RESUMO
BACKGROUND: Fanconi anemia (FA) is a rare genetic disorder and one of the most common inherited forms of aplastic anemia. FA is an autosomal recessive or X-linked genetic disorder that is characterized by typical physical malformations and haematopoietic anomalies. In most cases of FA, patients harbor homozygous or double heterozygous mutations in the FANCA (60-65%), FANCC (10-15%), FANCG (~ 10%), FANCD2 (3-6%) or FANCF (2%) genes in different ethnic populations, which leads to inherited bone marrow failure (IBMF). Hence, it is important to screen such mutations in correlation with clinical manifestations of FA in various ethnic populations. APPROACH: An 11 year old female pediatric patient of an East India family was presented with febrile illness, having thrombocytopenia with positive dengue IgM (Immunoglobulin M) and treated as a case of dengue hemorrhagic fever at the initial stage of diagnosis. Chromosomal breakage study was performed based on the abnormal physical examination, which showed 100% breaks, triradials, and quadrilaterals in mitomycin (MMC)-induced peripheral blood lymphocyte culture. Importantly, conventional cytogenetic assay in most of the bone marrow cells revealed an additional gain in chromosome 3q+ [46,XX,add(3)(q25)] and terminal loss in chr8p- [46,XX,del(8)(p23)], which might have a prognostic relevance in the outcomes of the FA patient. The bone marrow aspiration and biopsy were repeated and the results showed acute leukemia with 39% blast cells. Whole-genome sequencing analysis of the patient confirmed the presence of (exon 1; 496 > C-T) non-sense mutation leading to a truncated FANCF protein attributed to a stop codon at the amino acid position 166. CONCLUSION: The study reported the presence of a homozygous C-T exon 1 mutation in FANCF gene in the female pediatric patient from Odisha, India associated with FA. Furthermore, both parents were found to be carriers of FANCF gene mutation, as this allele was found to be in heterozygous state upon genome sequencing. The pathogenicity of the agent was robustly supported by the clinical phenotype and biochemical observations, wherein the patient eventually developed acute myeloid leukemia. The findings of the study infer the importance of early detection of FA and the associated mutations, which might lead to the development of acute myeloid leukemia.
Assuntos
Anemia de Fanconi , Leucemia Mieloide Aguda , Feminino , Humanos , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Proteínas de Ligação a DNA/genética , Mutação/genética , Éxons , Leucemia Mieloide Aguda/genéticaRESUMO
BACKGROUND: Fanconi anemia (FA) is an inherited bone marrow failure syndrome associated with characteristic dysmorphology primarily caused by biallelic pathogenic germline variants in any of 22 different DNA repair genes. There are limited data on the specific molecular causes of FA in different ethnic groups. METHODS: We performed exome sequencing and copy number variant analyses on 19 patients with FA from 17 families undergoing hematopoietic cell transplantation evaluation in Pakistan. The scientific literature was reviewed, and we curated germline variants reported in patients with FA from South Asia and the Middle East. RESULTS: The genetic causes of FA were identified in 14 of the 17 families: seven FANCA, two FANCC, one FANCF, two FANCG, and two FANCL. Homozygous and compound heterozygous variants were present in 12 and two families, respectively. Nine families carried variants previously reported as pathogenic, including two families with the South Asian FANCL founder variant. We also identified five novel likely deleterious variants in FANCA, FANCF, and FANCG in affected patients. CONCLUSIONS: Our study supports the importance of determining the genomic landscape of FA in diverse populations, in order to improve understanding of FA etiology and assist in the counseling of families.
Assuntos
Anemia de Fanconi/genética , Frequência do Gene , Adolescente , Ásia , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Exoma , Anemia de Fanconi/diagnóstico , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Feminino , Efeito Fundador , Humanos , Masculino , Oriente Médio , MutaçãoRESUMO
Fanconi anemia (FA) is a rare chromosomal instability syndrome with various clinical features and high cancer incidence. Despite being a DNA repair disorder syndrome and a frequently observed clinical hypersensitivity of FA patients towards ionizing radiation, the experimental evidence regarding the efficiency of radiation-induced DNA double-strand break (DSB) repair in FA is very controversial. Here, we performed a thorough analysis of the repair of radiation-induced DSBs in G1 and G2 in FA fibroblasts of complementation groups A, C, D1 (BRCA2), D2, E, F, G and P (SLX4) in comparison to normal human lung and skin fibroblasts. γH2AX, 53BP1, or RPA foci quantification after X-irradiation was combined with cell cycle markers. Cytogenetic analyses were performed on first metaphases after irradiation in G1 and by premature chromosome condensation after exposure in G2. Furthermore, the role of canonical-NHEJ and alternative-NHEJ for the fidelity of the repair of radiation-induced DSBs was examined. In FA fibroblasts, DSB repair was normal in G1 but compromised and more error-prone in the slow repair component of G2 as suggested by higher yields of radiation-induced γH2AX and 53BP1 foci as well as chromatid exchanges. However, RPA foci quantification in G2 indicated proficiency for homology-directed repair of DSBs in FA except for FA D1 (BRCA2). In lung fibroblasts, DSB repair in G1 was conducted with normal kinetics but elevated chromosome exchanges compared to skin fibroblasts. The overall repair of radiation-induced DSBs and the formation of chromosome exchanges in normal and FA fibroblasts in G1 and G2 were governed by canonical-NHEJ with no contribution of alternative-NHEJ. Together, we show impaired repair of radiation-induced DSBs in various FA complementation groups in the slow repair component of G2 that might promote the formation of potentially oncogenic aberrations and clinical radiation hypersensitivity.
Assuntos
Ciclo Celular , Aberrações Cromossômicas , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Anemia de Fanconi/metabolismo , Mutação , Reparo de DNA por Recombinação , Proteína BRCA2/genética , Células Cultivadas , DNA/metabolismo , DNA/efeitos da radiação , Anemia de Fanconi/genética , Anemia de Fanconi/fisiopatologia , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação E da Anemia de Fanconi/genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fibroblastos/efeitos da radiação , Histonas/metabolismo , Humanos , Cinética , Recombinases/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Raios XRESUMO
BACKGROUND/AIMS: Fanconi anemia complement group F (FANCF) is known to be involved in DNA repair, and the overexpression of FANCF protein leads to cell proliferation and ultimately to cancer. The purpose of this study was to assess whether FANCF methylation was associated with colorectal cancer (CRC). MATERIALS AND METHODS: A case-control experiment was conducted to study the association between FANCF methylation and CRC. We used quantitative methylation-specific PCR to measure the FANCF promoter methylation, and the percentage of methylation reference (PMR) to quantify the FANCF promoter methylation level. To investigate the effect of the selected FANCF fragment on gene expression regulation, we also performed a dual-luciferase reporter gene assay. RESULTS: The results indicated that FANCF methylation in CRC tumor tissues was significantly lower than that in the nontumor tissues (median PMR: 44.86% vs. 65.77%, p=0.00001). Analysis of receiver-operating characteristic curves showed that FANCF hypomethylation had a diagnostic value for CRC (area under curve [AUC]: 0.670, sensitivity: 55.8%, specificity: 71.7%, p=0.00001). The dual-luciferase reporter assay showed that the FANCF fragment upregulated gene expression (fold change: 1.93, p=0.002). CONCLUSION: Research demonstrates for the first time that FANCF hypomethylation is significantly associated with CRC risk. FANCF hypomethylation may ultimately increase the risk of CRC by upregulating the expression of FANCF.
