RESUMEN
Primary cutaneous anaplastic large cell lymphoma (pcALCL), a hematological neoplasm caused by skin-homing CD30+ malignant T cells, is part of the spectrum of primary cutaneous CD30+ lymphoproliferative disorders. To date, only a small number of molecular alterations have been described in pcALCL and, so far, no clear unifying theme that could explain the pathogenetic origin of the disease has emerged among patients. In order to clarify the pathogenetic basis of pcALCL, we performed high-resolution genetic profiling (genome/transcriptome) of this lymphoma (n=12) by using whole-genome sequencing, whole-exome sequencing and RNA sequencing. Our study, which uncovered novel genomic rearrangements, copy number alterations and small-scale mutations underlying this malignancy, revealed that the cell cycle, T-cell physiology regulation, transcription and signaling via the PI-3-K, MAPK and G-protein pathways are cellular processes commonly impacted by molecular alterations in patients with pcALCL. Recurrent events affecting cancer-associated genes included deletion of PRDM1 and TNFRSF14, gain of EZH2 and TNFRSF8, small-scale mutations in LRP1B, PDPK1 and PIK3R1 and rearrangements involving GPS2, LINC-PINT and TNK1. Consistent with the genomic data, transcriptome analysis uncovered upregulation of signal transduction routes associated with the PI-3-K, MAPK and G-protein pathways (e.g., ERK, phospholipase C, AKT). Our molecular findings suggest that inhibition of proliferation-promoting pathways altered in pcALCL (particularly PI-3-K/AKT signaling) should be explored as potential alternative therapy for patients with this lymphoma, especially, for cases that do not respond to first-line skin-directed therapies or with extracutaneous disease.
Asunto(s)
Linfoma Anaplásico de Células Grandes , Linfoma Anaplásico Cutáneo Primario de Células Grandes , Trastornos Linfoproliferativos , Neoplasias Cutáneas , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Proteínas Fetales , Humanos , Antígeno Ki-1 , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/patología , Trastornos Linfoproliferativos/patología , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas c-akt , Neoplasias Cutáneas/metabolismoRESUMEN
Primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma (pcAECyTCL) is a rare variant of cutaneous T-cell lymphoma with an aggressive clinical course and a very poor prognosis. Until now, neither a systematic characterization of genetic alterations driving pcAECyTCL has been performed, nor effective therapeutic regimes for patients have been defined. Here, we present the first highresolution genetic characterization of pcAECyTCL by using wholegenome and RNA sequencing. Our study provides a comprehensive description of genetic alterations (i.e., genomic rearrangements, copy number alterations and small-scale mutations) with pathogenic relevance in this lymphoma, including events that recurrently impact genes with important roles in the cell cycle, chromatin regulation and the JAKSTAT pathway. In particular, we show that mutually exclusive structural alterations involving JAK2 and SH2B3 predominantly underlie pcAECyTCL. In line with the genomic data, transcriptome analysis uncovered upregulation of the cell cycle, JAK2 signaling, NF-κB signaling and a high inflammatory response in this cancer. Functional studies confirmed oncogenicity of JAK2 fusions identified in pcAECyTCL and their sensitivity to JAK inhibitor treatment. Our findings strongly suggest that overactive JAK2 signaling is a central driver of pcAECyTCL, and consequently, patients with this neoplasm would likely benefit from therapy with JAK2 inhibitors such as Food and Drug Adminstration-approved ruxolitinib.
