NF-κB–driven suppression of FOXO3a contributes to … · NF-κB–driven suppression of FOXO3a...

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Correction MEDICAL SCIENCES Correction for NF-κBdriven suppression of FOXO3a con- tributes to EGFR mutation-independent gefitinib resistance,by Ching-Feng Chiu, Yi-Wen Chang, Kuang-Tai Kuo, Yu-Shiuan Shen, Chien-Ying Liu, Yang-Hao Yu, Ching-Chia Cheng, Kang-Yun Lee, Feng-Chi Chen, Min-Kung Hsu, Tsang-Chih Kuo, Jui-Ti Ma, and Jen-Liang Su, which appeared in issue 18, May 3, 2016, of Proc Natl Acad Sci USA (113:E2526E2535; first published April 18, 2016; 10.1073/pnas.1522612113). The authors note that Fig. 5 appeared incorrectly. The corrected figure and its legend appear below. This error does not affect the conclusions of the article. E654E655 | PNAS | January 24, 2017 | vol. 114 | no. 4 www.pnas.org

Transcript of NF-κB–driven suppression of FOXO3a contributes to … · NF-κB–driven suppression of FOXO3a...

Page 1: NF-κB–driven suppression of FOXO3a contributes to … · NF-κB–driven suppression of FOXO3a contributes to EGFR mutation-independent gefitinib resistance Ching-Feng Chiua,1,

Correction

MEDICAL SCIENCESCorrection for “NF-κB–driven suppression of FOXO3a con-tributes to EGFR mutation-independent gefitinib resistance,” byChing-Feng Chiu, Yi-Wen Chang, Kuang-Tai Kuo, Yu-Shiuan Shen,Chien-Ying Liu, Yang-Hao Yu, Ching-Chia Cheng, Kang-Yun Lee,Feng-Chi Chen, Min-Kung Hsu, Tsang-Chih Kuo, Jui-Ti Ma, andJen-Liang Su, which appeared in issue 18, May 3, 2016, of Proc NatlAcad Sci USA (113:E2526–E2535; first published April 18, 2016;10.1073/pnas.1522612113).The authors note that Fig. 5 appeared incorrectly. The corrected

figure and its legend appear below. This error does not affect theconclusions of the article.

E654–E655 | PNAS | January 24, 2017 | vol. 114 | no. 4 www.pnas.org

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Fig. 5. miR-155 is transcriptionally regulated by NF-κB of gefitinib-resistant lung cancer cells. (A) Real-time RT-PCR analysis of pri–miR-155, pre–miR-155, andmiR-155 expression in PC9/WT and PC9/GR cells. (B) Schematic description of serial deletion reporter constructs of the miR-155 promoter cloned into the pGL3-Basic vector (Left). PC9/WT and PC9/GR cells were transfected with various miR-155 promoter reporters, and the luciferase activity was measured by the dual-luciferase reporter assay (Right). (C) Schematic diagram showing that the NF-κB binding sequences or mutated versions in the miR-155 promoter (Upper), andluciferase activity was measured using the dual-luciferase reporter assay (Lower). (D) ChIP analysis of chromatin extracted from PC9/WT and PC9/GR usingpolyclonal antibodies directed against the p50, a subunit of NF-κB, followed by real-time RT-PCR analysis to confirm p50 occupancy at the miR-155 promoter.(E) Cytoplasmic and nuclear fractions from PC9/WT and PC9/GR cells were assessed for the presence of FOXO3a, p50, Lamin B (nuclear marker), and Tubulin(cytosolic marker) by Western blot analysis. (F) The FOXO3a and p50 protein expression of H292 cells with stable expression of the indicated transfectantswere measured by Western blot (Upper), and miR-155 expression by real-time RT-PCR (Lower), respectively. **P < 0.01 (two-tailed Student’s t test). (G) In vivotumor growth of H292 cells with stable expression of the indicated transfectants. Each point represents the mean ± SEM of tumor volumes of six mice in eachgroup. Tumor volume was calculated as indicated in Materials and Methods. *P < 0.05 and **P < 0.01 (two-tailed Student’s t test).

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NF-κB–driven suppression of FOXO3a contributes toEGFR mutation-independent gefitinib resistanceChing-Feng Chiua,1, Yi-Wen Changa,1, Kuang-Tai Kuob,c,1, Yu-Shiuan Shend, Chien-Ying Liue,f, Yang-Hao Yug,h,Ching-Chia Chenga, Kang-Yun Leei,j, Feng-Chi Chenk, Min-Kung Hsul, Tsang-Chih Kuom, Jui-Ti Maa,and Jen-Liang Sua,d,n,o,2

aNational Institute of Cancer Research, National Health Research Institutes, Miaoli County 35053, Taiwan; bDivision of Thoracic Surgery, Department ofSurgery, Shuang Ho Hospital, Taipei Medical University, New Taipei City 23561, Taiwan; cDivision of Thoracic Surgery, Department of Surgery, School ofMedicine, College of Medicine, Taipei Medical University, Taipei City 110, Taiwan; dDepartment of Biotechnology, Asia University, Taichung City 413,Taiwan; eSchool of Medicine, Chang Gung University, Taoyuan City 33305, Taiwan; fDivision of Pulmonary Oncology and Interventional Bronchoscopy,Department of Thoracic Medicine, Chang Gung Memorial Hospital, Taoyuan City 33305, Taiwan; gDivisions of Pulmonary and Critical Care Medicine,Department of Internal Medicine, China Medical University Hospital, Taichung City 404, Taiwan; hSchool of Medicine, China Medical University, TaichungCity 404, Taiwan; iDepartment of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei City 110, Taiwan; jDivision ofPulmonary Medicine, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, New Taipei City 23561, Taiwan; kInstitute of PopulationHealth Sciences, National Health Research Institutes, Miaoli County 35053, Taiwan; lDepartment of Biological Science and Technology, National Chiao TungUniversity, Hsinchu City 300, Taiwan; mInstitute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei City 10617, Taiwan;nGraduate Institute of Cancer Biology, China Medical University, Taichung City 404, Taiwan; and oCenter for Molecular Medicine, China Medical UniversityHospital, Taichung City 404, Taiwan

Edited by Napoleone Ferrara, University of California, San Diego, La Jolla, CA, and approved March 18, 2016 (received for review November 23, 2015)

