REVIEWS

HEDGEHOG SIGNALLING IN
CANCER FORMATION AND
MAINTENANCE
Marina Pasca di Magliano and Matthias Hebrok
The Hedgehog signalling pathway is essential for numerous processes during embryonic
development. Members of this family of secreted proteins control cell proliferation, differentiation
and tissue patterning in a dose-dependent manner. Although the overall activity of the pathway is
diminished after embryogenesis, recent reports show that the pathway remains active in some
adult tissues, including adult stem cells in the brain and skin. There is also evidence that
uncontrolled activation of the pathway results in specific types of cancer.

Recent reports that uncontrolled activation of the HH signalling pathway components

Hedgehog (HH) signalling pathway results in distinct
cancers of the brain, muscle and skin have received
significant attention. The interest is partly because of
the fact that deregulated HH signalling only seems to
cause tumours in a subset of adult cell types —
potentially a population of adult stem cells that
might require HH signalling for their proliferation
and maintenance. Furthermore, specific inhibition of
this pathway blocks tumour growth, indicating that
active HH signalling is essential for tumour survival.
Therefore, increased levels of HH signalling seem to
be both sufficient to initiate cancer formation and
required for tumour survival. Recent studies have
also shown that activation of HH signalling is
required for survival of other tumours, including pan-
creatic adenocarcinomas and small-cell lung carcino-
mas (SCLCs). Screens for HH signalling inhibitors
have led to the identification of reagents that block
signal transduction at different levels within the path-
way (TABLE 1), and several diverse antagonists of HH
Diabetes Center, signalling are available that could lead to new treat-
Department of Medicine, ment approaches for tumours that are difficult to treat
University of California, by conventional means. Increasing our understanding
San Francisco, California of the cell-specific mechanisms that control HH
94143, USA.
e-mail: signalling could provide clues to unravel the relation-
mhebrok@diabetes.ucsf.edu ship between regulated proliferation and uncontrolled
doi:10.1038/nrc1229 neoplasia in adult stem cells.
The Hh signalling pathway was first identified in a large
Drosophila screen for genes that were required for pat-
terning of the early embryo1. Analysis of the hedgehog
mutant, named after its prominent phenotype —
epidermal spikes in larval segments that normally are
devoid of these extensions — led to the cloning of the
hh gene. Subsequent studies showed that three members
of this family are present in mammals. These include
Sonic (Shh), Desert (Dhh) and Indian (Ihh), all of which
encode secreted proteins2. Hh ligands undergo post-
translational modifications, including autocatalytic
cleavage and coupling of cholesterol to the amino-
terminal peptide, which is the fragment that possesses
all of the signalling activity (detailed information about
Hh processing is described in REF. 2).
Interestingly, Hh signalling is mediated via a series of
inhibitory steps (FIG. 1). After secretion, the diffusion of
all three Hh ligands is limited by binding to the Hip1,
Patched1 (Ptch1) and Patched2 (Ptch2) transmembrane
receptors, all of which are expressed on Hh responsive
cells3–8. Although the exact details of ligand-receptor sig-
nalling are still under debate, the current model pro-
poses that in the absence of ligands, Ptch receptors block
the function of another transmembrane protein,
Smoothened (Smo), and that this inhibition is relieved
following ligand binding9. As a consequence, Smo
becomes active and initiates a signalling cascade that

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Summary Therefore, ligand-induced activation creates a negative-

