.
Cyclooxygenases and colorectal cancer
The first indication that COX enzymes might play a role in colorectal
tumorigenesis came from the observation that patients with Gardners
syndrome (which causes multiple intestinal polyposis) treated with
NSAIDs displayed a reduction in adenoma number (22). This was the
first clinical evidence that NSAIDs could be useful in the prevention
of cancer. Subsequently, in 1991, Thun et al. (23) undertook an epi-
demiological study, which reported that regular aspirin intake at low
doses reduces the risk of colorectal cancer, and suggested the link
between COX enzymes and cancer development. A large body of
evidence now indicates that elevated levels of COX-2 are present in
the majority of colorectal carcinomas (2428) and in a subset of
adenomas (24,25). The observation that COX-2 expression is up-
regulated in both pre-malignant as well as malignant colorectal tissue
is regarded as particularly significanthaving potential implications
for both the prevention and treatment of colorectal cancer (29).
Several lines of evidence now support a critical role for COX-2
during colorectal tumorigenesis. Most convincingly, in a randomized
double-blind placebo-controlled trial, administration of the COX-2-
selective NSAID celecoxib significantly decreased the occurrence of
sporadic colorectal adenomas (30). Notably, in this scenario, celecox-
ib might potentially act not only by suppressing the growth of existing
adenomas but also by preventing the formation of new adenomas (30).
Similarly, in a study of patients with familial adenomatous polyposis,
administration of celecoxib was associated with regression of
adenomas of both the colon and the rectum (31). By the same token,
studies have revealed that genetic disruption or pharmacological in-
hibition of COX-2 results in a substantial decrease in adenoma size
and number in murine models of intestinal tumorigenesis [(32,33),
respectively].
The pro-tumorigenic effects of COX-2 in the colorectum are largely
thought to be attributed to its role in producing PGE2. Indeed, in-
creased levels of PGE2 have been reported both in human colorectal
adenomas as well as carcinomas (34,35). PGE2 levels have also been
reported to increase in a size-dependent manner in the adenomas of
familial adenomatous polyposis patients (36) as well as in the adeno-
mas of ApcMin/ mice (37). Furthermore, in vivo studies have revealed
that prevention of adenoma development in familial adenomatous
polyposis patients is more effective when tissue prostaglandin levels
are reduced through NSAID treatment (38), and more specifically,
sequestration of PGE2 by administration of a PGE2 monoclonal anti-
378
body in mice has been demonstrated sufficient to retard the growth of
transplantable tumours in vivo (39). In addition, PGE2 has been found
to result in augmented incidence and multiplicity of carcinogen-
induced colon tumours in rats (40) and enhances intestinal adenoma
growth in ApcMin/ mice (41). Accumulating evidence from in vitro
studies has further substantiated the in vivo evidence and also pro-
vided insights into the mechanisms underlying the oncogenic role of
PGE2 (reviewed in ref. 42). Nevertheless, it is worth noting that while
good evidence exists suggesting that COX-2/PGE2 pathway inhibition
may be useful in the prevention of colorectal tumours, this may not be
the case under all circumstances. Indeed, COX-2/PGE2 signalling is
likely to act in concert with other important signalling pathways that
become deregulated in cancer, meaning that such pathways would
also need to be targeted in conjunction with the COX-2/PGE2 path-
way for efficient prevention (or treatment) of colorectal cancer.
While there is a large body of data indicating an important tumour-
promoting role for COX-2 during tumorigenesis, it is important to note
that some reports imply that the role of COX-2 and/or prostaglandins in
cancer might not be as straightforward as initially proposed. For exam-
ple, some NSAIDs produce antitumoral effects in vitro independently
of their ability to inhibit COX-2 or to reduce PGE2 synthesis (4345),
and furthermore, some NSAIDs actually induce COX-2 expression
(46,47). In addition, the COX-2/PGE2 pathway may even elicit what
might be considered counterintuitive effects under certain circumstan-
ces, by acting in a tumour-suppressive manner (4851). In one study,
the authors investigated the effects of exogenous administration of the
stable synthetic PGE2 analogue 16,16-dimethyl-PGE2 on tumour de-
velopment in ApcMin/ mice; unexpectedly, the authors observed reduc-
tions in both the number and size of intestinal tumours (48).
Furthermore, transgenic mice engineered to over-express COX-2 via
the keratin 14 promoter (resulting in COX-2 over-expression and ele-
vated PGE2 levels in the skin) were found to be more resistant, rather
than more sensitive, to the development of skin tumours induced by an
initiation/promotion protocol (49). These findings contrast with other
similar studies reporting tumour-promoting effects of COX-2/PGE2
signalling in mice (41,52,53), adding further layers of complexity
to the role of the COX-2/PGE2 pathway during intestinal tumour
development.
It is not clear what accounts for the apparent disparities between
these studies, but a number of possibilities can be considered. For
instance, it could be speculated that the precise level of PGE2 cells
are exposed to may result in different outcomes in a given experimen-
tal system. Indeed, we have previously reported that human colorectal
adenoma cells are growth stimulated by low concentrations of PGE2,
but growth inhibited by high PGE2 concentrations (54). Conversely,
colorectal carcinoma cells are growth stimulated by both low and high
concentrations of PGE2 (54). We hypothesize that during adenoma
progression, further eventssuch as over-expression of the EP4
receptormay be necessary for tumours to become growth stimulated
by high concentrations of PGE2. Therefore, it is possible that the use
of stable PGE2 (16,16-dimethyl-PGE2) (48) led to high levels and/or
accumulation of PGE2, resulting in the inhibition of adenoma growth.
Another interesting possibility, as noted earlier, is whether there could
perhaps be an important role for other genetic lesions occurring in
parallel to deregulation of the COX-2/PGE2 pathway; that is, addi-
tional changes might be required for COX-2 to promote tumorigenesis
efficiently. Thus, although much of data suggest an important role for
COX-2/PGE2 signalling in the promotion of tumorigenesis, further
research is still required in order to elucidate its precise roles in
specific cellular and experimental contexts.
