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1.
Redox Biol ; 38: 101766, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-33126057

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible disease characterized by an increase in differentiation of fibroblasts to myofibroblasts and excessive accumulation of extracellular matrix in lung tissue. Pharmacological activation of NRF2 has proved to be a valuable antifibrotic approach, however the detailed mechanisms of how NRF2 mediates antifibrotic function remain unclear. In this study, we found that the antifibrotic function of sulforaphane (SFN), an NRF2 activator, was largely dependent on LOC344887, a long noncoding RNA. Two functional AREs were identified in both the promoter and intron 1 of LOC344887, which defines LOC344887 as a novel anti-fibrotic NRF2 target gene. RNA-seq analysis revealed that LOC344887 controls genes and signaling pathways associated with fibrogenesis. Deletion or downregulation of LOC344887 enhanced expression of CDH2/N-cadherin, as well as a number of other fibrotic genes and blunted the antifibrotic effects of SFN. Furthermore, LOC344887-mediated downregulation of fibrotic genes may involve the PI3K-AKT signaling pathway, as pharmacologic inhibition of PI3K activity blocked the effects of LOC344887 knockdown. Our findings demonstrate that NRF2-mediated LOC344887 upregulation contributes to the antifibrotic potential of SFN by repressing the expression of CDH2 and other fibrotic genes, providing novel insight into how NRF2 controls the regulatory networks of IPF. This study provides a better understanding of the molecular mechanisms of NRF2 activators against pulmonary fibrosis and presents a novel therapeutic axis for prevention and intervention of fibrosis-related diseases.

2.
Semin Cancer Biol ; 61: 158-166, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31689495

RESUMO

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an autosomal dominant hereditary cancer syndrome with incomplete penetrance. It is caused by a germline amorphic allele of the FH gene, which encodes the TCA cycle enzyme, fumarate hydratase (FH). HLRCC patients are genetically predisposed to develop skin leiomyomas, uterine fibroids, and the aggressive kidney cancer of type 2 papillary morphology. Loss-of-heterozygocity at the FH locus that cause a complete loss of FH enzymatic function is always detected in these tumor tissues. Molecular pathway elucidation, genomic studies, and systematic genetics screens reported over the last two decades have identified several FH-inactivation driven pathways alterations, as well as rationally conceived treatment strategies that specifically target FH-/- tumor cells. These treatment strategies include ferroptosis induction, oxidative stress promotion, and metabolic alteration. As the fundamental biology of HLRCC continues to be uncovered, these treatment strategies continue to be refined and may one day lead to a strategy to prevent disease onset among HLRCC patients. With a more complete picture of HLRCC biology, the safe translation of experimental treatment strategies into clinical practice is achievable in the foreseeable future.

3.
J Biol Chem ; 294(48): 18131-18149, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31628195

RESUMO

The nuclear factor (erythroid 2)-like (NRF) transcription factors are a subset of cap'n'collar transcriptional regulators. They consist of three members, NRF1, NRF2, and NRF3, that regulate the expression of genes containing antioxidant-response elements (AREs) in their promoter regions. Although all NRF members regulate ARE-containing genes, each is associated with distinct roles. A comprehensive study of differential and overlapping DNA-binding and transcriptional activities of the NRFs has not yet been conducted. Here, we performed chromatin immunoprecipitation (ChIP)-exo sequencing, an approach that combines ChIP with exonuclease treatment to pinpoint regulatory elements in DNA with high precision, in conjunction with RNA-sequencing to define the transcriptional targets of each NRF member. Our approach, done in three U2OS cell lines, identified 31 genes that were regulated by all three NRF members, 27 that were regulated similarly by all three, and four genes that were differentially regulated by at least one NRF member. We also found genes that were up- or down-regulated by only one NRF member, with 84, 84, and 22 genes that were regulated by NRF1, NRF2, and NRF3, respectively. Analysis of the ARE motifs identified in ChIP peaks revealed that NRF2 prefers binding to AREs flanked by GC-rich regions and that NRF1 prefers AT-rich flanking regions. Thus, sequence preference, likely in combination with upstream signaling events, determines NRF member activation under specific cellular contexts. Our analysis provides a comprehensive description of differential and overlapping gene regulation by the transcriptional regulators NRF1, NRF2, and NRF3.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação da Expressão Gênica , Fator 1 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linhagem Celular , Humanos , Fator 1 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/genética , Fator 1 Nuclear Respiratório
4.
Mol Cell Biol ; 39(13)2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31010806

