gli and hedgehog in cancer: tumours, embryos and ... - Semantic Scholar

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GLI AND HEDGEHOG IN CANCER: TUMOURS, EMBRYOS AND STEM CELLS Ariel Ruiz i Altaba*, Pilar Sánchez* and Nadia Dahmane*‡ Do tumours arise from stem cells, or are they derived from more differentiated cells that, for some reason, begin to recapitulate developmental programmes? Inappropriate activation of the Sonic hedgehog–Gli signalling pathway occurs in several types of tumour, including those of the brain and the skin. Studies in these and other systems suggest that inappropriate function of the Gli transcription factors in stem or precursor cells might lead to the onset of a tumorigenic programme and that these factors are prime targets for anticancer therapies. ONTOGENY

The development of an organism from its earliest stages to maturity.

*The Skirball Institute, Developmental Genetics Program and Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, New York 10016, USA. ‡UMR 6156, Institute for Developmental Biology, Université de la Méditterannée-CNRS, Campus de Luminy, Case 907, 13 288 Marseille Cedex 09, France. Correspondence to A.R.A. e-mail: [email protected] doi:10.1038/nrc796

Cancer is a multitude of diseases that share an inappropriate increase in cell number, often owing to hyperproliferation. In the case of solid tumours, this is followed by abnormal morphogenesis. The histopathological diagnosis of many such tumours — for example, basal-cell carcinomas (BCCs) of the skin1 — is often based on patterns of tumour-cell aggregation or proliferation, the locations of tumour cells and the shape of the tumorigenic cells themselves. Tumours therefore seem to follow stereotypical developmental programmes that can be recognized morphologically and histologically. Contemporary understanding of molecular events during ONTOGENY sheds light on the cause and progression of cancer. In many cases, cancer seems to be caused by the deregulation of pathways that are normally involved in patterning cell groups, tissues or organs, which then affects cell fate and proliferation. So, can cancer be thought of as a disease of mispatterning? There is a growing body of evidence that links cancer with genes and pathways that are required for normal embryonic patterning, raising the possibility that loss of positional information is an important step in tumour formation. One pathway that is important in regulating patterning, proliferation, survival and growth in the embryo and the adult is the Sonic hedgehog (Shh)–Gli signalling pathway (reviewed in REFS 2–6; FIG. 1). When this pathway is activated or maintained inappropriately, various tumours can develop, including those in skin,

muscle and brain (reviewed in REFS 2,3). The events that initiate tumorigenesis might also be important for maintenance of the tumorigenic phenotype7–12, raising the question of what are the important events that initiate sporadic cancer. We subscribe to the hypothesis that cancer will be better understood, and so treated more effectively, if the molecular and cellular bases of the initiation events are elucidated and placed in a developmental context. In this sense, tumours can be seen as organs that normally do not develop, although the potential of their development exists all the time. We suggest that this potential could reside in stem cells, which are present in many, if not all, adult tissues, and that the need for the body to remain morphologically plastic, and therefore evolutionarily fit, by retaining stem cells in the adult has a price — tumorigenesis. The Shh–Gli pathway

Hedgehog (Hh) proteins are secreted glycoproteins that activate a membrane-receptor complex. This, in turn, by means of cytoplasmic signal transduction, activates Gli zinc-finger transcription factors (FIG. 1). This pathway has been the focus of much recent attention (reviewed in REFS 5,6), and the positive action of the pathway is tightly regulated by many inhibitors at different levels. For example, Shh signalling activates a receptor complex that is formed by Patched (Ptc) and Smoothened (Smo). Hh inhibits Ptc, which, in turn, normally inhibits Smo. Inhibition of Ptc by Hh activates

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Summary • The Sonic hedgehog (Shh) signal-transduction pathway is involved in the patterning, growth and survival of many cells and tissues, and its deregulation has been implicated in several cancers. • The pathway culminates in the activation of transcription factors of the Gli family. There are three such Gli proteins in vertebrates, with partially redundant functions, and Gli1 and Gli2 — both of which can mediate Hh signals — have been implicated in tumorigenesis. • The first hint that GLI1 activation might be involved in familial tumours came from the discovery that patched (PTCH, known as Ptc in mice), which inhibits the positive activation of GLI1, is mutated in basal-cell nevus syndrome (Gorlin’s syndrome) — a hereditary predisposition to basal-cell carcinomas, medulloblastomas and some other tumour types. This results in overexpression of positively activating GLI1 function. • The Shh–Gli pathway is also abnormally activated in sporadic cancers, including basal-cell carcinomas and medulloblastomas. Moreover, there is some evidence that Gli activation might be involved in the development of glioma and some other tumour types. Inhibition of Gli function might be a promising therapeutic target in these tumours. • In normal tissues, Gli is mainly active in precursor cells. This raises the possibility that tumours are derived from such cells, possibly even stem cells, which are unable to differentiate and/or to stop proliferating.

the Hh pathway and results in Gli activity (FIG. 1). The function of Ptc, and other inhibitors, is to silence the pathway in the absence of active Hh ligands. This implies that the Hh–Gli pathway must be off most of the time, and only active at the precise points and locations at which the Hh signals act. In mammals, there are three known Hh family members — Sonic, Desert and Indian Hh — and a large body of experiments have implicated them in the development of many tissues and organs, including skin, lung, brain, bone and blood (reviewed in REF. 5). Sonic hedgehog (Shh) is the most widely expressed, apparently the most potent13 and the one we focus on in this review. The Gli proteins

HOLOPROSENCEPHALY

A defect in the forebrain that is caused by abnormal dorso–ventral patterning of the anterior neural tube. FLOOR PLATE

Part of the neural tube that comprises the ventral cells closest to the midline. They, and the underlying notochord, secrete Sonic hedgehog, setting up a ventral–dorsal gradient of this morphogen in the neural tube. MESODERM

The middle germ layer of the developing embryo. It gives rise to the musculoskeletal, vascular and urinogenital systems, and to connective tissue (including that of the dermis).