COX-2, prostaglandins and their contribution to the hallmarks of
cancer
As mentioned previously, COX-2 is frequently over-expressed in co-
lorectal cancer, and PGE2 has been identified as the principal prosta-
noid promoting cell growth and survival in colorectal tumours. PGE2
is able to exert pleiotropic effects in colorectal tumours, promoting
proliferation, survival, angiogenesis, migration and invasion. Here,

we discuss the influence of the COX-2/PGE2 pathway on the hall-
marks of cancer (3).
Evasion of apoptosis
Apoptosis, the process of programmed cell death (55), is a critical
mechanism by which metazoan organisms control cell number, where
selective cell suicide enables the efficient removal of superfluous,
damaged or infected cells (for reviews, see refs 56,57). Apoptosis
plays an essential role during embryonic development and is required
for maintaining tissue homeostasis throughout the lifespan of meta-
zoan organisms. Disturbances to the apoptotic machinery that result in
excessive cell survival or cell death underlie a number of pathological
conditions, but most pertinent here, the failure to initiate cell death in
response to apoptotic stimuli is thought to play a central role in
tumorigenesis (58). Indeed, deregulated cell proliferation, coupled
with an acquired resistance to apoptosis, has been proposed to con-
stitute a platform both necessary and sufficient for tumour growth and
malignant progression (59,60).
While the acquired ability of tumour cells to evade apoptosis can
arise via a variety of mechanisms, the majority of such changes result
in an impaired ability of a cell to engage the intrinsic cell death
machinery (i.e. the mitochondrial pathway of apoptosis). This path-
way is governed by the ratio of pro-apoptotic and pro-survival BCL-2
family members that set a threshold for activation of the apoptotic
caspase cascade upstream of the mitochondria (61). Establishing the
connection between the receipt of extracellular signals and the acti-
vation of pathways such as the phosphatidylinositol-3-OH kinase
(PI3K)/AKT (also known as protein kinase B) and Ras-mitogen-
activated protein kinase (MAPK)/extracellular signal-regulated pro-
tein kinase (ERK) cascades to the suppression of apoptosis via the
intrinsic cell death machinery was a crucial finding of recent years
(62,63). Indeed, this is likely to represent the mechanism by which the
COX-2/PGE2 pathway regulates apoptosis, via the autocrine and/or
paracrine effects of PGE2 acting at one or more of the four cell-
surface EP receptors.
Although still not entirely clear, several mechanisms for the sup-
pression of apoptosis by the COX-2/PGE2 pathway have been sug-
gested, and engagement of one or more of the signalling pathways
downstream of EP receptor activation may be responsible for this pro-
survival response. Moreover, the ability of the COX-2/PGE2 pathway
to control an apoptosis evasion programme in tumour cells may differ
between cell types and depend on factors such as the tumour micro-
environment (for example, in hypoxic conditions). The initial indica-
tion that COX-2 might modulate the intrinsic pathway of apoptosis
came when its forced over-expression led to elevated levels of the pro-
survival protein BCL-2 and conferred increased resistance to the di-
etary fibre fermentation product butyrate-induced apoptosis in rat
intestinal epithelial cells (64). Interestingly, the first suggestion that
chemopreventive agents might produce their effects via the induction
of tumour cell apoptosis arose from studies with butyrate in colorectal
tumour cells (65); thus, the ability of COX-2 expression to suppress
butyrate-induced apoptosis suggests that the chemopreventive action
of COX-inhibiting NSAIDs may act in part by re-sensitizing cells to
apoptosis-inducing luminal factors. Further studies indicated that the
mechanism by which COX-2/PGE2 might suppress apoptosis and in-
crease the expression of BCL-2 was via activation of the Ras-MAPK/
ERK pathway (66). More recently, several studies indicate that the
COX-2/PGE2 pathway might alter apoptotic thresholds by engaging
a number of different signalling pathways. Indeed, PGE2 has been
reported to activate pro-survival pathways including the PI3K/AKT
pathway (67,68), ERK signalling (69), cyclic adenosine monophos-
phate (cAMP)/protein kinase A signalling (70) and activation of
epidermal growth factor receptor (EGFR) signalling (71,72). PGE2
has more recently been shown to promote cell survival in murine
intestinal adenomas by indirectly transactivating the nuclear peroxi-
some proliferator-activated receptor (PPAR)-delta via a PI3K/AKT-
dependent mechanism (52). However, while this and other evidence
(73) point to an important role for PPARd in intestinal tumour de-
The COX-2/PGE2 pathway
velopment, there remains an interesting debate as to the precise role
PPARd plays during intestinal tumorigenesis (74). It is also interesting
to note that in addition to the apoptosis-suppressive effects of COX-2-
derived PGE2, deregulated expression of the COX-2 protein itself
might also alter the susceptibility of cells to undergo apoptosis by
reducing the cellular pool of its substrate arachidonic acid, which can
stimulate apoptosis by stimulating ceramide production (45).
While the exact mechanism by which the COX-2/PGE2 pathway
suppresses apoptosis remains to be fully elucidated, it is clear that the
influence of the COX-2/PGE2 pathway has on this hallmark of cancer
plays an important role in aiding tumour progression. For instance,
under conditions of hypoxiaa situation conducive to the induction
of cell deathPGE2 appears to promote cell survival in colorectal
tumour cells by the stimulation of Ras-MAPK signalling (75). As well
as promoting the survival of cancer cells under harsh microenviron-
mental conditions, PGE2 might also play a crucial role in encouraging
the transition from adenoma to carcinoma. We have previously re-
ported that increased EP4 receptor expression occurs during the co-
lorectal adenomacarcinoma sequence (54) and that EP4 receptor
over-expression confers an anchorage-independent phenotype to oth-
erwise anchorage-dependent human colorectal adenoma cells (54).
