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Snail is one of the master regulators of the epithelial-mesenchymal transition (EMT) implicated in key tumor biological processes such as invasion and metastasizing. Besides the importance of genetic alterations in tumor cells it is obvious that also the micro environment affects the behavior of tumor cells. Hypoxia is one element in the tumor micro environment potentially affecting many important processes linked to clinical aggressiveness of a tumor. We therefore set out to investigate the regulation of EMT in hypoxia, by determining the role of the E-cadherin transcriptional repressor Snail, in breast cancer cell lines and primary breast tumors. Cell lysates and pellets from different time points of hypoxic exposure of the breast cancer cell lines MCF7, T47D and CAMA1 were analyzed by western blotting and immunohistochemistry, using antibodies for Snail and E-cadherin. The Snail antibody was validated for western blotting and immunhistochemistry by analyzes of cell lines transiently overexpressing Snail. At 0.1 % O2 (severe hypoxia) there was a clear increase in Snail protein content after approximately four hours in all three breast cancer cell lines. There was further a decrease in E-cadherin protein in MCF7 cells but no difference in migratory properties of these cell lines grown in hypoxia compared to normoxia. In order to test how the cell lines responded to a Snail increase, we overexpressed Snail in MCF7 and T47D cells. Interestingly, both cell lines were more migratory when overexpressing Snail but only MCF7 cells expressed lower levels of E-cadherin compared to control transfected cells. Further, transient siRNA transfection against Snail resulted in a slightly decreased migration compared to control transfection in MCF7 cells. These results indicate that hypoxia induces Snail but no migratory phenotype suggesting that hypoxic cells are partially pushed towards EMT. To further validate the importance of Snail in breast cancer and also delineate any potential links to clinical behaviors and HIF1α protein content, we analyzed a material of 350 primary breast cancer samples arranged in a tissue micro array. There was no direct link between Snail expression and HIF1α protein content but Snail expression correlated positively to lymph node status. This supports that increased expression of Snail protein in primary breast cancer might be important for the ability of tumors to metastasize, possibly through EMT.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA