Hedgehog genes and their role in BCC

Aberrant upregulation of the Hh signaling pathway is the primary cause of BCC in both Gorlin syndrome individuals and sporadic tumors and most of the time (about 75-80% of cases), the initiation event can be traced to homozygous inactivation of the Hh pathway tumor suppressor gene PTCH1 (Epstein, 2008).

PTCH1 gene

The PTCH1 gene has 23 exons, spans 47 kb of coding sequence, and encodes a 1447 amino acid, twelve-transmembrane protein (uniprot.org/uniprot/ Last accessed 4.11.2015).

Although the originally identified oncogenic events in PTCH1 were point mutations, it is now known that loss of heterozygosity (LOH) of the 9q22.32 region containing PTCH1 can occur. While studying chromosomal imbalances in BCC, Teh and collaborators (Teh et al.,

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2005) found that 93% of the studied tumors had LOH of the 9q region and that the great majority (62%) carried an inactivating mutation in the remaining PTCH1 allele. Most interestingly, a significant fraction of the tumors with LOH (38%) did not have a change in copy number. This was considered to be possibly caused by a somatic recombination event causing “uniparental disomy” for the 9q region. We now know this is indeed what happens, and this mechanism is called copy neutral loss of heterozygosity (cnLOH). Both LOHs and cnLOHs of the PTCH1 gene are important oncogenic driver events in BCCs (Pan et al., 2010, Epstein, 2008, Jayaraman et al., 2014).

Gorlin syndrome patients carry a predisposing germline mutation and therefore, acquired somatic events provide the “second hit” for the inactivation of the remaining wild type (wt) PTCH1 gene and the eventual development of BCC(for example, (Tate et al., 2014)). On the other hand, for a sporadic BCC to arise, the tumor initiating cells require homozygous inactivation of PTCH1 that can be the consequence of LOH and persistence of a mutated allele, of a cnLOH event, or of the co-occurrence of two pathogenic point mutations (Pan et al., 2010). No matter what the final mechanism is, the ultimate inactivation of the tumor suppressor PTCH1 leads to a continuing loss of inhibition over SMO that is then able to activate the Hh signaling pathway in the absence of Hh ligands.

The study of the distribution of disease-relevant point mutations along the PTCH1 gene and its protein product has shown that some regions tend to harbor mutations more than others. Lindstrom et al., (2006) carried out an evaluation of 284 mutations and 48 SNPs submitted to a carefully curated PTCH1 mutation database. The study of the distribution of mutations along the PTCH1 coding sequence shows that overall, damaging mutations tend to cluster in the regions encoding the two large extracellular and the intracellular loops, when compared to the distribution of frequent SNPs, much more common in the second half of the protein and the sterol-sensing domain (SSD, Figure 14).

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Figure 14. Distribution of mutations in the PTCH1 protein. a. Mutations in 86 sporadic BCCs.

b. Distribution of 31 exonic SNPs reported to the PTCH mutation database. Black circles represent splice mutations, blue triangles nonsense mutations, red triangles missense events, yellow triangles silent events. The thicker line corresponds to the SSD. Figures reproduced with minor modifications from Lindström et al., 2006.

It is also possible to evaluate the PTCH1 mutations that have been submitted to cancer mutation databases. For example, the COSMIC database (Forbes et al., 2015, Cosmic Database) currently contains mutation information for 32,184 tumors of different types and tissue of origin. The data available for BCC is based on candidate gene or mutation scan and not on exome or whole genome sequencing studies, and COSMIC contains limited mutation information for 632 BCCs. The mutational status of PTCH1 is available for 550 of them and 27% (147 samples) have a PTCH1 mutation predicted to be pathogenic (Table 3) (www.cancer.sanger.ac.uk Last accessed 4.11.2015).

SMO gene

Smothened (SMO), a G-protein-coupled receptor-like protein, is the second most common Hh gene mutated in BCCs, and it can drive tumor progression in the absence of PTCH1 mutations (Reifenberger et al., 2005). The SMO gene is located in chromosome 7q32.1 and contains 12 exons in 24 kb of genomic DNA (uniprot.org/uniprot/ Last accessed 4.11.2015). It encodes a seven-pace transmembrane protein of 787 amino acids length.