Asunto(s)
Linfoma Cutáneo de Células T , Neoplasias Cutáneas , Linfocitos T CD8-positivos/metabolismo , Humanos , Janus Quinasa 2/genética , Linfoma Cutáneo de Células T/genética , Piel/patología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Linfocitos T Citotóxicos/metabolismoRESUMEN
BACKGROUND: Individuals from melanoma-prone families have similar or reduced sun-protective behaviors compared to the general population. Studies on trends in sun-related behaviors have been temporally and geographically limited. METHODS: Individuals from an international consortium of melanoma-prone families (GenoMEL) were retrospectively asked about sunscreen use, sun exposure (time spent outside), sunburns, and sunbed use at several timepoints over their lifetime. Generalized linear mixed models were used to examine the association between these outcomes and birth cohort defined by decade spans, after adjusting for covariates. RESULTS: A total of 2407 participants from 547 families across 17 centers were analyzed. Sunscreen use increased across subsequent birth cohorts, and although the likelihood of sunburns increased until the 1950s birth cohort, it decreased thereafter. Average sun exposure did not change across the birth cohorts, and the likelihood of sunbed use increased in more recent birth cohorts. We generally did not find any differences in sun-related behavior when comparing melanoma cases to non-cases. Melanoma cases had increased sunscreen use, decreased sun exposure, and decreased odds of sunburn and sunbed use after melanoma diagnosis compared to before diagnosis. CONCLUSIONS: Although sunscreen use has increased and the likelihood of sunburns has decreased in more recent birth cohorts, individuals in melanoma-prone families have not reduced their overall sun exposure and had an increased likelihood of sunbed use in more recent birth cohorts. These observations demonstrate partial improvements in melanoma prevention and suggest that additional intervention strategies may be needed to achieve optimal sun-protective behavior in melanoma-prone families.
Asunto(s)
Melanoma , Neoplasias Cutáneas , Quemadura Solar , Humanos , Melanoma/epidemiología , Melanoma/prevención & control , Estudios Retrospectivos , Neoplasias Cutáneas/epidemiología , Neoplasias Cutáneas/prevención & control , Quemadura Solar/epidemiología , Quemadura Solar/prevención & control , Protectores Solares/uso terapéuticoRESUMEN
In burn patients, wound healing is often accompanied by hypertrophic scarring (HTS), resulting in both functional and aesthetic problems. HTSs are characterized by abundant presence of myofibroblasts (MFs) residing in the dermis. HTS development and MF persistence is primarily regulated by TGF-ß signalling. A promising method to target the transforming growth factor receptor I (TGFßRI; also known as activin-like kinase 5 (ALK5)) is by making use of exon skipping through antisense oligonucleotides. In HTS the distinguishing border between the papillary dermis and the reticular dermis is completely abrogated, thus exhibiting a one layered dermis containing a heterogenous fibroblast population, consisting of papillary fibroblasts (PFs), reticular fibroblasts (RFs) and MFs. It has been proposed that PFs, as opposed to RFs, exhibit anti-fibrotic properties. Currently, it is still unclear which fibroblast subtype is most affected by exon skipping treatment. Therefore, the aim of this study was to investigate the effect of TGFßRI inhibition by exon skipping in PF, RF and HTS fibroblast monocultures. Morphological analyses revealed the presence of a PF-like population after exon skipping in the different fibroblast cultures. This observation was further confirmed by the expression of genes specific for PFs, demonstrated by qPCR analyses. Further investigations on mRNA and protein level revealed that indeed MFs and to a lesser extent RFs are targeted by exon skipping. Furthermore, collagen gel contraction analysis showed that ALK5 exon skipping reduced TGF-ß- induced contraction together with decreased alpha-smooth muscle actin expression levels. In conclusion, we show for the first time that exon skipping primarily targets pro-fibrotic fibroblasts. This could be a promising step towards reduced HTS development of burn tissue.
Asunto(s)
Quemaduras , Cicatriz Hipertrófica , Receptor Tipo I de Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Actinas/genética , Quemaduras/patología , Quemaduras/terapia , Células Cultivadas , Cicatriz Hipertrófica/genética , Cicatriz Hipertrófica/patología , Cicatriz Hipertrófica/terapia , Exones , Fibroblastos/patología , Fibrosis , Humanos , Miofibroblastos/patología , Oligonucleótidos AntisentidoRESUMEN
Cutaneous T-cell lymphomas and leukemias (CTCLs) are a heterogeneous group of extranodal non-Hodgkin's lymphomas. These are characterized by an accumulation of malignant CD4+ T-lymphocytes in the skin, lymph nodes, and peripheral blood. Novel treatment options are needed for patients who progress to advanced stage disease. Cucurbitacin I has previously shown promising results in Sézary syndrome (Sz). A plethora of cucurbitacins, however, have not yet been tested in CTCL. Herein, we investigated the effect of cucurbitacin E and I in two CTCL cell lines. We show that both cucurbitacins decrease viability and cause apoptosis in these cell lines, although HuT-78 was more affected than SeAx (IC50 of 17.38 versus 22.01 µM for cucurbitacin E and 13.36 versus 24.47 µM for cucurbitacin I). Moreover, both cucurbitacins decrease viability of primary cells of a Sz patient (56.46% for cucurbitacin E and 59.07% for cucurbitacin I). Furthermore, while JAK2 inhibition leads to decreased viability in SeAx cells (IC50 of 9.98 and 29.15 µM for AZD1480 and ruxolitinib respectively), both JAK1 and JAK3 do not. This suggests that JAK2 has a preferential role in promoting survival. Western blotting in SeAx cells revealed that both cucurbitacins inhibit STAT3 activation (P < 0.0001), while only cucurbitacin I inhibits STAT5 activation (P = 0.05). This suggests that STAT3 plays a preferential role in the mechanism of action of these cucurbitacins. Nevertheless, a role of STAT5 and JAK2 cannot be excluded and should be explored further. This knowledge could contribute to the development of effective therapies for CTCL and other malignancies involving dysfunction of the JAK/STAT pathway.
