IKBKE Is Induced by STAT3 and Tobacco Carcinogen and Determines Chemosensitivity in Non-Small Cell Lung Cancer

ORIGINAL ARTICLE IKBKE is induced by STAT3 and tobacco carcinogen and determines chemosensitivity in non-small cell lung cancer

J Guo1, D Kim1, J Gao1, C Kurtyka1, H Chen1,CYu1,DWu1, A Mittal1, AA Beg2, SP Chellappan3, EB Haura4 and JQ Cheng1

Serine/threonine kinase IKBKE is a newly identified oncogene; however, its regulation remains elusive. Here, we provide evidence that IKBKE is a downstream target of signal transducer and activator of transcription 3 (STAT3) and that tobacco components induce IKBKE expression through STAT3. Ectopic expression of constitutively active STAT3 increased IKBKE mRNA and protein levels, whereas inhibition of STAT3 reduced IKBKE expression. Furthermore, expression levels of IKBKE are significantly associated with STAT3 activation and tobacco use history in non-small cell lung cancer (NSCLC) patients examined. In addition, we show induction of IKBKE by two components of cigarette smoke, nicotine and nicotine-derived nitrosamine ketone (NNK). Upon exposure to nicotine or NNK, cells express high levels of IKBKE protein and mRNA, which are largely abrogated by inhibition of STAT3. Characterization of the IKBKE promoter revealed two STAT3-response elements. The IKBKE promoter directly bound to STAT3 and responded to nicotine and NNK stimulation. Notably, enforcing expression of IKBKE induces chemoresistance, whereas knockdown of IKBKE not only sensitizes NSCLC cells to chemotherapy but also abrogates STAT3- and nicotine-induced cell survival. These data indicate for the first time that IKBKE is a direct target of STAT3 and is induced by tobacco carcinogens through STAT3 pathway. In addition, our study also suggests that IKBKE is an important therapeutic target and could have a pivotal role in tobacco-associated lung carcinogenesis.

Oncogene (2013) 32, 151--159; doi:10.1038/onc.2012.39; published online 13 February 2012 Keywords: IKBKE; STAT3; tobacco carcinogen; chemosensitivity; lung cancer

INTRODUCTION growth factor and environmental carcinogenesis such as nico- 20 The serine/threonine kinase IKBKE (also called IKKe and IKKi)isa tine. Many studies have also shown that diverse oncoproteins, non-canonical IkB kinase family member and regulates immune such as viral Src oncoprotein, ETK/BMX and an oncogenic form of 18,21,22 response.1,2 In response to inflammatory factor and viral infection, platelet-derived growth factor (PDGF), activate STAT3. In IKBKE is activated and subsequently phosphorylates interferon response to extra- and intra-cellular signals, STAT3 is phosphory- response factors 3 and 7 (IRF3 and IRF7) and signal transducer lated at tyrosine-705, which leads to STAT3 dimerization and and activator of transcription 1 (STAT1)2--5 as well as induces translocation to the nucleus where it binds to DNA and phosphorylation of p65/RelA.6,7 Activated IKEKE also regulates transcriptionally regulates a number of genes such as cyclin D1, 23 --26 CYLD,8 which is a deubiquitinase of several nuclear factor (NF)-kB bcl-xl, Mcl-1 and c-myc. Furthermore, STAT3 is frequently regulators, including tumor necrosis factor receptor-associated activated in a various types of human cancer and is required for 18,27 factor 2, 6 (TRAF2, TRAF6) and NEMO (IKK-g), to activate the cell transformation. canonical NF-kB pathway.9--11 We and others have recently In the current study, we demonstrated that IKBKE is a STAT3 demonstrated IKBKE direct phosphorylation of Akt-Thr308/ target gene. STAT3 transcriptionally induces IKBKE expression. We Ser473,12,13 leading to activation of Akt independent of PI3K, also showed the co-expression of pSTAT3 and IKBKE in non-small PDK1, mTORC2 and PH domain of Akt.12 IKBKE has been shown to cell lung cancer (NSCLC) specimens and association of elevated be frequently overexpressed in human malignancy.14 The IKBKE with smoking. Furthermore, protein and mRNA levels of potential role for IKBKE in breast cancer was first shown by IKBKE were induced by tobacco components nicotine and NNK. Sonenshein and her colleagues who demonstrated that IKBKE was Using genetic and pharmacological approaches, we demonstrated overexpressed in breast cancer cells lines, four of six breast that nicotine-induced IKBKE primarily depends upon STAT3. carcinomas patients and carcinogen-induced mouse mammary Enforcing expression of IKBKE induces chemoresistance and tumors.15 Subsequently, elevated levels of IKBKE were detected in knockdown of IKBKE sensitizes cells to chemotherapy and prostate cancer cell lines.6 Recent studies have shown that IKBKE is abrogates STAT3- and nicotine-induced cell survival. amplified/overexpressed in primary breast and ovarian cancers, with more frequent protein overexpression than DNA amplification,6,16,17 suggesting that IKBKE is regulated by aberrant RESULTS transcription. However, IKBKE has not been associated with STAT3 increases IKBKE expression at mRNA and protein levels environmental carcinogenesis to date. While the IKBKE locus chromosome 1q32.1 is amplified, over- STAT3 is a transcription factor and has a key role in many expression of IKBKE at mRNA and/or protein levels is much 18,19 cellular processes such as cell growth and apoptosis. It has common than its change at DNA level in human malignan- been well documented that STAT3 is activated by cytokine, cies.14,16,28 These prompted us to examine transcriptional regula-

