Research ArticleCancer

Stress hormones promote EGFR inhibitor resistance in NSCLC: Implications for combinations with β-blockers

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Science Translational Medicine  08 Nov 2017:
Vol. 9, Issue 415, eaao4307
DOI: 10.1126/scitranslmed.aao4307
  • Fig. 1. IL-6 is associated with resistance to EGFR TKIs and is induced by stress hormones.

    (A) IL-6 secretion in NSCLC cells with acquired resistance to EGFR TKIs. Data are means ± SD. (B) Median OS in NSCLC patients with high or low circulating concentrations of IL-6 treated with erlotinib. (C) mRNA expression of ADRB1,2,3 in 159 patient samples (left) and in 116 NSCLC cell lines (right). (D) NSCLC cells were stimulated with NE (24 hours). IL-6 secretion was determined by ELISA. *P ≤ 0.001. Bars are means ± SEM. (E) IL-6 mRNA expression after NE stimulation (10 μM for 3 hours). *P ≤ 0.001. Data are means ± SD. (F) NE-induced IL-6 secretion after treatment with the β-AR inhibitor propranolol (PPL; 1 μM) or the α-AR inhibitor phentolamine hydrochloride (1 μM). *P ≤ 0.01; one-way analysis of variance (ANOVA). Data are means ± SD. (G) IL-6 production after treatment with a β1-AR agonist (dobutamine; 50 μM) or a β2-AR agonist (salbutamol; 50 μM) for 24 hours. *P ≤ 0.02; one-way ANOVA. Data are means ± SD. (H) Effect of the adenylyl cyclase activator forskolin (10 μM) on IL-6 secretion. *P ≤ 0.0002. P value calculated by two-tailed Student’s t test. (I) Circulating concentrations of IL-6 in NSCLC patients with or without incidental pan β-blocker use (BB; *P = 0.02). Data are means ± SEM.

  • Fig. 2. β-ARs signal cooperatively with mutant EGFR and inactivate LKB1.

    (A) NSCLC cells harboring wild-type EGFR or EGFR-activating mutations were stimulated with NE (10 μM), and IL-6 secretion was measured by ELISA. *P ≤ 0.001. Data are means ± SD. (B) Coimmunoprecipitation of endogenous β2-AR and EGFR in HCC827 cells (EGFR mutant) after NE stimulation. (C) HCC827 (EGFR mutant) and A549 (EGFR wild-type) cells were transfected with control vector or a FLAG-tagged β2-AR expression vector. After treatment with NE, cells were immunostained with antibodies directed against EGFR or FLAG-tag and assessed by Duolink proximity ligation assay. Data are means ± SD. (D) Representative images from Duolink assay. Red foci indicate interactions between endogenous EGFR and FLAG-tagged β2-AR. Scale bar, 10 μm. (E) Heatmap depicting mean protein expression after treatment with NE (10 μM; 15 min). (F) Alterations in phosphorylation of LKB1 at the inhibitory site S428, AMPK activation, and mechanistic target of rapamycin (mTOR) activity (p70S6K) after NE stimulation as determined by RPPA. Data are means ± SD. (G) RPPA results were confirmed by Western blotting. (H) Western blot demonstrating the effect of NE (10 μM; 15 min) on the phosphorylation of LKB1 at the inhibitory site S428 and phosphorylation of mTOR and p70S6K in H1975 cells.

  • Fig. 3. β-AR signaling induces IL-6 in NSCLC cells via activation of PKC and CREB.

    (A) HCC827 and HCC4006 cells were treated with NE with or without Ro31-8220 (5 μM). LKB1S428 and p-CREBS133 were quantified by Western blotting, and (B) IL-6 was quantified by ELISA (*P ≤ 0.01; one-way ANOVA). Data are means ± SD. Representative data from two independent experiments performed in triplicate. (C) HCC827 cells were treated with a PKC inhibitor peptide and then stimulated with NE. LKB1S428 and p-CREBS133 were quantified by Western blotting. (D) LKB1 was stably overexpressed in HCC827 cells. After treatment with NE, IL-6 secretion was evaluated by ELISA. *P = 0.009; by two-tailed Student’s t test. Data are means ± SD. (E) NSCLC cells harboring EGFR-activating mutations (HCC827, HCC4006, H1650, and PC9) or wild-type EGFR (H460 and A549) were stimulated with NE, and p-CREBS133 was quantified by Western blotting. (F) HCC8227 and HCC4006 cells were treated with NE alone or in the presence of the CREBBP inhibitor SGC-CBP30 (1 μM), and IL-6 production was evaluated by ELISA. *P ≤ 0.01; one-way ANOVA. Data are means ± SEM.

  • Fig. 4. Stress hormones promote EGFR TKI resistance in vitro and in vivo.

