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MEDICAL SCIENCESCorrection for NF-Bdriven 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: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 theconclusions of the article.
E654E655 | PNAS | January 24, 2017 | vol. 114 | no. 4 www.pnas.org
PC9/WT PC9/GRIP: IgG
PC9/WT PC9/GRIP: p50
P3 P3/mut NF-B
mutant NF-B site
Relative luciferase activity (fold of PC9/WT)
0 5 10 15 20
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Fig. 5. miR-155 is transcriptionally regulated by NF-B of gefitinib-resistant lung cancer cells. (A) Real-time RT-PCR analysis of primiR-155, premiR-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-B 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-B, 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 Students 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 Students t test).
PNAS | January 24, 2017 | vol. 114 | no. 4 | E655
NF-Bdriven 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 3UTR of FOXO3a and was transcriptionally regulatedby NF-B, 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-B/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-B
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) (13). 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 1016 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 (1517), 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
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-B/miR-155. This finding highlights the potentialof targeting the NF-B/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.
E2526E2535 | PNAS | Published online April 18, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1522612113
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-B 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. S1CE). 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 01 representedlow expression of FOXO3a, and 23 represented highexpression. (Scale bars, 100 m.) (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