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1Department of Thoracic/Head and Neck Medical Oncology, University of Texas MDAnderson Cancer Center, Houston, TX 77030, USA. 2Department of BioinformaticsandComputational Biology, University of TexasMDAnderson Cancer Center, Houston,TX 77030, USA. 3Department of Biostatistics, University of Texas MD Anderson CancerCenter, Houston, TX 77030, USA. 4Department ofMolecular andCellular Oncology, Uni-versity of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 5AstraZeneca,Melbourn, SG8 6EE, UK. 6Department of Radiation Oncology, University of Texas MDAnderson Cancer Center, Houston, TX 77030, USA. 7Department of GynecologicOncology and Reproductive Medicine, University of Texas MD Anderson CancerCenter, Houston, TX 77030, USA. 8Massachusetts General Hospital Cancer Center,Boston, MA 02114, USA. 9Graduate Institute of Oncology, National Taiwan UniversityandNational TaiwanUniversityHospital, Taipei City 100, Taiwan. 10Solid TumorOncologyand Investigational Therapeutics, Levine Cancer Institute Carolinas HealthCare System,Charlotte,NC28204,USA. 11SectionofMedicalOncology, YaleCancerCenter andSmilowCancer Hospital, Yale, New Haven, CT 06510, USA. 12Department of Translational Molec-ular Pathology, University of TexasMDAndersonCancerCenter, Houston, TX77030, USA.*Corresponding author. Email: jheymach@mdanderson.org

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Stress hormones promote EGFR inhibitor resistance inNSCLC: Implications for combinations with b-blockersMonique B. Nilsson,1 Huiying Sun,1 Lixia Diao,2 Pan Tong,2 Diane Liu,3 Lerong Li,2 Youhong Fan,1

Alissa Poteete,1 Seung-Oe Lim,4 Kathryn Howells,5 Vincent Haddad,5 Daniel Gomez,6 Hai Tran,1

Guillermo Armaiz Pena,7 Lecia V. Sequist,8 James C. Yang,9 Jing Wang,2 Edward S. Kim,10

Roy S. Herbst,11 J. Jack Lee,3 Waun Ki Hong,1 Ignacio Wistuba,1,12 Mien-Chie Hung,4

Anil K. Sood,7 John V. Heymach1*

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance mediated by T790M-independentmechanisms remains a major challenge in the treatment of non–small cell lung cancer (NSCLC). We identified atargetable mechanism of EGFR inhibitor resistance whereby stress hormones activate b2-adrenergic receptors(b2-ARs) on NSCLC cells, which cooperatively signal with mutant EGFR, resulting in the inactivation of the tumorsuppressor, liver kinase B1 (LKB1), and subsequently induce interleukin-6 (IL-6) expression. We show that stressand b2-AR activation promote tumor growth and EGFR inhibitor resistance, which can be abrogated with b-blockersor IL-6 inhibition. IL-6 was associated with a worse outcome in EGFR TKI–treated NSCLC patients, and b-blocker usewas associated with lower IL-6 concentrations and improved benefit from EGFR inhibitors. These findings provideevidence that chronic stress hormones promote EGFR TKI resistance via b2-AR signaling by an LKB1/CREB (cyclicadenosine 3′,5′-monophosphate response element–binding protein)/IL-6–dependent mechanism and suggest thatcombinations of b-blockers with EGFR TKIs merit further investigation as a strategy to abrogate resistance.

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INTRODUCTIONEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors(TKIs) are effective therapies for non–small cell lung cancer (NSCLC)patients with EGFR-activating mutations. However, resistant diseaseinevitably emerges (1). Themost commonmechanism of resistance toEGFR TKIs is the acquisition of secondary EGFR T790M mutations(2, 3). Second- and third-generation EGFR inhibitors have shown activ-ity against T790M-driven resistance (4, 5). However, in nearly 50% ofcases, resistance is T790M-independent. Interleukin-6 (IL-6) has beenimplicated as a mediator of T790M-independent EGFR TKI resistance(6). The identification of targetable mechanisms of T790M-independentresistance is critical for improving outcomes in these patients.

Chronic stress results in increased production of stress hormonessuch as norepinephrine (NE) and epinephrine (E) from the adrenalmedulla and sympathetic neurons. The effects of stress hormones aremediated through binding to b1,2- or b1,2,3-adrenergic receptors (ARs)on target cells. TheARpathway contributes to tumor development andprogression in multiple malignancies including NSCLC (7–9). In lungcancer patients, psychological distress is a known predictor ofmortality

(10), and in preclinical models, b-AR signaling is associated with lungadenocarcinoma development and initiation of proliferative andinvasive cellular programs (7, 11, 12). AR signaling has been linkedto increased expression of cytokines including IL-6 (13).We previouslyreported that stress hormones includingNE and E activate ARs presenton ovarian cancer cells and increase tumor-derived IL-6 (14). Given thereports implicating IL-6 as a mediator of T790M-independent EGFRTKI resistance, we investigated whether stress hormones promote IL-6expression in NSCLC cells and whether activation of ARs facilitatesresistance to EGFR TKIs.

Here, we report that stress hormone activation of b2-AR promotesEGFR TKI resistance in cell lines and in mouse models of EGFR mu-tant NSCLC and that this can be blocked by anti–IL-6 antibodies orb-blockers. Using reverse phase protein array (RPPA) to identifysignaling pathways activated by b2-AR in NSCLC cells, we found thatb2-AR signaling inactivates the tumor suppressor liver kinase B1(LKB1), the loss ofwhich promotes therapeutic resistance andmetastasisin NSCLC (15). Analysis of clinical data sets revealed that high circulat-ing concentrations of IL-6 were associated with a worse progression-freesurvival (PFS) and overall survival (OS) in patients treated with EGFRinhibitors and that circulating IL-6 concentrationswere lower in patientsincidentally receiving b-blockers. Furthermore, our analysis of incidentalb-blocker use in the LUX-Lung3 study comparing afatinib with chemo-therapy in EGFR mutant NSCLC suggests that patients receivingb-blockers may receive greater benefit from EGFR-targeting agents.Collectively, these data suggest the need for clinical testing of EGFRTKIs in combination with b-blockers.

