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1.
Proc Natl Acad Sci U S A ; 118(34)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34408016

RESUMO

During malignant progression, epithelial cancer cells dissolve their cell-cell adhesion and gain invasive features. By virtue of its dual function, ß-catenin contributes to cadherin-mediated cell-cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bcl9 and Pygopus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of ß-catenin's transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)-PyMT mouse model of metastatic breast cancer, we compared the complete elimination of ß-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of ß-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of ß-catenin's transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial-mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by ß-catenin's transcriptional activities upon stimulation with Wnt3a or during TGF-ß-induced EMT. Our results uncouple the signaling from the adhesion function of ß-catenin and underline the importance of Wnt/ß-catenin-dependent transcription in malignant tumor progression of breast cancer.


Assuntos
Adesão Celular/fisiologia , Neoplasias Mamárias Animais/metabolismo , Transdução de Sinais/fisiologia , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo , Animais , Apoptose , Ciclo Celular , Movimento Celular , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Mamárias Animais/genética , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Metástase Neoplásica , Transcriptoma , Fator de Crescimento Transformador beta/farmacologia , Proteína Wnt3A/genética , beta Catenina/genética
2.
Br J Cancer ; 125(2): 164-175, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33824479

RESUMO

Heterogeneity within a tumour increases its ability to adapt to constantly changing constraints, but adversely affects a patient's prognosis, therapy response and clinical outcome. Intratumoural heterogeneity results from a combination of extrinsic factors from the tumour microenvironment and intrinsic parameters from the cancer cells themselves, including their genetic, epigenetic and transcriptomic traits, their ability to proliferate, migrate and invade, and their stemness and plasticity attributes. Cell plasticity constitutes the ability of cancer cells to rapidly reprogramme their gene expression repertoire, to change their behaviour and identities, and to adapt to microenvironmental cues. These features also directly contribute to tumour heterogeneity and are critical for malignant tumour progression. In this article, we use breast cancer as an example of the origins of tumour heterogeneity (in particular, the mutational spectrum and clonal evolution of progressing tumours) and of tumour cell plasticity (in particular, that shown by tumour cells undergoing epithelial-to-mesenchymal transition), as well as considering interclonal cooperativity and cell plasticity as sources of cancer cell heterogeneity. We review current knowledge on the functional contribution of cell plasticity and tumour heterogeneity to malignant tumour progression, metastasis formation and therapy resistance.


Assuntos
Neoplasias da Mama/patologia , Redes Reguladoras de Genes , Heterogeneidade Genética , Neoplasias da Mama/genética , Plasticidade Celular , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos
3.
J Mammary Gland Biol Neoplasia ; 24(1): 39-45, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30209717

RESUMO

Genetically engineered mouse models have become an indispensable tool for breast cancer research. Combination of multiple site-specific recombination systems such as Cre/loxP and Flippase (Flp)/Frt allows for engineering of sophisticated, multi-layered conditional mouse models. Here, we report the generation and characterization of a novel transgenic mouse line expressing a mouse codon-optimized Flp under the control of the mouse mammary tumor virus (MMTV) promoter. These mice show robust Flp-mediated recombination in luminal mammary gland and breast cancer cells but no Flp activity in non-mammary tissues, with the exception of limited activity in salivary glands. These mice provide a unique tool for studying mammary gland biology and carcinogenesis in mice.


Assuntos
Carcinogênese/genética , DNA Nucleotidiltransferases/genética , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/genética , Vírus do Tumor Mamário do Camundongo/genética , Animais , Carcinogênese/patologia , Progressão da Doença , Células Epiteliais/patologia , Feminino , Genes Reporter/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteínas Luminescentes/genética , Glândulas Mamárias Animais/citologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Microinjeções , Regiões Promotoras Genéticas/genética , Recombinação Genética , Glândulas Salivares/patologia , Microambiente Tumoral/genética , Proteína Vermelha Fluorescente
4.
Life Sci Alliance ; 5(2)2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34819356

RESUMO

In melanoma, a switch from a proliferative melanocytic to an invasive mesenchymal phenotype is based on dramatic transcriptional reprogramming which involves complex interactions between a variety of signaling pathways and their downstream transcriptional regulators. TGFß/SMAD, Hippo/YAP/TAZ, and Wnt/ß-catenin signaling pathways are major inducers of transcriptional reprogramming and converge at several levels. Here, we report that TGFß/SMAD, YAP/TAZ, and ß-catenin are all required for a proliferative-to-invasive phenotype switch. Loss and gain of function experimentation, global gene expression analysis, and computational nested effects models revealed the hierarchy between these signaling pathways and identified shared target genes. SMAD-mediated transcription at the top of the hierarchy leads to the activation of YAP/TAZ and of ß-catenin, with YAP/TAZ governing an essential subprogram of TGFß-induced phenotype switching. Wnt/ß-catenin signaling is situated further downstream and exerts a dual role: it promotes the proliferative, differentiated melanoma cell phenotype and it is essential but not sufficient for SMAD or YAP/TAZ-induced phenotype switching. The results identify epistatic interactions among the signaling pathways underlying melanoma phenotype switching and highlight the priorities in targets for melanoma therapy.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Melanoma/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Fatores de Transcrição/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Biomarcadores , Biomarcadores Tumorais , Proliferação de Células , Biologia Computacional , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Via de Sinalização Hippo , Humanos , Melanoma/etiologia , Melanoma/patologia , Modelos Biológicos , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Fenótipo , Interferência de RNA , RNA Interferente Pequeno/genética , Via de Sinalização Wnt
5.
STAR Protoc ; 3(2): 101438, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35707685

RESUMO

The various stages of epithelial-mesenchymal transition (EMT) generate phenotypically heterogeneous populations of cells. Here, we detail a dual recombinase lineage tracing system using a transgenic mouse model of metastatic breast cancer to trace and characterize breast cancer cells at different EMT stages. We describe analytical steps to label cancer cells at an early partial or a late full EMT state, followed by tracking their behavior in tumor slice cultures. We then characterize their transcriptome by five-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Luond et al. (2021).


