UV-specific mutations of the human patched gene in basal cell carcinomas from normal individuals and xeroderma pigmentosum patients

Germline mutations of the human patched gene, PTCH, are responsible for the nevoid basal cell carcinoma (NBCC) syndrome or Gorlin's syndrome, characterized by multiple skin cancers, internal cancers and severe developmental abnormalities. The patched gene codes for a developmental regulator protein implicated in the sonic hedgehog (SHH) signalling pathway which plays an important role in oncogenic transformation. Patched exhibits tumor suppression function and has been shown to be mutated in skin cancers isolated from DNA repair-proficient patients or from xeroderma pigmentosum (XP), a DNA repair-deficient syndrome.

We have reviewed and analyzed in detail the different mutation spectra found on the PTCH gene in these various models. The type and distribution of mutations are quite different between germline, sporadic and XP cancers. Among the germline alterations, there is a preponderance (70%) of rearrangements compared to other tumour types analysed where less than 30% of rearrangements is observed. Typical UV-induced mutations of the patched gene are found prominently in XP basal cell carcinomas (BCCs) and in particular, a significantly higher level (63%) of the UV signature tandem mutations is found compared to sporadic BCC (11%). The location of mutations along the PTCH protein delineates several important functional domains implicated in the biology of this transmembrane receptor.     

UV-specific mutations of the human patched gene in basal cell carcinomas from normal individuals and xeroderma pigmentosum patients

Germline mutations of the human patched gene, PTCH, are responsible for the nevoid basal cell carcinoma (NBCC) syndrome or Gorlin's syndrome, characterized by multiple skin cancers, internal cancers and severe developmental abnormalities. The patched gene codes for a developmental regulator protein implicated in the sonic hedgehog (SHH) signalling pathway which plays an important role in oncogenic transformation. Patched exhibits tumor suppression function and has been shown to be mutated in skin cancers isolated from DNA repair-proficient patients or from xeroderma pigmentosum (XP), a DNA repair-deficient syndrome.We have reviewed and analyzed in detail the different mutation spectra found on the PTCH gene in these various models. The type and distribution of mutations are quite different between germline, sporadic and XP cancers. Among the germline alterations, there is a preponderance (70%) of rearrangements compared to other tumour types analysed where less than 30% of rearrangements is observed. Typical UV-induced mutations of the patched gene are found prominently in XP basal cell carcinomas (BCCs) and in particular, a significantly higher level (63%) of the UV signature tandem mutations is found compared to sporadic BCC (11%). The location of mutations along the PTCH protein delineates several important functional domains implicated in the biology of this transmembrane receptor.     

Xeroderma pigmentosum and the role of UV-induced DNA damage in skin cancer

Xeroderma pigmentosum (XP) is a rare, autosomal recessive disease that is characterized by the extreme sensitivity of the skin to sunlight. Compared to normal individuals, XP patients have a more than 1000-fold increased risk of developing cancer on sun-exposed areas of the skin. Genetic and molecular analyses have revealed that the repair of ultraviolet (UV)-induced DNA damage is impaired in XP patients owing to mutations in genes that form part of a DNA-repair pathway known as nucleotide excision repair (NER). Two other diseases, Cockayne syndrome (CS) and the photosensitive form of trichothiodystrophy (TTD), are linked to a defect in the NER pathway. Strikingly, although CS and TTD patients are UV-sensitive, they do not develop skin cancer. The recently developed animal models that mimic the human phenotypes of XP, CS and TTD will contribute to a better understanding of the etiology of these diseases and the role of UV-induced DNA damage in the development of skin cancer.     

Synergistic interactions between XPC and p53 mutations in double-mutant mice: neural tube abnormalities and accelerated UV radiation-induced skin cancer

The significance of DNA repair to human health has been well documented by studies on xeroderma pigmentosum (XP) patients, who suffer a dramatically increased risk of cancer in sun-exposed areas of their skin [1] and [2]. This autosomal recessive disorder has been directly associated with a defect in nucleotide excision–repair (NER) [1] and [2]. Like human XP individuals, mice carrying homozygous mutations in XP genes manifest a predisposition to skin carcinogenesis following exposure to ultraviolet (UV) radiation [3], [4] and [5]. Recent studies have suggested that, in addition to roles in apoptosis [6] and cell-cycle checkpoint control [7] in response to DNA damage, p53 protein may modulate NER [8]. Mutations in the p53 gene have been observed in 50% of all human tumors [9] and have been implicated in both the early [10] and late [11] stages of skin cancer. To examine the consequences of a combined deficiency of the XPC and the p53 proteins in mice, we generated double-mutant animals. We document a spectrum of neural tube defects in XPC p53 mutant embryos. Additionally, we show that, following exposure to UV-B radiation, XPC p53 mutant mice have more severe solar keratosis and suffer accelerated skin cancer compared with XPC mutant mice that are wild-type with respect to p53.     

