RESUMO
Cell-autonomous circadian clocks coordinate tissue homeostasis with a 24-hourly rhythm. The molecular circadian clock machinery controls tissue- and cell type-specific sets of rhythmic genes. Disruptions of clock mechanisms are linked to an increased risk of acquiring diseases, especially those associated with aging, metabolic dysfunction and cancer. Despite rapid advances in understanding the cyclic outputs of different tissue clocks, less is known about how the clocks adapt to their local niche within tissues. We have discovered that tissue stiffness regulates circadian clocks, and that this occurs in a cell-type-dependent manner. In this Review, we summarise new work linking the extracellular matrix with differential control of circadian clocks. We discuss how the changes in tissue structure and cellular microenvironment that occur throughout life may impact on the molecular control of circadian cycles. We also consider how altered clocks may have downstream impacts on the acquisition of diseases.
Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/genética , Matriz Extracelular/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Mecanotransdução Celular , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Microambiente Celular/genética , Criptocromos/genética , Criptocromos/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Matriz Extracelular/química , Homeostase/genética , Humanos , Mamíferos , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
The proapoptotic Bcl-2 protein Bax is predominantly found in the cytosol of nonapoptotic cells and is commonly thought to translocate to mitochondria following an apoptotic stimulus. The current model for Bax activation is that BH3 proteins bind to cytosolic Bax, initiating mitochondrial targeting and outer-membrane permeabilization. Here, we challenge this and show that Bax is constitutively targeted to mitochondria but in nonapoptotic cells is constantly translocated back to the cytosol. Using live-cell spinning-disk confocal imaging with a combination of FLIP, FRAP, and photoactivatable GFP-Bax, we demonstrate that disrupting adhesion-dependent survival signals slows the rate of Bax's dissociation from mitochondria, leading to its accumulation on the outer mitochondrial membrane. The overall accumulation of mitochondrial Bax following loss of survival signaling sensitizes cells to proapoptotic BH3 proteins. Our findings show that Bax is normally in a dynamic equilibrium between cytosol and mitochondria, enabling fluctuations in survival signals to finely adjust apoptotic sensitivity.
Assuntos
Apoptose , Citosol/metabolismo , Mitocôndrias/metabolismo , Proteína X Associada a bcl-2/genética , Animais , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Membranas Mitocondriais/metabolismo , Transfecção , Proteína X Associada a bcl-2/metabolismoRESUMO
The circadian clock is an autonomous molecular feedback loop inside almost every cell in the body. We have shown that the mammary epithelial circadian clock is regulated by the cellular microenvironment. Moreover, a stiff extracellular matrix dampens the oscillations of the epithelial molecular clock. Here, we extend this analysis to other tissues and cell types, and identify an inverse relationship between circadian clocks in epithelia and fibroblasts. Epithelial cells from mammary gland, lung and skin have significantly stronger oscillations of clock genes in soft 3D microenvironments, compared to stiff 2D environments. Fibroblasts isolated from the same tissues show the opposite response, exhibiting stronger oscillations and more prolonged rhythmicity in stiff microenvironments. RNA analysis identified that a subset of mammary epithelial clock genes, and their regulators, are upregulated in 3D microenvironments in soft compared to stiff gels. Furthermore, the same genes are inversely regulated in fibroblasts isolated from the same tissues. Thus, our data reveal for the first time an intrinsic difference in the regulation of circadian genes in epithelia and fibroblasts.
Assuntos
Microambiente Celular/genética , Relógios Circadianos/genética , Mecanotransdução Celular/genética , Proteínas Circadianas Period/genética , Animais , Células Epiteliais/metabolismo , Feminino , Fibroblastos/metabolismo , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Camundongos , RNA/genética , Pele/crescimento & desenvolvimento , Pele/metabolismo , Células Estromais/metabolismoRESUMO
The role of the local microenvironment in influencing cell behavior is central to both normal development and cancer formation. Here, we show that sprouty 1 (SPRY1) modulates the microenvironment to enable proper mammary branching morphogenesis. This process occurs through negative regulation of epidermal growth factor receptor (EGFR) signaling in mammary stroma. Loss of SPRY1 resulted in up-regulation of EGFR-extracellular signal-regulated kinase (ERK) signaling in response to amphiregulin and transforming growth factor alpha stimulation. Consequently, stromal paracrine signaling and ECM remodeling is augmented, leading to increased epithelial branching in the mutant gland. By contrast, down-regulation of EGFR-ERK signaling due to gain of Sprouty function in the stroma led to stunted epithelial branching. Taken together, our results show that modulation of stromal paracrine signaling and ECM remodeling by SPRY1 regulates mammary epithelial morphogenesis during postnatal development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Epitélio/crescimento & desenvolvimento , Receptores ErbB/metabolismo , Matriz Extracelular/metabolismo , Glândulas Mamárias Animais/metabolismo , Proteínas de Membrana/metabolismo , Morfogênese , Comunicação Parácrina , Fosfoproteínas/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Anfirregulina/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Colágeno/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Epitélio/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ligantes , Masculino , Glândulas Mamárias Animais/efeitos dos fármacos , Proteínas de Membrana/deficiência , Camundongos Knockout , Camundongos Nus , Morfogênese/efeitos dos fármacos , Mutação/genética , Comunicação Parácrina/efeitos dos fármacos , Fosfoproteínas/deficiência , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Imagem com Lapso de Tempo , Fator de Crescimento Transformador alfa/farmacologiaRESUMO
BACKGROUND: Stem cells are precursors for all mammary epithelia, including ductal and alveolar epithelia, and myoepithelial cells. In vivo mammary epithelia reside in a tissue context and interact with their milieu via receptors such as integrins. Extracellular matrix receptors coordinate important cellular signalling platforms, of which integrins are the central architects. We have previously shown that integrins are required for mammary epithelial development and function, including survival, cell cycle, and polarity, as well as for the expression of mammary-specific genes. In the present study we looked at the role of integrins in mammary epithelial stem cell self-renewal. METHODS: We used an in vitro stem cell assay with primary mouse mammary epithelial cells isolated from genetically altered mice. This involved a 3D organoid assay, providing an opportunity to distinguish the stem cell- or luminal progenitor-driven organoids as structures with solid or hollow appearances, respectively. RESULTS: We demonstrate that integrins are essential for the maintenance and self-renewal of mammary epithelial stem cells. Moreover integrins activate the Rac1 signalling pathway in stem cells, which leads to the stimulation of a Wnt pathway, resulting in expression of ß-catenin target genes such as Axin2 and Lef1. CONCLUSIONS: Integrin/Rac signalling has a role in specifying the activation of a canonical Wnt pathway that is required for mammary epithelial stem cell self-renewal.
Assuntos
Autorrenovação Celular/fisiologia , Células Epiteliais/fisiologia , Integrinas/metabolismo , Neuropeptídeos/metabolismo , Células-Tronco/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Epitélio/fisiologia , Feminino , Integrinas/genética , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/fisiologia , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Organoides/fisiologia , Cultura Primária de Células/métodos , Transdução de Sinais/fisiologia , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Circadian rhythms maintain tissue homeostasis during the 24-h day-night cycle. Cell-autonomous circadian clocks play fundamental roles in cell division, DNA damage responses and metabolism. Circadian disruptions have been proposed as a contributing factor for cancer initiation and progression, although definitive evidence for altered molecular circadian clocks in cancer is still lacking. In this study, we looked at circadian clocks in breast cancer. METHODS: We isolated primary tumours and normal tissues from the same individuals who had developed breast cancer with no metastases. We assessed circadian clocks within primary cells of the patients by lentiviral expression of circadian reporters, and the levels of clock genes in tissues by qPCR. We histologically examined collagen organisation within the normal and tumour tissue areas, and probed the stiffness of the stroma adjacent to normal and tumour epithelium using atomic force microscopy. RESULTS: Epithelial ducts were disorganised within the tumour areas. Circadian clocks were altered in cultured tumour cells. Tumour regions were surrounded by stroma with an altered collagen organisation and increased stiffness. Levels of Bmal1 messenger RNA (mRNA) were significantly altered in the tumours in comparison to normal epithelia. CONCLUSION: Circadian rhythms are suppressed in breast tumour epithelia in comparison to the normal epithelia in paired patient samples. This correlates with increased tissue stiffness around the tumour region. We suggest possible involvement of altered circadian clocks in the development and progression of breast cancer.