This suggests an important role for a COX-2/PGE2/EP4-axis in the
suppression of apoptosis; indeed, in a recent complementary study,
Hull and colleagues (76) reported that EP4 receptor over-expression
results in the suppression of apoptosis and enhanced tumorigenic
behaviour of the human colon cancer cell line HT29 (76).
Finally, it is important to note that while the ability of the COX-2/
PGE2 pathway to suppress intrinsic cell death mechanisms might
facilitate cancer development, it may also confer a decreased sensi-
tivity of colorectal tumour cells to cytotoxic cancer treatments. For
example, it has previously been reported that PGE2 attenuates radia-
tion-induced apoptosis in colorectal cancer cells via activation of
EGFR/AKT signalling and a mechanism that prevents the transloca-
tion of pro-apoptotic Bax to the mitochondria (68). Observations such
as these merit further investigation into the potential of NSAIDs and/
or COX-2-selective inhibitors as adjuvants to current chemotherapy/
radiation regimens for patients with colorectal cancer.
Self-sufficiency in growth signals
The behaviour of normal cells in multicellular organisms is controlled
by the coordinated regulation of complex signalling pathways, which
transduce signals from growth factors and cytokines into decisions
that direct cell fate. It is thought that the deregulation of these signal-
ling networks underlies tumour initiation and progression, causing
cells to grow uncontrollably and unregulated by environmental cues.
Two major pathways frequently mutated in many human cancers,
including colorectal, are the Ras-MAPK- and the PI3K/AKT-signalling
pathways; activation of which stimulates cell growth, proliferation
and survival (reviewed in ref. 77,78). Deregulation of these signalling
pathways can occur by a variety of mechanisms, including alterations
to both upstream and downstream components of the pathways. Most
commonly, constitutive activation of the Ras-MAPK pathway occurs
via mutations to KRAS [in 50% of colorectal tumours (79)] or BRAF
[in 1020% of colorectal tumours (80)] genes, whereas deregulation
of the PI3K/AKT pathway occurs most commonly by PIK3CA mu-
tation [in 32% of colorectal tumours (81)], phosphatase and tensin
homolog mutations [in up to 20% of colorectal tumours (82)] or
mutations in the pleckstrin homology domain of AKT [in 6% of co-
lorectal tumours (83)].
As already mentioned, COX-2-derived PGE2 is able to signal via
the PI3K/AKT- and Ras-MAPK/ERK-signalling pathways to enhance
cell survival. An interesting possibility arising from such studies is the
prospect that the COX-2/PGE2 pathway represents an alternative
mechanism by which tumours acquire growth factor autonomyin
the absence of PI3K pathway- and MAPK pathway-activating muta-
tions. In other words, aberrant activation of the COX-2/PGE2 pathway
might phenocopy-activating mutations in the PI3K/AKT and/or Ras-
MAPK pathways, which could play an important role in promoting
tumour progression. This supports the notion that while the cohort of
379
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alterations to cancer-related genes and proteins might differ between
individual tumours, alterations to seemingly disparate genes may in
reality be functionally equivalent; bestowing one or more of the hall-
marks of cancer on cells by deregulating the same molecular pathway
(1). Indeed, deregulation of the COX-2/PGE2 pathway has been shown
to behave in a similar manner to constitutively active Ras in murine
intestinal adenomas, resulting in a positive feedback loop that boosts
COX-2 expression and further stimulation of tumour growth (41). This
mechanism might also operate during hypoxic (as well as normoxic)
conditions. In fact, we have previously reported that during hypoxia,
the transcription factor hypoxia-inducible factor-1 (HIF-1) directly up-
regulates COX-2 expression and increases PGE2 production (75). This
leads to activation of the Ras-MAPK pathway, which may act in a pos-
itive feedback loop to maintain an active pro-survival COX-2/PGE2
pathway during hostile microenvironmental conditions (75).
Insensitivity to anti-growth signals
As noted earlier, disturbances to normal tissue homeostasis that shift
the balance from a state of equilibrium towards increased cell growth
will invariably lead to the appearance of a neoplastic population of
cells (84). Under normal physiological conditions, in addition to the
strict control of apoptosis and growth-stimulatory signals, a further
crucial mechanism for maintaining tissue homeostasis is via the re-
straint of cell growth through the action of anti-proliferative signals
(3). These include soluble growth inhibitors, as well as membrane-
bound ligands, both of which act on cell-surface receptors that couple
to intracellular pathways to repress cell growth. Hanahan and Weinberg
(3) proposed two discrete mechanisms by which anti-growth signals
can block proliferation to restrain inappropriate cell growth and main-
tain tissue homeostasis. Importantly, both of these mechanisms are
frequently hijacked in colorectal cancers. Furthermore, deregulation
of the COX-2/PGE2 pathway may provide an additional and/or sec-
ondary mechanism by which tumour cells can attain this hallmark.
The first mechanism by which anti-growth signals can block pro-
liferation is by preventing cells from advancing through the G1 phase
of the cell cycle, which serves to maintain cells in a quiescent (G0)
state. The archetypal example of such an anti-proliferative signal is
the soluble signalling factor transforming growth factor-beta (TGFb),
which blocks progression through the G1 phase of the cell cycle via
the suppression of c-Myc and activation of cyclin-dependent kinase
inhibitors such as p15Ink4B and p21Cip1 (reviewed in ref. 85). Often,
cancer cells acquire the inability to respond to the growth-suppressive
effect of TGFb, which can occur as a result of inactivating mutations
in the downstream signalling effectors of TGFb signalling (e.g. loss of
SMAD function) or mutations to the receptors themselves (reviewed
in ref. 85). The latter mechanism is particularly relevant in colorectal
tumours with microsatellite instability, where TGFb type II receptor
mutations occur at a high frequency (86). However, not all cancer
cells exhibit mutational inactivation of the TGFb type II receptor,
and a further mechanism by which cancer cells lose TGFb respon-
siveness is via the down-regulation of the TGFb receptors. Indeed,
this could represent a mechanism by which the COX-2/PGE2 pathway
prevents the receipt of anti-growth signals since over-expression of
COX-2 has been reported to cause down-regulation of the TGFb type
II receptor (64).
