RESUMO
The original version of this Article incorrectly matched the supplementary files with their titles. This has been corrected. The following information has also been added to the Methods section.
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Metastatic breast cancer is an extremely complex disease with limited treatment options due to the lack of information about the major characteristics of metastatic disease. There is an urgent need, therefore, to understand the changes in cellular complexity and dynamics that occur during metastatic progression. In the current study, we analyzed the cellular and molecular differences between primary tumors and paired lung metastases using a syngeneic p53-null mammary tumor model of basal-like breast cancer. Distinct subpopulations driven by the Wnt- and/or STAT3 signaling pathways were detected in vivo using a lentiviral Wnt- and STAT3 signaling reporter system. A significant increase in the overlapping populations driven by both the Wnt- and STAT3 signaling pathways was observed in the lung metastases as compared to the primary tumors. Furthermore, the overlapping populations showed a higher metastatic potential relative to the other populations and pharmacological inhibition of both signaling pathways was shown to markedly reduce the metastatic lesions in established lung metastases. An analysis of the unique molecular features of the lung metastases revealed a significant association with immune response signatures. Specifically, Foxp3 gene expression was markedly increased and elevated levels of Foxp3 + Treg cells were detected in close proximity to lung metastases. Collectively, these studies illustrate the importance of analyzing intratumoral heterogeneity, changes in population dynamics, and the immune microenvironment during metastatic progression.
Assuntos
Neoplasias da Mama/imunologia , Fator de Transcrição STAT3/genética , Microambiente Tumoral/imunologia , Via de Sinalização Wnt/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Progressão da Doença , Feminino , Fatores de Transcrição Forkhead/imunologia , Humanos , Neoplasias Pulmonares/secundário , Camundongos , Metástase Neoplásica/genética , Metástase Neoplásica/imunologia , Linfócitos T Reguladores/imunologia , Proteína Supressora de Tumor p53/genéticaRESUMO
Functional abdominal pain disorders are highly prevalent in children. These disorders can be present in isolation or combined with organic diseases, such as celiac disease and inflammatory bowel diseases. Intestinal inflammation (infectious and non-infectious) predisposes children to the development of visceral hypersensitivity that can manifest as functional abdominal pain disorders, including irritable bowel syndrome. The new onset of irritable bowel syndrome symptoms in a patient with an underlying organic disease, such as inflammatory bowel disease, is clinically challenging, given that the same symptomatology may represent a flare-up of the inflammatory bowel disease or an overlapping functional abdominal pain disorder. Similarly, irritable bowel syndrome symptoms in a child previously diagnosed with celiac disease may occur due to poorly controlled celiac disease or the overlap with a functional abdominal pain disorder. There is little research on the overlap of functional abdominal disorders with organic diseases in children. Studies suggest that the overlap between functional abdominal pain disorders and inflammatory bowel disease is more common in adults than in children. The causes for these differences in prevalence are unknown. Only a handful of studies have been published on the overlap between celiac disease and functional abdominal pain disorders in children. The present article provides a review of the literature on the overlap between celiac disease, inflammatory bowel disease, and functional abdominal pain disorders in children and establish comparisons with studies conducted on adults.