Assuntos
Neoplasias da Mama/patologia , Mama/patologia , Relógios Circadianos/fisiologia , Epitélio/patologia , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Idoso , Mama/citologia , Estudos de Coortes , Colágeno/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Cultura Primária de Células , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
The mechanical properties of the cell nucleus change to allow cells to migrate, but how chromatin modifications contribute to nuclear deformability has not been defined. Here, we demonstrate that a major factor in this process involves epigenetic changes that underpin nuclear structure. We investigated the link between cell adhesion and epigenetic changes in T-cells, and demonstrate that T-cell adhesion to VCAM1 via α4ß1 integrin drives histone H3 methylation (H3K9me2/3) through the methyltransferase G9a. In this process, active G9a is recruited to the nuclear envelope and interacts with lamin B1 during T-cell adhesion through α4ß1 integrin. G9a activity not only reorganises the chromatin structure in T-cells, but also affects the stiffness and viscoelastic properties of the nucleus. Moreover, we further demonstrated that these epigenetic changes were linked to lymphocyte movement, as depletion or inhibition of G9a blocks T-cell migration in both 2D and 3D environments. Thus, our results identify a novel mechanism in T-cells by which α4ß1 integrin signaling drives specific chromatin modifications, which alter the physical properties of the nucleus and thereby enable T-cell migration.
Assuntos
Movimento Celular , Núcleo Celular/fisiologia , Epigênese Genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Integrina alfa4beta1/metabolismo , Linfócitos/imunologia , Animais , Adesão Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/química , Células HEK293 , Histonas/metabolismo , Humanos , Células Jurkat , Metilação , Camundongos Endogâmicos C57BL , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The myocyte enhancer factor 2 and histone deacetylase (MEF2-HDAC) axis is a master regulator of different developmental programs and adaptive responses in adults. In this paper, we have investigated the contribution of the axis to the regulation of epithelial morphogenesis, using 3D organotypic cultures of MCF10A cells as a model. We have demonstrated that MEF2 transcriptional activity is upregulated during acini formation, which coincides with exit from the proliferative phase. Upregulation of the transcription of MEF2 proteins is coupled to downregulation of HDAC7, which occurs independently from changes in mRNA levels, and proteasome- or autophagy-mediated degradation. During acini formation, the MEF2-HDAC axis contributes to the promotion of cell cycle exit, through the engagement of the CDK inhibitor CDKN1A. Only in proliferating cells can HDAC7 bind to the first intron of the CDKN1A gene, a region characterized by epigenetic markers of active promoters and enhancers. In cells transformed by the oncogene HER2 (ERBB2), acini morphogenesis is altered, MEF2 transcription is repressed and HDAC7 is continuously expressed. Importantly, reactivation of MEF2 transcriptional activity in these cells, through the use of a HER2 inhibitor or by enhancing MEF2 function, corrected the proliferative defect and re-established normal acini morphogenesis.