RESUMEN
BACKGROUND: In burn patients, wound healing is often accompanied by hypertrophic scar (HS) development, resulting in both functional and aesthetic problems. HSs are characterised by abundant presence of myofibroblasts that contribute to overproduction of extracellular matrix (ECM) that is regulated by the TGF-ß signalling pathway. Studies have shown that inhibition of TGF-ß receptors in fibrotic diseases reduces the fibrotic load. In the present study, we aim to inactivate ALK5, also known as TGF-ß receptor I, in human HS fibroblasts by exon skipping using antisense oligonucleotides (AONs). METHODS: HS biopsies were used to isolate and set up fibroblast monocultures. AONs targeting ALK5 were supplemented to the fibroblast cultures to induce exon skipping, while pharmacological ALK5 inhibition was induced using SB431542. AON delivery in HS fibroblasts was examined using immunofluorescence (IF), while TGF-ß signalling downstream targets, such as Smad2/3, PAI-1, ACTA2, COL1A1 and COL3A1, were analysed using touchdown polymerase chain reaction (PCR), quantitative PCR (qPCR), IF or western blotting. RESULTS: Our data clearly demonstrate that AONs were successfully delivered in the nuclei of HS fibroblasts and that functional exon skipping of ALK5 took place as confirmed with touchdown PCR and qPCR. In addition, exon skipping affected the expression of ECM-related genes, such as type I/III collagens, PAI-1 and CCN2. Moreover, AON treatment did not affect the migration of HS fibroblasts in a model for wound healing. CONCLUSION: Exon skipping is a promising tool to modulate the TGF-ß signalling pathway in HS. This would open a therapeutic window for the treatment of patients suffering from HSs.
RESUMEN
Quantifying T cells accurately in a variety of tissues of benign, inflammatory, or malignant origin can be of great importance in a variety of clinical applications. Flow cytometry and immunohistochemistry are considered to be gold-standard methods for T-cell quantification. However, these methods require fresh, frozen, or fixated cells and tissue of a certain quality. In addition, conventional and droplet digital PCR (ddPCR), whether followed by deep sequencing techniques, have been used to elucidate T-cell content by focusing on rearranged T-cell receptor (TCR) genes. These approaches typically target the whole TCR repertoire, thereby supplying additional information about TCR use. We alternatively developed and validated two novel generic single duplex ddPCR assays to quantify T cells accurately by measuring loss of specific germline TCR loci and compared them with flow cytometry-based quantification. These assays target sequences between the Dδ2 and Dδ3 genes (TRD locus) and Dß1 and Jß1.1 genes (TRB locus) that become deleted systematically early during lymphoid differentiation. Because these ddPCR assays require small amounts of DNA instead of freshly isolated, frozen, or fixated material, initially unanalyzable (scarce) specimens can be assayed from now on, supplying valuable information about T-cell content. Our ddPCR method provides a novel and sensitive way for quantifying T cells relatively fast, accurate, and independent of the cellular context.