1Department of Molecular Oncology, H Lee Moffitt Cancer Center, Tampa, FL, USA; 2Department of Immunology, H Lee Moffitt Cancer Center, Tampa, FL, USA; 3Department of Tumor Biology, H Lee Moffitt Cancer Center, Tampa, FL, USA and 4Department of Thoracic Oncology, H Lee Moffitt Cancer Center, Tampa, FL, USA. Correspondence: Dr JQ Cheng, Department of Molecular Oncology, H Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, SRB3, Tampa, FL 33612, USA. E-mail: jin.cheng@moffitt.org Received 13 August 2011; revised 1 January 2012; accepted 13 January 2012; published online 13 February 2012 Regulation of IKBKE by STAT3 and tobacco J Guo et al 152 tion of IKBKE. Using 50 race and EST (expression sequence tag) H157 and OVCAR-3 cells with STAT3 inhibitor and STAT3 shRNAs database analysis, we defined transcription start site of the IKBKE (Supplementary Figure S2). Conversely, ectopic expression of and isolated a 2.5-kb IKBKE promoter including intron 1 and exon 1, constitutively active STAT3 (STAT3-C) resulted in increase of IKBKE which does not encode IKBKE protein. Transcriptional element protein and mRNA in A549 cells in which endogenous pSTAT3 and analysis (http://www.genome.jp/tools/motif/) revealed 7 STAT3- IKBKE are low (Figures 1a and d). We also observed that IKBKE level binding sites (Supplementary Figure S1). Because frequent was elevated in A549 cells following expression of v-Src, especially activation of STAT3 has been detected in NSCLC,29,30 we co-expression of STAT3 and v-Src (Figure 1d), and treatment with investigated if modulation of STAT3 will affect IKBKE expression. IL6 (interleukin 6) (Figure 1e), two major STAT3 activators.18,31 Figure 1a shows pSTAT3 and IKBKE levels in 15 NSCLC cell lines These findings suggest that STAT3 upregulates IKBKE. examined. As H292 cells highly express pSTAT3 and IKBKE, we treated the cells with short hairpin RNA (shRNA)-STAT3 and STAT3 inhibitor JSI-124, and observed considerable decrease of IKBKE STAT3 binds to and activates IKBKE promoter protein and mRNA levels following inhibition of STAT3 (Figures 1b We further examined whether the IKBKE promoter is regulated by and c). These findings were further confirmed by treatment of STAT3. Luciferase reporter assay with a 2.5-kb IKBKE promoter

Y 292 1437 226 441 661 157 358 1355 K 460 2172 549 1299 1703 2122 H H H H H H H H U H H A H H H

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T3 1# 0 1 5 10 20 (h) FP TAT3 2# -G -S IKBKE A A-STA A N N N shR shR shR pSTAT3 IKBKE STAT3 STAT3 Actin Actin JSI-124 N 0 5 10 20(h) N IKBKE IKBKE RT-PCR GAPDH GAPDH RT-PCR

/STAT3 C T3-C C R TA IL6: 0 3 6 12 24 (h) Vector S v-S V-SR IKBKE IKBKE

pSTAT3 pSTAT3 Flag-STAT3 STAT3 HA-v-SRC

Actin

N IKBKE IKBKE

GAPDH GAPDH RT-PCR RT-PCR Figure 1. STAT3 transcriptionally regulates IKBKE. (a) Expression of IKBKE largely correlates with pSTAT3. Immunoblot analysis of a panel of NSCLC cell lines with the indicated antibodies. (b, c) Inhibition of STAT3 reduces IKBKE expression. H292 cells were transfected and treated with the indicated agents and then subjected to western blot (upper panels) and semi-quantitative RT--PCR (bottom panels) analyses. (d, e) Activation of STAT3 increases IKBKE expression. A549 cells were transfected with the indicated plasmids or treated with IL6. Expression of IKBKE was evaluated by immunoblot and RT--PCR analyses.

