1. Hedgehog signalling pathway

2. The Hedgehog signalling pathway plays a fundamental role in normal embryonic development
The Hedgehog pathway was discovered in fruit fly (Drosophila) and is conserved in vertebrates (including humans)1,2
The Hedgehog pathway is involved in cell growth and differentiation to control organ formation during embryonic development
Hedgehog signalling regulates embryonic development, ensuring that tissues reach their correct size and location, maintaining tissue polarity and cellular content2
In the skin, the Hedgehog pathway is critical for regulating hair follicle and sebaceous gland development3
Germline mutations in components of the Hedgehog signalling pathway results in a number of developmental abnormalities4,5
Hedgehog signalling normally remains inactive in most adult tissues2
The Hedgehog signalling pathway was originally discovered in fruit flies1,2
The pathway’s name originated from the discovery that mutations in a key component of the pathway caused a spiky-haired phenotype
The pathway is conserved in humans and other mammals
Mutations in genes involved in Hedgehog signalling are linked to multiple congenital malformations. These malformations include:3,4
Holoprosencephaly – incomplete separation of the ventral forebrain into cerebral hemispheres (associated with craniofacial abnormalities such as cleft lip and palate, a proboscis-like nasal structure and cyclopia)
Bone development abnormalities
Limb malformation such as preaxial polydactyly (extra thumb formation)
Hedgehog signalling normally remains inactive in most adult tissues but may be activated in the maintenance or repair of tissues1
Inappropriate reactivation of Hedgehog signalling can result in tumourigenesis2
References:
Nüsslein-Volhard C, Wieschaus E. Nature 1980;287:795–801
Scales SJ, de Sauvage FJ. Trends Pharmacol Sci 2009;30:303–12
Wilkie AO et al. Nat Rev Genet 2001;2:458–68
McMahon AP et al. Curr Top Dev Biol 2003;53:1–114
1. Nüsslein-Volhard C, Wieschaus E. Nature 1980;287:795–801
2. Scales SJ, de Sauvage FJ. Trends Pharmacol Sci 2009;30:303–12
3. Chiang C, et al. Dev Biol 1999;205:1–9
4. Wilkie AO et al. Nat Rev Genet 2001;2:458–68
5. McMahon AP et al. Curr Top Dev Biol 2003;53:1–114 2

3. Key components involved in Hedgehog signalling
The Hedgehog ligand, Hedgehog (Hh)
The Hedgehog ligand receptor, Patched (PTCH)
Initiates signal transduction of the Hedgehog pathway
Normally suppresses the activity of SMO
The cell surface signal transducer, Smoothened (SMO)
The downstream effectors,
the Gli transcription factors
Key components of the Hedgehog pathway are:
The Hedgehog ligand, Hedgehog (Hh)
The Hedgehog ligand receptor, Patched (PTCH)
The cell surface signal transducer, Smoothened (SMO)
The downstream effectors, the Gli transcription factors
Reference:
Scales SJ, de Sauvage FJ. Trends Pharmacol Sci 2009;30:303–12
Cytosolic complex of proteins including Suppressor of Fused (SuFu) and the Gli family of transcription factors. Activation leads to expression of specific genes that promote cell proliferation and differentiation
Normally suppressed by PTCH, preventing its activation of the Hedgehog signalling cascade
Scales SJ, de Sauvage FJ. Trends Pharmacol Sci 2009;30:303–12 3

4. When the Hedgehog pathway is inactive Patched inhibits Smoothened activity
No Hh ligand
No SMO-enabled signal transduction
The Hedgehog signalling pathway plays an essential role during embryonic development regulating tissue and organ formation1,2
Two key proteins of this pathway are Smoothened (SMO), which enables the cascade of Hedgehog signalling events, and the Hh ligand receptor Patched (PTCH), which acts as a suppressor of SMO1,3
The inhibitory effect of PTCH causes SMO to remain in endosomic vesicles, away from the cell surface1
Gli proteins suppress transcription of target genes when SMO is not activated1,3
References:
1. Rubin LL, de Sauvage FJ. Nat Rev Drug Discov 2006;5:1026–33
Beachy PA et al. Nature 2004;432:324–31
Caro I, Low JA. Clin Cancer Res 2010;16:3335–9
Inhibition
No intracellular signal transduction
In the absence of Hh ligand, PTCH inhibits SMO and the Hedgehog signalling pathway is suppressed 4

