title: High-content pSTAT3/1 imaging assays to screen for selective inhibitors of STAT3 pathway activation in head and neck cancer cell lines.
abstract: The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is hyperactivated in most cancers and represents a plausible therapeutic target. In the absence of STAT3-selective small-molecule inhibitors, we sought to develop pSTAT3/1 high-content imaging (HCS) assays to screen for selective inhibitors of STAT3 pathway activation in head and neck squamous cell carcinomas (HNSCC) tumor cell lines. Based on the expression of the interleukin-6 (IL-6)Rα and gp130 subunits of the IL-6 receptor complex and STAT3, we selected the Cal33 HNSCC cell line as our model. After developing image acquisition and analysis procedures, we rigorously investigated the cytokine activation responses to optimize the dynamic ranges of both assays and demonstrated that the pan-Janus kinase inhibitor pyridone 6 nonselectively inhibited pSTAT3 and pSTAT1 activation with 50% inhibition concentrations of 7.19 ± 4.08 and 16.38 ± 8.45 nM, respectively. The optimized pSTAT3 HCS assay performed very well in a pilot screen of 1,726 compounds from the Library of Pharmacologically Active Compounds and the National Institutes of Health clinical collection sets, and we identified 51 inhibitors of IL-6-induced pSTAT3 activation. However, only three of the primary HCS actives selectively inhibited STAT3 compared with STAT1. Our follow-up studies indicated that the nonselective inhibition of cytokine induced pSTAT3 and pSTAT1 activation by G-alpha stimulatory subunit-coupled G-protein-coupled receptor agonists, and forskolin was likely due to cyclic adenosine monophosphate-mediated up-regulation of suppressors of cytokine signaling 3. Azelastine, an H1 receptor antagonist approved for the treatment of seasonal allergic rhinitis, nonallergic vasomotor rhinitis, and ocular conjunctivitis, was subsequently confirmed as a selective inhibitor of IL-6-induced pSTAT3 activation that also reduced the growth of HNSCC cell lines. These data illustrate the power of a chemical biology approach to lead generation that utilizes fully developed and optimized HCS assays as phenotypic screens to interrogate specific signaling pathways.
authors.affiliation: 1 Department of Pharmaceutical Sciences, University of Pittsburgh , Pittsburgh, Pennsylvania.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Johnston
authors.forename: Malabika
authors.initials: M
authors.lastname: Sen
authors.forename: Yun
authors.initials: Y
authors.lastname: Hua
authors.forename: Daniel
authors.initials: D
authors.lastname: Camarco
authors.forename: Tong Ying
authors.initials: TY
authors.lastname: Shun
authors.forename: John S
authors.initials: JS
authors.lastname: Lazo
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Grandis
mesh_terms: D000970:Antineoplastic Agents / Q000008:administration & dosage*; D017209:Apoptosis / Q000187:drug effects; D001681:Biological Assay / Q000639:trends*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006258:Head and Neck Neoplasms / Q000378:metabolism* / Q000473:pathology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D057054:Molecular Imaging / Q000379:methods; D050794:STAT1 Transcription Factor / Q000378:metabolism*; D050796:STAT3 Transcription Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D000970:Antineoplastic Agents; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor