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2.
Am J Hum Genet ; 107(4): 622-635, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32946763

RESUMO

Quantifying the functional effects of complex disease risk variants can provide insights into mechanisms underlying disease biology. Genome-wide association studies have identified 39 regions associated with risk of epithelial ovarian cancer (EOC). The vast majority of these variants lie in the non-coding genome, where they likely function through interaction with gene regulatory elements. In this study we first estimated the heritability explained by known common low penetrance risk alleles for EOC. The narrow sense heritability (hg2) of EOC overall and high-grade serous ovarian cancer (HGSOCs) were estimated to be 5%-6%. Partitioned SNP heritability across broad functional categories indicated a significant contribution of regulatory elements to EOC heritability. We collated epigenomic profiling data for 77 cell and tissue types from Roadmap Epigenomics and ENCODE, and from H3K27Ac ChIP-seq data generated in 26 ovarian cancer and precursor-related cell and tissue types. We identified significant enrichment of risk single-nucleotide polymorphisms (SNPs) in active regulatory elements marked by H3K27Ac in HGSOCs. To further investigate how risk SNPs in active regulatory elements influence predisposition to ovarian cancer, we used motifbreakR to predict the disruption of transcription factor binding sites. We identified 469 candidate causal risk variants in H3K27Ac peaks that are predicted to significantly break transcription factor (TF) motifs. The most frequently broken motif was REST (p value = 0.0028), which has been reported as both a tumor suppressor and an oncogene. Overall, these systematic functional annotations with epigenomic data improve interpretation of EOC risk variants and shed light on likely cells of origin.


Assuntos
Carcinoma Epitelial do Ovário/genética , Proteínas Correpressoras/genética , Cistadenocarcinoma Seroso/genética , Elementos Facilitadores Genéticos , Histonas/genética , Proteínas do Tecido Nervoso/genética , Neoplasias Ovarianas/genética , Alelos , Sítios de Ligação , Carcinoma Epitelial do Ovário/diagnóstico , Carcinoma Epitelial do Ovário/patologia , Mapeamento Cromossômico , Proteínas Correpressoras/metabolismo , Cistadenocarcinoma Seroso/diagnóstico , Cistadenocarcinoma Seroso/patologia , Feminino , Predisposição Genética para Doença , Genoma Humano , Estudo de Associação Genômica Ampla , Histonas/metabolismo , Humanos , Padrões de Herança , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/patologia , Penetrância , Polimorfismo de Nucleotídeo Único , Risco
3.
Nat Genet ; 52(8): 790-799, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32690948

RESUMO

Epigenetic processes govern prostate cancer (PCa) biology, as evidenced by the dependency of PCa cells on the androgen receptor (AR), a prostate master transcription factor. We generated 268 epigenomic datasets spanning two state transitions-from normal prostate epithelium to localized PCa to metastases-in specimens derived from human tissue. We discovered that reprogrammed AR sites in metastatic PCa are not created de novo; rather, they are prepopulated by the transcription factors FOXA1 and HOXB13 in normal prostate epithelium. Reprogrammed regulatory elements commissioned in metastatic disease hijack latent developmental programs, accessing sites that are implicated in prostate organogenesis. Analysis of reactivated regulatory elements enabled the identification and functional validation of previously unknown metastasis-specific enhancers at HOXB13, FOXA1 and NKX3-1. Finally, we observed that prostate lineage-specific regulatory elements were strongly associated with PCa risk heritability and somatic mutation density. Examining prostate biology through an epigenomic lens is fundamental for understanding the mechanisms underlying tumor progression.


Assuntos
Neoplasias da Próstata/genética , Linhagem Celular , Linhagem Celular Tumoral , Progressão da Doença , Epigenômica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Masculino , Próstata/patologia , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Sequências Reguladoras de Ácido Nucleico/genética
4.
Nat Med ; 26(7): 1041-1043, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572266

RESUMO

Improving early cancer detection has the potential to substantially reduce cancer-related mortality. Cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) is a highly sensitive assay capable of detecting early-stage tumors. We report accurate classification of patients across all stages of renal cell carcinoma (RCC) in plasma (area under the receiver operating characteristic (AUROC) curve of 0.99) and demonstrate the validity of this assay to identify patients with RCC using urine cell-free DNA (cfDNA; AUROC of 0.86).


