Download Embryonic genes in cancer

Embryonic genes in cancer
Roser Calvo a and Harry A. Drabkin b
" Hospital Universitari Germans Trias i Pujol, Medical Oncology Service, Badalona (Barcelona), Spain
Division of Medical Oncology, University of Colorado Health Sciences Center, Denver, CO 80262, USA
b
Introduction
renal cancers [4]. Observations such as these led to
the concept of specific 'gatekeeper' genes, whose
inactivation (tumour suppressor genes) or activation
(proto-oncogenes) in particular cell types is necessary for tumour development. These are, in essence,
rate-limiting mutations effecting key regulatory pathways which occur in conjunction with other genetic
and epi-genetic mutational events.
Like APC mutations in colon cancer, Ptc is a good
example of the 'gatekeeper' of basal cell carcinoma
(BCC). Interestingly, many of the genes in the hh and
interacting Wnt/wg pathways are either oncogenes or
tumour suppressor genes [5] (Fig. 1). This is the case
for the oncogenes GUI and Wnt (homologues of cubitus interruptus (ci) and wingless (wg), respectively)
[6,7], or the tumour suppressor genes PTCH (human ptc) and APC (see below). Indeed, GUI was first
identified by and named for its involvement in glioma
formation [8], and Wntl is known to be involved in
murine mammary tumourigenesis [9]. These genes,
along with the Smad genes, which mediate TGFp1
signalling, are involved in the formation of a range
of tumour types.
The connection between developmental genes and
cancer has been a topic of great interest as the processes of proliferation, differentiation and tumourigenesis have long been thought to be inter-related.
Pioneered by genome-wide mutational screens in
Drosophila, and more recently coupled to powerful
molecular technologies, many genes responsible for
developmental alterations have now been incontrovertibly linked to human cancer. Among these are
genes of the segment polarity class, the majority of
which have been shown to encode components of the
hedgehog (hh) and wingless (wg) pathways. Genes
such as patched (ptc), a key regulator in Drosophila
embryonic development, provide important examples of how normal development and tumourigenesis
intertwine.
Oncogenes and tumour suppressor genes in
embryonic development and cancer
The notion that specific tumours within cancer
syndromes share mutations of critical genes with
their sporadic counterparts was established over a
decade ago. Indeed, the discovery of mutations in the
retinoblastoma gene (RE) in both hereditary and sporadic tumours [1] was followed by the observation of
mutations in the adenomatous polyposis coli (APC)
gene, not only in sporadic polyps [2] but in those patients affected by the familial adenomatous polyposis
(FAP) syndrome, in which patients develop multiple
adenomas and cancer. Likewise, RET, previously the
only proto-oncogene found to be responsible for an
inherited cancer syndrome, the multiple endocrine
neoplasia (MEN), was also found to be mutated in
sporadic medullary thyroid tumours [3]. More recently, activating mutations in the receptor tyrosine
kinase, MET, have been shown to be responsible for
some forms of hereditary and spontaneous papillary
Hedgehog signalling pathway: the patched gene in
development and cancer
The hh signalling pathway is a fundamental signal transduction system in embryonic development,
being responsible for the patterning of a range of
embryonic and adult tissues in both the fly and vertebrates. Hedgehog, which has three homologues in
mammals (Sonic, Desert and Indian), is a unique
cholesterol-tethered membrane ligand which binds
to its receptor, patched. Dysregulation of this pathway results in the formation of several tumour types
[6] and dysmorphology syndromes. From genetic
screens, ptc and smoothened (smo) were identified
as genes acting upstream of fused (fu), suppressor
of fused (su (fit)), costal-2 (cos-2) and ci (homol207
R. Calvo and HA. Drabkin
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Fig. 1. Segment polarity gene pathways include oncogenes (bold) and tumour suppressor genes (underlined). The mammalian ortologues
that act in HH signaling are shown. WNTs signal through mammalian DFZ2 homologues (HFZs) and cause the release of P-catenin/arm
from the destruction complex. Free P-catenin/arm translocates into the nucleus and stimulates cell division, perhaps working as other
mitogens through the activation of cyclin E/CDK2. TGF-P and BMPs signal through the TGF-P receptors (TGFBR) and through SMAD
proteins (DPC4 and others). TGF-P inhibits cell division through the repression of phosphorylation of pRB (see text for details). Figure is
modified from [5].