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normal development (Shh homozygous knockout mice and humans with SHH mutations are HOLOPROSEN17,18 CEPHALIC ), Gli1-knockout mice develop normally19. Gli2 and Gli3 knockouts, by contrast, have serious developmental abnormalities, and Gli1 mutations accentuate the Gli2-heterozygous phenotype19. Indeed, Gli proteins have partially redundant and partially distinct functions (FIG. 2; reviewed in REF. 6) and induce distinct targets16,20, indicating that they act in a combinatorial manner21 that is context dependent and, possibly, species specific. For example, Gli1 and Gli2 induce motor neurons in the frog spinal cord, whereas Gli3 represses this function. By contrast, Gli1 induces FLOORPLATE differentiation in the same species, whereas both Gli2 and Gli3 inhibit this function21. In mice, however, it is Gli2 that is primarily involved in floor-plate development. There seems to be a Gli code of action that is tightly regulated by Shh signalling. However, this code can be altered by modifying factors in a contextdependent manner. For example, in the early neural plate of frog embryos, expression of the transcription factor Zic2 modifies cell-fate decisions that are regulated by Gli proteins22. Perhaps what is crucial for a given cell is the overall state of Gli function, and Shh signalling might be just one of several ways to regulate it (FIG. 2). In this sense, it is important to note that Gli2 and Gli3 are also likely to respond to other signalling inputs as they are expressed in regions with few, if any,

Shh Cyclopamine Membrane Ptc

Smo

Agents that block transduction, transport or activation

Cytoplasm

Gli proteins are large (more than 1,000 amino acids), multifunctional transcription factors, and their activities are intricately regulated (reviewed in REFS 6,14). They reside in both the nucleus and the cytoplasm, where they are components of a multimolecular complex that is tethered to the cytoskeleton. The state of Shh signalling directly affects the fate of Gli proteins. In the absence of Shh, Gli proteins are cleaved by the proteasome, and carboxy-terminally truncated forms translocate to the nucleus, where they act as dominant transcriptional repressors6,14–16. Following Shh signalling, Gli repressor formation is inhibited and full-length labile activators of transcription are made instead. The three Gli proteins behave differently. In addition to being transcriptionally regulated in different ways, Gli1 does not seem to yield a strong repressor, whereas Gli2 and Gli3 do. Shh inhibits repressor formation by Gli3, but not by Gli2, and the formation of potent activators of Gli2, and perhaps of other Gli proteins, depends on Hh signalling (reviewed in REF. 6). Although loss of Shh function is not compatible with

Nucleus

Gli

Transcription

Anti-Gli agents

Gli target genes

Figure 1 | The Shh–Gli pathway and potential sites for blocking it with therapeutic agents. Sonic hegehog (Shh) acts on the membrane receptor complex formed by Patched (Ptc) and Smoothened (Smo) to inhibit the repression of Smo by Ptc. Smo is then thought to send the signal intracellularly through several cytoplasmic transduction steps (not shown), leading to the nuclear action of the Gli proteins, which regulate target genes. Thick blue arrows pointing up or down indicate activating or inactivating mutations, respectively, that might induce the pathway. Inhibitors of the pathway with potential therapeutic value (red lines) include: agents that block the action of Smo in the receptor complex, such as the plant alkaloid cyclopamine; agents that inhibit specific aspects of the transduction of the signal, including the nuclear import or activation of Gli proteins; and agents that specifically inhibit Gli function.

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— even neighbouring — cells? Although most of the targets are unknown, initial experiments are beginning to reveal some of them. For example, in the posterior mesoderm of frog embryos, Gli2 and Gli3 regulate the expression of Brachyury, which is necessary and sufficient for mesoderm specification; of Xhox3 (Evx1), which is necessary and sufficient for posterior identity; and of Wnt8 and Wnt11, which encode secreted factors that are required for posterior morphogenesis23,31. Gli proteins can, therefore, regulate cell fate at many levels. There is also evidence for the action of Shh at different times in the cell cycle (reviewed in REF. 6). In the cerebellum, Shh regulates D-type cyclins32,33, which are important G1/S regulators. Shh also regulates the proliferation of spinal-cord progenitor cells34, and it is required at two points in the cell cycle during spinalmotor-neuron differentiation, one being late in S phase35. Ptc might also inhibit cyclin B, a G2 cyclin36, and Shh signalling opposes epithelial cell-cycle arrest by Waf1 (also known as p21) — a negative regulator of cyclin-dependent kinases37.

b Shh

Shh

Gli1

Gli2

Gli3

Gli1

Gli2

Gli3

Wnt, Igf2, Pdgfrα signalling

Fgf

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d Gli1

Gli2 ?

Shh

Gli3

Hedgehog signalling in familial tumours

Shh

?

?

?

Rep

Act

Rep

Act

Rep

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Figure 2 | The Gli proteins and their functions. Relationships of the three Gli proteins and Sonic hedgehog (Shh) signalling, among themselves and in terms of target gene regulation. a | Shh can induce Gli1 and Gli2, whereas Shh–Gli1 seems to have a mutually repressive interaction with Gli3 in most contexts. Gli2 might also induce Gli1. b | The targets of Gli can overlap or be distinct. Fibroblast growth factor (Fgf) induces the expression of Gli2 and Gli3, whereas the Gli proteins induce expression of Wnt, insulin-like growth factor 2 (Igf2) and the platelet-derived growth factor receptor-α (Pdgfrα). Induction of Wnt, Igf2 or Pdgfrα leads to increased signalling of the respective pathways. c | Full-length Gli proteins can become activators or repressors of transcription. Repressor function is revealed after carboxy-terminal cleavage by the proteasome. d | The bimodal function of each Gli protein might result in the activation (Act) or repression (Rep) of different sets of target genes. It is not clear whether Gli1 can yield potent repressor derivatives. The mechanisms that regulate the formation of Gli1 activators and Gli2 repressors is unknown. Shh signalling induces formation of Gli2 activators and inhibits the formation of Gli3 repressors. The mechanisms that regulate the formation of Gli3 activators are also unclear.

