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1.
Nature ; 580(7803): 402-408, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32296183

RESUMO

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships1,2. Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome3, transcriptome4 and proteome5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.


Assuntos
Proteoma/metabolismo , Espaço Extracelular/metabolismo , Humanos , Especificidade de Órgãos , Mapeamento de Interação de Proteínas
2.
Proc Natl Acad Sci U S A ; 118(41)2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34607954

RESUMO

BRCA1 germline mutations are associated with an increased risk of breast and ovarian cancer. Recent findings of others suggest that BRCA1 mutation carriers also bear an increased risk of esophageal and gastric cancer. Here, we employ a Brca1/Trp53 mouse model to show that unresolved replication stress (RS) in BRCA1 heterozygous cells drives esophageal tumorigenesis in a model of the human equivalent. This model employs 4-nitroquinoline-1-oxide (4NQO) as an RS-inducing agent. Upon drinking 4NQO-containing water, Brca1 heterozygous mice formed squamous cell carcinomas of the distal esophagus and forestomach at a much higher frequency and speed (∼90 to 120 d) than did wild-type (WT) mice, which remained largely tumor free. Their esophageal tissue, but not that of WT control mice, revealed evidence of overt RS as reflected by intracellular CHK1 phosphorylation and 53BP1 staining. These Brca1 mutant tumors also revealed higher genome mutation rates than those of control animals; the mutational signature SBS4, which is associated with tobacco-induced tumorigenesis; and a loss of Brca1 heterozygosity (LOH). This uniquely accelerated Brca1 tumor model is also relevant to human esophageal squamous cell carcinoma, an often lethal tumor.


Assuntos
Proteína BRCA1/genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Perda de Heterozigosidade/genética , Proteína Supressora de Tumor p53/genética , 4-Nitroquinolina-1-Óxido/toxicidade , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Quinase 1 do Ponto de Checagem/metabolismo , Modelos Animais de Doenças , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/induzido quimicamente , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Mutação em Linhagem Germinativa/genética , Heterozigoto , Humanos , Perda de Heterozigosidade/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
3.
Mol Cell ; 54(6): 932-945, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24857551

RESUMO

Quiescence (G0) allows cycling cells to reversibly cease proliferation. A decision to enter quiescence is suspected of occurring early in G1, before the restriction point (R). Surprisingly, we have identified G2 as an interval during which inhibition of the protein phosphatase PP2A results in failure to exhibit stable quiescence. This effect is accompanied by shortening of the ensuing G1. The PP2A subcomplex required for stable G0 contains the B56γ B subunit. After PP2A inhibition in G2, aberrant overexpression of cyclin E occurs during mitosis and is responsible for overriding quiescence. Strikingly, suppression of Ras signaling re-establishes normal cyclin E levels during M and restores G0. These data point to PP2A-B56γ-driven Ras signaling modulation in G2 as essential for suppressing aberrant cyclin E expression during mitosis and thereby achieving normal G0 control. Thus, G2 is an interval during which the length and growth factor dependence of the next G1 interval are established.


Assuntos
Fase G1/genética , Fase G2/genética , Proteína Oncogênica p21(ras)/genética , Proteína Fosfatase 2/genética , Fase de Repouso do Ciclo Celular/fisiologia , Linhagem Celular , Ciclina E/biossíntese , Humanos , Células MCF-7 , Mitose/genética , Subunidades Proteicas/genética , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais/genética
4.
Proc Natl Acad Sci U S A ; 112(1): 232-7, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25535366

RESUMO

High-grade serous ovarian carcinoma (HGSOC) is the most common and aggressive form of epithelial ovarian cancer, for which few targeted therapies exist. To search for new therapeutic target proteins, we performed an in vivo shRNA screen using an established human HGSOC cell line growing either subcutaneously or intraperitoneally in immunocompromised mice. We identified genes previously implicated in ovarian cancer such as AURKA1, ERBB3, CDK2, and mTOR, as well as several novel candidates including BRD4, VRK1, and GALK2. We confirmed, using both genetic and pharmacologic approaches, that the activity of BRD4, an epigenetic transcription modulator, is necessary for proliferation/survival of both an established human ovarian cancer cell line (OVCAR8) and a subset of primary serous ovarian cancer cell strains (DFs). Among the DFs tested, the strains sensitive to BRD4 inhibition revealed elevated expression of either MYCN or c-MYC, with MYCN expression correlating closely with JQ1 sensitivity. Accordingly, primary human xenografts derived from high-MYCN or c-MYC strains exhibited sensitivity to BRD4 inhibition. These data suggest that BRD4 inhibition represents a new therapeutic approach for MYC-overexpressing HGSOCs.