Therapy with epidermal growth factor receptor (EGFR) tyrosinekinase inhibitors (EGFR-TKIs, such as gefitinib or erlotinib) signif-icantly prolongs survival time for patients with tumors harboringan activated mutation on EGFR; however, up to 40% of lung cancerpatients exhibit acquired resistance to EGFR-TKIs with an unknownmechanism. FOXO3a, a transcription factor of the forkhead family,triggers apoptosis, but the mechanistic details involved in EGFR-TKIresistance and cancer stemness remain largely unclear. Here, weobserved that a high level of FOXO3a was correlated with EGFRmutation-independent EGFR-TKI sensitivity, the suppression of can-cer stemness, and better progression-free survival in lung cancerpatients. The suppression of FOXO3a obviously increased gefitinibresistance and enhanced the stem-like properties of lung cancer cells;consistent overexpression of FOXO3a in gefitinib-resistant lung can-cer cells reduced these effects. Moreover, we identified that miR-155targeted the 3′UTR of FOXO3a and was transcriptionally regulatedby NF-κB, leading to repressed FOXO3a expression and increasedgefitinib resistance, as well as enhanced cancer stemness of lungcancer in vitro and in vivo. Our findings indicate that FOXO3a is asignificant factor in EGFR mutation-independent gefitinib resistanceand the stemness of lung cancer, and suggest that targeting the NF-κB/miR-155/FOXO3a pathway has potential therapeutic value in lungcancer with the acquisition of resistance to EGFR-TKIs.

EGFR | gefitinib | lung cancer | miR-155 | NF-κB

Small molecule inhibitors of receptor tyrosine kinase are cur-rently an important treatment for nonsmall cell lung cancer

(NSCLC), especially for tumors harboring an activated mutation ofepithelial growth factor receptor (EGFR) (1–3). Genetic mutationsof L858R or exon 19 deletions in ∼90% of EGFRmutations of lungcancer patients are associated with sensitivity to EGFR tyrosinekinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib;however, patients who received EGFR-TKI treatment showed aresponse for ∼10–16 mo (4). Increasing studies have reported sev-eral acquired resistance mechanisms, such as T790M site mutationof EGFR (5), hyperactivation of HER2 and receptor tyrosine ki-nase MET (6, 7), and somatic mutations in Kirsten rat sarcoma viraloncogene homolog (KRAS), BRAF, and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) (8), andindicated that minor subpopulations of cancer stem cells (CSC) areintrinsically more resistant to anticancer drugs or radiation and areresponsible for metastasis and recurrence to cancer therapies (9),including the EGFR-TKI resistance of lung cancers (10, 11). Un-fortunately, the intrinsic mechanisms of acquired resistance remainunclear for up to 40% of lung cancer patients (12). Therefore, it is

necessary to further clarify the underlying mechanism of EGFR-TKI resistance in lung cancer to improve the efficiency of theclinical treatment and develop new therapeutic strategies.The human FOXO family includes FOXO1, FOXO3a, FOXO4,

and FOXO6; FOXO3a is abundant in various tissues and is dif-ferent from the other three in its tissue specificity (13). Additionally,FOXO3a is a transcription factor that acts as a tumor suppressor byinducing cell cycle arrest, and the down-regulation of FOXO3a isinvolved in the tumorigenesis of various cancer types (14). Studiesindicate that FOXO3a activity is negatively regulated by oncogenickinases, such as AKT, IKK, and ERK (15–17), and the activation ofthese oncogenic kinases is associated with FOXO3a suppression,which triggers cancer progression. Because knockdown of FOXO3ain breast cancer results in a reduction of gefitinib-induced cell cyclearrest and cell death (18), and FOXO3a nuclear localization in-duced by metformin or SN-38 would down-regulate the propertiesof stem-like cells in breast and ovarian cancer cells (19), we spec-ulate that FOXO3a might be involved in resistance to EGFR-TKIs

Significance

Gefitinib is a small molecular inhibitor that targets EGFR tyrosinekinases (EGFR-TKI) and has been used as a first-line treatment foradvanced lung cancer. However, not all lung cancer patientsrespond to gefitinib treatment, and resistance to gefitinib hasbeen apparent for lung cancer patients who have undergonetreatment for a few months. We observed that FOXO3a ex-pression is inversely correlated with lung cancer patients whoresponded poorly to EGFR-TKI treatment and identified anunderlyingmechanism of FOXO3a in EGFRmutation-independentcancer stemness and gefitinib resistance through the epigeneticregulation of NF-κB/miR-155. This finding highlights the potentialof targeting the NF-κB/miR-155/FOXO3a pathway as a noveltherapeutic strategy for lung cancer with the acquisition of re-sistance to EGFR-TKIs.

Author contributions: J.-L.S. designed research; C.-F.C., Y.-W.C., K.-T.K., Y.-S.S., and C.-C.C.performed research; C.-F.C., Y.-W.C., K.-T.K., and J.-L.S. contributed new reagents/analytictools; C.-F.C., Y.-W.C., K.-T.K., Y.-S.S., C.-Y.L., Y.-H.Y., C.-C.C., K.-Y.L., F.-C.C., M.-K.H., T.-C.K.,J.-T.M., and J.-L.S. analyzed data; and C.-F.C. and J.-L.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1C.-F.C., Y.-W.C., and K.-T.K. contributed equally to this work.2To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1522612113/-/DCSupplemental.

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and the CSC properties of lung cancer and could be another path-way for cancer cells to survive by resisting gefitinib agents.Here, we found that FOXO3a was negatively correlated with

EGFR mutation-independent gefitinib resistance and reducedCSC properties in lung cancer. Moreover, we revealed the down-regulation of FOXO3a through NF-κB activation via miR-155,which confer gefitinib resistance and stemness in lung cancer.Our findings suggest that FOXO3a suppression contributes toacquired gefitinib resistance in NSCLC patients carrying anEGFR-activating mutation.

ResultsFOXO3a Expression Is Associated with EGFR-TKI Resistance and CancerStemness. To elicit the clinical significance of FOXO3a in EGFR-TKI resistance, we retrospectively collected and analyzed speci-mens from a cohort of 80 lung cancer patients who had receivedEGFR-TKIs (erlotinib or gefitinib), either as front line or salvagetreatment. The expression of FOXO3a in lung cancer tissues wasdetected by immunohistochemical (IHC) staining, and we observedthat the nuclear staining intensity of FOXO3a was stronger thancytoplasmic staining (Fig. 1A), and a higher proportion of patientswho responded to EGFR-TKI treatment expressed high FOXO3aprotein (Fig. 1B) and high FOXO3a mRNA (Fig. 1C) than thenonresponder group. In addition, high levels of FOXO3a were sig-

