The sonic hedgehog receptor patched associates with caveolin-1 in cholesterol-rich microdomains of the plasma membrane.

The Hedgehog signaling pathway is involved in early embryonic patterning as well as in cancer; however, little is known about the subcellular localization of the Hedgehog receptor complex of Patched and Smoothened. Since Hh has been found in lipid rafts in Drosophila, we hypothesized that Patched and Smoothened might also be found in these cholesterol-rich microdomains. In this study, we demonstrate that both Smoothened and Patched are in caveolin-1-enriched/raft microdomains. Immunoprecipitation studies show that Patched specifically interacts with caveolin-1, whereas Smoothened does not. Fractionation studies show that Patched and caveolin-1 can be co-isolated from buoyant density fractions that represent caveolae/raft microdomains and that Patched and caveolin-1 co-localize by confocal microscopy. Glutathione S-transferase fusion protein experiments show that the interaction between Patched and caveolin-1 involves the caveolin-1 scaffolding domain and a Patched consensus binding site. Immunocytochemistry data and fractionation studies also show that Patched seems to be required for transport of Smoothened to the membrane. Depletion of plasmalemmal cholesterol influences the distribution of the Hh receptor complex in the caveolin-enriched/raft microdomains. These data suggest that caveolin-1 may be integral for sequestering the Hh receptor complex in these caveolin-enriched microdomains, which act as a scaffold for the interactions with the Hh protein.

Identification of PATCHED mutations in medulloblastomas by direct sequencing.

Medulloblastoma is the most common malignant embryonic tumors of the central nervous system. The nevoid basal cell carcinoma syndrome (NBCCS), which is caused by mutations of PTCH gene on chromosome 9q22, accounts for about 2% of all medulloblastomas. Previous studies of PTCH in sporadic medulloblastomas using single strand conformational polymorphism (SSCP) detected mutations in about 10% of the tumors. In this study, we directly sequenced the PTCH gene in 20 sporadic medulloblastoma DNA samples. A nonsense mutation (Q694X) and a splice site alteration (2875+1G>A) were identified in two of the samples. The mutations are predicted to result in a truncated PTCH protein and aberrant splicing, respectively. In both cases, only the mutant alleles were identified, indicating that the mutations were associated with loss of the wild-type PTCH allele in the tumor cells. Several novel variants, including 1653T>C, 1672C>T, and 2292C>T, were also found in these tumor samples. One of the two mutations detected in this study had been missed by SSCP, suggesting that the true rate of PTCH mutations in sporadic medulloblastomas may be underestimated by SSCP screening. Nevertheless, the frequency of mutations in this study did not differ from previous reports.

Developmental genes and cancer: role of patched in basal cell carcinoma of the skin.

Many genes originally identified because of their role in embryonic development are also important in postnatal control of cell growth and differentiation. Mutations in some of these genes have been shown to cause cancer. Basal cell carcinoma (BCC) of the skin is the most common cancer in humans. More than 750000 new cases are diagnosed annually, and the incidence is rising. BCCs are slow-growing, locally invasive tumors that rarely metastasize but can result in extensive morbidity through local recurrence and tissue destruction. Epidemiologic studies suggest that sunlight (particularly UVB radiation) is a strong risk factor for BCC formation, although other factors are also involved. The nevoid basal cell carcinoma syndrome (NBCCS), a rare genetic disorder, is characterized by predisposition to BCCs and other tumors as well as to a wide range of developmental defects. NBCCS maps to chromosome 9q22.3, and loss of heterozygosity at this site in both sporadic and hereditary BCCs suggests that it functions as a tumor suppressor. The gene for NBCCS was recently cloned and is the human homologue of the Drosophila gene "patched." Genetic studies in Drosophila show that patched is part of the hedgehog signaling pathway, which is important in determining embryonic patterning and cell fate in multiple structures of the developing embryo. Human patched is mutated in both hereditary and sporadic BCCs, and inactivation of this gene is probably a necessary, if not sufficient, step for BCC formation. Delineation of the biochemical pathway in which patched functions may lead to rational medical therapy for BCCs and possibly for other tumors associated with NBCCS.

Pulsed-field gel electrophoresis and FISH mapping of chromosome 9q22: placement of a novel zinc finger gene within the NBCCS and ESS1 region.

Chromosome 9q22 is a gene-rich region to which several human disease loci have been mapped. Pulsed field gel electrophoresis (PFGE) and FISH were used to determine the order of and distance between 12 chromosome 9q22 markers flanked by D9S196 and D9S180. D9S780 and XPA were within 190 kb of each other and hybridized to the same 460-kb NotI fragment as D9S180. ZNF169, a novel kruppel-type gene, and D9S280 shared several PFGE fragments indicating that they are not more than 300 kb apart. Interphase FISH showed that COL15A1 lies distal to the region bounded by D9S180 and D9S196 and that ZNF169 is adjacent to D9S196. Based on the restriction fragment lengths in this region and estimates from FISH, the distance from D9S180 to D9S196 is not less than 2 Mb.