Assuntos
Povo Asiático/genética , Neoplasias Colorretais/etnologia , Neoplasias Colorretais/genética , Metilação de DNA/genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Área Sob a Curva , Estudos de Casos e Controles , China/etnologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Curva ROC , Fatores de RiscoRESUMO
Adriamycin(ADR) is still considered to be one of the most effective agents in the treatment of breast cancer (BrCa), its efficacy is compromised by intrinsic resistance or acquire characteristics of multidrug resistance. At present, there are few genetic alterations that can be exploited as biomarkers to guide targeted use of ADR in clinical. Therefore, exploring the determinants of ADR sensitivity is pertinent for their optimal clinical application. TP53 is the most frequently mutated gene in human BrCa, p53 mutation has been reported to be closely related to ADR resistance, whereas the underlying mechanisms that cause endogenous ADR resistance in p53-mutant BrCa cells are not completely understood. The aim of the present study was to investigate the potential roles of miRNA in the response to ADR in p53-mutated breast cancer. Here, we report that BrCa cells expressing mutp53 are more resistant to ADR than cells with wild-type p53 (wtp53). The DNA repair protein- Fanconi anemia complementation group F protein (FANCF) and the translesion synthesis DNA polymerase REV1 protein is frequently abundant in the context of mutant p53 of BrCa. By targeting two key factors, miR-30c increases the sensitivity of BrCa cells to ADR. Furthermore, p53 directly activates the transcription of miR-30c by binding to its promoter. Subsequent analyses revealed that p53 regulates REV1 and FANCF by modulating miR-30c expression. Mutation of the p53 abolished this response. Consistently, reduced miR-30c expression is highly correlated with human BrCa with p53 mutational status and is associated with poor survival. We propose that one of the pathways affected by mutant p53 to increase intrinsic resistance to ADR involves miR-30c downregulation and the consequent upregulation of FANCF and REV1. The novel miRNA-mediated pathway that regulates chemoresistance in breast cancer will facilitate the development of novel therapeutic strategies.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Doxorrubicina/farmacologia , Proteína do Grupo de Complementação F da Anemia de Fanconi/metabolismo , MicroRNAs/metabolismo , Nucleotidiltransferases/metabolismo , Proteína Supressora de Tumor p53/genética , Regiões 3' não Traduzidas , Animais , Antibióticos Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Mutação , Nucleotidiltransferases/genética , Transplante Heterólogo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by congenital anomalies, early-onset bone marrow failure, and a high predisposition to cancers. Up to know, different genes involved in the DNA repair pathway, mainly FANCA genes, have been identified to be affected in patients with FA. CASE PRESENTATION: Here, we report clinical, laboratory and genetic findings in a 3.5-year-old Iranian female patient, a product of a consanguineous marriage, who was suspicious of FA, observed with short stature, microcephaly, skin hyperpigmentation, anemia, thrombocytopenia and hypo cellular bone marrow. Therefore, Next Generation Sequencing was performed to identify the genetic cause of the disease in this patient. Results revealed a novel, private, homozygous frameshift mutation in the FANCF gene (NM_022725: c. 534delG, p. G178 fs) which was confirmed by Sanger sequencing in the proband. CONCLUSION: Such studies may help uncover the exact pathomechanisms of this disorder and establish the genotype-phenotype correlations by identification of more mutations in this gene. It is the first report of a mutation in the FANCF gene in Iranian patients with Fanconi anemia. This new mutation correlates with a hematological problem (pancytopenia), short stature, and microcephaly and skin hyperpigmentation. Until now, no evidence of malignancy was detected.