Assuntos
Testes Genéticos , Terapia de Alvo Molecular , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/terapia , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Fatores de Transcrição/metabolismo , Animais , Carcinoma Epitelial do Ovário , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Estudos de Associação Genética , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Proc Natl Acad Sci U S A ; 110(21): 8632-7, 2013 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-23657012

RESUMO

Germ-line mutations in PALB2 lead to a familial predisposition to breast and pancreatic cancer or to Fanconi Anemia subtype N. PALB2 performs its tumor suppressor role, at least in part, by supporting homologous recombination-type double strand break repair (HR-DSBR) through physical interactions with BRCA1, BRCA2, and RAD51. To further understand the mechanisms underlying PALB2-mediated DNA repair and tumor suppression functions, we targeted Palb2 in the mouse. Palb2-deficient murine ES cells recapitulated DNA damage defects caused by PALB2 depletion in human cells, and germ-line deletion of Palb2 led to early embryonic lethality. Somatic deletion of Palb2 driven by K14-Cre led to mammary tumor formation with long latency. Codeletion of both Palb2 and Tumor protein 53 (Trp53) accelerated mammary tumor formation. Like BRCA1 and BRCA2 mutant breast cancers, these tumors were defective in RAD51 focus formation, reflecting a defect in Palb2 HR-DSBR function, a strongly suspected contributor to Brca1, Brca2, and Palb2 mammary tumor development. However, unlike the case of Brca1-mutant cells, Trp53bp1 deletion failed to rescue the genomic instability of Palb2- or Brca2-mutant primary lymphocytes. Therefore, Palb2-driven DNA damage control is, in part, distinct from that executed by Brca1 and more similar to that of Brca2. The mechanisms underlying Palb2 mammary tumor suppression functions can now be explored genetically in vivo.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Síndromes Neoplásicas Hereditárias/metabolismo , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Modelos Animais de Doenças , Proteína do Grupo de Complementação N da Anemia de Fanconi , Feminino , Deleção de Genes , Humanos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Mutantes , Síndromes Neoplásicas Hereditárias/genética , Síndromes Neoplásicas Hereditárias/patologia , Proteínas Nucleares/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética
6.
J Biol Chem ; 289(35): 24617-29, 2014 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-25016020

RESUMO

PALB2 links BRCA1 and BRCA2 in homologous recombinational repair of DNA double strand breaks (DSBs). Mono-allelic mutations in PALB2 increase the risk of breast, pancreatic, and other cancers, and biallelic mutations cause Fanconi anemia (FA). Like Brca1 and Brca2, systemic knock-out of Palb2 in mice results in embryonic lethality. In this study, we generated a hypomorphic Palb2 allele expressing a mutant PALB2 protein unable to bind BRCA1. Consistent with an FA-like phenotype, cells from the mutant mice showed hypersensitivity and chromosomal breakage when treated with mitomycin C, a DNA interstrand crosslinker. Moreover, mutant males showed reduced fertility due to impaired meiosis and increased apoptosis in germ cells. Interestingly, mutant meiocytes showed a significant defect in sex chromosome synapsis, which likely contributed to the germ cell loss and fertility defect. Our results underscore the in vivo importance of the PALB2-BRCA1 complex formation in DSB repair and male meiosis.


Assuntos
Proteína BRCA1/metabolismo , Infertilidade Masculina/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Proteína BRCA1/química , Dano ao DNA , Reparo do DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi , Recombinação Homóloga , Humanos , Marcação In Situ das Extremidades Cortadas , Infertilidade Masculina/genética , Masculino , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Proteínas Supressoras de Tumor/química
8.
Sci Rep ; 9(1): 3952, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850678

RESUMO

Chromatin remodeler proteins exert an important function in promoting dynamic modifications in the chromatin architecture, performing a central role in regulating gene transcription. Deregulation of these molecular machines may lead to striking perturbations in normal cell function. The CHD7 gene is a member of the chromodomain helicase DNA-binding family and, when mutated, has been shown to be the cause of the CHARGE syndrome, a severe developmental human disorder. Moreover, CHD7 has been described to be essential for neural stem cells and it is also highly expressed or mutated in a number of human cancers. However, its potential role in glioblastoma has not yet been tested. Here, we show that CHD7 is up-regulated in human glioma tissues and we demonstrate that CHD7 knockout (KO) in LN-229 glioblastoma cells suppresses anchorage-independent growth and spheroid invasion in vitro. Additionally, CHD7 KO impairs tumor growth and increases overall survival in an orthotopic mouse xenograft model. Conversely, ectopic overexpression of CHD7 in LN-428 and A172 glioblastoma cell lines increases cell motility and invasiveness in vitro and promotes LN-428 tumor growth in vivo. Finally, RNA-seq analysis revealed that CHD7 modulates a specific transcriptional signature of invasion-related target genes. Further studies should explore clinical-translational implications for glioblastoma treatment.