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Activating missense mutations in SMO were first described in 1998 (Xie et al., 1998). Xie and collaborators were looking for additional mechanisms of activation of the Hh pathway besides PTCH1 mutations and scanned the full coding sequence of SMO with PCR of the exons and subsequent single strand conformation polymorphism (SSCP) tests, for activating mutations. In 47 tested sporadic BCC tumors, they identified two different mutations (p.R562G and p.W535L) in three tumors in the absence of PTCH1 mutations or

Table 3. Pathogenic mutations in BCC reported in the COSMIC database and Reifenberger et al., 2005. Data from Reifenberger et al., 2005 and COSMIC database, last accessed on 4.11.2015. *Pathogenicity evaluation automatically provided by COSMIC and performed with FATHMM: Functional Analysis through Hidden Markov Models (cancer.sanger.ac.uk, fathmm.biocompute.org.uk). N/A: mutational status not available.

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LOHs. The SMO variants were absent from the blood of the individuals and a control set of 100 samples, confirming their somatic status. When mutant SMO protein was transfected into mouse embryonic fibroblasts (MEF), density-dependent growth inhibition was impaired and cells lost their normal morphology when compared to control cells transfected with wild type SMO.

The authors suggest that these mutations may not impair the interaction of SMO with PTCH1 but instead cause its constitutive activation, since the p.W535L mutation occurs in a pocket conserved among several G-protein coupled receptors that keeps them in a latent state when unaltered (Xie et al., 1998). The relevance of the p.W535L mutation was reinforced by a later report of a collection of 97 sporadic BCCs where the mutation was found in 21% of cases, although the PTCH1 mutational status was not assessed (Lam et al., 1999).

Since the description of these two activating mutations, additional ones have been identified and have been shown to activate Hh signaling as well (Mao et al., 2006, Reifenberger et al., 1998). The mutational status of SMO in BCCs has been evaluated in 157 samples reported in COSMIC. 14% of the tumors have a SMO mutation and around half of them are predicted to be pathogenic (Table 3, www.cancer.sanger.ac.uk Last accessed 4.11.2015). SMO mutations can also be found in other cancer types, Hh-induced or not (Figure 15).

Figure 15. SMO mutations in cancer. Frequency of SMO mutations in different cancer types from COSMIC and current literature. Reproduced from Atwood et al, 2015.

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There has been a recent upsurge on the interest in SMO mutations, because Hh inhibitory molecules, specifically SMO inhibitors, have shown promising results in clinical trials and the first of them, vismodegib, has recently been approved by the FDA for the treatment of locally advanced or metastatic BCCs, as previously discussed (Fellner, 2012).

The majority of BCCs respond well to treatment with SMO inhibitors (Von Hoff et al., 2009, Sekulic et al., 2012, Dreno et al., 2014) but up to 30% of tumors do not improve with vismodegib treatment or relapse after initial positive response (Dreno et al., 2014, Chang and Oro, 2012, Sharpe et al., 2015, Atwood et al., 2015b).

To look for genetic mechanisms explaining this phenomenon, Sharpe et al. (2015) and Atwood (2015b) carried out whole exome sequencing (WES) of a total of 25 BCCs that were not responsive or that relapsed after treatment with vismodegib. They identified the primary driver as a PTCH1 mutation in 68% of the sporadic BCC samples and in 100% of the Gorlin syndrome tumors. The relapsed tumors with no PTCH1 mutations had SMO p.W535L, p.D473H or p.D473G mutations. These groups also studied BCCs before and after vismodegib treatment, identifying SMO driving mutations in the post-treatment samples in a significant fraction of tumoral cells (>60%, Figure 15) while in pre-treatment tumors, these mutations were found only at background level or not identifiable, suggesting drug induced selection of SMO mutant clones in the post-treatment tumors. They found several additional SMO mutations, such as p.V321M, p.T241M, and p.C469Y. Taking advantage of the recent availability of the structure of the SMO protein bound to teladegib, a SMO inhibitor (Wang et al., 2013), these two groups found that the SMO mutations either affect the conformation of the SMO drug-binding pocket (DBP) or are inside the pocket itself and impair the correct binding of the drug to it. Other SMO mutations not involved in the DBP but present in COSMIC, in other cancer types, or recurrent in the analyzed samples, also shown different levels of resistance to vismodegib and seem to cause the constitutive activation of SMO. With in-silico protein modeling or transfection assays of the mutants into smo^-/- MEFs, both groups agree that these mutations may destabilize the overall structure of the protein, reduce its affinity for antagonists, or impair the transmission of the Hh inhibitory signal, rendering the Hh pathway constitutively active (Sharpe et al., 2015, Atwood et al., 2015b).

