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[Cancer Research 64, 3559-3565, May 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Functional Analysis of Novel Sonic Hedgehog Gene Mutations Identified in Basal Cell Carcinomas from Xeroderma Pigmentosum Patients

Sophie Couvé-Privat1, Marc Le Bret3, Elisabeth Traiffort4, Sophie Queille1, Josée Coulombe4, Bakar Bouadjar5, Marie Françoise Avril2, Martial Ruat4, Alain Sarasin1 and Leela Daya-Grosjean1

1 Laboratoire Instabilité Génétique et Cancer, UPR2169 Centre National de la Recherche Scientifique, and 2 Département de Dermatologie, Institut Gustave Roussy, Villejuif Cedex, France; 3 Laboratoire de Biotechnologies et Pharmacologie Génétique Appliquée, UMR8113 Centre National de la Recherche Scientifique, Ecole Normale Supérieure Cachan, Cachan Cedex, France; 4 Institut de Neurobiologie Alfred Fessard, Institut Fédératif de Recherche 2118 and Laboratoire de Neurobiologie Cellulaire et Moleculaire, UPR 9040 Centre National de la Recherche Scientifique, Gif-sur-Yvette, France; and 5 Department of Dermato-Venerology, Centre Hospitalo-Universitaire, de Bab El Oued, Alger, Algeria


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Altered sonic hedgehog (SHH) signaling is crucial in the development of basal cell carcinomas (BCC), the most common human cancer. Mutations in SHH signal transducers, PATCHED and SMOOTHENED, have already been identified, but SHH mutations are extremely rare; only 1 was detected in 74 sporadic BCCs. We present data showing unique SHH mutations in BCCs from repair-deficient, skin cancer-prone xeroderma pigmentosum (XP) patients, which are characterized by high levels of UV-specific mutations in key genes involved in skin carcinogenesis, including PATCHED and SMOOTHENED. Thus, 6 UV-specific SHH mutations were detected in 5 of 33 XP BCCs. These missense SHH alterations are not activating mutations for its postulated proto-oncogene function, as the mutant SHH proteins do not show transforming activity and induce differentiation or stimulate proliferation to the same level as the wild-type protein. Structural modeling studies of the 4 proteins altered at the surface residues, G57S, G64K, D147N, and R155C, show that they do not effect the protein conformation. Interestingly, they are all located on one face of the compact SHH protein suggesting that they may have altered affinity for different partners, which may be important in altering other functions. Additional functional analysis of the SHH mutations found in vivo in XP BCCs will help shed light on the role of SHH in skin carcinogenesis. In conclusion, we report for the first time, significant levels of SHH mutations found only in XP BCCs and none in squamous cell carcinomas, indicating their importance in the specific development of BCCs.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hedgehog, first identified in Drosophila, belongs to a family of vertebrate morphogens that include Sonic, Desert, and Indian hedgehog, which regulate different events in embryonic differentiation and development (1 , 2) . The sonic hedgehog (SHH) gene encodes a ~45 kDa protein, which undergoes autocatalytic cleavage yielding an active ~20 kDa NH2-terminal fragment covalently bound to cholesterol and palmitoyl moieties. These lipid modifications are found to enhance the activity of SHHN proteins (3 , 4) . The cholesterol moiety probably allows the appropriate spatial distribution and also limits the diffusion of the secreted SHHN that remains cell associated.

Patched (Ptch), a multipass transmembrane protein, is the receptor for all of the hedgehog ligands (5 , 6) . In mammalians, at least two Ptch proteins, Ptch and Ptch2, are found with similar affinity for Shh (7) . The unbound Ptch receptor inhibits Smoothened (Smo), a G protein coupled-like receptor (8) . It is suggested that Ptch may inhibit Smo activity indirectly, via changes in distribution or concentration of endogenous small molecules (9) . Shh binding to Ptch releases the inhibition of Smo and initiates the signaling cascade activating several important genes including members of the transforming growth factor ß, Gli and Wnt family of proteins, as well as Ptch itself (10) . Recently identified, the Hedgehog-interacting protein (Hip) can attenuate Shh signaling due to its high binding affinity for Shh (11 , 12) .

