RESUMEN

Melanoma pathogenesis from normal neural crest-derived melanocytes is often fatal due to aggressive cell invasion throughout the body. The identification of signals that reprogram de-differentiated, metastatic melanoma cells to a less aggressive and stable phenotype would provide a novel strategy to limit disease progression. In this study, we identify and test the function of developmental signals within the chick embryonic neural crest microenvironment to reprogram and sustain the transition of human metastatic melanoma to a neural crest cell-like phenotype. Results reveal that co-culture of the highly aggressive and metastatic human melanoma cell line C8161 upregulate a marker of melanosome formation (Mart-1) in the presence of embryonic day 3.5 chick trunk dorsal root ganglia. We identify nerve growth factor (NGF) as the signal within this tissue driving Mart-1 re-expression and show that NGF receptors trkA and p75 cooperate to induce Mart-1 re-expression. Furthermore, Mart-1 expressing C8161 cells acquire a gene signature of poorly aggressive C81-61 cells. These data suggest that targeting NGF signaling may yield a novel strategy to reprogram metastatic melanoma toward a benign cell type.

RESUMEN

Neural crest cells are both highly migratory and significant to vertebrate organogenesis. However, the signals that regulate neural crest cell migration remain unclear. In this study, we test the function of differential screening-selected gene aberrant in neuroblastoma (DAN), a bone morphogenetic protein (BMP) antagonist we detected by analysis of the chick cranial mesoderm. Our analysis shows that, before neural crest cell exit from the hindbrain, DAN is expressed in the mesoderm, and then it becomes absent along cell migratory pathways. Cranial neural crest and metastatic melanoma cells avoid DAN protein stripes in vitro. Addition of DAN reduces the speed of migrating cells in vivo and in vitro, respectively. In vivo loss of function of DAN results in enhanced neural crest cell migration by increasing speed and directionality. Computer model simulations support the hypothesis that DAN restrains cell migration by regulating cell speed. Collectively, our results identify DAN as a novel factor that inhibits uncontrolled neural crest and metastatic melanoma invasion and promotes collective migration in a manner consistent with the inhibition of BMP signaling.

Asunto(s)

Proteínas Aviares/metabolismo , Movimiento Celular , Pollos/metabolismo , Melanoma/metabolismo , Cresta Neural/embriología , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas Aviares/genética , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Embrión de Pollo , Pollos/genética , Melanoma/genética , Melanoma/patología , Invasividad Neoplásica , Cresta Neural/patología , Proteínas Supresoras de Tumor/genética

RESUMEN

UNLABELLED: Metastatic dissemination drives the high mortality associated with melanoma. However, difficulties in visualizing in vivo cell dynamics during metastatic invasion have limited our understanding of these cell behaviors. Recent evidence has revealed that melanoma cells exploit portions of their ancestral embryonic neural crest emigration program to facilitate invasion. What remains to be determined is how embryonic microenvironmental signals influence invasive melanoma cell behavior, and whether these signals are relevant to human disease. To address these questions, we interrogated the role of the neural crest microenvironment in dictating the spatiotemporal pattern of melanoma cell invasion in the chick embryo using 2-photon time-lapse microscopy. Results reveal that both permissive and inhibitory neural crest microenvironmental signals regulate the timing and direction of melanoma invasion to coincide with the neural crest migration pattern. These cues include bidirectional signaling mediated through the ephrin family of receptor tyrosine kinases. We demonstrate that EphB6 reexpression forces metastatic melanoma cells to deviate from the canonical migration pattern observed in the chick embryo transplant model. Furthermore, EphB6-expressing melanoma cells display significantly reduced metastatic potential in a chorioallantoic membrane (CAM) metastasis assay. These data on melanoma invasion in the embryonic neural crest and CAM microenvironments identify EphB6 as a metastasis suppressor in melanoma, likely acting at the stage of intravasation. IMPLICATIONS: This article links cellular metastasis to behaviors observed in the ancestrally related embryonic neural crest and demonstrates the powerful influence of the embryonic microenvironment in regulating cell migratory behavior.

