Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 26
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Nat Methods ; 20(12): 1971-1979, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37884795

RESUMO

Brillouin microscopy is an emerging optical elastography technique capable of assessing mechanical properties of biological samples in a three-dimensional, all-optical and noncontact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated Brillouin scattering (SBS) process; however, current implementations require high pump powers, which prohibit applications to photosensitive or live imaging of biological samples. Here we present a pulsed SBS scheme that takes advantage of the nonlinearity of the pump-probe interaction. In particular, we show that the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. We demonstrate the low phototoxicity and high specificity of our pulsed SBS approach by imaging, with subcellular detail, sensitive single cells, zebrafish larvae, mouse embryos and adult Caenorhabditis elegans. Furthermore, our method permits observing the mechanics of organoids and C. elegans embryos over time, opening up further possibilities for the field of mechanobiology.


Assuntos
Caenorhabditis elegans , Microscopia , Animais , Camundongos , Peixe-Zebra , Luz , Lasers
2.
J Mammary Gland Biol Neoplasia ; 28(1): 17, 2023 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-37450065

RESUMO

On 8 December 2022 the organizing committee of the European Network for Breast Development and Cancer labs (ENBDC) held its fifth annual Think Tank meeting in Amsterdam, the Netherlands. Here, we embraced the opportunity to look back to identify the most prominent breakthroughs of the past ten years and to reflect on the main challenges that lie ahead for our field in the years to come. The outcomes of these discussions are presented in this position paper, in the hope that it will serve as a summary of the current state of affairs in mammary gland biology and breast cancer research for early career researchers and other newcomers in the field, and as inspiration for scientists and clinicians to move the field forward.


Assuntos
Neoplasias da Mama , Glândulas Mamárias Humanas , Humanos , Feminino , Mama , Biologia
4.
Mol Syst Biol ; 17(10): e10141, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34694069

RESUMO

Tumor relapse from treatment-resistant cells (minimal residual disease, MRD) underlies most breast cancer-related deaths. Yet, the molecular characteristics defining their malignancy have largely remained elusive. Here, we integrated multi-omics data from a tractable organoid system with a metabolic modeling approach to uncover the metabolic and regulatory idiosyncrasies of the MRD. We find that the resistant cells, despite their non-proliferative phenotype and the absence of oncogenic signaling, feature increased glycolysis and activity of certain urea cycle enzyme reminiscent of the tumor. This metabolic distinctiveness was also evident in a mouse model and in transcriptomic data from patients following neo-adjuvant therapy. We further identified a marked similarity in DNA methylation profiles between tumor and residual cells. Taken together, our data reveal a metabolic and epigenetic memory of the treatment-resistant cells. We further demonstrate that the memorized elevated glycolysis in MRD is crucial for their survival and can be targeted using a small-molecule inhibitor without impacting normal cells. The metabolic aberrances of MRD thus offer new therapeutic opportunities for post-treatment care to prevent breast tumor recurrence.


Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Feminino , Humanos , Camundongos , Recidiva Local de Neoplasia , Neoplasia Residual/genética
5.
Nat Methods ; 13(12): 997-1000, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27749839

RESUMO

We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure-function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies.


Assuntos
Proteínas de Fluorescência Verde/biossíntese , Complexos Multiproteicos/biossíntese , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Proteínas Virais/biossíntese , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Técnicas de Cultura de Células , Transferência Ressonante de Energia de Fluorescência/métodos , Código Genético , Vetores Genéticos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera , Proteínas Virais/química , Proteínas Virais/genética
6.
Genes Dev ; 23(14): 1677-88, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19605689