• Hedgehog (HH) signalling is required for cell differentiation and organ formation
during embryogenesis. In the adult, HH signalling remains active in some organs where
it has been implicated in the regulation of stem-cell maintenance and proliferation.
• HH signalling targets include genes that are important for cell proliferation — proto-
oncogenes — as well as growth factors.
• Misregulation of HH signalling has been shown to cause formation of basal-cell
carcinoma and medulloblastoma, and mutations of HH pathway components have
been found both in familial and sporadic cases. More recently, small-cell lung cancer
(SCLC) and pancreatic adenocarcinoma have been linked to HH signalling, providing
a molecular mechanism for these aggressive diseases.
• Importantly, HH signalling seems to be required not only for cancer initiation but also
for tumour growth and survival of medulloblastomas, SCLC and pancreatic
adenocarcinoma.
• HH inhibitors could provide novel therapeutic approaches for treatment of otherwise
hard to cure cancer types. Synthetic compounds have been identified that act as HH
inhibitors in a very specific manner.
results in the activation of Gli transcription factors —
the vertebrate homologous of the Drosophila Cubitus
interruptus, or Ci (reviewed in REF. 10).
Three vertebrate Gli genes — Gli1, Gli2 and Gli3 —
have been identified. They possess context-dependent,
distinct repressor and activator functions. Gli proteins
are post-translationally modified, and cleavage of the
whole proteins results in N-terminal-truncated activa-
tor and C-terminal-truncated repressor fragments. The
details of Gli activation remain obscure. However, evi-
dence indicates that in the absence of ligands, Gli pro-
teins are linked to the cytoskeleton by interaction with a
multiprotein complex that includes Fused (Fu) and
Suppressor of fused (SuFu)11. Following ligand binding,
Gli proteins translocate into the nucleus where they
control transcription of target genes. It is important to
note that several inhibitors of the pathway, including
Ptch and Hip1, are transcriptional target genes.
feedback loop that restricts the extent of Hh signalling.
As a consequence, Hh signalling is regulated at different
levels by components of the pathway — a peculiar phe-
nomenon that indicates that tight control of its activity
is crucial for proper function.
Natural Hh functions
Why is tight control of Hh signalling so important? One
reason is that Hh signalling regulates cell differentiation
and organ formation in a concentration-dependent
manner — properties that have been well studied dur-
ing embryonic development12. For example, during
neural-tube formation, Shh is expressed in the ventral
floorplate and directs the development of specific types
of neurons in a dose-dependent fashion13–16. Ectopically
increasing the activity of this pathway results in the
development of ventral, rather than dorsal, types of
neurons. This indicates that precise control of Hh activ-
ity is essential in regulating the appropriate localization
and number of distinct populations of neurons.
Within the developing intestinal tract, sharp borders
of Hh activity control patterning of organs in the fore-
midgut region17. Shh is expressed throughout the
epithelial layer of the developing digestive tract, but
expression is excluded from the area that gives rise to
the pancreas18,19. Ectopic activation of Hh signalling
within the pancreatic epithelium blocks normal pan-
creas development and results in transdifferentiation of
the pancreatic mesenchyme into the duodenal meso-
derm18,19. Interestingly, low-level Hh signalling seems to
be required for pancreas organogenesis and function, as
Ihh, Dhh, Smo, Ptch1 and Hip1 are expressed within
pancreatic epithelium, where they regulate insulin tran-
scription and secretion in cultured insulinoma cells20–23.
So, distinct tissues require specific levels of Hh sig-
nalling for proper function, and an increase or decrease
of pathway activity results in severe defects.

Table 1 | Inhibitors of Hedgehog signalling

Inhibitor Target Study results References
Anti-Shh antibody Shh Blocks Shh in vivo; can inhibit proliferation 76–78
of granule neuron precursors
Cyclopamine Smo Blocks Hh signalling in vivo; can inhibit growth 43,44,50,54,71,72
of medulloblastoma, small-cell lung cancer and
pancreatic cancer
KAAD-cyclopamine Smo Found to be a more powerful derivative 73
of cyclopamine
SANT1 Smo Tested in a cell-culture assay for inhibition 74
of Hh pathway activity (Gli-luciferase cell line)
SANT2 Smo Tested in a cell-culture assay for inhibition 74
of Hh pathway activity (Gli-luciferase cell line)
SANT3 Smo Tested in a cell-culture assay for inhibition 74
of Hh pathway activity (Gli-luciferase cell line)
SANT4 Smo Tested in a cell-culture assay for inhibition 74
of Hh pathway activity (Gli-luciferase cell line)
Cur61414 Smo Inhibits proliferation in an in vitro BCC model system 75
Forskolin PKA Blocks proliferation of granule neuron precursors 77
Gli-antisense Gli1 Prevents Gli1-induced tumour formation (Xenopus tadpole) 50
BCC, basal-cell carcinoma; Hh, hedgehog; PKA, protein Kinase A; Shh, Sonic Hedgehog; Smo, Smoothened.