Hh signals. For example, Gli2 and Gli3 can be regulated by fibroblast growth factor (Fgf) signalling in the embryonic MESODERM23, indicating that Gli proteins could integrate several signalling inputs. The Shh–Gli pathway seems to regulate many processes in cells. For instance, it can regulate survival in both differentiated and undifferentiated cell types24–26, induce floor-plate differentiation (reviewed in REF. 27) and induce proliferation of neuron precursors in the developing cerebellum28–30. How does the same pathway regulate different processes in different

Loss of, or interference with, Shh–Gli function results in a range of developmental defects, including holoprosencephaly, whereas its inappropriately maintained or ectopic function can lead to tumour formation. The first link between SHH signalling and tumour formation in humans was in familial cancers, as patients with basal-cell nevus syndrome (also known as Gorlin’s syndrome) — a familial condition38 that involves a predisposition to the development of BCCs, medulloblastomas and rhabdomyosarcomas — harbour PTCH mutations (the human orthologue of mouse Ptc )39,40. Direct evidence for a germ-line link between the Shh pathway and tumorigenesis was obtained from transgenic mouse models. Mice that lack Ptc function die as early embryos (~E9.5)41, but Ptc+/– mice — like patients with Gorlin’s syndrome — have an abnormally high frequency of cerebellar41 and muscle42 tumours. The penetrance of the tumorigenic phenotype in the cerebellum is strain dependent but, nevertheless, this result clearly established a causal relationship between loss of Ptc function and cancer. Moreover, tumours were more readily observed in a Trp53-null background (Trp53 encodes p53 in mice), indicating that loss of Ptc can synergize with loss of other tumour suppressors43. Ptcmutant mice, however, do not develop BCCs, although transgenic mouse embryos that misexpress Shh in the skin and irradiated Ptc+/– mice do develop lesions that resemble human BCCs44–46. But is inappropriate activation of the Shh pathway sufficient to induce sporadic tumour formation? Sporadic basal-cell carcinomas

The initial evidence that activation of the Shh–Gli pathway is involved in sporadic tumours came from the finding that transient misexpression of Gli1 (by means of mRNA injections) in the tadpole skin is sufficient to induce skin tumours that resemble BCCs on the basis of gene expression47 (FIG. 3). A histopathological analysis


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a Cleavage furrow V

Skin V

D

D

b

V

Brain V

D

D

d

c

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Head Neural tube

Somite Skin

Notochord

e Skin Tumour

Tail

Neural tube

Tumour

Figure 3 | The frog embryo as a model for tumorigenesis. a | When synthetic RNA that encodes Gli1 is injected into the ventral (V) animal pole of a two-cell frog embryo, tumours arise in the skin of the developing tadpole. b | By contrast, injection into the dorsal (D) animal pole of the frog embryo leads to brain tumours. c | Image of a 2.5-day-old tadpole showing the presence of a basal-cell-carcinoma-like skin tumour in the ventral epidermis. It is blue, owing to the presence of the product of the β-galactosidase reaction in cells that inherit the injected GLI1 mRNA along with the co-injected lacZ RNA. d | Cross-section of a central nervous system (CNS) tumour in a tadpole that has been injected with GLI1 mRNA and lacZ. Note the enormous CNS hyperplasia (blue). e | Cross-section of a 2.5-day-old tadpole after administration of bromodeoxyuridine (BrdU) to measure cell proliferation. Note the increase in BrdU+ cells in the tumour, as compared with the unaffected contralateral control (see REFS 11,47).

PRIMITIVE NEUROECTODERMAL TUMOUR

(PNET). A tumour with cells that resemble those of the neuroectoderm — part of the dorsal ectoderm that gives rise to the central nervous system. Medulloblastoma — the most common form of childhood brain tumour — is classified by some as a PNET.

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was not informative as tadpoles do not have a dermis, so patterns of epidermal-cell invasion cannot form47. Whether these tumours are neoplastic or might contain transformed cells remains unknown. The ability of Gli1 (REF. 48), and Gli2 (REF. 49), to induce tumour formation was later proved in mice. This showed that such skin tumours resemble human BCCs not only molecularly, but also histopathologically. The finding that Gli proteins can induce BCC-like tumours is very important because it indicates that activation of the most distal element of

the known Shh pathway leads to tumorigenesis. It also indicates that activation of a single transcription factor can lead to tumour formation. Moreover, the experiments in tadpoles show that the transient somatic misexpression of Gli1 leads to tumour formation47 (FIG. 3). Might all human sporadic BCCs derive from the inappropriate activation of the SHH–GLI pathway? If so, they should all express GLI1, as this is a loyal marker of the response of a cell to Hh signalling. Analyses of the expression of GLI1 in sporadic human BCCs and sporadic squamous-cell carcinomas (SCCs) showed that nearly all BCCs, but not SCCs, express GLI1 in the tumour masses as well as in the surrounding single cells47 (FIG. 4). These results, together with the expression of PTCH and SMOH in BCCs50–52 and the detection of mutations in PTCH and SMOH in sporadic BCCs 53–56, indicate that all sporadic BCCs have an active SHH–GLI pathway and that deregulated GLI function might be an important event in BCC formation. Moreover, it highlights GLI1 as a marker of BCC cells beyond the level that is normally achieved by histopathology47,57,58, pinpointing single cells that might be found beyond the ‘safe’ margins that are normally established during surgery; it also indicates a rational therapy for this very common type of skin cancer by targeting GLI function47. One important question that remains to be resolved is whether tumour grade is correlated with the level of GLI1 expression, although there is evidence that varying levels might correlate with grade in sarcomas59. What has been clearly shown, however, is that mice that misexpress Gli1 in the epidermis can develop not only BCC-like lesions, but also trichoepitheliomas — benign precancerous skin lesions48. If this is the case in humans, it remains unclear whether GLI1 induces a hyperproliferative state that then requires additional mutations for a bona fide BCC to develop46, or whether the microcontext of the cell that misexpresses GLI1 defines the nature of the lesion. Medulloblastomas