RESUMO

The nuclear factor (erythroid 2)-like 2 (NRF2 or NFE2L2) transcription factor regulates the expression of many genes that are critical in maintaining cellular homeostasis. Its deregulation has been implicated in many diseases, including cancer and metabolic and neurodegenerative diseases. While several mechanisms by which NRF2 can be activated have gradually been identified over time, a more complete regulatory network of NRF2 is still lacking. Here we show through a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen that a total of 273 genes, when knocked out, will lead to sustained NRF2 activation. Pathway analysis revealed a significant overrepresentation of genes (18 of the 273 genes) involved in autophagy. Molecular validation of a subset of the enriched genes identified 8 high-confidence genes that negatively regulate NRF2 activity irrespective of cell type: ATG12, ATG7, GOSR1, IFT172, NRXN2, RAB6A, VPS37A, and the well-known negative regulator of NRF2, KEAP1 Of these, ATG12, ATG7, KEAP1, and VPS37A are known to be involved in autophagic processes. Our results present a comprehensive list of NRF2 negative regulators and reveal an intimate link between autophagy and NRF2 regulation.


Assuntos
Redes Reguladoras de Genes , Fator 2 Relacionado a NF-E2/metabolismo , RNA Guia/farmacologia , Autofagia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Redes Reguladoras de Genes/efeitos dos fármacos , Genoma Humano , Homeostase , Humanos
6.
Proc Natl Acad Sci U S A ; 115(44): E10352-E10361, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30309964

RESUMO

NRF2 regulates cellular redox homeostasis, metabolic balance, and proteostasis by forming a dimer with small musculoaponeurotic fibrosarcoma proteins (sMAFs) and binding to antioxidant response elements (AREs) to activate target gene transcription. In contrast, NRF2-ARE-dependent transcriptional repression is unreported. Here, we describe NRF2-mediated gene repression via a specific seven-nucleotide sequence flanking the ARE, which we term the NRF2-replication protein A1 (RPA1) element (NRE). Mechanistically, RPA1 competes with sMAF for NRF2 binding, followed by interaction of NRF2-RPA1 with the ARE-NRE and eduction of promoter activity. Genome-wide in silico and RNA-seq analyses revealed this NRF2-RPA1-ARE-NRE complex mediates negative regulation of many genes with diverse functions, indicating that this mechanism is a fundamental cellular process. Notably, repression of MYLK, which encodes the nonmuscle myosin light chain kinase, by the NRF2-RPA1-ARE-NRE complex disrupts vascular integrity in preclinical inflammatory lung injury models, illustrating the translational significance of NRF2-mediated transcriptional repression. Our findings reveal a gene-suppressive function of NRF2 and a subset of negatively regulated NRF2 target genes, underscoring the broad impact of NRF2 in physiological and pathological settings.


