Elevated expression of TANK-binding kinase 1 PNAS PLUS enhances tamoxifen resistance in breast cancer Congwen Weia,1, Yuan Caob,c,1, Xiaoli Yanga,d,1, Zirui Zhenga, Kai Guana, Qiang Wangb, Yanhong Taie, Yanhong Zhanga, Shengli Maa, Ye Caoa, Xiaoxing Gea, Changzhi Xua, Jia Lia, Hui Yanf, Youguo Linga, Ting Songa, Lin Zhug, Buchang Zhangg, Quanbin Xua, Chengjin Huc,2, Xiu-wu Bianb,2, Xiang Hea,h,2, and Hui Zhonga,2 aState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Beijing 100850, China; bInstitute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400038, China; Departments of cLaboratory Medicine and ePathology, General Hospital of Jinan Military Region, Jinan, Shandong 250031, China; dGeneral Hospital of People’s Armed Police Hospital, Beijing 100039, China; fBeijing Institute of Pharmacology and Toxicology, Beijing 100850, China; gInstitute of Health Science, School of Life Sciences, AnHui University, Hefei, Anhui 230601, China; and hBeijing Institute of Disease Control and Prevention, Beijing 100071, China Edited by Jan-Åke Gustafsson, University of Houston, Houston, TX, and approved December 17, 2013 (received for review August 31, 2013) Resistance to antiestrogens is one of the major challenges in endocrine therapies (19, 20). For instance, ERα phosphorylation breast cancer treatment. Although phosphorylation of estrogen at Ser-118 has been suggested to be involved in protein turnover receptor α (ERα) is an important factor in endocrine resistance, the and directly associated with tamoxifen sensitivity (21–23). How- contributions of specific kinases in endocrine resistance are still ever, the contributions and mechanisms of specific kinase- not fully understood. Here, we report that an important innate mediated ERα phosphorylation in endocrine resistance are not immune response kinase, the IκB kinase-related TANK-binding ki- fully known. nase 1 (TBK1), is a crucial determinant of resistance to tamoxifen Here, we investigated the possible role of TBK1 protein in therapies. We show that TBK1 increases ERα transcriptional activ- tamoxifen resistance and found that phosphorylation by TBK1 at ity through phosphorylation modification of ERα at the Ser-305 the Ser-305 site stabilized ERα and modulated its transcriptional site. Ectopic TBK1 expression impairs the responsiveness of breast activity. Although ubiquitin-like domain (ULD)–mutated TBK1 cancer cells to tamoxifen. By studying the specimens from patients failedtoactivateIFN-β promoters, it retained the ability to with breast cancer, we find a strong positive correlation of TBK1 phosphorylate ERα, induce ERα transactivational activity, and α α with ER ,ER Ser-305, and cyclin D1. Notably, patients with modulate breast cancer cell growth. Moreover, ectopic expres- tumors highly expressing TBK1 respond poorly to tamoxifen treat- sion of TBK1 rendered breast cancer cells resistant to tamoxifen. ment and show high potential for relapse. Therefore, our findings Suppressing TBK1 with its pharmacological inhibitor BX795 suggest that TBK1 contributes to tamoxifen resistance in breast sensitized breast cancer cells to tamoxifen-induced cell death. cancer via phosphorylation modification of ERα. Administration of BX795 in conjunction with tamoxifen achieved synergistic inhibitory effects on tumors. The expression of TBK1 κ e e ANK-binding kinase 1 (TBK1) and I B kinase (IKK ) are was increased in patients with breast cancer and was positively Ttwo IKK-related serine/threonine kinases that display 64% correlated with ERα,ERα S305, and cyclin D1 expression. No- sequence identity and trigger the antiviral response of interfer- tably, patients with tumors highly expressing TBK1 responded ons (IFN) through NF-κB activation and interferon regulatory – poorly to tamoxifen treatment and showed a high potential transcription factor (IRF) 3/7 phosphorylation (1 3). In addition for relapse. Therefore, TBK1 is potentially a unique predictive to the proposed roles of IKK-related kinases in controlling transcription factors NF-κB and IRF, the involvement of TBK1 and IKKe in AKT-induced oncogenic transformation has been Significance demonstrated in a recent study (4). TBK1 is identified as a Ras- like (Ral) B effector in the Ral guanine nucleotide exchange We investigated the possible role of TANK-binding kinase 1 factor pathway that is required for Ras-induced transformation (TBK1) protein in tamoxifen resistance and found that phos- (5). IKKe acts downstream of the PI3K-AKT pathway and phorylation by TBK1 at the Ser-305 site stabilized estrogen cooperates with activated MEK to promote cellular trans- receptor α (ERα) and modulated its transcriptional activity. formation (6). IKKe has also been identified recently as a breast Ectopic expression of TBK1 rendered breast cancer cells re- cancer oncogene that is frequently amplified or overexpressed in sistant to tamoxifen. TBK1 inhibition sensitized breast cancer human breast cancer, and the phosphorylation