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

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


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 



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).


  1. 1.
    Frank DA (2007) STAT3 as a central mediator of neoplastic cellular transformation. Cancer Lett 251:199–210CrossRefGoogle Scholar
  2. 2.
    Germain D, Frank DA (2007) Targeting the cytoplasmic and nuclear functions of signal transducers and activators of transcription 3 for cancer therapy. Clin Cancer Res 13:5665–5669CrossRefGoogle Scholar
  3. 3.
    Jing N, Tweardy DJ (2005) Targeting Stat3 in cancer therapy. Anti-Cancer Drugs 16:601–607CrossRefGoogle Scholar
  4. 4.
    Johnston PA, Grandis JR (2011) STAT3 signaling: anticancer strategies and challenges. Mol Interv 11:18–26CrossRefGoogle Scholar
  5. 5.
    Quesnelle KM, Boehm AL, Grandis JR (2007) STAT-mediated EGFR signaling in cancer. J Cell Biochem 102:311–319CrossRefGoogle Scholar
  6. 6.
    Aggarwal BB, Kunnumakkara AB, Harikumar KB, Gupta SR, Tharakan ST, Koca C, Dey S, Sung B (2009) Signal transducer and activator of transcription-3, inflammation, and cancer: how intimate is the relationship? Ann N Y Acad Sci 1171:59–79CrossRefGoogle Scholar
  7. 7.
    Egloff AM, Grandis JR (2009) Improving response rates to EGFR-targeted therapies for head and neck squamous cell carcinoma: candidate predictive biomarkers and combination treatment with Src inhibitors. J Oncol 2009:896407, 12 pagesCrossRefGoogle Scholar
  8. 8.
    Heinrich PC, Behrmann I, Müller-Newen G, Schaper F, Graeve L (1998) Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochem J 334:297–314CrossRefGoogle Scholar
  9. 9.
    Heinrich PC, Behrmann I, Haan S, Hermanns HM, Müller-Newen G, Schaper F (2003) Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 374:1–20CrossRefGoogle Scholar
  10. 10.
    Leeman RJ, Lui VW, Grandis JR (2006) STAT3 as a therapeutic target in head and neck cancer. Expert Opin Biol Ther 6:231–241CrossRefGoogle Scholar
  11. 11.
    Ram PT, Iyengar R (2001) G protein coupled receptor signaling through the Src and Stat3 pathway: role in proliferation and transformation. Oncogene 20:1601–1606CrossRefGoogle Scholar
  12. 12.
    Silva CM (2004) Role of STATs as downstream signal transducers in Src family kinase-mediated tumorigenesis. Oncogene 23:8017–8023CrossRefGoogle Scholar
  13. 13.
    Wilks AF (2008) The JAK kinases; not just another kinase drug discovery target. Semin Cell Dev Biol 19:319–328CrossRefGoogle Scholar
  14. 14.
    Murray P (2007) The JAK-STAT signaling pathway: input and output integration. J Immunol 178:2632–2629Google Scholar
  15. 15.
    Seethala RR, Gooding WE, Handler PN, Collins B, Zhang Q, Siegfried JM, Grandis JR (2008) Immunohistochemical analysis of phosphotyrosine signal transducer and activator of transcription 3 and epidermal growth factor receptor autocrine signaling pathways in head and neck cancers and metastatic lymph nodes. Clin Cancer Res 14:1303–1309CrossRefGoogle Scholar
  16. 16.
    Sriuranpong V, Park JI, Amornphimoltham P, Patel V, Nelkin BD, Gutkind JS (2003) Epidermal growth factor receptor-independent constitutive activation of STAT3 in head and neck squamous cell carcinoma is mediated by the autocrine/paracrine stimulation of the interleukin 6/gp130 cytokine system. Cancer Res 63:2948–2956Google Scholar
  17. 17.
    Lufei C, Koh TH, Uchida T, Cao X (2007) Pin1 is required for the Ser727 phosphorylation-dependent Stat3 activity. Oncogene 26:7656–7664CrossRefGoogle Scholar
  18. 18.
    Schuringa J, Schepers H, Vellenga E, Kruijer W (2001) Ser727-dependent transcriptional activation by association of p300 with STAT3 upon IL-6 stimulation. FEBS Lett 495:71–76CrossRefGoogle Scholar
  19. 19.