RESUMO

The advent of targeted therapies for cancer has provoked interest in experimental models for the systematic study of oncogene dependence. To that end, we developed a three-dimensional (3D) culture system to analyze the responses of primary mouse mammary epithelial cells to the induction and deinduction of oncogenes. Mammary cells derived from normal virgin mice, or from tritransgenic mice (TetO-MYC;TetO-Kras(G12D);MMTV-rtTA) in which MYC and mutant Kras can be regulated by doxycycline, develop from single cells into polarized acini. Lumen formation occurs without apparent apoptosis, and the hollow spheres of cells enlarge by division, with metaphase plates oriented perpendicularly to the apical surface. When MYC and Kras(G12D) are induced, the acini enlarge and form solid, depolarized spheres. Upon deinduction of MYC and Kras(G12D) the solid structures regress, leaving a repolarized monolayer of viable cells. These cells display a phenotype consistent with progenitors of mammary epithelium: They exclude Hoechst dye 33342, and reform acini in 3D cultures and repopulate mammary fat pads more efficiently than cells harvested from uninduced acini. Moreover, cells in the surviving spheres retain the ability to respond to reinduction and thus may represent the type of cells that give rise to recurrent tumors.


Assuntos
Técnicas de Cultura de Células/métodos , Sobrevivência Celular/fisiologia , Regulação da Expressão Gênica , Glândulas Mamárias Animais/citologia , Oncogenes/genética , Transgenes/fisiologia , Animais , Antibacterianos/farmacologia , Apoptose , Benzimidazóis/metabolismo , Caspase 3/metabolismo , Divisão Celular , Doxiciclina/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Células-Tronco/citologia , Ativação Transcricional
7.
Cancer Cell ; 10(3): 227-39, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16959614

RESUMO

Erk/MAPK and TGFbeta signaling cause epithelial to mesenchymal transition (EMT) and metastasis in mouse mammary epithelial cells (EpH4) transformed with oncogenic Ras (EpRas). In trials to unravel underlying mechanisms, expression profiling for EMT-specific genes identified a secreted interleukin-related protein (ILEI), upregulated exclusively at the translational level. Stable overexpression of ILEI in EpH4 and EpRas cells caused EMT, tumor growth, and metastasis, independent of TGFbeta-R signaling and enhanced by Bcl2. RNAi-mediated knockdown of ILEI in EpRas cells before and after EMT (EpRasXT) prevented and reverted TGFbeta-dependent EMT, also abrogating metastasis formation. ILEI is overexpressed and/or altered in intracellular localization in multiple human tumors, an event strongly correlated to invasion/EMT, metastasis formation, and survival in human colon and breast cancer.


Assuntos
Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Citocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células-Tronco Mesenquimais/citologia , Proteínas de Neoplasias/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Citocinas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica/patologia , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neoplasias/metabolismo , Neoplasias/patologia , Prognóstico , Biossíntese de Proteínas/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo
9.
Dev Cell ; 58(7): 616-632.e6, 2023 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-36990090

RESUMO

3D cell cultures, in particular organoids, are emerging models in the investigation of healthy or diseased tissues. Understanding the complex cellular sociology in organoids requires integration of imaging modalities across spatial and temporal scales. We present a multi-scale imaging approach that traverses millimeter-scale live-cell light microscopy to nanometer-scale volume electron microscopy by performing 3D cell cultures in a single carrier that is amenable to all imaging steps. This allows for following organoids' growth, probing their morphology with fluorescent markers, identifying areas of interest, and analyzing their 3D ultrastructure. We demonstrate this workflow on mouse and human 3D cultures and use automated image segmentation to annotate and quantitatively analyze subcellular structures in patient-derived colorectal cancer organoids. Our analyses identify local organization of diffraction-limited cell junctions in compact and polarized epithelia. The continuum-resolution imaging pipeline is thus suited to fostering basic and translational organoid research by simultaneously exploiting the advantages of light and electron microscopy.


Assuntos
Técnicas de Cultura de Células em Três Dimensões , Microscopia , Organoides , Animais , Humanos , Camundongos , Técnicas de Cultura de Células em Três Dimensões/métodos , Microscopia Eletrônica , Organoides/diagnóstico por imagem , Organoides/fisiologia , Organoides/ultraestrutura , Neoplasias Colorretais/patologia
10.
Methods Mol Biol ; 2471: 185-194, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35175597