Assuntos
Dor Abdominal/epidemiologia , Gastroenteropatias/epidemiologia , Adolescente , Doença Celíaca/epidemiologia , Criança , Pré-Escolar , Humanos , Lactente , Doenças Inflamatórias Intestinais/epidemiologia , Síndrome do Intestino Irritável/complicações , PrevalênciaRESUMO
Cellular heterogeneity is a common feature in breast cancer, yet an understanding of the coexistence and regulation of various tumor cell subpopulations remains a significant challenge in cancer biology. In the current study, we approached tumor cell heterogeneity from the perspective of Wnt pathway biology to address how different modes of Wnt signaling shape the behaviors of diverse cell populations within a heterogeneous tumor landscape. Using a syngeneic TP53-null mouse model of breast cancer, we identified distinctions in the topology of canonical Wnt ß-catenin-dependent signaling activity and non-canonical ß-catenin-independent Ror2-mediated Wnt signaling across subtypes and within tumor cell subpopulations in vivo. We further discovered an antagonistic role for Ror2 in regulating canonical Wnt/ß-catenin activity in vivo, where lentiviral shRNA depletion of Ror2 expression augmented canonical Wnt/ß-catenin signaling activity across multiple basal-like models. Depletion of Ror2 expression yielded distinct phenotypic outcomes and divergent alterations in gene expression programs among different tumors, despite all sharing basal-like features. Notably, we uncovered cell state plasticity and adhesion dynamics regulated by Ror2, which influenced Ras Homology Family Member A (RhoA) and Rho-Associated Coiled-Coil Kinase 1 (ROCK1) activity downstream of Dishevelled-2 (Dvl2). Collectively, these studies illustrate the integration and collaboration of Wnt pathways in basal-like breast cancer, where Ror2 provides a spatiotemporal function to regulate the balance of Wnt signaling and cellular heterogeneity during tumor progression.
Assuntos
Neoplasias da Mama/genética , Proteínas Desgrenhadas/genética , Neoplasias Mamárias Animais/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Proteínas rho de Ligação ao GTP/genética , Quinases Associadas a rho/genética , Animais , Neoplasias da Mama/patologia , Adesão Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Mamárias Animais/patologia , Camundongos , Proteína Supressora de Tumor p53/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , Proteína rhoA de Ligação ao GTPRESUMO
Mechanisms regulating the transition of mammary epithelial cells (MECs) to mammary stem cells (MaSCs) and to tumor-initiating cells (TICs) have not been entirely elucidated. The p53 family member, p63, is critical for mammary gland development and contains transactivation domain isoforms, which have tumor-suppressive activities, and the ΔN isoforms, which act as oncogenes. In the clinic, p63 is often used as a diagnostic marker, and further analysis of the function of TAp63 in the mammary gland is critical for improved diagnosis and patient care. Loss of TAp63 in mice leads to the formation of aggressive metastatic mammary adenocarcinoma at 9-16 months of age. Here we show that TAp63 is crucial for the transition of mammary cancer cells to TICs. When TAp63 is lost, MECs express embryonic and MaSC signatures and activate the Hippo pathway. These data indicate a crucial role for TAp63 in mammary TICs and provide a mechanism for its role as a tumor- and metastasis-suppressor in breast cancer.
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Neoplasias da Mama/patologia , Carcinogênese , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Proteínas Quinases Ativadas por AMP , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Contagem de Células , Linhagem Celular Tumoral , Polaridade Celular , Transformação Celular Neoplásica , Transição Epitelial-Mesenquimal , Feminino , Técnicas de Silenciamento de Genes , Via de Sinalização Hippo , Humanos , Hiperplasia , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Animais/fisiopatologia , Camundongos , Células-Tronco Neoplásicas/patologia , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Regeneração , Transativadores/deficiência , Transativadores/genética , Transcrição GênicaRESUMO