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It is interesting to note that not all SMO variants found in resistant BCCs are activating mutations or impair drug binding. Mutations identified in the DBP, in the pivot region of the protein (region controlling the activation of SMO), or recurrent in COSMIC, were tested for pathogenicity. smo^-/- MEFs were transfected with Hh ligand and SMO proteins carrying the mutations, and the levels of Gli1 mRNA were then measured (as a proxy for Hh activation). A significant fraction of the mutations were shown to be neutral or even deleterious for the SMO protein. This finding suggest that not all tumor subclones carrying SMO mutations are driven by them, and that SMO can also have passenger mutations in BCC (Atwood et al., 2015a).

SUFU gene

The Homo sapiens suppressor of fused homolog (SUFU) gene is located in chromosome 10q24.32. It encodes a 484 amino acid protein. As previously mentioned, it binds and sequesters Gli transcription factors, the main effectors of the Hh pathway, and negatively regulates it (Stone et al., 1999).

Inactivating mutations in SUFU can cause GS, with skin manifestations of the disease but more frequently, medulloblastoma and mandibular keratocysts (Pastorino et al., 2009, Smith et al., 2014, Taylor et al., 2002). Mice with a deletion that removes the Sufu promoter and the first exon are, in the homozygous state, lethal at E9.5, and their MEF display full constitutional activation of the Hh pathway (Svard et al., 2006). This phenotype is very similar to that displayed by Ptch1 null mice. The heterozygous mice develop basal cell carcinomas and mandibular keratocysts as well as the rest of the signs of GS starting at 12 months of age. It is interesting to note that the Drosophila Sufu does not seem to be indispensable for Hh signaling but, given the results of Cooper et al. (2005) and Svärd et al.

(2006), mammalian, or at least murine Sufu, seems to play a more important role in Hh pathway regulation than its Drosophila counterpart.

The fraction of tumors with SUFU mutations in sporadic BCCs is lower than for SMO and PTCH1 (Reifenberger et al., 2005), and the number of BCCs in COSMIC tested for SUFU mutations is very small to draw any conclusions based on the mutations present in this database (Table 3). However, the proven relevance of SUFU mutations in GS and the

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phenotype observed in mice are strong evidence that SUFU mutations can be the primary driver in BCC. Furthermore, a small fraction of BCCs have focal loss of 10q in the region containing SUFU. Heterozygous deletions could cooperate with a co-occurring Gli2 amplification (Sharpe et al., 2015) or homozygous deletions of SUFU may confer resistance to vismodegib, since this phenomenon has been observed in medulloblastomas resistant to SMO inhibitors (Kool et al., 2014, Lee et al., 2007).

GLI genes

The first member of the glioblastoma-associated protein family (GLI) of zinc-finger transcription factors to be discovered was GLI1. It was isolated from a glioma, in which this gene was amplified, and was mapped to chromosome 12q13 (Kinzler et al., 1987). In following years, the other members of the family were identified and characterized through the study of proteins co-hybridizing to GLI1. GLI2 is located in chromosome 2q14, and GLI3 in chromosome 7p13 (Ruppert et al., 1990, Ruppert et al., 1988).

In mammals, GLI1 and GLI2 regulate proliferation though the activation of multiple genes, among them cyclins D1 and D2, and Hh genes PTCH1, GLI1 and GLI2 (Bai et al., 2002, Grachtchouk et al., 2011, Ng and Curran, 2011). GLI2 is the initial and main effector of the Hh pathway and directly promotes transcription and activation of GLI1 who, in a positive feedback loop, promotes the transcription of GLI2 and itself (Ikram et al., 2004, Regl et al., 2002).

