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OBJECTIVES: The pathological mechanisms of patients with Renal Cell Carcinoma (RCC) remain defined. This study aimed to evaluate relationships between the landscape of gene mutations and their clinical significance in RCC patients. METHODS: Tissue and peripheral blood samples of 42 patients with RCC were collected and performed for the Next Generation Sequencing (NGS) with Geneseeq PrimeTM 425-gene panel probes. Their landscapes of gene mutation were analyzed. We also carried out an evaluation of Tumor-Node-Metastasis (TNM) staging, RENAL nephelometry score, surgery, and targeted drug treatment of patients. Then we compared the correlations of landscape in gene mutations and the prognosis. RESULTS: The most common gene alternations, including BAP1, PBRM1, SETD2, CSF1R, NPM1, EGFR, POLE, RB1, and VHL genes, were identified in tissue and blood samples of 75% of patients. EGFR, POLE, and RB1 gene mutations frequently occurred in relapsed and metastatic patients. BAP1, CCND2, KRAS, PTPN11, ERBB2/3, JAK2, and POLE were presented in the patients with > 9 RENAL nephelometry score. Univariable analysis indicated that SETD2, BAP1, and PBRM1 genes were key factors for Disease-Free Survival (DFS). Multivariable analysis confirmed that mutated SETD1, NPM1, and CSF1R were critical factors for the Progression Free Survival (PFS) of RCC patients with target therapy. CONCLUSIONS: Wild-type PBRM1 and mutated BAP1 in patients with RCC were strongly associated with the outcomes of the patient. The PFS of the patients with SETD2, NPM1, and CSF1R mutations were significantly shorter than those patients without variants.
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Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Relevância Clínica , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/uso terapêutico , Mutação , Proteínas Nucleares/genética , Receptores ErbB/genética , Receptores ErbB/uso terapêuticoRESUMO
Abstract Objectives The pathological mechanisms of patients with Renal Cell Carcinoma (RCC) remain defined. This study aimed to evaluate relationships between the landscape of gene mutations and their clinical significance in RCC patients. Methods Tissue and peripheral blood samples of 42 patients with RCC were collected and performed for the Next Generation Sequencing (NGS) with Geneseeq PrimeTM 425-gene panel probes. Their landscapes of gene mutation were analyzed. We also carried out an evaluation of Tumor-Node-Metastasis (TNM) staging, RENAL nephelometry score, surgery, and targeted drug treatment of patients. Then we compared the correlations of landscape in gene mutations and the prognosis. Results The most common gene alternations, including BAP1, PBRM1, SETD2, CSF1R, NPM1, EGFR, POLE, RB1, and VHL genes, were identified in tissue and blood samples of 75% of patients. EGFR, POLE, and RB1 gene mutations frequently occurred in relapsed and metastatic patients. BAP1, CCND2, KRAS, PTPN11, ERBB2/3, JAK2, and POLE were presented in the patients with > 9 RENAL nephelometry score. Univariable analysis indicated that SETD2, BAP1, and PBRM1 genes were key factors for Disease-Free Survival (DFS). Multivariable analysis confirmed that mutated SETD1, NPM1, and CSF1R were critical factors for the Progression Free Survival (PFS) of RCC patients with target therapy. Conclusions Wild-type PBRM1 and mutated BAP1 in patients with RCC were strongly associated with the outcomes of the patient. The PFS of the patients with SETD2, NPM1, and CSF1R mutations were significantly shorter than those patients without variants.