A second mechanism by which cells can be instructed to curb their
proliferative potential involves the implementation of differentiation
programmes that instigate an irreversible post-mitotic (or terminally
differentiated) state (3). An example of such an anti-growth pathway
operates in the colorectal epithelium and is often deregulated in neo-
plasia. Under normal physiological conditions in the colonic crypt,
the long-lived stem cells continually give rise to rapidly proliferating
progenitor cells (also known as transit-amplifying cells), which com-
prise the proliferative region in the lower two-thirds of the crypt.
These progenitor cells are able to differentiate into the cell lineages
that occupy the upper third of the crypt: absorptive enterocytes, enter-
oendocrine cells and mucin-secreting goblet cells (87). Upon reaching
the top third of the crypt, committed progenitor cells undergo cell
380
cycle arrest, become differentiated and are eventually shed in to the
lumen. However, in colorectal tumours, aberrant activation of b-catenin
(i.e. the WNT pathway) serves to block this normal differentiation
programme and maintains cells in a progenitor-like state (88). Recent
evidence indicates that the COX-2/PGE2 pathway can activate the
APC/b-catenin pathway (89). Thus, in the context of colorectal can-
cer, where mutations in the APC/b-catenin pathway are common, the
COX-2/PGE2 pathway might enhance the activation of the APC/
b-catenin pathway to keep cells in a progenitor-like state. Further-
more, another attractive hypothesis would be that inappropriate acti-
vation of the COX-2/PGE2 pathway discourages differentiation in the
absence of APC/b-catenin mutations; in this scenario, the activation
of the APC/b-catenin pathway by PGE2 might even be able to con-
tribute to tumour initiation. In addition, the ability of PGE2 to switch
on b-catenin signalling might also contribute to the acquisition of an
immortalized phenotype, as discussed below.
Limitless replicative potential
The intrinsic capacities for self-renewal and limitless replicative po-
tential are characteristics thought to be shared by stem cells and
cancer cells (90). Because of these apparent similarities, it has been
proposed that cancer arises from the deregulation of pathways that
maintain the stem/progenitor cell phenotype in a given tissue (6,91).
As mentioned earlier, colorectal tumours are thought to originate from
such replication-competent cells (i.e. a proliferative progenitor cell or
stem cell) initiated by activating mutations in the APC/b-catenin
pathway. Perturbations to the APC/b-catenin pathway are detectable
in the earliest lesions of the colorectal epithelium (aberrant crypt foci)
and are thought to occur in virtually all colorectal tumours. This most
commonly arises by loss of the APC tumour suppressor gene, occur-
ring in 80% of all colorectal tumours (92). The tumour-suppressive
action of APC is thought to be mediated in large part by its ability to
regulate the levels of intracellular b-catenin, which transduces signal-
ling by the WNT ligand. The mutations in either APC or indeed other
components of the WNT pathway observed in colorectal tumours lead
to the formation of a constitutively active b-cateninT-cell factor
(TCF) complex that mimics active WNT signalling. Evidence sug-
gests that the proliferative compartments (stem cells and progenitor
cells) in intestinal crypts are maintained through activation of the
WNT pathway (88). Indeed, disruption of the WNT pathway by de-
letion of Tcf4 in mice results in loss of the stem cell and proliferative
compartments in the small intestine (93). This suggests that the WNT
pathway maintains the crypt stem/progenitor cell phenotype and that
the same pathway is active in both colorectal cancer cells and the
stem/progenitor cells of the intestinal crypt.
The recently discovered connection between the WNT pathway
and the COX-2/PGE2 pathway (89) indicates that PGE2 signalling
may be able to contribute to the crypt progenitor phenotype by acti-
vating b-catenin/TCF signalling in colorectal cancer cells. Gutkind
and colleagues (89) reported that upon engagement of the EP2 re-
ceptor, PGE2 stimulates a dual-signalling cascade involving activation
of the PI3K/AKT pathway by the EP2-associated G-protein bc sub-
units and the association of the G-protein as subunit with Axin (89).
This leads to the activation of b-catenin by a dual mechanism: first,
the activation of AKT inhibits glycogen synthase kinase-3 beta
(GSK3b), which reduces the inhibitory effect of GSK3b-mediated
phosphorylation on b-catenin; and second, the complex between the
EP2-associated G-protein as subunit and Axin promotes the release of
b-catenin from the AxinGSK3b complex, freeing b-catenin and
facilitating its nuclear accumulation (89). Thus, by activating the
b-catenin/TCF pathway, it is tempting to speculate that COX-2/
PGE2 signalling might contribute to limitless replicative potential
by promoting the acquisition of a progenitor or stem cell-like pheno-
type; although, at present, whether COX-2/PGE2 signalling is able to
promote such a phenotype is unknown.
In addition, it is also possible that the COX-2/PGE2 pathway, by
enhancing cell survival and growth, serves to prime cells for the
acquisition of further cellular alterations that contribute to immortal-
ization and the progression towards the full malignant phenotype.

Lastly, it is of interest to note that recent studies suggest a role for
COX-2/PGE2 signalling in the maintenance of haematopoietic stem
cell homeostasis (94) and in the suppression of embryonic stem cell
apoptosis (95). Whether the COX-2/PGE2 pathway influences the
behaviour of stem cells of the intestinal and colonic epithelium awaits
investigation.
Sustained angiogenesis
In the course of solid tumour development, it is well recognized that the
avascular tumour mass becomes dependent on angiogenesis for main-
tenance and progression, leading to the concept known as the angio-
genic switch (96). The initiation of this morphogenic process is
controlled by the relative balance of pro-angiogenic and anti-angio-
genic factors in a given tissue, such that a tipping of the balance towards
pro-angiogenic factors results in the stimulation of blood vessel forma-
tion (96). In the case of colorectal cancer, over-expression of COX-2 in
colon cancer cells induces the production of angiogenic factors such as
vascular endothelial growth factor (VEGF) and basic fibroblast growth
factor, which are instrumental in stimulating the formation of new
blood vesselsa requirement for tumours should they wish to develop
beyond a few millimetres in size. These pro-angiogenic effects of COX-
2 can be inhibited by NSAIDs, resulting in the inhibition of endothelial
cell proliferation, migration and vascular tube formation in vitro (97).
