TNF-driven adaptive response mediates resistance to EGFR inhibition in lung cancer
Ke Gong, … , Dawen Zhao, Amyn A. Habib
Ke Gong, … , Dawen Zhao, Amyn A. Habib
Published April 3, 2018
Citation Information: J Clin Invest. 2018;128(6):2500-2518. https://doi.org/10.1172/JCI96148.
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Research Article Therapeutics

TNF-driven adaptive response mediates resistance to EGFR inhibition in lung cancer

Abstract

Although aberrant EGFR signaling is widespread in cancer, EGFR inhibition is effective only in a subset of non–small cell lung cancer (NSCLC) with EGFR activating mutations. A majority of NSCLCs express EGFR wild type (EGFRwt) and do not respond to EGFR inhibition. TNF is a major mediator of inflammation-induced cancer. We find that a rapid increase in TNF level is a universal adaptive response to EGFR inhibition in NSCLC, regardless of EGFR status. EGFR signaling actively suppresses TNF mRNA levels by inducing expression of miR-21, resulting in decreased TNF mRNA stability. Conversely, EGFR inhibition results in loss of miR-21 and increased TNF mRNA stability. In addition, TNF-induced NF-κB activation leads to increased TNF transcription in a feed-forward loop. Inhibition of TNF signaling renders EGFRwt-expressing NSCLC cell lines and an EGFRwt patient-derived xenograft (PDX) model highly sensitive to EGFR inhibition. In EGFR-mutant oncogene-addicted cells, blocking TNF enhances the effectiveness of EGFR inhibition. EGFR plus TNF inhibition is also effective in NSCLC with acquired resistance to EGFR inhibition. We suggest concomitant EGFR and TNF inhibition as a potentially new treatment approach that could be beneficial for a majority of lung cancer patients.

Authors

Ke Gong, Gao Guo, David E. Gerber, Boning Gao, Michael Peyton, Chun Huang, John D. Minna, Kimmo J. Hatanpaa, Kemp Kernstine, Ling Cai, Yang Xie, Hong Zhu, Farjana J. Fattah, Shanrong Zhang, Masaya Takahashi, Bipasha Mukherjee, Sandeep Burma, Jonathan Dowell, Kathryn Dao, Vassiliki A. Papadimitrakopoulou, Victor Olivas, Trever G. Bivona, Dawen Zhao, Amyn A. Habib

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Figure 4

A TNF/NF-κB feed-forward loop regulates the expression of TNF in response to EGFR inhibition.

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A TNF/NF-κB feed-forward loop regulates the expression of TNF in respons...
(A) Inhibition of NF-κB using various chemical inhibitors — BMS-345541 (100 nM), 6-amino-4-(4-phenoxyphenylethylamino) quinazoline (QNZ; 1 μM), or sodium salicylate (5 mM) — inhibited erlotinib-induced upregulation of TNF in HCC827 cells as determined by qPCR. Cells were treated with NF-κB inhibitors for 1 hour and then 100 nM erlotinib for 24 hours. (B) Expression of a dominant-negative (DN) IκBα super-repressor mutant blocks erlotinib-induced upregulation of TNF in HCC827 cells. (C) Mithramycin A (MMA) (1 μM), an inhibitor of Sp1, failed to inhibit erlotinib-induced TNF upregulation. (D and E) The same experiment as in A and B was conducted in A549 cells. Expression of the dominant-negative IκBα super-repressor mutant was detected by Western blot. The mutant protein migrates more slowly on electrophoretic gels. (F–H) siRNA knockdown of TNFR1 in NSCLC cells inhibits erlotinib-induced upregulation of TNF mRNA as detected by qPCR. Silencing of TNFR1 was confirmed by Western blot. (I and J) Inhibition of TNFR signaling using etanercept (100 μg/ml) results in a block of erlotinib-induced TNF upregulation in HCC827 and A549 cells. (K) ChIP was carried out to assess the recruitment of the NF-κB p65 subunit onto the TNF promoter using primers specific to NF-κB binding region 1 on the TNF promoter. There is a substantially increased p65 antibody enrichment (percentage of input, compared with rabbit IgG) on the TNF promoter in both HCC827 and A549 cells in response to erlotinib treatment for 24 hours. Data represent the mean ± SEM. n = 3 biologically independent experimental replicates. *P < 0.05, **P < 0.01, ***P < 0.001, #not statistically significant, by Student’s t test for comparing 2 indicated groups, or 1-way ANOVA, Dunnett’s method, for comparing multiple groups with the same control (A and D). Western blot results are representative of at least 3 independent replicates.