Evidence for the involvement of the SHH–GLI pathway in familial brain tumours was originally derived from the finding that individuals with Gorlin’s syndrome who are heterozygous for PTCH1 not only develop BCCs at an early age and at frequencies than are higher than normal, but also show a higher than normal incidence of PRIMITIVE NEUROECTODERMAL TUMOURS (PNETs) of the cerebellum or medulloblastomas (for example, see REFS 39, 40, 60). Mice with a single functional Ptc allele also develop medulloblastomas4,41. But why? And why do these mice not develop BCCs? Cellular context: the epidermis and the cerebellum. BCCs have been described classically as follicular tumours1 — tumours with hair-follicle differentiation (reviewed in REF. 61). The origin of BCCs has been debated. One possibility is that they derive from follicular progenitors because hair follicles transiently express the GLI and SHH genes and use the pathway for normal development47,62–65. More specifically, they might derive




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Normal

Tumour

a Skin

b Basal-cell carcinoma

Hair folicles Tumour nodule

c Cerebellum

Pallisading nuclei

d Medulloblastoma

External germinal layer

Tumour mass

Internal granular layer

e Forebrain

f Glioblastoma multiforme

Neocortex

Tumour mass

Hippocampus

Figure 4 | Gli1 gene expression in normal skin and brain, and in tumours derived from these tissues. Expression of Gli1 in a | hair follicles; b | a basal-cell carcinoma (BCC); c | the cerebellum; d | a cerebellar medulloblastoma; e | the dorsal forebrain and f | a cerebral glioma. The endogenous normal expression (a, c, e) in a 5-day-old mouse pup shows the cellular context for the use of Gli function. Expression in precursor cells in the hair follicles and the forebrain continues into adulthood. GLI1 expression in adult human BCCs of the skin (b), paediatric cerebellar medulloblastoma (d) and adult cerebral gliomas (f) indicates activation of the Shh–Gli pathway, demarcates the tumour cells and indicates that the tumours are attempting to recapitulate steps in the development of their respective tissues of origin.

PURKINJE NEURON

An output cell of the cerebellum, which has a large cell body with a characteristic mass of highly branched dendrites, and a single axon that sends inhibitory signals to the cerebral cortex. These cells coordinate the development of the cerebellar cortex. GRANULE NEURON

A small interneuron in the cerebellum, which relays excitatory signals to Purkinje neurons.

from stem cells that are present in the bulge of the follicle66,67. However, it is also possible that they derive from non-follicular transit amplifying precursors that are present in the basal layer of the interfollicular epidermis. Might inappropriate activation of the SHH–GLI pathway in epidermal stem cells be responsible for BCC initiation? BCCs might be abnormal ‘organs’ that try to recapitulate GLI-dependent ontogenic steps in follicular differentiation1. BCCs could therefore be thought of as tumours that arise from mispatterning events in which an interfollicular progenitor gets inappropriate instructions to differentiate into an abnormal follicular-type cell. Alternatively, GLI+ cells from the hair follicle might form the tumours if mutations inappropriately maintain

or enhance GLI1 activity. The finding that BCCs rarely develop in epidermal regions that are devoid of follicles could be consistent with either possibility. So why do Ptc+/– mice not develop BCCs? Even if the tumours involve loss of heterozygosity or a second hit, the skin should be a prime target for environmental mutagens. Perhaps the answer lies in the fact that mice, unlike other mammals, simply do not sporadically develop BCCs, which begs the question of whether mouse lines are good models to study this type of skin cancer, even though BCCs can be induced by means of transgenic approaches. Nevertheless, Ptc+/– mice do have very small follicular tumours and can develop BCC-like lesions after being exposed to ultraviolet or ionizing radiation46. By contrast, non-irradiated Ptc+/– mice do develop medulloblastomas3,41. But why is the cerebellum affected? The finding that Shh is normally expressed in PURKINJE NEURONS of the cerebellar cortex opened up a new avenue of inquiry on brain and tumour development28–30. Shh controls the growth of the cerebellum, and it orchestrates the positioning and differentiation of several cell types (reviewed in REFS 2,3,6). Among other functions, Shh promotes proliferation of GRANULE-NEURON precursors in the external germinal layer (EGL) of the cerebellum. Medulloblastomas are predicted to arise when granule-neuron precursors inappropriately maintain an active Shh–Gli pathway28–30,41 (FIG. 5) and express Gli1 (FIG. 4), although they can also arise from other causes, such as deregulated p53 and retinoblastoma (Rb) function68. Normally, this signalling and patterning information is tightly regulated and when it persists inappropriately, precursor cells might be instructed to proliferate abnormally, thereby initiating a tumour. The regulation of proliferation in the EGL has two conflicting tasks: to fulfil the need for the cerebellum to grow to the appropriate size for a given species (FIG. 5), while preventing tumorigenesis. Endogenous secreted factors that might regulate the time at which outer EGL cells stop cycling could be useful as anticancer agents. Such agents could possibly be found in Bergman glia — cerebellar glial cells that inhibit proliferation of EGL cells69. A clear molecular understanding of how the Shh–Gli pathway operates in the cerebellar cortex is likely to not only reveal how this central nervous system (CNS) ‘organ’ is formed, but also provide clues for possible rational therapies. Many issues remain unresolved. For example, is activation of the SHH–GLI pathway sufficient to induce sporadic cerebellar tumours in humans? Several sporadic human medulloblastomas carry PTCH mutations53,56,70,71, so is loss of heterozygosity of PTCH sufficient to initiate a tumour? Is the pathway involved in initiation as well as maintenance of medulloblastomas? As Ptc+/– mice also develop rhabdomyosarcomas42, is loss of PTCH sufficient to induce these cancers in humans? Is activation of the pathway sufficient to induce different types of sarcoma59? What would the cells of origin be for these tumours? Would blocking the SHH–GLI pathway lead to tumour regression? Although many of these questions are being investigated, it seems that not all human PNETs


