RESUMO
PURPOSE: The phenotypic and genotypic landscapes in multifocal glioblastoma (MF GBM) cases can vary greatly among lesions. In a MF GBM patient, the rapid development of a secondary lesion was investigated to determine if a unique genetic signature could account for the apparent increased malignancy of this lesion. METHODS: The primary (G52) and secondary (G53) tumours were resected to develop patient derived models followed by functional assays and multiplatform molecular profiling. RESULTS: Molecular profiling revealed G52 was wild-type for TP53 while G53 presented with a TP53 missense mutation. Functional studies demonstrated increased proliferation, migration, invasion and colony formation in G53. CONCLUSION: This data suggests that the TP53 mutation led to gain-of-function phenotypes and resulted in greater overall oncogenic potential of G53.
Assuntos
Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Proteína Supressora de Tumor p53/genética , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/patologia , Mutação com Ganho de Função , Glioblastoma/patologia , Humanos , Antígeno Ki-67/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Células Tumorais CultivadasRESUMO
BACKGROUND: The O 6 -methylguanine methyltransferase (MGMT) gene is frequently unmethylated in patients with glioblastoma (GBM), rendering them non-responsive to the standard treatment regime of surgery followed by concurrent radiotherapy (RT) and temozolomide. Here, we investigate the efficacy of adding a PARP inhibitor, veliparib, to radiotherapy to treat MGMT unmethylated GBM. METHODS: The inhibition of PARP with veliparib (ABT-888), a potent and orally bioavailable inhibitor in combination with RT was tested on a panel of patient derived cell lines (PDCLs) and patient-derived xenografts (PDX) models generated from GBM patients with MGMT unmethylated tumors. RESULTS: The combination of veliparib and RT inhibited colony formation in the majority of PDCLs tested. The PDCL, RN1 showed significantly reduced levels of the homologous repair protein, Mre11 and a heightened response to PARP inhibition measured by increased apoptosis and decreased colony formation. The oral administration of veliparib (12.5 mg/kg, twice daily for 5 days in a 28-day treatment cycle) in combination with whole brain RT (4 Gy) induced apoptosis (Tunel staining) and decreased cell proliferation (Ki67 staining) in a PDX of MGMT unmethylated GBM. Significantly longer survival times of the PDX treated with the combination treatment were recorded compared to RT only or veliparib only. CONCLUSIONS: Our results demonstrate preclinical efficacy of targeting PARP at multiple levels and provide a new approach for the treatment of MGMT unmethylated GBM.
Assuntos
Benzimidazóis/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/radioterapia , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/radioterapia , Proteínas Supressoras de Tumor/genética , Animais , Apoptose/efeitos dos fármacos , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Terapia Combinada , Modelos Animais de Doenças , Feminino , Humanos , Camundongos Nus , Análise de SobrevidaRESUMO
PURPOSE: Constitutional MLH1 epimutations manifest as promoter methylation and silencing of the affected allele in normal tissues, predisposing to Lynch syndrome-associated cancers. This study investigated their frequency and inheritance. METHODS: A total of 416 individuals with a colorectal cancer showing loss of MLH1 expression and without deleterious germline mutations in MLH1 were ascertained from the Colon Cancer Family Registry (C-CFR). Constitutive DNA samples were screened for MLH1 methylation in all 416 subjects and for promoter sequence changes in 357 individuals. RESULTS: Constitutional MLH1 epimutations were identified in 16 subjects. Of these, seven (1.7%) had mono- or hemi-allelic methylation and eight had low-level methylation (2%). In one subject the epimutation was linked to the c.-27C>A promoter variant. Testing of 37 relatives from nine probands revealed paternal transmission of low-level methylation segregating with a c.+27G>A variant in one case. Five additional probands had a promoter variant without an MLH1 epimutation, with three showing diminished promoter activity in functional assays. CONCLUSION: Although rare, sequence changes in the regulatory region of MLH1 and aberrant methylation may alone or together predispose to the development of cancer. Screening for these changes is warranted in individuals who have a negative germline sequence screen of MLH1 and loss of MLH1 expression in their tumor.Genet Med 2013:15(1):25-35.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais/genética , Epigênese Genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Estudos de Coortes , Neoplasias Colorretais/patologia , Metilação de DNA , Feminino , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Mosaicismo , Proteína 1 Homóloga a MutL , Mutação , Linhagem , Sistema de Registros , Análise de Sequência de DNA , Adulto JovemRESUMO
Genomic imprinting first evolved in mammals around the time that humans last shared a common ancestor with marsupials and monotremes (180-210 million years ago). Recent comparisons of large imprinted domains in these divergent mammalian groups have shown that imprinting evolved haphazardly at various times in different lineages, perhaps driven by different selective forces. Surprisingly, some imprinted domains were formed relatively recently, using non-imprinted components acquired from unexpected genomic regions. Rearrangement and the insertion of retrogenes, small nucleolar RNAs, microRNAs, differential CpG methylation and control by non-coding RNA often accompanied the acquisition of imprinting. Here, we use comparisons between different mammalian groups to chart the course of evolution of two related epigenetic regulatory systems in mammals: genomic imprinting and X-chromosome inactivation.
Assuntos
Evolução Molecular , Impressão Genômica/fisiologia , Mamíferos , Animais , Proteínas Reguladoras de Apoptose , Cromossomos Humanos Par 14 , Proteínas de Ligação a DNA , Humanos , Marsupiais/genética , Modelos Biológicos , Monotremados/genética , Filogenia , Síndrome de Prader-Willi/genética , Proteínas/genética , Proteínas de Ligação a RNA , Seleção Genética , Inativação do Cromossomo XRESUMO
Genomic imprinting, representing parent-specific expression of alleles at a locus, raises many questions about how--and especially why--epigenetic silencing of mammalian genes evolved. We present the first in-depth study of how a human imprinted domain evolved, analyzing a domain containing several imprinted genes that are involved in human disease. Using comparisons of orthologous genes in humans, marsupials, and the platypus, we discovered that the Prader-Willi/Angelman syndrome region on human Chromosome 15q was assembled only recently (105-180 million years ago). This imprinted domain arose after a region bearing UBE3A (Angelman syndrome) fused with an unlinked region bearing SNRPN (Prader-Willi syndrome), which had duplicated from the non-imprinted SNRPB/B'. This region independently acquired several retroposed gene copies and arrays of small nucleolar RNAs from different parts of the genome. In their original configurations, SNRPN and UBE3A are expressed from both alleles, implying that acquisition of imprinting occurred after their rearrangement and required the evolution of a control locus. Thus, the evolution of imprinting in viviparous mammals is ongoing.
Assuntos
Impressão Genômica/genética , Marsupiais/genética , Ornitorrinco/genética , Alelos , Animais , Autoantígenos/genética , Mapeamento Cromossômico , Cromossomos de Mamíferos/genética , Genoma Humano/genética , Humanos , Hibridização in Situ Fluorescente , Camundongos , Ribonucleoproteínas Nucleares Pequenas/genética , Análise de Sequência de DNA , Homologia de Sequência , Ubiquitina-Proteína Ligases/genética , Proteínas Centrais de snRNPRESUMO
Effective treatments that extend survival of malignant brain tumor glioblastoma (GBM) have not changed in more than a decade; however, there exists a minority patient group (<5%) whose survival is longer than 3 yr. We herein present a case report of a long-term surviving 51-yr-old female diagnosed with a MGMT unmethylated GBM. The patient was progression-free for 23 mo. Fresh primary and recurrent tumor samples were collected and processed for patient-derived model development. Whole-genome sequencing (WGS) was performed concurrently with additional standard of care diagnostics. WGS revealed a hypermutated genotype in the germline tissue and in both the primary and recurrent tumor samples. Specific to the matched tumors, an average of 30 cancer driver genes were mutated. Noteworthy was the identification of a nonsynonymous mutation in the POLE gene. As a possible instigator of the hypermutational genotype observed in the tumors, we identified nonsynonymous germline mutations within the mismatch repair genes, MLH1 and PMS2 Mutations within these genes are often indicative of the pan-cancer phenotype known as Lynch syndrome; however, their pathogenicity remains unreported. We performed a drug screen of 165 compounds, which identified one compound, YM155, an experimental survivin inhibitor, that showed effectivity to the patient-derived cell lines of both tumors. Treatment selection based on a patient's genome to individualize treatment for GBM patients could potentially be useful in the clinic. This is a promising avenue for further translational research, with larger databases and integrated platforms to increase the efficiency of analyzing and interpreting the individual genomic data of GBM.
Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Imidazóis/farmacologia , Naftoquinonas/farmacologia , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/tratamento farmacológico , Reparo de Erro de Pareamento de DNA/genética , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Redes Reguladoras de Genes , Genótipo , Mutação em Linhagem Germinativa , Glioblastoma/diagnóstico por imagem , Glioblastoma/tratamento farmacológico , Humanos , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia , Fenótipo , Sequenciamento Completo do GenomaRESUMO
The CABARET trial (ACTRN12610000915055) reported no difference in overall survival (OS) between patients with recurrent glioblastoma (GBM) randomized to either bevacizumab monotherapy or bevacizumab plus carboplatin. However, a subset of patients showed durable responses and prolonged survival, with recorded survival times of over 30â¯months in five of 122 patients (4%). Patient selection for bevacizumab therapy would be enhanced if a predictive biomarker of response or survival could be identified; this biomarker sub-study attempted to identify novel biomarkers. Patients who opted to participate in this sub-study and who had adequate biospecimens for analysis (nâ¯=â¯54) were retrospectively evaluated for the expression of a series of tumor proteins. Immunohistochemistry (IHC) was used to measure the expression of 19 proteins previously implicated in cancer treatment response to bevacizumab. MGMT promoter methylation was also assessed. Tumor DNA from five patients with outlying survival duration ('poor' and 'exceptional' survivors) was subjected to whole genome sequencing (WGS). No single protein expression level, including VEGF-A, predicted OS in the cohort. WGS of poor and exceptional survivors identified a gain in Chromosome 19 that was exclusive to the exceptional survivors. Validation of this finding requires examination of a larger independent cohort.
Assuntos
Antineoplásicos/uso terapêutico , Bevacizumab/uso terapêutico , Biomarcadores Tumorais/análise , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Adulto , Idoso , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/metabolismo , Cromossomos Humanos Par 19/genética , Ensaios Clínicos Fase II como Assunto , Estudos de Coortes , Feminino , Glioblastoma/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos RetrospectivosRESUMO
PURPOSE: Methylation of the MGMT promoter is the major cause of O6-methylguanine methyltransferase deficiency in cancer and has been associated with the T variant of the promoter enhancer SNP rs16906252C>T. We sought evidence for an association between the rs16906252C>T genotype and increased risk of developing a subtype of colorectal cancer featuring MGMT methylation, mediated by genotype-dependent epigenetic silencing within normal tissues. EXPERIMENTAL DESIGN: By applying a molecular pathologic epidemiology case-control study design, associations between rs16906252C>T and risk for colorectal cancer overall, and colorectal cancer stratified by MGMT methylation status, were estimated using multinomial logistic regression in two independent retrospective series of colorectal cancer cases and controls. The test sample comprised 1,054 colorectal cancer cases and 451 controls from Sydney, Australia. The validation sample comprised 612 colorectal cancer cases and 245 controls from the Australasian Colon Cancer Family Registry (ACCFR). To determine whether rs16906252C>T was linked to a constitutively altered epigenetic state, quantitative allelic expression and methylation analyses were performed in normal tissues. RESULTS: An association between rs16906252C>T and increased risk of developing MGMT-methylated colorectal cancer in the Sydney sample was observed [OR, 3.3; 95% confidence interval (CI), 2.0-5.3; P < 0.0001], which was replicated in the ACCFR sample (OR, 4.0; 95% CI, 2.4-6.8; P < 0.0001). The T allele demonstrated about 2.5-fold reduced transcription in normal colorectal mucosa from cases and controls and was selectively methylated in a minority of normal cells, indicating that rs16906252C>T represents an expression and methylation quantitative trait locus. CONCLUSIONS: We provide evidence that rs16906252C>T is associated with elevated risk for MGMT-methylated colorectal cancer, likely mediated by constitutive epigenetic repression of the T allele. Clin Cancer Res; 22(24); 6266-77. ©2016 AACR.
Assuntos
Neoplasias Colorretais/genética , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Proteínas Supressoras de Tumor/genética , Idoso , Alelos , Estudos de Casos e Controles , Epigênese Genética/genética , Feminino , Genótipo , Humanos , Masculino , O(6)-Metilguanina-DNA Metiltransferase/genética , Regiões Promotoras Genéticas/genética , Estudos Retrospectivos , RiscoRESUMO
BACKGROUND: Promoter methylation of O(6)-methylguanine-DNA methyltransferase (MGMT) is an important predictive biomarker in glioblastoma. The T variant of the MGMT promoter-enhancer single nucleotide polymorphism (SNP; rs16906252) has been associated with the presence of MGMT promoter methylation in other cancers. We examined the association of the T allele of rs16906252 with glioblastoma development, tumor MGMT methylation, MGMT protein expression, and survival outcomes. METHODS: Two independent temozolomide-treated glioblastoma cohorts-one Australian (Australian Genomics and Clinical Outcomes of Glioma, n = 163) and the other American (University of California Los Angeles/Kaiser Permanente Los Angeles, n = 159)-were studied. Allelic bisulphite sequencing was used to determine if methylation was specific to the T allele. Additionally, we compared the incidence of the T allele between glioblastoma cases and matched controls to assess whether it was a risk factor for developing MGMT methylated glioblastoma. RESULTS: Carriage of the T allele of the rs16906252 SNP was associated with both MGMT methylation and low MGMT protein expression and predicted significantly longer survival in temozolomide-treated patients with both MGMT methylated and nonmethylated glioblastoma. Methylation was linked to the T allele, inferring that the T variant plays a key role in the acquisition of MGMT methylation. Carriage of the T allele was associated with a significantly elevated risk of developing glioblastoma (adjusted odds ratio, 1.96; P = .013), increasing further when glioblastoma was classified by the presence of MGMT methylation (adjusted odds ratio, 2.86; P = .001). CONCLUSIONS: The T allele of the rs16906252 SNP is a key determinant in the acquisition of MGMT methylation in glioblastoma. Temozolomide-treated patients with the rs16906252 T genotype have better survival, irrespective of tumor methylation status.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Dacarbazina/análogos & derivados , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Polimorfismo de Nucleotídeo Único , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Biomarcadores/metabolismo , Neoplasias Encefálicas/metabolismo , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Dacarbazina/uso terapêutico , Feminino , Genótipo , Glioblastoma/metabolismo , Humanos , Estimativa de Kaplan-Meier , Masculino , Metilação , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Fatores de Risco , Temozolomida , Resultado do Tratamento , Proteínas Supressoras de Tumor/metabolismoRESUMO
Germline mutations of the DNA mismatch repair genes MLH1, MSH2, MSH6 or PMS2, and deletions affecting the EPCAM gene adjacent to MSH2, underlie Lynch syndrome by predisposing to early-onset colorectal, endometrial and other cancers. An alternative but rare cause of Lynch syndrome is constitutional epimutation of MLH1, whereby promoter methylation and transcriptional silencing of one allele occurs throughout normal tissues. A dominantly transmitted constitutional MLH1 epimutation has been linked to an MLH1 haplotype bearing two single-nucleotide variants, NM_000249.2: c.-27C>A and c.85G>T, in a Caucasian family with Lynch syndrome from Western Australia. Subsequently, a second seemingly unrelated Caucasian Australian case with the same MLH1 haplotype and concomitant epimutation was reported. We now describe three additional, ostensibly unrelated, cancer-affected families of European heritage with this MLH1 haplotype in association with constitutional epimutation, bringing the number of index cases reported to five. Array-based genotyping in four of these families revealed shared haplotypes between individual families that extended across ≤2.6-≤6.4 megabase regions of chromosome 3p, indicating common ancestry. A minimal ≤2.6 megabase founder haplotype common to all four families was identified, which encompassed MLH1 and additional flanking genes and segregated with the MLH1 epimutation in each family. Our findings indicate that the MLH1 c.-27C>A and c.85G>T variants are borne on a European ancestral haplotype and provide conclusive evidence for its pathogenicity via a mechanism of epigenetic silencing of MLH1 within normal tissues. Additional descendants bearing this founder haplotype may exist who are also at high risk of developing Lynch syndrome-related cancers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Epigênese Genética , Genes Dominantes , Haplótipos , Proteínas Nucleares/genética , Mutação Puntual , População Branca/genética , Alelos , Estudos de Casos e Controles , Cromossomos Humanos Par 3 , Neoplasias Colorretais Hereditárias sem Polipose/genética , Variações do Número de Cópias de DNA , Metilação de DNA , Reparo de Erro de Pareamento de DNA , Análise Mutacional de DNA , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Proteína 1 Homóloga a MutL , Linhagem , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Constitutional epimutations of tumor suppressor genes manifest as promoter methylation and transcriptional silencing of a single allele in normal somatic tissues, thereby predisposing to cancer. Constitutional MLH1 epimutations occur in individuals with young-onset cancer and demonstrate non-Mendelian inheritance through their reversal in the germline. We report a cancer-affected family showing dominant transmission of soma-wide highly mosaic MLH1 methylation and transcriptional repression linked to a particular genetic haplotype. The epimutation was erased in spermatozoa but reinstated in the somatic cells of the next generation. The affected haplotype harbored two single nucleotide substitutions in tandem; c.-27C > A located near the transcription initiation site and c.85G > T. The c.-27C > A variant significantly reduced transcriptional activity in reporter assays and is the probable cause of this epimutation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Epigênese Genética , Inativação Gênica , Genes Dominantes , Proteínas Nucleares/genética , Regiões 5' não Traduzidas , Metilação de DNA , Ligação Genética , Humanos , Proteína 1 Homóloga a MutLRESUMO
OBJECTIVE: To elucidate the mechanism of the unique beneficial effect of intravitreal steroid therapy on diabetic macular edema, we investigated the effect of locally administered triamcinolone acetonide (TA) on the expression of vascular endothelial growth factor (VEGF)-A and its receptors in retinas of rats with streptozotocin (STZ)-induced diabetes. We then correlated the expression of these proteins with breakdown of the blood-retinal barrier (BRB). RESEARCH DESIGN AND METHODS: Thirty-two eyes of 16 diabetic and nondiabetic rats were divided into four groups. TA was injected into the vitreous of the right eye, and saline was injected into the left eye (control) 3.5 weeks after induction of diabetes. Retinas were harvested 48 h following treatment. mRNA and protein expression of VEGF-A, VEGF-A receptor 1 (fms-like tyrosine kinase [FLT]-1), and VEGF-A receptor 2 (fetal liver kinase [FLK]-1) were determined by real-time RT-PCR and immunohistochemistry. BRB permeability was quantitated by measuring extravasated endogenous albumin and retinal thickness. RESULTS: Diabetes-induced retinal thickness and albumin extravasation were significantly reduced in TA-treated diabetic retinas to a level similar to that in sham-treated nondiabetic eyes. A close correlation between albumin leakage and increased expression of both Vegf-a and Flk-1 was noted in the diabetic retinas. TA downregulated the expression of Vegf-a and Flk-1 but upregulated the expression of Flt-1. TA did not alter the expression of these genes in nondiabetic retinas. CONCLUSIONS: Intravitreal injection of TA stabilizes the BRB in association with regulation of Vegf-a, Flk-1, and Flt-1 expression in retinas in the early stages of diabetes.
Assuntos
Barreira Hematorretiniana/fisiologia , Diabetes Mellitus Experimental/fisiopatologia , Retinopatia Diabética/tratamento farmacológico , Glucocorticoides/uso terapêutico , Triancinolona Acetonida/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Barreira Hematorretiniana/efeitos dos fármacos , Diabetes Mellitus Experimental/genética , Retinopatia Diabética/genética , Progressão da Doença , Masculino , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacosRESUMO
Marsupials occupy a phylogenetic middle ground that is very valuable in genome comparisons of mammal and other vertebrate species. For this reason, whole genome sequencing is being undertaken for two distantly related marsupial species, including the model kangaroo species Macropus eugenii (the tammar wallaby). As a first step towards the molecular characterization of the tammar genome, we present a detailed description of the tammar karyotype, report the development of a set of molecular anchor markers and summarize the comparative mapping data for this species.