Abstract
BackgroundSpontaneous interleukin-6 (IL-6) production has been observed in various tumors and implicated in the pathogenesis, progression and drug resistance in cancer. However, the regulation of IL-6 autocrine production in cancer cells is not fully understood. IL-6 is auto-regulated in many types of cell. Two of the three major downstream pathways of IL-6, MEK/extracellular signal-related kinase (Erk) pathway and phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, have been shown to regulate IL-6 expression through the activation of AP-1 and NF-κB. However, it is not clear what the role of Janus kinase (Jak) 2/signal transducer and activator of transcription (Stat) 3 pathway. This study was designed to determine the role of Jak2/Stat3 pathway in the regulation of IL-6 autocrine production in cancer cells.ResultsInhibitors of Jak2/Stat3, MEK/Erk and PI3-K/Akt pathways down-regulated IL-6 secretion in the lung adenocarcinoma PC14PE6/AS2 (AS2) cells, which spontaneously secreted IL-6 and possessed constitutively activated Stat3. Transfection with dominant-negative Stat3, Stat3 siRNA, or Stat3 shRNA decreased IL-6 expression in AS2 cells. Conversely, transfection with constitutively-activated Stat3 increased the production of IL-6. In AS2 derived cells, resistance to paclitaxel was positively correlated with Stat3 activation status and the expression of IL-6, which is commonly secreted in drug resistant cancer cells. The pharmacological inhibition of NF-κB, PI3-K/Akt and MEK/Erk and the pharmacological inhibition and genetic inhibition (Stat3 siRNA) of Jak2/Stat3 pathway decreased IL-6 autocrine production in various drug resistant cancer cell lines and similarly decreased IL-6 autocrine production in clinically isolated lung cancer cells.ConclusionsThis study is the first to directly address the role Stat3 plays on the autocrine production of IL-6, which occurs through a positive-feedback loop. Our biochemical and genetic studies clearly demonstrated that Jak2/Stat3, in combination with other IL-6 downstream pathways, contributed frequently and substantially to IL-6 autocrine production in a broad spectrum of cancer cell lines as well as in clinical cancer samples. Our findings suggest that Stat3 could potentially be regulated to suppress IL-6 autocrine production in cancer cells to inhibit the progression of cancer and reduce drug resistance.
Highlights
Spontaneous interleukin-6 (IL-6) production has been observed in various tumors and implicated in the pathogenesis, progression and drug resistance in cancer
Jak2/signal transducer and activator of transcription 3 (Stat3) pathway positively regulated IL-6 autocrine production in AS2 cells To investigate whether Jak2/Stat3 as well as the other three IL-6 downstream pathways (PI3-K/Akt, MEK/extracellular signal-related kinase (Erk), and NF-B) known to be involved in IL-6 expression in various cells would act as an upstream regulator of IL-6 autocrine production in AS2 cells, we used Enzyme-linked immunosorbent assay (ELISA) to measure IL-6 secretion in one control AS2 group and in four different AS2 treatment groups each with one pathway (Jak2/Stat3, phosphatidylinositol 3-kinase (PI3-K)/Akt, MEK/Erk, or NF-B) pharmacologically inhibited by the inhibitors AG490, LY294002, U0126, or BAY11-7082, respectively
We found that, compared to the controls, MEK/Erk inhibitor and PI3-K/Akt inhibitor reduced IL-6 secretion in AS2 cells by about 80% and 90%, but NF-B inhibitor decreased it by only 20% (p < 0.05) (Figure 2A)
Summary
Spontaneous interleukin-6 (IL-6) production has been observed in various tumors and implicated in the pathogenesis, progression and drug resistance in cancer. Two of the three major downstream pathways of IL-6, MEK/extracellular signal-related kinase (Erk) pathway and phosphatidylinositol 3kinase (PI3-K)/Akt pathway, have been shown to regulate IL-6 expression through the activation of AP-1 and NF-B It is not clear what the role of Janus kinase (Jak) 2/signal transducer and activator of transcription (Stat) 3 pathway. It has been previously found that the functioning of endogenous Stat was inhibited when cells were transfected with S3F (a dominant-negative Stat mutant that cannot be tyrosine phosphorylated) or S3D (a dominant-negative Stat mutant that cannot bind to DNA) while an additional functioning of exogenous Stat was supplied when cells are transfected with S3C (a constitutively-active Stat mutant forced to form a dimer constitutively without stimulation) [2,10] The ability of these mutants to affect the functioning of Stat makes it possible to study the effect of Stat on gene regulation
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