Asunto(s)

Movimiento Celular/genética , Melanoma/genética , Invasividad Neoplásica/genética , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Animales , Embrión de Pollo , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/patología , Metástasis de la Neoplasia , Cresta Neural/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de la Familia Eph , Transducción de Señal

RESUMEN

Neural crest (NC) cells undergo an epithelial to mesenchymal transition (EMT) in order to exit from the dorsal neural tube. Similarly, ancestrally related melanoma cells employ an EMT-like event during the initial stages of metastasis to dissociate from surrounding keratinocytes. Whether or not the molecular pathogenesis and cellular dynamics of melanoma metastasis resemble the embryonic NC invasion program is unclear. Here, we highlight advances in our understanding of tumor cell behaviors and plasticity, focusing on the relationship between melanoma and the NC invasion programs. We summarize recent discoveries of NC cell guidance and emerging in vivo imaging strategies that permit single cell resolution of fluorescently labeled tumor cells, with a focus on our recently developed in vivo chick embryo transplant model. Crucial to the molecular pathogenesis of metastasis, we highlight advances in gene profiling of small cell numbers, including our novel ability to gather gene expression information during distinct stages of melanoma invasion. Lastly, we present preliminary details of a comparison of specific genetic pathways associated with the early phases of melanoma invasion and known NC induction and migration signals. Our results suggest that malignant melanoma cells hijack portions of the NC program to promote plasticity and facilitate metastasis. In summary, there is considerable power in combining an in vivo model system with molecular analysis of gene expression, within the context of established developmental signaling pathways, to identify and study the molecular mechanisms of metastasis.

Asunto(s)

Melanoma/patología , Cresta Neural/patología , Animales , Línea Celular Tumoral , Embrión de Pollo , Transición Epitelial-Mesenquimal , Perfilación de la Expresión Génica , Humanos , Melanoma/genética , Modelos Animales , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Cresta Neural/metabolismo

RESUMEN

The dynamic nature of the developing embryo makes it challenging to understand complex morphogenetic events using information from large-scale gene expression patterns. What would be more insightful is molecular profiling of small numbers of cells selectively surveyed at specific developmental stages. However, detecting gene expression profile information from small numbers of cells (<10) in homogenous tissue has remained a major challenge. Here, we describe the use of laser capture microdissection (LCM), immunohistochemistry (IHC), and RT-qPCR to extract gene profile information in distinct embryo tissue more precisely than is possible with any other method. We use the chick embryo model system and combine electroporation and dual-label IHC to specifically identify cells for harvest by LCM without significant degradation of total RNA. We describe the development of a pre-amplification protocol for small subpopulations of cells to produce sensitive RT-qPCR results. The gene-specific pre-amplification efficiently and linearly amplifies only gene transcripts of interest from the harvested material without the need for RNA isolation. By combining the above techniques with microfluidic RT-qPCR, we robustly analyze the expression of ∼300 genes from as few as 10 cells harvested by LCM. Together, this protocol presents a confident isolation and means of sensitive expression analysis of small cell numbers from tissues and overcomes a technical hurdle that limits gene profiling.

Asunto(s)

Pollos/genética , Perfilación de la Expresión Génica/métodos , Captura por Microdisección con Láser/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica/métodos

RESUMEN

Cancer cells must regulate plasticity and invasion to survive and metastasize. However, the identification of targetable mechanisms to inhibit metastasis has been slow. Signaling programs that drive stem and progenitor cells during normal development offer an inroad to discover mechanisms common to metastasis. Using a chick embryo transplant model, we have compared molecular signaling programs of melanoma and their embryonic progenitors, the neural crest. We report that malignant melanoma cells hijack portions of the embryonic neural crest invasion program. Genes associated with neural crest induction, delamination, and migration are dynamically regulated by melanoma cells exposed to an embryonic neural crest microenvironment. Specifically, we demonstrate that metastatic melanoma cells exploit neural crest-related receptor tyrosine kinases to increase plasticity and facilitate invasion while primary melanocytes may actively suppress these responses under the same microenvironmental conditions. We conclude that aberrant regulation of neural crest developmental genes promotes plasticity and invasiveness in malignant melanoma.

