RESUMEN
SignificanceTranscription-coupled repair (TCR) involves four core proteins: CSA, CSB, USP7, and UVSSA. CSA and CSB are mutated in the severe human neurocutaneous disease Cockayne syndrome. In contrast UVSSA is a mild photosensitive disease in which a mutated protein sequence prevents recruitment of USP7 protease to deubiquitinate and stabilize CSB. We deleted the UVSSA protein using CRISPR-Cas9 in an aneuploid cell line, HEK293, and determined the functional consequences. The knockout cell line was sensitive to transcription-blocking lesions but not sensitive to oxidative agents or PARP inhibitors, unlike CSB. Knockout of UVSSA also activated ATM, like CSB, in transcription-arrested cells. The phenotype of UVSSA, especially its rarity, suggests that many TCR-deficient patients and tumors fail to be recognized clinically.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas Portadoras/metabolismo , Reparación del ADN , Homeostasis , Transducción de Señal , Transcripción Genética , Alquilantes/farmacología , Secuencia de Aminoácidos , Proteínas Portadoras/química , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Células HEK293 , Humanos , Mutágenos/farmacología , Transducción de Señal/efectos de los fármacos , Rayos UltravioletaRESUMEN
Therapy of BRAF-mutant melanoma with selective inhibitors of BRAF (BRAFi) and MEK (MEKi) represents a major clinical advance but acquired resistance to therapy has emerged as a key obstacle. To date, no clinical approaches successfully resensitize to BRAF/MEK inhibition. Here, we develop a therapeutic strategy for melanoma using bromosporine, a bromodomain inhibitor. Bromosporine (bromo) monotherapy produced significant anti-tumor effects against established melanoma cell lines and patient-derived xenografts (PDXs). Combinatorial therapy involving bromosporine and cobimetinib (bromo/cobi) showed synergistic anti-tumor effects in multiple BRAFi-resistant PDX models. The bromo/cobi combination was superior in vivo to standard BRAFi/MEKi therapy in the treatment-naive BRAF-mutant setting and to MEKi alone in the setting of immunotherapy-resistant NRAS- and NF1-mutant melanoma. RNA sequencing of xenografts treated with bromo/cobi revealed profound down-regulation of genes critical to cell division and mitotic progression. Bromo/cobi treatment resulted in marked DNA damage and cell-cycle arrest, resulting in induction of apoptosis. These studies introduce bromodomain inhibition, alone or combined with agents targeting the mitogen activated protein kinase pathway, as a rational therapeutic approach for melanoma refractory to standard targeted or immunotherapeutic approaches.
Asunto(s)
Melanoma , Proteínas Proto-Oncogénicas B-raf , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Nucleares , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/metabolismo , Factores de TranscripciónRESUMEN
The invasive behavior of glioblastoma is essential to its aggressive potential. Here, we show that pleckstrin homology domain interacting protein (PHIP), acting through effects on the force transduction layer of the focal adhesion complex, drives glioblastoma motility and invasion. Immunofluorescence analysis localized PHIP to the leading edge of glioblastoma cells, together with several focal adhesion proteins: vinculin (VCL), talin 1 (TLN1), integrin beta 1 (ITGB1), as well as phosphorylated forms of paxillin (pPXN) and focal adhesion kinase (pFAK). Confocal microscopy specifically localized PHIP to the force transduction layer, together with TLN1 and VCL. Immunoprecipitation revealed a physical interaction between PHIP and VCL. Targeted suppression of PHIP resulted in significant down-regulation of these focal adhesion proteins, along with zyxin (ZYX), and produced profoundly disorganized stress fibers. Live-cell imaging of glioblastoma cells overexpressing a ZYX-GFP construct demonstrated a role for PHIP in regulating focal adhesion dynamics. PHIP silencing significantly suppressed the migratory and invasive capacity of glioblastoma cells, partially restored following TLN1 or ZYX cDNA overexpression. PHIP knockdown produced substantial suppression of tumor growth upon intracranial implantation, as well as significantly reduced microvessel density and secreted VEGF levels. PHIP copy number was elevated in the classical glioblastoma subtype and correlated with elevated EGFR levels. These results demonstrate PHIP's role in regulating the actin cytoskeleton, focal adhesion dynamics, and tumor cell motility, and identify PHIP as a key driver of glioblastoma migration and invasion.