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O asbesto apresenta grande utilidade comercial, no entanto, é o mais importante carcinógeno ocupacional. O mesotelioma maligno é o tumor mais frequente causado pela exposição ao asbesto, porém devido ao tempo de latência entre a exposição e o desenvolvimento da doença pode chegar a 30 anos, o desenvolvimento de políticas de prevenção é muito difícil. Além disso, as dificuldades de diagnóstico e ausência de terapias específicas, fazem com que este tumor tenha altas taxas de mortalidade, com sobrevida estimada em 8 meses. Estima-se que o Brasil ainda deverá observar um aumento na incidência deste tipo de tumor, resultado das décadas de exposição de trabalhadores da indústria do asbesto. Portanto, compreender os mecanismos moleculares associados ao mesotelioma maligno é fundamental para o desenvolvimento de novas estratégias terapêuticas e de diagnóstico. O objetivo deste trabalho foi caracterizar o perfil epidemiológico dos pacientes acometidos com mesotelioma maligno atendidos na instituição, caracterizar a via de sinalização PI3K/AKT/mtor, assim como identificar as alterações gênicas em amostras de mesotelioma disponíveis. O perfil epidemiológico encontrado do portador de mesotelioma pleural maligno é: homem, ECOG1, diagnosticado entre 65-74 anos, sem exposição ao asbesto, de estágio IV, tabagista, com tumor epitelioide, tratados com cirurgia e quimioterapia, sem histórico familiar de cancer, e com histórico pessoal de neoplasia, com progressão local e recidiva da doença. Enquanto o perfil do portador de mesotelioma peritonial maligno é: mulher, ECOG 1, diagnosticado entre 21 e 44 anos, sem exposição ao asbesto, estádio IV, não fumante, não etilista, apresentando tumor de histologia epitelioide, tratado com quimioterapia, sem histórico familiar de câncer, sem progressão da doença e sem recidiva, e com histórico de neoplasia anterior. A partir dos dados clínicos encontramos: indicativos de predominância de síndrome hereditária, relação entre expressão de BAP-1 e sobrevida global, expressão de BAP-1 e histologia dos tumores, e ausência de correlação entre a expressão de BAP-1 e a topografia do tumor. Foram coletados fragmentos de espécimes cirúrgicos de tumor de 5 pacientes para o estabelecimento de linhagens celulares. As células tumorais cultivadas apresentaram preservação de marcadores de mesotelioma como citoqueratina AE1/AE3, podoplanina e WT-1; e ativação das vias PI3K/AKT/mTOR. Destes 5 casos de mesotelioma maligno, conseguimos estabelecer com sucesso 3 linhagens celulares humana e 2 linhagens de xenoenxerto murinho. A partir do sequenciamento destas 3 linhagens encontramos 1220 alterações gênicas, dentre elas 81 inéditas e de potencialmente maligno. Encontramos também 11 alterações correspondentes com as 20 mais encontradas em mesotelioma maligno de acordo com o The Cancer Genome Atlas TCGA. Entretanto, apenas uma alteração em BAP1 foi identificada. Os resultados deste trabalho permitirão realizar futuros ensaios funcionais in vivo e in vitro para elucidar os mecanismos envolvidos no processo tumoral do mesotelioma maligno
The asbestos has great commercial utility. However, they are the most important occupational carcinogens. The malignant mesothelioma is the most common tumor caused by asbestos' exposure, however due to the latency time between exposure and the development of the disease can take up to 30 years, the development of prevention policies is very difficult. In addition, the difficulties of diagnosis and the absence of specific therapies, cause this tumor to have high mortality rates, with an estimated survival of 8 months. It is estimated that Brazil will still see an increase in the incidence of this type of tumor, as the result of decades of exposure of workers in the asbestos industry. Therefore, understanding the molecular mechanisms associated with malignant mesothelioma is essential for the development of new therapeutic and diagnostic strategies. The objective of this work was to characterize the epidemiological profile of patients affected with malignant mesothelioma treated at the institution, to characterize the PI3K/AKT/mTOR signaling pathway, as well as to identify the genetic alterations in available mesothelioma samples. The epidemiological profile found malignant pleural mesothelioma patients is: male, ECOG1, diagnosed between 6574 years old, without exposure to asbestos, stage IV, smoker, with epithelioid tumor, treated with surgery and chemotherapy, without family history of cancer, and with a personal history of neoplasia, with local progression and disease recurrence. While the profile of malignant peritoneal mesothelioma patient is: woman, ECOG 1, diagnosed between 21 and 44 years old, without exposure to asbestos, stage IV, nonsmoker, nonalcoholic, presenting epithelioid histology tumor, treated with chemotherapy, without family history of cancer, without progression of the disease and without recurrence, and with a history of previous neoplasia. From the clinical data we found: indicative of predominance of hereditary syndrome, relationship between BAP1 expression and overall survival, BAP1 expression and tumor histology, and absence of correlation between BAP1 expression and the topography of the tumor in our cohort. Fragments of surgical specimens of tumor were collected from 5 patients for the establishment of cell lines. Cultured tumor cells showed preservation of mesothelioma markers such as cytokeratin AE1/AE3, podoplanin and WT1; and activation of PI3K/AKT/mTOR pathway. Among these 5 cases of malignant mesothelioma, we were able to successfully establish 3 human cell lines and 2 murine xenograft lines. From the sequencing of these 3 lines we found 1220 gene alterations, 81 of them were unpublished and potentially malignant. We also found 11 changes corresponding to the 20 most commonly found in malignant mesothelioma according to the TCGA. However, only one change in BAP1 was identified. The results of this study will allow future tests in vivo and in vitro to elucidate the components involved in the malignant mesothelioma tumor process.
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Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Amianto , Mesotelioma MalignoRESUMO
Benign multicystic peritoneal mesothelioma (BMPM) is a rare peritoneal tumor diagnosed predominantly in pre-menopausal women. Associated risk factors include endometriosis and pelvic inflammatory disease in women, and prior abdominal surgery in both genders. To date, the pathogenesis of this disease remains controversial with possible etiologies, including a neoplastic versus a reactive process. Given the risk factors, some authors believe that this disease is secondary to a reactive process. However, because some studies describe cases where there is no prior surgical history or inflammatory milieu present, and because of this entity's predilection for recurrence, some authors believe the origin to be neoplastic. Some genetic and familial associations have also been reported. Malignant transformation is extremely rare, with only two cases reported in the literature, despite the recurrence potential. Like the etiology, the name of this entity is also controversial. Some authors prefer the term "peritoneal inclusion cyst (PCM)" instead of "benign cystic mesothelioma" and argue that the term mesothelioma should only be used when there is evidence of atypia. Most cases of BMPM are discovered incidentally. Others reflect sequela of tumor mass effect. It appears intra-operatively as large, multi-focal, cystic lesions in the peritoneal and pelvic cavity. Diagnosis is achieved through surgical sampling with histopathological examination. Immunobiologically, BMPM exhibits multiple small cystic spaces with flattened lining containing calretinin positive cells without atypical features, mitotic figures, or tissue invasion. Treatment includes cytoreductive surgery. Here we present a case of BMPM in a 60-year-old male - a rare disease in an uncommon patient population.
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Benign multicystic peritoneal mesothelioma (BMPM) is a rare peritoneal tumor diagnosed predominantly in pre-menopausal women. Associated risk factors include endometriosis and pelvic inflammatory disease in women, and prior abdominal surgery in both genders. To date, the pathogenesis of this disease remains controversial with possible etiologies, including a neoplastic versus a reactive process. Given the risk factors, some authors believe that this disease is secondary to a reactive process. However, because some studies describe cases where there is no prior surgical history or inflammatory milieu present, and because of this entity's predilection for recurrence, some authors believe the origin to be neoplastic. Some genetic and familial associations have also been reported. Malignant transformation is extremely rare, with only two cases reported in the literature, despite the recurrence potential. Like the etiology, the name of this entity is also controversial. Some authors prefer the term "peritoneal inclusion cyst (PCM)" instead of "benign cystic mesothelioma" and argue that the term mesothelioma should only be used when there is evidence of atypia. Most cases of BMPM are discovered incidentally. Others reflect sequela of tumor mass effect. It appears intra-operatively as large, multi-focal, cystic lesions in the peritoneal and pelvic cavity. Diagnosis is achieved through surgical sampling with histopathological examination. Immunobiologically, BMPM exhibits multiple small cystic spaces with flattened lining containing calretinin positive cells without atypical features, mitotic figures, or tissue invasion. Treatment includes cytoreductive surgery. Here we present a case of BMPM in a 60-year-old male - a rare disease in an uncommon patient population.
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Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Urogenitais/patologia , Mesotelioma Cístico/patologia , Linfangioma Cístico/patologia , Amianto , Fatores de RiscoRESUMO
Metalloproteinases are abundant enzymes in snake venoms of Viperidae family and are relevant in the pathophysiology of envenomation, by their hemorrhagic, fibrinogenolytic and inflammatory activities. From Bothrops asper snake venom was isolated the metalloproteinase BaP1, with molecular weight of 22.7 KDa, weak hemorrhagic action and containing only the catalytic domain of metalloproteinases. Previous studies of our group have shown that BaP1 induces important inflammatory events in vivo and in vitro, inducing the release of inflammatory mediators, such as TNF-α (tumor necrosis factor alpha) by macrophages, important cells of the immune system. ADAM-17 or TACE (TNF-α converting enzyme) is responsible for the release of active TNF-α from its precursor. However, the mechanisms involved in the processing of TNF-α, under the action of BaP1, are unknown. In the present study was demonstrated that macrophages of the RAW 264-7 lineage, pretreated or not with SN50 compound, the nuclear transcription factor NF-κB inhibitor, and stimulated with BaP1 (12.5 μg / mL) for a period of 6 hours, showed a significant decrease in protein expression and the release of TNF-α, when compared to the negative control (culture medium RPMI). Pretreatment of RAW 264-7 cells with the TACE inhibitor, TAPI-1 (200 nM), and incubation with BaP1 (12.5 μg / mL) or RPMI, for 3 h, significantly decreased TNF-α release, when compared to the control group (RPMI). The present study demonstrated that the activation of NF-κB is an important mechanism involved in the processing of TNF-α, induced by BaP1 in RAW 264-7 cells and the release of TNF-α induced by BaP1 is dependent on the action of ADAM-17.
As metaloproteinases são enzimas abundantes em venenos de serpentes da família Viperidae e relevantes na fisiopatologia do envenenamento, devido as suas atividades hemorrágica, fibrinogenolítica e inflamatória. A partir do veneno da serpente B. asper, foi isolada a metaloproteinase de veneno (MV) BaP1, com peso molecular de 22,7 KDa, de fraca ação hemorrágica e que contém apenas o domínio catalítico. Estudos anteriores realizados pelo nosso grupo, demonstraram que a BaP1 induz importantes eventos inflamatórios in vivo e in vitro, induzindo a liberação de mediadores inflamatórios, como o TNF-α (fator de necrose tumoral) em macrófagos, importantes células do sistema imune. A ADAM-17 ou TACE (enzima conversora de TNF-α) é a responsável pela liberação de TNF-α ativo, do seu percursor. Entretanto, os mecanismos envolvidos no processamento do TNF-α, sob ação da BaP1, são desconhecidos. No presente estudo foi demonstrado que macrófagos da linhagem RAW 264-7, pré-tratados ou não com o composto SN50, inibidor do fator de transcrição nuclear κB (NF-κB) e estimulados com a BaP1 (12,5 μg/mL) por um período de 6 horas, apresentaram uma diminuição significativa na expressão proteica e na liberação de TNF-α, quando comparados com o controle negativo (meio de cultura incompleto). O pré-tratamento das células RAW 264-7, com o inibidor da enzima TACE, TAPI-1 (200 nM), e incubação com a BaP1 (12,5 μg/mL) ou meio de cultura (RPMI) por um período de 3 h, diminuiu significativamente a liberação de TNF- α quando comparado ao grupo controle (RPMI). O presente trabalho demonstrou que a ativação do NF-κB é um dos mecanismos envolvidos no processamento do TNF-, induzido pela BaP1 nas células RAW 264-7 e a liberação de TNF-α induzida pela BaP1 é dependente da ação da ADAM-17.
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BACKGROUND: BRCA1 associated-protein 1 (BAP1) tumor predisposition syndrome is associated with an increased risk for malignant mesotheliomas, uveal and cutaneous melanomas, renal cell carcinomas, and singular cutaneous lesions. The latter are referred to as BAP1-inactivated melanocytic tumors (BIMTs). When multiple BIMTs manifest, they are considered potential markers of germline BAP1 mutations. CASE PRESENTATION: Here, we report a novel pathogenic BAP1 germline variant in a family with a history of BIMTs, cutaneous melanomas, and mesotheliomas. We also describe singular pathological aspects of the patient's BIMT lesions and their correlation with dermoscopic and reflectance confocal microscopy findings. CONCLUSIONS: This knowledge is crucial for the recognition of BIMTs by dermatologists and pathologists, allowing the determination of appropriate management for high-risk patients, such as genetic investigations and screening for potentially aggressive tumors.
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Melanoma/genética , Melanoma/patologia , Síndromes Neoplásicas Hereditárias/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Dermoscopia/métodos , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Masculino , Microscopia Confocal/métodos , Pessoa de Meia-Idade , Linhagem , Prognóstico , Melanoma Maligno CutâneoRESUMO
Human accidents with venomous snakes represent an overwhelming public health problem, mainly in rural populations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000 to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced by hyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyper immune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebite envenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have produced recombinant single chain variable fragment scFv against the venom of the pit viper Bothrops asper at high levels expressed transiently and stably in transgenic plants and in vitro cultures that is reactive to BaP1 (a metalloproteinase from B. asper venom). The yield from stably transformed plants was significantly (pâ¯>â¯0.05) higher than the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stable transformation were: transgenic callus 62⯵g/g (±2); biomass from cell suspension cultures 83⯵g/g (±0.2); culture medium from suspensions 71.75â¯mg/L (±6.18). The activity of scFvBaP1 was confirmed by binding and neutralization of the fibrin degradation induced by BnP1 toxins from B. neuwiedi and by Atroxlysin Ia from B. atrox venoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to be used in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms to be used in human therapy against snakebites.
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Metaloendopeptidases/antagonistas & inibidores , Planticorpos/farmacologia , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/farmacologia , Animais , Antivenenos/biossíntese , Antivenenos/farmacologia , Bothrops , Venenos de Crotalídeos/antagonistas & inibidores , Testes de Neutralização , Planticorpos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Human accidents with venomous snakes represent an overwhelming public health problem, mainly in ruralpopulations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced byhyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyperimmune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebiteenvenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have producedrecombinant single chain variable fragment scFv against the venom of the pit viperBothrops asperat high levelsexpressed transiently and stably in transgenic plants andin vitrocultures that is reactive to BaP1 (a metallo-proteinase fromB. aspervenom). The yield from stably transformed plants was significantly (p > 0.05) higherthan the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stabletransformation were: transgenic callus 62µg/g ( ± 2); biomass from cell suspension cultures 83µg/g ( ± 0.2);culture medium from suspensions 71.75 mg/L ( ± 6.18). The activity of scFvBaP1 was confirmed by binding andneutralization of thefibrin degradation induced by BnP1 toxins fromB. neuwiediand by Atroxlysin Ia fromB.atroxvenoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to beused in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms tobe used in human therapy against snakebites.
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BACKGROUND: Spitzoid tumors are a heterogeneous group of melanocytic neoplasms that frequently imposes diagnostic difficulties. Lately, several advances in molecular biology afforded significant discoveries on the pathogenesis of these tumors. BAP1 (BRCA-1 associated protein-1) inactivation and anomalous expression of kinase translocation-related proteins are among the main criteria launched by new classification proposals. Our aim was to systematically assess the immunoexpression of BAP1, ROS1 (receptor tyrosine kinase c-Ros oncogene 1), and ALK (anaplastic lymphoma receptor tyrosine kinase) proteins in an unpublished series of spitzoid tumors. METHODS: Retrospective study based on 47 formalin-fixed paraffin-embedded tissue samples from 3 different institutions. BAP1, ROS1, and ALK immunostains were performed in all cases. We included 27 Spitz tumors without significant abnormality, 15 atypical spitzoid tumors, and 5 spitzoid melanomas. RESULTS: We observed loss of BAP1 nuclear immunolabeling in 4.3% of evaluable cases (2/46), both of them atypical spitzoid tumors. The proportional frequency of BAP1-inactivated cases among atypical spitzoid tumors was 14.2% (2/14). No immunoexpression of ROS1 or ALK was found. CONCLUSIONS: Our study revealed 2 additional BAP1-inactived cases and described its respective frequency. The absence of anomalous expression of translocation-related proteins ALK and ROS1 in this series, composed predominantly of low-grade/low-risk tumors, indicates that translocated spitzoid lesions may not be as prevalent as initially suggested, at least in some populations. Furthermore, our findings encourage additional investigation on unequal occurrence of such immunomarkers among different diagnostic categories of spitzoid neoplasms.
