The role of hedgehog signalling in tumorigenesis - Stanford University

Cancer Letters 173 (2001) 1–7 www.elsevier.com/locate/canlet

Mini review

The role of hedgehog signalling in tumorigenesis Carol Wicking a,b,*, Edwina McGlinn a,b,c b

a Institute for Molecular Bioscience, The University of Queensland, St. Lucia 4072, Brisbane, Australia The CRC for Discovery of Genes for Common Human Disease, Cerylid, Swann Street, Richmond 3121, Melbourne, Australia c Department of Biochemistry, The University of Queensland, St. Lucia 4072, Brisbane, Australia

Accepted 5 July 2001

Abstract It has long been known from work in both Drosophila and vertebrate systems that the hedgehog signalling pathway is pivotal to embryonic development, but the past 5 years has seen an increase in our understanding of how members of this pathway are crucial to the processes of tumorigenesis. This important link was firmly established with the discovery that mutations in the gene encoding the hedgehog receptor molecule patched are responsible for both familial and sporadic forms of basal cell carcinoma (BCC), as well as a number of other tumour types. It is now known that a number of key members of the hedgehog cascade are involved in tumorigenesis, and dysregulation of this pathway appears to be a key element in the aetiology of a range of tumours. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hedgehog signalling; Patched; Basal cell carcinoma; Medulloblastoma

1. Introduction The hedgehog signalling cascade is pivotal to embryonic development, and is involved in patterning a diverse range of vertebrate structures including the developing neural tube, lungs, skin, axial skeleton, teeth, hair and limbs [1–5]. In addition, this pathway has more recently been shown to play a role in haematopoiesis [6]. The importance of the hedgehog pathway is underpinned by its high degree of conservation through evolution, and much of what is known about signalling in vertebrates has been inferred from studies in Drosophila. The role of the hedgehog pathway in tumorigenesis was established with the discovery that inactivating mutations in the patched gene, which encodes one component of the hedgehog recep* Corresponding author. Tel.: 161-733-654-560; fax: 161-733654-388. E-mail address: [email protected] (C. Wicking).

tor, is responsible for the inherited cancer predisposition disorder known as Gorlin or naevoid basal cell carcinoma syndrome (NBCCS), as well as sporadic BCCs [7,8]. Since that time several other tumour types have been shown to have mutations in key members of the hedgehog signalling pathway. These discoveries have highlighted the potential role of developmentally important genes in controlling cell growth and differentiation, and added to the increasing list of such genes whose aberrant function contributes to the process of tumorigenesis. 2. The hedgehog signalling pathway Studies in vertebrate systems have led to the formulation of a model for hedgehog signalling whereby reception at the cell surface occurs via a complex consisting of the patched and smoothened molecules [9,10]. According to this model patched, a 12-pass

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C. Wicking, E. McGlinn / Cancer Letters 173 (2001) 1–7

transmembrane protein, is the ligand-binding component of the receptor complex. Smoothened, a protein with homology to a serpentine G-protein coupled receptor, is responsible for transducing the hedgehog signal. In the absence of hedgehog binding, patched is thought to hold smoothened in an inactive state and thus inhibit signalling to downstream genes. With the binding of hedgehog, patched inhibition of smoothened is released and the signal is transduced (Fig. 1). In vitro binding studies in mammalian cell lines suggested that the inhibition of smoothened by patched is mediated by direct interaction, and that these two molecules form a heteromeric receptor complex regulating hedgehog signalling at the cell surface [9]. However, recent data suggests that patched inhibition of smoothened may occur through an indirect catalytic mechanism rather than by direct stoichiometric interaction (reviewed in Ref. [11]). In part this theory has arisen as a result of the observation that very little patched protein resides at the cell

surface at any given time, with much of the protein present in intracellular endocytic vesicles [12]. In addition, results from the Drosophila system suggest that in response to hedgehog signalling, smoothened is stabilised and accumulates at the cell membrane via phosphorylation, while patched is removed from the cell surface [13]. It has also been shown that patched mediates internalisation of sonic hedgehog by endocytosis in responsive neural precursor cells [14], a process that is thought to lead to degradation in the lysosome. While in Drosophila there is a single hedgehog molecule, in vertebrates there are three homologues known as Sonic (Shh), Desert (Dhh) and Indian hedgehog (Ihh), with Shh being the most widely expressed of the three. Hedgehog is a secreted molecule which undergoes autocatalytic cleavage to generate the active 19 kDa N-terminal fragment [15–17]. This fragment is modified by the addition of a cholesterol moiety at its C-terminus [18], and at least in the