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a Cerebellum Mouse

Chick

A

A P5

P

P D15

b Medulloblastoma

EGL

Tumour

Figure 5 | Morphological plasticity and tumorigenicity. a | Sagittal sections of the cerebellum of a 5-day-old (P5) mouse pup and that of a 15-day-old (D15) chick embryo differ greatly, in terms of the pattern and number of folia, but have similar dispositions of cell types. Only a central portion of the chick cerebellum is shown. b | Proposed mechanism for the growth of folia and the achievement of species-specific patterns. Differential Shh–Gli function might drive the regional growth of folia, in this case leading to the bifurcation of the folium tip. If the pathway is maintained inappropriately in external granular layer (EGL) precursors, a medulloblastoma will develop (right). Areas of high or abnormally maintained Shh–Gli function are shown by black arrows, which indicate growth. A, anterior; P, posterior.

show loss of PTCH heterozygosity72,73, raising the question of whether complete loss of function of PTCH is required for the development of medulloblastomas. Moreover, the significance of the level of expression of GLI1 or PTCH remains unknown. For example, desmoplastic medulloblastomas seem to express slightly more GLI1 and PTCH than non-desmoplastic ones74, but the meaning of this small increase is uncertain. Gliomas

VENTRICULAR ZONE

The layer of cells that immediately surrounds the cerebral ventricles. SUBVENTRICULAR ZONE

The layer of cells that is beneath the ventricular zone. Both the ventricular zone and the subventricular zone contain stem cells.

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Gliomas are CNS tumours with a very poor prognosis that are thought to derive from glial cells (see REFS 75,76 for recent reviews). Generally, gliomas can be divided into two classes: low-grade, slow-growing gliomas, and high-grade, fast-growing ones. The latter can arise de novo (primary) or derive from the low-grade (secondary) ones76,77. Aggressive high-grade gliomas in humans often show mutations in a small number of genes that are involved in growth-factor signalling, including those that encode the epidermal-growth-factor receptor (EGFR) and the lipid phosphatase PTEN. Are these prevalent mutations responsible for the development of the tumours? The finding that activated EGFR provides a growth advantage to cells in culture and after transplantation into the brains of mice78 supports this view. Similarly, the finding that development of aggressive glioma in mice can be achieved by overexpressing several genes — including

Akt, Ras and platelet-derived growth factor (Pdgf )79–82 — the products of which are involved in growth-factor signalling, would indicate that these oncogenes are involved in glioma development. Interestingly, the Shh–Gli pathway might also be involved in gliomagenesis. In addition to regulating cerebellar cortical development28–30, the Shh–Gli pathway also regulates the growth of the cerebral cortex and of the colliculi11. As in the cerebellum28, Shh might be transiently expressed in precursors, but from late embryogenesis and into postnatal development, Shh is expressed by cells in the cortical and tectal plates in a layer-specific manner. From here, it seems to regulate the proliferation of later-developing Gli1-expressing precursors in the 11 VENTRICULAR and SUBVENTRICULAR ZONES . The Shh–Gli pathway therefore regulates the growth of the dorsal brain, in contrast to its role in cell-fate determination in the early ventral neural tube (reviewed in REFS 2,27). In addition to the cerebral and cerebellar cortices and the colliculi, this pathway is also likely to control the development of granule cells of the hippocampus11. As all of these neuronal and glial precursor populations in the brain seem to express the Gli genes, could it be that brain tumours derive from Gli+ precursors? Analyses of various primary CNS tumour samples and tumour cell lines showed that nearly all tumours tested — including astrocytoma, oligodendroglioma, glioblastoma multiforme and PNETs — consistently express the Gli1 and Ptc genes11 (FIG. 4). Moreover, expression of Gli1 might not only be a marker of the origin of these varied brain tumours, but is also likely to indicate that tumour cells harbour an active Shh–Gli pathway. GLI1 was originally isolated as an amplified gene in a human glioma line83. So, it would make sense that gliomas express this gene. However, subsequent studies by various groups did not support a role for GLI1 in gliomagenesis84,85. All these studies looked for amplification or ‘overexpression’of the gene. GLI1 is located in a region that is sometimes amplified, but amplification is not the rule. Moreover, when analysing levels of GLI1 mRNA expression, some authors84 chose the cerebellum as the control for levels of expression in the normal brain. As we now know (for example, see REFS 28,86), the cerebellum expresses GLI1 at very high levels, so the fact that only very few samples had higher levels than cerebellar cells is not surprising. What we think is important is the inappropriate expression of GLI1 in time or space, not the amplification of the gene. Inappropriate GLI function might therefore be a crucial event in brain tumorigenesis, although whether the level of expression is correlated with the grade or subtype of the tumour remains unresolved. Experimental evidence for an involvement of Gli1 function in initiating brain tumorigenesis, possibly including glioma formation, derives from the ability of the transient somatic misexpression of human GLI1 in the developing tadpole CNS to give rise to tumours11 (FIG. 3). These tumours express platelet-derived growth-factor receptor-α (Pdgfrα) — a marker of oligodendrocyte precursors and of human gliomas — and form well after the injected RNA and its protein product have been




