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

  • Paul A. Johnston
  • Malabika Sen
  • Yun Hua
  • Daniel P. Camarco
  • Tong Ying Shun
  • John S. Lazo
  • Jennifer R. Grandis
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1683)

Abstract

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.

Key words

STAT3 pathway activation Head and neck cancer High content screening Imaging Image analysis 

Notes

Acknowledgments

This project has been funded in part with Federal Funds from the National Cancer Institute, National Institutes of Health, under Contract No. HSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.” NExT-CBC Project ID #1015, S08-221 Task Order 6 “STAT3 Pathway Inhibitor HCS ” (Grandis, PI), NCI Chemical Biology Consortium, Pittsburgh Specialized Application Center (PSAC) (Lazo & Johnston, co-PIs). The project was also supported in part by funds from the American Cancer Society (Grandis) and a Head and Neck Spore P50 award (Grandis, CA097190).

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Paul A. Johnston
    • 1
  • Malabika Sen
    • 3
  • Yun Hua
    • 1
  • Daniel P. Camarco
    • 1
  • Tong Ying Shun
    • 4
  • John S. Lazo
    • 5
    • 6
  • Jennifer R. Grandis
    • 2
    • 3
    • 7
  1. 1.Department of Pharmaceutical Sciences, School of PharmacyUniversity of PittsburghPittsburghUSA
  2. 2.University of Pittsburgh Cancer InstitutePittsburghUSA
  3. 3.Department of OtolaryngologyUniversity of PittsburghPittsburghUSA
  4. 4.University of Pittsburgh Drug Discovery InstitutePittsburghUSA
  5. 5.Department of PharmacologyUniversity of VirginiaCharlottesvilleUSA
  6. 6.Department of ChemistryUniversity of VirginiaCharlottesvilleUSA
  7. 7.Department of Otolaryngology - Head and Neck SurgeryUniversity of California, San FranciscoSan FranciscoUSA

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