Metastatic competence is contingent upon the aberrant activation of a latent embryonic program, known as the epithelial-mesenchymal transition (EMT), which bestows stem cell properties as well as migratory and invasive capabilities upon differentiated tumor cells. We recently identified the transcription factor FOXC2 as a downstream effector of multiple EMT programs, independent of the EMT-inducing stimulus, and as a key player linking EMT, stem cell traits and metastatic competence in breast cancer. As such, FOXC2 could serve as a potential therapeutic target to attenuate metastasis. However, as FOXC2 is a transcription factor, it is difficult to target by conventional means such as small-molecule inhibitors. Herein, we identify the serine/threonine-specific kinase p38 as a druggable upstream regulator of FOXC2 stability and function that elicits phosphorylation of FOXC2 at serine 367 (S367). Using an orthotopic syngeneic mouse tumor model, we make the striking observation that inhibition of p38-FOXC2 signaling selectively attenuates metastasis without impacting primary tumor growth. In this model, circulating tumor cell numbers are significantly reduced in mice treated with the p38 inhibitor SB203580, relative to vehicle-treated counterparts. Accordingly, genetic or pharmacological inhibition of p38 decreases FOXC2 protein levels, reverts the EMT phenotype and compromises stem cell attributes in vitro. We also identify the EMT-regulator ZEB1-known to directly repress E-cadherin/CDH1-as a downstream target of FOXC2, critically dependent on its activation by p38. Consistent with the notion that activation of the p38-FOXC2 signaling axis represents a critical juncture in the acquisition of metastatic competence, the phosphomimetic FOXC2(S367E) mutant is refractory to p38 inhibition both in vitro and in vivo, whereas the non-phosphorylatable FOXC2(S367A) mutant fails to elicit EMT and upregulate ZEB1. Collectively, our data demonstrate that FOXC2 regulates EMT, stem cell traits, ZEB1 expression and metastasis in a p38-dependent manner, and attest to the potential utility of p38 inhibitors as antimetastatic agents.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fatores de Transcrição Forkhead/metabolismo , Serina/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Feminino , Xenoenxertos , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Metástase Neoplásica , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Fosforilação , Ligação Proteica , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND: The epithelial to mesenchymal transition (EMT) has been implicated in metastasis and therapy resistance of carcinomas and can endow cancer cells with cancer stem cell (CSC) properties. The ability to detect cancer cells that are undergoing or have completed EMT has typically relied on the expression of cell surface antigens that correlate with an EMT/CSC phenotype. Alternatively these cells may be permanently marked through Cre-mediated recombination or through immunostaining of fixed cells. The EMT process is dynamic, and these existing methods cannot reveal such changes within live cells. The development of fluorescent sensors that mirror the dynamic EMT state by following the expression of bona fide EMT regulators in live cells would provide a valuable new tool for characterizing EMT. In addition, these sensors will allow direct observation of cellular plasticity with respect to the epithelial/mesenchymal state to enable more effective studies of EMT in cancer and development. RESULTS: We generated a lentiviral-based, dual fluorescent reporter system, designated as the Z-cad dual sensor, comprising destabilized green fluorescent protein containing the ZEB1 3' UTR and red fluorescent protein driven by the E-cadherin (CDH1) promoter. Using this sensor, we robustly detected EMT and mesenchymal to epithelial transition (MET) in breast cancer cells by flow cytometry and fluorescence microscopy. Importantly, we observed dynamic changes in cellular populations undergoing MET. Additionally, we used the Z-cad sensor to identify and isolate minor subpopulations of cells displaying mesenchymal properties within a population comprising predominately epithelial-like cells. The Z-cad dual sensor identified cells with CSC-like properties more effectively than either the ZEB1 3' UTR or E-cadherin sensor alone. CONCLUSIONS: The Z-cad dual sensor effectively reports the activities of two factors critical in determining the epithelial/mesenchymal state of carcinoma cells. The ability of this stably integrating dual sensor system to detect dynamic fluctuations between these two states through live cell imaging offers a significant improvement over existing methods and helps facilitate the study of EMT/MET plasticity in response to different stimuli and in cancer pathogenesis. Finally, the versatile Z-cad sensor can be adapted to a variety of in vitro or in vivo systems to elucidate whether EMT/MET contributes to normal and disease phenotypes.