Assuntos
Células Acinares/metabolismo , Células Epiteliais/metabolismo , Histona Desacetilases/metabolismo , Fatores de Transcrição MEF2/metabolismo , Linhagem Celular , Proliferação de Células/genética , Proliferação de Células/fisiologia , Imunoprecipitação da Cromatina , Imunofluorescência , Histona Desacetilases/genética , Humanos , Immunoblotting , Fatores de Transcrição MEF2/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
FGF signaling is essential for mammary gland development, yet the mechanisms by which different members of the FGF family control stem cell function and epithelial morphogenesis in this tissue are not well understood. Here, we have examined the requirement of Fgfr2 in mouse mammary gland morphogenesis using a postnatal organ regeneration model. We found that tissue regeneration from basal stem cells is a multistep event, including luminal differentiation and subsequent epithelial branching morphogenesis. Basal cells lacking Fgfr2 did not generate an epithelial network owing to a failure in luminal differentiation. Moreover, Fgfr2 null epithelium was unable to undergo ductal branch initiation and elongation due to a deficiency in directional migration. We identified FGF10 and FGF2 as stromal ligands that control distinct aspects of mammary ductal branching. FGF10 regulates branch initiation, which depends on directional epithelial migration. By contrast, FGF2 controls ductal elongation, requiring cell proliferation and epithelial expansion. Together, our data highlight a pleiotropic role of Fgfr2 in stem cell differentiation and branch initiation, and reveal that different FGF ligands regulate distinct aspects of epithelial behavior.
Assuntos
Epitélio/crescimento & desenvolvimento , Fator 10 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Morfogênese , Animais , Animais Recém-Nascidos , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Feminino , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ligantes , Glândulas Mamárias Animais/crescimento & desenvolvimento , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Nus , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Fosfatidilinositol 3-Quinases/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Regeneração/efeitos dos fármacos , Regeneração/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismoRESUMO
Epithelial cell adhesion to the surrounding extracellular matrix is necessary for their proper behavior and function. During pregnancy and lactation, mammary epithelial cells (MECs) receive signals from their interaction with laminin via ß1-integrin (ß1-itg) to establish apico-basal polarity and to differentiate in response to prolactin. Downstream of ß1-itg, the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both lactational differentiation and the establishment of apico-basal polarity. ILK is an adaptor protein that forms the IPP complex with PINCH and Parvins, which are central to its adaptor functions. However, it is not known how ILK and its interacting partners control tissue-specific gene expression. Expression of ILK mutants, which weaken the interaction between ILK and Parvin, revealed that Parvins have a role in mammary epithelial differentiation. This conclusion was supported by shRNA-mediated knockdown of the Parvins. In addition, shRNA knockdown of the Parvin-binding guanine nucleotide exchange factor αPix prevented prolactin-induced differentiation. αPix depletion did not disrupt focal adhesions, MEC proliferation, or polarity. This suggests that αPix represents a differentiation-specific bifurcation point in ß1-itg-ILK adhesive signaling. In summary, this study has identified a new role for Parvin and αPix downstream of the integrin-ILK signaling axis for MEC differentiation. J. Cell. Physiol. 231: 2408-2417, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Diferenciação Celular , Células Epiteliais/citologia , Integrina beta1/metabolismo , Glândulas Mamárias Animais/citologia , Proteínas dos Microfilamentos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Camundongos , Mutação/genética , Prolactina/farmacologia , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
High mammographic density is the most important risk factor for breast cancer, after ageing. However, the composition, architecture, and mechanical properties of high X-ray density soft tissues, and the causative mechanisms resulting in different mammographic densities, are not well described. Moreover, it is not known how high breast density leads to increased susceptibility for cancer, or the extent to which it causes the genomic changes that characterise the disease. An understanding of these principals may lead to new diagnostic tools and therapeutic interventions.
Assuntos
Densidade da Mama , Neoplasias da Mama/etiologia , Neoplasias da Mama/patologia , Biomarcadores , Neoplasias da Mama/epidemiologia , Feminino , Predisposição Genética para Doença , Humanos , Incidência , Glândulas Mamárias Humanas/diagnóstico por imagem , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Mamografia , Prognóstico , Risco , Células Estromais/metabolismo , Microambiente TumoralRESUMO
Circadian clocks respond to environmental time cues to coordinate 24-hour oscillations in almost every tissue of the body. In the breast, circadian clocks regulate the rhythmic expression of numerous genes. Disrupted expression of circadian genes can alter breast biology and may promote cancer. Here we overview circadian mechanisms, and the connection between the molecular clock and breast biology. We describe how disruption of circadian genes contributes to cancer via multiple mechanisms, and link this to increased tumour risk in women who work irregular shift patterns. Understanding the influence of circadian rhythms on breast cancer could lead to more efficacious therapies, reformed public health policy and improved patient outcome.
