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1.
Proc Natl Acad Sci U S A ; 117(31): 18600-18607, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32703806

RESUMO

Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulator of cell death-1 (rcd-1), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassa rcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1-1 and rcd-1-2, is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-1-dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible rcd-1-1/rcd-1-2 alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and rcd-1 This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.


Assuntos
Proteínas Fúngicas , Proteínas de Neoplasias , Piroptose/fisiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Células HEK293 , Humanos , Imunidade Inata/fisiologia , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Neurospora crassa/metabolismo
2.
Commun Biol ; 3(1): 374, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641750

RESUMO

The recent outbreak of infections and the pandemic caused by SARS-CoV-2 represent one of the most severe threats to human health in more than a century. Emerging data from the United States and elsewhere suggest that the disease is more severe in men. Knowledge gained, and lessons learned, from studies of the biological interactions and molecular links that may explain the reasons for the greater severity of disease in men, and specifically in the age group at risk for prostate cancer, will lead to better management of COVID-19 in prostate cancer patients. Such information will be indispensable in the current and post-pandemic scenarios.


Assuntos
Betacoronavirus , Infecções por Coronavirus/epidemiologia , Pandemias , Pneumonia Viral/epidemiologia , Neoplasias da Próstata/epidemiologia , Distribuição por Sexo , Antineoplásicos Hormonais/uso terapêutico , Antivirais/uso terapêutico , Betacoronavirus/fisiologia , Betacoronavirus/ultraestrutura , Comorbidade , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Suscetibilidade a Doenças , Reposicionamento de Medicamentos , Feminino , Previsões , Hormônios Esteroides Gonadais/fisiologia , Humanos , Masculino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/fisiologia , Peptidil Dipeptidase A/fisiologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Inibidores de Proteases/uso terapêutico , Receptores Virais/efeitos dos fármacos , Receptores Virais/fisiologia , Fatores de Risco , Serina Endopeptidases/biossíntese , Serina Endopeptidases/fisiologia , Estados Unidos/epidemiologia , Internalização do Vírus
3.
Am J Physiol Endocrinol Metab ; 319(2): E338-E344, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32574111

RESUMO

Obesity is associated with dyslipidemia and subclinical inflammation that promotes metabolic disturbances including insulin resistance and pancreatic ß-cell dysfunction. The nuclear protein, transcriptional regulator 1 (NUPR1) responds to cellular stresses and features tissue protective properties. To characterize the role of NUPR1 in endocrine pancreatic islets during inflammatory stress, we generated transgenic mice with ß-cell-specific Nupr1 overexpression (ßNUPR1). Under normal conditions, ßNUPR1 mice did not differ from wild type (WT) littermates and display normal glucose homeostasis and ß-cell mass. For induction of inflammatory conditions, mice were treated with multiple low-dose streptozotocin (mld-STZ) and/or fed a high-fat diet (HFD). All treatments significantly worsened glycaemia in WT mice, while ßNUPR1 mice substantially preserved insulin secretion and glucose tolerance. HFD increased ß-cell mass in all animals, with ßNUPR1 mice tending to show higher values. The improved outcome of ßNUPR1 mice was accompanied by decreased NF-κB activation and lymphocyte infiltration in response to mld-STZ. In vitro, isolated ßNUPR1 islets preserved insulin secretion and content with insignificantly low apoptosis during culture stress and IL-1ß exposure. These findings suggest that NUPR1 plays a vital role in the protection of ß-cells from apoptosis, related degradation of insulin storages and subsequent secretion during inflammatory and obesity-related tissue stress.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Dieta Hiperlipídica/efeitos adversos , Inflamação/fisiopatologia , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/fisiologia , Proteínas de Neoplasias/fisiologia , Estreptozocina/administração & dosagem , Animais , Apoptose/fisiologia , Glicemia/análise , Células Cultivadas , Proteínas de Ligação a DNA/genética , Feminino , Expressão Gênica , Homeostase , Inflamação/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Fatores Sexuais
4.
J Pharmacol Sci ; 143(3): 176-181, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32386905