Asunto(s)
Alelos , Mutación de Línea Germinal , Reacción en Cadena de la Polimerasa/métodos , Receptores de Antígenos de Linfocitos T/genética , Eliminación de Secuencia , Linfocitos T/metabolismo , Humanos , Reacción en Cadena de la Polimerasa/normas , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Lgr6+ cells have been identified as a novel class of proliferating (Ki67+) stem cells in mouse epidermis. We investigated their response to UV exposure in Lgr6-EGFP-Ires-CreERT2/R26R-LacZ haired and hairless mice and whether they become initiating cells of UV- or chemically induced skin tumors. UV overexposure erased Lgr6+ cells (EGFP+) from the interfollicular epidermis (IFE), but - as after wounding - they apparently repopulated the IFE from the hair follicles. Under sub-sunburn chronic UV exposure, Lgr6+ cells and their progeny (LacZ+ after pulse of tamoxifen) diminished strongly in the IFE. Although the inter-tumoral IFE clearly showed Lgr6 progeny, none of the UV- or chemically induced tumors (n = 22 and 41, respectively) appeared to be clonal expansions of Lgr6+ stem cells; i.e. no Lgr6+ cells or progeny in the proliferating tumor bulk. In checking for promoter methylation we found it to occur stochastically for the EGFP-Cre cassette. Lgr6 mRNA measured by qPCR was found to be diminished in skin tumors (also in UV tumors from wt type mice). The ratio of Lgr6/Ki67 was significantly reduced, pointing at a loss of Lgr6+ cells from the proliferative pool. Our data show that Lgr6+ cells are not major tumor-initiating cells in skin carcinogenesis.
Asunto(s)
Epidermis/metabolismo , Receptores Acoplados a Proteínas G/genética , Neoplasias Cutáneas/genética , Células Madre/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Proliferación Celular/efectos de la radiación , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/efectos de la radiación , Metilación de ADN/efectos de los fármacos , Metilación de ADN/efectos de la radiación , Epidermis/efectos de los fármacos , Epidermis/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Folículo Piloso/efectos de los fármacos , Folículo Piloso/metabolismo , Folículo Piloso/efectos de la radiación , Ratones Pelados , Ratones Transgénicos , Regiones Promotoras Genéticas/genética , Receptores Acoplados a Proteínas G/metabolismo , Piel/efectos de los fármacos , Piel/metabolismo , Piel/efectos de la radiación , Neoplasias Cutáneas/metabolismo , Rayos UltravioletaRESUMEN
Actively proliferating Lgr5+ skin stem cells are found deep in the hair follicle (HF). These cells renew the HF and drive its expansion in anagen phase. Their long residence and continuous mitotic activity make them prime candidates to transform into skin tumor-initiating cells. This was investigated by subjecting Lgr5-EGFP-Ires-CreERT2/R26R-LacZ mice (haired and hairless) to chemical and UV carcinogenic regimens. In the course of these regimens Lgr5+ cells (EGFP+) remained exclusively located in HFs, and in deep-seated cysts of hairless skin. In haired mice, progeny of Lgr5+ stem cells (LacZ+ after a pulse of tamoxifen) appeared in the interfollicular epidermis upon UV-induced sunburn and in TPA-induced hyperplasia. In hairless mice the progeny remained located in deep-seated cysts and in HF remnants. Progeny in hairless skin was only detected interfollicularly at a late stage, in between outgrowing tumors. Lgr5+ stem cells were absent in the ultimate tumor masses, and no tumor appeared to be a (clonal) expansion of Lgr5+ cells (52 tumors with tamoxifen at the start of carcinogenesis, 42 tumors with tamoxifen late during tumor outgrowth). In contrast to CD34/K15+ quiescent bulge stem cells, actively proliferating Lgr5+ stem cells do therefore not appear to be tumor drivers in experimental skin carcinogenesis.
Asunto(s)
Transformación Celular Neoplásica/patología , Folículo Piloso/patología , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Cutáneas/patología , Células Madre/patología , Animales , Epidermis/patología , Ratones , Ratones Pelados , Ratones TransgénicosRESUMEN
Differentiation between Sézary syndrome and erythrodermic inflammatory dermatoses can be challenging, and a number of studies have attempted to identify characteristic immunophenotypic changes and molecular biomarkers in Sézary cells that could be useful as additional diagnostic criteria. In this European multicenter study, the sensitivity and specificity of these immunophenotypic and recently proposed but unconfirmed molecular biomarkers in Sézary syndrome were investigated. Peripheral blood CD4(+) T cells from 59 patients with Sézary syndrome and 19 patients with erythrodermic inflammatory dermatoses were analyzed for cell surface proteins by flow cytometry and for copy number alterations and differential gene expression using custom-made quantitative PCR plates. Experiments were performed in duplicate in two independent centers using standard operating procedures with almost identical results. Sézary cells showed MYC gain (40%) and MNT loss (66%); up-regulation of DNM3 (75%), TWIST1 (69%), EPHA4 (66%), and PLS3 (66%); and down-regulation of STAT4 (91%). Loss of CD26 (≥80% CD4(+) T cells) and/or CD7 (≥40% CD4(+) T cells) and combination of altered expression of STAT4, TWIST1, and DNM3 or PLS3 could distinguish, respectively, 83% and 98% of patients with Sézary syndrome from patients with erythrodermic inflammatory dermatoses with 100% specificity. These additional diagnostic panels will be useful adjuncts in the differential diagnosis of Sézary syndrome versus erythrodermic inflammatory dermatoses.