nificantly correlated with better survival outcomes (SI Appendix, Fig.S1A), according to an analysis of the online PrognoScan database,and our collected cohort also observed that patients with a high levelof FOXO3a taking EGFR-TKI treatment lived for over a yearwithout tumor growth (progression-free survival; PFS of 13.0 mo)versus those with low levels of FOXO3a (PFS of 8.68 mo) (Fig. 1D),suggesting that FOXO3a plays a critical role in response to EGFR-TKIs and survival outcomes of lung cancer patients. We furtherfound that high expression of CSC markers, such as SRY (sex de-termining region Y)-box 2 (SOX2) and cell surface marker CD133,were significantly inversely correlated with FOXO3a expression(Fig. 1E and SI Appendix, Table S1) and positively with EGFR-TKInonresponders (SI Appendix, Fig. S1B). However, we analyzed therelationship between FOXO3a expression and EGFR mutationstatus of lung cancer and observed that FOXO3a is not significantlycorrelated with wild-type and mutated (exon 19 deletion, T790M orL858R) EGFR in our cohorts (Fig. 1G), and was consistent withobservations in the The Cancer Genome Atlas (TCGA), Gene-Expression Omnibus, and Oncomine databases (SI Appendix, Fig. S1C–E). These findings imply that FOXO3a might be involved inEGFR mutation-independent EGFR-TKI resistance in lung cancer.To further investigate whether FOXO3a expression is associatedwith EGFR mutation-independent EGFR-TKI resistance, we ana-lyzed the relationship of FOXO3a and gefitinib sensitivity in lung

Fig. 1. FOXO3a expression is associated with the re-sponse to gefitinib treatment and stemness in lungcancer. (A) IHC staining yielded scores ranging from 0 to3, which are representative of the amount of FOXO3ain lung cancer specimens. A score of 0–1 representedlow expression of FOXO3a, and 2–3 represented highexpression. (Scale bars, 100 μm.) (B) The percentages ofpatients with high expression (black bar) and low ex-pression of FOXO3a (gray bar) were assigned accordingto different responses to EGFR-TKIs (responder, n = 42;nonresponder, n = 38). Numbers near bars representthe percentage of patients for each condition. ***P <0.001 (Pearson’s χ2 test). (C) Expression of FOXO3amRNAwas assigned according to different responses toEGFR-TKIs. FOXO3a expression in tumors was classifiedaccording to the median of the individual ΔCt values ofpatient samples. The median of the individual ΔCtvalues of patient samples was used as the cut-off valuebetween high and low expression. Lower ΔCt valuesindicate higher expression of the gene. P = 0.003. (D)Mean PFS of patients with high levels of FOXO3a [n =45, PFS of 13 mo, 95% confidence interval (CI) = 10.33–15.68] and low levels of FOXO3a (n= 35, PFS of 8.68mo,95% CI = 6.59–10.77) taking EGFR-TKI treatment. P =0.016. (E and F) Patients with high SOX2 (E) and CD133(F) expression were accompanied by decreased ex-pression of FOXO3a protein. The expression levels ofFOXO3a were classified into two groups according tothe IHC scoring: low-expression group (score 0 and 1)and high-expression group (score 2 and 3). P valueswere determined by Pearson’s χ2 test. **P < 0.01.(G) The expression of FOXO3a between wild-type andmutant EGFR in lung cancer. The EGFR mutationalstatus of patients contains wild-type (n = 45), exon 19deletion (n = 28), exon 21 mutation (L858R andL861Q, n = 5), and T790M (n = 2). (H) Inverse corre-lation between FOXO3a expression (Upper) and IC50of gefitinib in human lung cancer cell lines (Lower). TheIC50 values were obtained by MTT assay as shown inSI Appendix, Table S2. r = −0.422; P = 0.030 (Pearson’scorrelation coefficient). (I) Expression levels of FOXO3a,FOXO1, and FOXO4 in PC9/WT and PC9/GR (Left), aswell as in HCC827/WT and HCC827/GR cells (Right),were analyzed by Western blot. Tubulin was used as aloading control.

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cancer cell lines and found that the FOXO3a protein level wasnegatively correlated with the IC50 of gefitinib in lung cancer cells(Fig. 1H and SI Appendix, Table S2). Compared with parental PC9/WT cells, gefitinib-resistant PC9 (PC9/GR) cells contained thesame exon 19 deletion of EGFR appeared to have a low level ofgefitinib sensitivity and FOXO3a expression but not FOXO1 orFOXO4 (Fig. 1I). Consistent effects were observed in parentalHCC827/WT cells and HCC827/GR cells with exon 19 deletion ofEGFR. These results suggest that the expression of FOXO3a waspositively correlated with gefitinib sensitivity and CSC suppressionof lung cancer in an EGFR mutation-independent manner.

Effects of FOXO3a Involved in Gefitinib Resistance and CSC Properties.To elucidate whether FOXO3a affects sensitivity to gefitinib,we generated PC9/GR with the overexpression of FOXO3a(Fig. 2A) and found that PC9/GR/FOXO3a cells significantlyincreased sensitivity to gefitinib (Fig. 2 B and C) and the effectsof FOXO3a were consistent in H460 and H292 cells (SI Appendix,Fig. S2 A–F). Additionally, FOXO3a-knockdown PC9/WT cells(PC9/WT/shFOXO3a) (Fig. 2D) exhibited increased resistance togefitinib and gefitinib-induced apoptotic population (Fig. 2 E andF), which was consistent in HCC827 and A549 cells (SI Appendix,Fig. S2 G and H). We also found that the expression of SOX2,Kruppel-like factor 4 (KLF4), Nanog homeobox (Nanog), theCD133+ and aldehyde dehydrogenases (ALDH)+ cells, sphereformation, and side population were significantly increased inPC9/GR cells compared with PC9/WT cells, and that the CSCproperties were increased in the FOXO3a-knockdown cells (Fig. 2

G and H and SI Appendix, Fig. S3 A–D). Moreover, the A549/shFOXO3a-bearing mice showed increased tumor volume inresponse to gefitinib resistance compared with the A549/shctrl-bearing mice (Fig. 2I), and FOXO3a expression was found in formedtumors (Fig. 2J). The above data suggest that FOXO3a was in-volved in the gefitinib response and the CSC characteristics oflung cancer cells.

miR-155 Targeting of FOXO3a Is Correlated with Gefitinib Sensitivity.To investigate whether FOXO3a was regulated by microRNAs,we first used five different algorithms (miRanda, DianamicroT,miRDB, miRWalk, and TargetScan) to filter out 13 candidatemicroRNAs commonly predicted to target to FOXO3a 3′UTR(Fig. 3A). Next, these microRNA candidates were validated byreal-time RT-PCR to confirm differential expression, and one ofthe identified candidates, miR-155, was significantly increased inPC9/GR cells compared with PC9/WT cells (Fig. 3B). Nevertheless,miR-380–3p and miR-495 were not detectable in the PC9/WT andPC9/GR cells. To further identify whether FOXO3a was a directtarget of miR-155, the binding sequence of miR-155 on FOXO3a3′UTR was predicted by miRanda software, and wild-typeFOXO3a 3′UTR (WT-3′UTR) and miR-155 binding site mutantFOXO3a 3′UTR (MT-3′UTR) reporters were transfectedinto PC9/WT cells at different miR-155–to–reporter ratios.Cotransfection with miR-155 and the WT-3′UTR reporter resultedin a significant inhibition of luciferase activity in a dose-dependentmanner, but not in the MT-3′UTR reporter (Fig. 3C). Westernblot analysis showed that the increased expression of miR-155