Assuntos
Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/genética , Metilação de DNA/genética , Detecção Precoce de Câncer , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/urina , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Ácidos Nucleicos Livres/urina , Epigenoma/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
5.
Nat Commun ; 11(1): 2020, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332753

RESUMO

The functional consequences of somatic non-coding mutations in ovarian cancer (OC) are unknown. To identify regulatory elements (RE) and genes perturbed by acquired non-coding variants, here we establish epigenomic and transcriptomic landscapes of primary OCs using H3K27ac ChIP-seq and RNA-seq, and then integrate these with whole genome sequencing data from 232 OCs. We identify 25 frequently mutated regulatory elements, including an enhancer at 6p22.1 which associates with differential expression of ZSCAN16 (P = 6.6 × 10-4) and ZSCAN12 (P = 0.02). CRISPR/Cas9 knockout of this enhancer induces downregulation of both genes. Globally, there is an enrichment of single nucleotide variants in active binding sites for TEAD4 (P = 6 × 10-11) and its binding partner PAX8 (P = 2×10-10), a known lineage-specific transcription factor in OC. In addition, the collection of cis REs associated with PAX8 comprise the most frequently mutated set of enhancers in OC (P = 0.003). These data indicate that non-coding somatic mutations disrupt the PAX8 transcriptional network during OC development.


Assuntos
Carcinoma Epitelial do Ovário/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Neoplasias Ovarianas/genética , Fator de Transcrição PAX8/metabolismo , Adulto , Idoso , Sítios de Ligação/genética , Carcinoma Epitelial do Ovário/patologia , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Epigênese Genética , Epigenômica , Feminino , Técnicas de Inativação de Genes , Humanos , Fatores de Transcrição Kruppel-Like/genética , Pessoa de Meia-Idade , Proteínas Musculares/metabolismo , Mutação , Neoplasias Ovarianas/patologia , Ovário/patologia , Polimorfismo de Nucleotídeo Único , RNA-Seq , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismo , Sequenciamento Completo do Genoma
7.
Cell Rep ; 29(11): 3726-3735.e4, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31825847

RESUMO

Fallopian tube secretory epithelial cells (FTSECs) are likely the main precursor cell type of high-grade serous ovarian cancers (HGSOCs), but these tumors may also arise from ovarian surface epithelial cells (OSECs). We profiled global landscapes of gene expression and active chromatin to characterize molecular similarities between OSECs (n = 114), FTSECs (n = 74), and HGSOCs (n = 394). A one-class machine learning algorithm predicts that most HGSOCs derive from FTSECs, with particularly high FTSEC scores in mesenchymal-type HGSOCs (padj < 8 × 10-4). However, a subset of HGSOCs likely derive from OSECs, particularly HGSOCs of the proliferative type (padj < 2 × 10-4), suggesting a dualistic model for HGSOC origins. Super-enhancer (SE) landscapes were also more similar between FTSECs and HGSOCs than between OSECs and HGSOCs (p < 2.2 × 10-16). The SOX18 transcription factor (TF) coincided with a HGSOC-specific SE, and ectopic overexpression of SOX18 in FTSECs caused epithelial-to-mesenchymal transition, indicating that SOX18 plays a role in establishing the mesenchymal signature of fallopian-derived HGSOCs.


Assuntos
Neoplasias Ovarianas/genética , Fatores de Transcrição SOXF/genética , Adulto , Idoso , Linhagem Celular , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal , Tubas Uterinas/metabolismo , Tubas Uterinas/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Aprendizado de Máquina , Pessoa de Meia-Idade , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Ovário/metabolismo , Ovário/patologia , RNA-Seq , Fatores de Transcrição SOXF/metabolismo , Análise de Célula Única , Transcriptoma
8.
Cell Rep ; 29(8): 2355-2370.e6, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31747605

RESUMO

Androgen-receptor (AR) inhibitors, including enzalutamide, are used for treatment of all metastatic castration-resistant prostate cancers (mCRPCs). However, some patients develop resistance or never respond. We find that the transcription factor CREB5 confers enzalutamide resistance in an open reading frame (ORF) expression screen and in tumor xenografts. CREB5 overexpression is essential for an enzalutamide-resistant patient-derived organoid. In AR-expressing prostate cancer cells, CREB5 interactions enhance AR activity at a subset of promoters and enhancers upon enzalutamide treatment, including MYC and genes involved in the cell cycle. In mCRPC, we found recurrent amplification and overexpression of CREB5. Our observations identify CREB5 as one mechanism that drives resistance to AR antagonists in prostate cancers.


Assuntos
Antagonistas de Receptores de Andrógenos/uso terapêutico , Proteína A de Ligação a Elemento de Resposta do AMP Cíclico/metabolismo , Antineoplásicos/uso terapêutico , Proteína A de Ligação a Elemento de Resposta do AMP Cíclico/genética , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Masculino , Fases de Leitura Aberta/genética , Feniltioidantoína/análogos & derivados , Regiões Promotoras Genéticas/genética , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo
9.
iScience ; 17: 242-255, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31307004

RESUMO

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of tumorigenesis, and yet their mechanistic roles remain challenging to characterize. Here, we integrate functional proteomics with lncRNA-interactome profiling to characterize Urothelial Cancer Associated 1 (UCA1), a candidate driver of ovarian cancer development. Reverse phase protein array (RPPA) analysis indicates that UCA1 activates transcription coactivator YAP and its target genes. In vivo RNA antisense purification (iRAP) of UCA1 interacting proteins identified angiomotin (AMOT), a known YAP regulator, as a direct binding partner. Loss-of-function experiments show that AMOT mediates YAP activation by UCA1, as UCA1 enhances the AMOT-YAP interaction to promote YAP dephosphorylation and nuclear translocation. Together, we characterize UCA1 as a lncRNA regulator of Hippo-YAP signaling and highlight the UCA1-AMOT-YAP signaling axis in ovarian cancer development.

10.
PLoS One ; 14(5): e0216997, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31125336

RESUMO

PURPOSE: Genome-wide-association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNPs) that are associated with an increased risk of breast cancer. Most of these studies were conducted primarily in postmenopausal breast cancer patients. Therefore, we set out to assess whether or not these breast cancer variants are also associated with an elevated risk of breast cancer in young premenopausal patients. METHODS: In 451 women of European ancestry who had prospectively enrolled in a longitudinal cohort study for women diagnosed with breast cancer at or under age 40, we genotyped 44 SNPs that were previously associated with breast cancer risk. A control group was comprised of 1142 postmenopausal healthy women from the Nurses' Health Study (NHS). We assessed if the frequencies of the adequately genotyped SNPs differed significantly (p≤0.05) between the cohort of young breast cancer patients and postmenopausal controls, and then we corrected for multiple testing. RESULTS: Genotyping of the controls or cases was inadequate for comparisons between the groups for seven of the 44 SNPs. 9 of the remaining 37 were associated with breast cancer risk in young women with a p-value <0.05: rs10510102, rs1219648, rs13387042, rs1876206, rs2936870, rs2981579, rs3734805, rs3803662 and rs4973768. The directions of these associations were consistent with those in postmenopausal women. However, after correction for multiple testing (Benjamini Hochberg) none of the results remained statistically significant. CONCLUSION: After correction for multiple testing, none of the alleles for postmenopausal breast cancer were clearly associated with risk of premenopausal breast cancer in this relatively small study.


Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Adulto , Alelos , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/patologia , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Receptores Estrogênicos/genética , Fatores de Risco
11.
Cell ; 174(2): 422-432.e13, 2018 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-29909987

RESUMO

Increased androgen receptor (AR) activity drives therapeutic resistance in advanced prostate cancer. The most common resistance mechanism is amplification of this locus presumably targeting the AR gene. Here, we identify and characterize a somatically acquired AR enhancer located 650 kb centromeric to the AR. Systematic perturbation of this enhancer using genome editing decreased proliferation by suppressing AR levels. Insertion of an additional copy of this region sufficed to increase proliferation under low androgen conditions and to decrease sensitivity to enzalutamide. Epigenetic data generated in localized prostate tumors and benign specimens support the notion that this region is a developmental enhancer. Collectively, these observations underscore the importance of epigenomic profiling in primary specimens and the value of deploying genome editing to functionally characterize noncoding elements. More broadly, this work identifies a therapeutic vulnerability for targeting the AR and emphasizes the importance of regulatory elements as highly recurrent oncogenic drivers.


Assuntos
Elementos Facilitadores Genéticos/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/metabolismo , Acetilação , Adulto , Idoso , Antineoplásicos/farmacologia , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Metilação de DNA , Edição de Genes , Histonas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/genética
12.
Nat Med ; 21(11): 1357-63, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26398868

RESUMO

The vast majority of disease-associated single-nucleotide polymorphisms (SNPs) mapped by genome-wide association studies (GWASs) are located in the non-protein-coding genome, but establishing the functional and mechanistic roles of these sequence variants has proven challenging. Here we describe a general pipeline in which candidate functional SNPs are first evaluated by fine mapping, epigenomic profiling, and epigenome editing, and then interrogated for causal function by using genome editing to create isogenic cell lines followed by phenotypic characterization. To validate this approach, we analyzed the 6q22.1 prostate cancer risk locus and identified rs339331 as the top-scoring SNP. Epigenome editing confirmed that the rs339331 region possessed regulatory potential. By using transcription activator-like effector nuclease (TALEN)-mediated genome editing, we created a panel of isogenic 22Rv1 prostate cancer cell lines representing all three genotypes (TT, TC, CC) at rs339331. Introduction of the 'T' risk allele increased transcription of the regulatory factor 6 (RFX6) gene, increased homeobox B13 (HOXB13) binding at the rs339331 region, and increased deposition of the enhancer-associated H3K4me2 histone mark at the rs339331 region compared to lines homozygous for the 'C' protective allele. The cell lines also differed in cellular morphology and adhesion, and pathway analysis of differentially expressed genes suggested an influence of androgens. In summary, we have developed and validated a widely accessible approach that can be used to establish functional causality for noncoding sequence variants identified by GWASs.


Assuntos
Cromossomos Humanos Par 6/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias da Próstata/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Alelos , Linhagem Celular Tumoral , Mapeamento Cromossômico , Proteínas de Ligação a DNA/genética , Epigenômica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Código das Histonas , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição de Fator Regulador X , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Nat Commun ; 6: 8234, 2015 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-26391404

RESUMO

Genome-wide association studies have reported 11 regions conferring risk of high-grade serous epithelial ovarian cancer (HGSOC). Expression quantitative trait locus (eQTL) analyses can identify candidate susceptibility genes at risk loci. Here we evaluate cis-eQTL associations at 47 regions associated with HGSOC risk (P≤10(-5)). For three cis-eQTL associations (P<1.4 × 10(-3), FDR<0.05) at 1p36 (CDC42), 1p34 (CDCA8) and 2q31 (HOXD9), we evaluate the functional role of each candidate by perturbing expression of each gene in HGSOC precursor cells. Overexpression of HOXD9 increases anchorage-independent growth, shortens population-doubling time and reduces contact inhibition. Chromosome conformation capture identifies an interaction between rs2857532 and the HOXD9 promoter, suggesting this SNP is a leading causal variant. Transcriptomic profiling after HOXD9 overexpression reveals enrichment of HGSOC risk variants within HOXD9 target genes (P=6 × 10(-10) for risk variants (P<10(-4)) within 10 kb of a HOXD9 target gene in ovarian cells), suggesting a broader role for this network in genetic susceptibility to HGSOC.


Assuntos
Estudos de Associação Genética , Proteínas de Neoplasias/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Locos de Características Quantitativas , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Cordão Nucal , Neoplasias Ovarianas/metabolismo , Ligação Proteica
14.
Nat Genet ; 47(8): 888-97, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26075790

RESUMO

Genome-wide association studies have identified several risk associations for ovarian carcinomas but not for mucinous ovarian carcinomas (MOCs). Our analysis of 1,644 MOC cases and 21,693 controls with imputation identified 3 new risk associations: rs752590 at 2q13 (P = 3.3 × 10(-8)), rs711830 at 2q31.1 (P = 7.5 × 10(-12)) and rs688187 at 19q13.2 (P = 6.8 × 10(-13)). We identified significant expression quantitative trait locus (eQTL) associations for HOXD9 at 2q31.1 in ovarian (P = 4.95 × 10(-4), false discovery rate (FDR) = 0.003) and colorectal (P = 0.01, FDR = 0.09) tumors and for PAX8 at 2q13 in colorectal tumors (P = 0.03, FDR = 0.09). Chromosome conformation capture analysis identified interactions between the HOXD9 promoter and risk-associated SNPs at 2q31.1. Overexpressing HOXD9 in MOC cells augmented the neoplastic phenotype. These findings provide the first evidence for MOC susceptibility variants and insights into the underlying biology of the disease.


Assuntos
Adenocarcinoma Mucinoso/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Neoplasias Ovarianas/genética , Polimorfismo de Nucleotídeo Único , Sequência de Bases , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromossomos Humanos Par 2/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Estudos de Associação Genética/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Locos de Características Quantitativas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco
15.
Philos Trans R Soc Lond B Biol Sci ; 368(1620): 20120363, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23650637

RESUMO

Breast cancer genome-wide association studies have pinpointed dozens of variants associated with breast cancer pathogenesis. The majority of risk variants, however, are located outside of known protein-coding regions. Therefore, identifying which genes the risk variants are acting through presents an important challenge. Variants that are associated with mRNA transcript levels are referred to as expression quantitative trait loci (eQTLs). Many studies have demonstrated that eQTL-based strategies provide a direct way to connect a trait-associated locus with its candidate target gene. Performing eQTL-based analyses in human samples is complicated because of the heterogeneous nature of human tissue. We addressed this issue by devising a method to computationally infer the fraction of cell types in normal human breast tissues. We then applied this method to 13 known breast cancer risk loci, which we hypothesized were eQTLs. For each risk locus, we took all known transcripts within a 2 Mb interval and performed an eQTL analysis in 100 reduction mammoplasty cases. A total of 18 significant associations were discovered (eight in the epithelial compartment and 10 in the stromal compartment). This study highlights the ability to perform large-scale eQTL studies in heterogeneous tissues.


Assuntos
Neoplasias da Mama/genética , Mama/citologia , Locos de Características Quantitativas , Adipócitos/metabolismo , Mama/metabolismo , Neoplasias da Mama/metabolismo , Biologia Computacional/métodos , Epitélio/metabolismo , Feminino , Estudos de Associação Genética , Heterogeneidade Genética , Marcadores Genéticos , Humanos , Mamoplastia , Valor Preditivo dos Testes , Fatores de Risco , Transcriptoma
16.
Cell ; 152(3): 633-41, 2013 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-23374354

RESUMO

Germline determinants of gene expression in tumors are infrequently studied due to the complexity of transcript regulation caused by somatically acquired alterations. We performed expression quantitative trait locus (eQTL)-based analyses using the multi-level information provided in The Cancer Genome Atlas (TCGA). Of the factors we measured, cis-acting eQTLs accounted for 1.2% of the total variation of tumor gene expression, while somatic copy-number alteration and CpG methylation accounted for 7.3% and 3.3%, respectively. eQTL analyses of 15 previously reported breast cancer risk loci resulted in the discovery of three variants that are significantly associated with transcript levels (false discovery rate [FDR] < 0.1). Our trans-based analysis identified an additional three risk loci to act through ESR1, MYC, and KLF4. These findings provide a more comprehensive picture of gene expression determinants in breast cancer as well as insights into the underlying biology of breast cancer risk loci.


Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Locos de Características Quantitativas , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos
17.
Cancer Res ; 70(18): 7325-35, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20807813

RESUMO

CRKL (CRK-like) is an adapter protein predominantly phosphorylated in cells that express the tyrosine kinase p210(BCR-ABL), the fusion product of a (9;22) chromosomal translocation causative for chronic myeloid leukemia. It has been unclear, however, whether CRKL plays a functional role in p210(BCR-ABL) transformation. Here, we show that CRKL is required for p210(BCR-ABL) to support interleukin-3-independent growth of myeloid progenitor cells and long-term outgrowth of B-lymphoid cells from fetal liver-derived hematopoietic progenitor cells. Furthermore, a synthetic phosphotyrosyl peptide that binds to the CRKL SH2 domain with high affinity blocks association of endogenous CRKL with the p210(BCR-ABL) complex and reduces c-MYC levels in K562 human leukemic cells as well as in mouse hematopoietic cells transformed by p210(BCR-ABL) or the imatinib-resistant mutant T315I. These results indicate that the function of CRKL as an adapter protein is essential for p210(BCR-ABL)-induced transformation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Processos de Crescimento Celular/fisiologia , Transformação Celular Neoplásica/patologia , Células-Tronco Hematopoéticas/patologia , Humanos , Interleucina-3/farmacologia , Células K562 , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fosforilação , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT5/metabolismo , Proteína bcl-X/metabolismo
18.
Mol Cell Biol ; 29(11): 3076-87, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19307307

RESUMO

The adapter protein CRKL is required for the normal development of multiple tissues that rely on fibroblast growth factor 8 (FGF8). The precise role of CRKL in receptor signaling has been unclear, however. To address this issue, we first modeled the three-dimensional structure of CRKL by molecular dynamics. By taking advantage of structural simulations, we performed in silico analysis of the interactions of the autophosphorylation sites of FGR receptor 1 (FGFR1) with the SH2 domain of CRKL or a highly related protein, CRK. As predicted by simulations, we confirm the specific physical interaction of phosphorylated Y463 (pY463) in FGFR1 with the CRKL SH2 domain at an affinity approximately 30-fold stronger than that of CRK. We also provide evidence that interactions outside of the core YXXP motif have a significant impact on physical association, which is consistent with predictions from molecular-dynamics simulations. Furthermore, we identify CRKL as an essential component of an FGF8-induced feed-forward loop permissive for efficient activation of the mitogen-activated protein kinase Erk1/2, as well as FGF8-induced anchorage-independent cell growth, using Crkl-deficient cells or a pY463 synthetic peptide. Although many cells generally require cell-matrix adhesion, our results demonstrate that CRKL permits cells to bypass the strict need for adhesion in response to FGF8 through direct interaction with receptor.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Retroalimentação Fisiológica/efeitos dos fármacos , Fator 8 de Crescimento de Fibroblasto/farmacologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Biologia Computacional , Simulação por Computador , Humanos , MAP Quinase Quinase 1/metabolismo , Camundongos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Peptídeos/química , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-crk/química , Proteínas Proto-Oncogênicas c-crk/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Relação Estrutura-Atividade , Quinases raf/metabolismo , Domínios de Homologia de src
19.
Toxicol Appl Pharmacol ; 216(2): 206-15, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16854443

RESUMO

Methylmercury (MeHg) is a ubiquitous environmental toxicant to which humans can be exposed by ingestion of contaminated food. MeHg has been suggested to exert its toxicity through its high reactivity to thiols, generation of arachidonic acid and reactive oxygen species (ROS), and elevation of free intracellular Ca(2+) levels ([Ca(2+)](i)). However, the precise mechanism has not been fully defined. Here we show that phosphatidylcholine-specific phospholipase C (PC-PLC) is a critical pathway for MeHg-induced toxicity in MDCK cells. D609, an inhibitor of PC-PLC, significantly reversed the toxicity in a time- and dose-dependent manner with concomitant inhibition of the diacylglycerol (DAG) generation and the phosphatidylcholine (PC)-breakdown. MeHg activated the group IV cytosolic phospholipase A(2) (cPLA(2)) and acidic form of sphingomyelinase (A-SMase) downstream of PC-PLC, but these enzymes as well as protein kinase C (PKC) were not linked to the toxicity by MeHg. Furthermore, MeHg produced ROS, which did not affect the toxicity. Addition of EGTA to culture media resulted in partial decrease of [Ca(2+)](i) and partially blocked the toxicity. In contrast, when the cells were treated with MeHg in the presence of Ca(2+) in the culture media, D609 completely prevented cell death with parallel decrease in [Ca(2+)](i). Our results demonstrated that MeHg-induced toxicity was linked to elevation of [Ca(2+)](i) through activation of PC-PLC, but not attributable to the signaling pathways such as cPLA(2), A-SMase, and PKC, or to the generation of ROS.


Assuntos
Cálcio/metabolismo , Poluentes Ambientais/toxicidade , Rim/metabolismo , Compostos de Metilmercúrio/toxicidade , Neuroblastoma/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quelantes/farmacologia , Diglicerídeos/metabolismo , Cães , Relação Dose-Resposta a Droga , Ácido Egtázico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Norbornanos , Fosfatidilcolinas/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Fosfolipases A/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Tiocarbamatos , Tionas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores
20.
Dev Cell ; 10(1): 71-80, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16399079

RESUMO

Deletions on chromosome 22q11.21 disrupt pharyngeal and cardiac development and cause DiGeorge and related human syndromes. CRKL (CRK-Like) lies within 22q11.21, and Crkl-/- mice have phenotypic features of 22q11 deletion (del22q11) syndromes. While human FGF8 does not localize to 22q11, deficiency of Fgf8 also generates many features of del22q11 syndrome in mice. Since Fgf8 signals via receptor-type tyrosine kinases, and Crk family adaptor proteins transduce intracellular signals downstream of tyrosine kinases, we investigated whether Crkl mediates Fgf8 signaling. In addition to discovering genetic interactions between Crkl and Fgf8 during morphogenesis of structures affected in del22q11 syndrome, we found that Fgf8 induces tyrosine phosphorylation of FgfRs 1 and 2 and their binding to Crkl. Crkl is required for normal cellular responses to Fgf8, including survival and migration, Erk activation, and target gene expression. These findings provide mechanistic insight into disrupted intercellular interactions in the pathogenesis of malformations seen in del22q11 syndrome.


Assuntos
Cromossomos Humanos Par 22 , Síndrome de DiGeorge/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Deleção de Genes , Proteínas Proto-Oncogênicas c-crk/deficiência , Transdução de Sinais/fisiologia , Animais , Apoptose , Western Blotting/métodos , Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Sistema Cardiovascular/embriologia , Sistema Cardiovascular/metabolismo , Contagem de Células/métodos , Células Cultivadas , Fatores Quimiotáticos/metabolismo , Síndrome de DiGeorge/genética , Modelos Animais de Doenças , Embrião de Mamíferos , Ativação Enzimática , Imunofluorescência/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Genótipo , Humanos , Camundongos , Camundongos Knockout , Modelos Biológicos , Crista Neural/metabolismo , Faringe/embriologia , Faringe/metabolismo , Fenótipo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Tempo
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