•<,
ogous to the vertebrate Gli genes). In Drosophila,
known downstream genetic targets of the hh pathway include wg (homologous to the vertebrate Wnt
genes), decapentaplegic (dpp, a member of the TGF{}
superfamily most homologous to the vertebrate bone
morphogenetic proteins, BMPs), as well as ptc itself. The term 'genetic targets' includes genes which
are both directly (cell-autonomous) as well as indirectly regulated. Recently, several other genes have
been shown to be responsive to hh signalling such
as hip (for hedgehog interacting protein) [10] and
WSB1/SWIP-1 [11]. Tumour formation occurs when
mutation of regulatory genes leads specifically to the
activation of downstream hh responsive genes.
Ptc was first identified in a search for genes
essential for embryonic development in flies [12].
Ptc is a component of the hh receptor complex
acting as a repressive component of the pathway.
Hedgehog protein (hh) must overcome pfc-induced
repression to activate hh target genes. The characterisation of another Drosophila segment polarity gene,
smo, added an important clue to the understanding
of the hh pathway and the way in which ptc functions. It is now clear that in the absence of hh, ptc
interacts with the seven-membrane spanning protein,
smo (smoothened), rendering it inactive. However,
when hh binds to ptc, inhibition of smo signalling is
released initiating a signalling cascade that includes
the intracellular components of the hh pathway, fu,
su (fu), cos-2 and the ci gene product. The end result
is the transcription of downstream target genes, ptc,
wg, and dpp (Fig. 2). Thus, in the absence of ptc
function, there is constitutive signalling from smo,
which results in the expression of downstream target
genes.
The recent identification of mutations in the human PTCH gene in BCCs indicated that HH signalling was important in human cutaneous carcinogenesis. Indeed, PTCH was identified as the locus
responsible for the Nevoid Basal Cell Carcinoma
Syndrome (NBCCS) or Gorlin's syndrome [13-15],
an autosomally inherited disorder in which patients
have multiple BCCs, various malformations, and
other tumours including medulloblastoma, ovarian
fibroma, meningioma, fibrosarcoma, rhabdomyosarcoma and cardiac fibroma. One PTCH allele is
mutated in the germline of these patients [1,16-18],
and the basal cell carcinomas in NBCCS individuals
arise with inactivation of the remaining PTCH allele,
consistent with PTCH acting as a tumour suppressor
gene. The finding of PTCH mutations in a proportion
of sporadic BCCs, in many cases with both alleles
inactivated by either mutation or loss of heterozygosity (LOH) [19], supports this putative function.
The estimated frequency of PTCH mutations in sporadic BCCs ranges from 12-38%, although this may
be a low estimate based on the mutation detection
method. To date, the molecular analysis of these
Embryonic genes in cancer
209
Fig. 2. The hedgehog signalling pathway, (a) In the absence of hedgehog (hh), the patched protein (ptc) inhibits signalling by smoothened
(smo) and the intracellular components of the hh pathway (Fused [Fu], Suppressor of Fused [Su(Fu)], Costal-2 [Cos2], and Cubitus
interruptus [Ci]), form a multimeric complex that associates with microtubules. The full-length activating form of the transcription factor
encoded by Ci (Ci 155 ) is cleaved to yield a repressing form (Ci 75 ). The activity of protein kinase A (PKA) appears to promote Ci
cleavage, possibly by phosphorylating Ci directly, (b) When hh binds to Ptc, the inhibition of smo is released. Smo activity promotes the
dissociation of the complex of segment polarity proteins (Fu, Su(Fu), Cos2, Ci), normally associated with the microtubules. Cleavage of
Ci is blocked and the full-length form of the protein (Ci 155 ) associates with the co-activator CBP to activate transcription of target genes.
In vertebrates the role of Ci is achieved by complex interactions involving three Gli genes.
mutations shows that most give rise to a truncated
form of the protein. In addition to mutations in the
PTCH gene, LOH in the region of chromosome 9q
encompassing PTCH is observed in upwards of 50%
ofBCCs[20].