RESUMO

The growth of organoid cultures from primary donor tissue is able to recapitulate the original tissue morphology, heterogeneity, and characteristics. Close study of these cultures grants a deeper understanding of the chain of events occurring during disease progression and healthy tissue development. While patient derived organoids are particularly suited to assay for novel treatment options, organoids obtained from model organisms are perfectly suited to establish in-depth analysis technology, including longitudinal imaging approaches, as well as proof of principle studies that rely on a steady source of primary tissue. All these approaches profit from advancements in technology to manipulate cells within an organoid.Here we present an optimized protocol to generate, culture, and transduce 3D acini obtained from mouse primary mammary epithelial cells via viral vectors. Applying this method, a few cells within the preserved organoid can be marked, changed, and tracked within an unaltered neighboring environment of non-transduced cells to better understand processes like, for instance, tumor initiation.


Assuntos
Células Acinares , Glândulas Mamárias Animais , Animais , Células Epiteliais , Glândulas Mamárias Animais/citologia , Camundongos , Organoides , Transdução Genética
11.
Commun Biol ; 4(1): 556, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976362

RESUMO

Preclinical breast tumor models are an invaluable tool to systematically study tumor progression and treatment response, yet methods to non-invasively monitor the involved molecular and mechanistic properties under physiologically relevant conditions are limited. Here we present an intravital mesoscopic fluorescence molecular tomography (henceforth IFT) approach that is capable of tracking fluorescently labeled tumor cells in a quantitative manner inside the mammary gland of living mice. Our mesoscopic approach is entirely non-invasive and thus permits prolonged observational periods of several months. The relatively high sensitivity and spatial resolution further enable inferring the overall number of oncogene-expressing tumor cells as well as their tumor volume over the entire cycle from early tumor growth to residual disease following the treatment phase. Our IFT approach is a promising method for studying tumor growth dynamics in a quantitative and longitudinal fashion in-vivo.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Microscopia Intravital/métodos , Tomografia Computadorizada por Raios X/métodos , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Fluorescência , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Tomografia/métodos , Carga Tumoral/fisiologia
12.
J Mol Med (Berl) ; 99(10): 1413-1426, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34129057

RESUMO

Expression of the phosphatase of regenerating liver-3 (PRL-3) is known to promote tumor growth in gastrointestinal adenocarcinomas, and the incidence of tumor formation upon inflammatory events correlates with PRL-3 levels in mouse models. These carcinomas and their onset are associated with the impairment of intestinal cell homeostasis, which is regulated by a balanced number of Paneth cells and Lgr5 expressing intestinal stem cells (Lgr5+ ISCs). Nevertheless, the consequences of PRL-3 overexpression on cellular homeostasis and ISC fitness in vivo are unexplored. Here, we employ a doxycycline-inducible PRL-3 mouse strain to show that aberrant PRL-3 expression within a non-cancerous background leads to the death of Lgr5+ ISCs and to Paneth cell expansion. A higher dose of PRL-3, resulting from homozygous expression, led to mice dying early. A primary 3D intestinal culture model obtained from these mice confirmed the loss of Lgr5+ ISCs upon PRL-3 expression. The impaired intestinal organoid formation was rescued by a PRL inhibitor, providing a functional link to the observed phenotypes. These results demonstrate that elevated PRL-3 phosphatase activity in healthy intestinal epithelium impairs intestinal cell homeostasis, which correlates this cellular mechanism of tumor onset with PRL-3-mediated higher susceptibility to tumor formation upon inflammatory or mutational events.Key messages• Transgenic mice homozygous for PRL-3 overexpression die early.• PRL-3 heterozygous mice display disrupted intestinal self-renewal capacity.• PRL-3 overexpression alone does not induce tumorigenesis in the mouse intestine.• PRL-3 activity leads to the death of Lgr5+ ISCs and Paneth cell expansion.• Impairment of cell homeostasis correlates PRL-3 action with tumor onset mechanisms.


Assuntos
Homeostase/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Mucosa Intestinal/patologia , Intestinos/patologia , Masculino , Camundongos , Camundongos Transgênicos , Organoides/metabolismo , Organoides/patologia , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Células-Tronco/patologia
13.
J Cell Biol ; 220(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34160561

RESUMO

Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)-SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.


Assuntos
Drosophila melanogaster/ultraestrutura , Células da Granulosa/ultraestrutura , Glândulas Mamárias Animais/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Coloração e Rotulagem/métodos , Células Tecais/ultraestrutura , Traqueia/ultraestrutura , Animais , Drosophila melanogaster/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Feminino , Expressão Gênica , Genes Reporter , Células da Granulosa/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Larva/metabolismo , Larva/ultraestrutura , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Glândulas Mamárias Animais/metabolismo , Camundongos , Microscopia Eletrônica de Varredura/instrumentação , Organoides/metabolismo , Organoides/ultraestrutura , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Células Tecais/metabolismo , Traqueia/metabolismo , Fluxo de Trabalho , Proteína Vermelha Fluorescente
14.
Elife ; 92020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32690136

RESUMO

Cancer clone evolution takes place within tissue ecosystem habitats. But, how exactly tumors arise from a few malignant cells within an intact epithelium is a central, yet unanswered question. This is mainly due to the inaccessibility of this process to longitudinal imaging together with a lack of systems that model the progression of a fraction of transformed cells within a tissue. Here, we developed a new methodology based on primary mouse mammary epithelial acini, where oncogenes can be switched on in single cells within an otherwise normal epithelial cell layer. We combine this stochastic breast tumor induction model with inverted light-sheet imaging to study single-cell behavior for up to four days and analyze cell fates utilizing a newly developed image-data analysis workflow. The power of this integrated approach is illustrated by us finding that small local clusters of transformed cells form tumors while isolated transformed cells do not.


There are now drugs to treat many types of cancer, but questions still remain around how these diseases start in the first place. Researchers think that tumor growth begins when a single cell suffers damage to certain sites in its DNA that eventually cause it to divide uncontrollably. That damaged cell, and its descendants, go on to form a lump, or tumor. The trouble with proving this theory is that it is hard to watch it happening in real time. Doctors usually only meet people with cancer when their tumors start to cause health problems. By this point, the tumors contain millions of cells. A way to watch the very beginnings of a cancer could reveal risk factors within a tissue that foster the growth of a tumor. But first, researchers need to test their theory about how the disease begins in the first place. One way to do this is to surround a single cancer cell with healthy cells and watch what happens next. To do this, Alladin, Chaible et al. took healthy cells from the breast tissue of mice and grew them in the laboratory into mini-organs called organoids. These organoids share a lot of features with actual mouse breast tissue; they can even make milk if given the right hormones. Once the organoids were ready, Alladin, Chaible et al then started modifying a small number of single cells inside them by switching on genes called oncogenes, which are known to drive cancer formation in humans. Using fluorescent proteins and a sheet of laser light it was possible to watch what happened to the cells over time. This revealed that, even though all the oncogene-driven single cells received the same signals, not all of them started to divide uncontrollably. In fact, a single modified cell had a low chance of forming a tumor on its own. The more oncogene-driven cells there were near to each other, the more likely they were to form tumors. Alladin, Chaible et al. think that this is because the healthy tissue interacts with the modified, oncogene-driven cells to suppress tumor formation. It is only when a larger number of modified cells group together and start to communicate with each other that they can override the inhibitory messages of the healthy tissue. How healthy tissue stops single modified cells from forming tumors is not yet clear. But, with this new mini-organ system, researchers now have the tools to investigate. In the future, this could lead to new strategies to stop cancer before it has a chance to get started.


Assuntos
Células Acinares/citologia , Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Rastreamento de Células/métodos , Células Epiteliais/citologia , Glândulas Mamárias Humanas/citologia , Células-Tronco Neoplásicas/citologia , Animais , Feminino , Humanos , Camundongos , Microscopia/métodos , Modelos Animais
15.
J Clin Invest ; 116(6): 1561-70, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16741576

