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1.
Breast Cancer Res ; 14(4): R98, 2012 Jul 02.
Article in English | MEDLINE | ID: mdl-22748014

ABSTRACT

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.


Subject(s)
Cell Communication , Neoplasms/metabolism , Neoplasms/pathology , Signal Transduction , Stromal Cells/metabolism , Transforming Growth Factor beta/metabolism , Animals , Cadherins/metabolism , Cell Line, Tumor , Cell Movement , Epithelial-Mesenchymal Transition/genetics , Fibroblasts/metabolism , Gene Expression Regulation, Neoplastic , Gene Knockout Techniques , Humans , Intercellular Junctions/metabolism , Mice , Neoplasms/genetics , Phenotype , Protein Serine-Threonine Kinases/metabolism , Protein Transport , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/genetics , beta Catenin/metabolism
2.
J Clin Invest ; 121(1): 226-37, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21135504

ABSTRACT

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.


Subject(s)
Breast Neoplasms/physiopathology , Integrin alpha2beta1/physiology , Mammary Neoplasms, Experimental/physiopathology , Tumor Suppressor Proteins/physiology , Animals , Base Sequence , Biomarkers, Tumor/genetics , Biomarkers, Tumor/physiology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , DNA Primers/genetics , Female , Genes, erbB-2 , Humans , In Vitro Techniques , Integrin alpha2beta1/deficiency , Integrin alpha2beta1/genetics , Kaplan-Meier Estimate , Male , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Knockout , Mice, Transgenic , Neoplasm Invasiveness , Neoplasm Metastasis/genetics , Neoplasm Metastasis/pathology , Neoplasm Metastasis/physiopathology , Neoplasm Metastasis/prevention & control , Prognosis , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Prostatic Neoplasms/physiopathology , Tumor Stem Cell Assay , Tumor Suppressor Proteins/genetics
3.
J Cell Biochem ; 89(4): 674-87, 2003 Jul 01.
Article in English | MEDLINE | ID: mdl-12858334

ABSTRACT

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.


Subject(s)
Cell Cycle/physiology , Integrases/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Antineoplastic Agents/pharmacology , Biological Transport , Blotting, Southern , Cell Cycle/genetics , Cell Membrane Permeability/genetics , Cell Membrane Permeability/physiology , Cell Size/physiology , Cells, Cultured , DNA, Bacterial/analysis , Escherichia coli/cytology , Escherichia coli/genetics , Escherichia coli/metabolism , Flow Cytometry , Genes, Reporter , Green Fluorescent Proteins , Integrases/drug effects , Integrases/genetics , Luminescent Proteins/metabolism , Recombinant Fusion Proteins/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombination, Genetic/physiology , Signal Transduction/genetics , Signal Transduction/physiology , Viral Proteins/drug effects , Viral Proteins/genetics
4.
Mol Cell Biol ; 23(15): 5301-7, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12861016

ABSTRACT

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.


Subject(s)
DNA-Binding Proteins/physiology , High Mobility Group Proteins/physiology , Alleles , Animals , Apoptosis , Blotting, Southern , Blotting, Western , Cell Division , Cell Survival , DNA-Binding Proteins/chemistry , Genes, p53 , Genetic Vectors , Genotype , High Mobility Group Proteins/chemistry , Homozygote , In Situ Nick-End Labeling , Mice , Models, Genetic , Mutation , Recombination, Genetic , Stem Cells/metabolism , Time Factors
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