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1.
Nat Commun ; 12(1): 1548, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33750780

RESUMO

Reconstituting artificial proto-cells capable of transducing extracellular signals into cytoskeletal changes can reveal fundamental principles of how non-equilibrium phenomena in cellular signal transduction affect morphogenesis. Here, we generated a Synthetic Morphogenic Membrane System (SynMMS) by encapsulating a dynamic microtubule (MT) aster and a light-inducible signaling system driven by GTP/ATP chemical potential into cell-sized liposomes. Responding to light cues in analogy to morphogens, this biomimetic design embodies basic principles of localized Rho-GTPase signal transduction that generate an intracellular MT-regulator signaling gradient. Light-induced signaling promotes membrane-deforming growth of MT-filaments by dynamically elevating the membrane-proximal tubulin concentration. The resulting membrane deformations enable recursive coupling of the MT-aster with the signaling system, which generates global self-organized morphologies that reorganize towards local external cues in dependence on prior shape. SynMMS thereby signifies a step towards bio-inspired engineering of self-organized cellular morphogenesis.


Assuntos
Sinais (Psicologia) , Lipossomos , Morfogênese/fisiologia , Células Artificiais , Fenômenos Biofísicos , Extensões da Superfície Celular/fisiologia , Centrossomo , Citoesqueleto/metabolismo , Humanos , Lipossomos/química , Microtúbulos/metabolismo , Proteínas Recombinantes , Transdução de Sinais , Estatmina/metabolismo , Biologia Sintética , Tubulina (Proteína)/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
2.
Nat Commun ; 11(1): 5105, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037191

RESUMO

RB1 mutational inactivation is a cancer driver in various types of cancer including lung cancer, making it an important target for therapeutic exploitation. We performed chemical and genetic vulnerability screens in RB1-isogenic lung cancer pair and herein report that aurora kinase A (AURKA) inhibition is synthetic lethal in RB1-deficient lung cancer. Mechanistically, RB1-/- cells show unbalanced microtubule dynamics through E2F-mediated upregulation of the microtubule destabilizer stathmin and are hypersensitive to agents targeting microtubule stability. Inhibition of AURKA activity activates stathmin function via reduced phosphorylation and facilitates microtubule destabilization in RB1-/- cells, heavily impacting the bipolar spindle formation and inducing mitotic cell death selectively in RB1-/- cells. This study shows that stathmin-mediated disruption of microtubule dynamics is critical to induce synthetic lethality in RB1-deficient cancer and suggests that upstream factors regulating microtubule dynamics, such as AURKA, can be potential therapeutic targets in RB1-deficient cancer.


Assuntos
Aurora Quinase A/genética , Neoplasias Pulmonares/genética , Microtúbulos/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Estatmina/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Nus , Microtúbulos/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteínas de Ligação a Retinoblastoma/metabolismo , Estatmina/genética , Mutações Sintéticas Letais , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
PLoS One ; 15(7): e0229193, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614890

RESUMO

BACKGROUND: Urine-based diagnostics indicated involvement of oncoprotein 18 (OP18) in bladder cancer. In cell culture models we investigated the role of OP18 for malignant cell growth. METHODS: We analyzed 113 urine samples and investigated two human BCa cell lines as a dual model: RT-4 and ECV-304, which represented differentiated (G1) and poorly differentiated (G3) BCa. We designed specific siRNA for down-regulation of OP18 in both cell lines. Phenotypes were characterized by cell viability, proliferation, and expression of apoptosis-related genes. Besides, sensitivity to cisplatin treatment was evaluated. RESULTS: Analysis of urine samples from patients with urothelial BCa revealed a significant correlation of the RNA-ratio OP18:uroplakin 1A with bladder cancer. High urinary ratios were mainly found in moderately to poorly differentiated tumors (grade G2-3) that were muscle invasive (stage T2-3), whereas samples from patients with more differentiated non-invasive BCa (G1) showed low OP18:UPK1A RNA ratios. Down-regulation of OP18 expression in ECV-304 shifted its phenotype towards G1 state. Further, OP18-directed siRNA induced apoptosis and increased chemo-sensitivity to cisplatin. CONCLUSIONS: This study provides conclusive experimental evidence for the link between OP18-derived RNA as a diagnostic marker for molecular staging of BCa in non-invasive urine-based diagnostics and the patho-mechanistic role of OP18 suggesting this gene as a therapeutic target.