RESUMO

Metastasis is the major cause of cancer morbidity, but strategies for direct interference with invasion processes are lacking. Dedifferentiated, late-stage tumor cells secrete multiple factors that represent attractive targets for therapeutic intervention. Here we show that metastatic potential of oncogenic mammary epithelial cells requires an autocrine PDGF/PDGFR loop, which is established as a consequence of TGF-beta-induced epithelial-mesenchymal transition (EMT), a faithful in vitro correlate of metastasis. The cooperation of autocrine PDGFR signaling with oncogenic Ras hyperactivates PI3K and is required for survival during EMT. Autocrine PDGFR signaling also contributes to maintenance of EMT, possibly through activation of STAT1 and other distinct pathways. Inhibition of PDGFR signaling interfered with EMT and caused apoptosis in murine and human mammary carcinoma cell lines. Consequently, overexpression of a dominant-negative PDGFR or application of the established cancer drug STI571 interfered with experimental metastasis in mice. Similarly, in mouse mammary tumor virus-Neu (MMTV-Neu) transgenic mice, TGF-beta enhanced metastasis of mammary tumors, induced EMT, and elevated PDGFR signaling. Finally, expression of PDGFRalpha and -beta correlated with invasive behavior in human mammary carcinomas. Thus, autocrine PDGFR signaling plays an essential role during cancer progression, suggesting a novel application of STI571 to therapeutically interfere with metastasis.


Assuntos
Comunicação Autócrina , Neoplasias da Mama , Neoplasias Mamárias Experimentais , Metástase Neoplásica , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Animais , Antineoplásicos/metabolismo , Apoptose , Benzamidas , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Ativação Enzimática , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Feminino , Humanos , Mesilato de Imatinib , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/genética , Vírus do Tumor Mamário do Camundongo/metabolismo , Mesoderma/fisiologia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , Piperazinas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Pirimidinas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas ras/metabolismo
16.
J Cell Biol ; 156(2): 299-313, 2002 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-11790801

RESUMO

Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras-transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor beta (TGFbeta) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFbeta receptor (TGFbeta-R) and oncogenic Ras signaling and is stabilized by autocrine TGFbeta production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFbeta alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFbeta-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFbeta-R and Raf/MAPK signaling.


Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Metástase Neoplásica , Proteína Oncogênica p21(ras)/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Epiteliais/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Peso Molecular , Mutação , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Proteína Oncogênica p21(ras)/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais/efeitos dos fármacos
17.
J Clin Invest ; 127(6): 2091-2105, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28504653

RESUMO

Tumor recurrence is the leading cause of breast cancer-related death. Recurrences are largely driven by cancer cells that survive therapeutic intervention. This poorly understood population is referred to as minimal residual disease. Here, using mouse models that faithfully recapitulate human disease together with organoid cultures, we have demonstrated that residual cells acquire a transcriptionally distinct state from normal epithelium and primary tumors. Gene expression changes and functional characterization revealed altered lipid metabolism and elevated ROS as hallmarks of the cells that survive tumor regression. These residual cells exhibited increased oxidative DNA damage, potentiating the acquisition of somatic mutations during hormonal-induced expansion of the mammary cell population. Inhibition of either cellular fatty acid synthesis or fatty acid transport into mitochondria reduced cellular ROS levels and DNA damage, linking these features to lipid metabolism. Direct perturbation of these hallmarks in vivo, either by scavenging ROS or by halting the cyclic mammary cell population expansion, attenuated tumor recurrence. Finally, these observations were mirrored in transcriptomic and histological signatures of residual cancer cells from neoadjuvant-treated breast cancer patients. These results highlight the potential of lipid metabolism and ROS as therapeutic targets for reducing tumor recurrence in breast cancer patients.