It is speculated that BCCs arise through the inappropriate activation of the Shh pathway in the nonfollicular epithelium caused in many cases by loss of
PTCH function in these human skin cells. Given that
constitutive activation of smo is likely to upregulate
transcription of hh target genes in the same way as
PTCH inactivation, it is not surprising that activating
mutations in the smo gene have been detected in 1020% of sporadic BCCs [21]. Since hedgehog itself is
primarily responsible for activation of this pathway,
it is feasible that it also may be mutated in associated
tumours. Indeed, a single recurrent mutation in Shh
was initially reported in a range of tumour types
including BCC [22] but subsequent studies failed to
detect this mutation, suggesting that it is extremely
rare [23,24].
Other tumours that carry PTCH mutations include
sporadic medulloblastomas and other primitive neuro-
ectodermal tumours [24] as well as the benign skin
lesions tricoepitheliomas, esophageal squamous cell
carcinomas and transitional cell carcinomas of the
bladder [25-27]. Interestingly, in the mouse loss of
only one copy (haploinsufficiency) appears to be sufficient for medulloblastoma development [28].
Wnt genes in growth control, development and
carcinogenesis
Among the most striking links between oncogenesis and development are those provided by wnt
genes. In particular, the discovery that activating
mutations in P-catenin are associated with a variety of human cancers has fuelled an extraordinary
explosion of interest into the relationship between
Wnt signalling and oncogenesis. In recent years,
major advances have been made in understanding
the wingless /wnt signalling pathway in Drosophila
and in vertebrates [29-32]. Wnt genes are sources
of differentiation-inducing signals during normal development events, but they also have the potential
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R. Calvo and HA. Drabkin
to promote carcinogenesis through local effects on
cell proliferation, particularly in the mammary gland.
Indeed, mis-regulation of wnt signalling not only can
cause developmental defects but is also implicated in
the genesis of several human cancers [29].
At the core of the Wnt pathway is P-catenin, a
multifunctional protein with independent roles in
cadherin-mediated cell adhesion and Wnt signal
transduction. P-catenin is central to the wnt signalling pathway and its activity is controlled by a
large number of binding partners that affect its stability and localisation, ultimately modulating several
developmental processes such as gene expression and
cell adhesion. Activating mutations in P-catenin and
in components regulating its stability contribute to
the formation of certain tumours.
P-Catenin displays a high degree of homology
with the Drosophila segment polarity gene armadillo
{arm). Its primary structure comprises an amino
terminus, important for regulating the stability of
P-catenin; a carboxy terminus, which functions as
a transcriptional activation domain, and a central
domain that consists of 12 repeats (referred to as
arm repeats), through which P-catenin will interact with its binding partners APC, TCF, E-cad and
Fascin (Fig. 3). The amino terminus domain of
P-catenin contains a series of serine and threonine
residues, which may be phosphorylated. Phosphorylation of these residues is thought to be the signal
for the degradation of P-catenin by the ubiquitinproteasome pathway. Interestingly, several colon car-
cinoma and melanoma cell lines contain elevated
levels of P-catenin; in many of these cell lines the
increased stability of P-catenin may be attributed to
point mutations in its amino terminus region that
change the serine and threonine residues, thereby
blocking its phosphorylation and subsequent degradation [33,34].
The W7VTs (human homologues of Drosophila
wg), with a critical role in the formation of many
differentiated cell types [31], are genes expressed
in cell-specific patterns in early development. WNTs
signal through mammalian homologues of the frizzled (fz) gene family (HFZs) of receptors [35]. A
prevailing model of the wnt signalling cascade is
summarised in Fig. 4 [36]. In the absence of a wnt
signal, P-catenin/arm level is low due to the degradation promoted by glycogen synthase kinase 3P
(GSK3) and APC. In the presence of a wnt signal,
a fz receptor is activated and it, in turn, activates
dishevelled (dsh). dsh then inactivates the GSK3 kinase, resulting in high levels of free P-catenin/arm in
the cytoplasm upon its release from the destruction
complex. P-Catenin then enters the nucleus where it
interacts with the transcription factors TCF/LEF-1
(T-Cell Factor in mammals and Xenopus and Lymphoid Enhancer Factor-1 in Drosophila) to modulate
transcription of wnt-responsive genes. The end result
is the stimulation of cell division.