Oncogene (2013) 151 --159 & 2013 Macmillan Publishers Limited Regulation of IKBKE by STAT3 and tobacco J Guo et al 153 6 ChIP 3 ChIP 2 ChIP 1 5 1 23 45 6 7 4 1# 3 -1200 -750 -400 +1 +911 activity 2 2# 1

Relative luciferase 3# 0 4# IKBKE-Luc: ++++ STAT3 response element STAT3-C: - + ++ - NF-B binding site DN-STAT3: ---+ PCR amplification of ChIP region

-actin -STAT3 2# Marker Input α α ChIP1 3# STAT3-C - 4# ChIP2

0 1 2 3 4 56 ChIP3 Relative luciferase activity

2 3 2# 2# 2#-2M 2#-3M 2#-2M 2#-2M/3M 2#-3M 2 STAT3-C 2M - 3 2#-2M/3M 3M 0 2 4 68 Relative luciferase activity Figure 2. STAT3 binds to and transactivates the IKBKE promoter. (a) The IKBKE promoter is activated by STAT3. A549 cells were transfected with IKBKE-Luc, b-galactosidase and various forms of STAT3. After incubation for 36 h, luciferase activity was measured and normalized to b-galactosidase. Results are mean±s.e. of three independent experiments performed in triplicate. (b) Diagram of a 2.1-kb IKBKE promoter and its deletion mutants based on putative STAT3-response elements. (c) The response elements 2 and 3 are required for STAT3 activation of the IKBKE promoter. Luciferase assay was performed as (a), with deletion mutants of IKBKE promoter. (d) STAT3 directly binds to the IKBKE promoter. A549 cells were transfected with the indicated plasmids and ChIP assay was performed as described under ‘Materials and methods’. STAT3 antibody was used for ChIP. ChIP with actin antibody was served as a negative control. The DNA before the immunoprecipitation was used as a positive control (input). (e) Mutation of STAT3-binding sites abrogated STAT3-stimulated IKBKE promoter activity. Two STAT3-binding sites were mutated individually and in combination (TT---GG; left). Luciferase reporter assay was performed as described above (right).

revealed that constitutively active but not dominant-negative in these sites. Furthermore, knockdown of STAT3 in H292 cells, STAT3 induces the promoter activity in a dose-dependent manner which express high levels of IKBKE and pSTAT3 (Figure 1), (Figure 2a). Since the IKBKE promoter contains seven putative significantly decreased STAT3 binding to the IKBKE promoter STAT3-response elements, we next determine which STAT3- (Supplementary Figure S3). To demonstrate if these two putative binding site(s) is required for response to STAT3. Deletion mutants STAT3-binding sites are required for STAT3 transactivation of the of pGL3-IKBKE promoter were created based on the STAT3- IKBKE promoter, we have mutated them individually and in response elements (Figure 2b). As shown in Figure 2c, removal of combination. As shown in Figure 2e, mutation of either site alone the first STAT3-binding site slightly increased basal and STAT3- moderately reduced STAT3-stimulated IKBKE promoter activity, induced promoter activity. However, after deletion of STAT3- whereas the IKBKE promoter with both site mutations failed to binding sites 2 and 3, the promoter failed to respond to STAT3. respond to STAT3-C. Moreover, knockdown of STAT3 in H292 cells To determine if STAT3 directly binds to the STAT3-response reduced basal wild type but not mutated IKBKE promoter activity elements within the IKBKE promoter in vivo, we carried out (Supplementary Figure S4). These results indicate that STAT3 chromatin immunoprecipitation (ChIP) assay, which detects directly binds to the response elements 2 and 3 of the IKBKE specific genomic DNA sequences that are associated with a promoter to transactivate IKBKE. particular transcription factor in intact cells. A549 cells were transfected with STAT3-C and then subjected to immunoprecipitation with STAT3 antibody. The STAT3-bound chromatin was Overexpression of IKBKE correlates with STAT3 activation and is subjected to PCR using oligonucleotide primers that amplify associated with smoking status in NSCLC region spanning STAT3-binding site within the IKBKE promoter Having demonstrated that IKBKE is a STAT3 target gene in cell (Figure 2b). In agreement with the findings from luciferase culture system, we asked if this regulation is seen in vivo.We reporter assay, the STAT3 antibody only pulled down STAT3- examined 98 NSCLC samples for protein expression of IKBKE and binding sites 2 and 3 (Figure 2d). In contrast, immunoprecipitation pSTAT3 (Figures 3a and b). Of the 98 lung tumors, 54 had with an irrelevant actin antibody resulted in the absence of bands overexpression of IKBKE and 64 had activation of STAT3. Of the 64