Aberrant SHH signaling is involved in the pathogenesis of two human diseases, Holoprosencephaly and the nevoid basal cell carcinoma (BCC) syndrome (13, 14, 15) . In nevoid BCC patients, altered SHH signaling is caused by germinal inactivating mutations of the PTCH gene resulting in developmental abnormalities and a predisposition to BCCs (14 , 15) . Somatic loss-of-function mutations of the tumor suppressor PTCH gene are also detected in sporadic BCCs, 50% of them being UV-specific mutations (16, 17, 18, 19, 20, 21) . Moreover, as could be expected by the antagonistic roles played by PTCH and SMO, some BCCs present activating mutations of the SMO gene (22 , 23) . To date, among several studies looking for SHH alterations, only one sporadic BCC in 74 BCCs, has been found to have a potential gain of function mutation, namely H133Y (23 , 24) . Another study, looking only for this mutation in 36 BCCs, found no alterations (25) . Thus, a role for SHH in human skin tumorigenesis has yet to be firmly established but seems likely as mice overexpressing SHH develop BCC-like skin cancers as well as many developmental anomalies (24) .

In the present study, we have analyzed for SHH alterations in BCCs and SCCs from xeroderma pigmentosum (XP) patients characterized by a defect in nucleotide excision repair resulting in a hyperphotosensitivity and a high incidence of skin cancers. We have already observed that XP skin tumors have higher levels of UV-induced mutations in key genes involved in skin carcinogenesis such as ras, p53, p16INK4a, and p14ARF, as well as in two SHH partners, PTCH and SMO, compared with sporadic skin cancers (26, 27, 28, 29, 30) . For the first time, we report data showing significant levels of SHH mutations found only in XP BCCs, none being detected in XP SCCs. Our findings indicate the importance of altered SHH function in the specific development of BCCs. We also present a structural and functional analysis of the SHH mutations found in vivo in XP BCCs and the unique sporadic BCC mutation, which will help elucidate the role of SHH in skin carcinogenesis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Samples and DNA Extraction.
Fourteen SCCs and 33 BCCs of 30 XP patients were obtained from different sources in France and North Africa. Among the 30 XP patients, 4 have been classified as belonging to the XP group C complementation group and 1 as an XP variant; the remaining patients are unclassified. Biopsies were snap-frozen and stored at –80°C. DNA was isolated as described previously (29) .

Single-Strand Conformational Polymorphism Analysis.
The DNA was amplified using the primers described by Oro et al. (24) for exons 1 and 2, and by Roessler et al. (13) for exon 3. Amplified products were analyzed at least twice for aberrantly migrating sequences in 0.5x mutation detection enhancement gels (FMC Bioproducts, Rockland, ME), with or without 10% glycerol, by electrophoresis at 6–10 W for 16–22 h at room temperature. The single-strand conformational polymorphism variant bands were eluted, amplified, purified, and sequenced with the Thermosequenase kit (Amersham Biosciences, Saclay, France).

Modeling.
The initial structure was taken from the murine SHH (Protein Data Bank entry: 1vhh; Ref. 31 ). The residue numbers were decremented by 1, and the atoms common to both threonine and serine were conserved at S67. Protons HE2 and HD1 in histidine residues 140 and 182 were suppressed to make them neutral and suppress clashes with the Zn2+ cation. Other point mutations have been generated by conserving as many atoms as possible, the missing atoms being automatically set by the AMBER program (32) . Structures were minimized with our quasi-Newtonian minimizer MORMIN (33) , using the AMBER force field (34) . The Zn cation was modeled by a sphere bearing the charge +2. Using such a force field, the Zn ligands had a planar configuration. To reproduce the tetragonal ligand conformation, we added a distance constraint between Zn and each of its four ligands (equilibrium distance 2.05 Å, force constant 50 kcal.Å–2) and angle constraints (equilibrium: 109.47 degrees, constant: 10 kcal.degrees–2). In vacuo molecular dynamics, the protein was stripped from its 118 water molecules, and the environment was simulated by a dielectric constant depending linearly with the distance separating the charges (e = 4r). In aqua molecular dynamics, the protein with its 118 bound water molecules has been embedded in a cubic box 54 x 54 x 54 Å3 in size filled with 3610 TIP3P water molecules, in periodic boundary conditions under a constant pressure of 1 atm and at a temperature of 300° K. The bound water molecules were then renamed to be undistinguishable from the solvent. Dynamics simulations were carried out with the AMBER SANDER module, and electrostatic interactions were calculated with the particle-mesh technique for Ewald sums with a 10 Å cutoff. Before production, the system was submitted to heating and cooling (35) .