Assuntos
Transição Epitelial-Mesenquimal , Neoplasias , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Camundongos , Camundongos Transgênicos , Transcriptoma
6.
Dev Cell ; 56(23): 3203-3221.e11, 2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34847378

RESUMO

Epithelial-mesenchymal transition (EMT) is a transient, reversible process of cell de-differentiation where cancer cells transit between various stages of an EMT continuum, including epithelial, partial EMT, and mesenchymal cell states. We have employed Tamoxifen-inducible dual recombinase lineage tracing systems combined with live imaging and 5-cell RNA sequencing to track cancer cells undergoing partial or full EMT in the MMTV-PyMT mouse model of metastatic breast cancer. In primary tumors, cancer cells infrequently undergo EMT and mostly transition between epithelial and partial EMT states but rarely reach full EMT. Cells undergoing partial EMT contribute to lung metastasis and chemoresistance, whereas full EMT cells mostly retain a mesenchymal phenotype and fail to colonize the lungs. However, full EMT cancer cells are enriched in recurrent tumors upon chemotherapy. Hence, cancer cells in various stages of the EMT continuum differentially contribute to hallmarks of breast cancer malignancy, such as tumor invasion, metastasis, and chemoresistance.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/secundário , Animais , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Análise de Sequência de RNA , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Oncogene ; 40(1): 12-27, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33046799

RESUMO

Despite major progress in breast cancer research, the functional contribution of distinct cancer cell clones to malignant tumor progression and metastasis remains largely elusive. We have assessed clonal heterogeneity within individual primary tumors and metastases and also during the distinct stages of malignant tumor progression using clonal tracking of cancer cells in the MMTV-PyMT mouse model of metastatic breast cancer. Comparative gene expression analysis of clonal subpopulations reveals a substantial level of heterogeneity across and also within the various stages of breast carcinogenesis. The intra-stage heterogeneity is primarily manifested by differences in cell proliferation, also found within invasive carcinomas of luminal A-, luminal B-, and HER2-enriched human breast cancer. Surprisingly, in the mouse model of clonal tracing of cancer cells, chemotherapy mainly targets the slow-proliferative clonal populations and fails to efficiently repress the fast-proliferative populations. These insights may have considerable impact on therapy selection and response in breast cancer patients.


Assuntos
Neoplasias da Mama/patologia , Rastreamento de Células/métodos , Perfilação da Expressão Gênica/métodos , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/patogenicidade , Receptor ErbB-2/genética , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Evolução Clonal , Progressão da Doença , Feminino , Redes Reguladoras de Genes , Humanos , Microdissecção e Captura a Laser , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/virologia , Camundongos , Metástase Neoplásica , Estadiamento de Neoplasias , Análise de Sequência de RNA
8.
Dev Cell ; 48(4): 539-553.e6, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30713070

RESUMO

Epithelial-mesenchymal transition (EMT) enables cells to gain migratory and invasive features underlined by major transcriptional and epigenetic reprogramming. However, most studies have focused on the endpoints of the EMT process, and the epistatic hierarchy of the transcriptional networks underlying EMT has remained elusive. We have used a siRNA-based, functional high-content microscopy screen to identify 46 (co)transcription factors ((co)TFs) and 13 miRNAs critically required for EMT in normal murine mammary gland (NMuMG) cells. We compared dynamic gene expression during EMT kinetics and used functional perturbation of critical (co)TFs and miRNAs to identify groups and networks of EMT genes. Computational analysis as well as functional validation experiments revealed interaction networks between TFs and miRNAs and delineated the hierarchical and functional interactions of multiple EMT regulatory networks in NMuMG cells.


Assuntos
Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Transição Epitelial-Mesenquimal/fisiologia , Redes Reguladoras de Genes/genética , Humanos , Camundongos
9.
Nat Commun ; 8(1): 1168, 2017 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-29079737

RESUMO

Epithelial tumour cells can gain invasive and metastatic capabilities by undergoing an epithelial-mesenchymal transition. Transcriptional regulators and post-transcriptional effectors like microRNAs orchestrate this process of high cellular plasticity and its malignant consequences. Here, using microRNA sequencing in a time-resolved manner and functional validation, we have identified microRNAs that are critical for the regulation of an epithelial-mesenchymal transition and of mesenchymal tumour cell migration. We report that miR-1199-5p is downregulated in its expression during an epithelial-mesenchymal transition, while its forced expression prevents an epithelial-mesenchymal transition, tumour cell migration and invasion in vitro, and lung metastasis in vivo. Mechanistically, miR-1199-5p acts in a reciprocal double-negative feedback loop with the epithelial-mesenchymal transition transcription factor Zeb1. This function resembles the activities of miR-200 family members, guardians of an epithelial cell phenotype. However, miR-1199-5p and miR-200 family members share only six target genes, indicating that, besides regulating Zeb1 expression, they exert distinct functions during an epithelial-mesenchymal transition.


Assuntos
Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/metabolismo , MicroRNAs/metabolismo , Metástase Neoplásica , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Mamárias Animais/genética , Camundongos , MicroRNAs/genética , Fenótipo , Fator de Crescimento Transformador beta/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética
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