& 2013 Macmillan Publishers Limited Oncogene (2013) 151 --159 Regulation of IKBKE by STAT3 and tobacco J Guo et al 154 NSCLC tumors α-IKBKE α-pSTAT3-Y705 N IKBKE

pSTAT3

Actin

Case 1 Case 1 pSTAT3 Total P value Low High

Low 22 22 44(44.9%) IKBKE High 12 42 54(55.1%) 0.006

Total 34(34.7%) 64(65.3%) 98 Case 2 Case 2

IKBKE Smoking* n P-value No overexpression Overexpression

Never smoker 21 15 (71.4%) 6 (28.6%) Continuous smoker 73 27 (37.0%) 46 (73.0%) (10 pack cigarettes/year) 0.014 Total 94 41 (43.6%) 53 (56.4%)

* 4 NSCLC patients with unknown smoking history. Figure 3. Co-expression of elevated levels of IKBKE and pSTAT3 in NSCLC. (a) Representative NSCLC specimens were immunoblotted with the indicated antibodies. (b) Immunohistochemistry was carried out with antibodies against IKBKE and pSTAT3-Y705. (c) w2 test analysis of IKBKE and pSTAT3 in 98 NSCLC specimens examined. The correlation is signiﬁcant (P ¼ 0.006). (d) Expression of IKBKE correlates with smoking status of NSCLC patients.

tumors with elevated pSTAT3, 42 (66%) also had elevated IKBKE treatment with nicotine (1 mM) and NNK (1 mM) for 12 h, we levels (P ¼ 0.006; Figure 3b). Immunohistochemistry of these performed immunoblot and reverse transcription (RT)--PCR analyses tumor samples showed that the co-expression of IKBKE and and observed that inhibition of STAT3 largely abrogates nicotine pSTAT3 are located specifically to the cancer cells and not to the and NNK-induced IKBKE expression (Figures 5a and b). Moreover, stroma (Figure 3c; Supplementary Figure S5). Furthermore, we basal levels of IKBKE were remarkably reduced by knockdown of found that the IKBKE overexpression is associated with patients’ STAT3 or by treatment with STAT3 inhibitor (Figure 5b). smoking history (P ¼ 0.014; Figure 3d). These data suggest that To further demonstrate that nicotine and NNK induce IKBKE and there is a significant relationship of co-expression of IKBKE and that this action is mediated by STAT3, we performed luciferase pSTAT3, which further supports STAT3 regulation of IKBKE. reporter assay and found that nicotine and NNK-stimulated IKBKE promoter activity in a dose-dependent manner. The promoter activity induced by nicotine and NNK was significantly reduced Nicotine and NNK induce IKBKE expression through STAT3 by inhibition of STAT3 (Figure 5c; Supplementary Figure S7). Tobacco smoke is the strongest documented tumor initiator and Furthermore, ChIP assay revealed that nicotine increased cap- promoter for lung cancer.32,33 Of the components in tobacco, ability of STAT3 binding to the IKBKE promoter. The STAT3-DNA- nicotine and NNK have been shown to activate multiple signaling binding activity induced by nicotine was abrogated by inhibition pathways including Akt/mammalian target of rapamycin (mTOR), of STAT3 (Figure 5d). Based on these findings, we concluded that extracellular signal-regulated kinase (ERK) and janus kinase (JAK)/ IKBKE is induced by nicotine and NNK through STAT3. STAT.34 --37 Since overexpression of IKBKE is associated with smoking, we reasoned that IKBKE could be induced by nicotine and NNK. To test this, A549 cells were serum starved for 12 h and then treated Ectopic expression of IKBKE induces chemoresistance and with nicotine (1 mM)orNNK(1mM) for different time points. Increase knockdown of IKBKE sensitizes NSCLC cells to apoptosis and of IKBKE protein expression was observed as early as 6 h of the reduces STAT3- and nicotine-induced cell survival treatment (Figures 4a and c), whereas mRNA level of the IKBKE was To evaluate if IKBKE is a therapeutic target in NSCLC, we elevated starting at 3 h (Figures 4b and d). We further confirmed ectopically expressed IKBKE in H661 cells, in which endogenous these findings using an additional cell line H1299 (Supplementary IKBKE is low (Figure 1a), and then treated the cells with various Figure S6). In agreement with a previous report,38 pSTAT3 but not chemotherapeutic agents. The cells transfected with vector and total STAT3 is induced by nicotine and NNK in a pattern similar to treated with dimethyl sulfoxide were used as control. Poly (ADP- IKBKE mRNA expression (Figures 4b and d). ribose) polymerase (PARP) cleavage and TUNEL assay showed that We next examined if STAT3 mediates nicotine and NNK-induced expression of IKBKE alone did not significantly induce cell survival IKBKE. A549 cells were transfected with shRNA of STAT3 or treated as compared with vector transfected cells (P ¼ 0.7; left panels of with STAT3 inhibitor before nicotine and NNK stimulation. The cells Figure 6a). However, the cells transfected with IKBKE became transfected with shRNA of green fluorescent protein (GFP) and resistance to cisplatin-, gemcitabine- and doxorubicin-induced treated with dimethyl sulfoxide were used as control. After apoptosis (Figure 6a). Furthermore, we infected endogenous

