Glucocorticoids (GC) induce apoptosis in lymphoblasts and are thus essential in the treatment of acute lymphoblastic leukemia (ALL). Their effects result from gene regulations via the GC receptor (NR3C1/GR), but it is unknown how these changes evolve, what the primary GR targets are, and to what extent responses differ between ALL subtypes and nonlymphoid malignancies. We delineated the transcriptional response to GC on the exon level in a time-resolved manner in a precursor B- and a T childhood ALL model employing Exon microarrays and combined this with genome-wide NR3C1-binding site detection using chromatin immunoprecipitation-on-chip technology. This integrative approach showed that the response was strongly influenced by kinetics and extent of GR autoinduction in both models. Although remarkable differences between the ALL systems were apparent, we defined a set of common response genes enriched in apoptosis-related processes. Globally, GR binding was higher for GC-induced vs. -repressed genes, suggesting that GR mediates gene repression by interaction with distant enhancers or by cross talk with other transcription factors. Exon level analysis defined several new GC-regulated transcript variants of genes, including ATP4B, GPR98, TBCD, and ZBTB16. Our study provides unprecedented insight into the transcriptional response to GC in ALL cells, essential to understand this biologically and clinically important phenomenon. We found evidence of cell type-specific as well as common responses, possibly related to apoptosis induction, and detected induction of novel transcript variants by GC in the investigated systems. Finally, we implemented a bioinformatic framework that might be useful for high-density microarray analyses to identify alternative transcript variant expression.
IntroductionThe Epithelial Cell Adhesion Molecule (EpCAM) has been shown to be strongly expressed in human breast cancer and cancer stem cells and its overexpression has been supposed to support tumor progression and metastasis. However, effects of EpCAM overexpression on normal breast epithelial cells have never been studied before. Therefore, we analyzed effects of transient adenoviral overexpression of EpCAM on proliferation, migration and differentiation of primary human mammary epithelial cells (HMECs).MethodsHMECs were transfected by an adenoviral system for transient overexpression of EpCAM. Thereafter, changes in cell proliferation and migration were studied using a real time measurement system. Target gene expression was evaluated by transcriptome analysis in proliferating and polarized HMEC cultures. A Chicken Chorioallantoic Membrane (CAM) xenograft model was used to study effects on in vivo growth of HMECs.ResultsEpCAM overexpression in HMECs did not significantly alter gene expression profile of proliferating or growth arrested cells. Proliferating HMECs displayed predominantly glycosylated EpCAM isoforms and were inhibited in cell proliferation and migration by upregulation of p27KIP1 and p53. HMECs with overexpression of EpCAM showed a down regulation of E-cadherin. Moreover, cells were more resistant to TGF-β1 induced growth arrest and maintained longer capacities to proliferate in vitro. EpCAM overexpressing HMECs xenografts in chicken embryos showed hyperplastic growth, lack of lumen formation and increased infiltrates of the chicken leukocytes.ConclusionsEpCAM revealed oncogenic features in normal human breast cells by inducing resistance to TGF-β1-mediated growth arrest and supporting a cell phenotype with longer proliferative capacities in vitro. EpCAM overexpression resulted in hyperplastic growth in vivo. Thus, we suggest that EpCAM acts as a prosurvival factor counteracting terminal differentiation processes in normal mammary glands.
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