Assuntos
Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Estudos de Associação Genética , Predisposição Genética para Doença/genética , Deleção de Sequência , Sequência de Bases , Pré-Escolar , Consanguinidade , Anemia de Fanconi/fisiopatologia , Proteína do Grupo de Complementação F da Anemia de Fanconi/metabolismo , Feminino , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Irã (Geográfico) , Pancitopenia/genética , Linhagem , Análise de Sequência de ProteínaRESUMO
OBJECTIVE: DNA repair pathways are potential targets of molecular therapy in cancer patients. The FANCD2, BRIP1, BRCA1/2, and FANCF genes are involved in homologous recombination DNA repair, which implicates their possible role in cell response to DNA-damaging agents. We evaluated a clinical significance of pre-treatment expression of these genes at mRNA level in 99 primary, advanced-stage ovarian carcinomas from patients, who later received taxane-platinum (TP) or platinum-cyclophosphamide (PC) treatment. METHODS: Gene expression was determined with the use of Real-Time PCR. The BRCA2 and BRIP1 gene sequence was investigated with the use of SSCP, dHPLC, and PCR-sequencing. RESULTS: Increased FANCD2 expression occurred to be a negative prognostic factor for all patients (PC+TP:HR 3.85, p = 0.0003 for the risk of recurrence; HR 1.96, p = 0.02 for the risk of death), and this association was even stronger in the TP-treated group (HR 6.7, p = 0.0002 and HR 2.33, p = 0.01, respectively). Elevated BRIP1 expression was the only unfavorable molecular factor in the PC-treated patients (HR 8.37, p = 0.02 for the risk of recurrence). Additionally, an increased FANCD2 and BRCA1/2 expression levels were associated with poor ovarian cancer outcome in either TP53-positive or -negative subgroups of the TP-treated patients, however these groups were small. Sequence analysis identified one protein truncating variant (1/99) in BRCA2 and no mutations (0/56) in BRIP1. CONCLUSIONS: Our study shows for the first time that FANCD2 overexpression is a strong negative prognostic factor in ovarian cancer, particularly in patients treated with TP regimen. Moreover, increased mRNA level of the BRIP1 is a negative prognostic factor in the PC-treated patients. Next, changes in the BRCA2 and BRIP1 genes are rare and together with other analyzed FA genes considered as homologous recombination deficiency may not affect the expression level of analyzed genes.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Adulto , Idoso , Biomarcadores Tumorais , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/terapia , Prognóstico , Modelos de Riscos Proporcionais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Fanconi anemia (FA) is a cancer predisposition syndrome characterized by congenital abnormalities, bone marrow failure, and hypersensitivity to aldehydes and crosslinking agents. For FA patients, gene editing holds promise for therapeutic applications aimed at functionally restoring mutated genes in hematopoietic stem cells. However, intrinsic FA DNA repair defects may obstruct gene editing feasibility. Here, we report on the CRISPR/Cas9-mediated correction of a disruptive mutation in Fancf. Our experiments revealed that gene editing could effectively restore Fancf function via error-prone end joining resulting in a 27% increased survival in the presence of mitomycin C. In addition, templated gene correction could be achieved after double strand or single strand break formation. Although templated gene editing efficiencies were low (≤6%), FA corrected embryonic stem cells acquired a strong proliferative advantage over non-corrected cells, even without imposing genotoxic stress. Notably, Cas9 nickase activity resulted in mono-allelic gene editing and avoidance of undesired mutagenesis. In conclusion: DNA repair defects associated with FANCF deficiency do not prohibit CRISPR/Cas9 gene correction. Our data provide a solid basis for the application of pre-clinical models to further explore the potential of gene editing against FA, with the eventual aim to obtain therapeutic strategies against bone marrow failure.