RESULTSAcquired resistance to EGFR TKIs is associated withincreased IL-6IL-6 is implicated as a driver of EGFR TKI resistance (6). We culturedHCC827 and HCC4006 (both EGFR mutant) cells with increasingconcentrations of erlotinib, an EGFR inhibitor, until resistant variants

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emerged. EGFR TKI–resistant (ER) vari-ants HCC827 ER-1, ER-3, and ER-6 andHCC4006 ER-3, ER-5, and ER-6 wereT790M-negative, as determined by quanti-tative polymerase chain reaction (PCR) (fig.S1). T790M-negative erlotinib-resistant cellswere also resistant to gefitinib, osimertinib,afatinib, and CO-1686 (fig. S2, A to D) andexpressed significantly higher IL-6 com-pared to parental cells (HCC827 ER-1,3,6versus HCC827, P = 0.005; HCC4006 ER-3,5,6 versus HCC4006, P = 0.0005; Fig.1A). IL-6 expression was minimal in cellswhere EGFR TKI resistance was asso-ciated with MET amplification (HCC827ER-2) or T790M (PC9 ER-8, ER-9, andER-11, and H1975; Fig. 1A).

Next, we analyzed pretreatment plas-ma concentrations of IL-6 by enzyme-linked immunosorbent assay (ELISA) in209 patients in the erlotinib arm of thephase 3 ZEST (ZACTIMAEfficacy Studyversus Tarceva; NCT00364351) (16) clinicalstudy. All patients had platinum-refractoryNSCLC and were not genotype-selected.In this population, high IL-6 (greater thanmedian) was associated with a significantlyworse OS compared to patients with lowerthanmedian IL-6 concentrations (P<0.0001;Fig. 1B and table S1). The median OS forerlotinib-treated patients with high IL-6was 4.8 months, whereas the median OSwas 11.5 months in patients with low IL-6.High IL-6 was also associated with a signif-icantly worse progression-free survival (P =0.0092; table S1 and fig. S2E). Smokershad higher concentrations of IL-6 thannonsmokers (table S2). The findings areconsistent with earlier preclinical studies(6) and suggest that higher IL-6 may beassociated with EGFR TKI resistance.

Stress hormones induce IL-6expression in NSCLC cells throughactivation of b2-ARNE and E are increased under stress con-ditions and can be substantially higherin tissues compared to circulating concen-trations (17, 18). Effects of stress hor-mones are mediated through binding toa-AR1,2 or b-AR1,2,3. We examinedADRB1, ADRB2, and ADRB3 gene ex-pression in 159 NSCLC clinical samplesand 116 lung cancer cell lines. Tumorsamples and cell lines tested positive forADRB1, ADRB2, and ADRB3 expression(Fig. 1C and fig. S3A). NSCLC cell linesharboring EGFR-activating mutationsexpressed constitutively phosphorylated

Fig. 1. IL-6 is associated with resistance to EGFR TKIs and is induced by stress hormones. (A) IL-6 secretion inNSCLC cells with acquired resistance to EGFR TKIs. Data are means ± SD. (B) Median OS in NSCLC patients with highor low circulating concentrations of IL-6 treated with erlotinib. (C) mRNA expression of ADRB1,2,3 in 159 patientsamples (left) and in 116 NSCLC cell lines (right). (D) NSCLC cells were stimulated with NE (24 hours). IL-6 secretionwas determined by ELISA. *P ≤ 0.001. Bars are means ± SEM. (E) IL-6 mRNA expression after NE stimulation (10 mMfor 3 hours). *P ≤ 0.001. Data are means ± SD. (F) NE-induced IL-6 secretion after treatment with the b-AR inhibitorpropranolol (PPL; 1 mM) or the a-AR inhibitor phentolamine hydrochloride (1 mM). *P ≤ 0.01; one-way analysis of variance(ANOVA). Data are means ± SD. (G) IL-6 production after treatment with a b1-AR agonist (dobutamine; 50 mM) or a b2-ARagonist (salbutamol; 50 mM) for 24 hours. *P ≤ 0.02; one-way ANOVA. Data are means ± SD. (H) Effect of the adenylylcyclase activator forskolin (10 mM) on IL-6 secretion. *P ≤ 0.0002. P value calculated by two-tailed Student’s t test. (I) Cir-culating concentrations of IL-6 in NSCLC patients with or without incidental pan b-blocker use (BB; *P = 0.02). Data aremeans ± SEM.

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EGFR (fig. S3B). We treated NSCLC cellsharboring EGFR-activating mutations(HCC827, HCC4006, and H3255) with1 or 10 mMNE for 24 hours andmeasuredIL-6 secretion by ELISA. NE induced amarked rise in IL-6 (Fig. 1D). IL-6mRNAexpression was significantly increased af-ter NE (10 mM) stimulation for 3 hours, asdetermined by quantitative real-time re-verse transcription PCR (P < 0.001; Fig. 1E).NE concentrations of 0.1 and 0.01 mM alsoincreased IL-6 (fig. S4, A and B). E andthe b-AR agonist isoproterenol (ISO) sim-ilarly induced IL-6 in EGFR mutantNSCLC cells; however, the effect on non-malignant human bronchial epithelialcells was minimal (fig. S4, C and D).