RESUMO

The volume-regulated anion channel (VRAC) plays a central role in maintaining cell volume in response to osmotic stress. Leucine-rich repeat-containing 8A (LRRC8A) was recently identified as an essential component of VRAC although other Cl- channels were also suggested to contribute to VRAC. VRAC is activated when a cell is challenged with a hypotonic environment or even in isotonic conditions challenged with different stimuli. It is not clear how VRAC is activated and whether activation of VRAC in hypotonic and isotonic conditions share the same mechanism. In this present study, we investigated relative contribution of LRRC8A and anoctamin 1(ANO1) to VRAC currents activated by fetal bovine serum (FBS) in isotonic condition, and studied the role of intracellular Ca2+ in this activation. We used CRISPR/Cas9 gene editing approach, electrophysiology, and pharmacology approaches to show that VRAC currents induced by FBS is mostly mediated by LRRC8A in HEK293 cells, but also with significant contribution from ANO1. FBS induces Ca2+ transients and these Ca2+ signals are required for the activation of VRAC by serum. These findings will help to further understand the mechanism in activation of VRAC.


Assuntos
Anoctamina-1/fisiologia , Cálcio/metabolismo , Tamanho Celular , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Canais de Ânion Dependentes de Voltagem/metabolismo , Canais de Ânion Dependentes de Voltagem/fisiologia , Animais , Proteína 9 Associada à CRISPR/genética , Bovinos , Canais de Cloreto/metabolismo , Canais de Cloreto/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , Células HEK293 , Humanos , Pressão Osmótica/fisiologia , Soro
5.
Life Sci ; 254: 117746, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376266

RESUMO

AIMS: Transmembrane 4 L six family member 1 (TM4SF1) is a small plasma membrane glycoprotein that is highly expressed in cancers. However, the role of TM4SF1 that plays in keloids remains unknown. We investigated the expression, function and the microRNA (miRNA) regulatory network of TM4SF1 in keloids. MAIN METHODS: Small interfering RNAs and lentivirus were used to alter the expression of TM4SF1 in fibroblasts. Dual-luciferase reporter assays were applied to determine the miRNA targets. Immunohistochemistry, western blotting, qRT-PCR, wound healing assays, Transwell assays, cell count kit-8 assays and flow cytometry were also employed in this study. KEY FINDINGS: TM4SF1 was frequently upregulated in human keloid fibroblasts (HKFs) compared with human normal skin fibroblasts (HSFs). The downregulation of TM4SF1 significantly inhibited proliferation and migration, and induced apoptosis in HKFs. Furthermore, si-TM4SF1 inhibited the AKT/ERK signaling. Meanwhile, the upregulation of TM4SF1 promoted proliferation, migration and the activation of AKT/ERK signaling in human foreskin fibroblasts (HFF-1). Moreover, TM4SF1 can be regulated by miRNAs, which have been validated to play important roles in keloids by posttranscriptional regulation of gene expression. After screening, we found miR-1-3p and miR-214-5p targeted TM4SF1, inhibited TM4SF1 expression, cell proliferation, migration, and induced apoptosis in HKFs. And the level of miR-1-3p and miR-214-5p were found lower in HKFs than in HSFs. SIGNIFICANCE: Our study demonstrates a novel regulatory mechanism by which miR-1-3p, miR-214-5p, and TM4SF1 are involved in proliferation, cell motility, and apoptosis, suggesting that they may be potential targets in therapies for keloids.


Assuntos
Antígenos de Superfície/fisiologia , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Queloide/patologia , MicroRNAs/metabolismo , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Humanos , Queloide/metabolismo
6.
Int J Nanomedicine ; 15: 2947-2955, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32425526

RESUMO

Introduction: ZnO quantum dots (QDs) have drawn much attention recently as they are Cd-free, low-cost, and have excellent optical properties. With the expanded production and application of ZnO nanoparticles, concerns about their potential toxicity have also been raised. Materials and Methods: We used RNA sequencing (RNA-seq) to analyze the global gene expression of liver and lung tissues after ZnO QDs treatment. Differentially expressed genes (DEGs) were screened, with a fold change >1.5 and padj <0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed, and padj <0.05 was considered significantly enriched. The RNA-seq results were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Results: A total of 47 and 218 genes were significantly differentially expressed in the liver and lung. Eight GO terms were enriched in the liver and lung, and retinol metabolism and the peroxisome proliferator-activated receptor (PPAR) signaling pathway were shared in different tissues. Discussion: According to DEGs and pathway enrichment analyses, inflammation might be induced in liver and lung tissues after intravenous injection of ZnO QDs. These findings will be helpful for future research and application of ZnO QDs.


Assuntos
Fígado/efeitos dos fármacos , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas Nucleares/efeitos dos fármacos , Pontos Quânticos/toxicidade , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Óxido de Zinco/toxicidade , Animais , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Fígado/fisiologia , Masculino , Camundongos , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Testes de Toxicidade , Ubiquitina-Proteína Ligases/fisiologia
7.
PLoS One ; 15(4): e0232047, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32339196

RESUMO

Spontaneous testicular teratomas (STTs) derived from primordial germ cells (PGCs) in the mouse embryonic testes predominantly develop in the 129 family inbred strain. Ter (spontaneous mutation) is a single nucleotide polymorphism that generates a premature stop codon of Dead end1 (Dnd1) and increases the incidence of STTs in the 129 genetic background. We previously found that DND1 interacts with NANOS2 or NANOS3 and that these complexes play a vital role in male embryonic germ cells and adult spermatogonia. However, the following are unclear: (a) whether DND1 works with NANOS2 or NANOS3 to regulate teratoma incidence, and (b) whether Ter simply causes Dnd1 loss or produces a short mutant DND1 protein. In the current study, we newly established a conventional Dnd1-knockout mouse line and found that these mice showed phenotypes similar to those of Ter mutant mice in spermatogenesis, oogenesis, and teratoma incidence, with a slight difference in spermiogenesis. In addition, we found that the amount of DND1 in Dnd1+/Ter embryos decreased to half of that in wild-type embryos, while the expression of the short mutant DND1 was not detected. We also found that double mutants for Dnd1 and Nanos2 or Nanos3 showed synergistic increase in the incidence of STTs. These data support the idea that Ter causes Dnd1 loss, leading to an increase in STT incidence, and that DND1 acts with NANOS2 and NANOS3 to regulate the development of teratoma from PGCs in the 129 genetic background. Thus, our results clarify the role of Dnd1 in the development of STTs and provide a novel insight into its pathogenic mechanism.


Assuntos
Células Germinativas Embrionárias/patologia , Proteínas de Neoplasias/fisiologia , Proteínas de Ligação a RNA/metabolismo , Teratoma/etiologia , Neoplasias Testiculares/etiologia , Testículo/patologia , Animais , Células Germinativas Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Oogênese , Proteínas de Ligação a RNA/genética , Espermatogênese , Teratoma/metabolismo , Teratoma/patologia , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologia , Testículo/metabolismo
8.
Science ; 367(6483): 1255-1260, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32165587

RESUMO

T cells maintain a quiescent state prior to activation. As inappropriate T cell activation can cause disease, T cell quiescence must be preserved. Despite its importance, the mechanisms underlying the "quiescent state" remain elusive. Here, we identify BTG1 and BTG2 (BTG1/2) as factors responsible for T cell quiescence. BTG1/2-deficient T cells show an increased proliferation and spontaneous activation due to a global increase in messenger RNA (mRNA) abundance, which reduces the threshold to activation. BTG1/2 deficiency leads to an increase in polyadenylate tail length, resulting in a greater mRNA half-life. Thus, BTG1/2 promote the deadenylation and degradation of mRNA to secure T cell quiescence. Our study reveals a key mechanism underlying T cell quiescence and suggests that low mRNA abundance is a crucial feature for maintaining quiescence.