The inhibitory effect of NSAIDs on angiogenesis in this context can be
rescued by adding exogenous PGE2 (98), suggesting that COX-2-
derived PGE2 is at least part responsible for the pro-angiogenic effects
of COX-2 over-expression; although it is important to note that pros-
taglandin-independent mechanisms of NSAID action on angiogenesis
were also reported (98). Consistent with this, PGE2 has been reported to
stimulate VEGF expression in colon cancer cells through the activation
of HIF-1, a key regulator of VEGF expression (99). Furthermore, in
vivo studies have also demonstrated an important role for the COX-2/
PGE2 pathway in angiogenesis. In mice, homozygous deletion of COX-
2 (but not COX-1) results in impeded growth of tumour xenografts and
is associated with a reduced tumour vascular density (100). Such ob-
servations are likely to be at least in part mediated by the resultant
reduction in PGE2, since the intestinal adenomas present in APCD716/
mice that are null for the PGE2 receptor EP2 are defective in VEGF
induction (101). One mechanism by which COX-2 might promote
tumour vascularization is via the production of PGE2 and prostaglan-
din I2, which have been shown to participate in inducing endothelial
cell spreading and migration by integrin aVb3-mediated activation of
the small guanosine 5#-triphosphatases Cdc42 and Rac (102). More
recently, PGE2 has been shown to regulate angiogenesis though the
modulation of chemokine receptor signalling: PGE2 can enhance
VEGF and basic fibroblast growth factor-induced chemokine recep-
tor-4 that is important for microvessel assembly in vivo (103). In
addition, PGE2 can induce the expression of the pro-angiogenic che-
mokine CXCL-1 in vivo (104). PGE2 may also work concomitantly
with the hypoxic tumour microenvironment to orchestrate the process
of angiogenesis, as discussed later (75).
Tissue invasion and metastasis
It is well established that the primary cause of cancer mortality is the
formation of distant metastases, making the capacity of tumour cells to
invade and metastasize one of the most pertinent hallmarks of cancer
from a therapeutic perspective. In order to achieve metastases, cancer
cells must exhibit a more motile, invasive phenotype, dissociate from
neighbouring cells within the tumour, invade through extracellular ma-
trix components and intravasate into local blood or lymphatics (re-
viewed in ref. 105). Having made their escape from the primary
tumour, cancer cells must then extravasate from the blood or lym-
phatics into the surrounding tissue in order to colonize distant sites.
Several lines of evidence indicate that COX-2 and the prostaglandins
play important roles in aiding these processesmore specifically,
PGE2 is thought to promote a more metastatic phenotype in colorectal
tumour cells. While inhibition of COX-2 in vivo can attenuate the
The COX-2/PGE2 pathway
metastatic potential of colorectal tumours in both humans (106) and
mice (107), over-expression of COX-2 in intestinal cells can modulate
their adhesive properties (64) and increase matrix metalloproteinase
activity to promote invasion (108). Further studies have demonstrated
that PGE2 promotes cytoskeletal reorganization and increases colorec-
tal cancer cell migration and invasion via PI3K signalling (67). The
stimulation of invasion and motility by PGE2 is dependent on the
intracellular Src-mediated transactivation of the EGFR (72). Further-
more, the hepatocyte growth factor receptor, c-Met, is also transacti-
vated by PGE2 in an EGFR-dependent fashion in colorectal cancer cells
(109). Hepatocyte growth factor/c-Met signalling is classically associ-
ated with loss of cellcell contact (or scattering) and invasive growth
(110). Transactivation of c-Met by PGE2 leads to increased nuclear
b-catenin accumulation, increased urokinase-type plasminogen activa-
tor receptor expression and invasion through matrigel (109). Further-
more, COX-2, c-Met and b-catenin are co-expressed at the invasive
front of colorectal tumour specimens (109). Although PGE2 is the most
abundant prostaglandin found in colorectal cancer tissue (35), PGF2a
has also been detected in colorectal tumours, and PGF2a can stimulate
motility in both colorectal carcinoma and adenoma cells and invasion
of colorectal carcinoma cells (111).
More recently, the significance of COX-2not only as a critical player
in tumour development but also as being necessary for dissemination of
cancer cells to other organswas demonstrated during an elegant series
of experiments in an in vivo model of breast cancer cell metastasis to the
lungs (112). In this study, using both genetic and pharmacological
approaches, the authors identified COX-2 as one of four key metastasis
progression genes, which collectively synergize to mediate both tu-
mour development and metastasis to other organs. Notably, the tumour
maintenance and progression functions of COX-2 were mediated solely
by the COX-2 expressed by the tumour cells themselvesas opposed to
COX-2 expression and PGE2 production by other cells (i.e. stromal
cells) in the tumour microenvironment (100,113). Therefore, these find-
ings contribute to the ongoing discussion concerning the roles of tu-
mour-derived and stroma-derived COX-2 during the different stages of
tumorigenesis and within specific cellular contexts (114).