Asunto(s)
Biomarcadores/análisis , Inmunofenotipificación/normas , Síndrome de Sézary/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos T CD4-Positivos/citología , Diagnóstico Diferencial , Europa (Continente) , Femenino , Citometría de Flujo , Dosificación de Gen , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inflamación , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Síndrome de Sézary/inmunología , Enfermedades de la Piel/diagnóstico , Enfermedades de la Piel/inmunologíaRESUMEN
EPHA4 belongs to the largest subfamily of receptor tyrosine kinases. In addition to its function during development, overexpression of EPHA4 in tumors has been correlated with increased proliferation, migration and poor survival. Several genome-wide transcription profiling studies have demonstrated high EPHA4 expression in Sézary syndrome (SS), a leukemic variant of cutaneous CD4+ T-cell lymphoma (CTCL) with an aggressive clinical course and poor prognosis. In this study we set out to explore the functional role of EPHA4 in SS. Both high EPHA4 mRNA and protein expression was found in circulating SS-cells of patients compared to healthy CD4+ T-cells. However, using a phosphospecific EPHA4 antibody, phosphorylation of the EPHA4 kinase domain was not detected in either circulating or skin residing SS cells. Moreover, treatment with the phosphatase inhibitor pervanadate did not result in detectable phosphorylation of the EPHA4 kinase domain, in either SS cells or in healthy CD4+ T-cells. Thus, the results from our study confirm high EPHA4 expression in SS cells both on the mRNA and protein levels, making EPHA4 a good diagnostic marker. However, the overexpressed EPHA4 does not appear to be functionally active and its overexpression might be secondary to other oncogenic drivers in SS, like STAT3 and TWIST1.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Proteínas Nucleares/metabolismo , Receptor EphA4/metabolismo , Factor de Transcripción STAT3/metabolismo , Síndrome de Sézary/metabolismo , Neoplasias Cutáneas/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Anciano , Western Blotting , Linfocitos T CD4-Positivos/patología , Estudios de Casos y Controles , Femenino , Humanos , Técnicas para Inmunoenzimas , Masculino , Proteínas Nucleares/genética , Fosforilación , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor EphA4/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/genética , Síndrome de Sézary/genética , Síndrome de Sézary/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Células Tumorales Cultivadas , Proteína 1 Relacionada con Twist/genéticaRESUMEN
Sézary syndrome is an aggressive cutaneous T-cell lymphoma. The malignant cells (Sézary cells) are present in skin, lymph nodes, and blood, and express constitutively activated signal transducer and activator of transcription (STAT)3. STAT3 can be activated by IL-21 in vitro and the IL-21 gene itself is a STAT3 target gene, thereby creating an autocrine positive feedback loop that might serve as a therapeutic target. Sézary cells underwent apoptosis when incubated with Stattic, a selective STAT3 inhibitor. STAT3 activation in Sézary cells did not affect expression of the supposed anti-apoptotic STAT3 target genes BCL2, BCL-xL, and SURVIVIN, whereas expression of (proto)oncogenes miR-21, TWIST1, MYC, and PIM1 was significantly increased. CD3/CD28-mediated activation of Sézary cells induced IL-21 expression, accompanied by STAT3 activation and increased proliferation. Blocking IL-21 in CD3/CD28-activated cells had no effects, whereas Stattic abrogated IL-21 expression and cell proliferation. Thus, specific inhibition of STAT3 is highly efficient in the induction of apoptosis of Sézary cells, likely mediated via the regulation of (proto)oncogenes. In contrast, blocking IL-21 alone seems insufficient to affect STAT3 activation, cell proliferation, or apoptosis. These data provide further insights into the pathogenic role of STAT3 in Sézary syndrome and strengthen the notion that STAT3 represents a promising therapeutic target in this disease.