Assuntos
Fator 2 Relacionado a NF-E2/genética , Proteína de Replicação A/genética , Proteínas Repressoras/genética , Transcrição Genética/genética , Ativação Transcricional/genética , Células A549 , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Genoma/genética , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética
7.
Sci Rep ; 8(1): 12846, 2018 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-30150714

RESUMO

Identification and characterization of somatic mutations in cancer have important prognostication and treatment implications. Genes encoding the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor and its negative regulator, Kelch-like ECH-associated protein 1 (KEAP1), are frequently mutated in cancer. These mutations drive constitutive NRF2 activation and correlate with poor prognosis. Despite its apparent significance, a comprehensive catalogue of somatic NRF2 mutations across different tumor types is still lacking. Here, we catalogue NRF2 mutations in The Cancer Genome Atlas (TCGA) database. 226 unique NRF2-mutant tumors were identified from 10,364 cases. NRF2 mutations were found in 21 out of the 33 tumor types. A total of 11 hotspots were identified. Of these, mutation to the R34 position was most frequent. Notably, R34 and D29 mutations were overrepresented in bladder, lung, and uterine cancers. Analyses of corresponding RNA sequencing data using a de novo derived gene expression classifier showed that the R34 mutations drive constitutive NRF2 activation with a selection pressure biased against the formation of R34L. Of all R34 mutants, R34L conferred the least degree of protein stabilization, suggesting a pro-tumor NRF2 half-life threshold. Our findings offer a comprehensive catalogue of NRF2 mutations in cancer that can help prognostication and NRF2 research.


Assuntos
Mutação com Ganho de Função , Fator 2 Relacionado a NF-E2/genética , Neoplasias/genética , Substituição de Aminoácidos , Linhagem Celular Tumoral , Biologia Computacional/métodos , Bases de Dados Genéticas , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias/metabolismo , Estabilidade Proteica , Análise de Sequência de DNA , Transdução de Sinais
8.
Cancer Sci ; 109(9): 2757-2766, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29917289

RESUMO

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a hereditary cancer syndrome characterized by inactivation of the Krebs cycle enzyme fumarate hydratase (FH). HLRCC patients are at high risk of developing kidney cancer of type 2 papillary morphology that is refractory to current radiotherapy, immunotherapy and chemotherapy. Hence, an effective therapy for this deadly form of cancer is urgently needed. Here, we show that FH inactivation (FH-/- ) proves synthetic lethal with inducers of ferroptosis, an iron-dependent and nonapoptotic form of cell death. Specifically, we identified gene signatures for compound sensitivities based on drug responses for 9 different drug classes against the NCI-60 cell lines. These signatures predicted that ferroptosis inducers would be selectively toxic to FH-/- cell line UOK262. Preferential cell death against UOK262-FH-/- was confirmed with 4 different ferroptosis inducers. Mechanistically, the FH-/- sensitivity to ferroptosis is attributed to dysfunctional GPX4, the primary cellular defender against ferroptosis. We identified that C93 of GPX4 is readily post-translationally modified by fumarates that accumulate in conditions of FH-/- , and that C93 modification represses GPX4 activity. Induction of ferroptosis in FH-inactivated tumors represents an opportunity for synthetic lethality in cancer.


Assuntos
Fumarato Hidratase/fisiologia , Leiomiomatose/enzimologia , Síndromes Neoplásicas Hereditárias/enzimologia , Neoplasias Cutâneas/enzimologia , Neoplasias Uterinas/enzimologia , Linhagem Celular Tumoral , Ativação Enzimática , Glutationa Peroxidase/fisiologia , Humanos , Leiomiomatose/patologia , Síndromes Neoplásicas Hereditárias/patologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Uterinas/patologia
9.
Antioxid Redox Signal ; 29(17): 1756-1773, 2018 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-28793787

