Reversibility of Epithelial-Mesenchymal Transition (EMT) Induced in Breast Cancer Cells by Activation of Urokinase Receptor-dependent Cell Signaling

Abstract

Hypoxia induces expression of the urokinase receptor (uPAR) and activates uPAR-dependent cell signaling in cancer cells. This process promotes epithelial-mesenchymal transition (EMT). uPAR overexpression in cancer cells also promotes EMT.In this study, we tested whether uPAR may be targeted to reverse cancer cell EMT. When MDA-MB 468 breast cancer cells were cultured in 1% O 2 , uPAR expression increased, as anticipated. Cell-cell junctions were disrupted, vimentin expression increased, and E-cadherin was lost from cell surfaces, indicating EMT. Transferring these cells back to 21% O 2 decreased uPAR expression and reversed the signs of EMT. In uPAR-overexpressing MDA-MB 468 cells, EMT was reversed by silencing expression of endogenously produced urokinase-type plasminogen activator (uPA), which is necessary for uPAR-dependent cell signaling, or by targeting uPAR-activated cell signaling factors, including phosphatidylinositol 3-kinase, Src family kinases, and extracellular signal-regulated kinase. MDA-MB 231 breast cancer cells express high levels of uPA and uPAR and demonstrate mesenchymal cell morphology under normoxic culture conditions (21% O 2 ). Silencing uPA expression in MDA-MB-231 cells decreased expression of vimentin and Snail, and induced changes in morphology characteristic of epithelial cells. These results demonstrate that uPAR-initiated cell signaling may be targeted to reverse EMT in cancer. Epithelial-mesenchymal transition (EMT)2 is a well recognized process in embryonic development (1). To facilitate migration of neural crest cells out of the neuroectoderm, N-cadherin-based cell adhesions are lost. Endocardial cells adopt a mesenchymal cell phenotype during formation of cardiac septa and valves. EMT also may be involved in metastasis of epithelial cell malignancies (2). In cancer cell EMT, E-cadherin protein and activity are decreased, causing disruption of cell-cell junctions and loss of cell polarity. At the same time, cancer cells that undergo EMT demonstrate increased expression of mesenchymal cell proteins such as vimentin. Loss of E-cadherin, in human malignancies, may reflect mutation of the E-cadherin gene or E-cadherin promoter hypermethylation (3-5). E-cadherin expression also may be repressed at the transcriptional level by Slug, Snail, or Twist (6 -9). Experimental regulation of E-cadherin expression controls cancer progression in mouse model systems (10 -13).Activation of the phosphatidylinositol 3-kinase (PI3K) and Akt promotes EMT in cancer cells (14). Akt phosphorylates and thereby inactivates glycogen synthase kinase-3␤ (GSK-3␤), increasing Snail activity by regulating Snail expression, degradation, and nuclear localization (6,7,15,16). Wnt signaling also regulates GSK-3␤ and thereby supports EMT by downstream effects on Snail and Slug (17). Extracellular signal-regulated kinase (ERK1/2) increases expression of Snail (18). Rac1 and its alternatively spliced variant, Rac1b, control Snail expression and activity, through a pathway that involves reactive oxygen species and NF-B...

ZEB1 Promotes Invasiveness of Colorectal Carcinoma Cells through the Opposing Regulation of uPA and PAI-1

“…In breast and squamous carcinoma cells, binding of uPA to its receptor triggers an EMT phenotype, including the expression of Snail1 (34,35). In this study, we found that uPA overexpression upregulates ZEB1, whereas inhibition of uPA expression by amiloride reduces ZEB1.…”

Signalling pathways involved in endocrine resistance in breast cancer and associations with epithelial to mesenchymal transition (Review)

“…hypoxia can promote EMT by inducing uPAR-dependent cell-signalling in cancer cells. MdA-MB 468 cells cultured in 1% O 2 exhibited increased uPAR expression, disruption of inter-cellular junctions, increased vimentin expression and loss of E-cadherin, indicating EMT (292). This effect could be reversed by shifting the cells to 21% O 2 .…”

Transcriptome profiling of a TGF-β-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies

“…This was confirmed using a Transwell invasion assay (Figure 6c), which showed that clusterin polyclonal IgG reduced the invasiveness of the 4T1 and PC3 cells by B60 and 25%, respectively. In addition, we also used the MDA-MB231LM2 cell line (Minn et al, 2005), which secretes clusterin (Figure 6e) and has been used in EMT studies (Buijs et al, 2007a(Buijs et al, , 2007bHunakova et al, 2009;Jo et al, 2009). We found that clusterin polyclonal IgG also reduced the invasiveness of these cells by B30%.…”