RESUMEN
DNA damage and oxidative stress play a critical role in photoageing. Seborrhoeic keratosis (SK) affects sunlight-exposed sites in aged individuals. This study examined the mechanism of photoageing in SK. The guanine deaminase gene, which is involved in purine metabolism, was upregulated with uric acid levels and p21 in SK. Guanine deaminase was detectable in keratinocytes. Repeated exposure to ultraviolet (UV) increased levels of guanine deaminase, together with DNA damage, such as γ-H2AX and cyclobutane pyrimidine dimer formation, generation of reactive oxygen species, and keratinocyte senescence, which were reversed by guanine deaminase knockdown. However, guanine deaminase overexpression and H2O2 formed γ-H2AX, but not cyclobutane pyrimidine dimer. Loss-of-function guanine deaminase mutants reduced the metabolic end-product uric acid, which was increased by exposure to exogenous xanthine. Repeated exposure to UV increased levels of uric acid. Exogenous uric acid increased cellular senescence, reactive oxygen species, and γ-H2AX, similar to guanine deaminase. Overall, guanine deaminase upregulation increased UV-induced keratinocyte senescence in SK, via uric acid mediated by reactive oxygen species followed by DNA damage.
Asunto(s)
Senescencia Celular , Guanina Desaminasa/genética , Guanina/metabolismo , Queratinocitos/fisiología , Queratosis Seborreica/enzimología , Rayos Ultravioleta , Adulto , Anciano , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Senescencia Celular/efectos de la radiación , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN/efectos de la radiación , Femenino , Guanina Desaminasa/metabolismo , Histonas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Dímeros de Pirimidina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Envejecimiento de la Piel/fisiología , Regulación hacia Arriba , Ácido Úrico/metabolismo , Ácido Úrico/farmacología , Xantina/farmacologíaRESUMEN
The enzyme 11ß-hydroxysteroid dehydrogenase (11ß-HSD) catalyzes the interconversion between hormonally active cortisol and inactive cortisone within cells. There are two isozymes: 11ß-HSD1 activates cortisol from cortisone and 11ß-HSD2 inactivates cortisol to cortisone. 11ß-HSD1 was recently discovered in skin, and we subsequently found that the enzyme negatively regulates keratinocyte proliferation. We verified 11ß-HSD1 and 11ß-HSD2 expression in benign and malignant skin tumors and investigated the role of 11ß-HSD in skin tumor pathogenesis. Randomly selected formalin-fixed sections of skin lesions of seborrheic keratosis (SK), squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) were stained with 11ß-HSD1 and 11ß-HSD2 antibodies, and 11ß-HSD expression was also evaluated in murine epidermis in which hyperproliferation was induced by 12-O-tetradecanoylphorbol-13 acetate (TPA). We observed that 11ß-HSD1 expression was decreased in all SK, SCC, and BCC lesions compared with unaffected skin. Conversely, 11ß-HSD2 expression was increased in SK and BCC but not in SCC. Overexpression of 11ß-HSD2 in keratinocytes increased cell proliferation. In the murine model, 11ß-HSD1 expression was decreased in TPA-treated hyperproliferative skin. Our findings suggest that 11ß-HSD1 expression is decreased in keratinocyte proliferative conditions, and 11ß-HSD2 expression is increased in basal cell proliferating conditions, such as BCC and SK. Assessing 11ß-HSD1 and 11ß-HSD2 expression could be a useful tool for diagnosing and characterizing skin tumors.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Carcinoma Basocelular/enzimología , Carcinoma de Células Escamosas/enzimología , Epidermis/enzimología , Regulación Enzimológica de la Expresión Génica , Queratosis Seborreica/enzimología , Adolescente , Adulto , Anciano , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Hidrocortisona/metabolismo , Queratinocitos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Acetato de Tetradecanoilforbol/farmacología , Adulto JovenRESUMEN
Malignant tumors result from the accumulation of genetic alterations in oncogenes and tumor suppressor genes. Much less is known about the genetic changes in benign tumors. Seborrheic keratoses (SK) are very frequent benign human epidermal tumors without malignant potential. We performed a comprehensive mutational screen of genes in the FGFR3-RAS-MAPK and phosphoinositide 3-kinase (PI3K)-AKT pathways from 175 SK, including multiple lesions from each patient. SK commonly harbored multiple bona fide oncogenic mutations in FGFR3, PIK3CA, KRAS, HRAS, EGFR, and AKT1 oncogenes but not in tumor suppressor genes TSC1 and PTEN. Despite the occurrence of oncogenic mutations and the evidence for downstream ERK/MAPK and PI3K pathway signaling, we did not find induction of senescence or a DNA damage response. Array comparative genomic hybridization (aCGH) analysis revealed that SK are genetically stable. The pattern of oncogenic mutations and X chromosome inactivation departs significantly from randomness and indicates that spatially independent lesions from a given patient share a clonal relationship. Our findings show that multiple oncogenic mutations in the major signaling pathways involved in cancer are not sufficient to drive malignant tumor progression. Furthermore, our data provide clues on the origin and spread of oncogenic mutations in tissues, suggesting that apparently independent (multicentric) adult benign tumors may have a clonal origin.