REVIEWS degraded, indicating that some endogenous components carry the ‘imprint’ of the action of the injected materials11. Could continued Gli activity be necessary for tumour maintenance? Analyses of the expression of endogenous Gli1 in frog embryos that had been injected with human GLI1 mRNA show that it is consistently expressed in the tumours that subsequently form. Because inhibition of endogenous Gli1 protein synthesis by an antisense oligonucleotide led to the inhibition of tumour formation by the co-injected human GLI1 mRNA11, these results indicate that the maintenance of tumorigenesis, which is initiated by human GLI1, requires the continuous action of the endogenous pathway. Direct experimental evidence that Shh–Gli signalling activity contributes to the maintenance of gliomas and other brain tumours comes from the finding that cyclopamine — a plant alkaloid that selectively inhibits the activity of the Shh receptor complex87,88 — acting at the level of SMOH89, inhibits the growth of several primary human gliomas as well as human medulloblastoma and glioma cell lines11. We reason that only a subset of the tumours responds to cyclopamine because only a fraction of all tumours will have activating mutations in the receptor complex. There are many other elements in the Shh–Gli pathway that could be affected, many of them acting downstream of the receptor complex, which should be insensitive to the action of this drug. It also remains possible that some brain tumours arise independently of Shh–Gli function and would therefore also be insensitive to the action of this drug. The idea that an active Shh–Gli pathway might be involved in brain tumour initiation and maintenance fits with recent results showing that tumours initiated by activated oncogenes in conditional mouse models remain dependent on the activity of these oncogenes7–10,12. It also has important therapeutic implications, as inhibiting GLI activity in established human tumours could, in principle, cause tumour regression. Future directions

Other tumours. The Shh–Gli signalling pathway is likely to be important in the initiation and/or maintenance of sporadic tumours that are derived from tissues or cell groups that use this pathway for cell proliferation during normal development. For example, Shh and the Gli genes are expressed in the lung; Gli2/3 mouse mutants have defects in lung development (reviewed in REF. 90), and it is conceivable that this pathway remains active in the adult, as it does in early postnatal animals91. As in the lung, the Shh–Gli pathway is active during prostate development92, and prostate carcinomas (reviewed in REF. 93) consistently express Gli1 (REF. 11), which is indicative of the presence of an active pathway. Gli function could therefore be important in lung and prostate cancer. How Gli1 might interact with, affect or respond to changes in prostate oncogenes or tumour suppressors remains to be determined. It is also unclear what regulates the appearance of cancer versus benign tumours and cysts after activation of this pathway48,94.

The involvement of Gli function in tumours in which Shh signalling seems not to be involved could result, in principle, from the effects of Fgf which, at least in one instance, can induce the expression of Gli2/3 (REF. 23). For example, activation of the Fgf and Wnt pathways can lead to mammary tumours in mice95,96. It is possible that, in this case, Fgfs induce Gli2/3 and these, in turn, induce Wnts as seen in amphibian development31. Ptc and Gli2 mutant mice show defects in mammary-gland morphogenesis97. Perhaps consistent with this proposal is the finding that human breast carcinomas do not consistently express GLI1 (GLI1 was expressed in only one out of nine primary tumours tested), but most samples express GLI2 and GLI3 (six out of nine and five out of seven, respectively; N.D., P.S. and A.R.A., unpublished observations). It is possible, therefore, that GLI proteins might be involved in tumour types in addition to those that are derived from tissues in which the Hh proteins are active. Can tumours arise from deregulation of one factor? Classical experiments indicate that cancer arises from the accumulation of mutations in the genome of a cell, affecting oncogenes or tumour-suppressor genes (reviewed in REF. 98). In this sense, Ptc can be viewed as a tumour suppressor39–41. But does complete loss of Ptc induce tumorigenesis? Analyses of the two PTCH alleles in various sporadic tumour samples shows that tumours do not have consistent loss of heterozygozity72,73,99. So how does heterozygosity in Ptc accelerate tumorigenesis? Classically speaking, where would the ‘other hit’ be? Possible answers to these questions could lie in the many functions of the Shh–Gli pathway and the many points at which it intersects the cell cycle (reviewed in REF. 6). For instance, there might be non-Gli mediators of aspects of Shh signalling100,101, and it could be speculated that Ptc might be haploinsufficient in terms of its proposed activity for sequestering cyclin B 36, thereby allowing a first hit to increase proliferation. Ptc mutant mice show an enormous increase in the frequency of medulloblastomas in a Trp53-null background43. Cyclin B action in G2, and loss of p53 — allowing hyperactivation of G1 cyclin–Cdk complexes and the survival of cycling cells — could conceivably cause tumorigenesis. However, there are many other possibilities. But could deregulated Gli1 function alone induce tumorigenesis or at least abnormal hyperproliferation — perhaps laying the foundation for additional mutations to trigger changes that are associated with malignant, transformed or neoplastic states? Direct tests should involve inducing Gli1 function in stem or progenitor cells and measuring proliferation, rather than transformation. Nevertheless, Gli1 seems to be sufficient to transform kidney epithelial cells in cooperation with adenovirus E1A102, resulting in the induction of a series of genes that are involved in cell proliferation, adhesion, signal transduction and apoptosis33,103. This indicates again that Gli1 acts through many targets at several levels.


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REVIEWS At least in some instances, it might also be possible to induce tumorigenesis by means of epigenetic alterations that lead to stable changes in gene expression. In our experiments, endogenous frog Gli1 can be activated by the transient production of human GLI1 protein11. This could imply that, in some cases, alterations in regulatory circuitry are sufficient to yield a transmissible change, without involving mutations in protein-coding sequences. Support for the existence of tumorigenic mechanisms, in addition to the classical mutations of oncogenes and tumour suppressors, also derives from methylation changes in regulatory regions104. Moreover, because oncogenic mutations can be found in normal cells that are adjacent to tumours105, these might not suffice in vivo to dictate a tumorigenic fate. In our case, inappropriate activation of Gli1 leads to a programme that abnormally maintains Gli1 function. How this occurs, however, remains unclear because Gli1-expressing cells do not consistently express Shh, indicating the lack of an autocrine signalling loop, and no cell-autonomous positive-feedback loops have been described for this pathway. If this occurs in brain tumours, for example, what role do the typical mutations that are observed in these tumours have? What is the significance of the clear and consistent alterations in genes such as Egfr and Pten? Stem cells and tumours. The two-step progression of primary to secondary gliomas indicates that at least two molecular components are involved (reviewed in REF. 76). One hypothesis that might be consistent with this twostep progression is the idea that tumour initiation derives from inappropriate activation of the Shh–Gli pathway in stem cells — cells with unlimited replicative potential, low proliferation rates and several possible fates. Hypothetical ‘tumour stem cells’ would differ from normal stem cells in that their terminal differentiation pathways would somehow be blocked (FIG. 6). Such cells would therefore be perfect targets for further oncogenic mutations, such as those affecting Pten or Egfr. Another possibility is that it is not the stem cells per se, but their progeny, that are targets for further oncogenic mutations. These cells, like their normal counterparts, might have lost the potential for unlimited renewal, but the accumulation of oncogenic mutations would confer a growth advantage. Secondary gliomas could conform to such a case. Therapies that are targeted at these mutations might be able to eliminate the bulk of the tumour, but would not affect the tumour stem cells, thereby allowing tumours to recur. Evidence for the possible presence of stem cells in non-solid tumours derives from studies on haematopoietic cancers (reviewed in REF. 106). For example, human acute myeloid leukaemia cells can induce tumours after transplantation at a low frequency, whereas differentiated cells that are derived from the same tumours cannot107, so presumably the leukaemogenic cells are undifferentiated precursors or stem cells. Interestingly, SHH induces proliferation of pluripotent blood-cell precursors108 and might also

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Tumour stem cell

Amplifying tumour precursor

Epigenetic patterning change

Classical mutation

Finite lifespan of lineages?

Figure 6 | Gli function and tumour stem cells. Hypothetical lineages and sites of action of alterations that lead to tumour formation. a | In the classical view, a progenitor cell is the target of mutations that activate the tumour programme, seen here as the rapid succession of cell divisions of amplifying tumour precursors that maintain an undifferentiated state. It is unclear whether all cells can regenerate the tumour after transplantation and whether these lineages have a finite lifespan. b | In this model, a patterning or epigenetic change, or possibly even a mutation, in a stem cell leads to the action of a genetic switch that provokes a transmissible change, leading to the slow abnormal growth of a tumour stem-cell population (thick arrows). This population can be the target of additional tumorigenic mutations that lead to the increased proliferation of amplifying tumour precursors. These cells might have finite replicative potential. In this case, the maintenance of the tumour might depend on the presence of tumour stem cells. c | A variation of b in which division of the tumour stem cell is asymmetrical, giving rise to an amplifying tumour progenitor with a higher proliferative capacity and to another tumour stem cell. As in b, classical mutations could induce further changes in the tumour phenotype, as cells with higher proliferative potential would be selected for. Nevertheless, these lineages might have a finite replicative potential, and the maintenance of the tumour could depend on the presence of tumour stem cells. In all cases, the initiating event might be crucial for tumour maintenance. Not shown in these diagrams is the possibility that tumour cells, even tumour stem cells, might derive from dedifferentiation events.




REVIEWS affect differentiation109, raising the possibility that the SHH–GLI pathway is also involved in haematopoietic ‘stem-cell’ tumours. Solid tumours can sometimes contain cells that differentiate. For instance, high-grade glioblastoma cells can differentiate abnormally to give rise to hair, muscle, epithelia or even teeth (reviewed in REFS 77,110,111) in a clonal tumour112. Similarly, gliosarcomas are thought to develop a muscle-cell component from a glioma origin113. Might the fates of these differentiating cells be limited to cell types that use the Shh–Gli pathway in normal development, including hair, muscle and teeth (for example, see REFS 11,47,62,114)? Perhaps these aberrant fate changes are transdifferentiation events that reflect the plasticity of stem or precursor cells. These ideas raise several questions. Are tumour stem cells formed by the acquisition of mutations or by epigenetic changes that establish inheritable traits? What would they give rise to if isolated and placed in a different context? Could these cells be manipulated to abort their tumorigenic programme? Could they be deprogrammed? Is the prolonged life of a tumour — understood as an organ that can regenerate — dependent on the presence of stem cells? Do most tumours depend on the activity of the initiating event? Questions like these are just beginning to address the problem of tumorigenesis as a patterning programme, and although we do not yet have any answers, there are at least precedents for the inappropriate reactivation of developmental processes in cancer. Perhaps most important is the finding that teratocarcinoma cells can contribute to normal development if placed in early mouse embryos 115. This indicates that, under some conditions, tumour cells can be deprogrammed to become normal and totipotent. Moreover, metastasis has many parallels with migratory ontogenic states: genes involved in developmental processes such as neural-crest formation are also involved in tumour invasion116. In addition, is not the fact that solid tumours can be classified morphologically in itself a taxonomical basis for the existence of diverse and consistent morphogenetic programmes? Is not the expression of genes that are involved in precursor proliferation in various tumours a clue to their origin11,74,117? And might these genes (such as Wnts)31,118–120 induce stereotypical patterns in tumours, as they do in normal development? Could we therefore think of tumours as organs that normally do not develop — that is, organs-in-formation that exist in all of us in a potential, ‘embryonic’ state? And if this were the case, would this not indicate that in the stem cells of each and every organ resides the potential for cancer? And that cancer cells can be, in principle, deprogrammed? Most cells cannot proliferate unless they already have mutations that prevent apoptosis or differentiation. But in some permissive contexts, such as in stem cells or precursors with long proliferation programmes, a single change might allow the maintenance of hyperproliferation and therefore tumour initiation. If this were the case, we could expect adult gliomas to derive originally

from adult neural stem cells11, which reside in several locations, including the subventricular zone of the lateral ventricle121, although such tumour stem cells could migrate (as cells in the rostral migratory stream normally do) and establish tumours in different areas. An alternative is that gliomas could arise from dedifferentiating glia, perhaps paralleling the dedifferentiation of cells that occurs in amphibians and other organisms during limb regeneration (reviewed in REF. 122). The many elements that, when deregulated, cause tumorigenesis (reviewed in REF. 98) could, in principle, be linked through common developmental and patterning pathways. For instance, Gli function could be related to Fgf, Egf (epidermal growth factor), Pten and Akt action. This possibility is based on the following observations: Gli proteins affect brain-precursor proliferation11; Gli2 and Gli3 can respond to Fgf 23; Fgf and Egf affect brain-precursor proliferation123; Pten normally acts to restrict precursor proliferation124; Pten regulates Akt phosphorylation and function (reviewed in REF. 75); and glioblastomas can arise in mice after the activation of Akt and Ras80. A connection between the Shh pathway and other glioma-inducing molecules might also not be farfetched (FIG. 2b). For example, Pdgf signalling can induce gliomagenesis in mice79,81, and alterations in this signalling pathway are often detected in human gliomas (for example, see REFS 75,76,125). Gli1 induces the upregulation of Pdgfrα in CNS tumours11, and Pdgfrα is also upregulated in BCCs58,126, which are induced by Gli1 (REFS 47,48). Upregulation of Pdgfrα should lead to an increase in Pdgf signalling81,127. Insulin-like growth factor (Igf) signalling has also been implicated in brain tumour development — in this case in tumour maintenance128,129 — and Igf2, which is a target of Shh signalling, is required for medulloblastoma and rhabdomyosarcoma development130. Last, Wnt signalling has similarly been implicated in various tumours, including colorectal carcinomas, melanomas, breast tumours and PNETs120,131, and Gli proteins regulate Wnt gene expression in a variety of contexts31. How misregulation of other genes that lead to gliomagenesis — such as those that cause the loss of Ink4a (also known as p16, and encoded by Cdkn2a), Arf (also known as p19, and also encoded by Cdkn2a) and p53 (reviewed in REF. 76), or the overexpression of others in gliomas and PNETs, such as Nmyc74 — could affect Shh–Gli function in stem cells, or vice versa, remains unclear. Finally, a link between the Shh–Gli pathway and Notch and its associated tumours132,133 might also be important as both Notch and Shh are mitogens for haemotopoietic stem cells108,134,135. Notch maintains neural stem cells and radial glia136, and both Shh and Notch signalling induce Hes expression — a gene that encodes a transcription factor that inhibits cerebellar granule-cell differentiation137. Tumorigenesis as the price for morphological plasticity. If inappropriate activation or maintenance of Gli1 function is sufficient to induce tumorigenesis, and its function is redundant in normal development, what


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REVIEWS is the selective advantage in keeping it? Apart from the possibility that it is just evolutionary junk, and nasty junk at that, it is likely to have a pivotal role in the life of the organism that has so far eluded study. For example, it could be a crucial factor for some aspect of brain function — something that would be difficult to study in model organisms that are placed in the usual environments. However, on the basis of what we already know, it is possible to propose that Gli1 has an important function in morphological plasticity during evolution. Perhaps Gli1 is indeed redundant with the other Gli proteins, because Gli1 knockout mice seem normal19, and a single organism can do without Gli1. But perhaps a healthy and redundant Gli family endows the species with an increased ability to modify the response to Hh signals, as well as to allow for greater versatility in the interpretation of these and other signals in different

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Acknowledgements We are grateful to J. L. Mullor, I. Carrera, A. Pellicer, M. Chao, V. Palma and Y. Gitton for comments. P.S. is a recipient of a Ramón Areces Foundation grant. N.D.’s laboratory is funded by an ATIPE grant from the Centre National de la Recherche Scientifique and grants from La Fondation pour la Recherche Médicale and L’Association pour la Recherche sur le Cancer. Our work described in this review was supported by grants from the March of Dimes, the Concern Foundation, the National Cancer Institute and National Institute of Neurological Disorders and Stroke, and a Pew Scholarship (to A.R.A.). Only a partial reference list is given due to journal restrictions.

Online links DATABASES The following terms in this article are linked online to: Cancer.gov: http://www.cancer.gov/cancer_information/ acute myeloid leukaemia | astrocytoma | brain tumour | breast carcinoma | colorectal carcinoma | medulloblastoma | melanoma | prostate carcinoma | rhabdomyosarcoma | skin carcinoma GenBank: http://www.ncbi.nih.gov/Genbank/ E1A LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ Akt | Cdkn2a | cyclin B | Desert hedgehog | D-type cyclins | Egf | Egfr | EGFR | Fgf | Gli | Gli1 | GLI1 | Gli2 | GLI2 | Gli3 | GLI3 | Hes | Hh | Igf1 | Igf2 | Indian hedgehog | Nmyc | Notch | Pdgf | Pdgfrα | Ptc | PTCH | Pten | PTEN | Ras | Rb | Shh | SHH | Smo | SMOH | Trp53 | Waf1 | Wnt | WNTs OMIM: http://www.ncbi.nlm.nih.gov/Omim/ basal-cell nevus syndrome FURTHER INFORMATION Encyclopedia of Life Sciences: http://www.els.net/ Hedgehog signalling Gorlin’s syndrome support group: http://www.gorlin-group.pwp.blueyonder.co.uk/ Way2Goal Sonic Hedgehog pathway: http://way2goal.com/shh.html Access to this interactive links box is free online.