Propranolol (PPL; b-AR inhibitor) butnot phentolamine hydrochloride (a-ARinhibitor) blocked NE-induced IL-6 (Fig.1F and fig. S4E). Treatment with a b2-AR agonist (salbutamol) but not a b1-ARagonist (dobutamine) increased IL-6(Fig. 1G). b-ARs activate the cyclic aden-osine 3′,5′-monophosphate (cAMP) sig-naling pathway though stimulation ofadenylyl cyclase (19). We treated NSCLCcells with an adenylyl cyclase activator,forskolin, and observed an increase inIL-6 similar to that induced by NE (Fig.1H). b-ARs can also activate Epac (20);however, treatment with 8-CPT-2Me-cAMP, a cAMP analog that specificallytargets Epac, did not affect IL-6 secretion(fig. S4F).

To determine whether b-blocker usemay be associated with a reduction ofIL-6 in patients, we measured circulatingconcentrations of IL-6 byELISA inpatientsamples from the BATTLE trial (21). IL-6concentrations were significantly lowerin patients using pan b-blockers com-pared to patients not receiving b-blockers,supporting the contribution of b-ARs inregulating IL-6 concentrations (P = 0.02;Fig. 1I).

b-AR signals cooperatively withmutant EGFRAlthough NE induced a robust increasein IL-6 in cells with EGFR-activatingmutations (HCC827, HCC4006, H3255,PC9, H1975, and H1650), the effect wasminimal in EGFR wild-type cells (A549,H441, Calu6, H460, H661, H23, HCC15,and H1993) (Fig. 2A). Similarly, salbuta-mol (a b2-AR agonist) and forskolin in-duced IL-6 expression in NSCLC cellswith mutant but not wild-type EGFR

Fig. 2. b-ARs signal cooperatively with mutant EGFR and inactivate LKB1. (A) NSCLC cells harboring wild-typeEGFR or EGFR-activating mutations were stimulated with NE (10 mM), and IL-6 secretion was measured by ELISA. *P ≤0.001. Data are means ± SD. (B) Coimmunoprecipitation of endogenous b2-AR and EGFR in HCC827 cells (EGFR mutant)after NE stimulation. (C) HCC827 (EGFRmutant) and A549 (EGFRwild-type) cells were transfectedwith control vector or aFLAG-tagged b2-AR expression vector. After treatment with NE, cells were immunostained with antibodies directedagainst EGFR or FLAG-tag and assessed by Duolink proximity ligation assay. Data are means ± SD. (D) Representativeimages from Duolink assay. Red foci indicate interactions between endogenous EGFR and FLAG-tagged b2-AR. Scalebar, 10 mm. (E) Heatmap depicting mean protein expression after treatment with NE (10 mM; 15 min). (F) Alterations inphosphorylationof LKB1 at the inhibitory site S428, AMPKactivation, andmechanistic target of rapamycin (mTOR) activity(p70S6K) after NE stimulation as determined by RPPA. Data aremeans ± SD. (G) RPPA results were confirmed byWesternblotting. (H) Western blot demonstrating the effect of NE (10 mM; 15min) on the phosphorylation of LKB1 at the inhibitorysite S428 and phosphorylation of mTOR and p70S6K in H1975 cells.

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(fig. S5, A and B). Dobutamine (a b1-AR agonist) treatment did not af-fect IL-6 secretion. b2-AR expression was similar across EGFR mutantand wild-type cell lines (fig. S3A). To determine whether EGFR in-teracts with b2-AR in cells with EGFR-activating mutations,HCC827 cells were treated with or without NE, lysates were immuno-precipitated (IP) with anti-EGFR antibodies, and blots were immuno-blotted (IB) with anti–b2-AR antibodies. We observed an interactionbetween endogenous b2-AR and EGFR in HCC827 cells (EGFR mu-tant) (Fig. 2B). A similar experimentwas performedusingHCC827 cellstransfected with a FLAG-tagged b2-AR expression vector. Lysates wereimmunoprecipitated with anti-EGFR antibodies, and blots were probedwith anti-FLAG antibodies.We observed an interaction between b2-ARand EGFR in HCC827 cells (EGFR mutant) (fig. S5C). To visualize thein situ interaction between EGFR and b2-AR, we performed a Duolinkproximity ligation assay. HCC827 (EGFR mutant) and A549 (EGFRwild-type) cells were transfectedwith a control vector or a FLAG-tagged

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b2-AR expression vector and treated with or without NE for 15 min.Confocal microscopy revealed NE-induced interactions between b2-AR and mutant EGFR but not wild-type EGFR (Fig. 2, C and D, redspots).

Stress hormone signaling inactivates the tumor suppressorLKB1 in a PKC-dependent mannerWe stimulated NSCLC cells with NE and conducted a proteomicanalysis by RPPA. NE induced LKB1S428 phosphorylation—a mod-ification that inhibits LKB1 function (Fig. 2, E and F) (22). LKB1plays a pivotal role in energy sensing, suppressing the mTOR path-way via activation of AMPK and TSC2 (23). NE concurrently de-creased AMPK activation and increased mTOR activity, as indicatedby p70S6K phosphorylation (Fig. 2, E to G). Total amounts of LKB1,AMPK, and p70S6K were unchanged (fig. S6A). RPPA data were con-firmed by Western blotting (Fig. 2G). The effect of NE on LKB1 in-

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activation was also observed at lowerconcentrations of NE (fig. S6B). NEhad a minimal effect on p-LKB1S428 intwo EGFR wild-type cell lines (A549andH661; fig. S6C). Forskolin induceda similar rise in p-LKB1S428 (fig. S6D).NE-induced inactivation of LKB1 wasabrogated by PPL (Fig. 2G). Similarly,in H1975 cells (EGFR mutant), NE stim-ulation induced a rise in p-LKB1S428

and increased p-mTOR and p-p70S6K(Fig. 2H).