Prokaryote Expression Vectors of SHH Mutant Proteins.
The point mutations of the altered SHH proteins were incorporated by site-directed mutagenesis into the wild-type SHH cDNA cloned in the pBS SK+ vector (pBS-SHH) following the manufacturer’s recommended protocol (Stratagene, Montigny le Bretonneux, France). Standard methods were used to clone the cDNA coding for the NH2-terminal domain of the wild-type and altered SHH proteins into a glutathione S-transferase gene fusion expression vector pGEX4T1 (Amersham Biosciences; Ref. 36 ). Thus, insertion of a 5' BamHI and a 3' EcoRI site, flanking the SHHN open reading frame (Cys-24 to Gly-197), allowed subcloning of the different mutations into the pGEX4T1 prokaryote expression vector, which were verified by restriction analysis and DNA sequencing. Competent Escheria coli strains were transformed with the expression vectors, and purification of the different SHHN proteins was carried out following the protocol described by Day et al. (36) for the murine SHHN protein. The purified SHHN wild-type and mutant proteins were quantified by Bradford assay (Bio-Rad, Marnes La Coquette, France).

Eukaryotic Expression Vectors and SHH Expression in Mammalian Cells.
Using the pBS-SHH vector, a 1618 bp HindIII-XbaI fragment, corresponding to the full length SHH cDNA, carrying the wild-type, H133Y, D147N, and R155C mutated sequences, was isolated and subcloned into a 5427 bp HindIII-XbaI fragment of the eukaryote expression vector, pcDNA3.1 (Invitrogen, Cergy Pontoise, France). A 532 bp HindIII-BstEII fragment carrying the G57S and G64K mutations from the pBS-SHH plasmid were subcloned into a 6513 bp HindIII-BstEII fragment of the pcDNA3.1 vector carrying the full-length SHH cDNA. Vector construction fidelity was verified by restriction analysis and sequencing.

Protein Expression in Mammalian Cells.
Mouse NIH 3T3 cells or human HEK 293 cells were transfected with the different SHH-pcDNA3.1 vectors. Stable transfectants secreting the SHH proteins were isolated using G418 selection. NIH 3T3 cells were used as feeder cells in the human keratinocyte proliferation assay. Conditioned medium from HEK 293 cells was collected from exponentially growing stable transfectants, filtered, and SHH protein concentrations quantified by Western analysis using the polyclonal 167Ab serum described previously (37) . Increasing concentrations of SHH protein were tested in the differentiation assay.

Proliferation of Cerebellar Granule Cell Precursors.
Cerebellar granule cell precursors were isolated according to the procedures described previously (38) with slight modifications. Briefly, cerebellar from P8 rats (Wistar) were removed, cut into small pieces, placed in Krebs-Ringer buffer (120 nM NaCl, 5 nM KCl, 1 mM KH2PO4, 25 mM NaHCO3, 15 mM glucose, and 0.04 mM phenol red) and digested with 250 µg/ml trypsin for 15 min at room temperature. The enzymatic digestion was stopped by addition of an equal volume of Krebs-Ringer buffer containing 250 µg/ml trypsin inhibitor in the presence of 80 µg/ml DNase (Sigma Aldrich, Saint Quentin Fallavier, France). The tissue was centrifuged (100 x g; 10 s), and the resulting pellet was resuspended using pipettes of decreasing pore size to obtain a single cell suspension. This suspension was centrifuged (200 x g; 5 min), and the resulting pellet was resuspended in Neurobasal Medium (Invitrogen) supplemented with 1 mM pyruvate, 2 mM L-glutamine, penicillin/streptomycin, and N2 supplement (Invitrogen), 60 µg/ml N-acetyl cysteine, and 100 µg/ml bovine serum albumin. Cerebellar cells were plated into 96-well plates at a density of 2 x 105 cells/well, and the recombinant human wild-type and mutant SHHN proteins (30 and 100 nM) were added immediately. All of the samples were tested in quadruplicates. The cells were cultured for 48 h at 37°C under 5% CO2, pulse labeled with [3H]thymidine (methyl, 1', 2'-[3H]thymidine; 110 Ci/mmol; Amersham Biosciences) for 12 h and harvested at 60 h onto filters using an automated cell harvester (Brandel). [3H]thymidine incorporation was quantified with a ß liquid scintillation counter (Wallac; Perkin-Elmer Life Science, Courtaboeuf, France).