Assuntos
Neoplasias da Mama/metabolismo , Recidiva Local de Neoplasia/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Lapatinib , Metabolismo dos Lipídeos , Redes e Vias Metabólicas , Camundongos , Recidiva Local de Neoplasia/prevenção & controle , Neoplasia Residual , Estresse Oxidativo , Progesterona/farmacologia , Quinazolinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transcriptoma , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Nat Genet ; 49(1): 65-74, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27869826

RESUMO

Extensive prior research focused on somatic copy-number alterations (SCNAs) affecting cancer genes, yet the extent to which recurrent SCNAs exert their influence through rearrangement of cis-regulatory elements (CREs) remains unclear. Here we present a framework for inferring cancer-related gene overexpression resulting from CRE reorganization (e.g., enhancer hijacking) by integrating SCNAs, gene expression data and information on topologically associating domains (TADs). Analysis of 7,416 cancer genomes uncovered several pan-cancer candidate genes, including IRS4, SMARCA1 and TERT. We demonstrate that IRS4 overexpression in lung cancer is associated with recurrent deletions in cis, and we present evidence supporting a tumor-promoting role. We additionally pursued cancer-type-specific analyses and uncovered IGF2 as a target for enhancer hijacking in colorectal cancer. Recurrent tandem duplications intersecting with a TAD boundary mediate de novo formation of a 3D contact domain comprising IGF2 and a lineage-specific super-enhancer, resulting in high-level gene activation. Our framework enables systematic inference of CRE rearrangements mediating dysregulation in cancer.


Assuntos
Variações do Número de Cópias de DNA/genética , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Substratos do Receptor de Insulina/genética , Fator de Crescimento Insulin-Like II/genética , Neoplasias/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Regiões Promotoras Genéticas
19.
Cell Rep ; 15(12): 2679-91, 2016 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-27292643

RESUMO

Chromosome instability (CIN) is associated with poor survival and therapeutic outcome in a number of malignancies. Despite this correlation, CIN can also lead to growth disadvantages. Here, we show that simultaneous overexpression of the mitotic checkpoint protein Mad2 with Kras(G12D) or Her2 in mammary glands of adult mice results in mitotic checkpoint overactivation and a delay in tumor onset. Time-lapse imaging of organotypic cultures and pathologic analysis prior to tumor establishment reveals error-prone mitosis, mitotic arrest, and cell death. Nonetheless, Mad2 expression persists and increases karyotype complexity in Kras tumors. Faced with the selective pressure of oncogene withdrawal, Mad2-positive tumors have a higher frequency of developing persistent subclones that avoid remission and continue to grow.


Assuntos
Instabilidade Cromossômica , Proteínas Mad2/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Oncogenes , Aneuploidia , Animais , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Células Cultivadas , Segregação de Cromossomos/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Camundongos , Mitose , Fenótipo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptor ErbB-2 , Fuso Acromático/metabolismo , Imagem com Lapso de Tempo , Transgenes
20.
Oncogene ; 22(46): 7155-69, 2003 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-14562044

RESUMO

Epithelial-to-mesenchymal transition (EMT), a switch of polarized epithelial cells to a migratory, fibroblastoid phenotype, is increasingly considered as an important event during malignant tumor progression and metastasis. To identify molecular players involved in EMT and metastasis, we performed expression profiling of a set of combined in vitro/in vivo cellular models, based on clonal, fully polarized mammary epithelial cells. Seven closely related cell pairs were used, which were modified by defined oncogenes and/or external factors and showed specific aspects of epithelial plasticity relevant to cell migration, local invasion and metastasis. Since mRNA levels do not necessarily reflect protein levels in cells, we used an improved expression profiling method based on polysome-bound RNA, suitable to analyse global gene expression on Affymetrix chips. A substantial fraction of all regulated genes was found to be exclusively controlled at the translational level. Furthermore, profiling of the above multiple cell pairs allowed one to identify small numbers of genes by cluster analysis, specifically correlating gene expression with EMT, metastasis, scattering and/or oncogene function. A small set of genes specifically regulated during EMT was identified, including key regulators and signaling pathways involved in cell proliferation, epithelial polarity, survival and trans-differentiation to mesenchymal-like cells with invasive behavior.


Assuntos
Transformação Celular Neoplásica/genética , Células Epiteliais/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Mesoderma/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Diferenciação Celular , Linhagem Celular , Análise por Conglomerados , Células Epiteliais/patologia , Feminino , Perfilação da Expressão Gênica , Genes ras , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/patologia , Mesoderma/patologia , Camundongos , Invasividade Neoplásica , Polirribossomos/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Transcrição Gênica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA