RESUMO
Mammalian NUMB is alternatively spliced generating four isoforms NUMB1-NUMB4 that can function as tumor suppressors. NUMB1-NUMB4 proteins, which normally determine how different cell types develop, are reduced in 21% of primary breast tumors. Our previous work has, however, indicated that two novel NUMB isoforms, NUMB5 and NUMB6 have the pro-oncogenic functions. Herein, we address a novel function of human NUMB isoform 6 (NUMB6) in promoting cancer cell migration and invasion. We found that NUMB6 induced expression of embryonic transcription factor Slug, which in turn actively repressed E-cadherin, prompting cells to undergo epithelial-mesenchymal transition (EMT). Low-metastatic breast cancer cells DB-7 stably expressing NUMB6, lost their epithelial phenotype, exhibited migratory and pro-invasive behavior, and ultimately elevated expression of mesenchymal markers. Among these markers, increased vimentin, ß-catenin, and fibronectin expression elicited metalloproteinase 9 (MMP9) production. Our results revealed that NUMB6-DB-7 cells have significantly increased level of Akt1 and Akt2 phosphorylation. Therefore, antagonizing Akt signaling using a chemical inhibitor LY294002, we found that NUMB6-induced Slug expression was reduced, and ultimately accompanied with decreased cell migration and invasion. In summary, this study identified a novel molecular determinant of breast cancer progression, uncovering a potential oncogenic role for the NUMB6 protein in cancer cell migration and invasion, coupled to the maintenance of mesenchymal-like cells. J. Cell. Biochem. 118: 237-251, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas de Membrana/genética , Invasividade Neoplásica , Células-Tronco Neoplásicas/patologia , Proteínas do Tecido Nervoso/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Bone morphogenetic signaling (BMP) is a key pathway during neurogenesis and depends on many downstream intermediators to carry out its signaling. One such signaling pathway utilizes neurotrophin receptor-interacting MAGE protein (NRAGE), a member of the melanoma-associated antigen (MAGE) family, to upregulate p38 mitogen activated protein kinase (p38(MAPK)) in response to cellular stress and activate caspases which are critical in leading cells to death. NRAGE consists of two conserved MAGE homology domains separated by a unique hexapeptide repeat domain. Although we have previously implicated NRAGE in inducing apoptosis in neural progenitors and P19 cells, a model system for neural progenitors, its domains have yet to be explored in determining which one may be responsible for setting up the signaling for apoptosis. Here, we overexpressed a series of deletion mutations in P19 cells to show that only those with at least half of the repeat domain, activated p38(MAPK) and underwent apoptosis offering intriguing incite into NRAGE's contribution in BMP apoptotic signaling.
Assuntos
Apoptose , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Neoplasias/química , Transdução de Sinais , Animais , Proteínas Morfogenéticas Ósseas/genética , Linhagem Celular , Sobrevivência Celular , Feminino , Humanos , Camundongos , Camundongos Endogâmicos ICR , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estrutura Terciária de Proteína , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Cellular interactions in development of the kidney are used as a model of reciprocal inductive events between epithelium and mesenchyme. Time- and labor-intensive methods have been developed to study this phenomenon. For example, in mice, the targeted disruption of genes in vivo has been used to modify the genetic program directing kidney development. However, gene targeting is a resource-intensive approach and alternative strategies for gene and protein modification in the kidney need to be developed. Herein, we have developed an efficient system for the delivery of antisense morpholino to alter normal protein expression. We describe the use of Endo-Porter to effectively deliver morpholinos to all parts and regions of the kidney explant. Also, we definitively show via confocal microscopy and Western blot analysis that the use of Endo-Porter in delivering antisense morpholinos is robust throughout the entire kidney explant, providing efficient suppression of protein expression. This method saves time and cost when compared with targeted disruption and is an improvement upon previous kidney organ culture methods.
Assuntos
Sistemas de Liberação de Medicamentos , Rim/citologia , Peptídeos , Animais , Fluoresceína/metabolismo , Corantes Fluorescentes/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Modelos Biológicos , Proteínas de Neoplasias/antagonistas & inibidores , Oligonucleotídeos Antissenso/química , Técnicas de Cultura de Órgãos , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
In normal growth and development, apoptosis is necessary to shape the central nervous system and to eliminate excess neurons which are not required for innervation. In some diseases, however, apoptosis can be either overactive as in some neurodegenerative disorders or severely attenuated as in the spread of certain cancers. Bone morphogenetic proteins (BMPs) transmit signals for regulating cell growth, differentiation, and apoptosis. Responding to BMP receptors stimulated from BMP ligands, neurotrophin receptor-mediated MAGE homolog (NRAGE) binds and functions with the XIAP-TAK1-TAB1 complex to activate p38(MAPK) and induces apoptosis in cortical neural progenitors. NRAGE contains a unique repeat domain that is only found in human, mouse, and rat homologs that we theorize is pivotal in its BMP MAPK role. Previously, we showed that deletion of the repeat domain inhibits apoptosis, p38(MAPK) phosphorylation, and caspase-3 cleavage in P19 neural progenitor cells. We also showed that the XIAP-TAB1-TAK1 complex is dependent on NRAGE for IKK-α/ß phosphorylation and NF-κB activation. XIAP is a major inhibitor of caspases, the main executioners of apoptosis. Although it has been shown previously that NRAGE binds to the RING domain of XIAP, it has not been determined which NRAGE domain binds to XIAP. Here, we used fluorescence resonance energy transfer (FRET) to determine that there is a strong likelihood of a direct interaction between NRAGE and XIAP occurring at NRAGE's unique repeat domain which we also attribute to be the domain responsible for downstream signaling of NF-κB and activating IKK subunits. From these results, we designed a small peptide modeled after the NRAGE repeat domain which we have determined inhibits NF-κB activation and apoptosis in P19 cells. These intriguing results illustrate that the paradigm of the NRAGE repeat domain may hold promising therapeutic strategies in developing pharmaceutical solutions for combating harmful diseases involving excessive downstream BMP signaling, including apoptosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/efeitos dos fármacos , MAP Quinase Quinase Quinases/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/química , Peptídeos/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Compartimento Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Microscopia Confocal , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Sequências Repetitivas de Aminoácidos , Transdução de Sinais/efeitos dos fármacosRESUMO
We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif) GTPase activating protein 1). Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5) and filopodia (NUMB6) in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer.
Assuntos
Desenvolvimento Fetal/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/genética , Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Transdução de Sinais/fisiologia , Animais , Western Blotting , Movimento Celular/genética , Polaridade Celular/genética , Embrião de Galinha , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais , Neurogênese/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Branching morphogenesis is a developmental process characteristic of many organ systems. Specifically, during renal branching morphogenesis, its been postulated that the final number of nephrons formed is one key clinical factor in the development of hypertension in adulthood. As it has been established that BMPs regulate, in part, renal activity of p38 MAP kinase (p38(MAPK)) and it has demonstrated that the cytoplasmic protein Neurotrophin Receptor MAGE homologue (NRAGE) augments p38(MAPK) activation, it was hypothesized that a decrease in the expression of NRAGE during renal branching would result in decreased branching of the UB that correlated with changes in p38(MAPK) activation. To verify this, the expression of NRAGE was reduced in ex vivo kidney explants cultures using antisense morpholino. Morpholino treated ex vivo kidney explants expression were severely stunted in branching, a trait that was rescued with the addition of exogenous GDNF. Renal explants also demonstrated a precipitous drop in p38(MAPK) activation that too was reversed in the presence of recombinant GDNF. RNA profiling of NRAGE diminished ex vivo kidney explants resulted in altered expression of GDNF, Ret, BMP7 and BMPRIb mRNAs. Our results suggested that in early kidney development NRAGE might have multiple roles during renal branching morphogenesis through association with both the BMP and GDNF signaling pathways.