Fig. 2. FOXO3a is required for gefitinib-inducedcell death and CSC markers of lung cancer cells.(A) FOXO3a expression was analyzed by Westernblot in PC9/WT, PC/GR, vector control, and FOXO3a-overexpressing PC9/GR (PC9/GR/FOXO3a) cells.(B) PC9/GR/FOXO3a and corresponding vector con-trol cells were treated with 4 μM gefitinib, and cellviability was measured by MTT assay and (C) cellapoptosis was detected by flow cytometric analysis,respectively. (D) Western blot analysis of FOXO3aexpression in PC9/WT, PC9/GR, shctrl control, andFOXO3a-knockdown of PC9/WT (PC9/WT/shFOXO3a#1and #2) cells. (E) The indicated concentrations ofgefitinib were added to these cells; cell viability wasmeasured by MTT assay, and (F) cell apoptosis wasdetected by flow cytometric analysis. (G) The effectsof FOXO3a expression mediated mRNA expressionof SOX2. Total RNA was harvested for the analysis ofSOX2 mRNA by real-time RT-PCR. (H) The effectsof FOXO3a expression mediated CD133+ cells. Cellswere harvested for flow cytometric analysis to detectCD133+ cells. The results are shown as the means ± SDof three independent experiments, each performed intriplicate. **P < 0.01 and ***P < 0.001 (two-tailedStudent’s t test). (I) Effects of FOXO3a depletion onthe tumor growth of A549 cells were evaluated in theNOD/SCID mice with xenografts after treatment withgefitinib for 2 wk. Tumor volume was calculated asindicated in Materials and Methods. Five mice foreach group; *P < 0.05 (two-tailed Student’s t test).(J) Western blot assay to confirm the expression ofFOXO3a in the indicated group of tumor samples.Tubulin was used as a loading control.

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clearly reduced the expression of FOXO3a protein (Fig. 3D),and the expression level of miR-155 was also inversely correlatedwith FOXO3a expression in lung cancer patients by an approachto the TCGA database (SI Appendix, Table S3). Moreover, wefound that FOXO3a-mediated gefitinib sensitivity was significantlyrepressed by miR-155 in PC9/WT cells (Fig. 3E) and observed apositive correlation between miR-155 expression and gefitinib re-sistance in lung cancer cells (Fig. 3F). PC9/WT cells with over-expression of miR-155 significantly increased the CSC properties,including the expression level of SOX2, KLF4, Nanog, the CD133+,and ALDH+ cells, sphere formation, and side population (Fig. 3 Gand H and SI Appendix, Fig. S3 E–H). These results suggest thatmiR-155 directly hinders FOXO3a expression and is involved inFOXO3a-mediated gefitinib sensitivity and cancer stemness.

miR-155 Represses FOXO3a-Mediated Gefitinib Sensitivity. To obtainmore detailed information on miR-155–repressed FOXO3a-mediated gefitinib sensitivity, we transfected PC9/GR cells withscramble control oligos (ctrl) or anti–miR-155. FOXO3a proteinlevels increased along with significantly reduced miR-155 ex-pression upon anti–miR-155 transfection in PC9/GR cells (Fig.4A). Moreover, the blockage of miR-155 expression increased

gefitinib sensitivity (Fig. 4B) and decreased the CSC properties(Fig. 4C and SI Appendix, Fig. S4 A–D). We established aFOXO3a-expressing construct lacking the FOXO3a 3′UTR andtransfected it into control- and miR-155–overexpressing PC9/WTcells. The transfection of the FOXO3a-expressing vector suc-cessfully restored the expression of FOXO3a protein suppressedby miR-155 in cells (Fig. 4D) and abolished miR-155–mediatedgefitinib sensitivity (Fig. 4E) and CSC phenotypes (Fig. 4F and SIAppendix, Fig. S4 E–H). Consistent with the observations made invitro, mice-bearing tumors derived from A549 cells with theoverexpression of miR-155 showed resistance to gefitinib treat-ment (Fig. 4G) because of FOXO3a depletion in formed tumors(Fig. 4H). By analyzing lung cancer tissue specimens, it was foundthat a higher proportion (66%) of patients who did not respond toEGFR-TKI treatment expressed a high level of miR-155 than theresponder group (Fig. 4I). Additionally, we observed an inversecorrelation between miR-155 and FOXO3a expression in lungcancer (Fig. 4J) and in the TCGA database (SI Appendix, TableS3). Taken together, these findings suggest that the expression ofmiR-155 is increased in patients who are considered to have poorresponse to EGFR-TKIs, and its expression is inversely correlatedwith FOXO3a expression.

Fig. 3. miR-155 is involved in FOXO3a-mediatedgefitinib sensitivity and CSC characteristics. (A) Sche-matic of the bioinformatic analyses of candidatemicroRNAs that targeted FOXO3a. The putativemicroRNAs targeting FOXO3a in miRWalk, TargetScan,miRanda, miRDB, and Diana-microT were searchedand 13 candidate microRNAs were filtered out forreal-time RT-PCR analysis. (B) Real-time RT-PCR anal-ysis of the differential expression of the 11microRNAs in PC9/WT compared with PC9/GR cells.(C) A schematic diagram representing the predictedmiR-155 binding sequences or mutated versions (Up-per). Luciferase activity (Lower) of wild-type FOXO3a-3′UTR (WT-3′UTR) and mutant-type FOXO3a-3′UTR(MT-3′UTR) reporter genes were measured using theDual-Luciferase Reporter Assay System in PC9/WT cellstransfected with miR-155 at different ratios. (D) Theeffects of miR-155 overexpression on endogenousFOXO3a protein expression in PC9/WT cells. FOXO3aand miR-155 expression were measured by Westernblot analysis and real-time RT-PCR, respectively. Tu-bulin was used as a loading control. (E) The functionsof miR-155 in regulating gefitinib sensitivity wereassayed by comparing PC9/WT/pLemiR and PC9/WT/miR-155 cells that were treated with or without 4 μMgefitinib. Cell viability was determined by MTT assay.(F) The correlation between miR-155 expression andthe IC50 of gefitinib with doses for the lung cancer celllines. (r = 0.512; P = 0.013; Pearson’s correlation co-efficient). Expression of miR-155 affected (G) SOX2mRNA levels by real-time RT-PCR and (H) CD133+ cellsby flow cytometric analysis in PC/WT cells with theexpression of pLemiR-155 and control vector. The re-sults are shown as the means ± SD of three in-dependent experiments, each performed in triplicate.*P < 0.05, **P < 0.01, and ***P < 0.001 (two-tailedStudent’s t test).

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NF-κB Is Involved in the Regulation of miR-155 Expression. The ex-pression of microRNA is controlled by factors that regulateprimary microRNA transcription, maturation, and degradation.We found that primary, precursor, and mature forms of miR-155were all up-regulated in PC9/GR cells compared with PC9/WTcells (Fig. 5A), thereby supporting the idea that miR-155 ex-pression may occur through transcriptional regulation. We fur-ther developed a series of reporter constructs containing variousregions of the miR-155 promoter (Fig. 5B, Left). A luciferasereporter assay showed that the region of the miR-155 promoterfrom −1,207 to −722 bp is required to maintain a high level ofmiR-155 promoter activity in PC9/GR cells compared with PC9/WT cells (Fig. 5B, Right). In addition, a previous study indicatedthat NF-κB could bind to a region of the miR-155 promoter (20).To further investigate whether NF-κB is vital for the regulationof miR-155 promoter activity in gefitinib-resistant cells, wild-typeor mutant putative NF-κB binding sites of miR-155 promoterwere transfected into PC9/WT and PC9/GR cells. As shown inFig. 5C, mutagenesis of the NF-κB binding site abolished theability of NF-κB to regulate the luciferase activity of the miR-155promoter in PC9/GR cells. To verify the direct binding of NF-κBon the miR-155 promoter in PC9/GR cells, we performed achromatin immunoprecipitation (ChIP) assay and showed thatp50, a subunit of NF-κB, increased the binding ability to themiR-155 promoter (Fig. 5D), and a high level of nuclear p50(Fig. 5E) was found in PC9/GR cells compared with PC9/WT

cells, resulting in decreased FOXO3a expression. Moreover,H292 cells with stable knockdown of p50 were shown to decreasemiR155 and increase FOXO3a expression but reduced FOXO3ain H292/shp50 cells with the overexpression of miR-155(Fig. 5F). Mice-bearing tumors derived from H292/shp50 cellsexhibited a significant reduction of tumor volume comparedwith H292/shctrl tumors, but the effects were reversed in micebearing H292/shp50/miR-155 tumors (Fig. 5G). These findingssuggest that NF-κB binds to the miR-155 promoter region andinduces miR-155 expression and subsequently decreasesFOXO3a expression, resulting in the increased tumorigenicity oflung cancer.

Inhibition of NF-κB Activity Enhances Gefitinib Sensitivity. We nextdetermined whether inhibited NF-κB activity causes PC9/GRcells to be more sensitive to gefitinib treatment. As shown in Fig.6A, treatment with NF-κB inhibitor led to the prevention ofnuclear translocation of p50 followed by increased FOXO3aexpression in the nucleus. Additionally, activation of NF-κB byTGF-β stimulus in PC9/WT cells increased miR-155 expression,decreased protein levels of FOXO3a and BCL-2–like protein 11(BIM), which is a FOXO3a-targeted proapoptotic protein, andresulted in enhancement of gefitinib resistance (SI Appendix, Fig.S5 A–C). To further investigate whether NF-κB inactivation wasassociated with a decrease in the transcriptional activity of miR-155, the reporter assay was used by the transfection of the

Fig. 4. miR-155 mediates gefitinib resistance andCSC of lung cancer cells. (A) FOXO3a and miR-155were measured by Western blot analysis (Upper) andreal-time RT-PCR (Lower) in PC9/WT, PC/GR, and PC9/GR cells with expressing anti–miR-155 (PC9/GR/anti-miR-155) and scramble control oligos (PC9/GR/ctrl),respectively. (B and C) Indicated cells were untreatedor treated with 4 μM gefitinib and analyzed for cellviability by MTT assay and CD133+ cells by flowcytometric analysis. **P < 0.01 and ***P < 0.001(two-tailed Student’s t test). (D) Western blot anal-ysis of FOXO3a expression in PC9/WT cells tran-siently transfected with miR-155 and FOXO3a. (Eand F) Indicated cells were untreated or treated with4 μM gefitinib and analyzed for cell viability by MTTassay and CD133+ cells by flow cytometric analysis.*P < 0.05 and ***P < 0.001 (two-tailed Student’st test). (G) Effects of miR-155 overexpression on thetumor growth of A549 cells were evaluated in theNOD/SCID mice with xenografts after treatment withgefitinib for 2 wk. Five mice in each group; **P <0.01 and n.s., not significant (two-tailed Student’st test). (H) FOXO3a and miR-155 expression weremeasured by Western blot analysis and real-timeRT-PCR in the indicated group of tumor samples,respectively. The results are shown as the means ±SD of three independent experiments, each per-formed in triplicate. ***P < 0.001 (two-tailed Stu-dent’s t test). (I) miR-155 expression in patients wasassigned according to different responses to EGFR-TKIs. miR-155 expression levels in tumors that wereclassified into high and low groups according tomedian of the individual ΔCt values of patientsamples. ***P < 0.001 (Pearson’s χ2 test). (J) The in-verse correlation between miR-155 and FOXO3a inlung cancer. n = 80; r = −0.329; P = 0.003 (Pearson’scorrelation coefficient).

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miR-155 promoter construct containing a wild-type or mutantNF-κB binding site in PC9/WT and PC9/GR cells. The resultsshowed the significantly decreased promoter activity of wild-type miR-155 reporter but not the mutant miR-155 reporterin PC9/GR cells after treatment with specific NF-κB in-hibitor (Fig. 6B), resulting in the increased expression ofFOXO3a and BIM and sensitivity of PC9/GR cells to gefitinibtreatment upon NF-κB inactivation (Fig. 6C). In addition tousing a NF-κB inhibitor, a knockdown approach of p50 wasalso performed and its knockdown efficiency was verified byWestern blot analysis, showing significantly decreased miR-155promoter activity and increased gefitinib sensitivity in PC9/GRcells (SI Appendix, Fig. S5 D–F). Additionally, the suppression

of p50 in H292 cells obviously increased gefitinib sensitivity(Fig. 6D) and repressed the CSC properties (Fig. 6 E–H), andthose effects of p50 knockdown were reversed by the over-expression of miR-155 in H292/shp50 cells. We further observedthat the expression of p50 was positively correlated with miR-155expression level in lung cancer patients (SI Appendix, Table S3)and was inversely correlated with FOXO3a expression in theOncomine database (SI Appendix, Table S4); it was also con-sistent with RelA/p65, another subunit of NF-κB (SI Appendix,Table S4). In combination, these results suggest that NF-κB ac-tivity is responsible for generating gefitinib resistance and CSCproperties through the suppression of FOXO3a via miR-155 inlung cancer.