RESUMO

SIGNIFICANCE: Iron and oxygen are intimately linked: iron is an essential nutrient utilized as a cofactor in enzymes for oxygen transport, oxidative phosphorylation, and metabolite oxidation. However, excess labile iron facilitates the formation of oxygen-derived free radicals capable of damaging biomolecules. Therefore, biological utilization of iron is a tightly regulated process. The nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor, which can respond to oxidative and electrophilic stress, regulates several genes involved in iron metabolism. Recent Advances: The bulk of NRF2 transcription factor research has focused on its roles in detoxification and cancer prevention. Recent works have identified that several genes involved in heme synthesis, hemoglobin catabolism, iron storage, and iron export are under the control of NRF2. Constitutive NRF2 activation and subsequent deregulation of iron metabolism have been implicated in cancer development: NRF2-mediated upregulation of the iron storage protein ferritin or heme oxygenase 1 can lead to enhanced proliferation and therapy resistance. Of note, NRF2 activation and alterations to iron signaling in cancers may hinder efforts to induce the iron-dependent cell death process known as ferroptosis. CRITICAL ISSUES: Despite growing recognition of NRF2 as a modulator of iron signaling, exactly how iron metabolism is altered due to NRF2 activation in normal physiology and in pathologic conditions remains imprecise; moreover, the roles of NRF2-mediated iron signaling changes in disease progression are only beginning to be uncovered. FUTURE DIRECTIONS: Further studies are necessary to connect NRF2 activation with physiological and pathological changes to iron signaling and oxidative stress. Antioxid. Redox Signal. 00, 000-000.


Assuntos
Homeostase , Ferro/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias/metabolismo , Animais , Humanos , Estresse Oxidativo , Transdução de Sinais
10.
Mol Carcinog ; 57(2): 182-192, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28976703

RESUMO

Targeting the transcription factor NRF2 has been recognized as a feasible strategy for cancer prevention and treatment, but many of the mechanistic details underlying its role in cancer development and progression are lacking. Therefore, careful mechanistic studies of the NRF2 pathway in cancer initiation and progression are needed to identify which therapeutic avenue-activation or inhibition-is appropriate in a given context. Moreover, while numerous reports confirm the protective effect of NRF2 activation against chemical carcinogenesis little is known of its role in cancer arising from spontaneous mutations. Here, we tested the effects of NRF2 modulation (activation by sulforaphane or inhibition by brusatol) in lung carcinogenesis using a chemical (vinyl carbamate) model in A/J mice and a genetic (conditional KrasG12D oncogene expression, to simulate spontaneous oncogene mutation) model in C57BL/6J mice. Mice were treated with NRF2 modulators before carcinogen exposure or KrasG12D expression to test the role of NRF2 in cancer initiation, or treated after tumor development to test the role of NRF2 in cancer progression. Lung tissues were analyzed to determine tumor burden, as well as status of NRF2 and KRAS pathways. Additionally, proliferation, apoptosis, and oxidative DNA damage were assessed. Overall, NRF2 activation prevents initiation of chemically induced cancer, but promotes progression of pre-existing tumors regardless of chemical or genetic etiology. Once tumors are initiated, NRF2 inhibition is effective against the progression of chemically and spontaneously induced tumors. These results have important implications for NRF2-targeted cancer prevention and intervention strategies.


Assuntos
Neoplasias Pulmonares/genética , Fator 2 Relacionado a NF-E2/genética , Animais , Anticarcinógenos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Progressão da Doença , Isotiocianatos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Quassinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Uretana/análogos & derivados , Uretana/farmacologia
11.
J Biol Chem ; 293(6): 2029-2040, 2018 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-29255090

RESUMO

Nuclear factor erythroid-2-related factor 1 (NRF1) and NRF2 are essential for maintaining redox homeostasis and coordinating cellular stress responses. They are highly homologous transcription factors that regulate the expression of genes bearing antioxidant-response elements (AREs). Genetic ablation of NRF1 or NRF2 results in vastly different phenotypic outcomes, implying that they play different roles and may be differentially regulated. Kelch-like ECH-associated protein 1 (KEAP1) is the main negative regulator of NRF2 and mediates ubiquitylation and degradation of NRF2 through its NRF2-ECH homology-like domain 2 (Neh2). Here, we report that KEAP1 binds to the Neh2-like (Neh2L) domain of NRF1 and stabilizes it. Consistently, NRF1 is more stable in KEAP1+/+ than in KEAP1-/- isogenic cell lines, whereas NRF2 is dramatically stabilized in KEAP1-/- cells. Replacing NRF1's Neh2L domain with NRF2's Neh2 domain renders NRF1 sensitive to KEAP1-mediated degradation, indicating that the amino acids between the DLG and ETGE motifs, not just the motifs themselves, are essential for KEAP1-mediated degradation. Systematic site-directed mutagenesis identified the core amino acid residues required for KEAP1-mediated degradation and further indicated that the DLG and ETGE motifs with correct spacing are insufficient as a KEAP1 degron. Our results offer critical insights into our understanding of the differential regulation of NRF1 and NRF2 by KEAP1 and their different physiological roles.


Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/genética , Fator 1 Nuclear Respiratório/química , Fator 1 Nuclear Respiratório/genética , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Proteólise
12.
Artigo em Inglês | MEDLINE | ID: mdl-29026837

RESUMO

Members of the NR4A subfamily of nuclear receptors make up a highly conserved, functionally diverse group of transcription factors implicated in a multitude of cellular processes such as proliferation, differentiation, apoptosis, metabolism and DNA repair. The gene nhr-6, which encodes the sole C. elegans NR4A nuclear receptor homolog, has a critical role in organogenesis and regulates the development of the spermatheca organ system. Our previous work revealed that nhr-6 is required for spermatheca cell divisions in late L3 and early L4 and spermatheca cell differentiation during the mid L4 stage. Here, we utilized chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to identify NHR-6 binding sites during both the late L3/early L4 and mid L4 developmental stages. Our results revealed 30,745 enriched binding sites for NHR-6, ~70% of which were within 3 kb upstream of a gene transcription start site. Binding sites for a cohort of candidate target genes with probable functions in spermatheca organogenesis were validated through qPCR. Reproductive and spermatheca phenotypes were also evaluated for these genes following a loss-of-function RNAi screen which revealed several genes with critical functions during spermatheca organogenesis. Our results uncovered a complex nuclear receptor regulatory network whereby NHR-6 regulates multiple cellular processes during spermatheca organogenesis.

13.
Mol Cell Biol ; 37(11)2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28289076

RESUMO

Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) cause a hereditary cancer syndrome known as hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC-associated tumors harbor biallelic FH inactivation that results in the accumulation of the TCA cycle metabolite fumarate. Although it is known that fumarate accumulation can alter cellular signaling, if and how fumarate confers a growth advantage remain unclear. Here we show that fumarate accumulation confers a chronic proliferative signal by disrupting cellular iron signaling. Specifically, fumarate covalently modifies cysteine residues on iron regulatory protein 2 (IRP2), rendering it unable to repress ferritin mRNA translation. Simultaneously, fumarate increases ferritin gene transcription by activating the NRF2 (nuclear factor [erythroid-derived 2]-like 2) transcription factor. In turn, increased ferritin protein levels promote the expression of the promitotic transcription factor FOXM1 (Forkhead box protein M1). Consistently, clinical HLRCC tissues showed increased expression levels of both FOXM1 and its proliferation-associated target genes. This finding demonstrates how FH inactivation can endow cells with a growth advantage.


Assuntos
Carcinoma de Células Renais/patologia , Ferritinas/genética , Fumarato Hidratase/metabolismo , Fumaratos/farmacologia , Neoplasias Renais/patologia , Leiomiomatose/patologia , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Sequência de Aminoácidos , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína Forkhead Box M1/metabolismo , Humanos , Espaço Intracelular/metabolismo , Proteína 2 Reguladora do Ferro/química , Proteína 2 Reguladora do Ferro/metabolismo , Neoplasias Renais/enzimologia , Neoplasias Renais/genética , Leiomiomatose/enzimologia , Leiomiomatose/genética , Modelos Biológicos , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ácido Succínico/metabolismo
14.
Mol Carcinog ; 56(5): 1493-1500, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28019675