Assuntos
Técnicas Biossensoriais , Células Epiteliais/citologia , Transição Epitelial-Mesenquimal , Células-Tronco Mesenquimais/citologia , Animais , Antígenos CD , Caderinas/genética , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Regiões Promotoras Genéticas , Fator de Crescimento Transformador beta1/farmacologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Proteína Vermelha FluorescenteRESUMO
Claudin-low tumors are a highly aggressive breast cancer subtype with no targeted treatments and a clinically documented resistance to chemotherapy. They are significantly enriched in cancer stem cells (CSCs), which makes claudin-low tumor models particularly attractive for studying CSC behavior and developing novel approaches to minimize CSC therapy resistance. One proposed mechanism by which CSCs arise is via an epithelial-mesenchymal transition (EMT), and reversal of this process may provide a potential therapeutic approach for increasing tumor chemosensitivity. Therefore, we investigated the role of known EMT regulators, miR-200 family of microRNAs in controlling the epithelial state, stem-like properties and therapeutic response in an in vivo primary, syngeneic p53(null) claudin-low tumor model that is normally deficient in miR-200 expression. Using an inducible lentiviral approach, we expressed the miR-200c cluster in this model and found that it changed the epithelial state, and consequently, impeded CSC behavior in these mesenchymal tumors. Moreover, these state changes were accompanied by a decrease in proliferation and an increase in the differentiation status. miR-200c expression also forced a significant reorganization of tumor architecture, affecting important cellular processes involved in cell-cell contact, cell adhesion and motility. Accordingly, induced miR200c expression significantly enhanced the chemosensitivity and decreased the metastatic potential of this p53(null) claudin-low tumor model. Collectively, our data suggest that miR-200c expression in claudin-low tumors offers a potential therapeutic application to disrupt the EMT program on multiple fronts in this mesenchymal tumor subtype, by altering tumor growth, chemosensitivity and metastatic potential in vivo.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Carboplatina/administração & dosagem , Doxorrubicina/administração & dosagem , MicroRNAs/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Neoplasias da Mama/patologia , Carboplatina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Claudinas/genética , Claudinas/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Metástase Neoplásica , Células-Tronco Neoplásicas/patologiaRESUMO
Despite radical surgery and multi-agent chemotherapy, less than one third of patients with recurrent or metastatic osteosarcoma (OS) survive. The limited efficacy of current therapeutic approaches to target tumor-initiating cells (TICs) may explain this dismal outcome. The purpose of this study was to assess the impact of modified T cells expressing a human epidermal growth factor receptor (HER2)-specific chimeric antigen receptor in the OS TIC compartment of human established cell lines. Using the sarcosphere formation assay, we found that OS TICs were resistant to increasing methotrexate concentrations. In contrast, HER2-specific T cells decreased markedly sarcosphere formation capacity and the ability to generate bone tumors in immunodeficient mice after orthotopic transplantation. In vivo, administration of HER2-specific T cells significantly reduced TICs in bulky tumors as judged by decreased sarcosphere forming efficiency in OS cells isolated from explanted tumors. We demonstrate that HER2-specific T cells target drug resistant TICs in established OS cell lines, suggesting that incorporating immunotherapy into current treatment strategies for OS has the potential to improve outcomes.
Assuntos
Neoplasias Ósseas/terapia , Imunoterapia Adotiva/métodos , Osteossarcoma/terapia , Receptor ErbB-2/imunologia , Linfócitos T/imunologia , Animais , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Epitopos de Linfócito T/imunologia , Humanos , Masculino , Metotrexato/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Osteossarcoma/imunologia , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
P190A and p190B Rho GTPase activating proteins (GAPs) are essential genes that have distinct, but overlapping roles in the developing nervous system. Previous studies from our laboratory demonstrated that p190B is required for mammary gland morphogenesis, and we hypothesized that p190A might have a distinct role in the developing mammary gland. To test this hypothesis, we examined mammary gland development in p190A-deficient mice. P190A expression was detected by in situ hybridization in the developing E14.5day embryonic mammary bud and within the ducts, terminal end buds (TEBs), and surrounding stroma of the developing virgin mammary gland. In contrast to previous results with p190B, examination of p190A heterozygous mammary glands demonstrated that p190A deficiency disrupted TEB morphology, but did not significantly delay ductal outgrowth indicating haploinsufficiency for TEB development. To examine the effects of homozygous deletion of p190A, embryonic mammary buds were rescued by transplantation into the cleared fat pads of SCID/Beige mice. Complete loss of p190A function inhibited ductal outgrowth in comparison to wildtype transplants (51% vs. 94% fat pad filled). In addition, the transplantation take rate of p190A deficient whole gland transplants from E18.5 embryos was significantly reduced compared to wildtype transplants (31% vs. 90%, respectively). These results suggest that p190A function in both the epithelium and stroma is required for mammary gland development. Immunostaining for p63 demonstrated that the myoepithelial cell layer is disrupted in the p190A deficient glands, which may result from the defective cell adhesion between the cap and body cell layers detected in the TEBs. The number of estrogen- and progesterone receptor-positive cells, as well as the expression levels of these receptors was increased in p190A deficient outgrowths. These data suggest that p190A is required in both the epithelial and stromal compartments for ductal outgrowth and that it may play a role in mammary epithelial cell differentiation.