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Nevo de Células Epitelioides e Fusiformes/diagnóstico , Proteínas Tirosina Quinases/análise , Proteínas Proto-Oncogênicas/análise , Receptores Proteína Tirosina Quinases/análise , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/patologia , Proteínas Supressoras de Tumor/análise , Ubiquitina Tiolesterase/análise , Adolescente , Adulto , Quinase do Linfoma Anaplásico , Biomarcadores Tumorais/análise , Criança , Pré-Escolar , Feminino , Humanos , Imuno-Histoquímica , Lactente , Masculino , Pessoa de Meia-Idade , Nevo de Células Epitelioides e Fusiformes/patologia , Estudos Retrospectivos , Adulto JovemRESUMO
Snakebites represent an important public health problem, with a great number of victims with permanent sequelae or fatal outcomes, particularly in rural, agriculturally active areas. The snake venom metalloproteases (SVMPs) are the principal proteins responsible for some clinically-relevant effects, such as local and systemic hemorrhage, dermonecrosis, and myonecrosis. Because of the difficulties in neutralizing them rapidly and locally by antivenoms, the search and design of small molecules as inhibitors of SVMPs are proposed. The Bothrops asper metalloprotease P1 (BaP1) is hereby used as a target protein and by High Throughput Virtual Screening (HTVS) approach, the free access virtual libraries: ZINC, PubChem and ChEMBL, were searched for potent small molecule inhibitors. Results from the aforementioned approaches provided strong evidences on the structural requirements for the efficient BaP1 inhibition such as the presence of the pyrimidine-2,4,6-trione moiety. The two proposed compounds have also shown excellent results in performed in vitro interaction studies against BaP1.
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Antídotos/química , Antídotos/farmacologia , Bothrops/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Pirimidinonas/química , Pirimidinonas/farmacologia , Venenos de Serpentes/antagonistas & inibidores , Animais , Simulação por Computador , Descoberta de Drogas , Metaloendopeptidases/metabolismo , Simulação de Acoplamento Molecular , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Clear cell renal cell carcinoma (ccRCC) is the major subtype of kidney cancer that is characterized by frequent inactivation of the von Hippel-Lindau (VHL) gene in 80-90% of the tumors. Recent reports using massive parallel sequencing technologies have discovered additional cancer driver genes. PBRM1 was found to be mutated in about 40% of ccRCC tumors, whereas BAP1 and SETD2 were each mutated in about 10-15% of ccRCC tumors. JARID1C and UTX, two histone H3 demethylases, were also found to harbor mutations in ccRCC, albeit at lower rates. ccRCC tumors display a high degree of intra-tumoral heterogeneity, with some mutations present in all cancer cells (ubiquitous), whereas others are subclonal. The VHL mutations were always ubiquitous in the tumors; PBRM1 mutations were also ubiquitous but to a lesser extent. On the contrary, mutations in BAP1, SETD2, JARID1C, and UTX were all subclonal, meaning that they were present in a subset of cancer cells in a tumor. The prognostic value of PBRM1 mutations in ccRCC is still controversial, whereas BAP1 mutations were tightly linked to worse clinical outcomes in multiple studies. The molecular functions of these newly identified cancer driver genes are discussed, and they were known readers, writers, or erasers of histone marks on histone H2 and H3 tails that are very close to each other, suggesting that these factors might functionally interact and affect common pathways. The studies on these newly identified tumor suppressors will shed light on ccRCC tumorigenesis and development, and will likely lead to development of novel therapeutic interventions for ccRCC patients.
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Carcinoma de Células Renais/genética , Transformação Celular Neoplásica/genética , Montagem e Desmontagem da Cromatina/genética , Cromatina/metabolismo , Neoplasias Renais/genética , Animais , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Camundongos , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
El melanoma maligno cutáneo (MMC) es un cáncer genéticamente heterogéneo, en cuya patogénesis participarían varios genes. Algunos de estos activan la vía MAP kinasa (BRAF, NRAS, KIT, NF1), mientras que otros confieren una mayor susceptibilidad a melanoma familiar, como CDKN2A, CDK4, MITF y BAP1. BAP1 (BRCA1-associated-protein 1) ha sido descrito como una proteína que se une a BRCA1 para inhibir el crecimiento celular. Actualmente se sabe que es producto de un gen supresor de tumores (denominado BAP1) y que actúa como una enzima con actividad deubiquitinasa, la cual se asocia a varios complejos de proteínas, regulando diversas vías celulares relacionadas con el ciclo celular, diferenciación y muerte celular, así como también gluconeogénesis y respuesta a daño del ADN. Tanto su actividad deubiquitinasa como su localización nuclear son relevantes para su función en la supresión de tumores.
