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1.
Oncogene ; 36(17): 2355-2365, 2017 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-27775073

RESUMO

Triple-negative breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Triple-negative tumors often display activated Wnt/ß-catenin signaling and most have impaired p53 function. We studied the interplay between p53 loss and Wnt/ß-catenin signaling in stem cell function and tumorigenesis, by deleting p53 from the mammary epithelium of K5ΔNßcat mice displaying a constitutive activation of Wnt/ß-catenin signaling in basal cells. K5ΔNßcat transgenic mice present amplification of the basal stem cell pool and develop triple-negative mammary carcinomas. The loss of p53 in K5ΔNßcat mice led to an early expansion of mammary stem/progenitor cells and accelerated the formation of triple-negative tumors. In particular, p53-deficient tumors expressed high levels of integrins and extracellular matrix components and were enriched in cancer stem cells. They also overexpressed the tyrosine kinase receptor Met, a feature characteristic of human triple-negative breast tumors. The inhibition of Met kinase activity impaired tumorsphere formation, demonstrating the requirement of Met signaling for cancer stem cell growth in this model. Human basal-like breast cancers with predicted mutated p53 status had higher levels of MET expression than tumors with wild-type p53. These results connect p53 loss and ß-catenin activation to stem cell regulation and tumorigenesis in triple-negative cancer and highlight the role of Met signaling in maintaining cancer stem cell properties, revealing new cues for targeted therapies.


Assuntos
Células-Tronco Neoplásicas/patologia , Neoplasias de Mama Triplo Negativas/patologia , Proteína Supressora de Tumor p53/deficiência , Animais , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Feminino , Deleção de Genes , Camundongos , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética , Proteína Supressora de Tumor p53/genética , beta Catenina/metabolismo
2.
Oncogene ; 33(34): 4286-95, 2014 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-24077284

RESUMO

The constitutive activation of ß-catenin signaling in the mammary basal epithelial cell layer in transgenic K5ΔNßcat mice leads to basal-type tumor development. Integrins of the ß1 family and integrin-mediated signaling events have an important role in breast tumor growth and progression. We show here that the deletion of α3ß1 integrin, a major laminin receptor, from the basal layer of the mammary epithelium of K5ΔNßcat mice completely prevented the tumorigenesis induced by ß-catenin signaling. Moreover, the depletion of α3ß1 integrin from a spontaneously transformed mouse mammary basal epithelial cell line (MEC) prevented the cells from forming colonies in soft agar and greatly reduced tumor development in orthotopic grafts. Inhibition of the integrin signaling intermediates Rac1 or PAK1 (P21-activated Kinase 1) in MEC affected tumor cell growth in soft agar, whereas the expression of activated forms of these effectors in α3-depleted cells rescued the capacity of these cells to grow in non-adherent conditions. Similarly, the tumorigenic potential of α3-depleted cells was restored by the expression of activated PAK1, as assessed by orthotopic transplantation assay. In three-dimensional Matrigel culture, MEC survival and proliferation were affected by the depletion of α3ß1 integrin, which also significantly decreased the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK). Our data suggest that the activation of signaling cascades downstream from α3ß1 and involving the Rac1/PAK1 pathway, MAPK and JNK, promotes prosurvival and proproliferative signals required for the malignant growth of basal mammary epithelial cells, providing further insight into the molecular mechanisms underlying breast cancer initiation and progression.


Assuntos
Carcinogênese/metabolismo , Integrina alfa3beta1/metabolismo , Sistema de Sinalização das MAP Quinases , Neoplasias Mamárias Experimentais/metabolismo , Neoplasia de Células Basais/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transplante de Neoplasias , Neoplasia de Células Basais/patologia , Neuropeptídeos/metabolismo , Ativação Transcricional , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
3.
Mol Cell Biol ; 24(19): 8649-61, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15367683

RESUMO

Both beta-catenin and plakoglobin can stimulate the expression of Lef/Tcf target genes in vitro. beta-Catenin is known to associate with Lef/Tcf factors and to participate directly in transactivation in vivo, whereas the role of plakoglobin in transcriptional regulation has been less studied. To analyze the functions of plakoglobin in vivo, we generated transgenic mice expressing in the epidermis N-terminally truncated plakoglobin (DeltaN122-PG) lacking the glycogen synthase kinase 3beta phosphorylation sites and therefore protected against degradation (transgenic line K5-DeltaN122-PG). The expression of DeltaN122-PG led to the formation of additional hair germs, hyperplastic hair follicles, and noninvasive hair follicle tumors, a phenotype reminiscent of that induced by expression of N-terminally truncated beta-catenin. However, if expressed in beta-catenin-null epidermis, DeltaN122-PG did not induce new hair follicle germs and follicular tumors. Thus, DeltaN122-PG cannot substitute for beta-catenin in its signaling functions in vivo and the phenotype observed in K5-DeltaN122-PG mouse skin must be due to the aberrant activation of beta-catenin signaling. On the other hand, the expression of DeltaN122-PG in beta-catenin-null skin significantly increased the survival rate of mutant mice, rescued differentiation, and limited excessive proliferation in the interfollicular epidermis, suggesting that plakoglobin may be involved in the intracellular signaling events essential for epidermal differentiation.


Assuntos
Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Epiderme/crescimento & desenvolvimento , Transativadores/metabolismo , Animais , Caderinas/metabolismo , Cistos/metabolismo , Proteínas do Citoesqueleto/genética , Desmoplaquinas , Epiderme/fisiologia , Genes Reporter , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/metabolismo , beta Catenina , gama Catenina
4.
J Biol Chem ; 276(32): 30350-8, 2001 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-11402039

RESUMO

In amphibians and birds, one of the first steps of neural crest cell (NCC) determination is expression of the transcription factor Slug. This marker has been used to demonstrate that BMP and Wnt molecules play a major role in NCC induction. However, it is unknown whether Slug expression is directly or indirectly regulated by these signals. We report here the cloning and characterization of three Xenopus Slug promoters: that of the Xenopus tropicalis slug gene and those of two Xenopus laevis Slug pseudoalleles. Although the three genes encode proteins with almost identical amino acid sequences and are expressed with similar spatiotemporal patterns, their 5'-flanking regions are quite different. A striking difference is a deletion in the X. tropicalis gene located precisely at the transcription initiation site that results in the X. tropicalis promoter being inefficient in X. laevis. Additionally, we identified two regions common to the three promoters that are necessary and sufficient to drive specific expression in NCCs. Interestingly, one of the common regulatory regions presents a functional Lef/beta-catenin-binding site necessary for specific expression. As the Lef.beta-catenin complex is a downstream effector of Wnt signaling, these results suggest that Xenopus Slug is a direct target of NCC determination signals.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Transativadores , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Alelos , Animais , Sítios de Ligação , Clonagem Molecular , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/química , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde , Hibridização In Situ , Íntrons , Proteínas Luminescentes/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Transdução de Sinais , Fatores de Transcrição da Família Snail , Fatores de Transcrição/biossíntese , Xenopus , Proteínas de Xenopus , beta Catenina
5.
EMBO Rep ; 2(5): 431-7, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11375936

RESUMO

Adhesion to extracellular matrix (ECM) induces intracellular signals that modulate cell proliferation, survival and differentiation. To study signalling events triggered by cell-ECM interactions in vivo we used transgenic mice exhibiting reduced mammary epithelial cell proliferation and increased apoptosis rates during the growth phase in pregnancy and lactation due to expression of a beta1-integrin dominant-negative mutant in the mammary gland epithelium. Here we show that ERK and JNK MAPKs were markedly less activated in lactating transgenic glands thereby accounting for the growth defects. The FAK pathway was not affected suggesting a mechanism of activation additional to the ECM signal. On the contrary, the significant decrease of Shc phosphorylation, Grb2 recruitment and the reduced phosphorylation level of Akt Thr308 and Akt substrates FKHR and Bad detected in transgenic glands show that activation of the Shc and the Akt pathways require intact cell-ECM interactions. These results provide an insight into the mechanisms of growth control by integrin-mediated adhesion that operate in vivo.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Células Epiteliais/fisiologia , Matriz Extracelular/metabolismo , Integrina beta1/metabolismo , Lactação , Sistema de Sinalização das MAP Quinases/fisiologia , Glândulas Mamárias Animais/fisiologia , Proteínas Serina-Treonina Quinases , Animais , Apoptose , Ativação Enzimática , Células Epiteliais/citologia , Feminino , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Marcação In Situ das Extremidades Cortadas , Integrina beta1/genética , Proteínas Quinases JNK Ativadas por Mitógeno , Glândulas Mamárias Animais/citologia , Camundongos , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Testes de Precipitina , Gravidez , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src
6.
Adv Exp Med Biol ; 480: 169-74, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10959424

RESUMO

To study the role of beta 1-integrins in mammary gland development we have generated transgenic mice expressing a dominant negative mutant of the beta 1-integrin chain in the mammary epithelium. The transgenic glands presented a delayed development in pregnancy and lactation due to decreased epithelial cell proliferation and increased apoptosis, whereas at the beginning of lactation, expression of milk proteins, WAP and beta-casein was diminished. In correlation with transgene expression, the basement membrane component, laminin, and the beta 4 integrin were accumulated at the lateral surface of luminal epithelial cells, revealing defects in polarization. Our data show that beta 1-integrins are involved in vivo in the control of proliferation, apoptosis, differentiation, and maintenance of baso-apical polarity of mammary epithelium.


Assuntos
Integrina beta1/fisiologia , Glândulas Mamárias Animais , Animais , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Polaridade Celular/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Lactação/fisiologia , Glândulas Mamárias Animais/embriologia , Glândulas Mamárias Animais/fisiologia , Camundongos , Camundongos Transgênicos , Gravidez
7.
J Cell Sci ; 112 ( Pt 7): 1035-44, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10198285

RESUMO

The mammary epithelium is composed of a luminal epithelium and a basal layer containing myoepithelial cells and undifferentiated precursors. Basal cells express specific protein markers, such as keratin 14 (K14) and P-cadherin. To study the factors that regulate the basal mammary epithelial cell phenotype, we have established two clonal derivatives of the mouse HC11 cell line, BC20 and BC44, expressing high levels of K14 and P-cadherin. Unlike the parental HC11 cells, these basal cells did not produce beta-casein in response to lactogenic hormone treatment; however their phenotype appeared to be plastic. Cultured in EGF-free medium, they exhibited enhanced cell-extracellular matrix adhesions and deficient cell-cell junctions, whereas long-term treatment with EGF induced a decrease of focal contact number and establishment of cell-cell junctions, resulting in downregulation of K14 and P-cadherin expression at the protein and mRNA levels. To determine whether cell-extracellular matrix interactions mediated by integrins have a role in the regulation of the expression of K14 and P-cadherin, the amounts of transcripts for the two proteins were analysed in the basal cells, which were plated on the function-blocking antibodies against beta1 and alpha6 integrin chains, on fibronectin and on laminin 5. The amount of P-cadherin transcript was 2- to 4-fold higher in cells plated on the function-blocking anti-integrin antibodies and on the extracellular matrix proteins, as compared to cells plated on poly-L-lysine, whereas the K14 transcript levels were not significantly modified in response to adhesion. The data demonstrate that integrin-mediated cell interaction with extracellular matrix is directly implicated in the control of P-cadherin expression, and that EGF and cell-extracellular matrix adhesion events are important regulators of the basal mammary epithelial cell phenotype.


Assuntos
Fator de Crescimento Epidérmico/fisiologia , Células Epiteliais/metabolismo , Matriz Extracelular/fisiologia , Glândulas Mamárias Animais/metabolismo , Transativadores , Animais , Northern Blotting , Caderinas/metabolismo , Caseínas/metabolismo , Adesão Celular , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Desmoplaquinas , Dexametasona/farmacologia , Citometria de Fluxo , Immunoblotting , Insulina/farmacologia , Integrinas/fisiologia , Queratina-14 , Queratinas/metabolismo , Camundongos , Microscopia de Fluorescência , Fenótipo , Prolactina/farmacologia , Regulação para Cima , alfa Catenina , beta Catenina
8.
EMBO J ; 17(8): 2139-47, 1998 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-9545227

RESUMO

The expression of a transgene coding for a chimeric molecule, containing the cytoplasmic and transmembrane domains of the beta1-integrin chain and the extracellular domain of the T-cell differentiation antigen CD4, was targeted to the mouse mammary gland by the mouse mammary tumor virus (MMTV) promoter. The chimera does not interact with the extracellular ligands; however, its expression in cultured cells was shown to interfere with focal adhesion kinase (FAK) phosphorylation following ligation of endogenous beta1-integrin. Therefore, expression of the transgenic protein on the cell surface should uncouple adhesion from intracellular events associated with the beta1-cytoplasmic domain and thus perturb beta1-integrin functions. Although most of the transgenic females were able to lactate, their mammary glands had a phenotype clearly distinct from that of wild-type mice. At mid-pregnancy and the beginning of lactation, transgenic glands were underdeveloped and the epithelial cell proliferation rates were decreased, while the apoptosis levels were higher than in wild-type glands. In lactation, the amounts of the whey acidic protein (WAP) and beta-casein gene transcripts were diminished, and the basement membrane component, laminin and the beta4-integrin chain accumulated at the lateral surface of luminal epithelial cells, revealing defects in polarization. Our observations prove that in vivo, beta1-integrins are involved in control of proliferation, apoptosis, differentiation and maintenance of baso-apical polarity of mammary epithelial cells, and therefore are essential for normal mammary gland development and function.


Assuntos
Integrina beta1/fisiologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Animais , Apoptose , Antígenos CD4/genética , Diferenciação Celular , Polaridade Celular , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Integrina beta1/genética , Lactação , Glândulas Mamárias Animais/anatomia & histologia , Glândulas Mamárias Animais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia
9.
J Bacteriol ; 174(22): 7458-62, 1992 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1429468

RESUMO

The nucleotide sequence of the slpA gene, which is responsible for the synthesis of the S-layer protein of Thermus thermophilus HB8, is described. This gene is transcribed as a unit in which the coding region is preceded by a 127-base-long leader mRNA sequence. The promoter region is also recognized by the RNA polymerase of Escherichia coli because of the presence of homologous -35 and -10 boxes. Homologies with other promoters from Thermus spp. are also presented.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias , Escherichia coli/genética , Genes Bacterianos , Regiões Promotoras Genéticas , Thermus thermophilus/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon/genética , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica
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