The LKB1 S428 residue can be phos-phorylated by protein kinase C (PKC)(24) and p90RSK (22), and activationof b-AR triggers activation of PKC(25) and the mitogen-activated proteinkinase (MAPK)/p90RSK pathways (fig.S6E) (26). Ro31-8220, an inhibitor ofPKC and p90RSK, reducedNE-inducedphosphorylation of PKC and LKB1S428

(Fig. 3A and fig. S6F) and abrogatedNE-induced IL-6 (Fig. 3B and fig. S6G).To distinguish between the potential con-tributions of the PKC and MAPK/p90RSK pathways, we treated HCC827cells with a control medium, a negativecontrol peptide, or a PKC inhibitor pep-tide (27) with orwithoutNE. PKC inhibi-tion blocked NE-induced p-LKB1S428

(Fig. 3C), whereas p90RSK knockdownfailed to inhibit NE-induced increases inp-LKB1S428 or IL-6 (fig. S6, H and I).b-AR signaling can also activate cAMP-dependent protein kinase (PKA) (28, 29).The PKA inhibitor H89 did not blockNE-induced p-LKB1S428 (fig. S6J). To in-vestigate the contribution of LKB1 in-activation in b-AR–induced IL-6, weoverexpressed LKB1 in HCC827 cells(fig. S6K). NE increased IL-6 in greenfluorescent protein (GFP) control cells butnot in cells overexpressing LKB1 (Fig. 3D).

Fig. 3. b-AR signaling induces IL-6 inNSCLC cells via activation of PKC and CREB. (A) HCC827 and HCC4006 cells weretreated with NE with or without Ro31-8220 (5 mM). 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 twoindependent experiments performed in triplicate. (C) HCC827 cells were treated with a PKC inhibitor peptide and thenstimulated with NE. LKB1S428 and p-CREBS133 were quantified by Western blotting. (D) LKB1 was stably overexpressedin HCC827 cells. After treatment with NE, IL-6 secretion was evaluated by ELISA. *P = 0.009; by two-tailed Student’st 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 mM), and IL-6 production was evaluated by ELISA. *P ≤ 0.01; one-way ANOVA. Data are means ± SEM.

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Stress hormones induce IL-6 expression in aCREB-dependent mannerTranscription of the IL6 gene is complex, involving multiple tran-scription factors including nuclear factor kB (NF-kB) and cAMP re-sponse element–binding protein (CREB). In HCC827 cells, NE

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treatment increased p-CREBS133 (fig. S7, A and B), and inhibition ofb-AR but not a-ARs abrogated NE-induced p-CREBS133 (fig. S7C).The NE-induced rise in p-CREBS133 in EGFR mutant cells was notobserved in EGFR wild-type cells (Fig. 3E). Activated CREB bindsCREB-binding proteins (CREBBPs), allowing transcription of target

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genes. SGC-CBP30 (CREBBP inhibitor)abrogated b2-AR–induced IL-6 (Fig. 3F).EGFR activation was not necessary forb-AR–mediated IL-6 expression, becauseerlotinib (1 mM) failed to inhibit NE-induced p-CREB and p-LKB1S428 (fig.S7D) and failed to block NE-induced IL-6expression (fig. S7E). NE had minimaleffects on NF-kB nuclear localization;NF-kB inhibition failed to block NE-induced IL-6 (fig. S8). Inhibition of PKCby RO31-8220 or a PKC inhibitor peptidediminished NE-induced rises in p-CREB(Fig. 3, A and C). In contrast, p90RSKknockdown failed to inhibit NE-inducedp-CREB (fig. S6H). NE increased p-CREBS133 in GFP control cells but not incells overexpressing LKB1 (fig. S6L), sug-gesting that CREB activation occurs down-stream of LKB1 suppression.

Stress hormones promote EGFR TKIresistance and growth ofNSCLC xenograftsWe treated EGFR mutant NSCLC celllines with NE 24 hours before the addi-tion of erlotinib. NE promoted erlotinibresistance in vitro (Fig. 4A). The additionof the b-AR inhibitor PPL or IL-6 neu-tralizing antibodies blocked the effecton EGFR TKI resistance (Fig. 4, B and C).

Next,we injectedHCC827andHCC4006cells into the flanks of nudemice. Chronicstress was initiated using an establishedrestraint model (30). Tumor volumeswere more than doubled in stressed micerelative to control mice (Fig. 4D; P≤ 0.04for both). Immunohistochemistry revealedincreased IL-6 expression in tumors fromstressed mice compared to controls (P =0.002; fig. S9, A and B). Tumor cell prolif-eration was not significantly different instressedmice, but they had reducedmicro-vessel density (MVD; fig. S9C). Treatmentof HCC827 tumor-bearing mice with ISOsignificantly increased IL-6 mRNA (P =0.02; Fig. 4E).

We established HCC827 xenograftsin female nudemice.Once tumors reached400mm3, animals were randomized to re-ceive erlotinib (100 mg/kg) alone or withdaily ISO. Erlotinib treatment resulted innear-complete tumor regression (Fig.4F). After prolonged erlotinib treatment,

Fig. 4. Stress hormones promote EGFR TKI resistance in vitro and in vivo. (A) EGFR mutant NSCLC cells werestimulated with NE (10 mM) 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. Cellviability 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 devel-oped 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 (b-AR agonist) for 3 days. Tumors were collected, and IL-6mRNA wasmeasured by quantitative PCR (control group n = 13; ISO group n = 7, run as technical duplicates). *P = 0.02 two-tailedStudent’s t test. Data are means ± SEM. (F) Erlotinib induced tumor regression in HCC827 xenografts. (G) After prolongederlotinib treatment, resistant disease emerged in mice treated with erlotinib plus ISO compared to mice receiving erlotinibalone (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.