Fig. 1. Proposed model for hedgehog signalling in Drosophila. In the absence of hedgehog (hh), the patched protein (ptc) inhibits signalling by smoothened (smo). Upon binding of hh this repression is released leading to dissociation of a complex of segment polarity proteins normally associated with the microtubules (Fu-fused, Su(fu)-suppressor of fused, cos-2-costal-2, ci-cubitis interruptus) allowing transcription of downstream target genes.


case of Shh, can also be N-terminally palmitoylated [19]. This highlights one level at which sterols are thought to play a role in hedgehog signalling. In addition, patched shares significant homology across several of its transmembrane domains with proteins known to be involved in cholesterol homeostasis, as well as the protein involved in Niemann-Pick C1 disease, a disorder resulting from defects in intracellular sterol transport [20,21]. Since this region has been shown in other proteins to be responsive to sterol levels, it has become known as the sterol sensing domain (SSD). While the function of the SSD in patched remains unknown, recent data from Drosophila suggests that it may play a role in mediating intracellular patched trafficking as a means of regulating smoothened [22,23]. The studies outlined above hint at the highly complex mechanisms involved in regulating the transduction of the hedgehog signal at the cell surface, and it is likely to be some time before the complete process is fully understood. Downstream mediation of the hedgehog signal is also the subject of much speculation, particularly in the vertebrate system. In Drosophila the cubitus interruptus (ci) gene encodes the zinc finger transcription factor primarily responsible for mediating the hedgehog signal and activating and repressing downstream genes [24]. Several other molecules are also known to act downstream of patched and smoothened in regulating the hedgehog signal. These include the kinesinlike protein costal-2, the serine/threonine kinase fused, and the novel protein suppressor of fused. These three molecules form a complex with fulllength ci at the microtubules in the absence of a hedgehog signal (Fig. 1). Under these conditions ci is cleaved to generate a 75 kDa N-terminal fragment which enters the nucleus and represses transcriptional activation of downstream genes. This processing of ci is thought to be mediated by phosphorylation of the full-length protein [25]. In the presence of hedgehog, cleavage of ci is inhibited, the microtubule complex dissociates and full-length ci matures into a labile transcriptional activator which enters the nucleus and results in activation of target gene transcription. In vertebrates there are three ci homologues, Gli 1,2 and 3. While it is likely that the role of ci in Drosophila is achieved by these three genes in the vertebrate system, the precise role of each of these remains unclear.

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Given the widespread role of hedgehog signalling it is not surprising that the mechanisms governing its regulation at the membrane, in the cytoplasm and in the nucleus form a complex and interconnected network. Regardless of these regulatory processes, the net result of activated hedgehog signalling is the transcriptional upregulation of a range of downstream target genes. Extrapolation from Drosophila predicts that these downstream targets in the vertebrate system are likely to include members of the Wnt and TGFb families (in particular genes encoding the bone morphogenetic proteins), and also patched itself (reviewed in Ref. [26]). However, a host of novel molecules will undoubtedly be regulated by hedgehog signalling in a cell and tissue specific manner. A number of such targets have already been identified in the vertebrate system, and the identification of additional hedgehog targets remains a major focus of research in this field.

3. Hedgehog signalling in tumorigenesis Key components of the hedgehog pathway have been implicated in both developmental abnormalities and in tumour formation. Individuals with NBCCS inherit one mutated copy of the patched gene, and this heterozygosity is responsible for the developmental abnormalities seen in this disorder. The majority of germline mutations in NBCCS are predicted to lead to premature truncation of the patched protein, and are thus assumed to represent null patched alleles [27]. As a result, it is thought that many of the heterozygous effects of patched mutation result from haploinsufficiency. Tumours in NBCCS individuals are likely to arise with inactivation of the remaining patched allele in a given cell, consistent with patched acting as a tumour suppressor gene. In contrast, sporadic or non-inherited tumours are thought to require inactivation of both alleles of patched in the same cell in a lifetime, an event which occurs far less frequently and generally at a greater age, depending on other genetic and environmental factors. To date inactivation of patched has been shown to be a major factor in basal cell carcinoma (BCC) formation, with mutations detected in between 12 and 40% of sporadic BCCs [28–31]. In addition, loss of heterozygosity for markers encompassing the