Assuntos
Neoplasias da Mama/etiologia , Relógios Circadianos , Animais , Mama/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação da Expressão Gênica , Humanos , Especificidade de Órgãos , Núcleo Supraquiasmático/fisiologia , Núcleo Supraquiasmático/fisiopatologiaRESUMO
BACKGROUND: High mammographic density is a therapeutically modifiable risk factor for breast cancer. Although mammographic density is correlated with the relative abundance of collagen-rich fibroglandular tissue, the causative mechanisms, associated structural remodelling and mechanical consequences remain poorly defined. In this study we have developed a new collaborative bedside-to-bench workflow to determine the relationship between mammographic density, collagen abundance and alignment, tissue stiffness and the expression of extracellular matrix organising proteins. METHODS: Mammographic density was assessed in 22 post-menopausal women (aged 54-66 y). A radiologist and a pathologist identified and excised regions of elevated non-cancerous X-ray density prior to laboratory characterization. Collagen abundance was determined by both Masson's trichrome and Picrosirius red staining (which enhances collagen birefringence when viewed under polarised light). The structural specificity of these collagen visualisation methods was determined by comparing the relative birefringence and ultrastructure (visualised by atomic force microscopy) of unaligned collagen I fibrils in reconstituted gels with the highly aligned collagen fibrils in rat tail tendon. Localised collagen fibril organisation and stiffness was also evaluated in tissue sections by atomic force microscopy/spectroscopy and the abundance of key extracellular proteins was assessed using mass spectrometry. RESULTS: Mammographic density was positively correlated with the abundance of aligned periductal fibrils rather than with the abundance of amorphous collagen. Compared with matched tissue resected from the breasts of low mammographic density patients, the highly birefringent tissue in mammographically dense breasts was both significantly stiffer and characterised by large (>80 µm long) fibrillar collagen bundles. Subsequent proteomic analyses not only confirmed the absence of collagen fibrosis in high mammographic density tissue, but additionally identified the up-regulation of periostin and collagen XVI (regulators of collagen fibril structure and architecture) as potential mediators of localised mechanical stiffness. CONCLUSIONS: These preliminary data suggest that remodelling, and hence stiffening, of the existing stromal collagen microarchitecture promotes high mammographic density within the breast. In turn, this aberrant mechanical environment may trigger neoplasia-associated mechanotransduction pathways within the epithelial cell population.
Assuntos
Neoplasias da Mama/genética , Colágeno/metabolismo , Glândulas Mamárias Humanas/anormalidades , Mamografia/métodos , Proteômica , Idoso , Animais , Densidade da Mama , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Moléculas de Adesão Celular/metabolismo , Colágeno/ultraestrutura , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Microscopia de Força Atômica , Pessoa de Meia-Idade , Ratos , Fatores de RiscoRESUMO
Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell-matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical-basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity.