Four proteins have been identified that directly
promote the degradation of P-catenin: GSK3, Axin,
APC, and P-TrCP/Slimb. These proteins comprise
a-catemn
Putative
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sites, mutated
in tumors
Interactions with:
APC
II
E-cadherin
TCF/LEF/Pan and Fascin
arm repeats
Trans-activation
domain
Ubiquitination
sites
Fig. 3. Structure of P-catenin. The central domain consists of 12 imperfect repeats, called arm repeats, that interact in an overlapping and
mutually exclusive manner with APC, TCF, E-cadherin and Fascin. The amino-terminus contains multiple phosphorylation sites which
are correlated with downregulation of P-catenin. Mutation of these potential phosphorylation sites or deletion of the amino-terminus
produces a more stable and hence constitutively active B-catenin protein.The carboxyl terminus contains a transcriptional activation
domain.
Embryonic genes in cancer
In the absence of a Wol/Wg signal
211
In the presence of a Wnt/Wg signal
Fig. 4. Wnt/P-catenin pathway, (a) In the absence if a Wnt signal, Dishevelled (Dsh) is inactive (Dshj) and Dmsophila Zeste-white 3 or
its mammalian homologue glycogen synthase kinase 3 (Zw3/GSK3) is active. GSK3 phosphorylates P-catenin. This phosphorylation is
facilitated by Axin and APC. Phosphorylation of P-catenin targets it for ubiquitination by P-TrCP resulting in the proteasomal degradation
of P-catenin. Meanwhile, some members of the TCF/LEF family of transcription factors are bound to their DNA-binding site in the
nucleus acting as repressors through interaction with the co-repressors, Groucho, CBP and CtBP. (b) In contrast, in the presence of a
Wnt signal, Dishevelled becomes activated (Dslu). Dsh, and GBP inhibit phosphorylation of P-catenin by GSK3. P-Catenin fails to be
phosphoiylated and thus is no longer targeted into the ubiquitin-proteasome pathway. Instead, p-catenin accumulates in the cytoplasm and
enters the nucleus. Nuclear P-catenin binds TCF/LEF and activates transcription of target genes.
the 'destruction' complex and act to maintain low
steady-state levels of P-catenin in the cell. GSK3, the
central player in this destruction complex, encodes
a Ser/Thr kinase which phosphorylates the aminoterminal serine and threonine residues of P-catenin
and thereby targets it for destruction, thus acting as a
negative regulator of the Wnt pathway. APC was first
identified as a tumour suppressor as mutations predispose individuals to develop colon carcinomas. It was
later shown that APC binds directly to P-catenin and
GSK3 [37]. Several colorectal carcinoma cell lines
contain mutant APC and elevated levels of P-catenin;
overexpression of wild-type APC in these cell lines
significantly reduces the level of free P-catenin, suggesting that APC is a negative regulator of the wnt
signalling pathway. Elevated free P-catenin in the
cytoplasm, as is the case when the cell receives a wnt
signal, leads to the nuclear accumulation of P-catenin
where it associates with transcription factors of the
TCF/LEF class and acts as a transcriptional activator.
Interestingly, in the absence of P-catenin, TCF still
can bind to its target DNA. In such a case, however,
TCF not only fails to activate transcription of target
genes but can function as a transcriptional repres-
sor by its binding of transcriptional repressors such
as groucho and the related TLEs (transducin-Iike
enhancer of split).