Fig. 5. miR-155 is transcriptionally regulated by NF-κB of gefitinib-resistant lung cancer cells. (A) Real-time RT-PCR analysis of pri–miR-155, pre–miR-155, andmiR-155 expression in PC9/WT and PC9/GR cells. (B) Schematic description of serial deletion reporter constructs of the miR-155 promoter cloned into the pGL3-Basic vector (Left). PC9/WT and PC9/GR cells were transfected with various miR-155 promoter reporters, and the luciferase activity was measured by the dual-luciferase reporter assay (Right). (C) Schematic diagram showing that the NF-κB binding sequences or mutated versions in the miR-155 promoter (Upper), andluciferase activity was measured using the dual-luciferase reporter assay (Lower). (D) ChIP analysis of chromatin extracted from PC9/WT and PC9/GR usingpolyclonal antibodies directed against the p50, a subunit of NF-κB, followed by real-time RT-PCR analysis to confirm p50 occupancy at the miR-155 promoter.(E) Cytoplasmic and nuclear fractions from PC9/WT and PC9/GR cells were assessed for the presence of FOXO3a, p50, Lamin B (nuclear marker), and Tubulin(cytosolic marker) by Western blot analysis. (F) The FOXO3a and p50 protein expression of H292 cells with stable expression of the indicated transfectantswere measured by Western blot (Upper), and miR-155 expression by real-time RT-PCR (Lower), respectively. **P < 0.01 (two-tailed Student’s t test). (G) In vivotumor growth of H292 cells with stable expression of the indicated transfectants. Each point represents the mean ± SEM of tumor volumes of six mice in eachgroup. Tumor volume was calculated as indicated in Materials and Methods. *P < 0.05 and **P < 0.01 (two-tailed Student’s t test).

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Physiological Effects of NF-κB/miR-155/FOXO3a Is Related to theGefitinib Response. To further assess the ability of NF-κB/miR-155/FOXO3a to affect gefitinib response in lung cancer, H292cells with stable knockdown of p50 (H292/shp50) were sub-cutaneously injected into nonobese diabetic (NOD)/SCID micewith gefitinib treatment after inoculation. Compared with the con-trol (H292/shctrl) group, tumors in the H292/shp50 group hadstatistically significantly less tumor volume after gefitinib treatmentfor 14 d (Fig. 7 A and B). In addition, the H292/shp50/pLemiR-155–bearing mice showed increased tumor volume and resistance togefitinib compared with the H292/shp50/pLemiR-bearing mice (Fig.7 A and B). We confirmed the expression of p50, FOXO3a, and

FOXO3a-targeted proapoptotic proteins, including BIM, tumornecrosis factor-related apoptosis inducing ligand (TRAIL), p53upregulated modulator of apoptosis (PUMA), and cell death sur-face receptor Fas ligand (FasL), in formed tumors by Western blot(Fig. 7C). We further examined whether these effects are involvedin cancer stemness in tumor cells. Consistent with the observationsmade in vitro, tumors derived from H292 cells with p50 knockdownsignificantly reduced the expression of SOX2, CD133, and Nanog,and preserved the effects by overexpressing miR-155 in H292/shp50 cells (Fig. 7D). These data demonstrate that the NF-κB/miR-155/FOXO3a axis is critical for gefitinib sensitivity and thesuppression of the cancer stemness of lung cancer (Fig. 7E).

Fig. 6. Inhibition of NF-κB activity results in reduced miR-155 expression and suppressed gefitinib resistance and CSC properties. (A) Western blot analysis ofp50, FOXO3a, Lamin B (nuclear marker), and Tubulin (cytosolic marker) in cytoplasmic and nuclear extracts of PC9/WT and PC9/GR cells treated with orwithout 2 μM NF-κB inhibitor. (B) Reporter assays of miR-155 promoter containing wild-type or mutant NF-κB binding site in PC9/WT and PC9/GR cells treatedwith NF-κB inhibitor. (C) Cell viability (Upper) was determined by MTT assay and expression of FOXO3a and BIM (Lower) were analyzed by Western blot inPC9/WT and PC9/GR cells treated with NF-κB inhibitor and 4 μM gefitinib. (D) Cell viability of gefitinib treatment was determined by MTT assay in H292 cellswith stable expression of the indicated transfectants. The results are shown as the means ± SD of three independent experiments, each performed intriplicate. **P < 0.01 (two-tailed Student’s t test). (E) Expression of miR-155 affected mRNA levels of SOX2, Nanog, and KLF4 by real-time RT-PCR, (F) ALDH+

and CD133+ cells by flow cytometric analysis, (G) sphere formation and (H) side population in H292/WT cells with p50 knockdown and miR-155 overexpression.The graph indicated the differences in the sphere numbers per microscopic field at 100× magnification (G, Upper) and that was calculated the percentage ofsphere forming cells compared with PC9/WT cells (Lower). The results are shown as the means ± SD of three independent experiments each performed intriplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 (two-tailed Student’s t test).

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DiscussionThe molecular mechanisms of acquired and intrinsic resistanceto EGFR-TKIs in the presence of EGFR-sensitizing mutations arerelatively unknown for up to 40% of patients. The presence ofT790M or other exon 20-resistant mutations has been reported inonly 5% of lung cancer patients with EGFR-TKI treatment (5).The activation of EGFR downstream signals, such as PIK3CA andKRAS mutations, seem to be indicators of resistance and poorsurvival in NSCLC patients with exposure to EGFR-TKI therapy,but only in 4.1% and 6.7% of patients, respectively (21). Resistanceto EGFR-TKIs have also been found in lung cancer patients with7.3% MET amplification (22) or with 5% EML4–ALK fusion inNSCLC patients (23). Recent studies showed that most NSCLCpatients with the EGFR mutation carry a proapoptotic protein,BIM, that restores apoptotic responses in oncogene-addicted can-cers and intrinsic resistance to EGFR-TKI therapy (24, 25), and itsmutation was detected in 12.5% of lung cancer patients in East

Asia (26). More importantly, FOXO3a has been found to increaseseveral target genes, such as TRAIL, PUMA, FasL, and BIM, whichis essential for the gefitinib-induced killing of NSCLC cells (14, 27),and the suppression of FOXO3a in breast cancer cells result in areduction of gefitinib-induced cell cycle arrest and cell death (18).These results are consistent with our observations, which showedthat 24% of lung cancer patients with high FOXO3a have a betterresponse to EGFR-TKIs and progression-free survival outcomes,suggesting that FOXO3a expression is involved in the regulation ofthe EGFR-TKI–induced apoptotic response and is a good markerfor predicting the therapeutic effects of EGFR-TKIs in lung cancer.Current studies reveal that NF-κB activation promotes re-

sistance to EGFR inhibitors and might be a possible mechanismand therapeutic strategy for EGFR-TKI resistance in cancertreatments (28). Additionally, the suppression of the Fas/NF-κBpathway specifically enhances erlotinib-induced cell death inEGFR mutant cells and tumor models (29). Consistently, we