Assuntos
Biomarcadores Tumorais/urina , RNA/urina , Estatmina/genética , Neoplasias da Bexiga Urinária/diagnóstico , Idoso , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/uso terapêutico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Musculares/secundário , Gradação de Tumores , Fenótipo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Estatmina/antagonistas & inibidores , Estatmina/metabolismo , Estatmina/urina , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Uroplaquina Ia/genética
4.
J Gastroenterol Hepatol ; 35(10): 1668-1675, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32250469

RESUMO

BACKGROUND AND AIM: The role of STMN1 in the development and progression of esophageal carcinoma is not yet determined. The present study aimed to systematically evaluate the correlation between STMN1 and prognosis of patients with esophageal carcinoma. METHODS: Electronic databases including PubMed, Embase, the Cochrane library, and Chinese Biomedical Literature Database (CBM) were searched to identify studies evaluating the impact of STMN1 on the survival of esophageal cancer patients, without the language limitation. Two investigators screened the literature according to the inclusion and exclusion criteria and evaluated the quality of the included studies. The combined analysis was performed using RevMan 5.3 software. RESULTS: A total of eight studies, involving 1240 esophageal carcinoma patients, were included in this retrospective design. Meta-analysis showed that esophageal carcinoma patients with low STMN1 had a superior overall survival and disease-free survival than those with high expression of STMN1. Compared with the high expression of STMN1, the 5-year survival rate was significantly higher in patients with low level of STMN1. Patients with high STMN1 expression had a higher risk of experiencing clinical grade III-IV disease, lymph node metastasis, and tumor invasion than those with low STMN1. CONCLUSION: STMN1 is an indicator for the prognosis of esophageal carcinoma patients.


Assuntos
Carcinoma/genética , Neoplasias Esofágicas/genética , Regulação da Expressão Gênica , Expressão Gênica , Estudos de Associação Genética , Estatmina/genética , Estatmina/metabolismo , Grupo com Ancestrais do Continente Asiático , Carcinoma/mortalidade , Carcinoma/patologia , Intervalo Livre de Doença , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida
5.
PLoS One ; 15(4): e0230814, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32251425

RESUMO

Microtubules are a major cytoskeletal component of neurites, and the regulation of microtubule stability is essential for neurite morphogenesis. ßPix (ARHGEF7) is a guanine nucleotide exchange factor for the small GTPases Rac1 and Cdc42, which modulate the organization of actin filaments and microtubules. ßPix is expressed as alternatively spliced variants, including the ubiquitous isoform ßPix-a and the neuronal isoforms ßPix-b and ßPix-d, but the function of the neuronal isoforms remains unclear. Here, we reveal the novel role of ßPix neuronal isoforms in regulating tubulin acetylation and neurite outgrowth. At DIV4, hippocampal neurons cultured from ßPix neuronal isoform knockout (ßPix-NIKO) mice exhibit defects in neurite morphology and tubulin acetylation, a type of tubulin modification which often labels stable microtubules. Treating ßPix-NIKO neurons with paclitaxel, which stabilizes the microtubules, or reintroducing either neuronal ßPix isoform to the KO neurons overcomes the impairment in neurite morphology and tubulin acetylation, suggesting that neuronal ßPix isoforms may promote microtubule stabilization during neurite development. ßPix-NIKO neurons also exhibit lower phosphorylation levels for Stathmin1, a microtubule-destabilizing protein, at Ser16. Expressing either ßPix neuronal isoform in the ßPix-NIKO neurons restores Stathmin1 phosphorylation levels, with ßPix-d having a greater effect than ßPix-b. Furthermore, we find that the recovery of neurite length and Stathmin1 phosphorylation via ßPix-d expression requires PAK kinase activity. Taken together, our study demonstrates that ßPix-d regulates the phosphorylation of Stathmin1 in a PAK-dependent manner and that neuronal ßPix isoforms promote tubulin acetylation and neurite morphogenesis during neuronal development.


Assuntos
Crescimento Neuronal/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/fisiologia , Estatmina/metabolismo , Tubulina (Proteína)/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Acetilação , Citoesqueleto de Actina/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Feminino , Hipocampo/metabolismo , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Neuritos/metabolismo , Neuritos/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Fosforilação/fisiologia , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia
6.
Sci Rep ; 10(1): 2914, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076022