Malignant cutaneous melanoma (MMC) is a genetically heterogeneous cancer and various genes participate in its pathogenesis. Some of these genes activate the MAP kinase pathway (BRAF, NRAS, KIT, NF1) and others are related to a higher susceptibility to familial melanoma like CDKN2A, CDK4, MITF y BAP1. BAP1 (BRCA1-associated protein 1) has been described as a BRCA1-binding protein inhibiting cell growth. This protein is a product of a gene with tumor suppressor activity, the protein being a deubiquitinase associated to multiple protein complexes regulating various cellular pathways, including the cell cycle, differentiation and cell death, as well as gluconeogenesis and DNA damage response. Both deubiquitinase activity and location to the nucleus are relevant to its tumor suppressor function.
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Humanos , Neoplasias Cutâneas/genética , Melanoma/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , MutaçãoRESUMO
Glycolic acid (GA) (2-Hydroxyethanoic acid) is widely used as chemical peeling agent in Dermatology and, more recently, as a therapeutic and cosmetic compound in the field of skin care and disease treatment. In this work we tested the inhibitory ability of glycolic acid on the enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P-I metalloproteinase from Bothrops asper venom, which induces a variety of toxic actions. Glycolic acid inhibited the proteolytic activity of BaP1 on azocasein, with an IC50 of 1.67 mM. The compound was also effective at inhibiting the hemorrhagic activity of BaP1 in skin and muscle in experiments involving preincubation of enzyme and inhibitor prior to injection. When BaP1 was injected i.m. and then, at the same site, different concentrations of glycolic acid were administered at either 0 or 5 min, 7 mM solutions of the inhibitor partially abrogated hemorrhagic activity when administered at 0 min. Moreover, glycolic acid inhibited, in a concentration-dependent manner, edema-forming activity of BaP1 in the footpad. In order to have insights on the mode of action of glycolic acid, UV-vis and intrinsic fluorescence studies were performed. Results of these assays suggest that glycolic acid interacts directly with BaP1 and chelates the Zn²âº ion at the active site. These findings were supported by molecular docking results, which suggested that glycolic acid forms hydrogen bonds with residues Glu143, Arg110 and Ala111 of the enzyme. Additionally, molecular modeling results suggest that the inhibitor chelates Zn²âº, with a distance of 3.58 Å, and may occupy part of substrate binding cleft of BaP1. Our results suggest that glycolic acid is a candidate for the development of inhibitors to be used in snakebite envenomation.
Assuntos
Bothrops , Edema/tratamento farmacológico , Glicolatos/farmacologia , Metaloendopeptidases/toxicidade , Venenos de Serpentes/toxicidade , Animais , Caseínas/metabolismo , Domínio Catalítico/efeitos dos fármacos , Quelantes/química , Hemorragia/tratamento farmacológico , Concentração Inibidora 50 , Metaloendopeptidases/antagonistas & inibidores , Camundongos , Simulação de Acoplamento Molecular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Proteólise/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/metabolismo , Mordeduras de Serpentes/tratamento farmacológico , Zinco/metabolismoRESUMO
The majority of snakebite envenomations in Central America are caused by the viperid species Bothrops asper, whose venom contains a high proportion of zinc-dependent metalloproteinases that play a relevant role in the pathogenesis of hemorrhage characteristic of these envenomations. Broad metalloproteinase inhibitors, such as the peptidomimetic hydroxamate Batimastat, have been shown to inhibit snake venom metalloproteinases (SVMP). However, the difficulty in having open public access to Batimastat and similar molecules highlights the need to design new inhibitors of SVMPs that could be applied in the treatment of snakebite envenomations. We have chosen the SVMP BaP1 as a model to search for new inhibitors using different strategies, that is, screening of the Prestwick Chemical Library and rational peptide design. Results from these approaches provide clues on the structural requirements for efficient BaP1 inhibition and pave the way for the design of new inhibitors of SVMP.
RESUMO
BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (Kd), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The Kds of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structurefunction relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.