Asunto(s)

Movimiento Celular , Melanoma/patología , Cresta Neural/embriología , Cresta Neural/patología , Animales , Diferenciación Celular , Línea Celular Tumoral , Microambiente Celular/genética , Embrión de Pollo , Progresión de la Enfermedad , Efrinas/genética , Efrinas/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Melanocitos/patología , Invasividad Neoplásica , Trasplante de Neoplasias , Cresta Neural/metabolismo , Receptores de la Familia Eph/genética , Receptores de la Familia Eph/metabolismo , Transducción de Señal/genética

RESUMEN

Cell proliferation is crucial to tissue growth and form during embryogenesis, yet dynamic tracking of cell cycle progression and cell position presents a challenging roadblock. We have developed a fluorescent cell cycle indicator and single cell analysis method, called CycleTrak, which allows for better spatiotemporal resolution and quantification of cell cycle phase and cell position than current methods. Our method was developed on the basis of the existing Fucci method. CycleTrak uses a single lentiviral vector that integrates mKO2-hCdt1 (30/120), and a nuclear-localized eGFP reporter. The single vector and nuclear localized fluorescence signals simplify delivery into cells and allow for rapid, automated cell tracking and cell cycle phase readout in single and subpopulations of cells. We validated CycleTrak performance in metastatic melanoma cells and identified novel cell cycle dynamics in vitro and in vivo after transplantation and 3D confocal time-lapse imaging in a living chick embryo.

Asunto(s)

Ciclo Celular , Rastreo Celular/métodos , Análisis de la Célula Individual/métodos , Animales , Embrión de Pollo , Fluorescencia , Vectores Genéticos , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Lentivirus , Imagen de Lapso de Tiempo

RESUMEN

Cathepsin D (CatD) is a lysosomal aspartyl endopeptidase originally considered a "house keeping enzyme" involved in the clearance of unwanted proteins. However, recent studies have revealed CatD's involvement in apoptosis and autophagy, thus signifying an important function in the proper development and maintenance of multi-cellular organs. In the mammary gland, matrix degradation and the remodeling process are orchestrated by proteolytic enzymes, but the role of CatD at distinct developmental stages has remained mostly unexplored. Based on our previous studies we sought to address the role of this endopeptidase in mammary gland development and remodeling. By employing a mouse model, we report a previously unidentified participation of CatD in different stages of mammary gland development. Our findings reveal that CatD undergoes distinct protein processing at different stages of mammary gland development, and this customized processing results in differential enzymatic activity (constitutive and low pH activatable) best fitting particular stage(s) of development. In addition, at the onset of involution the N-glycan structure of this endopeptidase switches from a mixed high mannose and hybrid structure to an almost exclusively high mannose type, but reverts back to the original N-glycan composition by day 4 of involution. Our findings illuminate (at least in part) the "raison d'être" for CatD's numerous and highly regulated proteolytic processing steps from the pro-form to the mature enzyme. In the mammary gland, specific cleavage product(s) perform specialized function(s) befitting each stage of remodeling. It is noteworthy that deregulated synthesis, secretion and glycosylation of CatD are hallmarks of cancer progression. Thus, identifying the role of CatD in a dynamic normal tissue undergoing highly regulated cycles of remodeling could provide valuable information illuminating the deregulation of CatD associated with cancer development and metastasis.

