High Content Imaging Assays for IL-6-Induced STAT3 Pathway Activation in Head and Neck Cancer Cell Lines.

In the canonical STAT3 signaling pathway, IL-6 receptor engagement leads to the recruitment of latent STAT3 to the activated IL-6 complex and the associated Janus kinase (JAK) phosphorylates STAT3 at Y705. pSTAT3-Y705 dimers traffic into the nucleus and bind to specific DNA response elements in the promoters of target genes to regulate their transcription. However, IL-6 receptor activation induces the phosphorylation of both the Y705 and S727 residues of STAT3, and S727 phosphorylation is required to achieve maximal STAT3 transcriptional activity. STAT3 continuously shuttles between the nucleus and cytoplasm and maintains a prominent nuclear presence that is independent of Y705 phosphorylation. The constitutive nuclear entry of un-phosphorylated STAT3 (U-STAT3) drives expression of a second round of genes by a mechanism distinct from that used by pSTAT3-Y705 dimers. The abnormally elevated levels of U-STAT3 produced by the constitutive activation of pSTAT3-Y705 observed in many tumors drive the expression of an additional set of pSTAT3-independent genes that contribute to tumorigenesis. In this chapter, we describe the HCS assay methods to measure IL-6-induced STAT3 signaling pathway activation in head and neck tumor cell lines as revealed by the expression and subcellular distribution of pSTAT3-Y705, pSTAT3-S727, and U-STAT3. Only the larger dynamic range provided by the pSTAT3-Y705 antibody would be robust and reproducible enough for screening.