RESUMO

The NRF2 pathway activates a cell survival response when cells are exposed to xenobiotics or are under oxidative stress. Therapeutic activation of NRF2 can also be used prior to insult as a means of disease prevention. However, prolonged expression of NRF2 has been shown to protect cancer cells by inducing the metabolism and efflux of chemotherapeutics, leading to both intrinsic and acquired chemoresistance to cancer drugs. This effect has been termed the "dark side" of NRF2. In an effort to combat this chemoresistance, our group discovered the first NRF2 inhibitor, the natural product brusatol, however the mechanism of inhibition was previously unknown. In this report, we show that brusatol's mode of action is not through direct inhibition of the NRF2 pathway, but through the inhibition of both cap-dependent and cap-independent protein translation, which has an impact on many short-lived proteins, including NRF2. Therefore, there is still a need to develop a new generation of specific NRF2 inhibitors with limited toxicity and off-target effects that could be used as adjuvant therapies to sensitize cancers with high expression of NRF2.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Quassinas/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Retículo Endoplasmático/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Quassinas/farmacocinética , Análise de Sequência de RNA
15.
Cancer Prev Res (Phila) ; 10(1): 67-75, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27923803

RESUMO

Cutaneous squamous cell carcinomas (cSCC) are among the most common and highly mutated human malignancies. Solar UV radiation is the major factor in the etiology of cSCC. Whole-exome sequencing of 18 microdissected tumor samples (cases) derived from SKH-1 hairless mice that had been chronically exposed to solar-simulated UV (SSUV) radiation showed a median point mutation (SNP) rate of 155 per Mb. The majority (78.6%) of the SNPs are C.G>T.A transitions, a characteristic UVR-induced mutational signature. Direct comparison with human cSCC cases showed high overlap in terms of both frequency and type of SNP mutations. Mutations in Trp53 were detected in 15 of 18 (83%) cases, with 20 of 21 SNP mutations located in the protein DNA-binding domain. Strikingly, multiple nonsynonymous SNP mutations in genes encoding Notch family members (Notch1-4) were present in 10 of 18 (55%) cases. The histopathologic spectrum of the mouse cSCC that develops in this model resembles very closely the spectrum of human cSCC. We conclude that the mouse SSUV cSCCs accurately represent the histopathologic and mutational spectra of the most prevalent tumor suppressors of human cSCC, validating the use of this preclinical model for the prevention and treatment of human cSCC. Cancer Prev Res; 10(1); 67-75. ©2016 AACR.


Assuntos
Carcinoma de Células Escamosas/terapia , Receptores Notch/genética , Neoplasias Cutâneas/terapia , Proteína Supressora de Tumor p53/genética , Animais , Carcinogênese/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Análise Mutacional de DNA/métodos , Exoma , Feminino , Humanos , Camundongos , Camundongos Pelados , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Domínios Proteicos/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Raios Ultravioleta/efeitos adversos
16.
Anticancer Res ; 35(12): 6639-53, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26637880

RESUMO

BACKGROUND/AIM: Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a rare autosomal dominant disorder characterized by fumarate hydratase (FH) gene mutation. It is associated with the development of very aggressive kidney tumors, characterized by early onset and high metastatic potential, and has no effective therapy. The aim of the study was to establish a new preclinical platform for investigating morphogenetic and metabolic features, and alternative therapy of metastatic hereditary papillary renal cell carcinoma type 2 (PRCC2). MATERIALS AND METHODS: Fresh cells were collected from pleural fluid of a patient with metastatic hereditary PRCC2. Morphogenetic and functional characteristics were evaluated via microscopy, FH gene sequencing analysis, real-time polymerase chaine reaction and enzymatic activity measurement. We performed bioenergetic analysis, gene-expression profiling, and cell viability assay with 19 anti-neoplastic drugs. RESULTS: We established a new in vitro model of hereditary PRCC2 - the NCCFH1 cell line. The cell line possesses a c.1162 delA - p.Thr375fs frameshift mutation in the FH gene. Our findings indicate severe attenuation of oxidative phosphorylation and glucose-dependent growth of NCCFH1 cells that is consistent with the Warburg effect. Furthermore, gene-expression profiling identified that the most prominent molecular features reflected a high level of apoptosis, cell adhesion, and cell signaling. Drug screening revealed a marked sensitivity of FH(-/-) cells to mitoxantrone, epirubicin, topotecan and a high sensitivity to bortezomib. CONCLUSION: We demonstrated that the NCCFH1 cell line is a very interesting preclinical model for studying the metabolic features and testing new therapies for hereditary PRCC2, while bortezomib may be a potential efficient therapeutic option.


Assuntos
Carcinoma de Células Renais/genética , Fumarato Hidratase/metabolismo , Adolescente , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Humanos , Técnicas In Vitro , Masculino
17.
Biochem Soc Trans ; 43(4): 650-6, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26551707

RESUMO

The nuclear erythroid 2-like 2 transcription factor (NRF2), is a major regulator of cellular redox balance. Although NRF2 activation is generally regarded as beneficial to human health, recent studies have identified that sustained NRF2 activation is over-represented in many cancers. This raises the question regarding the role of NRF2 activation in the development and progression of those cancers. This review focuses on the mechanisms and the effects of NRF2 activation in two hereditary cancer predisposition syndromes: hereditary leiomyomatosis and renal cell cancer (HLRCC) and hereditary tyrosinemia type 1 (HT1). Because the cancer initiating mutations in these hereditary syndromes are well defined, they offer a unique opportunity to explore the roles of NRF2 activation in the early stages of carcinogenesis. Over the years, a variety of approaches have been utilized to study the biology of HLRCC and HT1. In HLRCC, in vitro studies have demonstrated the importance of NRF2 activation in sustaining cancer cell proliferation. In the mouse model of HT1 however, NRF2 activation seems to protect cells from malignant transformation. In both HT1 and HLRCC, NRF2 activation promotes the clearance of electrophilic metabolites, enabling cells to survive cancer-initiating mutations. Biological insights gained from the hereditary syndromes' studies may shed light on to the roles of NRF2 activation in sporadic tumours.


Assuntos
Leiomiomatose/genética , Fator 2 Relacionado a NF-E2/genética , Neoplasias Cutâneas/genética , Tirosinemias/genética , Neoplasias Uterinas/genética , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Mutação , Síndromes Neoplásicas Hereditárias
18.
Eur Urol ; 68(1): 77-83, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25597018

RESUMO

BACKGROUND: Testicular germ cell tumors are the most common cancer diagnosed in young men, and seminomas are the most common type of these cancers. There have been no exome-wide examinations of genes mutated in seminomas or of overall rates of nonsilent somatic mutations in these tumors. OBJECTIVE: The objective was to analyze somatic mutations in seminomas to determine which genes are affected and to determine rates of nonsilent mutations. DESIGN, SETTING, AND PARTICIPANTS: Eight seminomas and matched normal samples were surgically obtained from eight patients. INTERVENTION: DNA was extracted from tissue samples and exome sequenced on massively parallel Illumina DNA sequencers. Single-nucleotide polymorphism chip-based copy number analysis was also performed to assess copy number alterations. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The DNA sequencing read data were analyzed to detect somatic mutations including single-nucleotide substitutions and short insertions and deletions. The detected mutations were validated by independent sequencing and further checked for subclonality. RESULTS AND LIMITATIONS: The rate of nonsynonymous somatic mutations averaged 0.31 mutations/Mb. We detected nonsilent somatic mutations in 96 genes that were not previously known to be mutated in seminomas, of which some may be driver mutations. Many of the mutations appear to have been present in subclonal populations. In addition, two genes, KIT and KRAS, were affected in two tumors each with mutations that were previously observed in other cancers and are presumably oncogenic. CONCLUSIONS: Our study, the first report on exome sequencing of seminomas, detected somatic mutations in 96 new genes, several of which may be targetable drivers. Furthermore, our results show that seminoma mutation rates are five times higher than previously thought, but are nevertheless low compared to other common cancers. Similar low rates are seen in other cancers that also have excellent rates of remission achieved with chemotherapy. PATIENT SUMMARY: We examined the DNA sequences of seminomas, the most common type of testicular germ cell cancer. Our study identified 96 new genes in which mutations occurred during seminoma development, some of which might contribute to cancer development or progression. The study also showed that the rates of DNA mutations during seminoma development are higher than previously thought, but still lower than for other common solid-organ cancers. Such low rates are also observed among other cancers that, like seminomas, show excellent rates of disease remission after chemotherapy.