Evasion of the antitumour immune response and its regulation by the
COX-2/PGE2 pathway
In addition to the six hallmarks of cancer originally proposed by
Hanahan and Weinberg, additional characteristics of tumours have
recently been proposed, one of which is the ability of tumours to
evade attack by the immune system (reviewed in ref. 115). Tumour-
specific antigens are capable of eliciting an antitumour immune re-
sponse involving cytotoxic CD8 T cells, resulting in tumour cell
lysis. However, in cancer patients, these antitumour immune re-
sponses are generally ineffective and increasing evidence suggests
that tumours evolve several strategies to escape tumour-specific im-
munity (116,117). In light of the known ability of PGE2 to modulate
multiple aspects of the immune response (118), this additional cancer
hallmark may therefore be influenced by inappropriate expression of
COX-2 and PGE2 production that occurs commonly in colorectal
tumours. Furthermore, COX-2 over-expression and the resulting in-
crease in PGE2 levels may represent a strategy adopted by tumours
that contributes to the evasion of tumour-specific immune response
(119). For example, PGE2 has been reported to shift the production of
cytokines by antigen-presenting dendritic cells, away from a Th1
(type 1 T cell) profile, leading to a reduced activation of antitumour
cytotoxic CD8 T cells (118120). This ability of PGE2 to suppress
these immune responses may allow tumour cells to escape immuno-
surveillance, adding to the already countless roles of the COX-2/PGE2
pathway during tumour development.
Emerging regulators of the COX-2/PGE2 pathway: PGE2 synthesis and
signalling as alternative chemopreventive/therapeutic targets
Until relatively recently, COX expression was believed to be the
major determinant of PGE2 levels, but recent data indicate that
381
A.Greenhough et al.
multiple levels of control exist for the regulation of PGE2 production
and turnover. In addition to regulation via COX enzymes, PGE2 syn-
thesis can be regulated via expression of specific prostaglandin
E synthases (PGES), which act on the COX product prostaglandin
H2 to produce PGE2 (reviewed in ref. 42,121). Moreover, in addition
to regulated synthesis, PGE2 levels are also determined by the rate of
its degradation, which can be regulated via the expression of 15-
hydroxyprostaglandin dehydrogenase (15-PGDH) and the prostaglan-
din transporter (PGT) (122). The cellular response to PGE2 can also
be regulated via the differential expression of the four PGE2 receptors
(EP14), which activate distinct downstream signalling responses (re-
viewed in ref. 42,123). An understanding of these further levels of
regulation controlling levels of (and response to) PGE2 may reveal
novel strategies for prevention and/or treatment of colorectal cancer.
There are currently three characterized PGES enzymes: cytosolic
PGES, microsomal PGES-1 and microsomal PGES-2 (reviewed
in ref. 42). Microsomal PGES-1 is, like COX-2, induced by pro-
inflammatory stimuli and up-regulated in colorectal tumours (124).
Moreover, loss of microsomal PGES-1 expression is reported to sup-
press intestinal neoplasia in APC-mutant mice (125), although the re-
verse has also been observed (126), possibly reflecting the knock-on
enhancement of synthesis of other prostaglandins with pro-tumorigenic
effects (such as PGF2a). Further work will be required to ascertain the
potential utility of PGES inhibition as an anticancer strategy in the colon.
As noted earlier, the four characterized receptors for PGE2 (EP14)
each show distinct downstream signalling effects. For example,
whereas activation of EP2 and EP4 receptors can both stimulate
cAMP production, activation of EP3 results in the inhibition of cAMP
production (reviewed in ref. 42). Recent evidence suggests a role for
the EP receptors in colorectal neoplasia. As previously discussed, we
have recently showed that up-regulation of EP4 occurs during human
colorectal tumorigenesis in vivo (54) and others have shown that
genetic or pharmacological inactivation of EP4 inhibits tumour
growth in a mouse model of intestinal neoplasia (127). Other PGE2
receptors are also probably play a role in colorectal neoplasia; for
example, homozygous deletion of the gene encoding the EP2 receptor
reduces adenoma size and number in ApcD716/ mice (101). In addi-
tion, EP3 has been shown to stimulate angiogenesis and growth of
tumours arising from implanted sarcoma cells in mice (128).
However, down-regulation of EP3 has been observed in colorectal
neoplasia and azoxymethane-induced tumorigenesis is enhanced in
EP3-null mice, suggesting a tumour-suppressive role for EP3 in the
intestine (129). It will be of interest to determine the contribution of
different PGE2 receptors to colorectal neoplasia and determine the
potential utility of specific EP antagonists in anticancer therapy in
the intestine (130,131).
Recent evidence suggests that deregulated catabolism of PGE2 also
plays a significant role in colorectal neoplasia. Prostaglandins are
essentially local-acting hormones with diverse effects in numerous
different tissues. Therefore, it is crucial that their catabolism is strictly
regulated in order to prevent them from reaching distant organs in the
bloodstream (reviewed in ref. 132). 15-PGDH is a cytosolic enzyme
that inactivates prostaglandins by oxidation to their 15-keto form
(reviewed in ref. 133) and was recently shown to act as a colorectal
tumour suppressor (134,135). In the normal human colon, 15-PGDH
is expressed in the non-proliferative epithelium at the top of the crypts
and loss of 15-PGDH expression and activity is seen in human co-
lorectal tumours (134,135). Several pathways deregulated in colorec-
tal neoplasia may contribute to this down-regulation as 15-PGDH is
negatively regulated by growth-stimulatory epidermal growth factor
signalling (up-regulated in colorectal neoplasia) and positively regu-
lated by TGFb (which colorectal cancer cells become resistant to)
(134,136). Functional evidence for the tumour suppressor activity of
15-PGDH in the intestine has been obtained using mouse models.
Loss of the gene encoding 15-PGDH in mice results in a doubling
of colonic mucosal PGE2 levels and promotes colorectal tumour
growth in ApcMin/ mice (137). Furthermore, restoring 15-PGDH
expression in colon cancer cells inhibits their ability to form tumours
in immune-deficient mice (134).
382
Prostaglandin synthesis and degradation are believed to be com-
partmentalized within the cell. This implies that following synthesis
and release, prostaglandins must be re-imported into the cell for deg-
radation by the cytoplasmic-localized 15-PGDH. Prostaglandins are
believed to traverse membranes poorly and their cellular uptake is
thought to occur mostly via PGT (also known as solute carrier organic
ion transporter family, member 2A1 or SLCO2A1) (reviewed in ref.