Assuntos
Proteínas Ativadoras de GTPase/fisiologia , Glândulas Mamárias Animais/embriologia , Glândulas Mamárias Animais/fisiologia , Proteínas Repressoras/fisiologia , Animais , Sequência de Bases , Primers do DNA/genética , Epitélio/embriologia , Feminino , Proteínas Ativadoras de GTPase/deficiência , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica no Desenvolvimento , Heterozigoto , Homozigoto , Hibridização In Situ , Glândulas Mamárias Animais/transplante , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Receptores de Esteroides/metabolismo , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Células Estromais/citologiaRESUMO
Significant advances in the stem-cell biology of several tissues, including the mammary gland, have occurred over the past several years. Recent progress on stem-cell fate determination, molecular markers, signalling pathways and niche interactions in haematopoietic, neuronal and muscle tissue may provide parallel insight into the biology of mammary epithelial stem cells. Taking advantage of approaches similar to those employed to isolate and characterize haematopoietic and epidermal stem cells, we have identified a mammary epithelial cell population with several stem/progenitor cell qualities. In this article, we review some recent data on mammary epithelial stem/progenitor cells in genetically engineered mouse models. We also discuss several potential molecular markers, including stem-cell antigen-1 (Sca-1), which may be useful for both the isolation of functional mammary epithelial stem/progenitor cells and the analysis of tumour aetiology and phenotype in genetically engineered mouse models. In different transgenic mammary tumour models, Sca-1 expression levels, as well as several other putative markers of progenitors including keratin-6, possess dramatically altered expression profiles. These data suggest that the heterogeneity of mouse models of breast cancer may partially reflect the selection or expansion of different progenitors.
Assuntos
Separação Celular/métodos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Humanas/citologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , HumanosRESUMO
Insulin receptor substrates (IRS) are central integrators of hormone, cytokine, and growth factor signaling. IRS proteins can be phosphorylated by a number of signaling pathways critical to normal mammary gland development. Studies in transgenic mice that overexpress IGF-I in the mammary gland suggested that IRS expression is important in the regulation of normal postlactational mammary involution. The goal of these studies was to examine IRS expression in the mouse mammary gland and determine the importance of IRS-1 to mammary development in the virgin mouse. IRS-1 and -2 show distinct patterns of protein expression in the virgin mouse mammary gland, and protein abundance is dramatically increased during pregnancy and lactation, but rapidly lost during involution. Consistent with hormone regulation, IRS-1 protein levels are reduced by ovariectomy, induced by combined treatment with estrogen and progesterone, and vary considerably throughout the estrous cycle. These changes occur without similar changes in mRNA levels, suggesting posttranscriptional control. Mammary glands from IRS-1 null mice have smaller fat pads than wild-type controls, but this reduction is proportional to the overall reduction in body size. Development of the mammary duct (terminal endbuds and branch points) is not altered by the loss of IRS-1, and pregnancy-induced proliferation is not changed. These data indicate that IRS undergo complex developmental and hormonal regulation in the mammary gland, and that IRS-1 is more likely to regulate mammary function in lactating mice than in virgin or pregnant mice.
