RESUMEN
Fibroblasts within the mammary tumor microenvironment are active participants in carcinogenesis mediating both tumor initiation and progression. Our group has previously demonstrated that genetic loss of phosphatase and tensin homolog (PTEN) in mammary fibroblasts induces an oncogenic secretome that remodels the extracellular milieu accelerating ErbB2-driven mammary tumor progression. While these prior studies highlighted a tumor suppressive role for stromal PTEN, how the adjacent normal epithelium transforms in response to PTEN loss was not previously addressed. To identify these early events, we have evaluated both phenotypic and genetic changes within the pre-neoplastic mammary epithelium of mice with and without stromal PTEN expression. We report that fibroblast-specific PTEN deletion greatly restricts mammary ductal elongation and induces aberrant alveolar side-branching. These mice concomitantly exhibit an expansion of the mammary epithelial stem cell (MaSC) enriched basal/myoepithelial population and an increase in in vitro stem cell activity. Further analysis revealed that NOTCH signaling, specifically through NOTCH3, is diminished in these cells. Mechanistically, JAGGED-1, a transmembrane ligand for the NOTCH receptor, is downregulated in the PTEN-null fibroblasts leading to a loss in the paracrine activation of NOTCH signaling from the surrounding stroma. Reintroduction of JAGGED-1 expression within the PTEN-null fibroblasts was sufficient to abrogate the observed increase in colony forming activity implying a direct role for stromal JAGGED-1 in regulation of MaSC properties. Importantly, breast cancer patients whose tumors express both low stromal JAG1 and low stromal PTEN exhibit a shorter time to recurrence than those whose tumors express low levels of either alone suggesting similar stromal signaling in advanced disease. Combined, these results unveil a novel stromal PTEN-to-JAGGED-1 axis in maintaining the MaSC niche, and subsequently inhibiting breast cancer initiation and disease progression.
Asunto(s)
Células Epiteliales/citología , Proteína Jagged-1/metabolismo , Glándulas Mamarias Animales/citología , Neoplasias Mamarias Animales/metabolismo , Fosfohidrolasa PTEN/fisiología , Células Madre/citología , Células 3T3 , Animales , Fibroblastos Asociados al Cáncer/metabolismo , Proliferación Celular , Células Epiteliales/patología , Femenino , Humanos , Proteína Jagged-1/deficiencia , Proteína Jagged-1/genética , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Ratones , Ratones Transgénicos , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/metabolismo , Receptor Notch3/metabolismo , Transducción de Señal , Células del Estroma/citología , Microambiente TumoralRESUMEN
This corrects the article DOI: 10.1038/onc.2016.383.
RESUMEN
Aldose reductase is an NADPH-dependent aldo-keto reductase best known as the rate-limiting enzyme of the polyol pathway that is implicated in the complications of diabetes. Aldose reductase appears to be involved in a variety of disease states other than diabetes, presumably due to its ability to catalyze the reduction of a broad spectrum of aldehydes, including some cytotoxic products of lipid peroxidation. Although the data regarding expression of aldose reductase in normal liver are conflicting, prior studies have suggested that the enzyme may be induced in diseased liver. The goal of these studies was to characterize expression of aldose reductase in normal and diseased human liver, using RT-PCR, Western analysis and immunohistochemistry. Aldose reductase transcripts and protein were detected at low levels in control human livers. In contrast, levels of aldose reductase mRNA and protein were increased in chronically diseased human livers. Immunohistochemistry demonstrated localization of aldose reductase in sinusoidal lining cells; dual immunofluorescence confocal microscopy with the macrophage marker, CD68, confirmed that the aldose reductase-positive sinusoidal lining cells were Kupffer cells. Abundant aldose reductase-positive, CD68-positive cells were present in the fibrous septa of cirrhotic livers, accounting for the increase in immunoreactive aldose reductase in diseased livers. Immunostaining of human lung, spleen and lymph node revealed that macrophages in those tissues also express aldose reductase. These data are the first to demonstrate that aldose reductase is expressed by human macrophages in various tissues and suggest that this enzyme may play a role in immune or inflammatory processes.
Asunto(s)
Aldehído Reductasa/metabolismo , Hepatopatías/enzimología , Hígado/enzimología , Aldehído Reductasa/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Secuencia de Bases , Estudios de Casos y Controles , ADN Complementario/genética , Expresión Génica , Humanos , Inmunohistoquímica , Macrófagos del Hígado/enzimología , Macrófagos del Hígado/patología , Hígado/citología , Hepatopatías/patología , Macrófagos/enzimología , Macrófagos/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We report the isolation and characterization of GP73, a novel 73kDa human Golgi protein. The GP73 cDNA was cloned by differential screening of a cDNA library derived from the liver of a patient with adult giant-cell hepatitis (GCH), a rare form of hepatitis with presumed viral etiology. In vitro transcription-translation studies indicate that GP73 is an integral membrane protein, and immunolocalization experiments using epitope-tagged GP73 demonstrate that the protein is localized to the Golgi apparatus. Northern blot analysis of RNA from multiple human tissues reveals a single GP73 mRNA transcript with a size of approximately 3.0kb. Immunohistochemical studies using rabbit polyclonal antisera directed against recombinant GP73 demonstrate that the protein is preferentially expressed by epithelial cells in many human tissues. In normal livers, GP73 is consistently present in biliary epithelial cells, whereas hepatocytes show little or no signal. In contrast, livers of patients with GCH display strong GP73 immunoreactivity in multinucleated hepatocytes. GP73 mRNA and protein are expressed in highly differentiated HepG2 hepatoma cells after infection with adenovirus in vitro. We conclude that GP73 represents a novel, epithelial cell-specific integral membrane Golgi protein that can be upregulated in response to viral infection.
Asunto(s)
Aparato de Golgi/metabolismo , Hepatitis Viral Humana/genética , Proteínas de la Membrana/genética , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN Complementario/química , ADN Complementario/genética , Femenino , Expresión Génica , Regulación de la Expresión Génica , Células Gigantes/virología , Hepatitis Viral Humana/virología , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Análisis de Secuencia de ADN , Distribución Tisular , Transcripción Genética , Transfección , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Stellate cells play an important role in the production and turnover of the normal extracellular matrix of the liver and are key effector cells in the hepatic fibrogenesis that occurs in response to liver injury. In the present study, we used a rat model of long term dietary iron supplementation to identify stellate cell genes that are expressed during chronic hepatic iron overload. Using a subtraction cloning strategy, we identified a rat isoform of the complement C4 protein gene whose expression was strongly induced in stellate cells after iron overload. Highly purified, cultured stellate cells synthesized the C4 precursor protein and released its subunits into the culture medium. The C4 protein secreted in vitro was biologically active in a C4-specific hemolytic assay. C4 mRNA expression was minimal in freshly isolated stellate cells and increased between days 3 and 7 of primary culture, coincident with the expression of smooth muscle alpha-actin (alpha-SMA), a marker of cellular activation. C4 expression was absent in strongly alpha-SMA-positive, passaged cells, but was induced by IFN-gamma, which simultaneously inhibited alpha-SMA expression. Our studies establish hepatic stellate cells as a previously unrecognized source of C4 and raise the possibility that complement protein expression by the cells plays a role in the hepatic injury response and in fibrogenesis. Our in vitro data point to the presence of two distinct stimulatory pathways for C4 expression in stellate cells that differ with regard to their sensitivity to IFN-gamma and their relationship to cellular activation.