of ERα by IKKe cells to tamoxifen-induced cell death. The expression of TBK1 contributes to tamoxifen resistance in breast cancer (7–9). In- was increased in subjects with breast cancer and was positively terestingly, TBK1 is also highly expressed in breast cancer (10), correlated with ERα,ERα Ser-305, and cyclin D1 expression. and knocking down TBK1 diminishes the viability of MCF-7 Subjects with tumors that highly expressed TBK1 had poor cells (9). However, the exact role of TBK1 in breast cancer re- responsiveness to tamoxifen treatment. Therefore, TBK1 is mains unclear. potentially a unique predictive marker of tamoxifen resistance Estrogen receptor α (ERα) is a nuclear receptor that exerts and a potential therapeutic target for breast cancer. a profound influence on the initiation and progression of breast cancer by regulating cell transformation, proliferation, and me- Author contributions: C.W., Yuan Cao, Q.X., C.H., X.H., and H.Z. designed research; – α C.W., Yuan Cao, X.Y., Z.Z., K.G., Q.W., S.M., Ye Cao, X.G., C.X., J.L., Y.L., T.S., and X.H. tastasis (11 13). For ER -positive patients with breast cancer, performed research; Yuan Cao, Q.W., Y.T., Y.Z., Ye Cao, H.Y., L.Z., and B.Z. contributed targeting the ER signaling pathway with tamoxifen, a selective new reagents/analytic tools; C.W., Yuan Cao, X.Y., Y.T., B.Z., C.H., X.-w.B., X.H., and H.Z. ER modulator, is efficacious in both prevention and treatment of analyzed data; and C.W., Yuan Cao, X.H., and H.Z. wrote the paper. breast cancer (14). Unfortunately, a substantial proportion of The authors declare no conflict of interest. patients are intrinsically resistant to this therapy, and a signifi- This article is a PNAS Direct Submission. cant number of patients with advanced disease eventually de- 1C.W., Yuan Cao, and X.Y. contributed equally to this work. – α velop acquired resistance to the treatment (15 18). ER is a key 2To whom correspondence may be addressed. E-mail: [email protected], bianxiuwu@263. determinant of breast cancer susceptibility to endocrine therapy. net, [email protected], or [email protected]. α Recent studies demonstrate that ER phosphorylation may have This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. MEDICAL SCIENCES had a significant impact on ERα signaling and its response to 1073/pnas.1316255111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1316255111 PNAS | Published online January 21, 2014 | E601–E610 Downloaded by guest on October 2, 2021 marker of tamoxifen resistance and a therapeutic target for including ERα-negative 293T (Fig. 1A), ERα-positive ZR-75-1 breast cancer. (Fig. S1A), MCF-7 (Fig. 1 B and C and Fig. S1B), and BT474 (Fig. S1C) cell lines, increased the transcription of a luciferase Results reporter containing the estrogen-responsive element (ERE). TBK1 Regulating the Transcriptional Activity of ERα Is Independent of TBK1 enhanced the transcriptional activity of ERα regardless of Its Role in the Innate Immune Response. To define the exact role of the presence of 17β-estradiol (E2) (Fig. 1B), suggesting that TBK1 in breast tumor growth, we investigated whether TBK1 the activation of ERα by TBK1 is both ligand-dependent and regulates estrogen signaling. TBK1 overexpression in cell lines, -independent. This effect required TBK1 kinase activity, because Fig. 1. TBK1 regulates the transcriptional activity of ERα independent of its role in the innate immune response. The 293T cells (A) or MCF-7 cells (B) were transfected with the ERE-Luc reporter, ERα, and TBK1 with or without E2. The Luc activity was measured 24 h later and normalized for transfection efficiency. #P < 0.01 and **P < 0.01 vs. control (Ctrl) Flag vector without E2 or with E2, respectively. IB, immunoblot. (C) MCF-7 cells were cotransfected with ERE-Luc together with TBK1, TBK1 L352A, I353A, or TBK1 K38A. Cells were treated with or without 1 μM BX795 for 2 h. The Luc activity was measured 24 h later and normalized for transfection efficiency. (D) MCF-7 cells were transfected with TBK1 siRNA oligos (siTBK1) and siRNA-resistant TBK1 (siTBK1/TBK1) expression plasmid. The Luc activity was measured 24 h later and normalized for transfection efficiency. (E) MCF-7 cells were transfected with pS2-Luc or catD-Luc together with TBK1 or TBK1 L352A, I353A. The Luc activity was measured 24 h later and normalized for transfection efficiency. (F) 293T cells were transfected with HA-ERα and different doses of Flag-TBK1. Whole-cell lysates were analyzed by immunoblotting with anti-Flag or anti-HA antibody. α-Tubulin was used as an equal loading control. (G) MCF-7 cells were transfected with Flag-TBK1 or its mutants in the presence or absence of 1 μM BX795 for 2 h. Whole-cell lysates were analyzed by immunoblotting with anti-Flag or anti-ERα antibody. α-Tubulin was used as an equal loading control. (H) MCF-7 cells were transfected with siTBK1 and siTBK1/TBK1 expression plasmid. ZR-75-1 cells were transfected with Flag-TBK1.