Assuntos
Sistemas CRISPR-Cas/genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Edição de Genes/métodos , Terapia Genética/métodos , Animais , Células Cultivadas , Reparo do DNA , Orelha , Fibroblastos , Camundongos , Células-Tronco Embrionárias MurinasRESUMO
OBJECTIVE: The expression of homologous recombination (HR) genes in high grade ovarian cancer (HGOC) samples from debulking surgeries were correlated to outcomes in patients selected for chemotherapy treatment regimens. STUDY DESIGN: RNA was extracted from 96 fresh frozen tumor samples from debulking surgeries from chemotherapy naïve patients with HGOC (primary derived surgeries (PDS), nâ¯=â¯55) or following neoadjuvant chemotherapy treatment (NACT), nâ¯=â¯41). The samples were selected for high tumor content by a gynecological pathologist, and cancer cell content was further confirmed using a percent tumor content covariate, and mutation score covariate analysis. Gene expression analysis was performed using a tailored NanoString-based Pancancer Pathway Panel. Cox proportional hazard regression models were used to assess the associations between the expression of 19 HR genes and survival. RESULTS: In the PDS group, over-expression of six HR genes (C11orf30, NBN, FANCF, FANCC, FANCB, RAD50) was associated with improved outcome, in contrast to the NACT group where four HR genes (BRCA2, TP53, FANCB, RAD51) were associated with worse outcome. With the adding extent of debulking as a covariate, three HR genes (NBN, FANCF, RAD50), and only one HR gene (RAD51) remained significantly associated with survival in PDS and NACT groups, respectively. CONCLUSION: Distinct HR expression profiles define subgroups associated with overall outcome in patients that are exposed to neoadjuvant chemotherapy and not only chemotherapy-naïve patients.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Endometrioide/genética , Procedimentos Cirúrgicos de Citorredução , Terapia Neoadjuvante , Neoplasias Císticas, Mucinosas e Serosas/genética , Neoplasias Ovarianas/genética , Reparo de DNA por Recombinação/genética , Hidrolases Anidrido Ácido , Idoso , Proteína BRCA1/genética , Proteína BRCA2/genética , Antígeno Ca-125/sangue , Carcinoma Endometrioide/sangue , Carcinoma Endometrioide/tratamento farmacológico , Carcinoma Endometrioide/patologia , Proteínas de Ciclo Celular/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana/sangue , Pessoa de Meia-Idade , Gradação de Tumores , Proteínas de Neoplasias/genética , Neoplasias Císticas, Mucinosas e Serosas/sangue , Neoplasias Císticas, Mucinosas e Serosas/tratamento farmacológico , Neoplasias Císticas, Mucinosas e Serosas/patologia , Proteínas Nucleares/genética , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Ovariectomia , PTEN Fosfo-Hidrolase/genética , Prognóstico , Modelos de Riscos Proporcionais , Rad51 Recombinase/genética , Proteínas Repressoras/genética , Taxa de Sobrevida , Transcriptoma , Proteína Supressora de Tumor p53/genéticaRESUMO
Approximately 5-10% of all pancreatic cancer patients carry a predisposing mutation in a known susceptibility gene. Since >90% of patients present with late stage disease, it is crucial to identify high risk individuals who may be amenable to early detection or other prevention. To explore the spectrum of hereditary pancreatic cancer susceptibility, we evaluated germline DNA from pancreatic cancer participants (n = 53) from a large hereditary cancer registry. For those without a known predisposition mutation gene (n = 49), germline next generation sequencing was completed using targeted capture for 706 candidate genes. We identified 16 of 53 participants (30%) with a pathogenic (P) or likely pathogenic (LP) variant that may be related to their hereditary pancreatic cancer predisposition; seven had mutations in genes associated with well-known cancer syndromes (13%) [ATM (2), BRCA2 (3), MSH2 (1), MSH6 (1)]. Many had mutations in Fanconi anemia complex genes [BRCA2 (3 participants), FANCF, FANCM]. Eight participants had rare protein truncating variants of uncertain significance with no other P or LP variants. Earlier age of pancreatic cancer diagnosis (57.5 vs 64.8 years) was indicative of possessing a P or LP variant, as was cancer family history (p values <0.0001). Our multigene panel approach for identifying known cancer predisposing genetic susceptibility in those at risk for hereditary pancreatic cancer may have direct applicability to clinical practice in cases with mutations in actionable genes. Future pancreatic cancer predisposition studies should include evaluation of the Fanconi anemia genes.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Predisposição Genética para Doença , Síndromes Neoplásicas Hereditárias/genética , Neoplasias Pancreáticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína BRCA2/genética , DNA Helicases/genética , Análise Mutacional de DNA , Anemia de Fanconi/genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Feminino , Testes Genéticos/métodos , Mutação em Linhagem Germinativa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Sistema de Registros , Adulto JovemRESUMO