5. Target gene expression
When Hedgehog ligand activates the Hedgehog pathway the cell responds by activating expression of target genes
Upon binding of Hh ligand to PTCH, the inhibitory effect on SMO is removed and SMO is localised to the cell surface1-3
SMO then activates a cytoplasmic complex of proteins, resulting in the phosphorylation of a family of transcription factors, Gli which translocate to the nucleus1-3
Phosphorylated Gli proteins are activators of transcription, controlling the expression of Hedgehog target genes that promote cell proliferation and differentiation1-3
References:
Rubin LL, de Sauvage FJ. Nat Rev Drug Discov 2006;5:1026–33
Caro I, Low JA. Clin Cancer Res 2010;16:3335–9
Epstein EH. Nat Rev Cancer 2008;8:743–54
Activation of the pathway is initiated by Hh ligand binding to PTCH, eventually resulting in target gene expression
Target gene expression 5

6. Abnormal Hedgehog pathway signalling plays an important role in the pathogenesis of certain types of cancer
Inappropriate reactivation of the Hedgehog pathway has been linked to several human cancers1
Two different mechanisms drive abnormal Hedgehog pathway signalling in different types of cancer:2
Ligand-independent signalling driven by mutations (e.g. BCC and medulloblastoma) Mutations in key pathway regulators (e.g. PTCH or SMO) cause SMO to be in a constitutively active state
Ligand-dependent signalling driven by overexpression of Hh ligand by tumour cells (e.g. ovarian cancer, colorectal cancer, pancreatic cancer)
Abnormal activation of the Hedgehog signalling pathway plays an important role in the pathogenesis of certain types of cancer1-3
Abnormal Hedgehog pathway signalling can lead to tumour cell proliferation via two different mechanisms:2
Mutations in key pathway regulators of Hedgehog signalling (e.g. PTCH or SMO) cause SMO to be in a constitutively active state: type 1 cancers
In BCC and medulloblastoma, mutations in key regulators of the Hedgehog pathway lead to increased cell proliferation and tumour development. This is known as mutation-dependent signalling and is ligand-independent
Overexpression of Hh ligand leads to upregulation of the Hedgehog signalling pathway: type 3 cancers
In ligand-dependent signalling, solid tumour cells overexpress Hh ligands that activate Hedgehog signalling in surrounding stromal cells. The stroma is hypothesised to respond by providing a growth-promoting microenvironment for the tumour. This signalling process is known as paracrine signalling and preclinical data suggest an importance of paracrine signalling in several types of cancer, including ovarian, colorectal and pancreatic cancers
References:
Scales SJ, de Sauvage FJ. Trends Pharmacol Sci 2009;30:303–12
Low JA, de Sauvage FJ. J Clin Oncol 2010;28:5321–6
Rudin CM. Cancer Prev Res 2010;3:1–3
1. Scales SJ, de Sauvage FJ. Trends Pharmacol Sci 2009;30:303–12
2. Low JA, de Sauvage FJ. J Clin Oncol 2010;28:5321–6
3. Rudin CM. Cancer Prev Res 2010;3:1–3 6

7. BCC and the Hedgehog signalling pathway
Abnormal activation of the Hedgehog signalling pathway is thought to play a critical role in the pathogenesis and progression of BCC, either by:1
Inactivating PTCH mutations, or;
Activating SMO mutations
Hedgehog pathway inhibitors may provide a new treatment option for patients with advanced BCC1
Abnormal Hedgehog pathway signalling is the key molecular driver of BCC1
Targeting the Hedgehog pathway may yield a novel therapeutic approach1
Reference
Epstein EH. Nat Rev Cancer 2008;8:743–54
1. Epstein EH. Nat Rev Cancer 2008;8:743–54 7

8. Target gene expression Tumour cell proliferation and/or cell survival
Mutation-driven Hedgehog signalling is involved in BCC: Inactivating PTCH mutations
In BCC that involves an inactivating PTCH mutation, the inhibitory effect on SMO is removed and SMO is localised to the cell surface1-3
SMO then activates a cytoplasmic complex of proteins, resulting in the phosphorylation of a family of transcription factors, Gli which translocate to the nucleus1-3
Phosphorylation of Gli proteins and their translocation to the nucleus results in the expression of target genes that promote tumourigenesis, tumour cell proliferation and survival1-3
References:
Rubin LL, de Sauvage FJ. Nat Rev Drug Discov 2006;5:1026–33
Caro I, Low JA. Clin Cancer Res 2010;16:3335–9
Epstein EH. Nat Rev Cancer 2008;8:743–54
Inhibition
Target gene expression
Inactivating mutations of PTCH lead to constitutive pathway activation
Tumour cell proliferation and/or cell survival