    Wen Z, Zhong Z, Darnell JE Jr (1995) Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82:241–250CrossRefGoogle Scholar
  20. 20.
    Wen Z, Darnell JE Jr (1997) Mapping of Stat3 serine phosphorylation to a single residue (727) and evidence that serine phosphorylation has no influence on DNA binding of Stat1 and Stat3. Nucleic Acids Res 25:2062–2067CrossRefGoogle Scholar
  21. 21.
    Herrmann A, Vogt M, Mönnigmann M, Clahsen T, Sommer U, Haan S, Poli V, Heinrich PC, Müller-Newen G (2007) Nucleocytoplasmic shuttling of persistently activated STAT3. J Cell Sci 120:3249–3261CrossRefGoogle Scholar
  22. 22.
    Liu L, McBride KM, Reich NC (2005) STAT3 nuclear import is independent of tyrosine phosphorylation and mediated by importin-alpha3. Proc Natl Acad Sci U S A 102:8150–8155CrossRefGoogle Scholar
  23. 23.
    Reich N, Liu L (2006) Tracking STAT nuclear traffic. Nat Rev Immunol 6:602–612CrossRefGoogle Scholar
  24. 24.
    Yang J, Chatterjee-Kishore M, Staugaitis SM, Nguyen H, Schlessinger K, Levy DE, Stark GR (2005) Novel roles of unphosphorylated STAT3 in oncogenesis and transcriptional regulation. Cancer Res 65:939–947Google Scholar
  25. 25.
    Yang J, Stark GR (2008) Roles of unphosphorylated STATs in signaling. Cell Res 18:443–451CrossRefGoogle Scholar
  26. 26.
    Ynag J, Liao X, Agarwal MK, Barnes L, Auron PE, Stark GR (2010) Unphosphorylated STAT3 accumulates in response to IL-6 and activates transcription by binding to NFkB. Genes Dev 21:1396–1408CrossRefGoogle Scholar
  27. 27.
    Brockstein B (2011) Management of recurrent head and neck cancer: recent progress and future directions. Drugs 71:1551–1559CrossRefGoogle Scholar
  28. 28.
    Goerner M, Seiwert TY, Sudhoff H (2010) Molecular targeted therapies in head and neck cancer—an update of recent developments. Head Neck Oncol 2:8–12CrossRefGoogle Scholar
  29. 29.
    Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K, Sivachenko A, Kryukov GV, Lawrence MS, Sougnez C, McKenna A, Shefler E, Ramos AH, Stojanov P, Carter SL, Voet D, Cortés ML, Auclair D, Berger MF, Saksena G, Guiducci C, Onofrio RC, Parkin M, Romkes M, Weissfeld JL, Seethala RR, Wang L, Rangel-Escareño C, Fernandez-Lopez JC, Hidalgo-Miranda A, Melendez-Zajgla J, Winckler W, Ardlie K, Gabriel SB, Meyerson M, Lander ES, Getz G, Golub TR, Garraway LA, Grandis JR (2012) The mutational landscape of head and neck squamous cell carcinoma. Science 333:1157–1160CrossRefGoogle Scholar
  30. 30.
    Boehm AL, Sen M, Seethala R, Gooding WE, Freilino M, Wong SM, Wang S, Johnson DE, Grandis JR (2008) Combined targeting of epidermal growth factor receptor, signal transducer and activator of transcription-3, and Bcl-X(L) enhances antitumor effects in squamous cell carcinoma of the head and neck. Mol Pharmacol 73:1632–1642CrossRefGoogle Scholar
  31. 31.
    Leeman-Neill RJ, Wheeler SE, Singh SV, Thomas SM, Seethala RR, Neill DB, Panahandeh MC, Hahm ER, Joyce SC, Sen M, Cai Q, Freilino ML, Li C, Johnson DE, Grandis JR (2009) Guggulsterone enhances head and neck cancer therapies via inhibition of signal transducer and activator of transcription-3. Carcinogenesis 30:1848–1856CrossRefGoogle Scholar
  32. 32.
    Johnston P, Sen M, Hua Y, Camarco D, Shun TY, Lazo JS, Grandis JR (2014) High-content pSTAT3/1 imaging assays to screen for selective inhibitors of STAT3 pathway activation in head and neck cancer cell lines. Assay Drug Dev Technol 12:55–79CrossRefGoogle Scholar
  33. 33.
    Johnston P, Sen M, Hua Y, Camarco DP, Shun TY, Lazo JS, Wilson GM, Resnick LO, LaPorte MG, Wipf P, Huryn DM, Grandis JR (2015) HCS campaign to identify selective inhibitors of IL-6-induced STAT3 pathway activation in head and neck cancer cell lines. Assay Drug Dev Technol 13:356–376CrossRefGoogle Scholar
  34. 34.
    LaPorte M, da Paz Lima DJ, Zhang F, Sen M, Grandis JR, Camarco D, Hua Y, Johnston PA, Lazo JS, Resnick LO, Wipf P, Huryn DM (2014) 2-Guanidinoquinazolines as new inhibitors of the STAT3 pathway. Bioorg Med Chem Lett 24:5081–5085CrossRefGoogle Scholar
  35. 35.
    LaPorte M, Wang Z, Colombo R, Garzan A, Peshkov VA, Liang M, Johnston PA, Schurdak ME, Sen M, Camarco DP, Hua Y, Pollock NI, Lazo JS, Grandis JR, Wipf P, Huryn DM (2016) Optimization of pyrazole-containing 1,2,4-triazolo-[3,4-b]thiadiazines, a new class of STAT3 pathway inhibitors. Bioorg Med Chem Lett 26:3581–3585CrossRefGoogle Scholar
  36. 36.
    Bauer V, Hieber L, Schaeffner Q, Weber J, Braselmann H, Huber R, Walch A, Zitzelsberger H (2010) Establishment and molecular cytogenetic characterization of a cell culture model of head and neck squamous cell carcinoma (HNSCC). Genes 1:338–412CrossRefGoogle Scholar
  37. 37.
    Gioanni J, Fischel JL, Lambert JC, Demard F, Mazeau C, Zanghellini E, Ettore F, Formento P, Chauvel P, Lalanne CM et al (1988) Two new human tumor cell lines derived from squamous cell carcinomas of the tongue: establishment, characterization and response to cytotoxic treatment. Eur J Cancer Clin Oncol 24:1445–1455CrossRefGoogle Scholar
  38. 38.
    Dudgeon D, Shinde SN, Shun TY, Lazo JS, Strock CJ, Giuliano KA, Taylor DL, Johnston PA, Johnston PA (2010) Characterization and optimization of a novel protein-protein interaction biosensor HCS assay to identify disruptors of the interactions between p53 and hDM2. Assay Drug Dev Technol 8:437–458CrossRefGoogle Scholar
  39. 39.
    Johnston PA, Shinde SN, Hua Y, Shun TY, Lazo JS, Day BW (2012) Development and validation of a high-content screening assay to identify inhibitors of cytoplasmic dynein-mediated transport of glucocorticoid receptor to the nucleus. Assay Drug Dev Technol 10:432–456CrossRefGoogle Scholar
  40. 40.
    Nickischer D, Laethem C, Trask OJ Jr et al (2006) Development and implementation of three mitogen-activated protein kinase (MAPK) signaling pathway imaging assays to provide MAPK module selectivity profiling for kinase inhibitors: MK2-EGFP translocation, c-Jun, and ERK activation. Methods Enzymol 414:389–418CrossRefGoogle Scholar
  41. 41.
    Trask O, Nickischer D, Burton A, Williams RG, Kandasamy RA, Johnston PA, Johnston PA (2009) High-throughput automated confocal microscopy imaging screen of a kinase-focused library to identify p38 mitogen-activated protein kinase inhibitors using the GE InCell 3000 analyzer. Methods Mol Biol 565:159–186CrossRefGoogle Scholar
  42. 42.
    Trask OJ Jr, Baker A, Williams RG et al (2006) Assay development and case history of a 32K-biased library high-content MK2-EGFP translocation screen to identify p38 mitogen-activated protein kinase inhibitors on the ArrayScan 3.1 imaging platform. Methods Enzymol 414:419–439CrossRefGoogle Scholar
  43. 43.
    Williams RG, Kandasamy R, Nickischer D et al (2006) Generation and characterization of a stable MK2-EGFP cell line and subsequent development of a high-content imaging assay on the Cellomics ArrayScan platform to screen for p38 mitogen-activated protein kinase inhibitors. Methods Enzymol 414:364–389CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Paul A. Johnston
    • 1
    Email author
  • 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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