Keratinocyte Proliferation Assay.
Primary foreskin keratinocyte cultures were grown on feeder layers of control NIH 3T3 cells containing the empty vector or stable transfectants, secreting the SHH wild-type or mutant proteins. The different NIH 3T3 cells were lethally irradiated with {gamma} ray (60 Gy) and seeded at a density of 4 x 105 cells/60 mm plate in Green medium, incubated for 12–16 h and inoculated with 5000 cells/plate of early passage foreskin keratinocytes. The medium was changed twice a week, and cells were fixed with 3.7% formaldehyde in PBS, stained with 1% rhodamine B at 10, 14, 17, and 21 days after plating, and keratinocyte colonies were compared (39) .

Cell Differentiation Assay.
The various SHH proteins were tested for their ability to induce differentiation of the mesenchymal C3H10T1/2 cells to an osteoblast lineage using the expression of alkaline phosphatase as a marker of differentiation (40) . Conditioned medium from HEK 293 cells expressing different SHH proteins were added at different concentrations to C3H10T1/2 cells cultured in 96-well plates. Alkaline phosphatase activity was measured after 5 days at 37°C using the p-nitrophenyl phosphate substrate (Sigma). RNA extraction was carried out using the Rneasy kit (Qiagen, Courtaboeuf, France). Ptch transcription was analyzed by reverse transcription-PCR as described by Outram et al. (41) .

Transformation Assay.
208F Fischer rat embryo fibroblasts were transfected with 10 µg of SHH wild-type and mutant protein expression vectors or a control empty vector using the calcium phosphate coprecipitation method. A positive control was carried out using oncogenic SV40 DNA. After transfection (24 h), cells were plated at a density of 106 cells/100 mm dish in DMEM containing 10% FCS. When cultures were confluent, cells were refed regularly with DMEM containing 5% FCS and 1 µM dexamethasone. Cultures were stained with Giemsa and foci of transformed cells scored ~2 weeks after transfection.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The SHH Gene Is Mutated in BCC from XP Patients.
We have studied the involvement of SHH in skin carcinogenesis by analyzing skin tumors from XP patients. BCCs and SCCs were screened for SHH alterations in all three exons of the SHH gene by single-strand conformational polymorphism followed by sequencing of bands showing altered mobility. We did not detect any SHH gene modifications in the 14 SCC samples, whereas we found 6 (18%) mutations in 5 among 33 XP BCCs (Table 1)Citation . All of the mutations are UV-specific, C->T transitions or CC->TT tandem substitutions. Four alterations are missense mutations (12%) in exons 1 and 2, in highly conserved codons, 57 and 64 in exon 1, and 147 and 155 in exon 2 at the NH2-terminal domain of the SHH protein (Fig. 1A)Citation . One silent mutation was found in exon 3, and a base substitution was detected in intron 1 near the junction to exon 1.


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  		Table 1 Mutations of SHH gene in BCCsa from patients with XP

 


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  		Fig. 1. A, localization of the missense sonic hedgehog mutations. The hatched section at the NH2-terminal domain indicates the signal peptide. B, space filling models of the fragment K38-K194 of the human sonic hedgehog NH2-terminal domain viewed from opposite directions. The color highlights: red, the five mutations; green, the sulfate anion; blue, the zinc ion.