Asunto(s)
Neoplasias Encefálicas/patología , Adhesiones Focales/patología , Glioblastoma/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neovascularización Patológica/patología , Citoesqueleto de Actina/metabolismo , Animales , Encéfalo/patología , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/genética , Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glioblastoma/irrigación sanguínea , Glioblastoma/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Microscopía Intravital , Ratones , Microscopía Confocal , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Neovascularización Patológica/genética , Imagen de Lapso de Tiempo , Vinculina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The identification and targeting of key molecular drivers of melanoma and breast and lung cancer have substantially improved their therapy. However, subtypes of each of these three common, lethal solid tumors lack identified molecular drivers, and are thus not amenable to targeted therapies. Here we show that pleckstrin homology domain-interacting protein (PHIP) promotes the progression of these "driver-negative" tumors. Suppression of PHIP expression significantly inhibited both tumor cell proliferation and invasion, coordinately suppressing phosphorylated AKT, cyclin D1, and talin1 expression in all three tumor types. Furthermore, PHIP's targetable bromodomain is functional, as it specifically binds the histone modification H4K91ac. Analysis of TCGA profiling efforts revealed PHIP overexpression in triple-negative and basal-like breast cancer, as well as in the bronchioid subtype of nonsmall cell lung cancer. These results identify a role for PHIP in the progression of melanoma and breast and lung cancer subtypes lacking identified targeted therapies. The use of selective, anti-PHIP bromodomain inhibitors may thus yield a broad-based, molecularly targeted therapy against currently nontargetable tumors.
Asunto(s)
Mama/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Dominios Homólogos a Pleckstrina/fisiología , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/fisiología , Ciclina D1/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
BACKGROUND: Mitotic rate is a strong, independent prognostic factor in patients with melanoma. However, incorporating it into the melanoma staging system has proved challenging. METHODS: The prognostic impact of mitotic rate was assessed in a melanoma cohort comprising 5050 patients from 2 geographically distinct populations. Computer-generated cut points for mitotic rate were constructed to determine its impact on melanoma-associated survival using Kaplan-Meier and multivariate regression analyses. The impact of mitotic rate also was assessed in randomly split training and validation sets. RESULTS: Mitotic rate had a nonlinear impact on survival, as evidenced by unequally spaced cut points. An index incorporating these cut points that was constructed from one population produced significantly more accurate predictions of survival in the other population than using the entire scale of mitotic rate. An index constructed from the combined cohort was found to be independently predictive of survival, with an impact comparable to that of ulceration. Optimal high-versus-low cut points for mitotic rate were generated separately for each T category (<2 mitoses/mm2 vs ≥2 mitoses/mm2 for T1 melanoma, <4 mitoses/mm2 vs ≥4 mitoses/mm2 for T2 melanoma, <6 mitoses/mm2 vs ≥6/mitoses/mm2 for T3 melanoma, and <7 mitoses/mm2 vs ≥7 mitoses/mm2 for T4 melanoma). Using Kaplan-Meier analysis, elevated mitotic rate was found to have an impact on survival comparable to that of ulceration within each T category. Application of the index for mitotic rate that was constructed from the training data set demonstrated an independent impact in the validation data set, with a significance similar to that of ulceration. CONCLUSIONS: The results of the current study demonstrated the comparable prognostic impact of mitotic rate and ulceration, providing support for its reincorporation into the T category.