In addition to its role in regulating gene expression, P-catenin can also affect cell adhesion. By
binding to both E-cadherin (E-cad) and a-catenin
simultaneously, P-catenin links the adherens junction
to the cytoskeleton of the cell. However, the degree
by which wnt signalling effects cell adhesion through
p-catenin is not yet clear. Certain Wnt ligands, such
as Wnt5a, affect cell migration and inter-cellular
adhesion in a calcium dependent manner through a
seemingly non-P-catenin mechanism. In an indirect
fashion, wnt signalling appears to modulate cell adhesion since the mouse E-cad promoter contains a
TCF-binding site and it has been shown [38] that
activation of the wnt signalling pathway leads to
transcriptional activation of E-cad. Whether this effect is mediated directly through the TCF/P-catenin
complex remains to be seen. APC may also have an
important role in cell adhesion [39], and like ptc, it
is a cytoplasmic molecule that controls a complex
signalling cascade. It seems reasonable to speculate
that the first effects of the lack of proper response of
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these proteins to signals from neighbouring cells may
be abnormal adhesion of the cells, resulting in abnormally proliferating clusters of cells from which other
genetic hits can occur, leading to tumour progression.
Wnt-1 was originally identified as a preferred integration site for the mouse mammary tumour virus
in breast carcinomas [40]. Although much evidence
points to the possibility of Wnt-1 acting as an oncogene, mutations in Wnt-1 have not been linked to
cancer in humans. However, since mutations in several components of the Wnt/P-catenin pathway are
implicated in tumour formation, it is likely that
Wnt-1, or other wnt proteins capable of activating
the Wnt/p-catenin pathway, may act as oncogenes
in humans. In addition, a number of studies provide evidence that WntSa is able to antagonise the
Wnt pathway [41]. The ability of WntSa to suppress transformation by Wnt-1 in vitro suggests that
WntSa may act as a tumour suppressor. However,
the role of WntSa in carcinogenesis remains unclear,
since current in vivo evidence appears to conflict
with the concept of WntSa as a tumour suppressor,
given the decrease in cell growth and proliferation
observed in mice after removal of the WntSa function, suggesting a positive role for this gene in the
regulation of cell growth and differentiation [42].
TGF-P pathway disruption in cancer
Besides WNTs, the hh signalling pathway influences
cellular proliferation via a different family of effector
proteins, the transforming growth factor-^ (TGF-P)
family and related cytokines —such as activins and
bone morphogenetic proteins (BMPs) [43]. These
molecules, by controlling the expression of cell cycle
regulators, cell adhesion molecules, and differentiation factors, regulate cell fate and are therefore
important for the development and maintenance of
most tissues [44]. Since the TGF-p cytokines were
first identified as inhibitors of epithelial cell proliferation [45], manifested by Gl-phase arrest, terminal differentiation, or induction of apoptosis, it has
been anticipated that understanding the mechanism
of TGF-p action would also shed light on the events
leading to neoplastic transformation.
Members of the TGF-p family of ligands (mammalian homologues of Drosophila dpp) signal
through heteromeric complexes of two transmembrane serine/threonine kinases, the type I and type II
receptors [46], which will interact with the SMAD
proteins, mediators of the TGF-P signalling. Upon
their phosphorylation in their carboxy-terminal domain by receptors, SMADs become activated; they
then associate to a second group of collaborating
SMAD proteins (co-SMAD) and move into the nucleus, where they interact with transcription factors,
such as FAST-1, and stimulate target gene expression. Thus, SMAD proteins directly transmit TGF-P
signals from the cell surface receptors to the nucleus
(Fig. 5). The only known member of the co-SMAD
group in vertebrates is smad 4, originally identified
as the product of the deleted in pancreatic cancer locus 4 (DPC4) tumour suppressor gene [47], which is
mutated or deleted in a high proportion of pancreatic
cancers and in a smaller proportion of other cancers.
Given the growth inhibitory effects of the TGF-P
signalling pathway, it is reasonable to speculate that
its disruption may predispose to or cause cancer.