Asunto(s)
Queratosis Seborreica/genética , Queratosis Seborreica/patología , Mutación/genética , Oncogenes/genética , Apoptosis/genética , Biomarcadores de Tumor , Proliferación Celular , Senescencia Celular , Células Clonales , Análisis Mutacional de ADN , Pruebas Genéticas , Genoma Humano/genética , Genotipo , Humanos , Queratosis Seborreica/enzimología , Proteínas Quinasas Activadas por Mitógenos/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismo , Proteínas ras/genéticaRESUMEN
Activating mutations of the p110 alpha subunit of PI3K (PIK3CA) oncogene have been identified in a broad spectrum of malignant tumors. However, their role in benign or preneoplastic conditions is unknown. Activating FGF receptor 3 (FGFR3) mutations are common in benign skin lesions, either as embryonic mutations in epidermal nevi (EN) or as somatic mutations in seborrheic keratoses (SK). FGFR3 mutations are also common in low-grade malignant bladder tumors, where they often occur in association with PIK3CA mutations. Therefore, we examined exons 9 and 20 of PIK3CA and FGFR3 hotspot mutations in EN (n = 33) and SK (n = 62), two proliferative skin lesions lacking malignant potential. Nine of 33 (27%) EN harbored PIK3CA mutations; all cases showed the E545G substitution, which is uncommon in cancers. In EN, R248C was the only FGFR3 mutation identified. By contrast, 10 of 62 (16%) SK revealed the typical cancer-associated PIK3CA mutations E542K, E545K, and H1047R. The same lesions displayed a wide range of FGFR3 mutations. Corresponding unaffected tissue was available for four EN and two mutant SK: all control samples displayed a WT sequence, confirming the somatic nature of the mutations found in lesional tissue. Forty of 95 (42%) lesions showed at least one mutation in either gene. PIK3CA and FGFR3 mutations displayed an independent distribution; 5/95 lesions harbored mutations in both genes. Our findings suggest that, in addition to their role in cancer, oncogenic PIK3CA mutations contribute to the pathogenesis of skin tumors lacking malignant potential. The remarkable genotype-phenotype correlation as observed in this study points to a distinct etiopathogenesis of the mutations in keratinocytes occuring either during fetal development or in adult life.
Asunto(s)
Queratosis Seborreica/genética , Mutación , Nevo/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Humanos , Queratosis Seborreica/enzimología , Nevo/enzimología , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genéticaRESUMEN
To elucidate involvement of proteinases in malignancy of keratinocytes, expression of cathepsin B, a cysteine proteinase, and cathepsin D, an aspartic proteinase, was ascertained in formalin-fixed paraffin-embedded specimens of normal skin, squamous cell carcinoma (SCC). Bowen's disease, seborrhoeic keratosis and basal cell carcinoma (BCC). Presence of procathepsin B and an intermediate form of cathepsin D was confirmed by Western blotting and enzyme activity analysis. Cathepsin B stained more intensely in SCC tumour cells than in normal epidermis; staining patterns were diffuse, granular or both. Diffuse and granular patterns (procathepsin B and mature enzyme, respectively) appeared in inner and outer parts of tumour islands, respectively. Five of 20 cases of Bowen's disease showed diffuse enhanced cathepsin B expression; 20 cases of seborrhoeic keratosis or BCC did not. Cathepsin D stained intensely in tumour cells of half the SCC cases. The staining manner and distribution of cathepsins B and D was similar in the cytoplasm of cancer cells. No enhanced staining of cathepsin D was seen in any cases of Bowen's disease, seborrhoeic keratosis, or BCC. Coexistence and localization of active mature forms of cathepsins B and D suggests that cooperation between the two enzymes may play an important part in invasion of SCC.
Asunto(s)
Carcinoma de Células Escamosas/enzimología , Catepsinas/metabolismo , Neoplasias Cutáneas/enzimología , Western Blotting , Enfermedad de Bowen/enzimología , Carcinoma Basocelular/enzimología , Catepsina B/metabolismo , Catepsina D/metabolismo , Epidermis/enzimología , Humanos , Queratosis Seborreica/enzimología , Invasividad NeoplásicaRESUMEN
The expression of nPKC eta, a Ca(++)-independent isoform of protein kinase C in normal human skin, and skin from patients with psoriasis, squamous cell carcinoma, basal cell epithelioma, nevus pigmentosus, and seborrheic keratosis, were examined by immunohistochemical staining using a polyclonal antibody raised against a synthetic peptide at a diverse region of the nPKC eta molecule. In normal epidermis, the strongest staining was observed in the uppermost granular layer with no staining of the spinous or basal layers. The inner layer of the intra-epidermal eccrine duct was also strongly stained. Weak staining was observed in several layers of the outer root sheath of the follicular infundibulum. No staining was detected in the inner root sheath of the hair follicles, hair matrix, sebaceous gland, eccrine gland, intradermal eccrine duct, arrectores pilorum, melanocytes, Langerhans cells, fibroblasts, or blood vessels. In psoriatic skin, stained keratinocytes were distributed in the suprabasal layers with the most being observed in the uppermost layer and the least in layers closed to the basal layer. In squamous cell carcinoma, weak staining was observed in the keratotic cells around horny pearls. In the basal cell epithelioma and nevus pigmentosus, the cells were not stained, whereas in seborrheic keratosis, cells that stained were located in the granular layer. We conclude from the evidence presented above that nPKC eta is expressed in close association with epidermal differentiation in normal skin and skin diseases.