RESUMO
TFEB-amplified renal cell carcinoma (RCC), which belongs to the MITF family of RCC, is characterized by genomic amplification at the 6p21.1 locus where the TFEB gene is located. The vascular endothelial growth factor A and cyclin D3 genes are also located at this same locus. When tumors lack classic morphologic features, they may be classified as "RCC not otherwise specified (NOS)." However, it is increasingly important to accurately diagnose the RCC subtype to define the patient's individual prognosis and select the subsequent therapeutic modalities, which now include targeted agents. Therefore, knowledge of the diagnostic features of TFEB-altered RCCs, such as t(6;11) RCCs and TFEB-amplified RCCs, is critical for identifying these tumors. Herein, we present an interesting case of TFEB-amplified RCC that was initially diagnosed as RCC NOS on biopsy of a renal tumor in a community practice setting with available molecular findings demonstrating CCND3 amplification. The genetic abnormality was "accidentally" detected due to the amplification of the colocated CCND3 gene at the 6p21 locus of the TFEB gene on a limited genetic sequencing panel. This case highlights the importance of molecular tests in accurately diagnosing RCC and carefully interpreting molecular findings in the context of histomorphologic features.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Amplificação de Genes , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Neoplasias Renais/diagnóstico , Neoplasias Renais/genética , Translocação Genética , Biomarcadores Tumorais/genética , Ciclina D3/genética , Ciclina D3/metabolismoRESUMO
Kynurenine (KYN), the most abundant metabolite of tryptophan, is classically associated with immune tolerance and tumor immune escape. In the last years, KYN is in the spotlight in other biological processes. Here, we showed that KYN inhibited tyrosinase expression and melanin content in primary human melanocyte and keratinocyte co-cultures. Furthermore, KYN decreased melanosome content in a 3D human skin reconstruction model. In these experiments, we used tyrosine + NH4 Cl to induce pigmentation. We compared the inhibitory effect of KYN on melanogenesis with the already known inhibitory effect promoted by IFN-γ. Since increased KYN production depends on the IFN-γ-inducible enzyme indoleamine-2,3-dioxygenase (IDO), we propose that part of the effect of IFN-γ on melanogenesis involves KYN production. From that, we tested if, during melanogenesis, changes in tryptophan metabolism would occur. For this purpose, we measured tryptophan, KYN and downstream products along with pigmentation. There were no significant changes in Trp metabolism, except for the high consumption of kynurenic acid. Our data identify the skin as a potential target for the action of KYN relevant for skin physiology and pigmentation. The results are discussed concerning the high production of KYN in skin inflammatory disorders and cancer.
Assuntos
Cinurenina , Triptofano , Técnicas de Cocultura , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Queratinócitos/metabolismo , Cinurenina/metabolismo , Melanócitos/metabolismo , Triptofano/farmacologiaRESUMO
We previously reported that intronic single nucleotide variations (SNVs) in MITF (c.938-325G>A, rs7623610) and CREB1 (c.303+373G>A, rs10932201) genes were associated with risk, aggressiveness, and prognosis of cutaneous melanoma (CM). In this study, we investigated the influence of the above SNVs in splicing patterns and efficiency. We constructed minigenes with wild type and variant alleles from MITF and CREB1 to assess the effect of the SNVs on splicing. The minigenes were transfected in the human melanoma cell line (SK-MEL-28). RT-PCR and DNA sequencing investigated the constructs' splicing patterns. Minigenes constructs' splicing efficiency and HNRNPA1 and SF1 splicing genes' expression were investigated by qPCR. We found that MITF and CREB1 SNVs did not alter the splicing pattern, but they influenced the splicing efficiency. MITF-A (p= 0.03) and CREB1-A (p= 0.005) variant minigenes yielded an increase of mRNA generated from the constructions. Additionally, lower mRNA levels of HNRNPA1 and SF1 were seen in the variant minigenes MITF-A (p= 0.04) and CREB1-A (p= 0.005). We described for the first time the potential importance of MITF rs7623610 and CREB1 rs10932201 SNVs in splicing efficiency and its relationship with CM.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Íntrons , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Mutação , Splicing de RNA , Neoplasias Cutâneas/genética , Alelos , Sequência de Bases , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Engenharia Genética/métodos , Humanos , Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Polimorfismo de Nucleotídeo Único , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Neoplasias Cutâneas/metabolismo , Melanoma Maligno CutâneoRESUMO
BACKGROUND: The microphthalmia-associated transcription factor gene (MITF) belongs to the MYC supergene family and plays an important role in melanocytes' homeostasis. Individuals harboring MITF germline pathogenic variants are at increased risk of developing cancer, most notably melanoma and renal cell carcinoma. CASE PRESENTATION: We describe a cohort of ten individuals who harbor the same MITF c.952G > A (p.Glu 318Lys), or p.E318K, germline pathogenic variant. Six carriers developed at least one malignancy (4 cases of breast cancer; 1 cervical cancer; 1 colon cancer; 1 melanoma; 1 ovarian/fallopian tube cancer). A significant phenotypic heterogeneity was found among these individuals and their relatives. Breast cancer was, overall, the most frequent malignancy observed in this case series, with 13 occurrences of 60 (21.67 %) total cancer cases described among the probands and their relatives. CONCLUSIONS: Our retrospective analysis data raise the hypothesis of a possible association of the MITF p.E318K pathogenic variant with an increased risk of breast cancer.
RESUMO
BACKGROUND: The generation of functional human epidermal melanocytes (HEM) from stem cells provides an unprecedented source for cell-based therapy in vitiligo. Despite the important efforts exerted to obtain melanin-producing cells from stem cells, pre-clinical results still lack the safety and scalability characteristics essential for their translational application. METHODS: Here, we report a rapid and efficient protocol based on defined culture conditions capable of differentiating adult adipose-derived stem cells (ADSC) to scalable amounts of proliferative melanocyte precursors (PreMel) within 30 days. PreMel were characterized in vitro through qPCR, Western blot, flow cytometry, biochemical assays, and in vivo assays in immunocompromised mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, or NSG). RESULTS: After 30 days of differentiation, the stem cell-derived PreMel were defined as CD105neg CD73low according to immunophenotypic changes in comparison with parental stem cell markers. In addition, expression of microphthalmia-associated transcription factor (MITF), active tyrosinase (TYR), and the terminal differentiation-involved premelanosome protein (PMEL) were detected. Furthermore, PreMel had the potential to synthesize melanin and package it into melanosomes both in vitro and in vivo in NSG mice skin. CONCLUSIONS: This study proposes a rapid and scalable protocol for the generation of proliferative melanocyte precursors (PreMel) from ADSC. These PreMel display the essential functional characteristics of bona fide HEM, opening a new path for an autologous cellular therapy for vitiligo patients.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular , Melanócitos/metabolismo , 5'-Nucleotidase/metabolismo , Adolescente , Adulto , Animais , Linhagem da Célula , Endoglina/metabolismo , Feminino , Humanos , Melaninas/metabolismo , Melanócitos/citologia , Melanócitos/transplante , Camundongos , Camundongos Endogâmicos NOD , Fator de Transcrição Associado à Microftalmia/metabolismo , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/metabolismo , Pele/patologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Vitiligo/patologia , Vitiligo/terapia , Adulto Jovem , Antígeno gp100 de Melanoma/metabolismoRESUMO
BACKGROUND: Hyperpigmentation disorders such as post-inflammatory hyperpigmentation are major concerns not only in light-skinned people but also in Asian populations with darker skin. The anti-tyrosinase and immunomodulatory effects of sericin have been known for decades. However, the therapeutic effects of sericin on hyperpigmentation disorders have not been well documented. METHODS: In this study, we used an in vitro model to study the anti-tyrosinase, tolerogenic, and anti-melanogenic effects of sericin on Staphylococcus aureus peptidoglycan (PEG)-stimulated melanocytes, dendritic cells (DCs), and artificial skin (MelanoDerm™). Enzyme-linked immunosorbent assay, conventional and immunolabeled electron microscopy, and histopathological studies were performed. RESULTS: The results revealed that urea-extracted sericin has strong anti-tyrosinase properties as shown by a reduction of tyrosinase activity in melanin pigments both 48 h and 10 days after allergic induction with PEG. Anti-inflammatory cytokines including interleukin (IL)-4, IL-10, and transforming growth factor-ß were upregulated upon sericin treatment (10, 20, and 50 µg/mL), whereas production of allergic chemokines, CCL8 and CCL18, by DCs was diminished 48 h after allergic induction with PEG. Moreover, sericin lowered the expression of micropthalmia-associated transcription factor (MITF), a marker of melanogenesis regulation, in melanocytes and keratinocytes, which contributed to the reduction of melanin size and the magnitude of melanin deposition. However, sericin had no effect on melanin transport between melanocytes and keratinocytes, as demonstrated by a high retention of cytoskeletal components. CONCLUSION: In summary, sericin suppresses melanogenesis by inhibition of tyrosinase activity, reduction of inflammation and allergy, and modulation of MITF function.
Assuntos
Hiperpigmentação/tratamento farmacológico , Queratinócitos/efeitos dos fármacos , Melanócitos/efeitos dos fármacos , Monofenol Mono-Oxigenase/antagonistas & inibidores , Sericinas/farmacologia , Células Cultivadas , Humanos , Hipersensibilidade , Inflamação , Queratinócitos/ultraestrutura , Melanócitos/ultraestrutura , Fator de Transcrição Associado à Microftalmia , Microscopia Eletrônica , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacosRESUMO
This paper deals with the molecular investigation of Waardenburg syndrome (WS) in a sample of 49 clinically diagnosed probands (most from southeastern Brazil), 24 of them having the type 1 (WS1) variant (10 familial and 14 isolated cases) and 25 being affected by the type 2 (WS2) variant (five familial and 20 isolated cases). Sequential Sanger sequencing of all coding exons of PAX3, MITF, EDN3, EDNRB, SOX10 and SNAI2 genes, followed by CNV detection by MLPA of PAX3, MITF and SOX10 genes in selected cases revealed many novel pathogenic variants. Molecular screening, performed in all patients, revealed 19 causative variants (19/49â¯=â¯38.8%), six of them being large whole-exon deletions detected by MLPA, seven (four missense and three nonsense substitutions) resulting from single nucleotide substitutions (SNV), and six representing small indels. A pair of dizygotic affected female twins presented the c.430delC variant in SOX10, but the mutation, imputed to gonadal mosaicism, was not found in their unaffected parents. At least 10 novel causative mutations, described in this paper, were found in this Brazilian sample. Copy-number-variation detected by MLPA identified the causative mutation in 12.2% of our cases, corresponding to 31.6% of all causative mutations. In the majority of cases, the deletions were sporadic, since they were not present in the parents of isolated cases. Our results, as a whole, reinforce the fact that the screening of copy-number-variants by MLPA is a powerful tool to identify the molecular cause in WS patients.
Assuntos
Variações do Número de Cópias de DNA , Mutação , Síndrome de Waardenburg/genética , Brasil , Éxons , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Mosaicismo , Análise de Sequência de DNA , Deleção de SequênciaRESUMO
BACKGROUND: Hyperpigmentation disorders such as post-inflammatory hyperpigmentation are major concerns not only in light-skinned people but also in Asian populations with darker skin. The anti-tyrosinase and immunomodulatory effects of sericin have been known for decades. However, the therapeutic effects of sericin on hyperpigmentation disorders have not been well documented. METHODS: In this study, we used an in vitro model to study the anti-tyrosinase, tolerogenic, and anti-melanogenic effects of sericin on Staphylococcus aureus peptidoglycan (PEG)-stimulated melanocytes, dendritic cells (DCs), and artificial skin (MelanoDerm™). Enzyme-linked immunosorbent assay, conventional and immunolabeled electron microscopy, and histopathological studies were performed. RESULTS: The results revealed that urea-extracted sericin has strong anti-tyrosinase properties as shown by a reduction of tyrosinase activity in melanin pigments both 48 h and 10 days after allergic induction with PEG. Anti-inflammatory cytokines including interleukin (IL)-4, IL-10, and transforming growth factor-p were upregulated upon sericin treatment (10, 20, and 50 µg/mL), whereas production of allergic chemokines, CCL8 and CCL18, by DCs was diminished 48 h after allergic induction with PEG. Moreover, sericin lowered the expression of micropthalmia-associated transcription factor (MITF), a marker of melanogenesis regulation, in melanocytes and keratinocytes, which contributed to the reduction of melanin size and the magnitude of melanin deposition. However, sericin had no effect on melanin transport between melanocytes and keratinocytes, as demonstrated by a high retention of cytoskeletal components. CONCLUSION: In summary, sericin suppresses melanogenesis by inhibition of tyrosinase activity, reduction of inflammation and allergy, and modulation of MITF function.