Assuntos
Estrogênios/farmacologia , Glândulas Mamárias Animais/fisiologia , Fosfoproteínas/genética , Progesterona/farmacologia , Transdução de Sinais/fisiologia , Tecido Adiposo/química , Tecido Adiposo/crescimento & desenvolvimento , Tecido Adiposo/fisiologia , Animais , Ciclo Estral/fisiologia , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Glândulas Mamárias Animais/química , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos , Ovariectomia , Fosfoproteínas/análise , Gravidez , Transdução de Sinais/efeitos dos fármacosRESUMO
Functional development of mammary epithelium during pregnancy depends on prolactin signaling. However, the underlying molecular and cellular events are not fully understood. We examined the specific contributions of the prolactin receptor (PrlR) and the signal transducers and activators of transcription 5a and 5b (referred to as Stat5) in the formation and differentiation of mammary alveolar epithelium. PrlR- and Stat5-null mammary epithelia were transplanted into wild-type hosts, and pregnancy-mediated development was investigated at a histological and molecular level. Stat5-null mammary epithelium developed ducts but failed to form alveoli, and no milk protein gene expression was observed. In contrast, PrlR-null epithelium formed alveoli-like structures with small open lumina. Electron microscopy revealed undifferentiated features of organelles and a perturbation of cell-cell contacts in PrlR- and Stat5-null epithelia. Expression of NKCC1, an Na-K-Cl cotransporter characteristic for ductal epithelia, and ZO-1, a protein associated with tight junction, were maintained in the alveoli-like structures of PrlR- and Stat5-null epithelia. In contrast, the Na-Pi cotransporter Npt2b, and the gap junction component connexin 32, usually expressed in secretory epithelia, were undetectable in PrlR- and Stat5-null mice. These data demonstrate that signaling via the PrlR and Stat5 is critical for the proliferation and differentiation of mammary alveoli during pregnancy.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Glândulas Mamárias Animais/citologia , Proteínas do Leite , Prenhez , Transativadores/fisiologia , Animais , Diferenciação Celular , Divisão Celular , Conexinas/metabolismo , Conexinas/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Crescimento Epidérmico/administração & dosagem , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/citologia , Feminino , Hormônio do Crescimento/administração & dosagem , Hormônio do Crescimento/metabolismo , Glândulas Mamárias Animais/anatomia & histologia , Glândulas Mamárias Animais/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Receptores da Prolactina/genética , Receptores da Prolactina/metabolismo , Receptores da Prolactina/fisiologia , Fator de Transcrição STAT5 , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto , Transativadores/genética , Transativadores/metabolismo , Proteína beta-1 de Junções ComunicantesRESUMO
The transplantation of primary mammary epithelial cells after adenovirus-Cre-mediated recombination provides a new approach for the study of specific gene function during mammary gland development and in breast cancer. Most mammary-gland-specific promoters identified to date are regulated by lactogenic hormones. They are expressed predominantly in lobuloalveolar cells during pregnancy and lactation, but not during early stages of ductal morphogenesis in the mammary epithelial cell progenitors, which are primarily implicated in tumorigenesis. In transgenic mice these promoters will continually or repeatedly express Cre depending on the hormonal environment precluding the definition of cell lineages. To circumvent these limitations, we have taken advantage of the unique regenerative capacity of mammary epithelium to reconstitute a mammary gland in an epithelium-cleared fat pad in conjunction with transient Cre expression using recombinant adenovirus in primary cultures. This approach was validated using mice carrying reporter constructs that exclusively express the LacZ gene after Cre-mediated deletion of a floxed DNA fragment. These studies demonstrated that, following recombination, cells that are marked as genetically manipulated contribute to the reconstitution of the mammary gland. The presence of beta-galactosidase-expressing cells in serial transplants of the primary outgrowths indicated that early progenitor or stem cells were successfully targeted. With the increased availability of floxed alleles, this approach should greatly facilitate the study of gene function during early stages of mammary gland development and in breast cancer.