 
Localization of Mutated Residues in the SHH Protein.
We next used protein modeling to evaluate the effect of these mutations on SHH protein conformation. A 1 ns molecular dynamics in aqua was first performed for the G64K protein. As the amino acid neighbors of G57S, G64K, H133Y, D147N, and R155C remained the same during the simulation and the same as in the wild-type protein, we turned to dynamics in vacuo, hoping to observe more rapid changes. The dynamics were simulated for each protein during 600 ps in vacuo, but no significant changes could be observed in this globular rigid protein. For instance, the side chain of R155C moved farther from the sulfate ion, which is found in the crystal structure (31) , but the backbone remained unchanged. As the sulfate anion is held by three arginine residues, R123, R153, and R155, it was suppressed in further 600 ps dynamics in vacuo. The same overall stability was observed for each protein. Only the motion amplitudes of the three arginines increased. The role of the sulfate anion is intriguing. In our simulations it could have escaped far from the protein, but this was never observed showing the great stability of the ionic complex. In fact, dynamic simulation without the sulfate ion present did not significantly modify the structure observed in its presence. In conclusion, molecular dynamic simulations indicate that the five mutations do not introduce large structural changes, possibly because the mutated residues lie at the surface of the protein. Furthermore, as shown in Fig. 1BCitation , all of the observed mutations lie on the same side of the protein indicating that the mutations could modify the interaction of SHH with different binding proteins.

Influence of Mutations on SHH Activity.
The mutation analysis in XP BCCs has permitted us to characterize four novel modified SHHN proteins. Apart from the H133Y modification described by Oro et al. (24) , no other altered SHH proteins have been characterized, and it was interesting to analyze the activity of these mutant proteins found in XP BCCs. The missense mutations were reproduced by site-directed mutagenesis and cloned into prokaryote and eukaryote expression vectors.

We first tested the wild-type and mutant G57S, G64K, D147N, and R155C NH2-terminal SHH protein moiety (C24 extending to G197) expressed in E. coli transformed with the different pGEX4T1 vector constructs (42) . When we tested the effect of SHHN on proliferation of primary cultures of cerebellar granule cell precursors (43, 44, 45) we found that in with 30 and 100 nM of wild-type SHHN there was a 5- and 10-fold increase in [3H]thymidine incorporation, respectively (data not shown). The activity of the G57S and G64K mutants was not significantly different from that of the wild-type protein. However, the activity of D147N and the R155C was reduced significantly (70% and 80%, respectively) when compared with the wild-type protein (Fig. 2)Citation . We also analyzed the proteins in the granule cell precursor culture medium by Western analysis at different times during the proliferation assay and found that all of the proteins were fairly stable except for SHHN-D147N, which is highly degraded after 36 h and completely degraded after 60 h of incubation compared with the other SHH proteins (data not shown).



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  		Fig. 2. Modulation of [3H]thymidine incorporation induced by wild-type or mutant human SHHN peptides in primary cultures of rat cerebellar granule cells. The values reported were determined from three to four independent primary cultures of cerebellar granule cells. Student’s t test has been performed between each mutant SHHN peptide and the wild-type SHHN peptide for each tested concentration. *, P < 0.01; **, P < 0.001; bars, ±SD.

 
To further analyze the mitogenic effect of SHH proteins, we carried out a test on the proliferation of foreskin keratinocyte cultures (39) . For this assay, stable NIH 3T3 transfectants were established after transfection with the eukaryote expression vector pcDNA3.1 vector carrying the different full-length SHH cDNAs. In parallel, we also analyzed the functional activity of the H133Y protein found mutated in a non-XP sporadic BCC by Oro et al. (24) . The secreted NH2-terminal moiety of the SHH proteins expressed by the stable NIH 3T3 transfectants, which undergo normal post-translational lipid modifications, were quantified by Western blotting. All of the wild-type and mutant SHH proteins were found secreted in the medium except for the D147N protein, which was found to be unstable like the SHHN D147N protein expressed in bacteria. Nevertheless, all of the NIH 3T3 stable transfectants were used as feeder layers for the culture of foreskin keratinocytes. Ten days after keratinocyte inoculation we found little difference between the cultures examined (Fig. 3)Citation but after 14 days, keratinocyte cultures on SHH producing feeders were remarkably different from those on control feeders without SHH. The keratinocyte colonies growing on feeders producing wild-type or mutant SHH proteins, including the unstable D147N protein, were larger and more abundant than the colonies observed on control (empty vector) feeder cells (Fig. 3)Citation .



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  		Fig. 3. Sonic hedgehog (SHH) stimulation of human keratinocyte proliferation. The keratinocytes were grown on NIH 3T3 feeder cells expressing wild-type (WT) or mutant SHH proteins or feeders transfected with empty vectors. Cultures were fixed and stained at 10, 14, 17, and 21 days after keratinocyte inoculation.

 
Next we found that increasing quantities of the purified SHHN proteins expressed in E. coli showed very little activity in inducing differentiation of C3H10T1/2 cells to an osteoblast lineage. To test whether this was due to the lack of lipid modifications normally found in SHH expressed in mammalian cells, we repeated the test using SHH proteins expressed by stable transfectants of HEK293 cells carrying the full-length SHH wild-type and mutant SHH cDNAs. Thus, equal amounts of SHH proteins found in the conditioned medium of HEK293 cells were tested on C3H10T1/2 cells except for the D147N protein, which again was not detectable. In fact, degraded protein intermediates found in the D147N-HEK293 cell extracts confirmed that degradation must occur before the SHHN moiety could be secreted (data not shown). Thus, we found that the G57S, G64K, as well as the H133Y mutant protein characterized in a sporadic non-XP BCC allowed differentiation of C3H10T1/2 cells with activities comparable with wild-type SHHN protein (Fig. 4)Citation ; a linear increase in differentiation up to 20 nM protein concentration is observed. However, the activity of R155C was lower because when tested at concentrations up to 60 nM it only induced 50% of the differentiation seen with the other SHHN proteins. To confirm that the differentiation observed here was via signaling of the SHH pathway, we also analyzed for modulation of transcription of the Ptch gene. We found that the SHH proteins induced differentiation by up-regulation of the Ptch gene as observed by the presence of increased levels of patched transcripts in C3H10T1/2 cells treated with SHH proteins (Fig. 5Citation ; Ref. 42 ).



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  		Fig. 4. Sonic hedgehog (SHH) induced differentiation of C3H10T1/2 cells. Shh proteins were incubated with the cells for 5 days and the resulting levels of alkaline phosphatase activity measured at 405 nm using the AP chromogenic substrate p-nitrophenyl phosphate. The results are expressed as mean (n >= 3); bars, ±SD.

 


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  		Fig. 5. PTCH expression in C3H10T1/2 cells. Cells were incubated with conditioned medium from HEK stable transfectants expressing different SHH proteins or transfected with control empty vector. Total RNA was analyzed for Ptch transcription 48 h after incubation. RNA from untreated control C3H10T1/2 cells was also analyzed. Levels of Ptch transcription were compared with transcription levels of the household GAPDH gene.

 
Finally, we tested for oncogenic properties of the mutant SHH proteins by testing for their ability to transform 208F rat embryo fibroblasts (46) . These cells have a regular flat morphology forming even monolayers and provide a good assay for oncogenic transformation. In this test we found that only a vector carrying SV40 large T-antigen DNA, known to be highly oncogenic, induced transformed foci in the 208F cells. All of the vectors carrying different mutant or wild-type SHH cDNA failed to induce transformation in this test as attested by the absence of foci (data not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our present study is the first to show the potential importance of the SHH protein in the specific formation of basal cell carcinomas by the detection of 6 SHH mutations in 5 BCCs among 33 from XP patients. To date only 1 SHH mutation has been identified in 74 sporadic BCCs analyzing all 3 exons of the SHH gene (23 , 24) . All of the SHH mutations are UV-specific occurring at bipyrimidic sequences, which are targets for UV-induced DNA lesions, and half of them are the UV signature tandem CC->TT transitions characteristic of the nucleotide excision repair-deficient XP patients. The hypermutability of XP cells by UV is correlated to the high predisposition to cutaneous cancers. Indeed, previous studies from our laboratory have already shown that XP tumors have significantly higher levels of UV-induced mutations in both proto-oncogenes (ras and SMO) and tumor suppressor genes (p53 p16INK4a, p14ARF, and PTCH) than the same types of tumors from the normal population (28, 29, 30 , 47) . Interestingly, all of the SHH mutations we detected are found together with alterations of either PTCH or SMO, members of the SHH pathway (Table 1)Citation . This could be due to clonal expansion of cells harboring the different mutated genes or that cells require modification of several key genes for tumor progression.

The SHH gene has already been paid particular attention in a number of studies (23 , 25 , 48) . Only Oro et al. (24) have detected a SHH alteration H133Y, in 1 of 43 sporadic BCCs as well as in a medulloblastoma and a breast carcinoma. This unique exon 2 mutation (gcCa397gcTa) is also a C->T transition located at a bipyrimidine sequence but is probably not a hot spot for UV-induced lesions in the SHH gene as it was not found in the XP BCCs and was identified in internal cancers not related with UV exposure.

All of the mutations we have identified here are located in the NH2-terminal domain of SHH known to retain the signaling activities of the protein, whereas the COOH-terminal domain is responsible for the intramolecular precursor processing (3 , 49 , 50) . The importance of SHH signaling in human development became evident by the discovery that germ-line mutations of the SHH gene are the cause of the Holoprosencephaly syndrome presenting forebrain malformation associated with mental retardation and craniofacial anomalies (13) . None of our mutations are identified in Holoprosencephaly where a variety of alterations are found.

This study is the first to identify several SHH mutants existing in skin cancers, and it was important to carry out a structural and functional analysis of the XP BCC SHH mutant proteins (Table 2)Citation . Our first approach was to analyze the biological activity of the SHHN moiety known to induce proliferation of cerebellar granule cell precursors (43, 44, 45) . We found that the prokaryote expressed G57S and G64K SHHN proteins were as active as wild-type protein, whereas the R155C and D147N proteins did not induce proliferation of the cerebellar granule cell precursors. However, all of the mutant proteins, including the D147N mutant, when expressed in the mammalian NIH 3T3 cells used as feeder cells, were like the wild-type SHH protein in enhancing keratinocyte proliferation. Thus, SHH activity depends on the lipid modifications of the SHHN protein in which a cholesterol molecule is covalently attached to the COOH-terminal glycine, and a palmitoyl moiety is found at the NH2-terminal cysteine. Indeed, the SHHN proteins expressed in bacteria do not have the lipid tethers covalently attached to its COOH terminus because of the absence of the SHH COOH-terminal autoprocessing domain in the prokaryote vector. Thus, our results clearly show the importance of normal maturation of the SHH protein to exhibit full biological activity.


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  		Table 2 Functional analysis of SHH proteins: summary of results

 
In our analysis of 33 XP BCCs, we have characterized 2 alterations, G57S and G64K, located in exon 1 and 2 alterations in exon 2, D147N and R155C of the SHH gene. Interestingly, these in vivo modifications are located in the vicinity of specific residues analyzed in vitro in other studies. Using a mapping strategy, Pepinsky et al. (51) demonstrated that the N50 and S156 surface residues must be in close proximity to the Ptch-binding site, and in vitro altered residues N50C and S156C in the protein resulted in a loss of Ptch binding activity whereas a N69C mutant protein behaved like wild-type protein. Fuse et al. (52) , looking for a putative catalytic hydrolase domain in SHHN, also altered a number of surface residues (N51A, V52A, T56A, and E168A) in the same protein, which behaved like wild-type protein in inducing ventral neural cell types or in suppressing dorsal markers in neural plate explants. The three functional tests we carried out show that the two exon 1 mutant proteins, G57S and G64K, also have activities comparable with the wild-type SHH protein.

In exon 2, the altered D147N residue we detect is situated at the zinc coordinating sites in the NH2 terminus of the SHH protein, a domain showing homology with a zinc-dependent hydrolase. It is still unclear whether this protein domain is implicated in the postulated hydrolase activity or if it is required for stabilizing the folded protein structure (36) . Day et al. (36) analyzing the zinc coordinating residues of SHHN have shown that D147 and H140 are important for maintaining full zinc occupancy. Modifications of these residues to alanine, D147A and H140A, resulted in a substantial reduction in protein stability, which was undetectable at the end of the tests when assayed for C3H10T1/2 differentiation or in the neural plate explant assay, suggesting proteolytic degradation (36) . The mutated SHH protein we have detected in a BCC modifying the same residue, D147N, was also found to be unstable when expressed in bacterial or mammalian cells. Nevertheless, the D147N mutant protein expressed by NIH 3T3 feeder layers, presumably before degradation occurs, can enhance keratinocyte proliferation to the same extent as the wild-type SHH protein, confirming an important structural and functional role of this residue at the zinc binding site. Therefore, our data showing the presence of an alteration in the highly conserved zinc domain clearly indicate its importance in possible modifications of the functional properties of the SHH protein.