Asunto(s)
Melanoma/genética , Índice Mitótico/métodos , Femenino , Humanos , Masculino , Melanoma/mortalidad , Estadificación de Neoplasias , PronósticoRESUMEN
Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are human photosensitive diseases with mutations in the nucleotide excision repair (NER) pathway, which repairs DNA damage from UV exposure. CS is mutated in the transcription-coupled repair (TCR) branch of the NER pathway and exhibits developmental and neurological pathologies. The XP-C group of XP patients have mutations in the global genome repair (GGR) branch of the NER pathway and have a very high incidence of UV-induced skin cancer. Cultured cells from both diseases have similar sensitivity to UV-induced cytotoxicity, but CS patients have never been reported to develop cancer, although they often exhibit photosensitivity. Because cancers are associated with increased mutations, especially when initiated by DNA damage, we examined UV-induced mutagenesis in both XP-C and CS cells, using duplex sequencing for high-sensitivity mutation detection. Duplex sequencing detects rare mutagenic events, independent of selection and in multiple loci, enabling examination of all mutations rather than just those that confer major changes to a specific protein. We found telomerase-positive normal and CS-B cells had increased background mutation frequencies that decreased upon irradiation, purging the population of subclonal variants. Primary XP-C cells had increased UV-induced mutation frequencies compared with normal cells, consistent with their GGR deficiency. CS cells, in contrast, had normal levels of mutagenesis despite their TCR deficiency. The lack of elevated UV-induced mutagenesis in CS cells reveals that their TCR deficiency, although increasing cytotoxicity, is not mutagenic. Therefore the absence of cancer in CS patients results from the absence of UV-induced mutagenesis rather than from enhanced lethality.
Asunto(s)
Síndrome de Cockayne/genética , Reparación del ADN , ADN/química , Mutación , Rayos Ultravioleta/efectos adversos , Xerodermia Pigmentosa/genética , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patología , ADN/metabolismo , Roturas del ADN de Doble Cadena , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Voluntarios Sanos , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Cultivo Primario de Células , Análisis de Secuencia de ADN , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/prevención & control , Xerodermia Pigmentosa/metabolismo , Xerodermia Pigmentosa/patologíaRESUMEN
Microphthalmia-associated transcription factor (MITF) plays a critical and complex role in melanocyte transformation. Although several downstream targets of MITF action have been identified, the precise mechanisms by which MITF promotes melanocytic tumor progression are incompletely understood. Recent studies identified an oncogenic role for the bromodomain plant homeodomain finger transcription factor (BPTF) gene in melanoma progression, in part through activation of BCL2, a canonical target of MITF signaling. Analysis of the BPTF promoter identified a putative MITF-binding site, suggesting that MITF may regulate BPTF expression. Overexpression of MITF resulted in up-regulation of BPTF in a panel of melanoma and melanocyte cell lines. shRNA-mediated down-regulation of MITF in melanoma cells was accompanied by down-regulation of BPTF and BPTF-regulated genes (including BCL2) and resulted in reduced proliferative capacity of melanoma cells. The suppression of cell growth mediated by MITF silencing was rescued by overexpression of BPTF cDNA. Binding of MITF to the BPTF promoter was demonstrated using ChIP analysis. MITF overexpression resulted in direct transcriptional activation of BPTF, as evidenced by increased luciferase activity driven by the BPTF promoter. These results indicate that BPTF transduces key prosurvival signals driven by MITF, further supporting its important role in promoting melanoma cell survival and progression.
Asunto(s)
Antígenos Nucleares/metabolismo , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Melanocitos/citología , Melanoma/patología , Factor de Transcripción Asociado a Microftalmía/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factores de Transcripción/metabolismo , Antígenos Nucleares/genética , Apoptosis , Sitios de Unión , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Técnica del Anticuerpo Fluorescente , Humanos , Luciferasas/metabolismo , Melanocitos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
Melanoma is difficult to treat once it becomes metastatic. However, the precise ancestral relationship between primary tumors and their metastases is not well understood. We performed whole-exome sequencing of primary melanomas and multiple matched metastases from eight patients to elucidate their phylogenetic relationships. In six of eight patients, we found that genetically distinct cell populations in the primary tumor metastasized in parallel to different anatomic sites, rather than sequentially from one site to the next. In five of these six patients, the metastasizing cells had themselves arisen from a common parental subpopulation in the primary, indicating that the ability to establish metastases is a late-evolving trait. Interestingly, we discovered that individual metastases were sometimes founded by multiple cell populations of the primary that were genetically distinct. Such establishment of metastases by multiple tumor subpopulations could help explain why identical resistance variants are identified in different sites after initial response to systemic therapy. One primary tumor harbored two subclones with different oncogenic mutations in CTNNB1, which were both propagated to the same metastasis, raising the possibility that activation of wingless-type mouse mammary tumor virus integration site (WNT) signaling may be involved, as has been suggested by experimental models.