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Interleucinas/fisiología , Factor de Transcripción STAT3/fisiología , Síndrome de Sézary/fisiopatología , Transducción de Señal/fisiología , Neoplasias Cutáneas/fisiopatología , Anciano , Anciano de 80 o más Años , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Óxidos S-Cíclicos/farmacología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptores de Interleucina-21/antagonistas & inhibidores , Receptores de Interleucina-21/efectos de los fármacos , Receptores de Interleucina-21/fisiología , Proteínas Recombinantes de Fusión/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/efectos de los fármacos , Síndrome de Sézary/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/tratamiento farmacológicoRESUMEN
Cutaneous melanoma, a type of skin tumor originating from melanocytes, often develops from premalignant naevoid lesions via a gradual transformation process driven by an accumulation of (epi)genetic lesions. These dysplastic naevi display altered morphology and often proliferation of melanocytes. Additionally, melanocytes in dysplastic naevi show structural mitochondrial and melanosomal alterations and have elevated reactive oxygen species (ROS) levels. For this study we performed genome-wide expression and proteomic analysis of melanocytes from dysplastic naevus (DNMC) and adjacent normal skin (MC) from 18 patients. Whole genome expression profiles of the DNMC and MC of each individual patient subjected to GO-based comparative statistical analysis yielded significantly differentially expressed GO classes including "organellar ribosome," "mitochondrial ribosome," "hydrogen ion transporter activity," and "prefoldin complex." Validation of 5 genes from these top GO classes revealed a heterogeneous differential expression pattern. Proteomic analysis demonstrated differentially expressed proteins in DNMC that are involved in cellular metabolism, detoxification, and cytoskeletal organization processes, such as GTP-binding Rho-like protein CDC42, glutathione-S-transferase omega-1 and prolyl 4-hydroxylase. Collectively these results point to deregulation of cellular processes, such as metabolism and protein synthesis, consistent with the observed elevated oxidative stress levels in DNMC potentially resulting in oxidative DNA damage in these cells.
RESUMEN
Sézary syndrome (SS) is an aggressive cutaneous T-cell lymphoma with CD4+ tumor cells localized in the skin, lymph nodes and peripheral blood. Characteristic molecular aberrancies in SS have been identified; however, paucity of functional models severely hampered the translation of these observations into pathogenic mechanisms, and subsequent validation of novel therapeutic targets. We therefore developed a mouse model for SS using intrahepatic injection of SS cells in newborn immunodeficient RAG2(-/-) γc(-/-) mice that are completely devoid of T-, B- and NK-cell activity. Injection of the SS cell line SeAx led to long-term and reproducible systemic repopulation of the mice. Injection of mice with the SS cell line HuT-78 led to the death of the mice owing to massive growth of internal tumors. Four weeks after injection of primary SS cells, human CD3+ T cells could be tracked back in the liver, peripheral blood, lymph nodes, spleen and skin of the mice, although the engraftment rate varied when using cells from different patients. In conclusion, we demonstrate that injection of SS cell lines or primary cells in newborn RAG2(-/-) γc(-/-) mice results in long-term systemic repopulation of the mice, thereby providing a novel mouse model for Sézary syndrome.
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Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Síndrome de Sézary/patología , Neoplasias Cutáneas/patología , Animales , Linfocitos T CD4-Positivos/trasplante , Humanos , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Antígeno Ki-67/metabolismo , Ganglios Linfáticos/metabolismo , Ratones , Ratones Noqueados , Ratones SCID , Trasplante de Neoplasias , Síndrome de Sézary/inmunología , Síndrome de Sézary/metabolismo , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismoRESUMEN
NOTCH signaling is important for development and tissue homeostasis and is activated in many human cancers. We investigated a role for NOTCH1 signaling in Sézary syndrome (SS), a cutaneous T-cell lymphoma in which CD4+ tumor cells (Sézary cells) are present in the skin, lymph nodes, and peripheral blood. We show consistent expression of activated NOTCH1 by Sézary cells isolated from peripheral blood of SS patients, as well as the SS-derived cell lines SeAx and HuT78. In addition, immunohistochemical stainings of skin biopsies from SS patients showed consistent expression of nuclear NOTCH1 and its downstream target hairy/enhancer of split-1 (HES1) by Sézary cells. We demonstrate that this persistent NOTCH1 activation is not caused by mutations in the coding regions of NOTCH1 and F-box and WD40 domain protein 7 (FBWX7) genes. Inhibition of NOTCH1 signaling by gamma secretase inhibitors decreased cellular viability and induced apoptosis of Sézary cells. These observations argue that NOTCH1 signaling is functionally involved in the pathogenesis of SS, and inhibition of NOTCH1 signaling represents a new therapeutic target for the treatment of SS.