Assuntos
Humanos , Queratinócitos/efeitos dos fármacos , Monofenol Mono-Oxigenase/antagonistas & inibidores , Hiperpigmentação/tratamento farmacológico , Sericinas/farmacologia , Melanócitos/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacos , Microscopia Eletrônica , Transdução de Sinais/efeitos dos fármacos , Queratinócitos/ultraestrutura , Células Cultivadas , Fator de Transcrição Associado à Microftalmia , Hipersensibilidade , Inflamação , Melanócitos/ultraestruturaRESUMO
Tietz syndrome and Waardenburg syndrome type 2A are allelic conditions caused by MITF mutations. Tietz syndrome is inherited in an autosomal dominant pattern and is characterized by congenital deafness and generalized skin, hair, and eye hypopigmentation, while Waardenburg syndrome type 2A typically includes variable degrees of sensorineural hearing loss and patches of de-pigmented skin, hair, and irides. In this paper, we report two unrelated families with MITF mutations. The first family showed an autosomal dominant pattern and variable expressivity. The second patient was isolated. MITF gene analysis in the first family demonstrated a c.648A>C heterozygous mutation in exon 8 c.648A>C; p. (R216S), while in the isolated patient, an apparently de novo heterozygous c.1183_1184insG truncating mutation was demonstrated in exon 10. All patients except one had bilateral reduced ocular anteroposterior axial length and a high hyperopic refractive error corresponding to posterior microphthalmos, features that have not been described as part of the disease. Our results suggest that posterior microphthalmos might be part of the clinical characteristics of Tietz/Waardenburg syndrome type 2A and expand both the clinical and molecular spectrum of the disease. © 2016 Wiley Periodicals, Inc.
Assuntos
Fator de Transcrição Associado à Microftalmia/genética , Microftalmia/genética , Mutação , Fenótipo , Síndrome de Waardenburg/genética , Alelos , Substituição de Aminoácidos , Criança , Pré-Escolar , Éxons , Fácies , Heterozigoto , Humanos , Masculino , Microftalmia/diagnóstico , Exame Físico , Síndrome de Waardenburg/diagnósticoRESUMO
Introducción: La incidencia de melanoma maligno se ha incrementado más rápido que cualquier otro tipo de cáncer, intensificando así la búsqueda de herramientas que faciliten la identificación temprana del melanoma. El factor de transcripción asociado con microftalmia (MITF) es conocido como el regulador maestro de melanocitos. En el presente estudio se analiza la expresión del gen MITF en sangre periférica de un grupo de individuos con melanoma, comparándola con un grupo de personas sin cáncer y en algunas líneas celulares. Materiales y métodos: Se extrajo ARN de 31 muestras de sangre periférica: 19 de pacientes con melanoma y 12 de personas sin ningún tipo de cáncer. Se cuantificaron niveles de expresión tanto para el gen MITF como para los genes de expresión constitutiva (b2M y GAPDH) mediante PCR tiempo real. Asimismo se evaluó la expresión de los mismos genes en cinco líneas celulares. Resultados: En todos los individuos se observó expresión del gen MITF, aunque no hubo diferencias estadísticamente significativas entre los niveles de expresión en los grupos de estudio (p=0.09). Sin embargo, la expresión de MITF en el grupo de pacientes con melanoma fue más variable que la observada en el grupo de personas sin cáncer. Asimismo, en la línea celular de adenocarcinoma gástrico se detectó expresión del gen MITF, no descrita hasta el momento. Conclusiones: Se encontraron niveles de expresión del gen MITF en sangre periférica tanto de personas con melanoma como en personas sin cáncer. Sin embargo, la variabilidad en los niveles de expresión del gen MITF observados en personas con melanoma, sugiere la posible presencia de células tumorales en circulación.
Background:The incidence of malign melanoma tumours has increased more rapidly than any other type of cancer; this has intensified the search for tools that facilitate early identification of melanoma. Microphthalmia associated transcription factor (MITF) is currently known as being a master melanocyte regulator; we analyse MITF gene expression in peripheral blood from individuals suffering from melanoma, compared to people without any type of cancer and ones cell lines. Materials and methods: Thirty one samples of peripheral blood were used: 19 from patients having melanoma and 12 from people without any cancer. RNA was then extracted from these samples. MITF and housekeeping genes (b2M and GAPDH) expression levels were then quantified by real-time PCR. Five cell lines were also used to determine the MITF expression Results: MITF gene expression could be observed in all individuals, though no statistically significant differences were found among expression levels in the groups studied (p=0.09). Even so, MITF expression in the group of patients suffering from melanoma was much more variable than that observed in the group of cancer-free people. Expression was detected in the cell line AGS (gastric adenocarcinoma), not yet described. Conclusions: MITF gene expression levels were detected in the peripheral blood of both people suffering from melanoma and people without any type of cancer. However, variability in the number of molecules in MITF gene expression was observe in people with melanoma, this suggest the presence of tumour cells in circulation.