9. Target gene expression Tumour cell proliferation and/or cell survival
Mutation-driven Hedgehog signalling is involved in BCC: Activating SMO mutations
In BCC that involves an activating SMO mutation, active SMO is localised to the cell surface where it activates the cytoplasmic complex of proteins, including the Gli family of transcription factors1-3
SMO then activates a cytoplasmic complex of proteins, resulting in the phosphorylation of a family of transcription factors, Gli which translocate to the nucleus1-3
Phosphorylation of Gli proteins and their translocation to the nucleus results in the expression of target genes that promote tumourigenesis, tumour cell proliferation and survival1-3
References:
Rubin LL, de Sauvage FJ. Nat Rev Drug Discov 2006;5:1026–33
Caro I, Low JA. Clin Cancer Res 2010;16:3335–9
Epstein EH. Nat Rev Cancer 2008;8:743–54
Inhibition
Target gene expression
Activating SMO mutations lead to constitutive pathway activation
Tumour cell proliferation and/or cell survival

10. Abnormal Hedgehog pathway signalling is synonymous with BCC
In BCC, abnormal Hedgehog pathway signalling is the key molecular driver of the disease1-3
More than 90% of BCCs have abnormal activation of Hedgehog pathway signalling4-6
Most BCC tumours have either inactivating mutations in PTCH or, less commonly, activating mutations in SMO3,7–9
As a result of inactivating PTCH mutations3,7,9 or activating SMO mutations,3,7,9 SMO moves to the cell surface leading to activation of the GLI family of transcription factors9
Activated GLI then moves to the nucleus and initiates the transcription of target genes9
Abnormal Hedgehog pathway signalling is the underlying molecular driver of BCC1-3
More than 90% of BCCs have abnormal activation of Hedgehog pathway signalling4-6
Inappropriate activation of the Hedgehog pathway, most commonly caused by mutations in the Hedgehog receptor, Patched, triggers the expression of specific genes that promote cell proliferation and survival2
As a result of inactivating PTCH mutations3,7,9 or activating SMO mutations,3,7,9 SMO moves to the cell surface where it activates the GLI family of transcription factors9
Activated GLI then moves to the nucleus and initiates the transcription of target genes9
References:
1. Bale AE, Yu KP Hum Mol Genet 2001;10:757–62
2. Hutchin ME, et al. Genes Dev 2005;19;214–23
3. Epstein EH. Nat Rev Cancer 2008;8:743–54
4. Teh MT, et al. Cancer Res 2005;65:8597–603
5. Kallassy M, et al. Cancer Res 1997;57:4731–5
6. Unden AB, et al. Cancer Res 1997;57:2336–40
7. Caro I, Low JA. Clin Cancer Res 2010;16:3335–9
8. Rudin CM. Cancer Prev Res 2010;3:1–3
9. Scales SJ. Trends Pharmacol Sci 2009;30:303–12
5. Kallassy M, et al. Cancer Res 1997;57:4731–5
6. Unden AB, et al. Cancer Res 1997;57:2336–40
7. Caro I, Low JA. Clin Cancer Res 2010;16:3335–9
8. Rudin CM. Cancer Prev Res 2010;3:1–3
9. Scales SJ. Trends Pharmacol Sci 2009;30:303–12
1. Bale AE, Yu KP Hum Mol Genet 2001;10:757–62
2. Hutchin ME, et al. Genes Dev 2005; 19:214–23
3. Epstein EH. Nat Rev Cancer 2008;8:743–54
4. Teh MT, et al. Cancer Res 2005;65:8597–603 10

11. Hereditary defects in PTCH predispose to BCC: Gorlin syndrome
Patient with Gorlin syndrome and multiple lesions4
Also known as basal cell nevus syndrome (BCNS)
Rare hereditary condition that predisposes the individual to develop multiple BCCs1
The severity of the disease is wide-ranging and it affects about 1 in 57,000 people (0.0018%)2
Gorlin syndrome occurs in individuals who inherit one defective copy of the PTCH gene
Leads to an array of congenital defects3
Preaxial polydactyly, immobile thumbs, short metacarpals, broad faces, rib defects, dental abnormalities, and high predisposition to certain malignancies such as medulloblastoma
Gorlin syndrome – also known as basal cell nevus syndrome (BCNS) – is a rare autosomal dominant condition characterised by multiple BCCs,1 jaw cysts and palmar or plantar pits in the palms of the hands and soles of the feet1
The severity of the disease is wide-ranging and it affects about 1 in 57,000 people (0.0018%)2
Occurs in individuals with one defective copy of the PTCH gene
Leads to an array of congenital defects3
Preaxial polydactyly, immobile thumbs, short metacarpals, broad faces, rib defects, dental abnormalities, and high predisposition to certain malignancies
References:
Roewert-Huber J et al. Br J Dermatol 2007;157:47–51
Farndon PA et al. Lancet 1992;339:581–2
McMahon AP et al. Curr Top Dev Biol 2003;53:1–114
Tang JY et al. Cancer Prev Res (Phila) 2010;3:25–34
Active BCC tumours are circled in green
Image reprinted by permission from the American Association for Cancer Research: Tang JY et al. Basal Cell Carcinoma Chemoprevention with Nonsteroidal
Anti-inflammatory Drugs in Genetically Predisposed PTCH1+/ - Humans and Mice. Cancer Prevention Research, 2010;3:25-34:doi: / CAPR
1. Roewert-Huber J et al. Br J Dermatol 2007;157:47–51
2. Farndon PA et al. Lancet 1992;339:581–2
3. McMahon AP et al. Curr Top Dev Biol 2003;53:1–114
4. Tang JY et al. Cancer Prev Res 2010;3:25–34
NB: if you wish to present, reproduce or adapt the image on this slide, please seek permission from the relevant publication house. 11