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Inactive Active
Ptch Ptch Hh
Smo
Hip Hip
Smo
?
Fused Fused
Sufu Sufu
Gli Gli
Gli
Target genes Target genes
Figure 1 | Hedgehog signalling pathway. In the absence of ligand, the Hh signalling pathway is
inactive (left). In this case, the transmembrane protein receptor Patched (Ptch) inhibits the activity of
Smoothened (Smo), a seven transmembrane protein. The transcription factor Gli, a downstream
component of Hh signalling, is prevented from entering the nucleus through interactions with
cytoplasmic proteins, including Fused and Suppressor of fused (Sufu). As a consequence,
transcriptional activation of Hh target genes is repressed. Activation of the pathway (right) is
initiated through binding of any of the three mammalian ligands —Sonic hedgehog, Desert
hedgehog or Indian hedgehog (all are represented as Hh in the figure) — to Ptch. Ligand binding
results in de-repression of Smo, thereby activating a cascade that leads to the translocation of the
active form of the transcription factor Gli to the nucleus. Nuclear Gli activates target gene
expression, including Ptch and Gli itself, as well as Hip, a Hh binding protein that attenuates ligand
diffusion. Other target genes that are important for the oncogenic function of the Hh pathway are
genes that are involved in controlling cell proliferation (cyclin D, cyclin E, Myc and components of
the epidermal-growth-factor pathway) and in angiogenesis (components of the platelet-derived-
growth-factor and vascular-epithelial-growth-factor pathway).
Cell-cycle regulation by Hh signalling
Whereas the requirements of Hh signalling during
embryogenesis have been studied in great detail2, less
attention has been paid to the role of the pathway in
adult tissues. Accumulating evidence indicates that Hh
activity remains in a subset of cells in mature organs,
and deregulated activity within these cells has been
implicated in tumour formation (reviewed in REF. 10).
One explanation for the role of Hh in tumorigenesis
comes from recent studies in which it was shown that, in
addition to controlling cell differentiation and tissue
patterning, Hh signalling also regulates the proliferation
of distinct cell types via direct activation of genes that
are involved in the progression of the cell cycle. In partic-
ular, cyclin D and cyclin E — proteins that are involved
in the G1–S transition — are known transcriptional
targets of Ci in Drosophila cells24, and Hh-dependent reg-
ulation of cyclin D1 and cyclin D2 has been confirmed in
mammalian cells25.
Further evidence for direct activation of the cell
cycle by Hh signalling comes from studies in which it
was shown that Ptch regulates the activity of cyclin B
— a part of the mitosis-promoting-factor (MPF)
complex26. MPF activation is required for G2–M tran-
sition in all cell types. However, interaction with Ptch
in the cytoplasm blocks cell proliferation by prevent-
ing nuclear localization of the activated complex.
Ligand-induced activation of the pathway leads to
NATURE REVIEWS | C ANCER
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nuclear localization of cyclin B by disruption of the
physical interaction between Ptch and cyclin B.
Finally, Shh blocks cell-cycle arrest that is mediated by
p21 — an inhibitor of cyclin-dependent kinases27.
These studies provide compelling evidence that
increased cell proliferation, a hallmark of tumour for-
mation, is mediated via direct interaction of the Hh
pathway with components of the cell-cycle machinery.
Hh and cancer
Pathway components that cause cancer. If constitu-
tive activation of Hh signalling induces tumorigene-
sis, it can be predicted that a subset of Hh-responsive
cancers should possess activating mutations in com-
ponents of the pathway (FIG. 2). In support of this
hypothesis, mutations in Shh have been identified in
a small percentage of basal-cell carcinoma (BCC),
medulloblastoma and also in one case of breast carci-
noma cells28. The role of Shh as a dominant oncogene
has further been shown in studies of mice and
humans, in which ectopic expression of Shh results in
BCC28,29. Similarly, constitutively active mutations of
SMO have been found in 10–20% of BCCs, and the
transcription factor GLI1 was originally identified as
a gene that was amplified in human glioma 30 — a
central nervous system (CNS) tumour that is thought
to be derived from glial cells (reviewed in REF. 31).
Ectopic expression of Gli1 or Gli2 in the skin of
Xenopus tadpoles or mice results in tumour forma-
tion, demonstrating that the most downstream com-
ponents of the pathway are sufficient to initiate
tumour growth32–34.
In addition, loss-of-function mutations in negative
regulators of the pathway, including PTCH1 and
SUFU, have been associated with tumorigenesis, indi-
cating that inhibitors of HH signalling act as tumour
suppressors. Mutations in SUFU have been associated
with an increased risk of medulloblastoma in
humans35, whereas mutations in PTCH1 are found in
patients with basal-cell nevus syndrome36,37 (BCNS,
also known as Gorlin’s syndrome). This syndrome is
characterized by a high incidence of BCCs and
medulloblastomas. It is important to note that
Ptch1+/– mice phenocopy many of the features that are
associated with BCNS, including the high frequency
of tumour development 38. So, a distinct subset of
tumours in mice and human is characterized by
mutations in Hh signalling components. More impor-
tantly, the observation that misregulation of HH
signalling occurs in familial cancer indicates that
deregulation is sufficient to cause tumour formation.
Moreover, sporadic BCCs and medulloblastomas are
often characterized by inactivation of PTCH1 or con-
stitutive activation of SMO39–42. Nonetheless, muta-
tions of HH signalling components have only been
identified in a subset of sporadic BCCs and medul-
loblastomas. Future studies will address whether
mutations in other HH-pathway genes and/or
mutations in signalling pathways that are unrelated to
HH cause formation of tumours that are marked by
activated HH signalling.
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BCC active during lung organogenesis, Hh signalling is nor-