Assuntos
Membrana Basal/metabolismo , Polaridade Celular , Células Epiteliais/fisiologia , Integrinas/metabolismo , Neoplasias/fisiopatologia , Animais , Humanos , Microtúbulos/metabolismo , Neoplasias/patologiaRESUMO
Understanding how cell cycle is regulated in normal mammary epithelia is essential for deciphering defects of breast cancer and therefore for developing new therapies. Signals provided by both the extracellular matrix and growth factors are essential for epithelial cell proliferation. However, the mechanisms by which adhesion controls cell cycle in normal epithelia are poorly established. In this study, we describe the consequences of removing the ß1-integrin gene from primary cultures of mammary epithelial cells in situ, using CreER. Upon ß1-integrin gene deletion, the cells were unable to progress efficiently through S-phase, but were still able to undergo collective two-dimensional migration. These responses are explained by the presence of ß3-integrin in ß1-integrin-null cells, indicating that integrins containing different ß-subunits exert differential control on mammary epithelial proliferation and migration. ß1-Integrin deletion did not inhibit growth factor signaling to Erk or prevent the recruitment of core adhesome components to focal adhesions. Instead the S-phase arrest resulted from defective Rac activation and Erk translocation to the nucleus. Rac inhibition prevented Erk translocation and blocked proliferation. Activated Rac1 rescued the proliferation defect in ß1-integrin-depleted cells, indicating that this GTPase is essential in propagating proliferative ß1-integrin signals. These results show that ß1-integrins promote cell cycle in mammary epithelial cells, whereas ß3-integrins are involved in migration.
Assuntos
Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Integrina beta1/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Movimento Celular/genética , Proliferação de Células , Células Cultivadas , Feminino , Citometria de Fluxo , Immunoblotting , Integrina beta1/genética , Masculino , Camundongos , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Breast cancer stem cells (BCSC) are presumed to be responsible for treatment resistance, tumor recurrence and metastasis of breast tumors. However, development of BCSC-targeting therapies has been held back by their heterogeneity and the lack of BCSC-selective molecular targets. Here, we demonstrate that RAC1B, the only known alternatively spliced variant of the small GTPase RAC1, is expressed in a subset of BCSCs in vivo and its function is required for the maintenance of BCSCs and their chemoresistance to doxorubicin. In human breast cancer cell line MCF7, RAC1B is required for BCSC plasticity and chemoresistance to doxorubicin in vitro and for tumor-initiating abilities in vivo. Unlike Rac1, Rac1b function is dispensable for normal mammary gland development and mammary epithelial stem cell (MaSC) activity. In contrast, loss of Rac1b function in a mouse model of breast cancer hampers the BCSC activity and increases their chemosensitivity to doxorubicin treatment. Collectively, our data suggest that RAC1B is a clinically relevant molecular target for the development of BCSC-targeting therapies that may improve the effectiveness of doxorubicin-mediated chemotherapy.
Assuntos
Neoplasias da Mama , Neoplasias Mamárias Animais , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Neoplasias Mamárias Animais/patologia , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologiaRESUMO
In mammary epithelial cells (MECs), prolactin-induced signaling and gene expression requires integrin-mediated cell adhesion to basement membrane (BM). In the absence of proper cell-BM interactions, for example, culturing cells on collagen-coated plastic dishes, signal propagation is substantially impaired. Here we demonstrate that the RhoA-Rok-myosin II pathway accounts for the ineffectiveness of prolactin signaling in MECs cultured on collagen I. Under these culture conditions, the RhoA pathway is activated, leading to downregulation of prolactin receptor expression and reduced prolactin signaling. Enforced activation of RhoA in MECs cultured on BM suppresses prolactin receptor levels, and prevents prolactin-induced Stat5 tyrosine phosphorylation and ß-casein expression. Overexpression of dominant negative RhoA in MECs cultured on collagen I, or inhibiting Rok activity, increases prolactin receptor expression, and enhances prolactin signaling. In addition, inhibition of myosin II ATPase activity by blebbistatin also exerts a beneficial effect on prolactin receptor expression and prolactin signaling, suggesting that tension exerted by the collagen substratum, in collaboration with the RhoA-Rok-myosin II pathway, contributes to the failure of prolactin signaling. Furthermore, MECs cultured on laminin-coated plastic have similar morphology and response to prolactin as those cultured on collagen I. They display high levels of RhoA activity and are inefficient in prolactin signaling, stressing the importance of matrix stiffness in signal transduction. Our results reveal that RhoA has a central role in determining the fate decisions of MECs in response to cell-matrix interactions.