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resistant disease emerged inmice treatedwith erlotinib plus ISO com-pared to mice receiving erlotinib alone (P = 0.018; Fig. 4G). Theaddition of PPL (P = 0.035), or the IL-6 antibody siltuximab (P =0.009), inhibited the effect of ISO on EGFR TKI resistance, indicat-ing a role for IL-6 in b-AR–mediated resistance.

b-Blocker use is associated with improved benefit fromafatinb in the LUX-Lung3 studyFinally, we evaluated the influence of incidental b-blocker use on treat-ment effect in the randomized phase 3 LUX-Lung3 study (31) comparingthe EGFR TKI afatinib with chemotherapy in EGFRmutation–positiveNSCLCpatients. In patients not receiving b-blockers, afatinib improvedPFS time, with a median PFS of 11.1 and 6.9 months for afatinib andchemotherapy, respectively (log-rank test P = 0.001; Fig. 5, left), and ahazard ratio (HR) of 0.60, corresponding to a 40% reduction in the like-lihood of progression for afatinib. In patients receiving b-blockers,afatinibwas associatedwith a greater relative PFS benefit, with amedianPFS of 13.6 and 2.5months for afatinib and chemotherapy, respectively(HR = 0.25, log-rank test P = 0.0001; Fig. 5, right), corresponding to a75% reduction in the likelihood of progression. Although the analysis ofthese randomized trials was exploratory and limited by the modestnumber of patients receiving b-blockers, the findings are consistentwiththe preclinical data that b blockade could delay the emergence of EGFRTKI resistance.

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DISCUSSIONAcquired resistance to EGFR-targeted therapies is amajor challenge inthe treatment of NSCLC patients. The development of second- andthird-generation EGFR inhibitors with activity against T790M-positiveEGFR mutants is a clear advancement for targeting resistance. How-ever, effective strategies are needed to delay and target resistant diseasethat emerges through T790M-independent mechanisms. This studyrevealed that stress hormones act on b2-AR on NSCLC tumor cells andpromote EGFR inhibitor resistance through the up-regulationof IL-6 (fig.S10). By using a high-throughput proteomic analysis, we identified amechanism of LKB1- and CREB-dependent induction of IL-6 that could

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be inhibited by b-blockers. Our preclinical findings were supported byclinical data indicating that high plasma concentrations of IL-6 are asso-ciated with a worse response to EGFR inhibitors, and b-blocker use isassociated with lower circulating concentrations of IL-6. Finally,our analysis of the phase 3 LUX-Lung3 study suggests that b-blockerusemay improve response to EGFR inhibitors inNSCLCpatients withEGFR-activating mutations.

The b-AR pathway contributes to tumor progression in a variety ofmalignancies, enhancing tumor cell proliferation and invasion (7, 32–36),inducing vascular and lymphatic remodeling (37), and enhancing lymphnode metastasis (37). In lung cancer patients, psychological distress is apredictor of mortality (10). Retrospective clinical analyses have indi-cated that b-blocker use is associated with a reduced risk of cancerdevelopment (38) and decreased breast cancer–specific (39) and pros-tate cancer–specific mortality (40), but whether b-blockers improveclinical outcome remains controversial. Whereas first-generationb-blockers (PPL, nadolol, and timolol) are nonselective and thusblock b1- and b2-ARs, second-generation cardioselective b-blockers(metoprolol, acebutolol, and busoprolol) have activity against b1-ARsbut not b2-ARs. In the treatment of patients with cardiovascularconditions, cardioselective b-blockers are more frequently used ineffort tominimize adverse effects. Here, we show that b2-ARs are high-ly expressed on NSCLC tumor cells and that stress hormones activatesignaling pathways that contribute to therapeutic resistance in NSCLCcell lines though b2-AR but not b1-AR. These findings indicate that panb-blockers but not cardioselective b-blockers may improve the clinicaloutcome of this patient population. Moreover, there were differentialeffects between cells with or without EGFR-activating mutations. It isfeasible that the conflicting clinical data regarding the effect ofb-blocker use on clinical outcome are related to the specificity of theb-blocker used and the mutational context of the tumors. Note thatthe tobacco-specific nitrosamine NKK is a high-affinity agonist forb1-ARs and b2-ARs (12, 41). Therefore, it is feasible that the establishedassociation between smoking status and IL-6 expression occurs in partthrough activation of the adrenergic pathway as described here.

Findings from this study and others (6) indicate that IL-6 is a keydriver of T790M-independent EGFR TKI resistance. The present report

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highlights the regulation of IL-6 by stresshormones, but smoking status and comor-bidities including obesity and diabetes arealso associatedwith chronic inflammationand enhanced expression of IL-6 (42–44)and thusmay affect the emergence of ther-apeutic resistance.Moreover, the deleteriouseffects of chronic stress on the clinical out-come of EGFR mutant NSCLC patientsmay extend beyond the induction of IL-6.Stress hormone–mediated activation of b2-ARs can further activate the b-arrestinpathway, triggering degradation of p53and the accumulation of DNA damage(45). In addition, b-arrestins are thoughtto facilitate GPCR (heterotrimeric GTP-binding protein–coupled receptor)–mediated transactivation of EGFR incancer cells (46).

Earlier studies in other settings havesuggested that interactions betweenb-AR and mutant EGFR could occur.

Fig. 5. b-Blocker use is associated with improved benefit from afatinib in LUX-Lung3. Analysis of incidentalb-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.

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Many GPCRs use EGFR as an intermediate signaling protein (47).b-AR transactivates EGFR, resulting in incomplete downstreamsignaling. However, when EGFR and b-AR are simultaneously acti-vated, broader signal transduction pathways are initiated (48). Inanimal models of carcinogen-induced lung adenocarcinoma, activa-tion of both b-AR and EGFR is observed, and data suggest receptorcross-talk (41). Our data indicate that the effects of stress hormoneson lung cancer cells may be heightened in cells with EGFR-activatingmutations.

