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We have previously reported that the activation of PI3k/Akt survival pathway correlated with resistance of colorectal cancer cells (CRC) to IFN-β induced cell death. Here, we report the IFN-β induced activation of stat3 and its modulation to PI3k/Akt pathway. We evaluated two CRC cell lines, SW620, more resistant cell line to IFN-β induced cell death (IC50>3000 u/ml), and SW480, more IFN-β sensitive cell line (IC50<800 u/ml). The rapid tyrosine phosphorylation of stat3 was observed following IFN-β treatment not only in SW620 but also in SW480. The phenomenon that IFN-β induced activation of stat3 resulted in activation of PI3k/Akt in SW620 and inactivation in SW480 prompted us investigates the role of activation of stat3 in regulation of Akt pathway. We therefore generated a stable SW620 cell clone SW620Y705F that overexpresses the dominant negative stat3 mutant Y705F. Cell viability demonstrated that this clone was more sensitive to IFN-β treatment compared to the parent cell line. Immunoblotting showed an abrogation of the induction of the phosphorylation of Akt by IFN-β in this clone. These data indicate that IFN-β induced tyrosine phosphorylation of stat3 play a role in Akt activation. We further evaluated the DNA binding and promoter activity of stat3 in order to determine whether activation of stat3 increases its transcriptional activity and activation of Akt. Surprisely, our results showed that IFN-β induced increase in DNA binding and promoter activity of stat3 in the more sensitive SW480 cell line, but decrease in the more resistant SW620 cell line. The immunoblotting demonstrated rapid nuclear localization of stat3 following IFN-β treatment in SW480, but not in SW620 cells. Additionally, an increase of PI3K P55α subunit, which has been identified as a switch of apoptosis and direct target of stat3, was detected in SW480 cells following IFN-β treatment. However, no increase, even a decrease in P55α protein level was observed in SW620 cells. Co-immunoprecipitation confirmed the direct interaction between stat3 and P55α during IFN signaling. Taken together, our data indicated that IFN-β induced activation and nuclear translocalization of stat3 play an important role in cellular response to IFN-β. The blockade of nuclear translocation of stat3 in SW620 may result in decrease of DNA binding activity of stat3 and in turn reduced the induction of PI3K P55 subunit. Moreover, the accumulation of phosphorylated stat3 in cytoplasm due to its blockade of nuclear translocation may be facilitating the activation of PI3K/Akt pathway and resultant cell survival. This regulation of nuclear translocation of stat3 need to be further investigated in order to better understand IFN-β therapy.

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