Assuntos
Proteínas Imediatamente Precoces/fisiologia , Ativação Linfocitária , Proteínas de Neoplasias/fisiologia , Estabilidade de RNA , RNA Mensageiro/química , Linfócitos T/imunologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Células Cultivadas , Proteínas Imediatamente Precoces/genética , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Poliadenilação , Proteínas Supressoras de Tumor/genética
9.
Cancer Res ; 80(8): 1644-1655, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32094299

RESUMO

Glioblastoma multiforme (GBM) and other solid malignancies are heterogeneous and contain subpopulations of tumor cells that exhibit stem-like features. Our recent findings point to a dedifferentiation mechanism by which reprogramming transcription factors Oct4 and Sox2 drive the stem-like phenotype in glioblastoma, in part, by differentially regulating subsets of miRNAs. Currently, the molecular mechanisms by which reprogramming transcription factors and miRNAs coordinate cancer stem cell tumor-propagating capacity are unclear. In this study, we identified miR-486-5p as a Sox2-induced miRNA that targets the tumor suppressor genes PTEN and FoxO1 and regulates the GBM stem-like cells. miR-486-5p associated with the GBM stem cell phenotype and Sox2 expression and was directly induced by Sox2 in glioma cell lines and patient-derived neurospheres. Forced expression of miR-486-5p enhanced the self-renewal capacity of GBM neurospheres, and inhibition of endogenous miR-486-5p activated PTEN and FoxO1 and induced cell death by upregulating proapoptotic protein BIM via a PTEN-dependent mechanism. Furthermore, delivery of miR-486-5p antagomirs to preestablished orthotopic GBM neurosphere-derived xenografts using advanced nanoparticle formulations reduced tumor sizes in vivo and enhanced the cytotoxic response to ionizing radiation. These results define a previously unrecognized and therapeutically targetable Sox2:miR-486-5p axis that enhances the survival of GBM stem cells by repressing tumor suppressor pathways. SIGNIFICANCE: This study identifies a novel axis that links core transcriptional drivers of cancer cell stemness to miR-486-5p-dependent modulation of tumor suppressor genes that feeds back to regulate glioma stem cell survival.


Assuntos
Neoplasias Encefálicas/patologia , Sobrevivência Celular , Proteína Forkhead Box O1/genética , Genes Supressores de Tumor , Glioblastoma/patologia , MicroRNAs/metabolismo , Proteínas de Neoplasias/fisiologia , PTEN Fosfo-Hidrolase/genética , Fatores de Transcrição SOXB1/fisiologia , Animais , Proteína 11 Semelhante a Bcl-2/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/radioterapia , Morte Celular , Desdiferenciação Celular/genética , Linhagem Celular Tumoral , Reprogramação Celular/fisiologia , Repressão Epigenética , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , MicroRNAs/administração & dosagem , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Nanopartículas/administração & dosagem , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação , Células-Tronco Neurais , Fator 3 de Transcrição de Octâmero/metabolismo , Tolerância a Radiação , Distribuição Aleatória , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transfecção/métodos , Carga Tumoral , Ensaio Tumoral de Célula-Tronco/métodos , Regulação para Cima
10.
Bull Cancer ; 107(1): 41-47, 2020 Jan.
Artigo em Francês | MEDLINE | ID: mdl-31916995

RESUMO

A growing number of studies suggest a tumor suppressor role for the SWI/SNF complex involved in the remodeling of chromatin. Alterations of this complex have been found in many tumors of different origins, with topographic, morphologic and phenotypic diversity. Notably, they define 2 types of thoracic tumors: SMARCA4-deficient non-small cell lung carcinoma and SMARCA4-deficient sarcoma. Some clinical features appear to be common to both, such as intrathoracic localization, smoking exposure, male predominance and poor prognosis. However, the histological distinction between these two entities is sometimes difficult and it is not excluded that these entities belong to the same tumor spectrum with different degrees of differentiation. The therapy of these tumors is not yet codified. These tumors do not seem associated with oncogenic driver mutations allowing the prescription of targeted therapy, but immunotherapy has been shown to be effective in rare reported cases. More specific treatments using EZH2 inhibitors also seem promising in SMARCA4 deficient sarcomas.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Montagem e Desmontagem da Cromatina , DNA Helicases/deficiência , Proteínas de Neoplasias/deficiência , Proteínas Nucleares/deficiência , Sarcoma/genética , Neoplasias Torácicas/genética , Fatores de Transcrição/deficiência , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Quimiorradioterapia , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Terapia Combinada , Procedimentos Cirúrgicos de Citorredução , DNA Helicases/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Neoplasias do Mediastino/genética , Neoplasias do Mediastino/patologia , Neoplasias do Mediastino/terapia , Terapia de Alvo Molecular , Complexos Multiproteicos/efeitos dos fármacos , Complexos Multiproteicos/fisiologia , Invasividade Neoplásica , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/fisiologia , Proteína SMARCB1/fisiologia , Sarcoma/patologia , Sarcoma/terapia , Neoplasias Torácicas/patologia , Neoplasias Torácicas/terapia , Fatores de Transcrição/fisiologia
11.
Int J Cancer ; 146(1): 208-222, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31251822

RESUMO

The role of Fyn-related kinase (FRK) in malignant tumors remains controversial. Our study investigated the function of FRK in lung cancer. Immunohistochemistry staining and generating a knockout of FRK by CRISPR/Cas9 in H1299 (FRK-KO-H1299) cells were strategies used to explore the role of FRK. Immunohistochemistry staining indicated that FRK expression was elevated in 223 lung cancer tissues compared to 26 distant normal lung tissues. FRK contributed to poor survival status in lung cancer patients and acted as a predictor for poor prognosis of lung cancer. Knockout of FRK by CRISPR/Cas9 markedly inhibited proliferation, invasion, colony formation and epithelial-mesenchymal transition (EMT) process in the lung cancer cell line H1299. Further exploration indicated that FRK-KO damaged the stemness phenotype of H1299 by inhibiting CD44 and CD133 expression. Seahorse detection and a U-13 C flux assay revealed that FRK-KO induced metabolism reprogramming by inhibiting the Warburg effect and changing the energy type in H1299 cells. Epidermal growth factor stimulation recovered the expression of FRK and biological functions, metabolic reprogramming and stemness phenotype of H1299 cells. FRK plays an oncogenic role in lung cancer cells via a novel regulation mechanism of enhancing the stemness of H1299 cells by inducing metabolism reprogramming, which finally promotes EMT and metastasis. Our study also indicates that FRK could be used as a potential therapeutic target for drug development.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/fisiologia , Células-Tronco Neoplásicas/patologia , Oncogenes , Proteínas Tirosina Quinases/fisiologia , Sistemas CRISPR-Cas , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Prognóstico , Proteínas Tirosina Quinases/genética
12.
Soft Matter ; 15(48): 9829-9839, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31728468

RESUMO

Membrane curvature has recently been recognized as an active regulator of cellular function, with several protein families identified as sensors and generators of membrane curvature. Amongst them, the inverse Bin/Amphiphysin/Rvs (I-BAR) domain family has been implicated in the sensing and generation of membrane structures with negative membrane curvature e.g. filopodia or dendritic spines. However, to date, quantitative biophysical investigations of I-BAR domains have mostly taken place in reconstitution. Here, we use fluorescence microscopy to quantitatively investigate membrane curvature sensing and generation by I-BARs in filopodia of living cells. As a model system, we selected two prototypic members of the I-BAR family, the insulin receptor substrate p53 and missing-in-metastasis. Our data demonstrated how I-BARs sense negative membrane curvature in the complex environment of live cells by revealing a dependence on membrane curvature for both their binding affinity to membranes and their saturation density. The non-monotonic dependence of protein sorting with negative membrane curvature allowed us to apply previously developed thermodynamic models to provide estimates of the effective intrinsic curvature and bending rigidity of the two I-BARs bound at the plasma membrane. Our results agree with studies performed on the insulin receptor substrate p53 in reconstitution. To quantitate membrane curvature generation by I-BARs we measured how their overexpression reduces the peak and the width of the size distribution of filopodia, resulting in filopodia populations with smaller and more uniform diameters. Our findings provide a quantitative biophysical insight in the ability of I-BARs to sense and generate negative membrane curvature in the crowded environment of living cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Membrana Celular/fisiologia , Proteínas dos Microfilamentos/fisiologia , Modelos Biológicos , Proteínas de Neoplasias/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Pseudópodes/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Células PC12 , Domínios Proteicos , Ratos
13.
Proc Natl Acad Sci U S A ; 116(47): 23625-23635, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31690663