In-situ hybridization experiments have shown that GLI2 is expressed in the interfollicular epidermis and hair follicles in both normal skin and BCC (Ikram et al., 2004). Furthermore, the overexpression of Shh, Gli1, Gli2, or an active form of Gli2 in epidermis from transgenic mice shows that elevated levels of these proteins are capable of inducing BCC-like tumors in mice (Grachtchouk et al., 2011, Grachtchouk et al., 2000, Dahmane et al., 2001). These two observations suggest an important role for GLI2 in the regulation of the Hh pathway in epithelial growth in both normal and oncogenic states.Although elevated expression of GLI genes has been observed in BCCs, their amplification or activating mutations, have not been found to be associated to the progression of this cancer type.

55 of 299 2.4.2. The TP53 gene in BCC

The cellular tumor antigen p53 (TP53) gene, the most mutated gene in human cancers (Vogelstein et al., 2000), is involved in cell cycle regulation, acting as a negative activator of cell division and controlling genes involved in this process (uniprot.org/uniprot/ Last accessed 4.11.2015). It was discovered in the late 70’s and was categorized as an oncogene (Eliyahu et al., 1984, Jenkins et al., 1984) however, Baker and collaborators (Baker et al., 1989), when studying colorectal cancers with 17q deletions and the genes included in this segment, noted that a significant fraction of their tumors had an heterozygous 17q deletion spanning TP53. While the remaining allele was capable of producing p53 protein, they found that the allele also harbored a missense mutation that rendered the protein non-functional, putting TP53 forward as the first identified tumor suppressor gene (Vogelstein et al., 2000), based on the Knudson two-hit hypothesis.

Individuals with a germline TP53 inactivating mutation have Li-Fraumeni syndrome(Srivastava et al., 1990, Li and Fraumeni, 1969), a disease characterized by an increased risk of developing several cancer types at an early age (Kamihara et al., 2014).

Furthermore, p53 knock-out mice also develop a large number of tumors at an earlier age (Donehower et al., 1992), showing that the absence of an active copy of the gene (tumor suppressor scenario) is what usually renders a cell oncogenic and not necessarily the presence of a mutant copy (oncogene scenario). There has been however, work on the putative oncogenic function of TP53 mutations and some specific ones, which can account for a significant fraction of all missense mutations in TP53 (about 30%), have a behavior consistent with a gain of function profile (Olive et al., 2004).

Damaging mutations in TP53 are frequently found in non-cancerous skin (Ling et al., 2001, Giglia-Mari and Sarasin, 2003). Immunohistochemistry (IHC) (Jonason et al., 1996) and next-generation sequencing (NGS) studies (Martincorena et al., 2015) estimate that around 3-5% of sun-exposed skin cells carry a TP53 mutation. Interestingly, the average size of clones with a TP53 mutation in normal skin (0.33 mm²) is significantly bigger (q=0.009) than clones in the same regions harboring neutral mutations (0.15 mm²), suggesting that TP53 mutations do confer a growth advantage to carrier cells, even when a tumor has not yet developed (Martincorena et al., 2015).

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The TP53 gene is frequently found mutated in BCCs, and it is estimated that between 40-70% of the tumors harbor inactivating events in this gene (Reifenberger et al., 2005, Ling et al., 2001). Inactivation of TP53 through LOH has been reported to occur in BCCs but to a lesser extent (8% of cases)(Reifenberger et al., 2005, Ziegler et al., 1993), and biallelic point mutations are often the mechanism of inactivation (Ziegler et al., 1993, Ponten et al., 1997, Ling et al., 2001). The TP53 mutational status has been evaluated in 594 BCCs present in the COSMIC database, and 39% of them have at least one TP53 mutation, the majority of which are predicted to have a deleterious effect on the protein product (Table 3, www.cancer.sanger.ac.uk Last accessed 4.11.2015).

Giglia-Mari and collaborators (Giglia-Mari and Sarasin, 2003), when analyzing the distribution of TP53 mutations in BCC and other skin cancers from the Institut Curie p53 database (http://p53.fr/), identified aminoacid 248 to be frequently mutated in all the skin cancers studied (BCC, SCC and melanoma). There were as well, three BCC-specific hotspots (codons 177, 196 and 245) (Figure 16). Aminoacid 177, known to be slowly repaired after UV irradiation (Tornaletti and Pfeifer, 1994), is frequently mutated in BCC, while the other two codons are also frequently mutated in breast, colon, lung and head and neck malignancies, among others. Codon 245 is mutated in some cases of Li-Faumeni syndrome as well.

Figure 16. TP53 mutations in BCC. Distribution of mutations in the p53 protein in BCCs.

Only mutational hotspots are labeled, with their position and the codon sequence.

Reproduced from Giglia-Mari et al, 2003.

57 of 299 2.5. Mutational signatures in BCC

Although the genome-wide mutational signatures of BCC have not been studied until very recently (see Results and Discussion sections), the signatures on the main BCC driver genes have been known for some time. For example, in BCCs as well as in other non-melanoma skin cancers (NMSC), TP53 is known to have a clear UV-signature, with most mutations being C>T transitions in a pyrimidine context (Dumaz et al., 1993, Ziegler et al., 1994, Bodak et al., 1999). This profile is indistinguishable from that observed in SCC, also characterized by a strong UV-light signature, while melanoma seems to have a higher frequency of A>G mutations that are not caused by UV-light and are probably the consequence of reactive oxygen species (ROE). When the mutational signatures of TP53 in melanoma and NMSC were compared, the differences were statistically significant (p<5x10

-3), while in the case of BCC and SCC, no significant differences were observed (p<0.3, Figure 17a)(Giglia-Mari and Sarasin, 2003).

On the other hand the UV-light signature in PTCH1 for sporadic BCCs is less strong than that observed for TP53 (Figure 17b). Only 50% of the mutations are of the C>T/CC>TT type (p=1.2x10^-8). In Gorlin syndrome cases, the fraction is reduced to 13% (Lindstrom et al., 2006).

Figure 17. Mutational signature in BCC. a. Mutational signature for TP53 in BCC= skin basal cell carcinoma, SCC= squamous cell carcinoma of the skin, and melanoma. Redrawn with modification from Giglia-Mari et al, 2003. b. Mutational signature for PTCH1 in sporadic and Gorlin syndrome patients. Drawn with information from Lindstrom et al., 2006.

a b

58 of 299 2.6. BCC treatment and management options

Most tumors are localized and small when detected, and they are usually removed surgically in an ambulatory procedure. The most traditional surgery is an excision with margin evaluation. After excision, microscopic examination of part of the border takes place to verify the full inclusion of the tumor in the excisate (NIH-BCC treatment, 2015). A very popular alternative for cosmetically sensitive areas is Mohs micrographic surgery. In this procedure, the narrowest margins possible are taken. The tumor is microscopically delineated and serial radial resections are made, with each one being evaluated by microscopy, until all the tumoral cells are excised (NIH-BCC treatment, 2015, Smeets et al., 2004).

Radiation therapy can be used in patients that would otherwise require extensive surgery or when the tumors are in areas with small amount of tissue, such as the nose or ear. It is also used in lesions that recur after surgical removal but should be avoided in patients with diseases predisposing to skin cancer, such as Gorlin syndrome and xeroderma pigmentosa (Caccialanza et al., 2003, Fellner, 2012). Cryosurgery, electrodessication or topical fluorouracil are unusual treatments but may be used in superficial lesions or when other methods are contraindicated (NIH-BCC treatment, 2015).

Advanced or metastatic BCC cannot be treated with the aforementioned methods and affected patients usually die within 8 months (Fellner, 2012). For these cases, the FDA approved at the beginning of 2012 vismodegib (Erivedge, developed by Genentech), a drug

Advanced or metastatic BCC cannot be treated with the aforementioned methods and affected patients usually die within 8 months (Fellner, 2012). For these cases, the FDA approved at the beginning of 2012 vismodegib (Erivedge, developed by Genentech), a drug