    (A) EGFR mutant NSCLC cells were stimulated with NE (10 μM) for 24 hours and then treated with erlotinib. Cell viability was determined by MTS assay. *P < 0.0001; by two-tailed Student’s t test. Bars are means ± SD. (B) HCC827 cells were treated with PPL (*P ≤ 0.03) or (C) IL-6 neutralizing antibodies (*P < 0.001) before NE stimulation. After 24 hours, cells were treated with erlotinib for 5 days. Cell viability was evaluated by MTS assay. (D) Ten mice per cell line were injected subcutaneously (control n = 5; stress n = 5). Chronic stress was induced via restraint. For HCC4006, all mice developed tumors. For HCC827, four and two mice developed tumors in the stress and control groups, respectively. Data are means ± SEM. *P < 0.04; two-tailed Student’s t test. (E) HCC827 tumor-bearing mice were treated with ISO (β-AR agonist) for 3 days. Tumors were collected, and IL-6 mRNA was measured by quantitative PCR (control group n = 13; ISO group n = 7, run as technical duplicates). *P = 0.02 two-tailed Student’s t test. Data are means ± SEM. (F) Erlotinib induced tumor regression in HCC827 xenografts. (G) After prolonged erlotinib treatment, resistant disease emerged in mice treated with erlotinib plus ISO compared to mice receiving erlotinib alone (P = 0.018). PPL (P = 0.035) or the IL-6 antibody siltuximab (Silt, P = 0.009) blocked the effect. *P = 0.018; means ± SEM.

  • Fig. 5. β-Blocker use is associated with improved benefit from afatinib in LUX-Lung3.

    Analysis of incidental β-blocker use in the LUX-Lung3 clinical study comparing the EGFR TKI afatinib with chemotherapy in EGFR mutant–positive NSCLC. E, number of events; N, number of patients; pem, pemetrexed; cis, cisplatin.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/415/eaao4307/DC1

    Fig. S1. Frequency of T790M EGFR secondary mutations in erlotinib-resistant cells.

    Fig. S2. Resistance of HCC827 ER and HCC4006 ER cells to EGFR TKIs.

    Fig. S3. ADRB expression and EGFR status in NSCLC cell lines.

    Fig. S4. Induction of IL-6 after β-AR activation.

    Fig. S5. Differential effects of β2-AR signaling on EGFR mutant and wild-type cells.

    Fig. S6. Stress hormone–induced effects on LKB1, PKC, and CREB.

    Fig. S7. Effect of EGFR inhibition on β-AR–induced p-LKB1, p-CREB, and IL-6.

    Fig. S8. Effect of β-AR signaling on NF-κB activity.

    Fig. S9. Effect of chronic stress on IL-6 expression and MVD in NSCLC xenografts.

    Fig. S10. The effects of stress hormones on EGFR inhibitor resistance in NSCLC.

    Table S1. PFS and OS of NSCLC patients with high or low IL-6 treated with erlotinib.

    Table S2. Smoking status of patients with high or low circulating IL-6 in the ZEST trial.

    Table S3. Lung cancer cell lines used in ADRB gene expression analysis.

  • Supplementary Material for:

    Stress hormones promote EGFR inhibitor resistance in NSCLC: Implications for combinations with β-blockers

    Monique B. Nilsson, Huiying Sun, Lixia Diao, Pan Tong, Diane Liu, Lerong Li, Youhong Fan, Alissa Poteete, Seung-Oe Lim, Kathryn Howells, Vincent Haddad, Daniel Gomez, Hai Tran, Guillermo Armaiz Pena, Lecia V. Sequist, James C. Yang, Jing Wang, Edward S. Kim, Roy Herbst, J. Jack Lee, Waun Ki Hong, Ignacio Wistuba, Mien-Chie Hung, Anil K. Sood, John V. Heymach*

    *Corresponding author. Email: jheymach{at}mdanderson.org

    Published 8 November 2017, Sci. Transl. Med. 9, eaao4307 (2017)
    DOI: 10.1126/scitranslmed.aao4307

    This PDF file includes:

    • Fig. S1. Frequency of T790M EGFR secondary mutations in erlotinib-resistant cells.
    • Fig. S2. Resistance of HCC827 ER and HCC4006 ER cells to EGFR TKIs.
    • Fig. S3. ADRB expression and EGFR status in NSCLC cell lines.
    • Fig. S4. Induction of IL-6 after β-AR activation.
    • Fig. S5. Differential effects of β2-AR signaling on EGFR mutant and wild-type cells.
    • Fig. S6. Stress hormone–induced effects on LKB1, PKC, and CREB.
    • Fig. S7. Effect of EGFR inhibition on β-AR–induced p-LKB1, p-CREB, and IL-6.
    • Fig. S8. Effect of β-AR signaling on NF-κB activity.
    • Fig. S9. Effect of chronic stress on IL-6 expression and MVD in NSCLC xenografts.
    • Fig. S10. The effects of stress hormones on EGFR inhibitor resistance in NSCLC.
    • Table S1. PFS and OS of NSCLC patients with high or low IL-6 treated with erlotinib.
    • Table S2. Smoking status of patients with high or low circulating IL-6 in the ZEST trial.
    • Table S3. Lung cancer cell lines used in ADRB gene expression analysis.

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