RESUMO

Studies indicate that stathmin expression associates with PI3K activation in breast cancer, suggesting stathmin as a marker for targetable patient subgroups. Here we assessed stathmin in relation to tumour proliferation, vascular and immune responses, BRCA1 germline status, basal-like differentiation, clinico-pathologic features, and survival. Immunohistochemical staining was performed on breast cancers from two series (cohort 1, n = 187; cohort 2, n = 198), and mass spectrometry data from 24 cases and 12 breast cancer cell lines was examined for proteomic profiles. Open databases were also explored (TCGA, METABRIC, Oslo2 Landscape cohort, Cancer Cell Line Encyclopedia). High stathmin expression associated with tumour proliferation, p53 status, basal-like differentiation, BRCA1 genotype, and high-grade histology. These patterns were confirmed using mRNA data. Stathmin mRNA further associated with tumour angiogenesis, immune responses and reduced survival. By logistic regression, stathmin protein independently predicted a BRCA1 genotype (OR 10.0, p = 0.015) among ER negative tumours. Cell line analysis (Connectivity Map) implied PI3K inhibition in tumours with high stathmin. Altogether, our findings indicate that stathmin might be involved in the regulation of tumour angiogenesis and immune responses in breast cancer, in addition to tumour proliferation. Cell data point to potential effects of PI3K inhibition in tumours with high stathmin expression.


Assuntos
Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/imunologia , Estatmina/genética , Proteína BRCA1/genética , Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mutação em Linhagem Germinativa/genética , Humanos , Estimativa de Kaplan-Meier , Modelos Logísticos , Invasividade Neoplásica , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Estrogênicos/metabolismo , Estatmina/metabolismo
7.
Lab Invest ; 100(6): 812-823, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31949244

RESUMO

Glioblastoma multiforme (GBM) is characterized by highly invasive growth, which leads to extensive infiltration and makes complete tumor excision difficult. Since cytoskeleton proteins are related to leading processes and cell motility, and through analysis of public GBM databases, we determined that an actin-interacting protein, zyxin (ZYX), may involved in GBM invasion. Our own glioma cohort as well as the cancer genome atlas (TCGA), Rembrandt, and Gravendeel databases consistently showed that increased ZYX expression was related to tumor progression and poor prognosis of glioma patients. In vitro and in vivo experiments further confirmed the oncogenic roles of ZYX and demonstrated the role of ZYX in GBM invasive growth. Moreover, RNA-seq and mass-spectrum data from GBM cells with or without ZYX revealed that stathmin 1 (STMN1) was a potential target of ZYX. Subsequently, we found that both mRNA and protein levels of STMN1 were positively regulated by ZYX. Functionally, STMN1 not only promoted invasion of GBM cells but also rescued the invasion repression caused by ZYX loss. Taken together, our results indicate that high ZYX expression was associated with worse prognosis and highlighted that the ZYX-STMN1 axis might be a potential therapeutic target for GBM.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Invasividade Neoplásica/patologia , Zixina , Animais , Biomarcadores Tumorais , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Linhagem Celular Tumoral , Movimento Celular/genética , Técnicas de Silenciamento de Genes , Glioblastoma/diagnóstico , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Humanos , Camundongos , Camundongos Endogâmicos NOD , Prognóstico , Estatmina/análise , Estatmina/genética , Estatmina/metabolismo , Zixina/análise , Zixina/genética , Zixina/metabolismo
8.
Br J Cancer ; 122(3): 434-444, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31806880

RESUMO

BACKGROUND: Stathmin mediates cell migration and invasion in vitro, and metastasis in vivo. To investigate stathmin's role on the metastatic process, we performed integrated mRNA-miRNA expression analysis to identify pathways regulated by stathmin. METHODS: MiRNA and gene arrays followed by miRNA-target-gene integration were performed on stathmin-depleted neuroblastoma cells (CtrlshRNA vs. Stmn Seq2shRNA). The expression of the predicted target PTPN14 was evaluated by RT-qPCR, western blot and immunohistochemistry. Gene-silencing technology was used to assess the role of PTPN14 on proliferation, migration, invasion and signalling pathway. RESULTS: Stathmin levels modulated the expression of genes and miRNA in neuroblastoma cells, leading to a deregulation of migration and invasion pathways. Consistent with gene array data, PTPN14 mRNA and protein expression were downregulated in stathmin- depleted neuroblastoma cells and xenografts. In two independent neuroblastoma cells, suppression of PTPN14 expression led to an increase in cell migration and invasion. PTPN14 and stathmin expression did not act in a feedback regulatory loop in PTPN14- depleted cells, suggesting a complex interplay of signalling pathways. The effect of PTPN14 on YAP pathway activation was cell-type dependent. CONCLUSIONS: Our findings demonstrate that stathmin levels can regulate PTPN14 expression, which can modulate neuroblastoma cell migration and invasion.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neuroblastoma/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Estatmina/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Humanos , Camundongos , Camundongos SCID , MicroRNAs/metabolismo , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Transplante de Neoplasias , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Estatmina/metabolismo
9.
Arch Virol ; 165(1): 69-85, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31705208