Recent advances have been made in the understanding of Fanconi anemia (FA), a hereditary disease that increases the risk for head and neck squamous cell carcinomas (HNSCC) by 500- to 700-fold. FA patients harbour germline mutations in genes of cellular DNA repair pathways that are assumed to facilitate the accumulation of mutations during HNSCC development. Mutations in these FA genes may also contribute to HNSCC in general. In the present study, we analysed three FA genes; FANCF, FANCG and BRIP1, that are involved in the repair of DNA inter strand cross-links, in HNSCC and their potential role for patient survival. We measured loss of heterozygosity (LOH) mutations at eight microsatellite loci flanking three FA genes in 54 HNSCC of the oral cavity and corresponding blood samples. Survival analyses were carried out using mutational data and clinical variables. LOH was present in 17% (FANCF region), 41% (FANCG region) and 11% (BRIP1 region) of the patients. Kaplan-Meier survival curves and log-rank tests indicated strong clinical predictors (lymph node stages with decreased survival: p=2.69e-12; surgery with improved survival: p=0.0005). LOH in the FANCF region showed a weaker association with decreased overall survival (p=0.006), which however, did not hold in multivariate analyses. LOH may predominantly indicate copy number gains in FANCF and losses in FANCG and BRIP1. Integration of copy number data and gene expression proved difficult as the available sample sets did not overlap. In conclusion, LOH in FA genes appears to be a common feature of HNSCC development seen here in 57% of patients and other mutation types may increase this mutation frequency. We suggest larger patient cohorts would be needed to test the observed association of LOH in FANCF and patient survival comprehensively.
Assuntos
Carcinoma de Células Escamosas/genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Neoplasias de Cabeça e Pescoço/genética , RNA Helicases/genética , Idoso , Carcinoma de Células Escamosas/patologia , Intervalo Livre de Doença , Anemia de Fanconi/patologia , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Estimativa de Kaplan-Meier , Perda de Heterozigosidade/genética , Masculino , Pessoa de Meia-Idade , Boca/patologia , Mutação , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Fanconi anemia is a heterogeneous genetic disorder that is characterized by progressive bone marrow failure, congenital anomalies, and markedly increased risk for malignancies. Mutations in the FANCF (FA-F) gene represent approximately 2% of affected patients. Currently, information on the phenotypic findings of patients with Fanconi anemia from biallelic mutations in FANCF is limited. Here, we report three patients who illustrate the clinical variability within the FA-F group. This analysis suggests a more severe phenotype for those with the common c.484_485delCT mutation. © 2016 Wiley Periodicals, Inc.
Assuntos
Alelos , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Estudos de Associação Genética , Mutação , Fenótipo , Biomarcadores , Criança , Pré-Escolar , Anemia de Fanconi/terapia , Evolução Fatal , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Lactente , Masculino , Análise de Sequência de DNARESUMO
OBJECTIVE: To assess the 5' CpG island methylation of Fanconi anemia, complementation group F (FANCF) gene in epithelial ovarian cancer (EOC) tissues and normal ovarian tissues and to investigate the relationship between FANCF methylation and clinicopathologic features and prognosis of EOC. METHODS: The experiment was performed with 112 EOC tissue samples (case group) and 60 normal ovarian tissues (control group). With methylation-specific polymerase chain reaction (MSP), FANCF methylation status of cases and controls was assessed. And the association between FANCF methylation and the clinicopathological features of EOC was investigated with univariate survival analysis and Cox regression model analysis. RESULTS: The methylation-positive rate of the case group was significantly higher than that of the control group (P = 0.015). The FANCF promoter methylation rates showed significant differences in the comparisons stratified by age, International Federation of Gynecology and Obstetrics (FIGO) staging, histopathological classification, and lymph node metastasis (all P < .05). Univariate survival analysis showed there were significant differences in mean survival time between the groups based on FIGO staging, histopathological classification, lymph node metastasis, and FANCF methylation (all P < .05). Cox regression model analysis suggested that FIGO staging and FANCF methylation were independent risk factors for EOC prognosis. CONCLUSION: CpG island methylation of FANCF gene promoter region is strongly associated with the susceptibility and clinicopathologic features of EOC. The FIGO staging and FANCF methylation are independent risk factors for EOC prognosis.