Assuntos
Seminoma/genética , Neoplasias Testiculares/genética , Adenosina Trifosfatases/genética , Adolescente , Adulto , Caderinas/genética , Estudos de Casos e Controles , Caseína Quinase II/genética , Proteínas Cromossômicas não Histona/genética , Variações do Número de Cópias de DNA , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Exoma/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Taxa de Mutação , Fosfatidilinositol 3-Quinases/genética , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de DNA , Proteínas Supressoras de Tumor/genética
19.
Cancer Res ; 74(24): 7430-41, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25339352

RESUMO

Oncogenic KRAS mutations found in 20% to 30% of all non-small cell lung cancers (NSCLC) are associated with chemoresistance and poor prognosis. Here we demonstrate that activation of the cell protective stress response gene NRF2 by KRAS is responsible for its ability to promote drug resistance. RNAi-mediated silencing of NRF2 was sufficient to reverse resistance to cisplatin elicited by ectopic expression of oncogenic KRAS in NSCLC cells. Mechanistically, KRAS increased NRF2 gene transcription through a TPA response element (TRE) located in a regulatory region in exon 1 of NRF2. In a mouse model of mutant KrasG12D-induced lung cancer, we found that suppressing the NRF2 pathway with the chemical inhibitor brusatol enhanced the antitumor efficacy of cisplatin. Cotreatment reduced tumor burden and improved survival. Our findings illuminate the mechanistic details of KRAS-mediated drug resistance and provide a preclinical rationale to improve the management of lung tumors harboring KRAS mutations with NRF2 pathway inhibitors.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Resistência a Medicamentos/genética , Fator 2 Relacionado a NF-E2/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/administração & dosagem , Resistência a Medicamentos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Camundongos , Mutação , Fator 2 Relacionado a NF-E2/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas p21(ras) , Quassinas/administração & dosagem , Elementos de Resposta/genética
20.
Cancer Res ; 73(7): 2044-51, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23365135

RESUMO

Sustained activation of the stress-regulated transcription factor NRF2 (NFE2L2) is a prominent feature of many types of cancer, implying that mutations driving NRF2 may be important to tumor progression. In hereditary type 2 papillary renal cell carcinoma (PRCC2, also known as hereditary leiomyomatosis and renal cell cancer), NRF2 activation is a direct consequence of the accumulation of intracellular fumarate, a result of fumarate hydratase (FH) inactivation, but it is not clear how NRF2 may be activated in sporadic forms of PRCC2. Here we show that somatic mutations in NRF2, CUL3, and SIRT1 are responsible for driving the NRF2 activation phenotype in sporadic PRCC2. Transcriptome sequencing revealed the expression pattern of mutant alleles of NRF2, CUL3, and SIRT1 and also confirmed NRF2 activation in clinical specimens. Our results show a convergence in somatic mutations in sporadic PRCC2 with FH mutation in hereditary PRCC2.


Assuntos
Carcinoma de Células Renais/genética , Proteínas Culina/genética , Neoplasias Renais/genética , Mutação/genética , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Sequência de Aminoácidos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Células Cultivadas , Proteínas Culina/metabolismo , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Técnicas Imunoenzimáticas , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Homologia de Sequência de Aminoácidos , Sirtuína 1/metabolismo
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