Assuntos
Adenoviridae/genética , Mama/metabolismo , Epitélio/metabolismo , Técnicas Genéticas , Integrases/metabolismo , Recombinação Genética , Proteínas Virais/metabolismo , Animais , Mama/patologia , Linhagem Celular , Linhagem da Célula , Células Cultivadas , DNA/metabolismo , Epitélio/patologia , Feminino , Deleção de Genes , Genótipo , Imuno-Histoquímica , Camundongos , Modelos Genéticos , Células-Tronco , Fatores de Tempo , beta-Galactosidase/metabolismoRESUMO
Epidemiological studies have consistently shown that an early full-term pregnancy is protective against breast cancer. We hypothesize that the hormonal milieu that is present during pregnancy results in persistent changes in the pattern of gene expression in the mammary gland, leading to permanent changes in cell fate that determine the subsequent proliferative response of the gland. To investigate this hypothesis, we have used suppression subtractive hybridization to identify genes that are persistently up-regulated in the glands of E- and progesterone (P)-treated Wistar-Furth rats 28 d after steroid hormone treatment compared with age-matched virgins. Using this approach, a number of genes displaying persistent altered expression in response to previous treatment with E and P were identified. Two markers have been characterized in greater detail: RbAp46 and a novel gene that specifies a noncoding RNA (designated G.B7). Both were persistently up-regulated in the lobules of the regressed gland and required previous treatment with both E and P for maximal persistent expression. RbAp46 has been implicated in a number of complexes involving chromatin remodeling, suggesting a mechanism whereby epigenetic factors responsible for persistent changes in gene expression may be related to the determination of cell fate. These results provide the first support at the molecular level for the hypothesis that hormone-induced persistent changes in gene expression are present in the involuted mammary gland.
Assuntos
Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glândulas Mamárias Animais/fisiologia , Progesterona/farmacologia , RNA não Traduzido/genética , Animais , Northern Blotting , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/genética , Clonagem Molecular , Estradiol/sangue , Feminino , Hibridização In Situ/métodos , Glândulas Mamárias Animais/efeitos dos fármacos , Dados de Sequência Molecular , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/genética , Perfenazina/farmacologia , Gravidez , Prolactina/sangue , RNA não Traduzido/metabolismo , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Regulação para CimaRESUMO
The distal region (-830 to -720 bp) of the rat whey acidic protein (WAP) gene contains a composite response element (CoRE), which has been demonstrated previously to confer mammary gland-specific and hormonally regulated WAP gene expression. Point mutations in the binding sites for specific transcription factors present within this CoRE have demonstrated the importance of both nuclear factor I (NFI) and STAT5 as well as cooperative interactions with the glucocorticoid receptor (GR) in the regulation of WAP gene expression in the mammary gland of transgenic mice. This study reports the characterization of NFI gene expression during mammary gland development and the identification and cloning of specific NFI isoforms (NFI-A4, NFI-B2, and NFI-X1) from the mouse mammary gland during lactation. Some but not all of these NFI isoforms synergistically activate WAP gene transcription in cooperation with GR and STAT5, as determined using transient cotransfection assays in JEG-3 cells. On both the WAP CoRE and the mouse mammary tumor virus long terminal repeat promoter, the NFI-B isoform preferentially activated gene transcription in cooperation with STAT5A and GR. In contrast, the NFI-A isoform suppressed GR and STAT cooperativity at the WAP CoRE. Finally, unlike their interaction with the NFI consensus binding site in the adenovirus promoter, the DNA-binding specificities of the three NFI isoforms to the palindromic NFI site in the WAP CoRE were not identical, which may partially explain the failure of the NFI-A isoform to cooperate with GR and STAT5A.