Assuntos
Metilação de DNA , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Predisposição Genética para Doença , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Regiões Promotoras Genéticas , Fatores Etários , Carcinoma Epitelial do Ovário , Estudos de Casos e Controles , Ilhas de CpG , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática/genética , Metástase Linfática/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Epiteliais e Glandulares/mortalidade , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Prognóstico , Taxa de SobrevidaRESUMO
BACKGROUND: Cisplatin is one of the most commonly used chemotherapy agent for lung cancer. The therapeutic efficacy of cisplatin is limited by the development of resistance. In this study, we test the effect of RNA interference (RNAi) targeting Fanconi anemia (FA)/BRCA pathway upstream genes on the sensitivity of cisplatin-sensitive (A549 and SK-MES-1) and -resistant (A549/DDP) lung cancer cells to cisplatin. RESULT: Using small interfering RNA (siRNA), knockdown of FANCF, FANCL, or FANCD2 inhibited function of the FA/BRCA pathway in A549, A549/DDP and SK-MES-1 cells, and potentiated sensitivity of the three cells to cisplatin. The extent of proliferation inhibition induced by cisplatin after knockdown of FANCF and/or FANCL in A549/DDP cells was significantly greater than in A549 and SK-MES-1 cells, suggesting that depletion of FANCF and/or FANCL can reverse resistance of cisplatin-resistant lung cancer cells to cisplatin. Furthermore, knockdown of FANCL resulted in higher cisplatin sensitivity and dramatically elevated apoptosis rates compared with knockdown of FANCF in A549/DDP cells, indicating that FANCL play an important role in the repair of cisplatin-induced DNA damage. CONCLUSION: Knockdown of FANCF, FANCL, or FANCD2 by RNAi could synergize the effect of cisplatin on suppressing cell proliferation in cisplatin-resistant lung cancer cells through inhibition of FA/BRCA pathway.
Assuntos
Proteína BRCA1 , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Proteína do Grupo de Complementação F da Anemia de Fanconi , Proteína do Grupo de Complementação L da Anemia de Fanconi , Neoplasias Pulmonares , Interferência de RNA , Transdução de Sinais , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/antagonistas & inibidores , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação F da Anemia de Fanconi/antagonistas & inibidores , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/antagonistas & inibidores , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
The Fanconi anemia (FA)associated proteins FANCF and FANCD2 are important components of the FA pathway of DNA crosslink repair. FANCF and FANCD2 have been found to be involved in drugresistant multiple myeloma, ovarian cancer, nonsmallcell lung cancer, and head and neck cancer. However, it is unclear whether these two genes participate in adriamycin (ADR)resistant leukemia. Therefore, the aim of the current study was to investigate FANCF and FANCD2 expression in drugresistant and drugsensitive leukemia cells. Western blot analysis revealed enhanced FANCF expression and monoubiquitination of FANCD2 in ADRresistant cells. Additionally, it was observed that drugresistant cells had reduced DNA damage compared with drugsensitive cells. The results of this study indicate that the FA pathway may confer leukemia resistance to ADR via enhanced DNA interstrand crosslink repair.
Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação F da Anemia de Fanconi/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , Leucemia/metabolismo , Leucemia/patologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacosRESUMO
The Fanconi anemia/BRCA (FA/BRCA) pathway plays a vital role in DNA damage repair induced by DNA cross-linking agents and is closely related to drug response in cancer treatment. Here we demonstrate that the FA/BRCA pathway contributes to acquired drug resistance in adriamycin (ADR)-resistant leukemia cell lines, and disruption of this pathway partially reverses the drug resistance. We observed that ADR-resistant cells have reduced DNA interstrand cross-links (ICL) compared with ADR-sensitive cells. Western blot studies demonstrated enhanced FA protein expression in ADR-resistant cells. Using siRNA to knock down FANCF in K562/R drug-resistant cells showed increases in sensitivity to ADR and ADR-induced DNA damage, and demonstrated a direct relationship between the FA/BRCA pathway and drug sensitivity. Overexpression of FANCF in K562 drug-sensitive cells partially reproduced the drug-resistant phenotype. These results show that the FA/BRCA pathway is involved in acquired ADR resistance of leukemia cells. The FA/BRCA pathway may be a new target to reverse ADR resistance in leukemia treatment.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Doxorrubicina/farmacologia , Proteína do Grupo de Complementação F da Anemia de Fanconi/metabolismo , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Western Blotting , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Dano ao DNA , Reparo do DNA , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Expressão Gênica , Humanos , Células K562 , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.