RESUMO

Herpesviruses are predicted to express more than 80 proteins during their infection cycle. The proteins synthesized by the immediate early genes and early genes target signaling pathways in host cells that are essential for the successful initiation of a productive infection and for latency. In this study, proteomic and phosphoproteomic tools showed the occurrence of changes in Madin-Darby bovine kidney cells at the early stage of the infection by bovine herpesvirus 1 (BoHV-1). Proteins that had already been described in the early stage of infection for other herpesviruses but not for BoHV-1 were found. For example, stathmin phosphorylation at the initial stage of infection is described for the first time. In addition, two proteins that had not been described yet in the early stages of herpesvirus infections in general were ribonuclease/angiogenin inhibitor and Rab GDP dissociation inhibitor beta. The biological processes involved in these cellular responses were repair and replication of DNA, splicing, microtubule dynamics, and inflammatory responses. These results reveal pathways that might be used as targets for designing antiviral molecules against BoHV-1 infection.


Assuntos
Infecções por Herpesviridae/metabolismo , Herpesvirus Bovino 1/patogenicidade , Proteômica/métodos , Proteínas Virais/metabolismo , Animais , Bovinos , Linhagem Celular , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 1/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Espectrometria de Massas , Fosforilação , Mapas de Interação de Proteínas , Estatmina/metabolismo , Replicação Viral
10.
Rhinology ; 58(1): 74-79, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31710049

RESUMO

BACKGROUND: Inverted papilloma (IP) is a locally destructive benign tumour of the sinonasal mucosa with a tendency for malignant transformation. Stathmin and epidermal growth factor receptor (EGFR) are important markers in cancer prognosis. Here we investigate if expression of stathmin and EGFR correlate to dysplasia, recurrence and HPV in IP. METHODS: 98 patients with IP diagnosed 2000-2010 were analyzed for stathmin and EGFR by immunohistochemistry (IHC) and HPV by polymerase chain reaction assay (PCR). RESULTS: All IPs expressed stathmin while its expression was absent or weak in normal mucosa. Dysplasia was present in 26,7% of IPs with high stathmin expression while only 7.4% of IPs with low stathmin expression showed dysplasia. Stathmin positive IPs showed a trend towards earlier recurrences. 57.1% of IP expressed EGFR but no significant association was seen between EGFR-positivity and recurrence or dysplasia. EGFR was expressed by 91.7% of the HPV-positive IPs compared to 52,3% of the HPV negative IPs. CONCLUSIONS: EGFR expression is significantly higher in HPV positive IP. Stathmin is expressed by all IP tumour cells. Stathmin was also associated with dysplasia and a trend towards a correlation between stathmin positivity and recurrence was found. Stathmin and EGFR might therefore be considered therapeutic targets.


Assuntos
Papiloma Invertido/diagnóstico , Infecções por Papillomavirus/diagnóstico , Neoplasias dos Seios Paranasais/diagnóstico , Estatmina/metabolismo , Receptores ErbB/metabolismo , Humanos , Recidiva Local de Neoplasia
11.
Cell Commun Signal ; 17(1): 159, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783876

RESUMO

BACKGROUND: Members of the karyopherin superfamily serve as nuclear transport receptors/adaptor proteins and provide exchange of macromolecules between the nucleo- and cytoplasm. Emerging evidence suggests a subset of karyopherins to be dysregulated in hepatocarcinogenesis including karyopherin-α2 (KPNA2). However, the functional and regulatory role of KPNA2 in liver cancer remains incompletely understood. METHODS: Quantitative proteomics (LC-MS/MS, ~ 1750 proteins in total) was used to study changes in global protein abundance upon siRNA-mediated KPNA2 knockdown in HCC cells. Functional and mechanistic analyses included colony formation and 2D migration assays, co-immunoprecipitation (CoIP), chromatin immunoprecipitation (ChIP), qRT-PCR, immmunblotting, and subcellular fractionation. In vitro results were correlated with data derived from a murine HCC model and HCC patient samples (3 cohorts, n > 600 in total). RESULTS: The proteomic approach revealed the pro-tumorigenic, microtubule (MT) interacting protein stathmin (STMN1) among the most downregulated proteins upon KPNA2 depletion in HCC cells. We further observed that KPNA2 knockdown leads to reduced tumor cell migration and colony formation of HCC cells, which could be phenocopied by direct knockdown of stathmin. As the underlying regulatory mechanism, we uncovered E2F1 and TFDP1 as transport substrates of KPNA2 being retained in the cytoplasm upon KPNA2 ablation, thereby resulting in reduced STMN1 expression. Finally, murine and human HCC data indicate significant correlations of STMN1 expression with E2F1/TFPD1 and with KPNA2 expression and their association with poor prognosis in HCC patients. CONCLUSION: Our data suggest that KPNA2 regulates STMN1 by import of E2F1/TFDP1 and thereby provide a novel link between nuclear transport and MT-interacting proteins in HCC with functional and prognostic significance.