Asunto(s)

Catepsina D/metabolismo , Glándulas Mamarias Animales/enzimología , Glándulas Mamarias Animales/fisiología , Aciltransferasas/genética , Aciltransferasas/metabolismo , Animales , Apoptosis , Autofagia , Membrana Basal/enzimología , Membrana Basal/fisiología , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/metabolismo , Matriz Extracelular/enzimología , Matriz Extracelular/fisiología , Femenino , Técnica del Anticuerpo Fluorescente , Regulación Enzimológica de la Expresión Génica , Glicosilación , Humanos , Lactancia , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , N-Acetilglucosaminiltransferasas , Reacción en Cadena de la Polimerasa , Embarazo , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismo

RESUMEN

Vertebrate development is best studied in an intact embryo model, but a robust interface between time-lapse microscopy and in vivo embryo health and maintenance can be difficult to achieve in model systems that rely on external factors for life support. This protocol presents a system for in ovo culture and time-lapse imaging of fluorescently labeled cells within living avian embryos, using a Teflon membrane that is oxygen-permeable and liquid-impermeable. The protocol describes the Teflon membrane assembly (the assembly size can be changed to fit smaller eggs, such as those of the quail), its interface with the egg window, and the use of an upright microscope and heated chamber. The use of the system is demonstrated in chick embryos by following individual fluorescently labeled neural crest cells, a multipotent stem cell-like population that differentiates into a wide range of derivatives and travels extensively throughout the embryo. By combining in ovo culture with confocal or two-photon four-dimensional time-lapse imaging, embryo health can be maintained for up to 5 d, and neural crest cell behaviors can be visualized for long periods of time (approximately 36 h). This technique has been adapted to study somitogenesis.

Asunto(s)

Aves/embriología , Embrión no Mamífero/embriología , Imagenología Tridimensional/métodos , Óvulo , Animales , Calibración , Embrión de Pollo , Embrión no Mamífero/citología , Desarrollo Embrionario , Calor , Membranas Artificiales , Óvulo/crecimiento & desarrollo , Politetrafluoroetileno , Codorniz/embriología

RESUMEN

The neural crest serve as an excellent model to better understand mechanisms of embryonic cell migration. Cell tracing studies have shown that cranial neural crest cells (CNCCs) emerge from the dorsal neural tube in a rostrocaudal manner and are spatially distributed along stereotypical, long distance migratory routes to precise targets in the head and branchial arches. Although the CNCC migratory pattern is a beautifully choreographed and programmed invasion, the underlying orchestration of molecular events is not well known. For example, it is still unclear how single CNCCs react to signals that direct their choice of direction and how groups of CNCCs coordinate their interactions to arrive at a target in an ordered manner. In this review, we discuss recent cellular and molecular discoveries of the CNCC migratory pattern. We focus on events from the time when CNCCs encounter the tissue adjacent to the neural tube and their travel through different microenvironments and into the branchial arches. We describe the patterning of discrete cell migratory streams that emerge from the hindbrain, rhombomere (r) segments r1-r7, and the signals that coordinate directed migration. We propose a model that attempts to unify many complex events that establish the CNCC migratory pattern, and based on this model we integrate information between cranial and trunk neural crest development.

Asunto(s)

Movimiento Celular/fisiología , Cresta Neural/citología , Cresta Neural/embriología , Rombencéfalo/citología , Rombencéfalo/embriología , Animales , Región Branquial/embriología , Región Branquial/metabolismo , Cresta Neural/metabolismo , Rombencéfalo/metabolismo , Transducción de Señal , Cráneo/metabolismo

