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1.
Proc Natl Acad Sci U S A ; 114(5): E707-E716, 2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28096336

RESUMO

Aberrant activation of ß-catenin through its activity as a transcription factor has been observed in a large proportion of human malignancies. Despite the improved understanding of the ß-catenin signaling pathway over the past three decades, attempts to develop therapies targeting ß-catenin remain challenging, and none of these targeted therapies have advanced to the clinic. In this study, we show that part of the challenge in antagonizing ß-catenin is caused by its dual functionality as a cell adhesion molecule and a signaling molecule. In a mouse model of basal ErbB2 receptor tyrosine kinase 2 (ErbB2)-positive breast cancer (ErbB2KI), which exhibits aberrant ß-catenin nuclear signaling, ß-catenin haploinsufficiency induced aggressive tumor formation and metastasis by promoting the disruption of adherens junctions, dedifferentiation, and an epithelial to mesenchymal transition (EMT) transcriptional program. In contrast to the accelerated tumor onset observed in the haploid-insufficient ErbB2 tumors, deletion of both ß-catenin alleles in the ErbB2KI model had only a minor impact on tumor onset that further correlated with the retention of normal adherens junctions. We further showed that retention of adherens junctional integrity was caused by the up-regulation of the closely related family member plakoglobin (γ-catenin) that maintained both adherens junctions and the activation of Wnt target genes. In contrast to the ErbB2KI basal tumor model, modulation of ß-catenin levels had no appreciable impact on tumor onset in an ErbB2-driven model of luminal breast cancer [murine mammary tumor virus promoter (MMTV-NIC)]. These observations argue that the balance of junctional and nuclear ß-catenin activity has a profound impact on tumor progression in this basal model of ErbB2-positive breast cancer.


Assuntos
Neoplasias Mamárias Experimentais/patologia , Receptor ErbB-2/metabolismo , beta Catenina/genética , Animais , Transição Epitelial-Mesenquimal , Feminino , Haploinsuficiência , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos Transgênicos , RNA Interferente Pequeno/genética , Receptor ErbB-2/genética , Transdução de Sinais , Células Tumorais Cultivadas , gama Catenina/genética
2.
Nat Rev Cancer ; 7(5): 389-97, 2007 05.
Artigo em Inglês | MEDLINE | ID: mdl-17446858

RESUMO

One-third of patients with breast cancer overexpress the ERBB2 receptor tyrosine kinase, which is associated not only with a more aggressive phenotype but also reduced responsiveness to hormonal therapies. Over the past two decades, many ERBB2 mouse models for breast cancer have conclusively shown that this receptor has a causal role in breast cancer development. These mouse models have also enabled the mechanisms controlling tumour growth, angiogenesis, metastasis, dormancy and recurrence in ERBB2-positive breast cancer to be elucidated. In addition, a mouse model has recently been described that accurately recapitulates many of the hallmarks associated with the early stages of the human disease.


Assuntos
Neoplasias da Mama/etiologia , Modelos Animais de Doenças , Neoplasias Mamárias Experimentais/etiologia , Receptor ErbB-2/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Amplificação de Genes , Genes erbB-2 , Instabilidade Genômica , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Dados de Sequência Molecular , Mutação
3.
Mol Cell Proteomics ; 11(9): 710-23, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22665516

RESUMO

Chaperones and foldases in the endoplasmic reticulum (ER) ensure correct protein folding. Extensive protein-protein interaction maps have defined the organization and function of many cellular complexes, but ER complexes are under-represented. Consequently, chaperone and foldase networks in the ER are largely uncharacterized. Using complementary ER-specific methods, we have mapped interactions between ER-lumenal chaperones and foldases and describe their organization in multiprotein complexes. We identify new functional chaperone modules, including interactions between protein-disulfide isomerases and peptidyl-prolyl cis-trans-isomerases. We have examined in detail a novel ERp72-cyclophilin B complex that enhances the rate of folding of immunoglobulin G. Deletion analysis and NMR reveal a conserved surface of cyclophilin B that interacts with polyacidic stretches of ERp72 and GRp94. Mutagenesis within this highly charged surface region abrogates interactions with its chaperone partners and reveals a new mechanism of ER protein-protein interaction. This ability of cyclophilin B to interact with different partners using the same molecular surface suggests that ER-chaperone/foldase partnerships may switch depending on the needs of different substrates, illustrating the flexibility of multichaperone complexes of the ER folding machinery.