Interestingly, the other exon 2 mutation we have characterized modifies an arginine residue, R155, which, together with R123 and R125, seems to hold the sulfate anion integrated in the SHH crystal structure (31) , which may be important for stabilizing the SHH protein. The role of the sulfate ion is intriguing because, for example, one can imagine that in vivo, a phosphate anion could occupy the sulfate site. Two modifications in the near vicinity of the R155 residue were also previously analyzed independently by two groups (51 , 52) . In the analysis by Fuse et al. (52) , the murine Shh protein was altered at R154A and S157A, corresponding to human residues R153 and S156, and resulted in only a mild effect on Patched binding but showed a reduced signaling activity in the neural plate assay. Pepinsky et al. (51) altered the human 156 residue from serine to a cysteine and found it less able to differentiate C3H10T1/2 cells and with a weaker binding affinity for Ptch. The R155C protein we have characterized also shows a greatly reduced capacity for differentiation of C3H10T1/2 cells although it induces keratinocyte proliferation like the wild-type protein. Moreover, R155C showed no proliferative activity in cerebellar granule cell precursors, which may be due to differences in protein maturation, as discussed above.

Among the many SHH studies analyzing modified SHH residues published since 1997, little has been reported concerning the function of the SHH H133Y mutant protein, the first to be identified in a BCC. The murine H135 residue, which corresponds to human H134, was shown to stabilize a potential tetrahedral intermediate in the protein, and a mutant H135A protein was found to retain the capacity to bind Ptch and to induce neural floor plate formation (52) . Our results analyzing the modified human residue H133Y also show it to have an activity very similar to that of the wild-type SHH protein in both the differentiation of C3H10T1/2 cells and in the stimulation of keratinocyte proliferation.

Interestingly, all of our functional assays have tested for SHH activity involving the major SHH pathway partners PTCH, SMO, and GLI as confirmed by the up-regulation of Ptch transcripts we see in C3H10T1/2 cells treated with the different SHH proteins. Indeed, our structural analysis indicates that the mutations may be located at strategic domains of the SHH protein, which may not implicate PTCH binding. Surprisingly, although it has been postulated that SHH is a proto-oncogene, the mutated SHH proteins exhibited no transforming activity. However, the SHH protein is known to be involved in a variety of different functions, which may indirectly play a role in cancer progression. Thus, we wonder whether the clustered location of the altered surface residues found to be on one side of the SHH protein in our protein modeling study may affect the other functions via different interactions with various partners.

In conclusion, our study showing significant levels of SHH mutations in BCCs from XP patients clearly points to a role for deregulated SHH signaling in the specific genesis of basal cell carcinomas, none having been detected in XP squamous cell carcinomas. Moreover, mutation of either PTCH or SMO are found associated with each of the SHH mutations in our XP BCCs and suggest an additive effect of the oncogenic potential of these mutations resulting in BCC skin carcinogenesis.


   ACKNOWLEDGMENTS
 
We thank Dr. Jean Claude Ehrhart, (Laboratoire Instabilité Génétique et Cancer, UPR 2169, Institut Gustave Roussy, Villejuif, France) for helpful discussions and critical reading of the manuscript. The protein modeling was made possible through access to the SGI O2000 multiprocessor of the Pôle Parallélisme IdF Sud.


   FOOTNOTES
 
Grant support: Association de Recherche sur le Cancer (Villejuif, France), the Ligue Nationale Contre le Cancer (Créteil, France), and the Groupement des Enterprises Françaises dans la Lutte Contre le Cancer (Charenton, France).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Leela Daya-Grosjean, Laboratoire Instabilité Génétique et Cancer, UPR2169 Centre National de la Recherche Scientifique, Institut Gustave Roussy, 39, rue Camille 1 Desmoulins, 94805 Villejuif Cedex, France. Phone: 33-1-42-11-63-25; Fax: 33-1-42-11-50-08; E-mail: daya{at}igr.fr

Received 12/24/03. Revised 2/17/04. Accepted 3/ 8/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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