Retrovirus-mediated DNA repair gene transfer into xeroderma pigmentosum cells: Perspectives for a gene therapy

The rare hereditary disease xeroderma pigmentosum (XP) is clinically characterized by extreme sun sensitivity and an increased predisposition for developing skin cancer. Cultured cells from XP patients exhibit hypersensitivity to ultraviolet (UV) radiation due to the defect in nucleotide excision repair (NER), and other cellular abnormalities. Seven genes identified in the classical XP forms, XPA to XPG, are involved in the NER pathway. In view of developing a strategy of gene therapy for XP, we devised recombinant retrovirus-carrying DNA repair genes for transfer and stable expression of these genes in cells from XP patients. Results showed that these retroviruses are efficient tools for transducing XP fibroblasts and correcting repair-defective cellular phenotypes by recovering normal UV survival, unscheduled DNA synthesis, and RNA synthesis after UV irradiation, and also other cellular abnormalities resulting from NER defects. These results imply that the first step of cellular gene therapy might be accomplished successfully.     

Mutations in the Hedgehog Pathway Genes SMO and PTCH1 in Human Gastric Tumors

The causal role of the hedgehog pathway in cancer has been best documented in basal cell carcinoma of the skin. To assess potential DNA alterations of the hedgehog pathway in gastric cancer, we sequenced SMO and PTCH1 genes in a set of 39 gastric tumors. Tumors were classified by histology based on the Lauren classification and Sanger sequencing was performed to obtain full length coding sequences. Genomic instability was evident in these tumors as a number of silent or missense mutations were found. In addition to those that are potential germline polymorphisms, we found three SMO missense mutations, and one PTCH1 frameshift mutation that are novel and have not been documented in basal cell carcinoma. Mutations were found in both intestinal and diffuse type gastric tumors as well as in tumors that exhibit both intestinal and diffuse features. mRNA expression of hedgehog pathway genes was also examined and their levels do not indicate unequivocal higher pathway activity in tumors with mutations than those without. In summary, SMO and/or PTCH1 mutations are present at low frequency in different histologic subtypes of gastric tumors and these do not appear to be driver mutations.     

Genetic evolution of nevus of Ota reveals clonal heterogeneity acquiring BAP1 and TP53 mutations

Melanoma presents molecular alterations based on its anatomical location and exposure to environmental factors. Due to its intrinsic genetic heterogeneity, a simple snapshot of a tumor's genetic alterations does not reflect the tumor clonal complexity or specific gene–gene cooperation. Here, we studied the genetic alterations and clonal evolution of a unique patient with a Nevus of Ota that developed into a recurring uveal‐like dermal melanoma. The Nevus of Ota and ulterior lesions contained GNAQ mutations were c‐KIT positive, and tumors showed an increased RAS pathway activity during progression. Whole‐exome sequencing of these lesions revealed the acquisition of BAP1 and TP53 mutations during tumor evolution, thereby unmasking clonal heterogeneity and allowing the identification of cooperating genes within the same tumor. Our results highlight the importance of studying tumor genetic evolution to identify cooperating mechanisms and delineate effective therapies.     

Systems biology approach for mutational and site-specific structural investigation of DNA repair genes for xeroderma pigmentosum

Xeroderma pigmentosum (XP) is a rare genetic skin disorder caused due to the extreme sensitivity for ultraviolet (UV) radiations. On its exposure, DNA acquires damages leading to skin and often neurological abnormalities. The DNA repair implicated in fixing UV-induced damages is NER and mutations in genes involved in NER and TLS form the basis of XP. The analyses of such mutations are vital for understanding XP and involved cancer genetics to facilitate the identification of crucial biomarkers and anticancer therapeutics. We detected the deleterious nsSNPs and examined them at structure-level by altering the structure, estimating secondary structure, solvent accessibility and performing site specific analysis. Crucial phosphorylation sites were also identified for their role in the disorder. These mutational and structural analyses offer valuable insight to the fundamental association of genetic mutations with phenotypic variations in XP and will assist experimental biologists to evaluate the mutations and their impact on genome.     