Assuntos
Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Mapas de Interação de Proteínas , Animais , Ciclofilinas/metabolismo , Células Epiteliais , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Imunoglobulina G/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/química , Peptidilprolil Isomerase/metabolismo , Ratos
4.
Cancer Res ; 67(16): 7579-88, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17699761

RESUMO

ErbB-2 overexpression and amplification occurs in 15% to 30% of human invasive breast carcinomas associated with poor clinical prognosis. Previously, we have shown that four ErbB-2/Neu tyrosine-autophosphorylation sites within the cytoplasmic tail of the receptor recruit distinct adaptor proteins and are sufficient to mediate transforming signals in vitro. Two of these sites, representing the growth factor receptor binding protein 2 (Grb2; Neu-YB) and the Src homology and collagen (Shc; Neu-YD) binding sites, can induce mammary tumorigenesis and metastasis. Here, we show that transgenic mice bearing the two other ErbB-2 autophosphorylation sites (Neu-YC and Neu-YE) develop metastatic mammary tumors. A detailed comparison of biological profiles among all Neu mutant mouse models revealed that Neu-YC, Neu-YD, and Neu-YE mammary tumors shared similar pathologic and transcriptional features. By contrast, the Neu-YB mouse model displayed a unique pathology with a high metastatic potential that correlates with a distinct transcriptional profile, including genes that promote malignant tumor progression such as metalloproteinases and chemokines. Furthermore, Neu-YB tumor epithelial cells showed abundant intracellular protein level of the chemokine CXCL12/SDF-1alpha, which may reflect the aggressive nature of this Neu mutant mouse model. Taken together, these findings indicate that activation of distinct Neu-coupled signaling pathways has an important impact on the biological behavior of Neu-induced tumors.


Assuntos
Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sítios de Ligação , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Quimiocina CXCL12 , Quimiocinas CXC/biossíntese , Quimiocinas CXC/genética , Ativação Enzimática , Proteína Adaptadora GRB2/metabolismo , Perfilação da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Transcrição Gênica
5.
Mol Biol Cell ; 15(12): 5492-502, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15483057

RESUMO

We have determined the transcriptional response of the budding yeast Saccharomyces cerevisiae to cold. Yeast cells were exposed to 10 degrees C for different lengths of time, and DNA microarrays were used to characterize the changes in transcript abundance. Two distinct groups of transcriptionally modulated genes were identified and defined as the early cold response and the late cold response. A detailed comparison of the cold response with various environmental stress responses revealed a substantial overlap between environmental stress response genes and late cold response genes. In addition, the accumulation of the carbohydrate reserves trehalose and glycogen is induced during late cold response. These observations suggest that the environmental stress response (ESR) occurs during the late cold response. The transcriptional activators Msn2p and Msn4p are involved in the induction of genes common to many stress responses, and we show that they mediate the stress response pattern observed during the late cold response. In contrast, classical markers of the ESR were absent during the early cold response, and the transcriptional response of the early cold response genes was Msn2p/Msn4p independent. This implies that the cold-specific early response is mediated by a different and as yet uncharacterized regulatory mechanism.


Assuntos
Adaptação Fisiológica/genética , Temperatura Baixa , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Metabolismo dos Carboidratos , Análise por Conglomerados , Genes Fúngicos/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , Fatores de Tempo , Transcrição Gênica/genética
6.
Gene ; 344: 43-51, 2005 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-15656971

RESUMO

We have developed a set of vectors that have enhanced capabilities for efficiently constructing and expressing differentially tagged fusion proteins using Drag&Drop cloning in the yeast Saccharomyces cerevisiae. The pGREG vectors are based on the pRS series with an additional general kanR selection marker. In vivo homologous recombination is used to introduce genes of interest into galactose-inducible expression vectors (pGREGs), permitting the formation of amino-terminal fusions. The vectors all contain common regions for recombination that flank the stuffer fragment. Introduction of common recombination sequences at the end of PCR fragments will permit the cloning of genes without the need for specific restriction sites. In this process, the selectable stuffer HIS3 gene is replaced by successful gene integration, and a screen for loss of the selection marker identifies potential recombinants. Due to the modular structure of the vectors, genes introduced into one vector can be readily transferred by in vivo recombination to all other members of the vector system, thus permitting rapid and easy Drag&Drop construction of a series of tagged proteins. The pGREG series combines features for expression, tagging, integration, localization and library construction with the advantage of obtaining immediate results from sub-sequent experiments. This Drag&Drop system also allows efficient cloning and expression of heterologous genes in large-scale experiments.