Assuntos
Fator de Transcrição E2F1/metabolismo , Neoplasias Hepáticas/genética , Estatmina/genética , Fator de Transcrição DP1/metabolismo , alfa Carioferinas/metabolismo , Fator de Transcrição E2F1/genética , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Transdução de Sinais/genética , Estatmina/metabolismo , Fator de Transcrição DP1/genética , Células Tumorais Cultivadas , alfa Carioferinas/genética
12.
Virchows Arch ; 475(6): 771-779, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31686194

RESUMO

The aim of this study was to review the histopathological, phenotypic, and molecular characteristics of pediatric-type follicular lymphoma (PTFL) and to assess the diagnostic value of novel immunohistochemical markers in distinguishing PTFL from follicular hyperplasia (FH). A total of 13 nodal PTFLs were investigated using immunohistochemistry, fluorescence in situ hybridization (FISH), and PCR and were compared with a further 20 reactive lymph nodes showing FH. Morphologically, PTFL cases exhibited a follicular growth pattern with irregular lymphoid follicles in which the germinal centers were composed of numerous blastoid cells showing a starry-sky appearance. Immunohistochemistry highlighted preserved CD10 (13/13) and BCL6 (13/13) staining, CD20 (13/13) positivity, a K light chain predominance (7/13), and partial BCL2 expression in 6/13 cases (using antibodies 124, E17, and SP66). The germinal center (GC)-associated markers stathmin and LLT-1 were positive in most of the cases (12/13 and 12/13, respectively). Interestingly, FOXP-1 was uniformly positive in PTFL (12/13 cases) in contrast to reactive GCs in FH, where only a few isolated positive cells were observed. FISH revealed no evidence of BCL2, BCL6, or MYC rearrangements in the examined cases. By PCR, clonal immunoglobulin gene rearrangements were detected in 100% of the tested PTFL cases. Our study confirmed the unique morphological and immunophenotypic features of PTFL and suggests that FOXP-1 can represent a novel useful diagnostic marker in the differential diagnosis between PTFL and FH.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Linfoma de Células B/patologia , Linfoma Folicular/metabolismo , Linfoma Folicular/patologia , Proteínas Repressoras/metabolismo , Adolescente , Adulto , Criança , Diagnóstico Diferencial , Humanos , Imuno-Histoquímica/métodos , Imunofenotipagem/métodos , Linfoma de Células B/diagnóstico , Linfoma de Células B/metabolismo , Linfoma Folicular/diagnóstico , Masculino , Estatmina/metabolismo , Adulto Jovem
13.
Aging (Albany NY) ; 11(18): 7817-7829, 2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31546234

RESUMO

PRL-3, an oncogenic dual-specificity phosphatase, is overexpressed in 50% of acute myeloid leukemia patients. Stathmin has been identified as a downstream target of PRL-3 in colorectal cancer. However, the correlation between PRL-3 and stathmin in myeloid leukemia is unclear. In this study, we revealed the positive correlation between PRL-3 and stathmin in myeloid leukemia. Knockdown of the PRL-3 gene by shRNA reduced the expression of downstream stathmin, suppressed cell proliferation, induced G2/M arrest and cell apoptosis, and inhibited migration and invasion in myeloid leukemia cells. Moreover, our study was the first to provide evidence that silencing PRL-3 increased the phosphorylation level in Ser16, Ser25, Ser38, and Ser63 of stathmin, and in turn inhibited the STAT3 and STAT5 signaling in myeloid leukemia cells. This evidence points to a promoted role for PRL-3 in the progression of myeloid leukemia, and PRL-3 could be a possible new treatment target.


Assuntos
Leucemia Mieloide/genética , Proteínas de Neoplasias/genética , Proteínas Tirosina Fosfatases/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Estatmina/metabolismo , Apoptose/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Leucemia Mieloide/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , RNA Interferente Pequeno
14.
Cell Stem Cell ; 25(3): 342-356.e7, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31422913

RESUMO

The gastric corpus epithelium is the thickest part of the gastrointestinal tract and is rapidly turned over. Several markers have been proposed for gastric corpus stem cells in both isthmus and base regions. However, the identity of isthmus stem cells (IsthSCs) and the interaction between distinct stem cell populations is still under debate. Here, based on unbiased genetic labeling and biophysical modeling, we show that corpus glands are compartmentalized into two independent zones, with slow-cycling stem cells maintaining the base and actively cycling stem cells maintaining the pit-isthmus-neck region through a process of "punctuated" neutral drift dynamics. Independent lineage tracing based on Stmn1 and Ki67 expression confirmed that rapidly cycling IsthSCs maintain the pit-isthmus-neck region. Finally, single-cell RNA sequencing (RNA-seq) analysis is used to define the molecular identity and lineage relationship of a single, cycling, IsthSC population. These observations define the identity and functional behavior of IsthSCs.


Assuntos
Células-Tronco Adultas/citologia , Mucosa Gástrica/citologia , Estômago/citologia , Células-Tronco Adultas/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Autorrenovação Celular , Células Cultivadas , Mucosa Gástrica/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Estatmina/metabolismo , Nicho de Células-Tronco
15.
Hum Mol Genet ; 28(22): 3742-3754, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31363739

RESUMO

Spinal muscular atrophy (SMA) is a devastating infantile genetic disorder caused by the loss of survival motor neuron (SMN) protein that leads to premature death due to loss of motor neurons and muscle atrophy. The approval of an antisense oligonucleotide therapy for SMA was an important milestone in SMA research; however, effective next-generation therapeutics will likely require combinatorial SMN-dependent therapeutics and SMN-independent disease modifiers. A recent cross-disease transcriptomic analysis identified Stathmin-1 (STMN1), a tubulin-depolymerizing protein, as a potential disease modifier across different motor neuron diseases, including SMA. Here, we investigated whether viral-based delivery of STMN1 decreased disease severity in a well-characterized SMA mouse model. Intracerebroventricular delivery of scAAV9-STMN1 in SMA mice at P2 significantly increased survival and weight gain compared to untreated SMA mice without elevating Smn levels. scAAV9-STMN1 improved important hallmarks of disease, including motor function, NMJ pathology and motor neuron cell preservation. Furthermore, scAAV9-STMN1 treatment restored microtubule networks and tubulin expression without affecting tubulin stability. Our results show that scAAV9-STMN1 treatment improves SMA pathology possibly by increasing microtubule turnover leading to restored levels of stable microtubules. Overall, these data demonstrate that STMN1 can significantly reduce the SMA phenotype independent of restoring SMN protein and highlight the importance of developing SMN-independent therapeutics for the treatment of SMA.


Assuntos
Atrofia Muscular Espinal/genética , Estatmina/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Infusões Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/metabolismo , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/fisiopatologia , Fenótipo , Estatmina/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo
16.
Virology ; 535: 83-101, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31299491

RESUMO

Genomic instability is a hallmark of many cancers; however, the molecular etiology of chromosomal dysregulation is not well understood. The human T-cell leukemia virus type-1 (HTLV-1) oncoprotein Tax activates NF-κB-signaling and induces DNA-damage and aberrant chromosomal segregation through diverse mechanisms which contribute to viral carcinogenesis. Intriguingly, Stathmin/oncoprotein-18 (Op-18) depolymerizes tubulin and interacts with the p65RelA subunit and functions as a cofactor for NF-κB-dependent transactivation. We thus hypothesized that the dissociation of p65RelA-Stathmin/Op-18 complexes by Tax could lead to the catastrophic destabilization of microtubule (MT) spindle fibers during mitosis and provide a novel mechanistic link between NF-κB-signaling and genomic instability. Here we report that the inhibition of Stathmin expression by the retroviral latency protein, p30II, or knockdown with siRNA-stathmin, dampens Tax-mediated NF-κB transactivation and counters Tax-induced genomic instability and cytotoxicity. The Tax-G148V mutant, defective for NF-κB activation, exhibited reduced p65RelA-Stathmin binding and diminished genomic instability and cytotoxicity. Dominant-negative inhibitors of NF-κB also prevented Tax-induced multinucleation and apoptosis. Moreover, cell clones containing the infectious HTLV-1 ACH. p30II mutant provirus, impaired for p30II production, exhibited increased multinucleation and the accumulation of cytoplasmic tubulin aggregates following nocodozole-treatment. These findings allude to a mechanism whereby NF-κB-signaling regulates tubulin dynamics and mitotic instability through the modulation of p65RelA-Stathmin/Op-18 interactions, and support the notion that p30II enhances the survival of Tax-expressing HTLV-1-transformed cells.


