Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas
Sizhi Paul Gao, … , Bayard Clarkson, Jacqueline F. Bromberg
Sizhi Paul Gao, … , Bayard Clarkson, Jacqueline F. Bromberg
Published December 3, 2007
Citation Information: J Clin Invest. 2007;117(12):3846-3856. https://doi.org/10.1172/JCI31871.
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Research Article

Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas

Abstract

Persistently activated or tyrosine-phosphorylated STAT3 (pSTAT3) is found in 50% of lung adenocarcinomas. pSTAT3 is found in primary adenocarcinomas and cell lines harboring somatic-activating mutations in the tyrosine kinase domain of EGFR. Treatment of cell lines with either an EGFR inhibitor or an src kinase inhibitor had no effect on pSTAT3 levels, whereas a pan-JAK inhibitor (P6) blocked activation of STAT3 and inhibited tumorigenesis. Cell lines expressing these persistently activated mutant EGFRs also produced high IL-6 levels, and blockade of the IL-6/gp130/JAK pathway led to a decrease in pSTAT3 levels. In addition, reduction of IL-6 levels by RNA interference led to a decrease in tumorigenesis. Introduction of persistently activated EGFR into immortalized breast epithelial cells led to tumorigenesis, IL-6 expression, and STAT3 activation, all of which could be inhibited with P6 or gp130 blockade. Furthermore, inhibition of EGFR activity in multiple cell lines partially blocked transcription of IL-6 and concurrently decreased production and release of IL-6. Finally, immunohistochemical analysis revealed a positive correlation between pSTAT3 and IL-6 positivity in primary lung adenocarcinomas. Therefore, mutant EGFR could activate the gp130/JAK/STAT3 pathway by means of IL-6 upregulation in primary human lung adenocarcinomas, making this pathway a potential target for cancer treatment.

Authors

Sizhi Paul Gao, Kevin G. Mark, Kenneth Leslie, William Pao, Noriko Motoi, William L. Gerald, William D. Travis, William Bornmann, Darren Veach, Bayard Clarkson, Jacqueline F. Bromberg

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Figure 8

EGFR tyrosine kinase inhibition reduces de novo production of IL-6.

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EGFR tyrosine kinase inhibition reduces de novo production of IL-6. (A) ...
(A) 11-18 and H1650 cells were treated with a medium change (MC) with ZD (MC+ZD), no MC with ZD (No MC+ZD), or MC with DMSO (MC+D). Levels of IL-6 were measured by ELISA at the indicated times after the addition of ZD (mean ± SD). (B) Shown are extracts isolated from 11-18 and H1650 cells treated as described above after 16 hours and analyzed for pSTAT3, STAT3, and α-tubulin. (C) Human IL-6 mRNA levels from 11-18 cells treated with DMSO or ZD for 16 hours after a medium change were determined by RT-PCR and normalized to β-actin. The same mRNA samples were analyzed by quantitative real-time PCR (QPCR), and the IL-6 mRNA levels (normalized to hypoxanthine-guanine phosphoribosyltransferase [HPRT]) are shown below. (D) NIH3T3 cells were cotransfected with an IL-6 reporter construct, a TK-Renilla construct (for transfection/loading control), and either a pB vector (baseline activity) or a pB-ΔEGFR expression construct. Twenty-four hours after transfection, DMSO or ZD was added, and an additional 24 hours later, cells were lysed and subjected to firefly and Renilla luciferase activity measurements. The bars show fold induction over the baseline activity (mean ± SD).