Assuntos
Clonagem Molecular/métodos , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Regulação Fúngica da Expressão Gênica , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
7.
Mol Cancer Ther ; 13(8): 2030-9, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24994770

RESUMO

Upregulation of HER2 is a hallmark of 20% to 30% of invasive breast cancers, rendering this receptor an attractive target for cancer therapy. Although HER2-targeting agents have provided substantial clinical benefit as cancer therapeutics, there is a need for the development of new agents aiming at circumventing anti-HER2 resistance. On the basis of the approved antibody pertuzumab, we have created a panel of bispecific FynomAbs, which target two epitopes on HER2. FynomAbs are fusion proteins of an antibody and a Fyn SH3-derived binding protein. One bispecific FynomAb, COVA208, was characterized in detail and showed a remarkable ability to induce rapid HER2 internalization and apoptosis in vitro. Moreover, it elicited a strong inhibition of downstream HER2 signaling by reducing HER2, HER3, and EGFR levels in vitro and in vivo. Importantly, COVA208 demonstrated superior activity in four different xenograft models as compared with the approved antibodies trastuzumab and pertuzumab. The bispecific FynomAb COVA208 has the potential to enhance the clinical efficacy and expand the scope of HER2-directed therapies, and delineates a paradigm for designing a new class of antibody-based therapeutics for other receptor targets.


Assuntos
Anticorpos/farmacologia , Antineoplásicos/farmacologia , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Animais , Apoptose , Proliferação de Células , Humanos , Células MCF-7 , Camundongos Endogâmicos C57BL , Transporte Proteico , Receptor ErbB-2/imunologia , Receptor ErbB-3/metabolismo , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Cancer Res ; 73(14): 4474-87, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23720052

RESUMO

Although ERBB2 amplification and overexpression is correlated with poor outcome in breast cancer, the molecular mechanisms underlying the aggressive nature of these tumors has not been fully elucidated. To investigate this further, we have used a transgenic mouse model of ErbB2-driven tumor progression (ErbB2(KI) model) that recapitulates clinically relevant events, including selective amplification of the core erbB2 amplicon. By comparing the transcriptional profiles of ErbB2(KI) mammary tumors and human ERBB2-positive breast cancers, we show that ErbB2(KI) tumors possess molecular features of the basal subtype of ERBB2-positive human breast cancer, including activation of canonical ß-catenin signaling. Inhibition of ß-catenin-dependent signaling in ErbB2(KI)-derived tumor cells using RNA interference impaired tumor initiation and metastasis. Furthermore, treatment of ErbB2(KI) or human ERBB2-overexpressing tumor cells with a selective ß-catenin/CBP inhibitor significantly decreased proliferation and ErbB2 expression. Collectively, our data indicate that ERBB2-mediated breast cancer progression requires ß-catenin signaling and can be therapeutically targeted by selective ß-catenin/CBP inhibitors.


Assuntos
Neoplasias da Mama/patologia , Neoplasias Mamárias Experimentais/patologia , Receptor ErbB-2/metabolismo , beta Catenina/metabolismo , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Receptor ErbB-2/genética , Transdução de Sinais , Transcrição Gênica , beta Catenina/genética
9.
Cancer Res ; 70(20): 7776-87, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20924104