Here, we uncovered a mechanism of LKB1 inactivation in NSCLCcells, where intact LKB1 is inactivated by stress hormone signaling.Although the LKB1/STK11 tumor suppressor is frequently lost atthe genomic level, or mutated, in NSCLC, the mechanism reportedhere may be particularly important in tumors with EGFR muta-tions because they nearly always have intact LKB1 (49). Consistentwith our proposed model, in cardiac myocytes, b-AR stimulationinactivates the LKB1 target AMPK (50) as part of metabolic changesinduced by catecholamines. Our data provide a detailed mechanisticunderstanding of how this could occur in nonmalignant cells and indi-cate that NSCLC cells may usurp this physiological process. LKB lossor inactivation has been linked to an enhanced CREB gene expressionsignature (51) and an immunosuppressive tumor microenvironmentcharacterized by increased infiltration of myeloid-derived tumor sup-pressor cells and increased IL-6 (52). Studies have found an associa-tion between reduced LKB1/AMPK pathway signaling and increaseddistant metastasis (15, 53). In an analysis of 722 NSCLC patientsreceiving definitive radiotherapy, b-blocker use was strongly associatedwith reduced distant metastasis (8). Studies are warranted to determinewhether stress hormones promote lung cancer metastasis through in-activation of LKB1.

Our analysis also demonstrated that b-AR activation stimulates theMAPK pathway in EGFR mutant NSCLC cells. The effects of b-ARsignaling on MAPK are notable because reactivation of the MAPKpathway has been established as a mechanism of EGFR TKI resistance(54, 55).

Our preclinical findings indicate that stress hormones act on b2-ARon NSCLC cells, inducing increases in IL-6 and, in turn, resistance toEGFR TKIs. Our in vitro observations were supported by an animalmodel of NSCLC, where b-AR activation promoted the emergence oferlotinib-resistant disease, which was blocked by IL-6 neutralizing anti-bodies or b-blockers. To provide clinical validation, we analyzed the ef-fect of incidental b-blocker use on benefit from afatinib versuschemotherapy in NSCLC patients with EGFR mutations in the LUX-Lung3 study. In patients receiving b-blockers, afatinib appeared to pro-duce a larger improvement in PFS compared to patients not receivingb-blockers. However, interpreting this effect is difficult because patientsreceiving b-blockers also had aworse clinical response to chemotherapy(Fig. 5). Although our analysis of the LUX-Lung3 study supports ourhypothesis that b-blocker use may improve clinical response to EGFRinhibitors, definitive clinical proof of this hypothesis would requiredirect clinical testing of EGFR TKIs alone and in combination withb-blockers. Given the potential benefits highlighted here, as well as thefavorable tolerability profile and low cost of these drugs, we feel thatsuch clinical testing merits consideration.

In summary, this study reveals a mechanism of EGFR inhibitorresistance and advances the understanding of how chronic stressmay influence disease progression. These findings have immediateclinical implications because many well-tolerated and inexpensiveb-blockers are available. These data support the future clinical

Nilsson et al., Sci. Transl. Med. 9, eaao4307 (2017) 8 November 2017

testing of b-blockers in combination with EGFR-targetingagents.

MATERIALS AND METHODSStudy designThe objectives of this study were to (i) determine whether IL-6 ex-pression was associated with T790M-negative resistance to EGFRTKIs and (ii) determine whether stress hormones could signal onNSCLC tumor cells and promote EGFR TKI resistance through theup-regulation of IL-6. To investigate the effects of b-AR signalingon NSCLC cells and the effects of b-AR activation on EGFR TKIresistance, we used quantitative real-time PCR, in vitro assays,Western blotting, RPPA, ELISAs, immunohistochemistry, and xeno-graft models of EGFR mutant NSCLC. All animal studies were ap-proved by the Institutional Animal Care and Use Committee at theUniversity of Texas MD Anderson Cancer Center in accordance withNational Institutes of Health guidelines. Animals were randomly as-signed to control or treatment groups. No statistical method was usedto predetermine the sample size. Investigators were not blinded for thepreclinical experiments.

Cell lines and reagentsCell lines (table S3) were maintained in 10% fetal bovine serum (FBS;Sigma) RPMI medium. NE, E, ISO, phentolamine hydrochloride,salbutamol, dobutamine, and forskolin were obtained from Sigma-Aldrich. 8-pCPT-2-Me-cAMP was obtained from Thermo FisherScientific. QNZ (EVP4593), SGC-CBP30, and RO31-8220 were obtainedfrom Selleck Chemicals. H89 was obtained from Tocris Chemicals. IL-6neutralizing antibodies were obtained from R&D Systems. Erlotinib andsiltuximabwere obtained fromthe institutional pharmacy at theUniversityof TexasMDAndersonCancer Center. LKB1 expression vectors were ob-tained fromAddgene. The PKC inhibitor peptide 19–36 and control pep-tides (EMDMillipore) were used at a concentration of 100 mg/ml inice-cold permeabilization buffer for 5 min. Cells recovered in serum-freemedium for 2 hours before NE treatment. The following antibodies wereused for Western blotting: IKB-a (4814; Cell Signaling), p-CREBS133

(9198; Cell Signaling), pEGFR (3777; Cell Signaling), EGFR (1005; SantaCruz Biotechnology Inc.), p-LKB1S428 (3482 and 3051; Cell Signal-ing), p-p90RSK (9344; Cell Signaling), p-AMPK (2535; Cell Signal-ing), p-p70S6K (9205; Cell Signaling), p-ERK (9101; Cell Signaling),vinculin (v9131; 1:10,000; Sigma-Aldrich), and b-actin (A5441;1:10,000; Sigma-Aldrich). For immunohistochemical studies, thefollowing antibodies were used: anti-mouse CD31 antibody (1:400;PharMingen), Ki67 (Dako), and Alexa 594–conjugated secondaryantibody (Molecular Probes).