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Receptor Notch1/metabolismo , Síndrome de Sézary/metabolismo , Transducción de Señal/fisiología , Neoplasias Cutáneas/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Benzodiazepinonas/farmacología , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Femenino , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Oligopéptidos/farmacología , Fosfohidrolasa PTEN/genética , Estructura Terciaria de Proteína , Receptor Notch1/química , Receptor Notch1/genética , Factor de Transcripción STAT3/metabolismo , Síndrome de Sézary/tratamiento farmacológico , Síndrome de Sézary/genética , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Factor de Transcripción HES-1 , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Mycosis fungoides (MF) is the most common type of primary cutaneous T-cell lymphoma (CTCL). To identify a molecular signature characteristic of MF tumor stage, we used a bioinformatic approach involving meta-analysis of publicly available gene expression data sets combined with previously generated gene expression data. Results for a selection of genes were further refined and validated by quantitative PCR and inclusion of additional controls. With this approach, we identified a profile specific for MF tumor stage, consisting of 989 aberrantly expressed genes, the majority of which (718 genes) are statistically significantly more expressed in MF compared with normal skin, inflamed skin, and normal T cells. As expected, the signature contains genes reflecting the highly proliferative characteristic of this T-cell malignancy, including altered expression of cell cycle and kinetochore regulators. We uncovered details of the immunophenotype, suggesting that MF originates from IL-32-producing cells and identified previously unreported therapeutic targets and/or diagnostic markers, for example, GTSF1 and TRIP13. Loss of expression of the NF-κB inhibitor, NFKBIZ, may partly explain the enhanced activity of NF-κB, which is a hallmark of MF and other CTCLs.
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Proteínas Portadoras/biosíntesis , Perfilación de la Expresión Génica , Linfoma Cutáneo de Células T/genética , Linfoma Cutáneo de Células T/metabolismo , Micosis Fungoide/genética , Micosis Fungoide/metabolismo , Proteínas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Biología Computacional/métodos , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas I-kappa B , Inmunofenotipificación , Interleucinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Modelos Genéticos , FN-kappa B/metabolismo , Proteínas Nucleares/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas/genética , ARN Mensajero/metabolismoAsunto(s)
Linfocitos T CD4-Positivos/metabolismo , MicroARNs/metabolismo , Síndrome de Sézary/genética , Síndrome de Sézary/metabolismo , Dermatitis Atópica/complicaciones , Dermatitis Exfoliativa/genética , Dermatitis Exfoliativa/metabolismo , Dinaminas/genética , Humanos , MicroARNs/genética , Análisis de Secuencia de ARNRESUMEN
Sézary syndrome (SS) is a cutaneous T-cell lymphoma (CTCL) with malignant CD4+ T cells (SS cells) in skin, lymph nodes, and blood. Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in SS cells, whereas this activation is lost upon in vitro culturing, indicating that STAT3 activation observed in vivo is the result of activating factors in the micromilieu of the malignant cells. We investigated which factors are involved in STAT3 activation in SS, focusing on cytokines of the common γ-chain family because of their crucial role in T-cell activation. Furthermore, downstream effects of STAT3 signaling in SS cells were assayed. In SS cells, STAT3 was strongly activated by IL-21, and increased expression of IL-21 and its receptor chains was observed in peripheral blood SS cells. IL-21 and IL-21R protein expression was detectable on neoplastic cells in SS skin biopsies. Using short-term culturing experiments, we demonstrate that IL-21 itself and the α-chain of the IL-2 receptor are STAT3 target genes in SS cells, thereby rendering cells more sensitive to stimulation with the T-cell proliferation and activating cytokine IL-2. Combined, our data point toward a pivotal role for an autocrine positive feedback loop involving IL-21 and consequent persistent STAT3 activation in the pathogenesis of SS, thereby indicating IL-21 and IL-21R as new therapeutical targets.