Asunto(s)
Melanoma/patología , Filogenia , Humanos , Melanoma/genética , Metástasis de la NeoplasiaRESUMEN
Cockayne syndrome (CS) is a human DNA repair-deficient disease that involves transcription coupled repair (TCR), in which three gene products, Cockayne syndrome A (CSA), Cockayne syndrome B (CSB), and ultraviolet stimulated scaffold protein A (UVSSA) cooperate in relieving RNA polymerase II arrest at damaged sites to permit repair of the template strand. Mutation of any of these three genes results in cells with increased sensitivity to UV light and defective TCR. Mutations in CSA or CSB are associated with severe neurological disease but mutations in UVSSA are for the most part only associated with increased photosensitivity. This difference raises questions about the relevance of TCR to neurological disease in CS. We find that CSB-mutated cells, but not UVSSA-deficient cells, have increased levels of intramitochondrial reactive oxygen species (ROS), especially when mitochondrial complex I is inhibited by rotenone. Increased ROS would result in oxidative damage to mitochondrial proteins, lipids, and DNA. CSB appears to behave as an electron scavenger in the mitochondria whose absence leads to increased oxidative stress. Mitochondrial ROS, however, did not cause detectable nuclear DNA damage even when base excision repair was blocked by an inhibitor of polyADP ribose polymerase. Neurodegeneration in Cockayne syndrome may therefore be associated with ROS-induced damage in the mitochondria, independent of nuclear TCR. An implication of our present results is that mitochondrial dysfunction involving ROS has a major impact on CS-B pathology, whereas nuclear TCR may have a minimal role.
Asunto(s)
Núcleo Celular/patología , Daño del ADN , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Fibroblastos/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/efectos de la radiación , Etidio/análogos & derivados , Etidio/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Fibroblastos/efectos de la radiación , Histonas/metabolismo , Humanos , Peróxido de Hidrógeno/toxicidad , Mitocondrias/efectos de los fármacos , Mitocondrias/efectos de la radiación , Oxidación-Reducción/efectos de los fármacos , Oxidación-Reducción/efectos de la radiación , Proteínas de Unión a Poli-ADP-Ribosa , Rotenona/toxicidad , Rayos UltravioletaRESUMEN
Mutations in genes on the nucleotide excision repair pathway are associated with diseases, such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy, that involve skin cancer and developmental and neurological symptoms. These mutations cause the defective repair of damaged DNA and increased transcription arrest but, except for skin cancer, the links between repair and disease have not been obvious. Widely different clinical syndromes seem to result from mutations in the same gene, even when the mutations result in complete loss of function. The mapping of mutations in recently solved protein structures has begun to clarify the links between the molecular defects and phenotypes, but the identification of additional sources of clinical variability is still necessary.
Asunto(s)
Síndrome de Cockayne/genética , Reparación del ADN/genética , Variación Genética/genética , Síndromes de Tricotiodistrofia/genética , Xerodermia Pigmentosa/genética , Animales , HumanosRESUMEN
Cockayne syndrome (CS) is a rare autosomal recessive neurodegenerative disease that is associated with mutations in either of two transcription-coupled DNA repair genes, CSA or CSB. Mice with a targeted mutation in the Csb gene (Cs-b(m/m)) exhibit a milder phenotype compared with human patients with mutations in the orthologous CSB gene. Mice mutated in Csb were crossed with mice lacking Xpc (Xp-c(-/-)), the global genome repair gene, to enhance the pathological symptoms. These Cs-b(m/m).Xp-c(-/-) mice were normal at birth but exhibited progressive failure to thrive, whole-body wasting, and ataxia and died at approximately postnatal day 21. Characterization of Cs-b(m/m).Xp-c(-/-) brains at postnatal stages demonstrated widespread reduction of myelin basic protein (MBP) and myelin in the sensorimotor cortex, the stratum radiatum, the corpus callosum, and the anterior commissure. Quantification of individual axons by electron microscopy showed a reduction in both the number of myelinated axons and the average diameter of myelin surrounding the axons. There were no significant differences in proliferation or oligodendrocyte differentiation between Cs-b(m/m).Xp-c(-/-) and Cs-b(m/+).Xp-c(-/-) mice. Rather, Cs-b(m/m).Xp-c(-/-) oligodendrocytes were unable to generate sufficient MBP or to maintain the proper myelination during early development. Csb is a multifunctional protein regulating both repair and the transcriptional response to reactive oxygen through its interaction with histone acetylase p300 and the hypoxia-inducible factor (HIF)1 pathway. On the basis of our results, combined with that of others, we suggest that in Csb the transcriptional response predominates during early development, whereas a neurodegenerative response associated with repair deficits predominates in later life.