mally downregulated in the mature organ, and only few
BCC Hh-responsive cells remain. However, adult tissue
Medulloblastoma retains the capacity to respond to Hh signals, as Shh
Rhabdomyosarcoma Ptch Hh
and Ptch expression are upregulated in regenerating tis-
Smo
BCC Pancreatic adenocarcinoma sue following chemically induced lung injury 43.
Interestingly, HH expression is also increased in a sub-
Hip
set of SCLC. Cancer cell lines that are derived from
? Oesophagus and human tumours express SHH, as well as GLI1, indicat-
stomach cancer
Fused ing that an autocrine mechanism is maintaining active
Sufu HH signalling within these cells. More importantly,
Medulloblastoma
Gli Pka
inhibition of HH signalling via treatment with
cyclopamine — a naturally occurring cholesterol ana-
Small-cell lung cancer logue that inhibits SMO and functions through inter-
Tumour proliferation
and survival action with the HEPTAHELICAL BUNDLE47 — arrests the cell
BCC Gli
cycle at G0–G1 and induces apoptosis in SCLC43.
Glioma In a manner that is similar to that seen in the lung,
Hh signalling is active during pancreas organogenesis17
and low-level expression of Hip1, Ptch1, Smo, Ihh and
Dhh has been detected within mature islets and cultured
Figure 2 | Hedgehog pathway and cancer. Misregulation of Hedgehog (Hh) signalling causes β-cell lines20,21,23. Studies in transgenic mice that carry
cancer in different tissues. Ptch mutations that are associated with basal-cell carcinoma (BCC), as the bacterial lacZ gene under control of the Ptch1 pro-
well as with medulloblastoma and rhabdomyosarcoma. Cancer associated mutations are usually moter show that low-level β-galactosidase activity is also
loss-of-function alleles, so Ptch can be considered to be a tumour suppressor. Similarly, loss-of-
found in pancreatic-duct cells20, the cell type that is
function mutations in Suppressor of fused (Sufu) have also been identified in some
medulloblastoma cells. Constitutively active forms of Smo are oncogenic and can function
believed to be responsible for adenocarcinoma growth46.
independently of ligand binding to Ptch, leading to BCC. An oncogenic form of Shh has been Whereas the expression level of PTC1 is below the
associated with BCC, whereas ectopic expression of Gli has been shown to cause glioma. Gli is threshold that can be detected by immunohistochem-
inhibited by protein kinase A (Pka). Misregulation of Hh signalling has also been associated with istry in human pancreatic samples, Hh signalling also
pancreatic adenocarcinoma, oesophageal and stomach cancer, and small-cell lung cancer. Little is seems to be involved in pancreatic cancer progression,
known about the molecular mechanisms by which Hh signalling is upregulated in these tumours. as expression of signalling components is progressively
increased in pancreatic intraepithelial neoplasia
(PanIN) and pancreatic adenocarcinomas44. Moreover,
Specificity of Hh-induced tumour types. Until recently, ectopic expression of Shh under control of the pancre-
increased Hh signalling had been linked to only a small atic and duodenal homeobox gene 1 (Pdx1) promoter
subset of tumours in the brain, skin and muscle10 in transgenic mice results in formation of PanIN-1 and
(FIG. 2; TABLE 2). Several recent studies indicate that ele- PanIN-2 lesions44. PanIN-1 lesions are characterized by
vation of the pathway causes cancers in other organs, loss of cuboidal morphology of pancreatic-duct cells,
HEPTAHELICAL BUNDLE
including the lungs, gastrointestinal tract and pan- mucin accumulation and papillary growth; nuclear
A transmembrane domain of
the Smoothened protein that is
creas43,44. These findings are particularly important, as abnormalities, including enlargement and some loss of
composed of seven α-helical both SCLC and pancreatic adenocarcinoma are highly polarity occur in PanIN-2 lesions.
stretches. aggressive tumours with poor prognosis45,46. Although Notably, the histological progression of pancreatic
neoplasia in these Pdx1–Shh transgenic mice is
accompanied by the induction of Erbb2 (also known
Table 2 | Animal models of Hedgehog-dependent tumours as Her2/neu) expression, and mutations of the
proto-oncogene KRas 44 that have previously been
Animal model Phenotype Species References associated with pancreatic adenocarcinomas46,48,49.
Shh overexpression BCC Mouse 38 However, Pdx1–Shh transgenic mice die at around
in the skin K14 promoter
three weeks of age, and therefore cannot be used to
Ptch inactivation Medulloblastoma and other Mouse 28 test whether prolonged Hh upregulation results in
tumours, mice larger than normal
metastatic cancer. Further experiments involving the
Smo-M2 overexpression BCC Mouse 41 transient activation of Hh signalling in adult pan-
in the skin K5 promoter
creas are required to clearly establish that increased
Gli1 overexpression BCC, trichoepithelioma Mouse 32,33
in the skin
levels of this protein are sufficient to cause pancreatic
adenocarcinoma formation.
Gli2 overexpression BCC Mouse 34
in the skin Recent evidence also indicates that deregulated Hh
signalling not only causes tumour formation, but is also
Gli2 overexpression Skin tumours with Xenopus 32
in the skin BCC-like characteristics required for tumour maintenance, as transformed cells
continue to depend on Hh activity for survival and
Gli1 overexpression Hyperproliferation of Xenopus 50
in the brain progenitor cells growth. Analysis of 26 human pancreatic adenocarci-
BCC, basal-cell carcinoma; K14, cytokeratin 14; K5, cytokeratin 5; Ptch, Patched; Smo, noma cell lines showed that all lines express HH target
Smoothened. genes, and that treatment with cyclopamine induced