Assuntos
Glândulas Mamárias Animais/metabolismo , Miosina Tipo II/metabolismo , Prolactina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Sequência de Bases , Adesão Celular , Diferenciação Celular , Células Cultivadas , Microambiente Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/efeitos dos fármacos , Camundongos , Miosina Tipo II/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Prolactina/genética , Receptores da Prolactina/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTPRESUMO
Ectodysplasin A receptor (EDAR) is a death receptor in the Tumour Necrosis Factor Receptor (TNFR) superfamily with roles in the development of hair follicles, teeth and cutaneous glands. Here we report that human Oestrogen Receptor (ER) negative breast carcinomas which display squamous differentiation express EDAR strongly. Using a mouse model with a high Edar copy number, we show that elevated EDAR signalling results in a high incidence of mammary tumours in breeding female mice. These tumours resemble the EDAR-high human tumours in that they are characterised by a lack of oestrogen receptor expression, contain extensive squamous metaplasia, and display strong ß-catenin transcriptional activity. In the mouse model, all of the tumours carry somatic deletions of the third exon of the CTNNB1 gene that encodes ß-catenin. Deletion of this exon yields unconstrained ß-catenin signalling activity. We also demonstrate that ß-catenin activity is required for transformed cell growth, showing that increased EDAR signalling creates an environment in which ß-catenin activity can readily promote tumourigenesis. Together, this work identifies a novel death receptor oncogene in breast cancer, whose mechanism of transformation is based on the interaction between the WNT and Ectodysplasin A (EDA) pathways.
Assuntos
Receptores da EctodisplasinaRESUMO
Apoptosis is controlled by a signaling equilibrium between prosurvival and proapoptotic pathways, such that unwanted apoptosis is avoided, but when required it occurs rapidly and efficiently. Many apoptosis regulators display dual roles, depending upon whether a cell has received an apoptotic stimulus or not. Here, we identify a novel and unexpected function for X-linked inhibitor of apoptosis (XIAP) that occurs when apoptosis is triggered under physiological conditions. We show that in response to loss of survival signals provided by cell adhesion, endogenous XIAP translocates from the cytosol into a mitochondrial 400-kDa complex and that this occurs very early in the apoptosis process. Membrane-associated XIAP induces mitochondrial outer membrane permeabilization leading to cytochrome c and Smac release, which is dependent on Bax and Bak. Thus, although XIAP suppresses apoptosis in healthy cells, our data indicate that XIAP may contribute to it in response to a proapoptotic signal such as loss of extracellular matrix-dependent survival signaling. We suggest that, as with Bcl-2 family proteins, more diverse functions for XIAP exist than previously identified. Moreover, switching the function of proteins from anti- to proapoptotic forms may be a common theme in the efficient execution of cell death.
Assuntos
Apoptose/fisiologia , Citosol/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Citocromos c/genética , Citocromos c/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Permeabilidade , Transporte Proteico/fisiologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
The expression of tissue-specific genes during mammary gland differentiation relies on the coincidence of two distinct signaling events: the continued engagement of beta1 integrins with the extracellular matrix (ECM) and a hormonal stimulus from prolactin (Prl). How the integrin and Prl receptor (PrlR) systems integrate to regulate milk protein gene synthesis is unknown. In this study, we identify Rac1 as a key link. Dominant-negative Rac1 prevents Prl-induced synthesis of the milk protein beta-casein in primary mammary epithelial cells cultured as three-dimensional acini on basement membrane. Conversely, activated Rac1 rescues the defective beta-casein synthesis that occurs under conditions not normally permissive for mammary differentiation, either in beta1 integrin-null cells or in wild-type cells cultured on collagen. Rac1 is required downstream of integrins for activation of the PrlR/Stat5 signaling cascade. Cdc42 is also necessary for milk protein synthesis but functions via a distinct mechanism to Rac1. This study identifies the integration of signals provided by ECM and hormones as a novel role for Rho family guanosine triphosphatases.