Assuntos
Produtos do Gene tax/metabolismo , Instabilidade Genômica , Interações Hospedeiro-Patógeno , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Fuso Acromático/metabolismo , Estatmina/metabolismo , Fator de Transcrição RelA/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Mapas de Interação de Proteínas
17.
Cell Syst ; 9(2): 167-186.e12, 2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31302154

RESUMO

Neuroepithelial stem cells (NSC) from different anatomical regions of the embryonic neural tube's rostrocaudal axis can differentiate into diverse central nervous system tissues, but the transcriptional regulatory networks governing these processes are incompletely understood. Here, we measure region-specific NSC gene expression along the rostrocaudal axis in a human pluripotent stem cell model of early central nervous system development over a 72-h time course, spanning the hindbrain to cervical spinal cord. We introduce Escarole, a probabilistic clustering algorithm for non-stationary time series, and combine it with prior-based regulatory network inference to identify genes that are regulated dynamically and predict their upstream regulators. We identify known regulators of patterning and neural development, including the HOX genes, and predict a direct regulatory connection between the transcription factor POU3F2 and target gene STMN2. We demonstrate that POU3F2 is required for expression of STMN2, suggesting that this regulatory connection is important for region specificity of NSCs.


Assuntos
Células-Tronco Neurais/metabolismo , Rombencéfalo/embriologia , Medula Espinal/embriologia , Diferenciação Celular/genética , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Neurais/fisiologia , Células Neuroepiteliais , Neurogênese , Neurônios/metabolismo , Fatores do Domínio POU/genética , Fatores do Domínio POU/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Estatmina/genética , Estatmina/metabolismo , Transcriptoma/genética
18.
J Vasc Surg ; 70(6): 2021-2031.e1, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30929966

RESUMO

OBJECTIVE: Restenosis limits the durability of all cardiovascular reconstructions. Vascular smooth muscle cell (VSMC) proliferation drives this process, but an intact, functional endothelium is necessary for vessel patency. Current strategies to prevent restenosis employ antiproliferative agents that affect both VSMCs and endothelial cells (ECs). Knockdown of the myristoylated alanine-rich C kinase substrate (MARCKS) arrests VSMC proliferation and paradoxically potentiates EC proliferation. MARCKS knockdown decreases expression of the kinase interacting with stathmin (KIS), increasing p27kip1 expression, arresting VSMC proliferation. Here, we seek to determine how MARCKS influences KIS protein expression in these two cell types. METHODS: Primary human coronary artery VSMCs and ECs were used for in vitro experiments. MARCKS was depleted by transfection with small interfering RNA. Messenger RNA was quantitated with the real-time reverse transcription polymerase chain reaction. Protein expression was determined by Western blot analysis. Ubiquitination was determined with immunoprecipitation. MARCKS and KIS binding was assessed with co-immunoprecipitation. Intimal hyperplasia was induced in CL57/B6 mice with a femoral artery wire injury. MARCKS was knocked down in vivo by application of 10 µM of small interfering RNA targeting MARCKS suspended in 30% Pluronic F-127 gel. Intimal hyperplasia formation was assessed by measurement of the intimal thickness on cross sections of the injured artery. Re-endothelialization was determined by quantitating the binding of Evans blue dye to the injured artery. RESULTS: MARCKS knockdown did not affect KIS messenger RNA expression in either cell type. In the presence of cycloheximide, MARCKS knockdown in VSMCs decreased KIS protein stability but had no effect in ECs. The effect of MARCKS knockdown on KIS stability was abrogated by the 26s proteasome inhibitor MG-132. MARCKS binds to KIS in VSMCs but not in ECs. MARCKS knockdown significantly increased the level of ubiquitinated KIS in VSMCs but not in ECs. MARCKS knockdown in vivo resulted in decreased KIS expression. Furthermore, MARCKS knockdown in vivo resulted in decreased 5-ethynyl-2'-deoxyuridine integration and significantly reduced intimal thickening. MARCKS knockdown enhanced endothelial barrier function recovery 4 days after injury. CONCLUSIONS: MARCKS differentially regulates the KIS protein stability in VSMCs and ECs. The difference in stability is due to differential ubiquitination of KIS in these two cell types. The differential interaction of MARCKS and KIS provides a possible explanation for the observed difference in ubiquitination. The effect of MARCKS knockdown on KIS expression persists in vivo, potentiates recovery of the endothelium, and abrogates intimal hyperplasia formation.