RESUMO

Myocardin-related transcription factor B (MRTFB) is a candidate tumor-suppressor gene identified in transposon mutagenesis screens of the intestine, liver, and pancreas. Using a combination of cell-based assays, in vivo tumor xenograft assays, and Mrtfb knockout mice, we demonstrate here that MRTFB is a human and mouse colorectal cancer (CRC) tumor suppressor that functions in part by inhibiting cell invasion and migration. To identify possible MRTFB transcriptional targets, we performed whole transcriptome RNA sequencing in MRTFB siRNA knockdown primary human colon cells and identified 15 differentially expressed genes. Among the top candidate tumor-suppressor targets were melanoma cell adhesion molecule (MCAM), a known tumor suppressor, and spindle apparatus coiled-coil protein 1 (SPDL1), which has no confirmed role in cancer. To determine whether these genes play a role in CRC, we knocked down the expression of MCAM and SPDL1 in human CRC cells and showed significantly increased invasion and migration of tumor cells. We also showed that Spdl1 expression is significantly down-regulated in Mrtfb knockout mouse intestine, while lower SPDL1 expression levels are significantly associated with reduced survival in CRC patients. Finally, we show that depletion of MCAM and SPDL1 in human CRC cells significantly increases tumor development in xenograft assays, further confirming their tumor-suppressive roles in CRC. Collectively, our findings demonstrate the tumor-suppressive role of MRTFB in CRC and identify several genes, including 2 tumor suppressors, that act downstream of MRTFB to regulate tumor growth and survival in CRC patients.


Assuntos
Adenocarcinoma/genética , Proteínas de Ciclo Celular/metabolismo , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/fisiologia , Fatores de Transcrição/fisiologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Antígeno CD146/metabolismo , Movimento Celular , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Técnicas de Silenciamento de Genes , Genes Supressores de Tumor , Células HCT116 , Células HT29 , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Interferência de RNA , RNA Interferente Pequeno/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
14.
Proc Natl Acad Sci U S A ; 116(47): 23691-23697, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31685623

RESUMO

Predictive biomarkers for tumor response to neoadjuvant chemotherapy are needed in breast cancer. This study investigates the predictive value of 280 genes encoding proteins that regulate microtubule assembly and function. By analyzing 3 independent multicenter randomized cohorts of breast cancer patients, we identified 17 genes that are differentially regulated in tumors achieving pathological complete response (pCR) to neoadjuvant chemotherapy. We focused on the MTUS1 gene, whose major product, ATIP3, is a microtubule-associated protein down-regulated in aggressive breast tumors. We show here that low levels of ATIP3 are associated with an increased pCR rate, pointing to ATIP3 as a predictive biomarker of breast tumor chemosensitivity. Using preclinical models of patient-derived xenografts and 3-dimensional models of breast cancer cell lines, we show that low ATIP3 levels sensitize tumors to the effects of taxanes but not DNA-damaging agents. ATIP3 silencing improves the proapoptotic effects of paclitaxel and induces mitotic abnormalities, including centrosome amplification and multipolar spindle formation, which results in chromosome missegregation leading to aneuploidy. As shown by time-lapse video microscopy, ATIP3 depletion exacerbates cytokinesis failure and mitotic death induced by low doses of paclitaxel. Our results favor a mechanism by which the combination of ATIP3 deficiency and paclitaxel treatment induces excessive aneuploidy, which in turn results in elevated cell death. Together, these studies highlight ATIP3 as an important regulator of mitotic integrity and a useful predictive biomarker for a population of chemoresistant breast cancer patients.


Assuntos
Aneuploidia , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Neoplasias/fisiologia , Paclitaxel/farmacologia , Proteínas Supressoras de Tumor/fisiologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Citocinese/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Estudos Multicêntricos como Assunto/estatística & dados numéricos , Terapia Neoadjuvante , Invasividade Neoplásica/genética , Transplante de Neoplasias , Interferência de RNA , Ensaios Clínicos Controlados Aleatórios como Assunto/estatística & dados numéricos , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/ultraestrutura , Taxoides/farmacologia , Imagem com Lapso de Tempo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética
15.
Nat Struct Mol Biol ; 26(11): 999-1012, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31611688

RESUMO

Protein arginine methyltransferase 5 (PRMT5) has emerged as a promising cancer drug target, and three PRMT5 inhibitors are currently in clinical trials for multiple malignancies. In this study, we investigated the role of PRMT5 in human acute myeloid leukemia (AML). Using an enzymatic dead version of PRMT5 and a PRMT5-specific inhibitor, we demonstrated the requirement of the catalytic activity of PRMT5 for the survival of AML cells. We then identified PRMT5 substrates using multiplexed quantitative proteomics and investigated their role in the survival of AML cells. We found that the function of the splicing regulator SRSF1 relies on its methylation by PRMT5 and that loss of PRMT5 leads to changes in alternative splicing of multiple essential genes. Our study proposes a mechanism for the requirement of PRMT5 for leukemia cell survival and provides potential biomarkers for the treatment response to PRMT5 inhibitors.


Assuntos
Leucemia Mieloide Aguda/enzimologia , Proteínas de Neoplasias/fisiologia , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/fisiologia , Processamento de RNA , Animais , Antineoplásicos/farmacologia , Arginina/análogos & derivados , Arginina/biossíntese , Arginina/metabolismo , Catálise , Linhagem Celular Tumoral , Sobrevivência Celular , Inibidores Enzimáticos/farmacologia , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/genética , Metilação , Camundongos , Terapia de Alvo Molecular , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/genética , Processamento de Proteína Pós-Traducional/genética , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/deficiência , Proteômica , Fatores de Processamento de Serina-Arginina/metabolismo
16.
Proc Natl Acad Sci U S A ; 116(47): 23534-23541, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31591207

RESUMO

Mitochondrial superoxide dismutase (SOD2) suppresses tumor initiation but promotes invasion and dissemination of tumor cells at later stages of the disease. The mechanism of this functional switch remains poorly defined. Our results indicate that as SOD2 expression increases acetylation of lysine 68 ensues. Acetylated SOD2 promotes hypoxic signaling via increased mitochondrial reactive oxygen species (mtROS). mtROS, in turn, stabilize hypoxia-induced factor 2α (HIF2α), a transcription factor upstream of "stemness" genes such as Oct4, Sox2, and Nanog. In this sense, our findings indicate that SOD2K68Ac and mtROS are linked to stemness reprogramming in breast cancer cells via HIF2α signaling. Based on these findings we propose that, as tumors evolve, the accumulation of SOD2K68Ac turns on a mitochondrial pathway to stemness that depends on HIF2α and may be relevant for the progression of breast cancer toward poor outcomes.


Assuntos
Neoplasias da Mama/patologia , Autorrenovação Celular/fisiologia , Proteínas de Neoplasias/fisiologia , Células-Tronco Neoplásicas/fisiologia , Superóxido Dismutase/fisiologia , Acetilação , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Neoplasias da Mama/metabolismo , Reprogramação Celular , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Peróxido de Hidrogênio/metabolismo , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/enzimologia , Invasividade Neoplásica , Proteínas de Neoplasias/química , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/química
17.
Gynecol Oncol ; 155(2): 340-348, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31477279

RESUMO

OBJECTIVE: To determine the involvement of homeobox D9 (HOXD9) in the survival, proliferation, and metastasis of cervical cancer cells through regulating the expression of human papillomavirus (HPV) 16 E6/E7 genes using the P97 promoter. METHODS: One hundred cases of cervical cancer (CC), CC cell lines SKG-I, SKG-II, SKG-IIIa, SKG-IIIb, HeLa, and SiHa, and a human tumor xenograft mouse model were used to examine the roles of HOXD9 in CC. Knockdown experiments employed RNA interference of HOXD9. qPCR, functional assays, western blotting, DNA microarray, and luciferase and ChIP assays were applied for assessments. RESULTS: All CC cell lines expressed HOXD9 mRNA and protein. In uterine CC, HOXD9 gene expression was significantly higher than in normal cervical tissues. A positive correlation of lymphovascular space invasion and lymph node metastasis with high levels of HOXD9 expression was found in patient samples. HOXD9-knockdown cells in the mouse xenograft model only formed small or no tumors. Knockdown of HOXD9 markedly reduced CC cell proliferation, migration and invasion, induced apoptosis, increased P53 protein expression, and suppressed HPV E6/E7 expression by directly binding to the P97 promoter of HPV16 E6/E7 genes. A positive correlation between HOXD9 and HPV16 E6 expression was found in CC patients. CONCLUSIONS: HOXD9 promotes HPV16 E6 and E7 expression by direct binding to the P97 promoter, which enhances proliferation, migration, and metastasis of CCr cells. Our results suggest that HOXD9 could be a prognostic biomarker and potential therapeutic target in CC.