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patched locus has been detected in more than 50% of BCCs, suggesting that in many tumours one of the patched alleles is inactivated by deletion [32]. Like germline mutations in NBCCS, a high proportion of mutations in sporadic BCCs are predicted to lead to premature protein termination [29]. While a number of these are C- . T substitutions typical of ultravioletB irradiation involvement [29], there is epidemiological data indicating a role for factors other than UV-B in BCC induction [33]. Mutations in patched have also been detected in BCCs associated with Xeroderma Pigmentosum (XP), an autosomal recessive disorder characterised by hyperphotosensitivity and skin cancer predisposition [34]. In addition to BCCs, the patched gene has also been implicated in the aetiology of a range of other tumours including medulloblastoma [31,35], squamous cell carcinomas of the oesophagus [36], transitional cell carcinomas of the bladder [37], and the benign skin lesions trichoepitheliomas [38]. It has also been shown that mice heterozygous for a null allele of patched develop soft tissue tumours of the muscle at a high frequency [39], indicating that hedgehog signalling may play a role in the pathogenesis of human rhabdomyosarcoma. Several other members of the hedgehog pathway have also been shown to have a role in tumour formation. Most notably, activating mutations in smoothened which have the same downstream effects as inactivation of patched, have been detected in 10– 20% of BCCs [40,41]. In particular, one common mutation (Trp535Leu) has been detected in a number of independent studies in up to 20% of BCCs analysed [40,42]. Activating mutations in smoothened have also been detected in primitive neuroectodermal tumours [41]. The Gli genes have also been implicated in tumour formation, with Gli1 first being named for its involvement in the brain tumour glioma. In addition, although mutations have yet to be found in human tumours, mice overexpressing either Gli1 or Gli2 develop BCC-like lesions [43,44]. From what we know of the regulation of the hedgehog signalling pathway, combined with mutational analysis in tumours, it is clear that tumour development is associated with activation of hedgehog signalling in specific cells. In the case of BCCs this seems to be the major factor involved, with a high percentage of BCCs known to have either inactivating mutations

in patched or activating mutations in smoothened. This has lead to the hypothesis that dysregulation of the hedgehog pathway is a requirement for BCC formation, and this conforms with the notion of the hedgehog pathway as the ‘gatekeeper’ of BCC development. The finding that constitutive activation of hedgehog signalling is the key to tumour formation suggests that the identity of downstream target genes in particular cell/tissue types will provide insight into the neoplastic phenotype. While this is likely to be a contributing factor, the recent discovery that patched interacts with cyclin B1 suggests that some tumorigenic effects of hedgehog signalling may arise from a more direct effect on cell cycle progression [45]. 4. Possible therapies for hedgehog-related BCCs Based on the premise that tumours arise as a result of activation of the hedgehog pathway, therapies involving inhibition of hedgehog signalling might be expected to suppress tumour growth. One agent which has arisen as a possible treatment for BCCs is a teratogenic steroidal alkaloid known as cyclopamine. This compound is derived from the Veratrum lily species and its teratogenic effects were first seen in grazing ewes whose offspring were born with holoprosencephaly, a defect whereby the forebrain fails to divide into hemispheres [46,47]. This defect is also seen in both humans and mice with inactivating mutations in the Sonic hedgehog gene [48,49]. It has since been shown that cyclopamine acts by inhibiting hedgehog signalling [50], and that it can specifically reverse the effects of oncogenic smoothened and patched mutations [51]. To date there appears to be no adverse effects of exposure of adults to this teratogenic compound, making it a feasible therapeutic agent for BCCs and possibly other tumours associated with dysregulation of hedgehog signalling. 5. Conclusions Dysregulation of hedgehog signalling plays a major role in tumorigenesis, particularly in the development of the common skin tumour basal cell carcinoma. The complex network of mechanisms governing the tissue specific regulation of this signalling cascade remain to be fully elucidated, but it seems clear that tumorigen-


esis is promoted by the constitutive activation of downstream targets of the hedgehog signal. A major challenge for the future involves modulation of hedgehog signalling in a way that it is not detrimental to the adult but will result in effective treatments for tumours associated with aberrant signalling by this pathway.

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Acknowledgements C.W. is supported by an R. Douglas Wright Award from the Australian National Health and Medical Research Council.

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