This idea has been confirmed in recent studies. Inactivation of the type II TGF-P receptor has been
detected in some tumour types and may be associated with the RER+ (replication error) phenotype
[48]. These data suggest that type II TGF-P receptor
may function as a tumour suppressor gene. A significant number of inactivating mutations have also
been found in SMAD genes derived from human
cancers, smad 4/DPC4 is either deleted or mutated
in a large proportion of pancreatic cancers [47]. smad
2 is also mutated in a number of colon and head
and neck carcinomas [49-51]. The majority of the
identified missense mutations map within the carboxy-terminal domain of the protein, which not only
mediates receptor-mediated phosphorylation and activation of SMADs but contributes essential proteinprotein interactions to the SMAD pathway and therefore trans-activation functions. Thus, tumourigenic or
developmentally inactivating missense mutations in
the carboxy-terminal domain abolish specific SMAD
interactions that are vital for the pathway. Tumourderived missense mutations have also been identified
in the amino-terminal domain of the protein. This
region is involved in the process of self-inhibition
of SMADs, which occur through direct interaction
between the amino-terminal and carboxy-terminal
domains. Tumour-derived mutations in the aminotenninal domain of smad 2 or smad 4/DPC4 increase
their affinity for their respective carboxy-terminal domains, thereby preventing smad 2-smad 4 association
and TGF-P signalling [52]. In addition, many cancers, including most pancreatic tumours, show allelic
loss at a site on chromosome 18q that contains the
genes DPC4 and DCC {deleted in colorectal cancer),
a tumour suppressor for colorectal cancer.
Finally, TGF-P signals can regulate the retinoblastoma protein (pRB) in the nucleus through the regulation of pl5/pl6. Diverse inactivating mutations in
the RB gene occur in many types of cancer, and func-
213
Embryonic genes in cancer
Ligands
TGF-S
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BMP
Extracellular
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Cytoplasm
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Smad3
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Biological responses
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Apoptosis
Cell cycle arrest
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Fig. 5. TGF-P signalling pathway. The TGF-p family of ligands binds to and activates distinct combinations of type I and type n
serine-threonine kinase receptors leading to a transient association with specific receptor-activatable SMADs. These SMADs (Smad 1,
5, 8, 2, and 3, in vertebrates) become phosphorylated (p) by the activated type I receptors, then associate with a co-SMAD (e.g. Smad
4) and move into the nucleus. In the nucleus the SMAD complexes associate with DNA-binding transcriptions factors (e.g. FAST-1,
or in this hypothetical setting, A, B, and C) leading to active transcriptional complexes and stimulation of target gene expression. The
combination of activated target genes and the particular cellular environment will determine the kind of biological response. (Figure is
adapted from [46].)
tional pRB can suppress the tumourigenicity of these
cells. Whereas the phosphorylated pRB protein forms
complexes with the E2F transcription factor and activates genes required for cells to pass the restriction
point in the Gl phase of the cell cycle [53], underphosphorylated pRB prevents progression through
the cell cycle. TGF-P may inhibit growth by preventing pRB phosphorylation. The pRB is normally
phosphorylated by at least two cyclin/cyclin dependent kinases (CDK) complexes, which in turn can
be inhibited by other proteins such as pl5 and pi6,
both identified as tumour suppressor genes. Such repressors, pl5 and pi6, through their interaction with
the cyclin D-CDK4/6 complex, prevent pRB phosphorylation and cell cycle progression, helping to
explain why the loss of p l 5 / p l 6 is a common event
in tumours. TGF-p is known to affect the expression
and/or activity of pl5/pl6, potential effectors of
TGF-P-mediated cell cycle arrest [54]. In addition,
the CDK inhibitor p21 can block the activity of both
CDK2 and CDK4/6. DNA damage due to chemical
agents or radiation leads to the accumulation of p53
protein. p53 induces the expression of p21 and prevents the cell from entering S phase with a damaged
genome. Cells that cannot repair their DNA can be
induced to undergo apoptosis.
Homeoboxes, homeodomains and cancer
Another group of genes first isolated as determinants
of development, the homeobox-encoding genes, have
also been shown to have either oncogenic or tumour suppressor potential. Homeobox genes control
anterior-posterior cell fates in multiple tissues of
organisms as different as flies, mice, worms and vertebrates [55]. Individual genes within the homeobox
gene family share a 183-nucleotide DNA segment
termed the homeobox [56]. The homeobox encodes
a 61-amino acid protein segment called the homeodomain, a region able to bind specific DNA sequences. Homeodomain proteins have been shown
to be transcription factors, with either positive or
negative effects on the expression of target genes,
which will lead to the formation of characteristic
structures along the body axis. The search for homeobox downstream targets is currently an important
avenue in the investigation of homeobox gene func-
214
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Fig. 6. Hox genes nomenclature system. Hox genes in vertebrates are clustered in four complexes (A-D). Based on sequence similarity
Hox genes can be sorted into 13 'paralog' groups, each group having, in most cases, a representative in each complex. The order of
paralogs along the chromosome is maintained in the four complexes. Genes within the complexes are numbered according to the paralog
group they belong, from 1 (at the 3' end) to 13 (at the 5' end). In several cases a representative of a paralog group is absent from a
complex, in which case the corresponding gene number is omitted (blank spaces in the figure). H = nomenclature for human. M =
nomenclature for mouse. The genes required farthest anterior in the developing animal are at the 3' end of the complex. Genes with more
posterior domains of action are located farther 5' in the complex. Thus, in the figure, paralog groups are demarcated by 'anterior' and
'posterior'. Drosophila counterpart homeotic genes are also shown.
tion. Few homeobox target genes are known in flies
and almost none in vertebrates, so the mechanism
by which homeobox genes control morphogenesis
remains incompletely understood. Most Hox targets
identified to date have been found in the visceral
mesoderm of Drosophila, where they are required
for the development of the midgut constrictions. A
hierarchy of target genes regulated by the Hox genes
Ultrabithorax (Ubx) and abdominal A (abdA) to dictate morphogenetic decisions in the midgut has been
identified. In particular, these target molecules include WG and DPP proteins (components involved
in transducing wg and dpp signals), and the transcription factor Teashirt (Tsh) [57]. To date, a plethora
of mammalian homeobox genes have been reported,
among which 38 are located in four clusters (A-D)
and are referred to as 'Hox' genes. Each cluster
contains 9 to 11 genes which are numbered beginning with those located at the 3' end of the complex
(Fig. 6).
In cancer, the deregulation of homeobox genes has
been most convincingly demonstrated in leukemia.
For example, the pre-B cell t(l;19) translocation
fuses E2A with the homeobox gene, Pbxl, and the
T-cell t(10;14) results in overexpression of Hoxll
[58-60]. In acute myelogenous leukaemia, the human
Trithorax or MLL gene, which normally helps to
maintain Hox gene expression, is a frequent target
of chromosomal rearrangements. Recently, Joh et
al. [61], demonstrated that a chimeric MLL-LTG9
protein led to the inhibition of HoxA7, HoxB7 and
HoxC9 expression in mouse 32Dcl3 myeloid cells.
In an experimental setting, a high proportion of mice
transplanted with bone marrow cells overexpressing
either HoxB8, A9, A10 or B3 eventually developed
AML [62-64].
In solid tumours, rearrangements of Hox genes
have not been reported. However, expression surveys have noted differences between normal and tumour samples in kidney, colon and lung carcinomas
[65-68]. In melanomas, Card et al. [69] demonstrated
that HOXB7 was constitutively expressed in 25/25
melanoma cell lines and that antisense HOXB7 inhibited cellular proliferation and the expression of
the basic fibroblast growth factor (bFGF). In the gut,
the caudal homologue, Cdx-2, functions as a tumour
suppressor gene. Not only is its activity downregulated by mutant Ras [70] but mice lacking Cdx-2
develop multiple gastrointestinal polyps [71]. Moreover, in one case of a human colon carcinoma both
Embryonic genes in cancer
CDX2 alleles were mutated [72]. While expression
and alterations of Cdx-2 were previously confined to
the gut, the human CDX2 gene has recently been involved in a t(12;13) chromosomal translocation along
with TEL [73,74] .
Further analysis of HOX genes in different cancer
types combined with other interactive components
such as the WAT genes will lead not only to a better understanding of carcinogenesis but will provide
important prognostic and diagnostic molecular markers. Also, since these pathways are interactive during
normal development, an integrated approach to their
analysis should be particularly informative.
Cyclooxygenases: their role in inflammation,
cancer, and development
The cyclooxygenase (COX) enzymes catalyse a key
step in the conversion of arachidonate to PGH2
(prostaglandin H2), the immediate substrate for a series of cell specific prostaglandin and thromboxane
synthesis. Prostaglandins play key roles in several biologic processes such as regulation of immune function, kidney development, reproductive biology, and
gastrointestinal integrity [75]. COX-1 and COX-2,
the two COX isoforms, differ mainly in their expression patterns; while COX-1 is expressed in most
tissues, COX-2 is absent but is induced by numerous physiological stimuli. Cyclooxygenase activity
can be inhibited by nonsteroidal anti-inflammatory
drugs (NSAIDs), the prototype of which is aspirin.
Although it has been suggested that COX-1 is the
'housekeeping' isoform of cyclooxygenase, and that
COX-2 acts in a pro-inflammatory fashion, being
rapidly inducible in response to numerous stimuli,
the pro-inflammatory role of COX-2 has recently
been questioned since it has been demonstrated that
COX-2 generated prostaglandins may actually enhance resolution of inflammation [76].
Phenotypic analysis of Coxl and Coxl null mice
yields information about the role of cyclooxygenases
in development, and it is clear that the role of these
genes in a developing organism is much more complicated than was once thought. Indeed, given that
COX-1 generated prostaglandins appear to be cytoprotective to the gastric mucosa, it was hypothesised
that Coxl null mice might exhibit gastric pathology.
Surprisingly, disruption of Coxl not only did not
result in gastrointestinal abnormalities but null mice
were generally healthy [77]. In contrast, Cox2 null
mice show reproductive anomalies and defects in
kidney development [78-80].
Some of the same processes occur in both uterine
215
decidualisation (sometimes referred to as pseudomalignant state) and cancer, allowing COX-2 parallels
to be established between cancer and development.
Indeed, during blastocyst implantation and decidualisation, a vascular network must be established to
support the nutritional needs of the developing embryo. In a similar way, expansion of a tumour mass
requires a vascular network to support its metabolic
demands. There is evidence that COX-2 generated
prostaglandins participate in angiogenesis, common
to both development and cancer. It has also been
demonstrated that maximal tumour expansion occurs
when the tumour escapes immunologic surveillance.
Like tumour cells, the blastocyst, also considered
foreign material, must escape immunologic surveillance to survive. In this regard, COX-2 generated
prostaglandins have been demonstrated to be immunosuppressive.
There is ample genetic and pharmacologic evidence to implicate COX-2 in neoplasia. It has been
found that NSAIDs, inhibitors of cyclooxygenase,
are chemopreventive for colon cancer. If NSAIDs do
reduce the risk of developing colon cancer, by what
mechanism do they achieve this effect? Certainly,
the COX enzymes are known targets of NSAIDs. If
COX were shown to contribute to tumour growth,
then one might expect that levels of downstream
metabolites (i.e. prostaglandins) would be increased
in tumours, and that there would be abnormal COX
expression. Consistent with this hypothesis, COX-2
is overexpressed in 50% of benign polyps and 8 0 85% of adenocarcinomas [81]. Mice heterozygous
for an APC mutant allele develop hundreds of intestinal polyps. Interestingly, offspring from cox2 null by
ApcA116 matings exhibit an 86% reduction in polyp
number when compared to offspring from control
animals [82], thus providing genetic evidence that
COX-2 contributes to tumour formation or growth.
The precise contribution of COX-2 to neoplastic
growth has not been elucidated. However, there is
some evidence that COX-2 may blunt the apoptotic
response in tumour cells and play a direct role in tumour cell growth. Additionally, there is evidence that
COX-2 may indirectly modulate tumour expansion
since it has been demonstrated that COX-2 induces
angiogenesis in vitro and can also downregulate natural killer T-cell function.
Summary
Over the past several years, great strides have been
made in our understanding of the mechanisms and
functions of many signalling pathways involved in
216
R. Calvo and HA. Drabkin
both development and oncogenesis. It is now clear
that some proteins/genes essential for cell differentiation can also provide signals for growth control,
suggesting that these genes may be mutated in tumours; this knowledge can help further the search for
new targets for chemotherapy. However, many questions about how dysregulation of signalling pathways
leads to neoplasia are still unanswered. With the
dramatic increase in the number of researchers examining those questions, answers will not be far
away.
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