Pre-malignant transformation of normal mammary epithelial cells compromises tensional homeostasis at cell-cell junctions

• Main Author: Poon, Tak Kwong
• Language: English
• Published: University of British Columbia 2015
• Online Access: http://hdl.handle.net/2429/54566

Cell-cell junctions regulate the form and function of epithelial tissues, in part, by mechanically coupling adjacent cells together. Unlike normal cells, pre-malignant cells are capable of mechanically uncoupling these junctions in response to motogenic factors such that the cells become invasive and, ultimately malignant. Therefore, I asked whether the mechanical responses of cell-cell junctions to increases in intracellular tension are altered in pre-malignant mammary epithelial cells in the absence of such motogenic factors. In an effort to answer this question I altered the intracellular tension on the cell-cell junctions of normal (EpH4) and pre-malignant oncogenic ras-transformed (EpRas) mammary epithelial cells either chronically, by altering the density of cells attached to a rigid substratum, or acutely, by physically extending (i.e. ‘stretching’) confluent monolayers of cells attached to a compliant silicone rubber substrate. When intracellular tension was chronically increased, the tension-sensitive protein zyxin relocalized to cell-cell junctions in normal, but not pre-malignant cells. The zyxin relocalization in normal cells was associated with a junctional increase in the phosphorylated form of myosin light chain 2 (MLC2) suggesting that it may involve actomyosin contractility. The same differential in zyxin relocalization and phosphorylated MLC2 accumulation occurred when the intracellular tension was acutely increased in the two cell types. This differential was blocked by Rho-ROCK inhibition which indicates that it may be dependent on actomyosin contractility. In addition, apical actin structure reorganization occurred when intracellular tension was acutely increased in the normal cells that did not occur in the pre-malignant cells. Taken together, these observations led me to conclude that the ability of cell-cell junctions to respond in a mechanosensory-appropriate manner to changes in intracellular tension is compromised in ras-transformed pre-malignant mammary epithelial cells. Acute pharmacologic inhibition of oncogenic Ras-mediated increases in MAPK and/or PI3K signalling did not correct this compromised response. Therefore, this compromised mechanosensitivity, which may functionally contribute to the ability of pre-malignant cells to become invasive in response to motogenic factors, may be initiated by long term epigenetic changes that occur under conditions of stable oncogenic transformation. === Medicine, Faculty of === Graduate