Assuntos
Proteínas de Homeodomínio/fisiologia , Proteínas de Neoplasias/fisiologia , Infecções por Papillomavirus/genética , Regiões Promotoras Genéticas/genética , Neoplasias do Colo do Útero/virologia , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Papillomavirus Humano 16/genética , Humanos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Proteínas Oncogênicas Virais/metabolismo , Oncogenes , Proteínas E7 de Papillomavirus/metabolismo , Fenótipo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/genética
19.
Pediatr Cardiol ; 40(7): 1401-1409, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31367953

RESUMO

Integrin receptors enable cells to sense and respond to their chemical and physical environment. As a class of membrane receptors, they provide a dynamic, tightly regulated link between the extracellular matrix or cellular counter-receptors and intracellular cytoskeletal and signaling networks. They enable transmission of mechanical force across the plasma membrane, and particularly for cardiomyocytes, may sense the mechanical load placed on cells. Talins and Kindlins are two families of FERM-domain proteins which bind the cytoplasmic tail of integrins, recruit cytoskeletal and signaling proteins involved in mechano-transduction, and those which synergize to activate integrins, allowing the integrins to physically change and bind to extracellular ligands. In this review, we will discuss the roles of talin and kindlin, particularly as integrin activators, with a focus on cardiac myocytes.


Assuntos
Integrinas/genética , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Talina/genética , Animais , Coração/fisiologia , Humanos , Integrinas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , Talina/metabolismo
20.
Hematol Oncol ; 37(4): 360-367, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31359442

RESUMO

De novo CD5-positive diffuse large B-cell lymphoma (CD5+ DLBCL) is increasingly recognized as a distinct pathologic phenomenon with a specific clinical picture. However, CD5+ DLBCL has not been studied on a large scale in China. In this study, we show that CD5+ DLBCL occurs at a low frequency (9.2%). Comparison of clinical characteristics of CD5+ vs CD5- DLBCL showed that CD5+ DLBCL was more frequently elderly (>60 years) and had B symptoms, high-performance status, stage III-IV, an IPI score >2 and bone marrow involvement. Patients with CD5+ DLBCL had tumours with a higher prevalence of BCL-2 and p53 overexpression than CD5- DLBCL. Patients with CD5+ DLBCL had inferior progression-free survival (PFS) and overall survival (OS) than did patients with CD5- DLBCL. For CD5+ DLBCL, the patients who were treated with rituximab showed significantly better PFS and OS than those treated without rituximab. However, patients treated with RCHOP showed similar PFS and OS when compared with the group treated with intensive therapy. In addition, patients with p53 and CD5 co-expression had the worst PFS and OS. In conclusion, CD5+ DLBCL was associated with unfavorable clinicopathologic variables and with inferior survival. CD5+ DLBCL has a high frequency of p53 overexpression, and CD5 augments the negative effect of p53 overexpression in DLBCL.


Assuntos
Antígenos CD5/análise , Linfoma Difuso de Grandes Células B/metabolismo , Proteínas de Neoplasias/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Etoposídeo/administração & dosagem , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Prednisona/administração & dosagem , Prognóstico , Intervalo Livre de Progressão , Modelos de Riscos Proporcionais , Fatores de Risco , Rituximab/administração & dosagem , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Regulação para Cima , Vincristina/administração & dosagem , Adulto Jovem
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