Fig. 7. NF-κB/miR-155–mediated FOXO3a suppression is required for gefitinib resistance and stemness of lung cancer in vivo. (A) Mice were implantedsubcutaneously with the indicated cell lines for ∼100 mm3 and were treated with vehicle and gefitinib for 14 d. Each point represents the mean ± SEM oftumor volumes of six mice in each group. Tumor volume was calculated as indicated in Materials and Methods. *P < 0.05 and n.s., not significant (two-tailedStudent’s t test). (B) The indicated tumors with vehicle and gefitinib treatment for 14 d were dissected from the surrounding tissue. (C) Western blot assay toconfirm the expression of FOXO3a, p50, BIM, TRAIL, PUMA, and FasL in the indicated group of tumor samples. Tubulin was used as a loading control. (D) Therelative expression of SOX2, CD133, and Nanog in the indicated group of tumor samples were measured by real-time RT-PCR analysis. The results are shown asthe means ± SD of three independent experiments each performed in triplicate. *P < 0.05 and **P < 0.01 (two-tailed Student’s t test). (E) Model of theregulatory signaling networks of NF-κB/miR-155/FOXO3a in gefitinib resistance and CSC properties of lung cancer. NF-κB activates the transcription of miR-155, promoting the targeting of the 3′UTR of FOXO3a and decreasing the expression of FOXO3a.

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found that NF-κB–driven miR-155 expression suppressed theFOXO3a protein level in gefitinib-resistant lung cancer, andthe suppression of NF-κB activity and expression contributed tothe resensitivity to gefitinib treatment in vitro and in vivo, im-plying that the combination of both the NF-κB and EGFR in-hibitors might be a potential therapeutic strategy to improvetumors with resistance to EGFR-TKIs. In addition to the factthat NF-κB–activating signal cascades promote resistance tochemotherapy through the transcriptional induction of multidrugresistance gene-1 (30), which promotes the CSC properties oflung cancer (31), increasing the expression of CSC markers haspreviously been considered to associate with acquired EGFR-TKI resistance and metastasis in lung cancer (32, 33). CD133expression is linked to a resistant phenotype in NSCLC andsuggests that the detection of CD133+ cells may be useful topredict the efficacy of cytotoxic therapy for lung cancer (34). Inaddition, SOX2 expression maintained the self-renewal of lungcancer cells and was positively correlated with the metastaticprogression of NSCLC (35, 36), suggesting that CD133 andSOX2 expression plays an important role in studying the prop-erties of cancer stem cells and the therapeutic response in lungcancer. Our study observed that SOX2 and CD133 expressionwere positively correlated with EGFR-TKI nonresponders andlow expression levels of FOXO3a in lung cancer patients, and wefound that NF-κB/miR-155 decreased FOXO3a expression toenhance CSC characteristics in vitro and in vivo, indicating thatthe NF-κB–driven suppression of FOXO3a plays a crucial role inCSC properties and EGFR-TKI resistance of lung cancer.miR-155 is transcribed from the host gene, BIC (B-cell in-

tegration cluster), also termedMIR155HG gene, which is locatedon chromosome 21 (37). Previous studies reported that TGF-β–mediated Smad4 expression induces miR-155 promoter ac-tivity and enriches miR-155 expression to contribute to cell mi-gration and invasion (38), and found that NF-κB, STAT5, orCCAAT/enhancer binding protein beta (C/EBPβ) could bind tomiR-155 promoter region and induce miR-155 expression incolon cancer, cutaneous T-cell lymphoma, or fat cells (20, 39,40). miR-155 has been considered an “oncomicroRNA” bytargeting several tumor suppressors, including SOCS1, FOXO3a,RhoA, C/EBPβ, PP2A/C, and von Hippel–Lindau tumor suppres-sor (VHL), and it has been found to promote the EMT, invasion,metastasis, growth, and angiogenesis of cancer cells (20, 41–43). Inaddition, the up-regulation of miR-155 has been reported to pro-mote tumor angiogenesis by targeting VHL and is associated withpoor prognosis for breast cancer (43); additionally, the loss of VHLhas been reported to induce NF-κB activity (44). These findingssupport our results that miR-155 decreased VHL expression, whichin turn activated NF-κB; this may contribute to the positive feed-back activation of miR-155 and promote gefitinib resistance andcancer stemness. Collectively, the significance of this negative cor-relation between miR-155 and FOXO3a of lung cancer indicatesthat miR-155 may be a critical therapeutic target in lung cancer,especially in EGFR-TKI–resistant patients.A previous study indicated an important role for NF-κB in

regulating the miR-155 expression of immune cell developmentand function (45); however, the role of NF-κB–mediated miR-155in cancer progression and gefitinib resistance and cancer stemnesswere not investigated. Consistent with previous studies, our find-ings used a computational design to search the microRNA data-base, and we identified and verified that miR-155 is able to inducegefitinib resistance by directly inhibiting FOXO3a at the post-transcriptional level and that NF-κB increases gefitinib resistanceby inducing miR-155 expression. Moreover, miR-140 was found tobe a critical suppressor, inhibiting NF-κB activity (46, 47). In ad-dition to miR-155, we also found that miR-140 was significantlyreduced in resistant cells (PC9/GR), which may confer synergisticeffects with miR-155 in mediating gefitinib resistance.

In this study, we found that NF-κB/miR-155 decreased FOXO3aexpression to enhance CSC characteristics and resistance to gefitinibin lung cancer, suggesting that FOXO3a could be an independentprognostic marker of lung cancer and that the NF-κB/miR-155/FOXO3a axis plays an important role in lung cancer patientswith acquired resistance to EGFR-TKIs.

Materials and MethodsSpecimens. Lung cancer specimens were obtained from the China MedicalUniversity Hospital with Institutional Review Board approval (CMUH102-REC2-035), and written informed consent was obtained from all patients.Between December 2005 and September 2011, a total of 80 cases of stage IVlung adenocarcinoma patients, including 37 males and 43 females with an ageranging from31 to 85 y (median, 69 y), were enrolled in this retrospective study.Patients who had ever received EGFR-TKI (erlotinib or gefitinib) therapy be-tween 5.7 and 37.5 mo were evaluated for their clinical response according toRECIST 1.1 criteria (48), and patients who had progressive disease and stabledisease for 4 mo or less were regarded as EGFR-TKI nonresponders (n = 38).The status of complete response, partial response, and stable disease for morethan 4 mo were classified as EGFR-TKIs responders (n = 42). Details of EGFRmutational analysis, immunohistochemical staining, and real-time PCR quan-tification of lung cancer are provided in SI Appendix, Materials and Methods.