Oncogene (2013) 151 --159 & 2013 Macmillan Publishers Limited Regulation of IKBKE by STAT3 and tobacco J Guo et al 155

Nicotine (1μM) 0 3 6 12 24 hNNK (1μM): 0 3 6 12 24 h

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STAT3 STAT3

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μ Nicotine (1 M) NNK (1μM) M0 1 3 61224h 03 6 1224h IKBKE IKBKE mRNA mRNA GAPDH GAPDH

Figure 4. Nicotine and NNK induce IKBKE expression. (a-- d) Following serum starvation for 12 h, A549 cells were treated with 1 mM nicotine or NNK and then subjected to immunoblot (a, c) and RT--PCR (b, d) analyses.

shRNA-GFPshRNA-STAT3JSI-124 DMSO2# shRNA-GFP shRNA-STAT3JSI-124 2# Nicotine (1 μM): -++++ NNK (1 μM): -+-+-+

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GAPDH GAPDH RT-PCR W.B RT-PCR W.B

6.0 M Input α-IgG α-STAT3 5.0 Nicotine: - ++++- ++ 4.0 JSI-124: ---+ - --+ 3.0 shRNA-STAT3 2#: - + ---+ -- RLU 2.0

1.0 1.0 0.4 3.1 0.8 0 STAT3 Nicotine: - +++++ ++ pSTAT3 JSI-124: ---+ - (A549) shRNA-STAT3: ----+ Figure 5. STAT3 mediates nicotine/NNK-induced IKBKE expression. (a, b) Inhibition of STAT3 decreases nicotine- and NNK-induced IKBKE. H358 cells were transfected with STAT3 shRNA or treated with STAT3 inhibitor. Immunoblot (upper panels) and RT--PCR (bottom panels) were performed following treatment with/without 1 mM nicotine (a) and 1 mM NNK (b) for 12 h. (c) Nicotine induces the IKBKE promoter activity via STAT3. A549 cells were transfected with IKBKE-Luc together with/without shRNA of STAT3 or treated with/without STAT3 inhibitor before exposure to nicotine as described above. Luciferase reporter assay was performed as described in Figure 2. (d) Nicotine induces STAT3 binding to the IKBKE promoter, which is inhibited by depletion of STAT3. Following treatment with the indicated agents, A549 cells were subjected to ChIP assay.

IKBKE-high H292 cells with lentiviruses expressing shRNA-IKBKE or tively active STAT3-C or were treated with nicotine together with control shRNA and observed that basal level of apoptosis was not and without shRNA-IKBKE. In agreement with previous studies,35,39 signiﬁcantly affected by knockdown of IKBKE alone (P ¼ 0.06) as cells expressing STAT3-C or treated with nicotine became compared with the cells treated with control shRNA. Yet, the resistance to gemcitabine (Figure 6c). However, knockdown depletion of IKBKE considerably sensitized the cells to chemother- of IKBKE largely abrogates STAT3-C and nicotine protected cells apeutic drug-induced apoptosis (Figure 6b). These data suggest from gemcitabine-induced cell death (Figure 6c). Furthermore, that IKBKE has a role in chemosensitivity in NSCLC. we examined the effect of IKBKE on nicotine-induced cell survival Since IKBKE is upregulated by STAT3 and nicotine, we further in two more IKBKE-low cell lines H1299 and H661. Figure 6d and investigated if STAT3- and nicotine-induced cell survival is Supplementary Figure S8 showed that nicotine protected both mediated by IKBKE. A549 cells were transfected with constitu- cell lines from the gemcitabine- and cisplatin-induced cell death

& 2013 Macmillan Publishers Limited Oncogene (2013) 151 --159 Regulation of IKBKE by STAT3 and tobacco J Guo et al 156 DMSO CDDP Gem DOX DMSO CDDP Gem DOX Vector: +- +-- + + - Ctr. shRNA: ++++- --- IKBKE: - + - + - ++- IKBKE-shRNA: - + - + - ++-

IKBKE IKBKE

Cleaved Cleaved PARP PARP Actin Actin

40 45 P<0.003 P<0.002 40 Control P<0.001 35 IKBKE sh Control 35 30 P<0.001 sh IKBKE 30 P<0.002 25 P<0.006 25 20 20 15 % apoptosis

% apoptosis 15 10 10 P=0.7 P=0.06 5 5 0 0 DMSO CDDP Gem DOX DMSO CDDP Gem DOX (H661) (H292)

60 p<0.05 40 DMSO p<0.04 ** ** 50 35 Nicotine ** 30 40 * 25 p<0.01 p<0.01

30 p<0.03 p<0.02 20 15 20 10 5 10 % TUNEL positive cells % TUNEL positive cells 0 0 shRNA-IKBKE: - - + - - + STAT3-C: - -- - ++ sh-GFP sh-GFP sh-GFP sh-IKBKE Nicotine: - + + --- sh-IKBKE sh-IKBKE DMSO Gemcitabine CDDP (A549/Gemcitabine) (H1299) Figure 6. Enforcing expression of IKBKE induces chemoresistance whereas knockdown of IKBKE sensitizes NSCLC cells to chemotherapy and reduces anti-apoptotic function of STAT3- and nicotine. (a) Expression of IKBKE renders cells resistance to chemotherapeutic drug-induced apoptosis. H661 cells expressing low endogenous IKBKE were transfected with IKBKE and then treated with the indicated agents (e.g., cisplatin 20 mM, gemcitabine 10 mM and doxorubicin 1.0 mM) for 36 h (top panel). Apoptosis was assayed by PARP cleavage (panel 2) and TUNEL (bottom panel). (b) Depletion of IKBKE enhances apoptosis induced by chemotherapeutic drugs. IKBKE was knocked down in high-IKBKE H292 cells. After treatment with the indicated agents, the programmed cell death was evaluated by PARP cleavage and TUNEL assay for three times in triplicate. (c, d) STAT3- and nicotine-induced cell survival was largely abrogated by knockdown of IKBKE. A549 (c) and H1299 (d) cells were transfected and treated with the indicated plasmids and agents and then subjected to TUNEL assay. Single asterisk represents Po0.05 and double asterisks indicate Po0.01.

and that this protection was considerably reduced by knock- agent-induced cell death and reduces STAT3-induced cell survival. down of IKBKE. These results indicate that IKBKE is an important These ﬁndings suggest that IKBKE could be an important mediator of STAT3- and nicotine-induced cell survival and therapeutic target in NSCLC. chemoresistance. The tobacco smoke is the strongest documented tumor initiator and promoter for lung cancer.32,33 Traditional models of tobacco- related tumorigenesis are genocentric, in that tobacco components DISCUSSION promote carcinogenesis through a multistep processes that Current treatments for NSCLC include surgery, radiotherapy and involve exposure to and activation of carcinogens such as NNK chemotherapy. Patients with locally advanced disease that are or polyaromatic hydrocarbons, which leads to formation of DNA surgically unrespectable will be subjected to radiation and adducts and mutations in key genes such as K-ras, p53 or p16. chemotherapy. Chemoradiotherapy improves the survival rate of Recent studies have shown that tobacco components can also NSCLC patients; however, the overall median 5-year survival rate is promote lung tumorigenesis through modulation of signal still only B15%.40 The major reason is that the patients eventually transduction pathways that regulate cell proliferation, transforma- develop chemoresistance and radioresistance. Therefore, there is tion and survival. For example, nicotine and NNK are potent an unmet need to understand molecular mechanism of the agonists of nicotinic acetylcholine receptor nicotinic acetylcholine chemoresistance and radioresistance and further develop tar- receptor (nAChR), which are expressed in lung epithelial cells and geted therapy by identiﬁcation new promising target(s). Activation can activate multiple signaling cascades such as Akt/mTOR,42,43 of STAT3 is a common change in NSCLC and represents an mitogen-activated protein kinase (MAPK)28 and janus kinase/ attractive therapeutic target for anti-cancer drug discovery.41 Our STAT3 pathways.44 In the current study, we demonstrated that study indicates that the IKBKE is a STAT3 target gene. STAT3 overexpression of IKBKE is associated with tobacco smoking and directly binds to the IKBKE promoter and induces IKBKE transcrip- that IKBKE protein and mRNA levels are increased in response to tion. Co-expression of pSTAT3 and IKBKE was observed in primary nicotine and NNK. In addition, depletion of IKBKE reduces nicotine NSCLCs. Ectopic expression of IKBKE induces chemoresistance and protection of NSCLC cells from apoptosis as well as nicotine- knockdown of IKBKE sensitizes NSCLC cells to chemotherapeutic induced chemoresistance. Thus, we provide the evidence that