Assuntos
Humanos , Antineoplásicos/farmacologia , Movimento Celular/genética , Proliferação de Células/genética , /genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistência a Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Interferência de RNA , RNA Interferente PequenoRESUMO
Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.
Assuntos
Antineoplásicos/farmacologia , Movimento Celular/genética , Proliferação de Células/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistência a Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Interferência de RNA , RNA Interferente PequenoRESUMO
In the present study, we downregulated FANCF expression by small interfering RNA (siRNA) in OVCAR ovarian cancer cells to address the effects of decreased FANCF expression on the function of the Fanconi anemia (FA)/breast cancer susceptibility gene (BRCA) pathway. Furthermore, we investigated whether this method increases the sensitivity of OVCAR3 cells to adriamycin (ADM) and the possible mechanism(s). We found that silencing of FANCF inactivated the FA/BRCA pathway by decreasing the monoubiquitination and focus formation of FANCD2 and reduced the function of the FA/BRCA pathway, resulting in the inhibition of cell proliferation, increased cell apoptosis and DNA damage in OVCAR3 cells. Moreover, we observed that silencing of FANCF enhanced the antiproliferative effect of ADM in OVCAR3 cells and increased ADM intracellular accumulation consequently sensitizing OVCAR3 cells to ADM. Furthermore, silencing of FANCF increased cell apoptosis of OVCAR3 cells which was caused by decreased mitochondrial membrane potential (MMP)-induced DNA damage, activated Jun N-terminal kinase (JNK), increased release of cytochrome c, increased expression of cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP) dependent on JNK activation following treatment of ADM. Collectively, we confirm that silencing of FANCF sensitizes OVCAR3 ovarian cancer cells to ADM, suggesting that FANCF may serve as a potential target for therapeutic strategies in the treatment of ovarian cancer.
Assuntos
Apoptose/efeitos dos fármacos , Doxorrubicina/farmacologia , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , RNA Interferente Pequeno/genética , Apoptose/genética , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Terapia Combinada , Citocromos c/genética , Citocromos c/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação F da Anemia de Fanconi/metabolismo , Feminino , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Fanconi anemia complementation group-F (FANCF) is a key factor to maintain the function of FA/BRCA, a DNA-damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. In this study, we examined the effects and mechanisms of FANCF-RNAi on the sensitivity of breast cancer cells to mitoxantrone (MX). FANCF silencing by FANCF-shRNA blocked functions of FA/BRCA pathway through inhibition of FANCD2 mono-ubiquitination in breast cancer cell lines MCF-7 and T-47D. In addition, FANCF shRNA inhibited cell proliferation, induced apoptosis, and chromosome fragmentation in both breast cancer cells. We also found that FANCF silencing potentiated the sensitivity to MX in breast cancer cells, accompanying with an increase in intracellular MX accumulation and a decrease in BCRP expression. Furthermore, we found that the blockade of FA/BRCA pathway by FANCF-RNAi activated p38 and JNK MAPK signal pathways in response to MX treatment. BCRP expression was restored by p38 inhibitor SB203580, but not by JNK inhibitor SP600125. FANCF silencing increased JNK and p38 mediated activation of p53 in MX-treated breast cancer cells, activated the mitochondrial apoptosis pathway. Our findings indicate that FANCF shRNA potentiates the sensitivity of breast cancer cells to MX, suggesting that FANCF may be a potential target for therapeutic strategies for the treatment of breast tumors.