Asunto(s)
Comunicación Autocrina , Interleucinas/fisiología , Factor de Transcripción STAT3/metabolismo , Síndrome de Sézary/inmunología , Neoplasias Cutáneas/inmunología , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Humanos , Interleucina-2/farmacología , Interleucinas/análisis , Interleucinas/genética , Masculino , Persona de Mediana Edad , Receptores de Interleucina-21/análisis , Síndrome de Sézary/etiologíaRESUMEN
MicroRNAs (miRNAs) are small RNAs that control gene expression, and are involved in the regulation of fundamental biological processes including development, cell differentiation, proliferation, and apoptosis. miRNAs regulate gene expression in normal hematopoiesis, and aberrant miRNA expression might contribute to leukomogenesis. Specifically, miR-21 is abundantly expressed in various tumors including leukemia and lymphoma, and is functionally involved in oncogenic processes. We investigated a role for miR-21 in Sézary Syndrome (SS), a cutaneous T-cell lymphoma (CTCL) with CD4+ tumor cells (Sézary cells) present in the skin, lymph nodes, and peripheral blood. It was shown previously that SS is characterized by constitutively activated signal transducer and activator of transcription 3 (STAT3) signaling. In this study we show by chromatin immunoprecipitation that miR-21 is a direct STAT3 target in Sézary cells. Stimulation of Sézary cells or healthy CD4+ T cells with the common-γ chain cytokine IL-21 results in a strong activation of STAT3, and subsequent upregulation of miR-21 expression. Both pri- and mature miR-21 expression are increased in Sézary cells when compared with CD4+ T cells from healthy donors. Silencing of miR-21 in Sézary cells results in increased apoptosis, suggesting a functional role for miR-21 in the leukomogenic process. Consequently, miR-21 might represent a therapeutic target for the treatment of SS.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , MicroARNs/metabolismo , Factor de Transcripción STAT3/metabolismo , Síndrome de Sézary/metabolismo , Neoplasias Cutáneas/metabolismo , Anciano , Apoptosis/fisiología , Linfocitos T CD4-Positivos/patología , Línea Celular Tumoral , Femenino , Humanos , Interleucinas/metabolismo , Masculino , Síndrome de Sézary/patología , Transducción de Señal/fisiología , Neoplasias Cutáneas/patologíaRESUMEN
This study was designed to identify highly recurrent genetic alterations typical of Sézary syndrome (Sz), an aggressive cutaneous T-cell lymphoma/leukemia, possibly revealing pathogenetic mechanisms and novel therapeutic targets. High-resolution array-based comparative genomic hybridization was done on malignant T cells from 20 patients. Expression levels of selected biologically relevant genes residing within loci with frequent copy number alteration were measured using quantitative PCR. Combined binary ratio labeling-fluorescence in situ hybridization karyotyping was done on malignant cells from five patients. Minimal common regions with copy number alteration occurring in at least 35% of patients harbored 15 bona fide oncogenes and 3 tumor suppressor genes. Based on the function of the identified oncogenes and tumor suppressor genes, at least three molecular mechanisms are relevant in the pathogenesis of Sz. First, gain of cMYC and loss of cMYC antagonists (MXI1 and MNT) were observed in 75% and 40% to 55% of patients, respectively, which were frequently associated with deregulated gene expression. The presence of cMYC/MAX protein heterodimers in Sézary cells was confirmed using a proximity ligation assay. Second, a region containing TP53 and genome maintenance genes (RPA1/HIC1) was lost in the majority of patients. Third, the interleukin 2 (IL-2) pathway was affected by gain of STAT3/STAT5 and IL-2 (receptor) genes in 75% and 30%, respectively, and loss of TCF8 and DUSP5 in at least 45% of patients. In sum, the Sz genome is characterized by gross chromosomal instability with highly recurrent gains and losses. Prominent among deregulated genes are those encoding cMYC, cMYC-regulating proteins, mediators of MYC-induced apoptosis, and IL-2 signaling pathway components.