Detection of IL-6NSCLC cells (200,000 cells per well in six-well plates) were treated with1ml of serum-free mediumwith or without NE, E, or ISO for 24 hours,and medium was collected. IL-6 ELISA (R&D Systems) was performedaccording to the manufacturer’s instructions. For IL-6 staining, anti-bodies (1:50; Millipore) were used on frozen tumor sections. For inhib-itor studies, cells were pretreated with inhibitors for 1 hour before NEstimulation. The doses of catecholamines used for our studies wereselected to reflect physiologic conditions of these hormones at the levelof the tumor. Whereas circulating plasma concentrations of NE rangefrom 10 to 1000 pM in a normal individual and may reach as high as100 nM in conditions of stress (56), catecholamine concentrations in

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some tissues are at least 100 times higher andmay reach concentrationsas high as 10 mM (17, 18).

ADRB gene expression profilingThe MD Anderson cohort was obtained from the Profiling ofResistance patterns and Oncogenic Signaling Pathways in Evaluationof Cancers of the Thorax (PROSPECT) study. Surgically resected tumors,collected between 2006 and 2010, from 189 patients were included inPROSPECT, and of these, 152 were lung adenocarcinomas. Gene ex-pression analysis and sequencing of select genes were conducted asreported elsewhere (57–59).

ImmunoprecipitationCellswere stably transfectedwith FLAG-B2ADRor control vector usinglentivirus. Cells were serum-starved for 1 hour and then treatedwithNE(10 mM) for 10 min. Cells were washed with phosphate-buffered saline(PBS) and incubated in dithiobis succinimidyl propionate (DSP;ThermoScientific; 1mMinPBS) cross-linker solution and incubated on ice.After2 hours, tris-HCl stop solution was added at a final concentration of10 mM and incubated for 15 min. Cells were lysed in protein lysisbuffer. Coimmunoprecipitation was performed using Pierce cross-linkimmunoprecipitation kit according to the manufacturer’s instructions(cat. #26147). After elution, DSP cross-linking was quenched using20 mM DTT (3′-deoxythymidine 5′-triphosphate) loading buffer in1%SDS, 60mMtris-HCl, and 10%glycerol, boiling at 100°C for 10min.

Duolink II fluorescence assayDuolink II fluorescence assay was performed as previously described(60). Cells were fixed with 4% paraformaldehyde and permeabilizedwith 0.5% Triton X-100 and then blocked in 5% FBS in PBS and incu-bated with mouse anti-EGFR (1:2000 dilution; Thermo Scientific) andrabbit anti-Flag (1:400 dilution; Cell Signaling) antibodies for 1 hour.The Duolink II PLA probe (Sigma) was used to detect the signals.

Reverse phase protein arrayNSCLCcellswere treatedwith 10mMNE for 15min, andprotein lysateswere collected. RPPA slides were printed from lysates, and RPPA studieswere performed and analyzed as previously described (53, 61–63).

Cell viability assaysNSCLC cells (2000 cells per well in 96-well plates) were treated withincreasing concentrations of EGFR TKIs. After 5 days, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assays were performed. To evaluate theeffect of NE on EGFR TKI resistance, we seeded cells in 24-wellplates (40,000 cells per well) and treated them with NE for 24 hours,and then, erlotinib was added to the culture medium. After 5 days, cellviability was measured by MTS assay. PPL (1 mM) or IL-6 antibodies(1 mg/ml) were added 1 hour before the addition of NE.

MiceCancer cells (1 × 106) were injected subcutaneously into female nudemice. Chronic stress was induced using a restraint-stress procedure(64). To test the effect of ISO on IL-6, once tumors reached a volumeof 400 mm3, animals received daily ISO treatment (10 mg/kg, intra-peritoneally) for 3 days. Tumors were processed for RNA collectionand quantitative real-time PCR. For erlotinib resistance studies, 1 × 106

HCC827 cellswere injected subcutaneously intonudemice.Once tumorsreached 400 mm3, animals (n = 5 mice per group) received vehicle, ISO

Nilsson et al., Sci. Transl. Med. 9, eaao4307 (2017) 8 November 2017

(10 mg/kg, daily, intraperitoneally), PPL (10 mg/kg, daily, intra-peritoneally), or siltuximab (20 mg/kg, twice weekly, intraperitoneally).Erlotinib was given at a dose of 100 mg/kg (oral gavage) on a 4-week-on/2-week-off schedule.

Clinical analysisBiospecimens were obtained after patients gave informed consent,under protocols approved by institutional reviewboards at all participatinginstitutions. Studies were conducted in accordance with theDeclarationof Helsinki. The PROSPECT data set has been detailed previously (65)and includes patients who underwent surgical resection of NSCLCwithcurative intent between 1996 and 2008 at theMDAndersonCancerCenter.In the PROSPECT study, most of the cases were lung adenocarcinomas.Clinical outcomes from BATTLE (NCT00409968, NCT00411671,NCT00411632, NCT00410059, NCT00410189), the LUX-Lung3 study(NCT00949650) (31), and ZEST (NCT00364351) (16) have been reported.We analyzed baseline plasma samples collected during the BATTLEclinical trial (21) and the phase 3 ZEST clinical study and were blindedto clinical outcome. Each sample was analyzed in duplicate. For theanalysis of circulating concentrations of IL-6 in the BATTLE trial,185 patients received no b-blockers and 3 patients received panb-blockers.The groups were compared using a two-tailed Student’s t test.

StatisticsFor the in vitro studies, statistical analysiswas performedusing Student’st test (two-tailed) or one-way ANOVA. P≤ 0.05 was considered statis-tically significant. For the RPPA data, ANOVA was used on a protein-by-protein basis. The resulting P values, computed from F statistic, weremodeled using the b-uniform mixture model and used to determine afalse discovery rate cutoff to identify significantly differentiallyexpressed proteins. For in vivo studies, we fitted a linear model (withtreatment and time effects) using the generalized least squares methodto account for repeated-measurements correlation. Dunnett’s test wasused to adjust formultiple comparisons in comparingmany treatmentswith a single control.

SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/9/415/eaao4307/DC1Fig. 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 b-AR activation.Fig. S5. Differential effects of b2-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 b-AR–induced p-LKB1, p-CREB, and IL-6.Fig. S8. Effect of b-AR signaling on NF-kB 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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Acknowledgments: We thank E. Roarty for the helpful scientific advice and editorialassistance. The hemagglutinin-tagged b2-AR expression vector was a gift from M. Tuvim(University of Texas MD Anderson Cancer Center). J. Minna (University of Texas SouthwesternMedical Center) provided the NSCLC cell lines. C. Frohn (Boehringer Ingelheim) assistedwith acquisition of the LL3 clinical data. Funding: This work was supported by LUNGevityFoundation; Lung SPORE grant 5 P50 CA070907; Lung Cancer Moon Shot Program, NIH CancerCenter Support Grant (CA016672); and 1R01 CA190628, Hallman Fund, Bruton EndowedChair in Tumor Biology, Stading Fund for EGFR inhibitor resistance, the Fox Lung EGFRInhibitor, the Rexanna Foundation for Fighting Lung Cancer. Additional support was providedthrough CA109298 and the American Cancer Society Research Professor award (A.K.S.).Author contributions: M.B.N. designed the research, performed the experiments, interpretedthe data, and wrote the manuscript. H.S. performed the in vitro and in vivo experiments.A.P. assisted with the animal experiments. G.A P. performed the animal experiments usingrestraint, which was supervised by A.K.S. Y.F. performed the RPPA analysis. L.L., L.D., P.T.,and D.L. performed the statistical and computational analysis, and J.W. and J.J.L. supervisedand designed the analysis. S.-O.L. performed the Duolink assays, and M.-C.H supervised thework and provided direction and insight. K.H. and V.H. conducted the statistical analysesrelated to the ZEST clinical study. D.G. assisted with the data interpretation. H.T. analyzed andsupervised the evaluation of circulating levels of IL-6 in patient samples from the ZEST andBATTLE clinical studies. L.V.S. and J.C.Y. conducted the LUX-Lung3 clinical study. R.S.H., E.S.K.,W.K.H., and I.W. conducted and designed the BATTLE study and assisted with the datainterpretation. J.V.H. supervised the work, designed the research, interpreted the data, andwrote the manuscript. Competing interests: K.H. and V.H. are employees of AstraZeneca.L.V.S. is a noncompensated consultant for Boehringer Ingelheim, Clovis, Merrimack, Novartis,and Taiho and a compensated consultant for AZ and Ariad and receives institutionalsupport from Boehringer Ingelheim for the LL3 clinical trial. J.C.Y. received honoraria forspeech or participated in the compensated advisory boards of Boehringer Ingelheim, Eli Lilly,Bayer, Roche/Genentech, Chugai, Astellas, MSD, Merck Serono, Pfizer, Novartis, ClovisOncology, Celgene, Innopharma, Merrimack, Yuhan Pharmaceuticals, BMS, OnoPharmaceuticals, Daiichi Sankyo, AstraZeneca, and Hansoh Pharmaceuticals and was part of anuncompensated advisory board of AstraZeneca. J.V.H. has received honoraria and paidconsulting from AstraZeneca and Boehringer Ingelheim and has received research fundingfrom AstraZeneca. R.S.H. has received honoraria and paid consulting from AbbviePharmaceuticals, AstraZeneca, Biodesix, Bristol-Myers Squibb, Eli Lilly and Company, EMDSerrano, Genentech/Roche, Heat Biologics, Halozyme, Infinity Pharmaceuticals, Merck andCompany, Neon Therapeutics, NextCure, Novartis, Pfizer, Sanofi, Seattle Genetics, and ShirePLC, and he has received research funding from AstraZeneca, Eli Lilly and Company, andMerck and Company. M.-C. H. has received research funding from StCube and Hengrui.A.K.S. has received paid consulting from Kiyatec, research funding from M-Trap, and is aBioPath stock holder. Data and materials availability: Affymetrix microarray results werepreviously published and archived at the Gene Expression Omnibus (GEO) repository. Cell linedata are available at GEO accession no. GSE4824. PROSPECT gene expression data areavailable at GEO accession no. GSE42127.

Submitted 19 July 2017Accepted 11 October 2017Published 8 November 201710.1126/scitranslmed.aao4307

Citation: M. B. Nilsson, H. Sun, L. Diao, P. Tong, D. Liu, L. Li, Y. Fan, A. Poteete, S.-O. Lim,K. Howells, V. Haddad, D. Gomez, H. Tran, G. A. Pena, L. V. Sequist, J. C. Yang, J. Wang,E. S. Kim, R. S. Herbst, J. J. Lee, W. K. Hong, I. Wistuba, M.-C. Hung, A. K. Sood,J. V. Heymach, Stress hormones promote EGFR inhibitor resistance in NSCLC: Implicationsfor combinations with b-blockers. Sci. Transl. Med. 9, eaao4307 (2017).

10 of 10

-blockersβcombinations with Stress hormones promote EGFR inhibitor resistance in NSCLC: Implications for

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

DOI: 10.1126/scitranslmed.aao4307, eaao4307.9Sci Transl Med

blocked this mechanism of resistance and may become a useful adjunct to lung cancer therapy regimens.−−a common class of drugs used in humans−−-blockersβinhibitors, a key therapy for this disease. Conversely,

-adrenergic receptors on cancer cells, activating a signaling cascade that promotes tumor resistance to EGFR2βunderlying mechanism and a potential intervention. The authors found that stress hormones activate

small cell lung cancer, providing insights into the− investigated this phenomenon in nonet al.body. Nilsson considering that both the diagnosis of cancer and the associated treatments are quite stressful for the mind and

Common wisdom holds that stress is not good for cancer patients, but it can be difficult to avoid-blockersβDestressing cancer with

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