Oncogene (2013) 151 --159 & 2013 Macmillan Publishers Limited Regulation of IKBKE by STAT3 and tobacco J Guo et al 157 IKBKE is induced by tobacco carcinogen and mediates tobacco Western blot, immunohistochemistry and RT--PCR analysis action in promoting lung cancer cell survival. Western blot analysis was performed as described previously.12 Briefly, Previous studies demonstrated that IKBKE is induced by a panel cells and the frozen tissues were lysed and equal amounts of protein were of inflammation factors or cytokines including lipopolysacccharide immunoblotted with appropriate antibody indicated in the legend of 45 (LPS), IL6, IL-1b and interferon-g at transcriptional levels, Figures 1--6. Detection was carried out with the ECL Western Blotting suggesting involvement of transcription factors in regulation of Analysis System (Amersham, Piscataway, NJ, USA) and quantified with 15 IKBKE. In fact, Eddy et al. showed that the tumors and Image J program (NIH, Bethesda, MD, USA). Immunohistochemistry with mammary glands derived from a casein kinase 2 catalytic IKBKE and pSTAT3 antibodies were described previously.16,49 subunit a transgenic mouse model of mammary adenocarcinoma Total RNA was extracted using TRIzol buffer (Invitrogen) according to exhibit higher levels of IKBKE. Likewise, overexpression of the manufacturer’s instruction. RT--PCR was performed as previously casein kinase 2 in Michigan Cancer Foundation (MCF)-10F cells described.17 Primers are IKBKE, 50-CAGGGCTTGGCTACAACGAG-30 (forward) also increased IKBKE expression. These results suggested and 50-GATGTCCAGGAGGTCAGATGC-30 (reverse) and GAPDH, 50-CATG that casein kinase 2 has an important role in regulating TTCGTCATGGGTGTGAACCA-30 (forward) and 50-AGTGATGGCATGGACTGTG IKBKE. Furthermore, NF-kB has been demonstrated to activate GTCAT-30 (reverse). IKBKE promoter.46 Our data show that enforcing expression of STAT3-C or constitutively active Src increases, whereas inhibition of STAT3 represses IKBKE transcription. Cloning of the human IKBKE promoter Interestingly, recent evidence indicates that STAT3 collaborates Transcriptional start site of human IKBKE gene was determined by EST with NF-kB to regulate certain gene expressions through binding to analysis and 50 race. A 2.5-kb IKBKE promoter including exon 1 and intron 1 adjacent sites in the regions of shared target genes and/or was generated by PCR using genomic DNA isolated from A549 cells. The direct interaction between STAT3 with p65/RelA and p50, which PCR primers used for the amplified IKBKE promoter are 50-GGAAGATCT leads to recruitment of p300 to NF-kB complex and acetylation of AGACCAACCTGCTCAATC-30 (forward) and 50-CCCAAGCTT CTTTAACCC p65/RelA.47 The STAT3-response element number 2 in the TTTCCTGTTTGTC-30 (reverse). Forward primers used for deletion mutants IKBKE promoter is close to NF-kB-binding site (for example, 187 bp and the different reverse primers are 50-CGGGATCC ATGGGAAAGTCCCTC apart; Figure 2b; Supplementary Figure S1), suggesting that STAT3 CAAC-30,50-CGGGATCC CCCAGGCAGCTTTCCACT-30 and 50-CGGGATCC and NF-kB could work together to transcriptionally regulate IKBKE. In GCTACCAGGAGGCTAAGAAC-30. The PCR products were cloned to BglII/ fact, we have examined the effect of inhibition of NF-kBon HindIII sites of pGL3-Luciferase vector and the constructs were confirmed IKBKE expression induced by STAT3-C and nicotine and observed by DNA sequencing. that blocking NF-kB, unlike inhibition of STAT3, only moderately reduces nicotine-stimulated IKBKE. Furthermore, constitutively Luciferase reporter assay and ChIP assay activated STAT3-induced IKBKE was not compromised by inhibition of NF-kB (Supplementary Figure S9). It has been shown that nicotine For reporter assay, the cells were seeded in a 12-well plate and transfected simultaneously activates NF-kB and STAT3 pathways.48 Thus, with IKBKE-Luc, b-gal and additional plasmid as described in the legend of our findings suggest that STAT3 regulates IKBKE independent NF- Figures 2 and 5. The amount of DNA in each transfection was kept kBandthatNF-kB and STAT3 seem to individually regulate IKBKE in constant by the addition of empty vector pCMV-Tag3B. Thirty-six hours response to nicotine with STAT3 playing more predominant role than after transfection, luciferase was assayed according to the manufacturer’s NF-kB. protocol (Promega, Madison, WI, USA). The luciferase activity was normal- In summary, we identified IKBKE as a direct target of STAT3 and ized to that of b-gal. ChIP assay was performed essentially as previously described.50 Briefly, nicotine/NNK-induced IKBKE via STAT3 in human NSCLC cells. 