ABSTRACT
Epithelial to mesenchymal transition (EMT) is implicated in the progression of primary tumours towards metastasis and is likely caused by a pathological activation of transcription factors regulating EMT in embryonic development. To analyse EMT-causing pathways in tumourigenesis, we identified transcriptional targets of the E-cadherin repressor ZEB1 in invasive human cancer cells. We show that ZEB1 repressed multiple key determinants of epithelial differentiation and cell-cell adhesion, including the cell polarity genes Crumbs3, HUGL2 and Pals1-associated tight junction protein. ZEB1 associated with their endogenous promoters in vivo, and strongly repressed promotor activities in reporter assays. ZEB1 downregulation in undifferentiated cancer cells by RNA interference was sufficient to upregulate expression of these cell polarity genes on the RNA and protein level, to re-establish epithelial features and to impair cell motility in vitro. In human colorectal cancer, ZEB1 expression was limited to the tumour-host interface and was accompanied by loss of intercellular adhesion and tumour cell invasion. In invasive ductal and lobular breast cancer, upregulation of ZEB1 was stringently coupled to cancer cell dedifferentiation. Our data show that ZEB1 represents a key player in pathologic EMTs associated with tumour progression.
Subject(s)
Breast Neoplasms/pathology , Cell Differentiation , Cell Polarity , Colonic Neoplasms/pathology , Cytoskeletal Proteins/antagonists & inhibitors , Homeodomain Proteins/metabolism , Membrane Glycoproteins/antagonists & inhibitors , Membrane Proteins/antagonists & inhibitors , Nucleoside-Phosphate Kinase/antagonists & inhibitors , Transcription Factors/metabolism , Adult , Aged , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cadherins/metabolism , Chromatin Immunoprecipitation , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Disease Progression , Down-Regulation , Epithelium/metabolism , Epithelium/pathology , Gene Expression Profiling , Homeodomain Proteins/genetics , Humans , Immunoblotting , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Fluorescence , Middle Aged , Neoplasm Invasiveness/pathology , Nucleoside-Phosphate Kinase/genetics , Nucleoside-Phosphate Kinase/metabolism , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Snail Family Transcription Factors , Transcription Factors/genetics , Tumor Cells, Cultured , Zinc Finger E-box-Binding Homeobox 1ABSTRACT
OBJECTIVES: The activator protein-1 (AP-1) is a dimeric transcription factor formed by members of the Jun and Fos protein family. AP-1 plays a role in a variety of physiological functions including cell proliferation and differentiation, although both c-Jun and c-Fos have also been implicated in oncogenic transformation and tumor progression. To further elucidate the role of AP-1 in breast cancer, we have investigated the expression of the AP-1 proteins c-Jun, JunB, JunD, phosphorylated c-Jun, c-Fos, Fral, Fra2 and the tumor supressor protein p53. METHODS: Protein expression was evaluated on a breast cancer tissue microarray with 58 lymph node positive or negative breast cancer specimens, 29 corresponding lymph node metastases, and 11 tissue samples from surrounding tumor-free tissue, each cored as triplicate. Jun and Fos protein family expression was evaluated by immunohistochemistry and was correlated with clinicopathological parameters. RESULTS: High expression levels were observed for c-Jun, JunD, c-Fos and Fra2, whereas JunB and Fral exhibited lower staining. c-Jun protein expression was correlated to Fral staining (p = 0.007, Kendall's Tau) and Fral was further associated with c-Fos (p < 0.001), JunD (p = 0.001) and Fra2 (p = 0.011) expression. JunD expression correlated with c-Fos (p < 0.001), JunB (p = 0.035) and c-Jun (p = 0.05). Activated c-Jun correlated with c-Fos expression (p = 0.041). JunB was negatively correlated to tumor stage, (p = 0.093, corr coeff. = -0.293, Spearman's correlation) but was significantly increased in nodal negative tumors (p = 0.004, Mann Whitney test). In addition, increased Fral expression showed a trend towards an increased overall survival (p = 0.077, RR = 0.534, Cox regression). CONCLUSION: Our results suggest an important role for JunB and Fral in the biological behavior of malignant breast tumors.