RESUMEN

Interferon regulatory factor 6 (IRF6) is a non-canonical member of the interferon regulatory factor family of transcription factors. We recently identified IRF6 as a novel Maspin-interacting protein in mammary epithelial cells. Maspin is a tumor suppressor in the breast and has also been implicated in mammary gland morphogenesis. To explore a possible role for IRF6 in conjunction with Maspin during mammary gland growth and differentiation, we examined the expression of IRF6 and Maspin during post-utero mammary gland development using a combination of in vitro and in vivo approaches. The data revealed that the expression of IRF6 and Maspin is temporally and spatially regulated throughout mammary gland development, with maximal expression of both proteins occurring in fully differentiated, lactating lobuloalveolar cells. We further show that IRF6 adopts a lumenal localization pattern following complete epithelial cell polarization and present new evidence for the secretion of IRF6 into the milk. These results support the hypothesis that IRF6 and Maspin are important for mammary epithelial cell differentiation, and advance our understanding of the Maspin-IRF6 partnership during normal mammary gland development.

Asunto(s)

Factores Reguladores del Interferón/metabolismo , Serpinas/metabolismo , Animales , Western Blotting , Técnicas de Cultivo de Célula , Línea Celular , Células Epiteliales/metabolismo , Femenino , Humanos , Inmunohistoquímica , Glándulas Mamarias Humanas/metabolismo , Ratones

RESUMEN

Post utero development of the mammary gland is a complex developmental process characterized by states of rapid cell proliferation (branching morphogenesis) followed by functional differentiation (lactation) and the consequent apoptosis (involution) of the secretory mammary epithelial cell. This process is cyclical, such that involution returns the mammary gland to a near-virgin-like state capable of responding to morphogenic cues with each consecutive pregnancy. Importantly, many of the regulatory processes which oversee mammary gland development are corrupted or otherwise compromised during the development of breast cancer. For example, Interferon Regulatory Factor 6 (IRF6) is a novel protein with growth inhibitory properties that was initially identified in mammary epithelial cells through its interaction with maspin, a known tumor suppressor in normal breast tissue. Recent findings from our laboratory suggest that IRF6 functions synergistically with maspin to regulate mammary epithelial cell differentiation by acting on the cell cycle. This perspective focuses on the possible involvement of IRF6 in promoting differentiation by regulating exit from the cell cycle and entry into the G(0) phase of cellular quiescence, and how these new findings shed light on normal mammary gland development and the initiation and progression of breast cancer.

Asunto(s)

Neoplasias de la Mama/metabolismo , Mama/metabolismo , Células Epiteliales/metabolismo , Factores Reguladores del Interferón/metabolismo , Serpinas/metabolismo , Proteínas 14-3-3/metabolismo , Animales , Apoptosis/fisiología , Ciclo Celular , Diferenciación Celular/fisiología , Femenino , Humanos , Quinasa I-kappa B/metabolismo , Ratones , Ratones Noqueados

RESUMEN

In this study we examined the ability of interferon-gamma (IFN-gamma) to regulate mammary epithelial cell growth and gene expression, with particular emphasis on two genes: Maspin (a member of serine protease inhibitor superfamily), and the lysosomal aspartyl endopeptidase cathepsin D (CatD). The protein products of these genes are critically involved in regulation of multitude of biological functions in different stages of mammary tissue development and remodeling. In addition, the expression of Maspin is down-regulated in primary breast cancer and is lost in metastatic disease, while CatD is excessively produced and aberrantly secreted by breast cancer cells. We report that IFN-gamma receptors are expressed in mammary epithelial cells, and receptor engagement by IFN-gamma transduces the IFN-gamma signal via Stat-1 resulting in decreased vacuolar pH. This change in vacuolar pH alters CatD protein processing and secretion concurrent with increased Maspin secretion. In addition, IFN-gamma exerts a suppressive effect on cell growth and proliferation, and induces morphological changes in mammary epithelial cells. Our studies also reveal that breast cancer cells, which are devoid of Maspin, are refractory to IFN-gamma with respect to changes in vacuolar pH and CatD. However, Maspin transfection of breast cancer cells partially sensitizes the cells to IFN-gamma's effect, thus providing new therapeutic implications.

Asunto(s)

Autofagia/efectos de los fármacos , Catepsina D/metabolismo , Células Epiteliales/enzimología , Interferón gamma/farmacología , Glándulas Mamarias Humanas/efectos de los fármacos , Glándulas Mamarias Humanas/enzimología , Vacuolas/enzimología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Neoplasias de la Mama/patología , Línea Celular , Células Epiteliales/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Electrónica de Transmisión , Fenotipo , ARN Mensajero/genética , Vacuolas/efectos de los fármacos

RESUMEN

Interferon regulatory factor 6 (IRF6) is a novel and unique member of the IRF family of transcription factors. IRF6 has not been linked to the regulatory pathways or functions associated with other IRF family members, and the regulation and function of IRF6 remain unknown. We recently identified a protein interaction between IRF6 and the tumor suppressor maspin. To gain insight into the biological significance of the maspin-IRF6 interaction, we examined the regulation and function of IRF6 in relation to maspin in normal mammary epithelial cells. Our results demonstrate that in quiescent cells, IRF6 exists primarily in a nonphosphorylated state. However, cellular proliferation leads to rapid IRF6 phosphorylation, resulting in proteasome-dependent IRF6 degradation. These data are supported in situ by the increased expression of IRF6 in quiescent, differentiated lobuloalveolar cells of the lactating mammary gland compared to its expression in proliferating ductal and glandular epithelial cells during pregnancy. Furthermore, the reexpression of IRF6 in breast cancer cells results in cell cycle arrest, and the presence of maspin augments this response. These data support a model in which IRF6, in collaboration with maspin, promotes mammary epithelial cell differentiation by facilitating entry into the G(0) phase of the cell cycle.

Asunto(s)

Adenocarcinoma/patología , Neoplasias de la Mama/patología , Factores Reguladores del Interferón/fisiología , Glándulas Mamarias Animales/citología , Fase de Descanso del Ciclo Celular/fisiología , Adenocarcinoma/metabolismo , Animales , Neoplasias de la Mama/metabolismo , División Celular , Línea Celular Tumoral/citología , Línea Celular Tumoral/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Factores Reguladores del Interferón/genética , Lactancia/fisiología , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Embarazo , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/fisiología , Serpinas/fisiología

RESUMEN

The microenvironment is being increasingly recognized as a critical component in tumor progression and metastases. As such, the bi-directional signaling of extracellular mediators that promote tumor growth within the microenvironment is a focus of intense scrutiny. Interestingly, there are striking similarities between the phenotypes of aggressive tumor and embryonic stem cells, particularly with respect to specific signaling pathways underlying their intriguing plasticity. Here, we demonstrate the epigenetic influence of the hESC microenvironment on the reprogramming of aggressive melanoma cells using an innovative 3-D model. Specifically, our laboratory has previously demonstrated the redifferentiation of these melanoma cells to a more melanocyte-like phenotype (Postovit et al., Stem Cells 24(3):501-505, 2006), and now we show the loss of VE-Cadherin expression (indicative of a plastic vasculogenic phenotype) and the loss of Nodal expression (a plasticity stem cell marker) in tumor cells exposed to the hESC microenvironment. Further studies with the 3-D culture model revealed the epigenetic influence of aggressive melanoma cells on hESCs resulting in the down-regulation of plasticity markers and the emergence of phenotype-specific genes. Additional studies with the aggressive melanoma conditioned matrix microenvironment demonstrated the transdifferentiation of normal melanocytes into melanoma-like cells exhibiting a vasculogenic phenotype. Collectively, these studies have advanced our understanding of the epigenetic influence associated with the microenvironments of hESCs and aggressive melanoma cells, and shed new light on their therapeutic implications. Moreover, we have a better appreciation of the convergence of embryonic and tumorigenic signaling pathways that might stimulate further consideration of targeting Nodal in aggressive tumor cells resulting in a down-regulation of tumorigenic potential and plasticity.