Assuntos
Células Endoteliais/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/fisiologia , Estatmina/metabolismo , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Células Endoteliais/citologia , Humanos , Hiperplasia/metabolismo , Técnicas In Vitro , Leupeptinas/farmacologia , Camundongos , Camundongos Endogâmicos , Ligação Proteica , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia
19.
J Proteomics ; 202: 103364, 2019 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-31009804

RESUMO

Odontoblastic/osteogenic differentiation of human dental pulp stem cells (hDPSCs) is a key factor in tooth and pulp regeneration, but its mechanism still remains unknown. The purpose of this research is to look into the mechanism by which Stathmin affects the proliferation and odontoblastic/osteogenic differentiation of hDPSCs, and whether the Wnt/ß- catenin is related to this regulation. First, the Stathmin expression was inhibited by lentiviral vector, after that the transcriptome sequencing technology was used to screen the differentially expressed genes, then we found Wnt5a which related to the regulation of Wnt/ß-catenin was regulated. Comparing with hDPSC in the control group, the shRNA-Stathmin group inhibited proliferation and odontoblastic/osteogenic differentiation. The result of molecular analysis indicated that the Wnt/ß-catenin was inhibited when Stathmin was silenced. After that, the shRNA-Stathmin group were added with LiCl (activator of Wnt/ß-catenin), and the Wnt/ß-catenin was significantly activated in ß-catenin. After activation of the Wnt/ß-catenin, the proliferation of hDPSCs was significantly increased and the expression of genes related to odontoblastic/osteogenic differentiation was also significantly increased. Taken together, these findings reveal for the first time that the Stathmin-Wnt/ß-catenin plays a positive regulatory role in hDPSC proliferation and odontoblastic/osteogenic differentiation. SIGNIFICANCE: Transcriptome sequencing revealed that Stathmin interacts with Wnt/ß-catenin signaling pathway-related proteins such as Wnt5a. At the same time, experiments have confirmed that Stathmin protein can affect the proliferation and odontogenetic differentiation of hDPSCs.The innovation of this paper is to link the Stathmin and Wnt/ß-catenin signaling pathways for the first time, to explore the interaction of Stathmin and Wnt/ß-catenin signaling pathways and the mechanism of this regulation on human dental pulp stem cells (hDPSCs) of odontoblastic/osteogenic differentiation and proliferation function. Especially for the regulation of odontoblastic/osteogenic differentiation, we have verified this mechanism at the molecular level and characterization leveland this regulation also provides new ideas for dental pulp tissue engineering. At the same time, more than 3000 proteins related to the change of Stathmin level were screened by transcriptome sequencing technology, which provided a possibility to further exploration of the regulation mechanism of Stathmin on various aspects of cell biological characteristics.


Assuntos
Diferenciação Celular , Proliferação de Células , Polpa Dentária/metabolismo , Odontoblastos/metabolismo , Osteogênese , Estatmina/metabolismo , Células-Tronco/metabolismo , Via de Sinalização Wnt , Polpa Dentária/citologia , Humanos , Odontoblastos/citologia , Células-Tronco/citologia , beta Catenina/metabolismo
20.
Int J Biol Markers ; 34(2): 108-116, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30966849

RESUMO

BACKGROUND: The prognostic value of Stathmin 1 (STMN1) in malignant solid tumors remains controversial. Thus, we conducted this meta-analysis to summarize the potential value of STMN1 as a biomarker for predicting overall survival in patients with solid tumor. METHODS: We systematically searched eligible studies in PubMed, Web of Science, and EMBASE from the establishment date of these databases to September 2018. Hazard ratio (HR) and its 95% confidence interval (CI) was used to assess the association between STMN1 expression and overall survival. RESULTS: A total of 25 studies with 4625 patients were included in this meta-analysis. Our combined results showed that high STMN1 expression was associated with poor overall survival in solid tumors (HR = 1.85, 95% CI 1.55, 2.21). In general, our subgroup and sensitivity analyses demonstrated that our combined results were stable and reliable. However, from the results of the subgroups we found that high STMN1 expression was not related to overall survival in colorectal cancer and endometrial cancer anymore, suggesting that much caution should be taken to interpret our combined result, and more studies with large sample sizes are required to further explore the prognostic value of STMN1 expression in the specific type of tumors, especially colorectal cancer and endometrial cancer. CONCLUSIONS: STMN1 could serve as a prognostic biomarker and could be developed as a valuable therapeutic target for patients with solid tumors. However, due to the limitations of the present meta-analysis, this conclusion should be taken with caution. Further studies adequately designed are required to confirm our findings.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias/mortalidade , Estatmina/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Prognóstico , Taxa de Sobrevida
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