7 Depletion of IKBKE largely abrogates STAT3- and nicotine-induced soluble chromatin was prepared from a total of 2 Â 10 asynchronously cell survival and sensitizes NSCLC cells to apoptosis. These findings growing A549 cells, which were serum starved for 24 h, and then treated not only provide mechanism by which IKBKE is induced by with or without nicotine and STAT3 inhibitor for 12 h. The pre-cleared tobacco carcinogen but also may help to develop a strategy for chromatin solution was subjected to immunoprecipitation with either intervention of NSCLC by targeting IKBKE. STAT3 antibody or IgG. Following multiple washes, the antibody--protein-- DNA complex was eluted from the beads. The DNA was purified and subjected to PCR with primers flanking the STAT3 potential binding sites. MATERIALS AND METHODS The sequences of the PCR primers are ChIP-1, 50-CCCAGGCAGCTTTCCACT 0 0 0 Cell culture, transfection, lung tumor specimens, plasmids and TAG-3 (forward) and 5 -GAACTGGGGCCCTTCTGGTAAT-3 (reverse); ChIP-2, antibodies 50-AGCAGTCTCTGAAGAGCATGGG-30 (forward) and 50-CACTCACCCGCAG AGTAACAGC-30 (reverse) and ChIP-3, 50-GGTGGAACATGAGGATCTC-30 NSCLC cells were cultured in RPMI 1640 containing 10% fetal calf (forward) and 50-GAATGTTGGAGGGACTTT-30 (reverse). serum. The studies using nicotine, NNK, anti-cancer drugs were performed in the cells that were rendered quiescent by serum starvation for 12 h, thereafter, were stimulated with 1 mM nicotine or 1 mM NNK Assays for detection of programmed cell death (Sigma, St Louis, MO, USA) in the presence or absence of the indicated NSCLC cells were serum starved for 24 h and stimulated with 1 mM nicotine drugs for different time described in the legend of Figures 4--6. in the presence or absence of chemotherapeutic agents for time indicated The transfection was carried out with Lipofectamine 2000 (Invitrogen, in the legend of Figure 6. Apoptosis was measured by TUNEL assay Carlsbad, CA, USA) according to the manufacturer’s instruction. (Promega) and caspase3/7 activity (Roche, New York, NY, USA) following Knockdown of STAT3 or IKBKE was processed by specific shRNA got from the manufacture’s instruction. Open Biosystem (Lafayette, CO, USA). The primary human NSCLC specimens were procured at the Moffitt Cancer Center under the institutional review board approved protocol. The tissues were snap Statistical analysis frozen and stored at À70 1C. For luciferase activity, cell survival and apoptosis, the experiments were HA-STAT3, STAT3-C, DN-STAT3 and v-Src plasmids were obtained repeated at least three times in triplicate. The data are represented by from Dr Richard Jove (Beckman Research Institute, Duarte, CA, USA). mean values±s.d. Differences between control and testing cells were Antibodies against pSTAT3-Y705 and STAT3 were purchased from evaluated by Student’s t-test. The correlation of IKBKE with smoker status Cell Signaling (Danvers, MA, USA). Anti-IKBKE antibody was from Sigma. or pSTAT3 was analysis by w2 analysis. All analyses were performed with STAT3 inhibitor JSI-124 was purchased from Calbiochem (San Diego, SPSS software, version 13.0 (USF, Tampa, FL, USA) and Pp0.05 was CA, USA). considered statistically significant.

& 2013 Macmillan Publishers Limited Oncogene (2013) 151 --159 Regulation of IKBKE by STAT3 and tobacco J Guo et al 158 CONFLICT OF INTEREST 22 Wen X, Lin HH, Shih HM, Kung HJ, Ann DK. Kinase activation of the non-receptor The authors declare no conflict of interest. tyrosine kinase Etk/BMX alone is sufficient to transactivate STAT-mediated gene expression in salivary and lung epithelial cells. J Biol Chem 1999; 274: 38204--38210. ACKNOWLEDGEMENTS 23 Barre B, Vigneron A, Coqueret O. The STAT3 transcription factor is a target for the Myc and riboblastoma proteins on the Cdc25A promoter. J Biol Chem 2005; 280: We are grateful to Tissue Procurement, DNA Sequence and Image Core Facilities at 15673 --15681. H Lee Moffitt Cancer Center for providing cancer specimens, sequencing and cell 24 Epling-Burnette PK, Liu JH, Catlett-Falcone R, Turkson J, Oshiro M, Kothapalli R apoptosis analysis as well as Fumi Kinose for helping provide lung cancer cell lines et al. 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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

& 2013 Macmillan Publishers Limited Oncogene (2013) 151 --159