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EFFECT OF PTEN DOWNREGULATION ON PROLIFERATION, INVASION AND DIFFERENTIATION OF HUMAN COLON CANCER CELLS
MJ Langlois, N Rivard
CIHR Team on Digestive Epithelium, University of Sherbrooke
PTEN dephosphorylates phosphatidylinositols resulting from phosphatidylinositol 3-kinase (PI3K) activation. Germinal mutations of PTEN have been identified in juvenile polyposis syndrome, a disease characterized by the development of hamartomatous polyps in the digestive tract and associated with an increased risk of cancer. However, little is known about the specific role of PTEN in human intestinal epithelial cells. OBJECTIVE: To investigate the role of PTEN in human colorectal cancer cells.
METHODS: PTEN expression was analyzed by Western blot and immunofluorescence in normal epithelial crypt cells (HIEC) and in many colorectal cell lines: Caco-2/15, DLD-1, HCT116, HT29, LoVo, Colo205 and SW480. Caco-2/15 cells, which spontaneously differentiate into an enterocyte phenotype, were infected with a recombinant lentivirus expressing a shRNA which specifically knocked-down PTEN. The impact of PTEN down-regulation on proliferation (cell counting), migration/invasion (matrigel-coated transwells) and differentiation (expression of sucrase-isomaltase and villin, polarization, expression of cell-cell adhesion proteins, barrier function) was analyzed.
RESULTS: 1. PTEN is expressed and is nuclear in all cancer cell lines but at a reduced level in comparison to normal epithelial cells. 2. PTEN protein is localized into the nucleus of subconfluent proliferating cells whereas it localizes at the cell-cell contacts of post-confluent differentiating Caco-2/15 cells. 3. The lentiviral infection of a shRNA which knocked-down PTEN expression significantly stimulates proliferation rate and invasion capacity of Caco-2/15 cells. 4. Loss of PTEN expression leads to a decrease in p21Cip and p27Kip1 protein levels and to an increase of phosphorylated Akt and cyclin D2 protein. 5. Regarding differentiation, loss of PTEN expression decreases levels of CDX2, HNF-1, villin and sucrase-isomaltase and reduces alkaline phosphatase activity. 6. Electron microscopy analysis shows that loss of PTEN inhibits cell polarization and brush border development. In addition, strong reduction in claudin 1, 3 and 4 expression is observed as well as a decrease in transepithelial resistance. CONCLUSION: These results indicate that endogenous PTEN is involved in the control of proliferation, migration/invasion and differentiation of human colon cancer cells.
Supported by CIHR MT144-05 to NR and CIHR Team grant