12. Summary
The Hedgehog signalling pathway plays a fundamental role in normal embryonic development
In most adult tissues, the Hedgehog pathway normally remains inactive
Abnormal activation of the Hedgehog pathway signalling plays an important role in the pathogenesis of certain types of cancer
In BCC, abnormal Hedgehog pathway signalling is the key molecular driver of the disease
Hedgehog pathway mutations, most commonly in PTCH, drive abnormal Hedgehog pathway signalling in BCC
More than 90% of BCCs have abnormal activation of Hedgehog pathway signalling
Hedgehog pathway inhibitors may provide a new treatment option for patients with advanced BCC

13. References The Hedgehog signalling pathway
Bale AE, Yu KP. The hedgehog pathway and basal cell carcinomas. Hum Mol Genet 2001;10:757–762. Beachy PA, et al. Tissue repair and stem cell renewal in carcinogenesis. Nature 2004;432:324–331. Caro I, Low JA. The role of the hedgehog signaling pathway in the development of basal cell carcinoma and opportunities for treatment. Clin Cancer Res 2010;16:3335–3339. Chiang C, et al. Essential role for Sonic hedgehog during hair follicle morphogenesis. Dev Biol 1999;205:1–9. Epstein EH. Basal cell carcinomas: attack of the hedgehog. Nat Rev Cancer 2008;8:743–754. Farndon PA, et al. Location of gene for Gorlin syndrome. Lancet 1992;339:581–582. Hutchin ME, et al. Sustained Hedgehog signaling is required for basal cell carcinoma proliferation and survival: conditional skin tumorigenesis recapitulates the hair growth cycle. Genes Dev 2005;19;214–223. Kallassy M, et al. Patched (ptch)-associated preferential expression of smoothened (smoh) in human basal cell carcinoma of the skin. Cancer Res 1997;57:4731–4735. Low JA, de Sauvage FJ. Clinical experience with Hedgehog pathway inhibitors. J Clin Oncol 2010;28:5321–5326. McMahon AP, et al. Developmental roles and clinical significance of hedgehog signaling. Curr Top Dev Biol 2003;53:1–114. Nüsslein-Volhard C, Wieschaus E. Mutations affecting segment number and polarity in Drosophila. Nature 1980;287:795–801. Roewert-Huber J, et al. Epidemiology and aetiology of basal cell carcinoma. Br J Dermatol 2007;157:47–51. Rubin LL, de Sauvage FJ. Targeting the Hedgehog pathway in cancer. Nat Rev Drug Discov 2006;5:1026–1033. Rudin CM. Beyond the scalpel: targeting hedgehog in skin cancer prevention. Cancer Prev Res 2010;3:1–3. Scales SJ, de Sauvage FJ. Mechanisms of Hedgehog pathway activation in cancer and implications for therapy. Trends Pharmacol Sci 2009;30:303–312. Tang JY, et al. Basal cell carcinoma chemoprevention with nonsteroidal anti-inflammatory drugs in genetically predisposed PTCH1+/- humans and mice. Cancer Prev Res 2010;3:25–34. Teh MT, et al. Genomewide single nucleotide polymorphism microarray mapping in basal cell carcinomas unveils uniparental disomy as a key somatic event. Cancer Res 2005;65:8597–8603. Unden AB, et al. Human patched (PTCH) mRNA is overexpressed consistently in tumor cells of both familial and sporadic basal cell carcinoma. Cancer Res 1997;57:2336–2340. Wilkie AO, et al. Genetics of craniofacial development and malformation. Nat Rev Genet 2001;2:458–468.