Mutations in the human homologue of the Drosophila patched gene in Caucasian and African-American nevoid basal cell carcinoma syndrome patients.

The nevoid basal cell carcinoma syndrome (NBCCS), or Gorlin syndrome, is a multisystem autosomal dominant disorder. The salient features of this syndrome include multiple basal cell carcinomas, palmar and/or plantar pits, odontogenic keratocysts, skeletal and developmental anomalies, and ectopic calcification. Other features include such tumors as ovarian fibromas and medulloblastomas. There is extensive interfamilial as well as intrafamilial variability with respect to the manifestation and severity of the phenotype. Alterations in the human homologue (PTCH) of the Drosophila segment polarity gene patched have been identified in NBCCS patients as well as tumors associated with this syndrome. We report several mutations in this gene in NBCCS patients and present the clinical phenotypes of the individuals in whom these mutations were identified.

The role of the human homologue of Drosophila patched in sporadic basal cell carcinomas.

Basal cell carcinoma (BCC) is the most common cancer in humans. The majority of sporadic BCCs have allele loss on chromosome 9q22 implying that inactivation of a tumour suppressor in this region is an important step in BCC formation. The gene for nevoid basal cell carcinoma syndrome (NBCCS), an autosomal dominant disorder characterized by multiple BCCs, maps to the same region and is presumed to be the tumour suppressor inactivated at this site. NBCCS has been identified recently and encodes a protein with strong homology to the Drosophila segment polarity gene, patched. Analysis of Drosophila mutants indicates that patched interacts with the hedgehog signalling pathway, repressing the expression of various hedgehog target genes including wingless, decapentaplegic and patched itself. Using single strand conformational polymorphism (SSCP) to screen human patched in 37 sporadic BCCs, we detected mutations in one-third of the tumours. Direct sequencing of two BCCs without SSCP variants revealed mutations in those tumours as well suggesting that inactivation of patched is probably a necessary step in BCC development. Northern blots and RNA in situ hybridization showed that patched is expressed at high levels in tumour cells but not normal skin suggesting that mutational inactivation of the gene leads to overexpression of mutant transcript owing to failure of a negative feedback mechanism.

Molecular analysis of chromosome 9q deletions in two Gorlin syndrome patients.

Gorlin syndrome is an autosomal dominant disorder characterized by multiple basal cell carcinomas, medulloblastomas, ovarian fibromas, and a variety of developmental defects. All affected individuals share certain key features, but there is significant phenotypic variability within and among kindreds with respect to malformations. The gene (NBCCS) maps to chromosome 9q22, and allelic loss at this location is common in tumors from Gorlin syndrome patients. Two recessive cancer-predisposition syndromes, xeroderma pigmentosum group A (XPAC) and Fanconi anemia group C (FACC), map to the NBCCS region; and unusual, dominant mutations in these genes have been proposed as the cause of Gorlin syndrome. This study presents cytogenetic and molecular characterization of germ-line deletions in one patient with a chromosome 9q22 deletion and in a second patient with a deletion of 9q22-q3l. Both have typical features of Gorlin syndrome plus additional findings, including mental retardation, conductive hearing loss, and failure to thrive. That Gorlin syndrome can be caused by null mutations (deletions) rather than by activating mutations has several implications. First, in conjunction with previous analyses of allelic loss in tumors, this study provides evidence that associated neoplasms arise with homozygous inactivation of the gene. In addition, dominant mutations of the XPAC and FACC1 genes can be ruled out as the cause of Gorlin syndrome, since the two patients described have null mutations. Finally, phenotypic features that show variable expression must be influenced by genetic background, epigenetic effects, somatic mutations, or environmental factors, since these two patients with identical alterations (deletions) of the Gorlin syndrome gene have somewhat different manifestations of Gorlin syndrome.

Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome.

The nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by multiple basal cell carcinomas (BCCs), pits of the palms and soles, jaw keratocysts, a variety of other tumors, and developmental abnormalities. NBCCS maps to chromosome 9q22.3. Familial and sporadic BCCs display loss of heterozygosity in this region, consistent with the gene being a tumor suppressor. A human sequence (PTC) with strong homology to the Drosophila segment polarity gene, patched, was isolated from a YAC and cosmid contig of the NBCCS region. Mutation analysis revealed alterations of PTC in NBCCS patients and in related tumors. We propose that a reduction in expression of the patched gene can lead to the developmental abnormalities observed in the syndrome and that complete loss of patched function contributes to transformation of certain cell types.

Relationship between sunlight exposure and a key genetic alteration in basal cell carcinoma.