Assuntos
Movimento Celular , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Glioblastoma/patologia , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias
9.
Cancer Discov ; 9(4): 526-545, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30709805

RESUMO

Although the majority of BRAF-mutant melanomas respond to BRAF/MEK inhibitors, these agents are not typically curative. Moreover, they are largely ineffective in NRAS- and NF1-mutant tumors. Here we report that genetic and chemical suppression of HDAC3 potently cooperates with MAPK pathway inhibitors in all three RAS pathway-driven tumors. Specifically, we show that entinostat dramatically enhances tumor regression when combined with BRAF/MEK inhibitors, in both models that are sensitive or relatively resistant to these agents. Interestingly, MGMT expression predicts responsiveness and marks tumors with latent defects in DNA repair. BRAF/MEK inhibitors enhance these defects by suppressing homologous recombination genes, inducing a BRCA-like state; however, addition of entinostat triggers the concomitant suppression of nonhomologous end-joining genes, resulting in a chemical synthetic lethality caused by excessive DNA damage. Together, these studies identify melanomas with latent DNA repair defects, describe a promising drug combination that capitalizes on these defects, and reveal a tractable therapeutic biomarker. SIGNIFICANCE: BRAF/MEK inhibitors are not typically curative in BRAF-mutant melanomas and are ineffective in NRAS- and NF1-mutant tumors. We show that HDAC inhibitors dramatically enhance the efficacy of BRAF/MEK inhibitors in sensitive and insensitive RAS pathway-driven melanomas by coordinately suppressing two DNA repair pathways, and identify a clinical biomarker that predicts responsiveness.See related commentary by Lombard et al., p. 469.This article is highlighted in the In This Issue feature, p. 453.


Assuntos
Reparo do DNA/genética , Genes ras/genética , MAP Quinase Quinase Quinases/genética , Melanoma/genética , Humanos , Proteínas Proto-Oncogênicas B-raf
10.
Mol Biotechnol ; 58(6): 404-14, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27126696

RESUMO

Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6.


Assuntos
Fator VIII/química , Fator VIII/metabolismo , Furina/metabolismo , Animais , Células CHO , Cricetulus , Fator VIII/genética , Humanos , Pró-Proteína Convertases/metabolismo , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/metabolismo , Subtilisinas/metabolismo
11.
Oncotarget ; 6(32): 33120-33, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26431549

RESUMO

Glioblastoma multiforme is the most common and lethal of the central nervous system glial-derived tumors. RECK suppresses tumor invasion by negatively regulating at least three members of the matrix metalloproteinase family: MMP-9, MMP-2, and MT1-MMP. A positive correlation has been observed between the abundance of RECK expression in tumor samples and a more favorable prognosis for patients with several types of tumors. In the present study, novel alternatively spliced variants of the RECK gene: RECK-B and RECK-I were isolated by RT-PCR and sequenced. The expression levels and profiles of these alternative RECK transcripts, as well as canonical RECK were determined in tissue samples of malignant astrocytomas of different grades and in a normal tissue RNA panel by qRT-PCR. Our results show that higher canonical RECK expression, accompanied by a higher canonical to alternative transcript expression ratio, positively correlates with higher overall survival rate after chemotherapeutic treatment of GBM patients. U87MG and T98G cells over-expressing the RECK-B alternative variant display higher anchorage-independent clonal growth and do not display modulation of, respectively, MMP-2 and MMP-9 expression. Our findings suggest that RECK transcript variants might have opposite roles in GBM biology and the ratio of their expression levels may be informative for the prognostic outcome of GBM patients.


Assuntos
Neoplasias Encefálicas/genética , Proteínas Ligadas por GPI/genética , Glioblastoma/genética , Adulto , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Feminino , Proteínas Ligadas por GPI/metabolismo , Genes Supressores de Tumor , Glioblastoma/metabolismo , Humanos , Masculino , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , Isoformas de Proteínas , Splicing de RNA
12.
Nat Commun ; 5: 5496, 2014 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-25400221

RESUMO

BRCA1-a breast and ovarian cancer suppressor gene-promotes genome integrity. To study the functionality of BRCA1 in the heterozygous state, we established a collection of primary human BRCA1(+/+) and BRCA1(mut/+) mammary epithelial cells and fibroblasts. Here we report that all BRCA1(mut/+) cells exhibited multiple normal BRCA1 functions, including the support of homologous recombination- type double-strand break repair (HR-DSBR), checkpoint functions, centrosome number control, spindle pole formation, Slug expression and satellite RNA suppression. In contrast, the same cells were defective in stalled replication fork repair and/or suppression of fork collapse, that is, replication stress. These defects were rescued by reconstituting BRCA1(mut/+) cells with wt BRCA1. In addition, we observed 'conditional' haploinsufficiency for HR-DSBR in BRCA1(mut/+) cells in the face of replication stress. Given the importance of replication stress in epithelial cancer development and of an HR defect in breast cancer pathogenesis, both defects are candidate contributors to tumorigenesis in BRCA1-deficient mammary tissue.


Assuntos
Replicação do DNA/fisiologia , Genes BRCA1/fisiologia , Haploinsuficiência/fisiologia , Animais , Mama/citologia , Células Cultivadas , Centrossomo/fisiologia , Replicação do DNA/genética , Feminino , Haploinsuficiência/genética , Heterozigoto , Humanos , Camundongos , RNA Satélite/genética , RNA Satélite/fisiologia , Rad51 Recombinase/genética , Rad51 Recombinase/fisiologia , Reparo de DNA por Recombinação/genética , Reparo de DNA por Recombinação/fisiologia , Polos do Fuso/genética , Polos do Fuso/fisiologia
13.
Cancer Discov ; 3(8): 894-907, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23650262

RESUMO

Hereditary breast cancers stem from germline mutations in susceptibility genes such as BRCA1, BRCA2, and PALB2, whose products function in the DNA damage response and redox regulation. Autophagy is an intracellular waste disposal and stress mitigation mechanism important for alleviating oxidative stress and DNA damage response activation; it can either suppress or promote cancer, but its role in breast cancer is unknown. Here, we show that similar to Brca1 and Brca2, ablation of Palb2 in the mouse mammary gland resulted in tumor development with long latency, and the tumors harbored mutations in Trp53. Interestingly, impaired autophagy, due to monoallelic loss of the essential autophagy gene Becn1, reduced Palb2-associated mammary tumorigenesis in a Trp53-wild-type but not conditionally null background. These results indicate that, in the face of DNA damage and oxidative stress elicited by PALB2 loss, p53 is a barrier to cancer development, whereas autophagy facilitates cell survival and tumorigenesis.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Autofagia , Carcinogênese , Neoplasias Mamárias Experimentais/patologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Apoptose , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Proteína Beclina-1 , Senescência Celular , Dano ao DNA , Modelos Animais de Doenças , Proteína do Grupo de Complementação N da Anemia de Fanconi , Feminino , Heterozigoto , Humanos , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Knockout , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/metabolismo
14.
Cancer Discov ; 2(4): 366-375, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22576213

RESUMO

UNLABELLED: DNA repair competency is one determinant of sensitivity to certain chemotherapy drugs, such as cisplatin. Cancer cells with intact DNA repair can avoid the accumulation of genome damage during growth and also can repair platinum-induced DNA damage. We sought genomic signatures indicative of defective DNA repair in cell lines and tumors and correlated these signatures to platinum sensitivity. The number of subchromosomal regions with allelic imbalance extending to the telomere (N(tAI)) predicted cisplatin sensitivity in vitro and pathologic response to preoperative cisplatin treatment in patients with triple-negative breast cancer (TNBC). In serous ovarian cancer treated with platinum-based chemotherapy, higher levels of N(tAI) forecast a better initial response. We found an inverse relationship between BRCA1 expression and N(tAI) in sporadic TNBC and serous ovarian cancers without BRCA1 or BRCA2 mutation. Thus, accumulation of telomeric allelic imbalance is a marker of platinum sensitivity and suggests impaired DNA repair. SIGNIFICANCE: Mutations in BRCA genes cause defects in DNA repair that predict sensitivity to DNA damaging agents, including platinum; however, some patients without BRCA mutations also benefit from these agents. NtAI, a genomic measure of unfaithfully repaired DNA, may identify cancer patients likely to benefit from treatments targeting defective DNA repair.


Assuntos
Desequilíbrio Alélico , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Telômero/genética , Antineoplásicos , Linhagem Celular Tumoral , Aberrações Cromossômicas , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Genes BRCA1 , Humanos , Modelos Biológicos , Mutação , Neoplasias Ovarianas/genética , RNA Mensageiro/genética
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