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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

  • www.pnas.org/cgi/doi/10.1073/pnas.1621230114

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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

    CORR

    ECTION

    CORR

    ECTION

    www.pnas.org/cgi/doi/10.1073/pnas.1621230114

  • 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