BACKGROUND: Basal cell carcinoma (BCC) of the skin is the most common cancer in humans. Epidemiologic studies implicate sunlight exposure as one risk factor, but the limited association between BCCs and UVB radiation (i.e., UV radiation of a wavelength of 280-320 nm) suggests that additional factors must be involved. At the molecular level, not much is known about the role of specific environmental agents in the pathogenesis of BCCs. Point mutations of the types produced by UVB radiation are seen in the p53 gene (also known as TP53; chromosome 17p) of 40%-56% of BCCs. Loss of heterozygosity (LOH) on chromosome 9q22, however, is the most frequent genetic alteration in these tumors, and its causative agent is unknown. PURPOSE: We investigated whether the genetic alteration in chromosome 9 is common to all clinical subtypes of BCCs and whether inactivation of this putative tumor suppressor is related to sunlight exposure. The presence of UVB radiation-related point mutations in the p53 gene was used as an internal control for sunlight exposure to the precursor cells. METHODS: Tumor and blood samples were obtained from skin cancer patients by a surgeon who used Mohs' micrographic surgical technique. Clinical information on each tumor included location, size, histologic, subtype and whether it was primary or recurrent and sporadic or hereditary. Sixty BCCs from 58 patients were evaluated for LOH with 12 polymorphic markers that span chromosome 9. A subset of 18 tumors was evaluated for point mutations in exons 2-11 of the p53 gene, and a subset of 26 tumors was evaluated for LOH by use of a polymorphism in exon 4 of the p53 gene. Associations between tumor characteristics and molecular alterations were tested by a two-tailed chi-squared analysis or a two-tailed Fisher's exact test, depending on sample size. RESULTS: In a clinically diverse series of 47 informative tumors, 32 (68%) showed LOH for chromosome 9q markers, irrespective of histologic characteristics or clinical behavior. Forty-four (94%) of the 47 tumors were from sun-exposed areas of the body, defined as the head and neck in both sexes, shoulders or chest in males, and legs in females. No association was found between chromosome 9q LOH and sunlight exposure, as assessed by either the location of tumors on the body or the presence of UVB radiation-related p53 mutations. Of note, there was a striking difference between the frequency of LOH on chromosome 17p (two [12.5%] of 16 informative tumors) and on chromosome 9q (32 [68%] of 47 informative tumors; P < .001). CONCLUSIONS: Inactivation of a gene on chromosome 9q22 may be a necessary event for basal cell carcinogenesis. The pathogenesis of mutations in this gene may involve factors other than sunlight in a large proportion of tumors. IMPLICATIONS: The limited association between sunlight exposure and BCC incidence may reflect an etiologic contribution of additional environmental agents.

Nevoid basal cell carcinoma syndrome.

The nevoid basal cell carcinoma syndrome is an autosomal dominant disorder that predisposes to basal cell carcinomas of the skin, ovarian fibromas, and medulloblastomas. Unlike other hereditary disorders associated with cancer, it features widespread developmental defects. Laboratory studies of radiation sensitivity and chromosome instability over the past 20 years have generally yielded negative or inconclusive results. Recently, screening for allelic loss in sporadic and hereditary basal cell carcinomas, hereditary ovarian fibromas, and sporadic medulloblastomas provided evidence for a tumor suppressor gene on chromosome 9q, important in all three tumor types. Demonstration of a chromosome 9q deletion in an unusual patient with this syndrome and genetic linkage studies in large kindreds indicated that the nevoid basal cell carcinoma syndrome gene maps to the exact same location lost in tumors. These data show that tumors arise with homozygous inactivation of the gene and imply that it normally functions as a tumor suppressor. In contrast, hemizygous germ-line mutations lead to multiple congenital anomalies.

Developmental defects in Gorlin syndrome related to a putative tumor suppressor gene on chromosome 9.

Gorlin syndrome is an autosomal dominant disorder that predisposes to basal cell carcinomas of the skin, ovarian fibromas, and medulloblastomas. Unlike other hereditary disorders associated with cancer, it features widespread developmental defects. To investigate the possibility that the syndrome is caused by mutation in a tumor suppressor gene, we searched for loss of heterozygosity in 16 sporadic basal cell carcinomas, 2 hereditary basal cell carcinomas, and 1 hereditary ovarian fibroma and performed genetic linkage studies in five Gorlin syndrome kindreds. Eleven sporadic basal cell carcinomas and all 3 hereditary tumors had allelic loss of chromosome 9q31, and all informative kindreds showed tight linkage between the Gorlin syndrome gene and a genetic marker in this region. Loss of heterozygosity at this chromosomal location, particularly in hereditary tumors, implies that the gene is homozygously inactivated and normally functions as a tumor suppressor. In contrast, hemizygous germline mutations lead to multiple congenital anomalies.