RESUMEN

Maspin (Mammary Serine Protease Inhibitor) was first reported in 1994 as a serpin with tumor suppressive properties. Maspin was initially isolated through subtractive hybridization and differential display analysis as a 42-kDa protein that is expressed in normal mammary epithelial cells but reduced or absent in breast carcinomas (Zou et al., 1994). Further research led to maspin's characterization as a class II tumor suppressor based on its ability to inhibit cell invasion, promote apoptosis, and inhibit angiogenesis (Sheng et al., 1996; Zhang et al., 2000b; Jiang et al., 2002). Since then, efforts have been made to characterize maspin's tumor suppressive mechanisms. In particular, researchers have studied maspin localization, the regulation of maspin expression, and more recently, maspin protein interactions. By elucidating these mechanisms, researchers are beginning to understand the complex, pleiotropic nature of maspin and the pathways through which maspin exerts its tumor suppressive properties. These new findings not only further enhance our understanding of cancer biology but also provide an avenue to develop maspin's potential as a diagnostic marker for cancer progression, and as a potentially powerful therapeutic agent in the fight against breast cancer.

Asunto(s)

Genes Supresores de Tumor , Neoplasias , Serpinas , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Humanos , Factores Reguladores del Interferón/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Patológica , Análisis de Secuencia por Matrices de Oligonucleótidos , Serpinas/genética , Serpinas/metabolismo , Serpinas/uso terapéutico , Distribución Tisular , Técnicas del Sistema de Dos Híbridos

RESUMEN

Since its reported discovery in 1994, maspin (mammary serine protease inhibitor) has been characterized as a class II tumor suppressor by its ability to promote apoptosis and inhibit cell invasion. Maspin is highly expressed in normal mammary epithelial cells but reduced or absent in aggressive breast carcinomas. However, despite efforts to characterize the mechanism(s) by which maspin functions as a tumor suppressor, its molecular characterization has remained somewhat elusive. Therefore, in an attempt to identify maspin-interacting proteins and thereby gain insight into the functional pathways of maspin, we employed a maspin-baited yeast two-hybrid system and subsequently identified Interferon Regulatory Factor 6 (IRF6) as a maspin-binding protein. IRF6 belongs to the IRF family of transcription factors, which is best known for its regulation of interferon and interferon-inducible genes following a pathogenic stimulus. Although many of the IRF family members have been well characterized, IRF6 remains poorly understood. We report that IRF6 is expressed in normal mammary epithelial cells and that it directly associates with maspin in a yeast two-hybrid system and in vitro. The interaction occurs via the conserved IRF protein association domain and is regulated by phosphorylation of IRF6. We have shown that, similar to maspin, IRF6 expression is inversely correlated with breast cancer invasiveness. We further demonstrated that the transient re-expression of IRF6 in breast cancer cells results in an increase of N-cadherin and a redistribution of vimentin commensurate with changes in cell morphology, suggestive of an epithelial-to-mesenchymal transition event. Concomitantly, we showed that maspin acts as a negative regulator of this process. These findings help to elucidate the molecular mechanisms of maspin and suggest an interactive role between maspin and IRF6 in regulating cellular phenotype, the loss of which can lead to neoplastic transformation.

Asunto(s)

Neoplasias de la Mama/patología , Mama/fisiología , Genes Supresores de Tumor/fisiología , Factores Reguladores del Interferón/metabolismo , Inhibidores de Serina Proteinasa/fisiología , Serpinas/fisiología , Secuencia de Aminoácidos , Mama/citología , Cadherinas/biosíntesis , Transformación Celular Neoplásica , Células Epiteliales/fisiología , Femenino , Perfilación de la Expresión Génica , Humanos , Factores Reguladores del Interferón/biosíntesis , Datos de Secuencia Molecular , Fenotipo , Fosforilación , Reacción en Cadena de la Polimerasa , Células Tumorales Cultivadas , Técnicas del Sistema de Dos Híbridos , Vimentina/metabolismo , Levaduras/genética