Construction of Expression Vectors, Plasmids, and Anti-miR. Full-length humanFOXO3a was amplified by PCR using cDNA of HeLa cells and cloned into theNheI and NotI sites of pCDH-CMV-MCS-EF1-copGFP+Puro (System Biosciences).The hsa-miR-155 stem-loop sequence plus its up- and downstream-flankingregions was amplified by PCR using genomic DNA of HeLa cells and cloned intothe XhoI and NotI sites of pLemiR-empty vector (Open Biosystem). All primersequences of cDNA and microRNA constructs are shown in SI Appendix, TableS5 (lowercase is representative of the additional sequence, and the restrictionenzyme site is underlined). All constructs were confirmed by DNA sequencing.The lentiviral FOXO3a shRNA clones TRCN0000010334 and TRCN0000235488,p50 shRNA clones TRCN0000006518 and TRCN0000006520, the pLKO.1-shLucvector TRCN0000072244 that was shRNA against luciferase acted as controls;the pMD2.G plasmid and pCMVdeltaR8.91 plasmid were purchased from theNational RNAi Core Facility at Academia Sinica, Taipei, Taiwan. The pRL-TK,pGL3-basic, and pmirGLO Dual-Luciferase vectors were obtained from Prom-ega. Anti–miR-155, the microRNA inhibitor (Ambion), was a single-strandedchemically modified oligonucleotide designed to inhibit endogenous miR-155.The anti-miR negative control is a random sequence that has been extensivelytested in human cell lines and tissues and was validated to produce no iden-tifiable effects on known microRNA function.

ChIP Assay. The ChIP assay was performed with the EZ ChIP kit (Millipore) aspreviously described (49). Briefly, cells were fixed with 1% formaldehyde,washed, and lysed. The cell lysates were sonicated to shear DNA to sizes of 300–1,000 bp. Protein–DNA complexes were precipitated with either nonimmune IgGor target protein p50 (Abcam) overnight at 4 °C with rotation. After reversecross-link of protein–DNA complexes to free DNA, real-time RT-PCR were per-formed with the LightCycler 480 (Roche) by using LightCycler FastStart DNAMaster SYBR Green I kit (Roche) and specific primers (SI Appendix, Table S5).Cycling conditions were 95 °C for 10 min followed by 50 cycles of 95 °C for 15 s,60 °C for 1 min. A dissociation procedure was performed to generate a meltingcurve for confirmation of amplification specificity. The relative occupancy of theimmunoprecipitated factor at a locus was estimated by using the comparativethreshold method (50) and expressed as fold-enrichment versus PC9/WT cells.

Cellular Fractionation. The cytosolic and nuclear fractions were extracted aspreviously described (51). Briefly, cells were washed twice with ice-cold PBS,harvested by scraping with a rubber policeman, and lysed in buffer A(20 mM Hepes, pH 7.0, 10 mM KCl, 2 mM MgCl2, 0.5% Nonidet P-40, 1 mMNa3VO4, 10 mM NaF) containing protease inhibitor mixture (Roche). After10-min incubation on ice, cells were homogenized by 15–20 strokes in a tightlyfitting Dounce homogenizer and centrifuged 5 min at 1,500 × g to sedimentthe nuclei. The supernatant is the cytosolic fraction. To remove contaminationfrom cytoplasmic membranes, the nuclear pellet was washed three times withbuffer A. To extract nuclear proteins, the isolated nuclei were resuspended inNETN lysis buffer (20 mM Tris·HCl, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40,and 1 mM EDTA) containing protease inhibitor mixture (Roche) and the mix-ture was sonicated briefly to aid nuclear lysis. After centrifugation at 16,100 × gfor 20 min at 4 °C, the nuclear lysates were collected. Cytosolic fraction andnuclear fractions were analyzed by Western blot (SI Appendix).

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Animal Studies. All animal work was done in accordance with a protocolapproved by the Institutional Animal Care and Use Committee of NationalHealth Research Institutes. Four- to 6-wk-old NOD.CB17-Prkdcscid/J severecombined immunodeficient (NOD/SCID) male mice (supplied by LASCO) wereused for tumor growth in a xenograft studies. Next, 5 × 106 cells/100 μL in PBSmixed with equal volume of Matrigel (BD Biosciences) were subcutaneouslyinjected into the back of mice. When tumor volumes reached ∼100 mm3, asdetermined by measuring tumor length and width using calipers and calcu-lating volume through the equation [1/2 (length × width2)], mice were ran-domly allocated into groups of six mice to receive gefitinib or vehicle by oralgavage at 100 mg/kg every 2 d and tumor volumes were measured every 2 d.

Statistical Analysis. All statistical analyses were performed with Prism 6software. Data of in vitro experiments were approximately normally dis-tributed and presented as the means ± SD from at least three independentexperiments, each performed in triplicate. Statistical evaluation of varianceamong experimental groups was similar and performed with a two-tailedStudent’s t test for comparison between two groups. Pearson’s χ2 test was

used to compare the relationship between FOXO3a, miR-155 expression, andclinicopathological factors.

ACKNOWLEDGMENTS. We thank the National RNAi Core Facility (AcademiaSinica, Taiwan) for providing specific shRNAs; Ms. Fang-Yu Tsai, Dr. I-ShouChang, and Dr. Shih-Sheng Jiang of the Taiwan Bioinformatics Institute CoreFacility for assistance in using Oncomine (National Core Facility Program forBiotechnology, NSC 100-2319-B-400-001). The results shown here are in partbased upon data generated by the TCGA Research Network (cancergenome.nih.gov/). We thank the Cell Sorting Core Facility of the First Core Laboratoryand the Center of Genomic Medicine, National Taiwan University, for theservices they provided. This work was supported by Ministry of Science andTechnology (MOST) (National Science Council; NSC) of Taiwan Grants NSC101-2320-B-400-016-MY3, NSC 102-2314-B-038-028-MY3, MOST 103-2314-B-038-059, MOST 104-2314-B-038-002, MOST 104-2321-B-400-018-MY3, andMOST 104-2320-B-400-015-MY3 and by National Health Research Institutes of TaiwanGrants CA-102-PP-41, CA-103-PP-35, CA-104-PP-12, MOHW104-TDU-B-212-124-008, and CA-105-PP-12.

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Chiu et al. PNAS | Published online April 18, 2016 | E2535

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