132). Indeed, in HeLa cells, co-expression of both 15-PGDH and PGT
is required for metabolism of exogenously added PGE2 (122). Simi-
larly to 15-PGDH, PGT was recently shown to be down-regulated in
colorectal neoplasia in both humans and ApcMin/ mice (138), sug-
gesting that PGE2 signalling can be further controlled via regulated
uptake.
It would be therapeutically desirable to increase prostaglandin deg-
radation in colorectal tumours but at present relatively little is known
about the potential for therapeutic re-expression of 15-PGDH and
PGT. However, PGT expression can be induced by the histone deace-
tylase inhibitor trichostatin A and the histone demethylating agent
5-azacytidine; two classes of drug relevant to cancer therapy (138).
Moreover, histone deacetylase inhibitors and 5-azacytidine can also
up-regulate 15-PGDH (139). Future advancements in our understand-
ing of prostaglandin catabolism in the intestine should allow insight
into the potential for targeting 15-PGDH and PGT in colorectal cancer
prevention and therapy.
Regulation of COX-2 expression by the tumour microenvironment
COX-2 up-regulation has been reported in a number of different
tumour types (reviewed in ref. 140), and accordingly there has been
a great deal of interest in attempting to achieve a greater understand-
ing of the regulatory networks that control COX-2 expression.
Whereas activating mutations in the PTGS2 gene (the gene encoding
COX-2 in humans) have not yet been described, there are several
known mechanisms underlying enhanced COX-2 expression in tu-
mour cells. Principally, these mechanisms include deregulated
growth factor signalling and oncogene activation. Examples of these
mechanisms include activation of the WNT pathway (141,142) and
the Ras-MAPK pathway (41,143) signalling via growth factor re-
ceptors including EGFR (144), TGFb receptors (145), c-Met (146)
and gastrin receptors (147). More recently, our laboratory has shown
that COX-2 can be induced by the hypoxic microenvironment in
colorectal tumour cells derived from both adenomas and carcinomas
(75). This up-regulation of COX-2 is transcriptional and is mediated
by the master regulator of transcription during hypoxia, HIF-1.
COX-2 has also been reported to be up-regulated in lung cancer cells
via a mechanism that is dependent on HIF-1 (148). COX-2 up-
regulation during hypoxia has also been described previously in
endothelial cells (149), monocytes (150) and corneal epithelial
cells (151); whereas in endothelial cells, COX-2 up-regulation ap-
pears to be mediated by nuclear factor-kappa B (149), in corneal
epithelial cells COX-2 over-expression was linked to PPARa/b
activation (151).
The role of COX-2 and PGE2 during hypoxia
As noted above, the HIF-1-dependent COX-2 up-regulation during
hypoxia is associated with an increase in PGE2 levels (75). Given
the pivotal role of PGE2 in colorectal tumorigenesis, these findings
build on the already known roles of PGE2 by demonstrating a role for
PGE2 in the promotion of cell survival under hypoxic conditionsa
property that tumour cells must acquire in order to propagate and
progress in vivo (152). Although the mechanism for this pro-survival
attribute of PGE2 is not yet defined, it is likely to involve the alteration
of apoptotic thresholds by modulating the intrinsic cell death machin-
ery; for example, by increasing BCL-2 expression, which has been
previously demonstrated to promote cell survival under hypoxic con-
ditions (153). The ability of hypoxia (154) and PGE2 (155) to activate
nuclear factor-kappa B may also be important in enhancing cell sur-
vival under hypoxic conditions. PGE2 may also confer alternative

survival strategies to cells via the up-regulation of pro-survival factors
such as inhibitor of apoptosis-2 (156), which has been shown to be up-
regulated by hypoxia (157).
In addition to promoting cell survival under hypoxic conditions, the
amplification of the COX-2/PGE2 pathway in hypoxic colorectal can-
cer cells may have important implications for stimulating angiogen-
esis, a process that is considered critical for solid tumour growth and
progression (96). While bearing in mind the important separate roles
of both hypoxia and COX-2/PGE2 signalling in the promotion of
tumour angiogenesis, we suggest that during hypoxia PGE2 signalling
may be integrated to act concordantly in the regulation of angiogen-
esis. Indeed, increased levels of PGE2 appear to enhance HIF-1 tran-
scriptional activity and VEGF production in hypoxic colorectal
cancer cells (75). Another possible level for the integration between
PGE2 and hypoxia signalling in the modulation of angiogenesis could
occur at the level of Src; a key factor in hypoxia-induced VEGF and
PGE2-mediated transactivation of EGFR that has implications for
colorectal tumour progression (72). Furthermore, as mentioned pre-
viously, PGE2 has been shown to enhance the expression of chemokine
receptor-4 (103), whose expression has been shown to be regulated by
HIF-1/hypoxia (158,159). The ability of PGE2 to enhance HIF-1 tran-
scriptional activity stands to be a potentially important observation.
Accordingly, PGE2 may promote the expression of several HIF-1 tar-
gets (in addition to VEGF) that are crucially involved in tumorigenesis.
If it turns out to be the case, PGE2 may then be regarded as a key
positive modulator of the transcriptional response to hypoxia, which
is believed to be critical for the progression of solid tumours including
colorectal cancer (99). The HIF-1-mediated transcriptional response
provides both short- and long-term adaptive strategies, including cell
survival, metabolic shift, angiogenesis, invasion and metastasis. There-
fore, further investigations into cross talk between HIF-1 and PGE2
signalling may provide greater insight into the mechanisms that pro-
mote adaptation to hypoxia and hence tumour progression.
Interplay between the COX-2/PGE2 pathway, HIF-1 and b-catenin
signalling
One of the mechanisms by which COX-2/PGE2 signalling influences
the hallmarks of cancer is via the ability of PGE2 to simulate the
activation of b-catenin, which leads to the stimulation of cell growth
and proliferation via its interaction with TCF-4 in the nucleus (89).
However, while in one study we found that COX-2/PGE2 levels are
increased in response to hypoxia (75), we also found that hypoxia
results in a transient cell cycle arrest that involves the inhibition of
b-catenin/TCF-4 activity (160). In the latter study, we found that the
cell cycle arrest induced by hypoxia in colorectal cancer cells results
from the direct binding of HIF-1a to b-catenin; this results in the
suppression of b-catenin/TCF-4 activity and a subsequent shutting
down of cell proliferation via the c-Myc-p21-axis (160). Furthermore,
we also revealed a novel role for b-catenin in the enhancement of HIF-
1 transcriptional activity, resulting in cell survival and adaptation to
the hypoxic microenvironment. Hypoxia, therefore, appears to shift
the balance from b-catenin signalling and co-activation of TCF-4 to
b-catenin interacting with and enhancing of HIF-1 activitythereby
favouring cell quiescence and survival over proliferation. This sce-
nario illustrates an intriguing dynamicity in b-catenin function in re-
sponse to conditions of low oxygen tension. The ability of b-catenin to
interact with and enhance HIF-1 transcriptional activity represents
a novel function for b-catenin in colorectal tumorigenesis that is
applicable to hypoxic conditions and may have implications for other
aspects of carcinogenesis including metabolic adaptation, invasion and
metastasis.
Together, our findings (75), with those of Castellone et al. (89),
suggest on the one hand, a selective role for PGE2 in the stimulation of
cell proliferation that is restricted to normoxic conditions. On the
other hand, during hypoxia, the proliferative effect of PGE2 may be
overridden as a result of HIF-1 competing with TCF-4 for b-catenin,
causing the inhibition of b-catenin/TCF-4 activity and cell cycle arrest
(160). Therefore, we propose a model in which hypoxia favours the
pro-survival (anti-apoptotic) and pro-angiogenic functions of PGE2,
The COX-2/PGE2 pathway
whereas hampering the pro-proliferative effect of PGE2. The net ef-
fect of this selective strategy is enhanced tumour cell survival due to
a cell cycle arrest that enables the mitigation of hypoxia-induced
cellular stresses, which would otherwise compromise cell survival if
proliferation was allowed to continue during hypoxic conditions. This
scenario may represent a spatialtemporal switching in PGE2 signal-
ling and function during colorectal tumorigenesis. This model is sum-
marized in Figure 1.
The role of PGE2 in the activation of b-catenin/TCF signalling also
suggests that PGE2 might serve an important function at early stages
of colorectal tumorigenesis. This highlights the possibility that basal
PGE2 levels may switch on the b-catenin-signalling cascade, which in
turn would promote cell proliferation that may contribute to tumour
initiation. Given that COX-2 is not induced at the very early stages of
colorectal tumorigenesis, COX-1 might be the source of PGE2 during
tumour initiation, perhaps enhancing b-catenin/TCF-4 signalling
upon the loss of APC. This possibility is consistent with the findings
that loss of either COX-1 or COX-2 decreases tumour burden in
mouse models of intestinal tumorigenesis (53). This may also explain
why inhibition of both COX-1 and COX-2 enzymes (for example with
aspirin or sulindac) is as effectiveif not potentially more soas
selective COX-2 inhibitors as chemopreventive agents for colorectal
cancer.
Concluding remarks
Compelling evidence gained from mechanistic studies with cancer
cell lines, mouse models of intestinal tumorigenesis and a number
of clinical trials with both non-selective and COX-2-selective
NSAIDs support an important role for the COXprostaglandin path-
way in the development of colorectal tumours. Furthermore, these
studies have validated the COX-2/PGE2 pathway as a bona fide target
for cancer chemoprevention and therapy. Evidence suggests that the
effectiveness of suppressing the COX-2/PGE2 pathway in cancer pre-
vention and therapy stems from its role in driving the hallmarks of
cancer, without which tumours cannot sustain their growth and
development.
Recent clinical trials involving highly selective COX-2 inhibitors as
chemopreventive agents for colorectal cancer have cast doubt on the
suitability of such drugs for long-term use due to increased risks of
adverse cardiovascular events (30,161). Notwithstanding, the COX-2
Fig. 1. Under normoxic conditions, COX-2 expression can be induced
through the activation of the oncogenic pathways such as the Ras-MAPK
pathway. Furthermore, COX-2-derived PGE2 promotes cell proliferation at
least in part via the stimulation of b-catenin/TCF-4 activity, as well as via
enhancement of Ras-MAPK signalling. During hypoxia, b-catenin is
displaced from TCF-4 (which results in TCF-4 repression) via its interaction
with HIF-1, and b-catenin enhances HIF-1 transcriptional activity. In
addition, COX-2 is up-regulated during hypoxia via HIF-1 activation,
resulting in increased levels of PGE2. Both enhanced HIF-1 transcriptional
activity and increased PGE2 levels promote cell survival leading to
adaptation to hypoxia. Whereas b-catenin acts as essential activator of TCF-4
in normoxia, b-catenin results in the enhancement of HIF-1 transcriptional
activity during hypoxia. These effects are likely to be transient and reversible
(dynamic) depending on the tumour microenvironment and fluctuations in
oxygen levels.
383
A.Greenhough et al.
selective inhibitor celecoxib remains a clinically relevant agent for
colorectal cancer prevention and therapy, and further studies will be
required to establish the usefulness of NSAIDs in chemoprevention
particularly in patients at a high risk from colorectal cancer. Given the
importance of COX-2/PGE2 in influencing the hallmarks of cancer, it
will also be important to determine the applicability of NSAIDs as
adjuvants to current chemotherapy/radiation regimens, not only for
colorectal cancer patients but also for patients with other tumour types
displaying deregulated COX-2 expression.
Future studies into the emerging players within the COX-2/PGE2
pathway may reveal novel approaches for more safely targeting this
pathway for both cancer chemoprevention and therapy.
Funding
Cancer Research UK programme; The Citrina Foundation; The John
James Bristol Foundation.
Acknowledgements
Conflict of Interest Statement: None declared.
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Received September 8, 2008; revised December 4, 2008;
accepted January 4, 2009