Human skin reconstructed in vitro as a model to study the keratinocyte, the fibroblast and their interactions: photodamage and repair processes

The protective role of the skin is provided by the two major compartments of the skin, dermis and epidermis. Both are affected in the long term by consequences of sun exposure such as skin photoaging and cancer development. Characterization of UV-induced skin response at cellular and molecular levels is needed for prevention or correction of these long term effects. The human skin reconstructed in vitro, comprising both a living dermal equivalent and a fully differentiated epidermis represents a predictive tool to characterize wavelength and cell type specific biological damage together with tissular distribution. While UVB directly affects epidermis, inducing DNA lesions and apoptotic sunburn keratinocytes, UVA radiation can directly target the dermal compartment through ROS generation, dermal fibroblasts alterations and extracellular matrix (ECM) modifications. Interactions between the two compartments have also been found, especially for MMP1 induction. In the normal population, photodamage can be repaired through specialized systems. Using skin cells from Xeroderma pigmentosum (XP, a photosensitive and cancer-prone disease), a DNA-repair deficient skin has been developed in vitro. Specific features due to intrinsic XP cell phenotype have been discovered, some of them being indicative of early steps of neoplasia and suggesting a particular role for stroma-epithelium interactions. Finally, human reconstructed skin can be used for approaches designed to regenerate photodamaged skin. The dermal-epidermal junction (DEJ), which is crucial for skin cohesion, is drastically altered in photo-aged skin. The three-dimensional skin model allowed to visualize the improving effects of vitamin C on the DEJ. Modified skin models, lacking one cell type, allowed us to determine the cellular origin of the different markers, their spatial localization, and the respective roles and interactions of keratinocytes and fibroblasts during DEJ formation. All together these studies give a global and tissular view concerning the effects of UV light on skin cells and emphazise the interest of such models for general aspects of cellular biology. By allowing the control of cells used to reconstruct the model and their origin, these studies make it possible to assess the respective role of the two major cellular actors of the skin as well as their interactions. Ongoing research about incorporating other cell types may certainly give rise to even more relevant models.     

Differential role of basal keratinocytes in UV-induced immunosuppression and skin cancer

Cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) comprise major UV-induced photolesions. If left unrepaired, these lesions can induce mutations and skin cancer, which is facilitated by UV-induced immunosuppression. Yet the contribution of lesion and cell type specificity to the harmful biological effects of UV exposure remains currently unclear. Using a series of photolyase-transgenic mice to ubiquitously remove either CPDs or 6-4PPs from all cells in the mouse skin or selectively from basal keratinocytes, we show that the majority of UV-induced acute effects to require the presence of CPDs in basal keratinocytes in the mouse skin. At the fundamental level of gene expression, CPDs induce the expression of genes associated with repair and recombinational processing of DNA damage, as well as apoptosis and a response to stress. At the organismal level, photolyase-mediated removal of CPDs, but not 6-4PPs, from the genome of only basal keratinocytes substantially diminishes the incidence of skin tumors; however, it does not affect the UVB-mediated immunosuppression. Taken together, these findings reveal a differential role of basal keratinocytes in these processes, providing novel insights into the skin's acute and chronic responses to UV in a lesion- and cell-type-specific manner.     

Hedgehog signaling in normal urothelial cells and in urothelial carcinoma cell lines

Constitutive activation of hedgehog signaling, often caused by PTCH1 inactivation and leading to inappropriate activation of GLI target genes, is crucial for the development of several human tumors including basal cell carcinoma of the skin and medulloblastoma. The PTCH1 gene at 9q22 is also considered as a candidate tumor suppressor in transitional cell carcinoma (TCC), of which >50% show LOH in this region. However, only rare mutations have been found in PTCH1. We have therefore investigated GLI-dependent promoter activity and expression of hedgehog pathway components in TCC cell lines and proliferating normal urothelial cells. Normal urothelial cells cultured in serum-free medium, but not TCC lines exhibited low, but significant promoter activity under standard growth conditions. Accordingly, GLI1-3 and PTCH1 mRNAs were expressed at moderate levels, and sonic hedgehog (SHH) mRNA expression was low to undetectable. In co-transfection experiments GLI1 increased promoter activity significantly in one TCC line and further in normal urothelial cells, but less strongly in other TCC lines. Expression patterns of GLI factor mRNAs did not correlate with inducibility. No significant effects of SHH or cyclopamine on proliferation were observed, ruling out autocrine effects. However, SHH induced GLI-dependent promoter activity in normal urothelial cells. Taken together, our data suggest that the hedgehog pathway is weakly active in normal adult urothelial cells and of limited importance in TCC.     

Genetic tumor archeology: microdissection and genetic heterogeneity in squamous and basal cell carcinoma

Carcinogenesis is a multi-step series of somatic genetic events. The complexity of this multi-hit process makes it difficult to determine each single event and the definitive outcome of such events. To investigate the genetic alterations in cancer-related genes, sensitive and reliable detection methods are of major importance for generating relevant results. Another critical issue is the quality of starting material which largely affects the outcome of the analysis. Microdissection of cells defined under the microscope ensures a selection of representative material for subsequent genetic analysis. Skin cancer provides an advantageous model for studying the development of cancer. Detectable lesions occur early during tumor progression, facilitating molecular analysis of the cell populations from both preneoplastic and neoplastic lesions. Alterations of the p53 tumor suppressor gene are very common in non-melanoma skin cancer, and dysregulation of p53 pathways appear to be an early event in the tumor development. A high frequency of epidermal p53 clones has been detected in chronically sun-exposed skin. The abundance of clones containing p53 mutated keratinocytes adjacent to basal cell (BCC) and squamous cell carcinoma (SCC) suggests a role in human skin carcinogenesis. Studies using p53 mutations as a clonality marker have suggested a direct link between actinic keratosis, SCC in situ and invasive SCC. Microdissection-based studies have also shown that different parts of individual BCC tumors can share a common p53 mutation yet differ with respect to additional alterations within the p53 gene, consistent with subclonal development within tumors. Here, we present examples of using well-defined cell populations, including single cells, from complex tissue in combination with molecular tools to reveal features involved in skin carcinogenesis.     

UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency

Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (-/-) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP.     

Variability in nucleotide excision repair and cancer risk: a review

Cancer initiation is classically associated with the induction of mutations on specific oncogenes or tumor suppressor genes, due to the presence of unrepaired DNA lesions produced by endogenous or exogenous genotoxic agents. Among several DNA repair pathways, the nucleotide excision repair (NER) is the most important and versatile one in removing the bulky adducts induced by physical and chemical carcinogens. Xeroderma pigmentosum (XP), characterized by a deficiency in NER and an over 1000-fold increased risk of skin cancer, represents a paradigm to understand the role of unrepaired lesion in the development of cancer. We reviewed here several NER assays used in epidemiological studies investigating the association between DNA repair efficiency and cancer risk. Reduced DNA repair could contribute to the development of cutaneous basal cell carcinoma (BCC), although discordant results have been reported. More consistent findings were observed between cellular sensitivity towards genotoxic agents and smoking-related cancers.     

Effect of DNA repair on the cytotoxicity and mutagenicity of polycyclic hydrocarbon derivatives in normal and xeroderma pigmentosum human fibroblasts

The cytotoxicity of the “K-region” epoxides as well as several other reactive metabolites or chemical derivatives of polycyclic hydrocarbons was compared in normally-repairing human diploid skin fibroblasts and in fibroblasts from a classical xeroderma pigmentosum (XP) patient (XP2BE) whose cells have been shown to carry out excision repair of damage induced in DNA by ultraviolet (UV) radiation at a rate approx. 20% that of normal cells. Each compound tested exhibited a 2- to 3-fold greater cytotoxicity in this XP strain than in the normal strain. To determine whether this difference in survival reflected a difference in the capacity of the strains to repair DNA damage caused by such hydrocarbon derivatives, we compared the cytotoxic effect of several “K-region” epoxides in two additional XP strains, each with a different capacity for repair of UV damage. The ration of the slopes of the survival curves for each of the XP strains to that of the normal strain, following exposure to each epoxide, was very similar to that which we had previously determined for their respective UV curves, suggesting that human cells repair damage induced in DNA by exposure to hydrocarbon derivatives with the same system used for UV-induced lesions.To determine whether the deficiency in rate of excision repair in this classical XP strain (XP2BE) causes such cells to be abnormally susceptible to mutations induced by “K-region” epoxides of polycyclic hydrocarbons, we compared them with normal cells for the frequency of induced mutations to 8-azaguanine resistance. The XP cells were two to three times more susceptible to mutations induced by the “K-region” epoxide of benzo(a)pyrene (BP), 7,12-dimethylbenz(a)anthracene (DMBA), and dibenz(a,h)anthracene (DBA). Evidence also was obtained that cells from an XP variant patient are abnormally susceptible to mutations induced by hydrocarbon epoxides and, as is the case following exposure to UV, are abnormally slow in converting low molecular weight DNA, synthesized from a template following exposure to hydrocarbon epoxides, into large-size DNA.     

Shh Signaling and Pancreatic Cancer: Implications for Therapy?

Hedgehog signaling has been implicated in the development of several human cancers, including small cell lung carcinomas, medulloblastomas, basal cell carcinomas, and digestive tract tumors. Elevated levels of pathway components are observed in pancreatic ductal adenocarcinoma (PDAC) precursor lesions, and these levels increase further as lesions progress to more advanced stages. Yet the mechanisms by which hedgehog signaling contributes to pancreatic tumorigenesis were poorly understood. We recently published results showing that activated hedgehog signaling enhances the proliferation and survival of pancreatic duct epithelial cells, the presumptive target cells for PDAC development. We also demonstrated that sonic hedgehog (Shh) expression, in cooperation with loss of the Trp53 and Ink4a/Arf tumor suppressor loci, was sufficient to initiate the formation of early pancreatic lesions. Furthermore, Shh signaling enhanced K-Ras-mediated pancreatic tumorigenesis and reduced the dependence of tumor cells on the sustained activation of Ras-stimulated signaling pathways. Here we discuss the significance of these findings and the implications for therapy.