Asunto(s)
Síndrome de Cockayne/genética , Vaina de Mielina/química , Animales , Conducta Animal , Peso Corporal , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Ratones , Modelos Genéticos , Mutación , Vaina de Mielina/genética , Oligodendroglía/citología , Tamaño de los Órganos , Transcripción GenéticaRESUMEN
Protein-trafficking pathways are targeted here in human melanoma cells using methods independent of oncogene mutational status, and the ability to up-regulate and down-regulate tumor treatment sensitivity is demonstrated. Sensitivity of melanoma cells to cis-diaminedichloroplatinum II (cDDP, cis-platin), carboplatin, dacarbazine, or temozolomide together with velaparib, an inhibitor of poly (ADP ribose) polymerase 1, is increased by up to 10-fold by targeting genes that regulate both protein trafficking and the formation of melanosomes, intracellular organelles unique to melanocytes and melanoma cells. Melanoma cells depleted of either of the protein-trafficking regulators vacuolar protein sorting 33A protein (VPS33A) or cappuccino protein (CNO) have increased nuclear localization of cDDP, increased nuclear DNA damage by platination, and increased apoptosis, resulting in increased treatment sensitivity. Depleted cells also exhibit a decreased proportion of intracellular, mature melanosomes compared with undepleted cells. Modulation of protein trafficking via cell-surface signaling by binding the melanocortin 1 receptor with the antagonist agouti-signaling protein decreased the proportion of mature melanosomes formed and increased cDDP sensitivity, whereas receptor binding with the agonist melanocyte-stimulating hormone resulted in an increased proportion of mature melanosomes formed and in decreased sensitivity (i.e., increased resistance) to cDDP. Mutation of the protein-trafficking gene Hps6, known to impair the formation of mature melanosomes, also increased cDDP sensitivity. Together, these results indicate that targeting protein-trafficking molecules markedly increases melanoma treatment sensitivity and influences the degree of melanosomes available for sequestration of therapeutic agents.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/genética , Melanoma/tratamiento farmacológico , Melanosomas/efectos de los fármacos , Proteínas de Transporte Vesicular/deficiencia , Secuencia de Aminoácidos , Carboplatino/farmacología , Línea Celular Tumoral , Reparación del ADN , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Humanos , Immunoblotting , Microscopía Electrónica , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación/genética , Transporte de Proteínas/genética , Interferencia de ARN , Receptor de Melanocortina Tipo 1/metabolismo , Temozolomida , Regulación hacia Arriba/efectos de los fármacos , Proteínas de Transporte Vesicular/genéticaRESUMEN
Nucleotide-excision repair diseases exhibit cancer, complex developmental disorders and neurodegeneration. Cancer is the hallmark of xeroderma pigmentosum (XP), and neurodegeneration and developmental disorders are the hallmarks of Cockayne syndrome and trichothiodystrophy. A distinguishing feature is that the DNA-repair or DNA-replication deficiencies of XP involve most of the genome, whereas the defects in CS are confined to actively transcribed genes. Many of the proteins involved in repair are also components of dynamic multiprotein complexes, transcription factors, ubiquitylation cofactors and signal-transduction networks. Complex clinical phenotypes might therefore result from unanticipated effects on other genes and proteins.
Asunto(s)
Neoplasias/genética , Xerodermia Pigmentosa/genética , Niño , Síndrome de Cockayne/genética , Síndrome de Cockayne/fisiopatología , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Discapacidades del Desarrollo/genética , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Humanos , Neoplasias/etiología , Neoplasias/fisiopatología , Transcripción Genética/genética , Xerodermia Pigmentosa/complicaciones , Xerodermia Pigmentosa/fisiopatología , Proteína de la Xerodermia Pigmentosa del Grupo AAsunto(s)
Química , Premio Nobel , Reparación del ADN , Historia del Siglo XX , Historia del Siglo XXI , Reino Unido , Estados UnidosRESUMEN
The phosphorylation of H2Ax on its S139 site, γH2Ax, is important during DNA double-strand repair and is considered necessary for assembly of repair complexes, but its functional role after other kinds of DNA damage is less clear. We have measured the survival of isogenic mouse cell lines with the H2Ax gene knocked out, and replaced with wild-type or mutant (S139A) H2Ax genes, exposed to a range of agents with varied mechanisms of DNA damage. Knockout and mutant cells were sensitive to γ-rays, etoposide, temozolamide, and endogenously generated reactive oxygen species, each of which can include double-strand breaks among their spectra of DNA lesions. The absence or mutation of H2Ax had no influence on sensitivity to cisplatin or mitomycin C. Although UV light induced the highest levels of γH2Ax, mutation of S139 had no influence on UV sensitivity or the UV DNA damage response. Complete loss of H2Ax reduced the survival of cells exposed to UV light and reduced pChk1 induction, suggesting that sites other than S139 may impact the ATR-pChk1 pathway. The relative intensity of γH2Ax measured in Western blots in wild-type cells did not correlate with the functional importance of γH2Ax. The use of γH2Ax as a general biomarker of DNA damage is therefore potentially misleading because it is not an unambiguous indicator of double-strand breaks, and a significant fraction of DNA repair, especially involving nucleotide excision or crosslink repair, can occur without functional involvement of γH2Ax.
Asunto(s)
Daño del ADN , Reparación del ADN/genética , Histonas/fisiología , Animales , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Técnicas de Inactivación de Genes , Histonas/genética , Ratones , Mutación , Fosforilación , Proteínas Quinasas/metabolismo , TransgenesRESUMEN
Squamous cell carcinomas (SCCs) are one of the most frequent forms of human malignancy, but, other than TP53 mutations, few causative somatic aberrations have been identified. We identified NOTCH1 or NOTCH2 mutations in ~75% of cutaneous SCCs and in a lesser fraction of lung SCCs, defining a spectrum for the most prevalent tumor suppressor specific to these epithelial malignancies. Notch receptors normally transduce signals in response to ligands on neighboring cells, regulating metazoan lineage selection and developmental patterning. Our findings therefore illustrate a central role for disruption of microenvironmental communication in cancer progression. NOTCH aberrations include frameshift and nonsense mutations leading to receptor truncations as well as point substitutions in key functional domains that abrogate signaling in cell-based assays. Oncogenic gain-of-function mutations in NOTCH1 commonly occur in human T-cell lymphoblastic leukemia/lymphoma and B-cell chronic lymphocytic leukemia. The bifunctional role of Notch in human cancer thus emphasizes the context dependency of signaling outcomes and suggests that targeted inhibition of the Notch pathway may induce squamous epithelial malignancies.
Asunto(s)
Carcinoma de Células Escamosas/genética , Comunicación Celular/genética , Neoplasias Pulmonares/genética , Receptor Notch1/genética , Receptor Notch2/genética , Transducción de Señal/genética , Neoplasias Cutáneas/genética , Secuencia de Bases , Codón sin Sentido/genética , Ensayo de Cambio de Movilidad Electroforética , Humanos , Escala de Lod , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Previous studies showed conflicting and inconsistent results regarding the effect of anatomic location of the melanoma on sentinel lymph node (SLN) positivity and/or survival. This study was conducted to evaluate and compare the effect of the anatomic locations of primary melanoma on long-term clinical outcomes. METHODS: All consecutive cutaneous melanoma patients (n=2,079) who underwent selective SLN dissection (SLND) from 1993 to 2009 in a single academic tertiary-care medical center were included. SLN positive rate, disease-free survival (DFS), and overall survival (OS) were determined. Kaplan-Meier survival, univariate, and multivariate analyses were performed to determine predictive factors for SLN status, DFS, and OS. RESULTS: Head and neck melanoma (HNM) had the lowest SLN-positive rate at 10.8% (16.8% for extremity and 19.3% for trunk; P=0.002) but had the worst 5-year DFS (P<0.0001) and 5-year OS (P<0.0001) compared with other sites. Tumor thickness (P<0.001), ulceration (P<0.001), HNM location (P=0.001), mitotic rate (P<0.001), and decreasing age (P<0.001) were independent predictive factors for SLN-positivity. HNM with T3 or T4 thickness had significantly lower SLN positive rate compared with other locations (P≤0.05). Also, on multivariate analysis, HNM location versus other anatomic sites was independently predictive of decreased DFS and OS (P<0.001). By Kaplan-Meier analysis, HNM was associated significantly with the worst DFS and OS. CONCLUSIONS: Primary melanoma anatomic location is an independent predictor of SLN status and survival. Although HNM has a decreased SLN-positivity rate, it shows a significantly increased risk of recurrence and death as compared with other sites.
Asunto(s)
Extremidades/patología , Neoplasias de Cabeza y Cuello/mortalidad , Melanoma/mortalidad , Biopsia del Ganglio Linfático Centinela , Neoplasias Cutáneas/mortalidad , Extremidades/cirugía , Femenino , Estudios de Seguimiento , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/cirugía , Humanos , Metástasis Linfática , Masculino , Melanoma/patología , Melanoma/cirugía , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/cirugía , Tasa de Supervivencia , Centros de Atención TerciariaRESUMEN
UV irradiation induces histone variant H2AX phosphorylated on serine 139 (gammaH2AX) foci and high levels of pan-nuclear gammaH2AX staining without foci, but the significance of this finding is still uncertain. We examined the formation of gammaH2AX and 53BP1 that coincide at sites of double-strand breaks (DSBs) after ionizing radiation. We compared UV irradiation and treatment with etoposide, an agent that causes DSBs during DNA replication. We found that during DNA replication, UV irradiation induced at least three classes of gammaH2AX response: a minority of gammaH2AX foci colocalizing with 53BP1 foci that represent DSBs at replication sites, a majority of gammaH2AX foci that did not colocalize with 53BP1 foci, and cells with high levels of pan-nuclear gammaH2AX without foci of either gammaH2AX or 53BP1. Ataxia-telangiectasia mutated kinase and JNK mediated the UV-induced pan-nuclear gammaH2Ax, which preceded and paralleled UV-induced S phase apoptosis. These high levels of pan-nuclear gammaH2AX were further increased by loss of the bypass polymerase Pol eta and inhibition of ataxia-telangiectasia and Rad3-related, but the levels required the presence of the damage-binding proteins of excision repair xeroderma pigmentosum complementation group A and C proteins. DSBs, therefore, represent a small variable fraction of UV-induced gammaH2AX foci dependent on repair capacity, and they are not detected within high levels of pan-nuclear gammaH2AX, a preapoptotic signal associated with ATM- and JNK-dependent apoptosis during replication. The formation of gammaH2AX foci after treatment with DNA-damaging agents cannot, therefore, be used as a direct measure of DSBs without independent corroborating evidence.
Asunto(s)
Apoptosis , Núcleo Celular/metabolismo , Roturas del ADN de Doble Cadena , Regulación de la Expresión Génica , Histonas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Línea Celular , Separación Celular , ADN Polimerasa Dirigida por ADN/metabolismo , Citometría de Flujo , Humanos , Cinética , Fase S , Proteína 1 de Unión al Supresor Tumoral P53 , Rayos UltravioletaRESUMEN
XPC, the main damage-recognition protein responsible for nucleotide excision repair of UVB damage to DNA, is lost or mutated in xeroderma pigmentosum group C (XP-C), a rare inherited disease characterized by high incidence and early onset of non-melanoma and melanoma skin cancers. The high incidence of skin cancers in XP-C patients suggests that loss of expression of XPC protein might also provide a selective advantage for initiation and progression of similar cancers in non XP-C patients in the general population. To test whether XPC is selectively lost in squamous cell carcinomas from non XP-C patients, we examined XPC expression by immunohistochemistry on a tissue microarray with 244 tissue cores, including in situ and invasive squamous-cell carcinomas (SCCs), keratoacanthoma (KA), and normal skin samples from both immunocompetent and immunosuppressed patients. We found that XPC expression was lost in 49% of invasive squamous cell carcinomas from immunocompetent patients and 59% from immunosuppressed patients. Loss of expression was correlated with deletions of chromosomal 3p and mutations in the XPC gene. The XPC gene is consequently inactivated or lost in almost half of squamous cell carcinomas from non XP-C patients. Loss or mutation of XPC may be an early event during skin carcinogenesis that provides a selective advantage for initiation and progression of squamous cell carcinomas in non XP-C patients.