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas de Ligação a DNA/metabolismo , Proteínas do Leite/genética , Proteínas do Leite/metabolismo , Receptores de Glucocorticoides/metabolismo , Transativadores/metabolismo , Fatores de Transcrição , Transcrição Gênica , Adenoviridae/genética , Processamento Alternativo , Animais , Sítios de Ligação , Ligação Competitiva , Western Blotting , Mama/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células Cultivadas , Clonagem Molecular , DNA/metabolismo , Feminino , Regulação da Expressão Gênica , Lactação , Luciferases/metabolismo , Camundongos , Modelos Genéticos , Fatores de Transcrição NFI , Proteínas Nucleares , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA/metabolismo , Receptores de Glucocorticoides/genética , Elementos de Resposta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , Fator de Transcrição STAT5 , Fatores de Tempo , Transfecção , Proteína 1 de Ligação a Y-Box , beta-Galactosidase/metabolismoRESUMO
Previously, we reported that whereas both signal transducers and activators of transcription (STAT) 5A and STAT5B can be activated with respect to tyrosine phosphorylation and DNA binding potential by Src kinase, only STAT5B was translocated to the nucleus, where it presumably activates unique downstream responses. To help elucidate the functional consequences of STAT5B activation by v-src, the properties of stably transfected NIH-3T3 cells containing both an intact and a dominant negative, COOH-terminal-truncated isoform of STAT5B were investigated. STAT5B enhanced the transforming potential of v-Src as reflected by both an increase in focus formation and growth in soft agar. STAT5B also enhanced v-Src-induced cell cycle progression and cell motility in NIH-3T3 cells. Furthermore, the dominant negative, COOH-terminal-truncated isoform of STAT5B was able to partially suppress v-Src-mediated cell transformation. These results support the hypothesis that STAT5B may enhance Src/Abl-induced tumorigenesis. Accordingly, the equilibrium between STAT5B and STAT5A and their naturally occurring truncated forms may therefore play a key role in the etiology of certain cancers.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas do Leite , Proteína Oncogênica pp60(v-src)/metabolismo , Transativadores/metabolismo , Células 3T3 , Ágar , Animais , Ciclo Celular , Divisão Celular , Movimento Celular , Núcleo Celular/metabolismo , Sobrevivência Celular , Meios de Cultura Livres de Soro/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Citometria de Fluxo , Genes Dominantes , Camundongos , Fenótipo , Plasmídeos/metabolismo , Isoformas de Proteínas , Transporte Proteico , Fator de Transcrição STAT5 , Transdução de Sinais , Fatores de Tempo , Transativadores/química , Transativadores/genética , Transfecção , Transformação GenéticaRESUMO
The transcription factor, CCAAT/enhancer binding protein beta (C/EBPbeta), regulates the expression of genes involved in proliferation and terminal differentiation. Dimerization of the dominant-negative C/EBPbeta-liver-enriched inhibitory protein (LIP) isoform with the C/EBPbeta-liver-enriched activating protein (LAP) isoform inhibits the transcriptional activation of genes involved in differentiation. Consequently, an increase in LIP levels may inhibit terminal differentiation and lead to proliferation. C/EBPbeta-LIP and LAP are crucial for mammary gland development (G. W. Robinson et al., Genes Dev., 12: 1907-1916, 1998; T. N. Seagroves et al., Genes Dev., 12: 1917-1928, 1998) and are also overexpressed in breast cancer (B. Raught et al., Cancer Res., 56: 4382-4386. 1996; C. A. Zahnow et al., J. Natl. Cancer Inst., 89: 1887-1891, 1997); however, little is known about how these isoforms differentially regulate cell cycle progression. To address this question, C/EBPbeta-LIP was overexpressed in both the mammary glands of transgenic mice and in cultured TM3 mammary epithelial cells. Here we report that the involuted mammary glands from transgenic mice overexpressing C/EBPbeta-LIP contain both focal and diffuse alveolar hyperplasia and, less frequently, contain mammary intraepithelial neoplasias (high grade) and invasive and noninvasive carcinomas. Likewise, cultured TM3 cells, stably expressing C/EBPbeta-LIP, showed an increase in proliferation and foci formation attributable to a reentry into S-phase during cellular confluence. These results demonstrate that C/EBPbeta-LIP can induce epithelial proliferation and the formation of mammary hyperplasias and suggest that a C/EBPbeta-LIP-initiated growth cascade may be susceptible to additional oncogenic hits, which could result in the initiation and progression of neoplasia.