STAT3 positively regulates IL-6 expression in lung cancer cells

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IL-6 is a pleiotropic cytokine produced by many different cell types. It plays multiple roles largely depending on down-stream Jak/STAT3 pathway. Spontaneous production of IL-6 has been observed in various tumors. Furthermore, constitutive activation of STAT3 has also been shown in most of them. Upregulation of IL-6 has been associated with cancer pathogenesis, tumor progression, antiapoptosis, drug resistance, and poor clinical outcomes. Most of the studies emphasized the importance of NF-κB and AP-1 on IL-6 regulation. Others pointed out the potential roles of HIF-1α and p53. Though STAT3 decoy has been shown to non-specifically interrupted the expression of a series of cytokines including IL-6, the role of STAT3 in mediating IL-6 autocrine in cancer cells has not been clearly demonstrated. To answer this question we used lung adenocarcinoma cell line-PC14PE6/AS2 (AS2), in which STAT3 is constitutively active. We showed a time-dependent IL-6 autocrine in the AS2 cell. The Erk inhibitor (U0126) decreased about 80% of IL-6 secretion from AS2 cell but the NF-κB inhibitor (BAY11-7082) had no evident effect. Because blocking the activity of Jak/STAT3 signal by AG490 diminished IL-6 secretion by more than 60%, we speculated that Jak/STAT3 pathway may regulate IL-6 expression. To reveal the role of STAT3 in IL-6 secretion, we established several AS2-derived cell lines. AS2-S3C (S3C) cell was transfected with plasmids containing constitutively activated form of STAT3, AS2-S3D (S3D) cell was transfected with plasmids containing dominant-negative STAT3 with mutation at DNA binding domain, and AS2-S3F (S3F) cell was transfected with plasmids containing dominant-negative STAT3 with mutation at tyrosine phosphorylation site. The S3C cell expressed higher IL-6 mRNA and a 10-fold higher IL-6 secretion than did the control cells. The S3D and S3F cells showed a 50% decrease of IL-6 secretion than did the control cells. These results strongly suggested the role of STAT3 in regulating IL-6 expression. To confirm this, we introduced siRNA against STAT3 into AS2 cell. Knocking down STAT3 expression by siRNA decreased IL-6 secretion in AS2 cells. There was no inhibition on AS2 proliferation observed in the siRNA knock-down study. In summary, we have demonstrated the positive-feedback control of IL-6/STAT3 axis other than the well-known NF-κB and AP-1 pathway in a lung adenocarcinoma model.