RESUMO

Using transgenic mouse models of breast cancer that ablate Src homology and collagen A (ShcA) expression or oncogene-coupled ShcA signaling, we previously showed that this adaptor is critical for mammary tumor onset and progression. We now provide the first evidence that ShcA regulates mammary tumorigenesis, in part, through its ability to regulate the adaptive immune response. Inactivation of ShcA signaling within tumor cells results in extensive CD4(+) T-cell infiltration and induction of a humoral immune response in mammary tumors. This is associated with a robust CTL response in preneoplastic lesions that are deficient in ShcA signaling. Moreover, mammary tumor progression of ShcA-deficient hyperplasias is accelerated in a T cell-deficient background. We also uncover a clinically relevant correlation between high ShcA expression and low CTL infiltration in human breast cancers. Finally, we define a novel ShcA-regulated immune signature that functions as an independent prognostic marker of survival in human epidermal growth factor receptor 2(+) and basal breast cancers. We reveal a novel role for tumor cell-derived ShcA in the establishment and maintenance of an immunosuppressive state.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Receptores Proteína Tirosina Quinases/fisiologia , Proteínas Adaptadoras da Sinalização Shc/genética , Animais , Neoplasias da Mama/imunologia , Linfócitos T CD4-Positivos/patologia , Feminino , Humanos , Terapia de Imunossupressão , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Paridade , Gravidez , Modelos de Riscos Proporcionais , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Proteínas Adaptadoras da Sinalização Shc/deficiência , Proteínas Adaptadoras da Sinalização Shc/fisiologia , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/patologia , Resultado do Tratamento
10.
J Biol Chem ; 284(28): 19018-26, 2009 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-19435886

RESUMO

Overexpression and/or amplification of the ErbB-2 oncogene as well as inactivation of the PTEN tumor suppressor are two important genetic events in human breast carcinogenesis. To address the biological impact of conditional inactivation of PTEN on ErbB-2-induced mammary tumorigenesis, we generated a novel transgenic mouse model that utilizes the murine mammary tumor virus (MMTV) promoter to directly couple expression of activated ErbB-2 and Cre recombinase to the same mammary epithelial cell (MMTV-NIC). Disruption of PTEN in the mammary epithelium of the MMTV-NIC model system dramatically accelerated the formation of multifocal and highly metastatic mammary tumors, which exhibited homogenous pathology. PTEN-deficient/NIC-induced tumorigenesis was associated with an increase in angiogenesis. Moreover, inactivation of PTEN in the MMTV-NIC mouse model resulted in hyperactivation of the phosphatidylinositol 3'-kinase/Akt signaling pathway. However, like the parental strain, tumors obtained from PTEN-deficient/NIC mice displayed histopathological and molecular features of the luminal subtype of primary human breast cancer. Taken together, our findings provide important implications in understanding the molecular determinants of mammary tumorigenesis driven by PTEN deficiency and ErbB-2 activation and could provide a valuable tool for testing the efficacy of therapeutic strategies that target these critical signaling pathways.


Assuntos
Neoplasias da Mama/metabolismo , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/fisiologia , Receptor ErbB-2/fisiologia , Animais , Neoplasias da Mama/patologia , Feminino , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Metástase Neoplásica , Neovascularização Patológica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
11.
Cancer Res ; 68(7): 2122-31, 2008 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-18381417

RESUMO

Loss of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and amplification or elevated expression of ErbB-2 are both involved in human breast cancer. To directly test the importance of these genetic events in mammary tumorigenesis, we have assessed whether mammary-specific disruption of PTEN could cooperate with activation of ErbB-2. Transgenic mice expressing ErbB-2 under the transcriptional control of its endogenous promoter (ErbB-2(KI)) were interbred with mice carrying conditional PTEN alleles and an MMTV/Cre transgene. Loss of one or both PTEN alleles resulted in a dramatic acceleration of mammary tumor onset and an increased occurrence of lung metastases in the ErbB-2(KI) strain. Tumor progression in PTEN-deficient/ErbB-2(KI) strains was associated with elevated ErbB-2 protein levels, which were not due to ErbB-2 amplification or to a dramatic increase in ErbB-2 transcripts. Moreover, the PTEN-deficient/ErbB-2(KI)-derived mouse mammary tumors display striking morphologic heterogeneity in comparison with the homogeneous pathology of the ErbB-2(KI) parental strain. Therefore, inactivation of PTEN would not only have a dramatic effect on ErbB-2-induced mammary tumorigenesis but would also lead to the formation of mammary tumors that, in part, display pathologic and molecular features associated with the basal-like subtype of primary human breast cancer.


Assuntos
Neoplasias Mamárias Experimentais/genética , PTEN Fosfo-Hidrolase/genética , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Cromossomos de Mamíferos , Modelos Animais de Doenças , Amplificação de Genes , Deleção de Genes , Humanos , Perda de Heterozigosidade , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , PTEN Fosfo-Hidrolase/deficiência , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética
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