RESUMEN
INTRODUCTION: Transforming growth factor beta (TGF-ß) has a dual role during tumor progression, initially as a suppressor and then as a promoter. Epithelial TGF-ß signaling regulates fibroblast recruitment and activation. Concurrently, TGF-ß signaling in stromal fibroblasts suppresses tumorigenesis in adjacent epithelia, while its ablation potentiates tumor formation. Much is known about the contribution of TGF-ß signaling to tumorigenesis, yet the role of TGF-ß in epithelial-stromal migration during tumor progression is poorly understood. We hypothesize that TGF-ß is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and invasion. METHODS: Fluorescently labeled murine mammary carcinoma cells, isolated from either MMTV-PyVmT transforming growth factor-beta receptor II knockout (TßRII KO) or TßRIIfl/fl control mice, were combined with mammary fibroblasts and xenografted onto the chicken embryo chorioallantoic membrane. These combinatorial xenografts were used as a model to study epithelial-stromal crosstalk. Intravital imaging of migration was monitored ex ovo, and metastasis was investigated in ovo. Epithelial RNA from in ovo tumors was isolated by laser capture microdissection and analyzed to identify gene expression changes in response to TGF-ß signaling loss. RESULTS: Intravital microscopy of xenografts revealed that mammary fibroblasts promoted two migratory phenotypes dependent on epithelial TGF-ß signaling: single cell/strand migration or collective migration. At epithelial-stromal boundaries, single cell/strand migration of TßRIIfl/fl carcinoma cells was characterized by expression of α-smooth muscle actin and vimentin, while collective migration of TßRII KO carcinoma cells was identified by E-cadherin+/p120+/ß-catenin+ clusters. TßRII KO tumors also exhibited a twofold greater metastasis than TßRIIfl/fl tumors, attributed to enhanced extravasation ability. In TßRII KO tumor epithelium compared with TßRIIfl/fl epithelium, Igfbp4 and Tspan13 expression was upregulated while Col1α2, Bmp7, Gng11, Vcan, Tmeff1, and Dsc2 expression was downregulated. Immunoblotting and quantitative PCR analyses on cultured cells validated these targets and correlated Tmeff1 expression with disease progression of TGF-ß-insensitive mammary cancer. CONCLUSION: Fibroblast-stimulated carcinoma cells utilize TGF-ß signaling to drive single cell/strand migration but migrate collectively in the absence of TGF-ß signaling. These migration patterns involve the signaling regulation of several epithelial-to-mesenchymal transition pathways. Our findings concerning TGF-ß signaling in epithelial-stromal interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment.
Asunto(s)
Comunicación Celular , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal , Células del Estroma/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal/genética , Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Uniones Intercelulares/metabolismo , Ratones , Neoplasias/genética , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/genética , beta Catenina/metabolismoRESUMEN
The high-mobility-group (HMG) SSRP1 protein is a member of a conserved chromatin-remodeling complex (FACT/DUF/CP) implicated in DNA replication, basal and regulated transcription, and DNA repair. To assist in the functional analysis of SSRP1, the Ssrp1 gene was targeted in murine embryonic stem cells, and the mutation was introduced into the germ line. Embryos homozygous for the targeted allele die soon after implantation, and preimplantation blastocysts are defective for cell outgrowth and/or survival in vitro. The Ssrp1 mutation was also crossed into a p53 null background without affecting growth and/or survival defects caused by loss of Ssrp1 function. Thus, Ssrp1 appears to encode nonredundant and p53-independent functions that are essential for cell viability.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas del Grupo de Alta Movilidad/fisiología , Alelos , Animales , Apoptosis , Southern Blotting , Western Blotting , División Celular , Supervivencia Celular , Proteínas de Unión al ADN/química , Genes p53 , Vectores Genéticos , Genotipo , Proteínas del Grupo de Alta Movilidad/química , Homocigoto , Etiquetado Corte-Fin in Situ , Ratones , Modelos Genéticos , Mutación , Recombinación Genética , Células Madre/metabolismo , Factores de TiempoRESUMEN
Integrins regulate cell-cell and cell-matrix adhesion and thereby play critical roles in tumor progression and metastasis. Although work in preclinical models suggests that ß1 integrins may stimulate metastasis of a number of cancers, expression of the ß1 subunit alone has not been shown to be a useful prognostic indicator in human cancer patients. Here we have demonstrated that the α2ß1 integrin suppresses metastasis in a clinically relevant spontaneous mouse model of breast cancer. These data are consistent with previous studies indicating high expression of α2ß1 integrin in normal breast epithelium and loss of α2ß1 in poorly differentiated breast cancer. They are also consistent with our systematic analysis of microarray databases of human breast and prostate cancer, which revealed that decreased expression of the gene encoding α2 integrin, but not genes encoding α1, α3, or ß1 integrin, was predictive of metastatic dissemination and decreased survival. The predictive value of α2 expression persisted within both good-risk and poor-risk cohorts defined by estrogen receptor and lymph node status. Thus, the α2ß1 integrin functionally inhibits breast tumor metastasis, and α2 expression may serve as an important biomarker of metastatic potential and patient survival.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Integrina alfa2beta1/fisiología , Neoplasias Mamarias Experimentales/fisiopatología , Proteínas Supresoras de Tumor/fisiología , Animales , Secuencia de Bases , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/fisiología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Cartilla de ADN/genética , Femenino , Genes erbB-2 , Humanos , Técnicas In Vitro , Integrina alfa2beta1/deficiencia , Integrina alfa2beta1/genética , Estimación de Kaplan-Meier , Masculino , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Invasividad Neoplásica , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/fisiopatología , Metástasis de la Neoplasia/prevención & control , Pronóstico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/fisiopatología , Ensayo de Tumor de Célula Madre , Proteínas Supresoras de Tumor/genéticaRESUMEN
Protein transduction has been widely used to analyze biochemical processes in living cells quantitatively and under non-steady-state conditions. The present study analyzed the effects of cell cycle on the uptake and activity of cell-permeant Cre recombinase proteins. Previous studies had suggested that the efficiency of recombination and/or protein transduction varied among individual cells, even within a clonal population. We report here that cells in the G1 phase of the cell cycle undergo recombination at a lower rate than cells at other phases of the cell cycle, and that this variation results largely from differences in protein uptake, associated with differences in cell size. These results have implications regarding the mechanism of protein transduction and identify a source of heterogeneity that can influence the response of individual cells to cell-permeant proteins.