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Box 1 | Hedgehog inhibitors

The Hedgehog (Hh) pathway can be blocked at different levels, and Hh inhibitors could serve as attractive anticancer
agents because of their specific effects on a small number of cells in adult tissues. Several Hh-specific antagonists
have therefore been identified and tested. Inhibition of ligand activity has been reported with antibodies (Ab)
directed against Sonic Hedgehog (Shh)15 (see a), and similar strategies might be considered for treating tumours that
are shown to require continuous ligand activity for survival43. Several specific Smoothened (Smo) inhibitors have
been identified (see b). Cyclopamine, a natural alkaloid derivative that is isolated from a plant of the lily family
Veratum californicum, represents the first member of a class of small chemical compounds that specifically inhibit
the Hh pathway70–72. It is a potent teratogen that specifically inhibits Smo activity by binding to its heptahelical
bundle47. Treatment of mice that carry Hh-dependent tumours with cyclopamine results in growth inhibition and
regression of cancerous tissue, but does not affect the health of treated animals. So, Hh inhibition causes little, if any,
toxic effects on cells that do not depend on Hh signalling 44,54.
Cyclopamine, however, is difficult to synthesize in large quantities and therefore is not applicable as a therapeutic
agent — a factor that might also apply to a modified and more effective version of this compound, KAAD-
cyclopamine73. Two large-scale screens for small-molecule inhibitors have identified several compounds that bind to
Smo, including several that potently block a
constitutively activated form of Smo that is known to
b a
cause BCCs (SANT1–4, Cur61414)74,75. In addition, Cyclopamine Anti-Shh Ab
two additional compounds were isolated that seem to KAAD-cyclop
inhibit the pathway downstream of Smo. Although SANT1–4
Cur61414
these reagents have not been characterized in detail,
these results are encouraging, as they indicate that Hh Ptch Hh
signalling can be blocked with small compounds at Smo
different levels within the pathway. This is particularly
important as mutations in proteins that lie
Hip
downstream of Smo can be tumorigenic35.
Other compounds that block Gli activity could be used
to treat a wide variety of Hh-dependent tumours. c
Forskolin Pka Gli
Protein kinase A (Pka) maintains the Gli transcription
factors in an inactive state, so activation of Pka with Gli antisense
agonists such as forskolin would prevent Gli-mediated
activation of target-gene transcription. Gli can also be
Gli
inhibited at the RNA level by targeting its transcripts
with antisense oligonucleotides — an approach that has
been used successfully in Xenopus. These and other Target genes
related compounds might provide a novel way of
treating Hh-responsive tumours.

apoptosis and loss of proliferation in 50% of the lines
tested44. The observation that only half of the cell lines
responded to cyclopamine treatment could indicate that
the non-responsive lines have developed activating
mutations in components downstream of SMO — a
hypothesis that is supported by previous studies in
which only a subset of glioma cell lines were noted to be
responsive to cyclopamine-mediated inhibition of HH
signalling50. Interestingly, most pancreatic cancer cell
lines that were tested were positive for SHH expression
by reverse transcription-PCR, indicating that tumour
formation and growth might be elicited by an autocrine
mechanism51. Similarly, other tumours that are derived
from the digestive tract (oesophagus, stomach, biliary
tract, but not colon) are also marked by increased levels
of HH pathway activity and increased levels of SHH lig-
and expression. As expected, cell lines that are derived
from these gastrointestinal tumours are also susceptible
to cylopamine-mediated growth inhibition51.
It is interesting to note that Hh signalling remains
active in some pancreatic adenocarcinoma cell lines
that were originally isolated from liver metastases of
primary pancreatic tumours, and that were subse-
quently re-derived during several rounds of injection
into nude mice. More importantly, metastatic cells
remain susceptible to cyclopamine treatment, both in
cell culture and after xenotransplantation into nude
mice44. Although it is unknown at present if increased
Hh signalling facilitates tumour metastasis, these find-
ings are exciting, as inhibition of the pathway could
present novel avenues for therapy of primary and
metastatic tumours (BOX 1). This is particularly impor-
tant as the high frequency of metastasis in pancreatic
adenocarcinomas during early stages of the disease,
often before diagnosis, is one of the complications that
contribute to low survival rates46.
Only a few familial cases of pancreatic adenocarci-
nomas have been described so far, and the involvement
of HH signalling in these cases not been addressed.
One family (family X) has been identified in which
pancreatic adenocarcinomas occur with a very high
frequency. The genomic location of the syndrome has
recently been mapped to the chromosomal region
4q32-34 (REF. 52). Interestingly, HIP1, which encodes an

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inhibitor of HH signalling, is located immediately
adjacent to this region, raising the possibility that a
hypomorphic mutation in HIP1 could activate HH
signalling in pancreatic tissue. Although further stud-
ies are required to address this hypothesis, it is impor-
tant to note that HIP1 expression is lost in most
human pancreatic adenocarcinoma cell lines44. In
addition, Hip1–/– mice have increased levels of Hh sig-
nalling during pancreas development23. Most Hip1–/–
mice, however, die shortly after birth53, and no pancre-
atic lesions have been found in Hip+/– mice that survive
to adulthood. Sequence analysis of the HIP1 gene in
DNA samples from members of family X might there-
fore be required to determine whether mutations in
HH inhibitors are associated with familial forms of
pancreatic adenocarcinomas.
Target genes and interactions
Recent studies mark Hh signalling as a key contribu-
tor to cancer formation and maintenance in a distinct
but restricted set of cell types43,44,54. Improving our
understanding of the mechanism that regulate Hh
signalling and that of its target genes could lead to
new diagnostic and therapeutic approaches. As men-
tioned above, Hh signalling controls cell-cycle pro-
gression by regulating cyclin expression and activity.
Moreover, Hh signalling regulates the expression of
the oncogene n-Myc in the nervous system 55 and
could regulate Myc expression in other tissues. Myc
transcription factors are important inducers of cell
proliferation, and cyclopamine treatment of a medul-
loblastoma cell line decreases expression of c-Myc,
l-Myc, and n-Myc genes 54. Therefore, constitutive
activation of Hh signalling could maintain the prolif-
erative state of cells through deregulated control of
the cell cycle.
Other transcriptional targets of Hh signalling are
of particular interest, as they are also genes that have
been found to be upregulated during tumorigenesis.
In Drosophila, Hh signalling promotes epidermal
growth factor (Egf) signalling by inducing its expres-
sion, along with expression of Egf receptors56.
Interestingly, the activation of the Egf-receptor path-
way is considered to be an early event in pancreas
tumorigenesis. Autocrine signalling of the Egf path-
way becomes activated during early stages of adeno-
carcinoma formation, and sustained expression of the
Egf ligand Tgf-α in Trp53-mutant mice results in
adenocarcinoma formation49,57,58.
The Ras–Erk (extracellular-signal-regulated kinase)
pathway, which is associated with cell proliferation, is
activated by platelet-derived growth factor (Pdgf) sig-
nalling59,60. The Pdgf receptor-α (Pdgfr-α) is expressed
at high levels in human and murine BCC61. The inter-
action between Hh and Pdgf signalling has been shown
in cultured murine fibroblasts, BCC cells and CNS
tumours50. In these cells, ectopic expression of Gli1
increases Pdgfr-α expression, whereas inhibition of the
Hh pathway reduces Pdgfr-α levels61. Therefore, Hh
signalling controls many important pathways that have
been associated with tumorigenesis.
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Although we have learned much about the down-
stream target genes of Hh signalling, few studies have
addressed the upstream regulation of Hh signalling
during cancer formation. A recent study presents
intriguing evidence that Notch signalling — another
pathway that is known to regulate cell differentiation
and proliferation — regulates Gli2 expression in
mouse skin62. Inactivation of the Notch1 gene in epi-
dermis induces sustained expression of Gli2 and
causes formation of BCC-like tumours. By contrast,
recent evidence indicates that Notch pathway activa-
tion is involved in pancreatic cancer formation63,
although an interaction between Notch and Hh sig-
nalling has not been described in this tissue. In the
skin, Notch-dependent transformation is associated
with the activation of β-catenin and Lef1 — two
markers of active Wnt signalling. Upregulation of
WNT expression has previously been observed in
human BCCs64, indicating that tumour progression is
mediated via interaction of distinct signalling path-
ways that regulate organ development during embryo-
genesis. Further studies will be required to determine
if these interactions might open new avenues for
treatment of Hh-responsive tumours64.
Future directions
Adult stem cells, Hh signalling and cancer. One of the
most important unresolved questions in cancer biol-
ogy concerns the identity of cells that become
tumorigenic. Striking similarities between cancer and
stem cells have been previously reported, as both cell
types have the potential for unlimited self-renewal.
Hh signalling is active in and required to maintain
stem-cell or precursor populations in several organs,
and deregulation is known to result in tumorigenesis
(BOX 2). Increasing evidence also indicates that, at least
in some organs, uncontrolled Hh signalling results in
unregulated self-renewal of progenitor cells. In skin,
Hh signalling is required for hair morphogenesis
during embryonic development. In the mature tissue,
the multipotent skin and hair stem cells transiently
express Ptch during the proliferation phase 65.
Multipotent cells then give rise to two progenitor
populations — epithelial progenitors (which do not
express Ptch and give rise to the stratified epithelium)
and hair progenitors (which continue to express Ptch
while they proliferate and then differentiate into the
different cell populations of the hair follicle). The
level of Hh signalling, which is mainly mediated by
Gli2 (REF. 25), seems to be crucial — loss of Hh sig-
nalling prevents proliferation, whereas increased Hh
signalling results in formation of BCCs (BOX 2).
Within the adult lung, Hh expression is limited to
small patches of epithelial cells43. Expression becomes
transiently activated during acute airway epithelial regen-
eration after tissue injury, indicating that the pathway
might mark neuroendocrine progenitor cells within the
lung epithelium. SCLCs possess many primitive neuroen-
docrine features, and some SCLCs require Hh signalling
for tumour maintenance. The similarities beween Hh
signalling during neuroendocrine-cell regeneration and
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Box 2 | Hh signalling, stem cells and cancer

Hedgehog (Hh) signalling is important for the maintenance of the hair follicle. The hair-follicle stem cells or multipotent
progenitors (MPs, light yellow) give rise to both epithelium progenitors (EPs, orange) and to hair-follicle progenitors
(HPs, red). The EPs proliferate and subsequently differentiate into stratified epithelium , whereas the HPs give rise to the
hair follicle (dark yellow). The MPs express Ptch (which indicates that they are responding to Hh signalling) transiently
when they proliferate at the beginning of each hair-follicle cycle. The HPs express Ptch when they proliferate, but Ptch
becomes downregulated once they undergo differentiation. The expression pattern of Ptch is consistent with a role of
Hh signalling in maintaining the stem-cell/progenitor-cell compartment. Basal-cell carcinomas of the skin are thought
to derive from the hair follicle — in particular from the HP cells. Failure to downregulate Hh activity at the appropriate
time could start the series of events that will lead to cancer. The cells that express Ptch are outlined in blue. A dashed blue
outline indicates transient expression of Ptch.
In the pancreas, Ptch expression is found in duct and islet cells (red). Although still controversial, evidence indicates
that ducts harbour progenitor cells (yellow — pancreatic stem cells that can give rise to the other pancreatic cell types,
such as exocrine and endocrine cells). So far, it is not known whether all duct cells have the potential to differentiate into
other lineages (islets and EXOCRINE ACINI), or whether a distinct set of a few multipotent progenitor cells are located within
ducts. Pancreatic adenocarcinomas (purple) are believed to derive from duct cells and activation of Hh signalling is
observed in human adenocarcinomas44. Transgenic mice that overexpress Shh in the pancreas show precancerous
lesions, and continued activity of the Hh pathway is required for proliferation and survival of the cancer cells once the
tumour has formed.

Skin (adult) Pancreas (adult)

Epithelium

Hh signalling
Hair follicle upregulation
Pancreatic
adenocarcinoma

EP
Differentiation
Duct
Basal-cell
carcinoma
Pancreatic
stem cell? Self-renewal
MP

Differentiation

HP

Islet

Hh signalling Proliferation
upregulation

Exocrine acini

EXOCRINE ACIN
Alveolar structures that are
formed by the cells that produce
and release pancreatic digestive
enzymes in the lumen of
collecting pancreatic ducts.
SCLC formation indicate that deregulation of the
pathway in epithelial precursors is involved in tumour
formation. Similarly, the duct structures that are believed
to harbour adult pancreatic progenitor cells express Ptch1
(REF. 20). Although conclusive evidence is lacking, cells
within or attached to pancreatic ducts are thought to give
rise to endocrine and exocrine cells during regeneration66,
and endocrine cells that are located in epithelial struc-
tures known as islets of Langerhans continue to express
Ptch1. Although the issue is still controversial, pancreatic
adenocarcinomas are thought to arise from duct cells67,68,
indicating that Hh expression could mark pancreatic
progenitor cells and control their proliferative potential.
Identification of these cells would be important for both
generating more differentiated β-cells to treat diabetics, as
well as to better understand the molecular and cellular
principles that result in adenocarcinoma formation.
HH signalling is essential for numerous processes
during organ development and maintenance of organ
function. However, its ability to regulate cell differenti-
ation and renewal in a dose-dependent manner also
means that deregulation of this pathway can result in
uncontrolled cell proliferation. Fortunately, specific
inhibitors of the pathway are available for basic
research, and those with therapeutic potential are
being developed. However, it should be noted that

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REVIEWS

detailed molecular analysis of tumour types is required
to determine which patients will respond to anti-HH
therapy. Although all tested pancreatic adenocarci-
noma cell lines seem to express HH signalling compo-
nents, only five out of ten SCLC tumours express both
SHH and GLI1 (REF. 43). So, analysis of tumour gene-
expression profiles69 might be useful in determining
which tumour types have activated HH signalling and
therefore be useful in predicting the outcome of
potential treatments with HH inhibitors.
Discovering the dual role of this pathway — on one
hand its requirement for normal organ development
and function, and on the other hand its association
with tumorigenesis — has proven that the study of sig-
nalling pathways in the developing embryo can lead to
important insights into disease progression and treat-
ment. In the case of Hh signalling, this knowledge
could lead to new therapeutic approaches to treat
tumours that have poor prognoses. Finally, Hh
signalling interacts with other embryonic signalling
pathways that are known to be involved in cancer for-
mation. Analysis of these connections should provide
important insights into the molecular causes of cancer
formation and growth.

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Acknowledgements
We would like to dedicate this manuscript to the memory of Ira
Herskowitz, who inspired us to contemplate about the connec-
tion between embryonic signalling pathways and cancer. We
would like to thank all members of the Hebrok laboratory for
stimulating discussions. In particular, we would like to thank
P. Heiser and J. Lau as well as H. Kawahira, D. Cano and
M. Tzanakakis for critical reading of the manuscript. Work in
M. H.’s laboratory was supported by grants from the Juvenile
Diabetes Research Foundation, the Hillblom Foundation and the
National Institutes of Health.
Competing interests statement
The authors declare that they have no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
Cancer.gov: http://cancer.gov/
pancreatic adenocarcinomas | SCLC
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/
Ci | Dhh | Fu | Gli1 | Gli2 | Gli3 | hh | Hip1 | Ihh | Ptch1 | Ptch2 | Shh |
Smo | SuFu
OMIM: http://www.ncbi.nlm.nih.gov/omim/
BCNS
Access to this interactive links box is free online.
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