Glibenclamide Targets Sulfonylurea

Published OnlineFirst July 24, 2019; DOI: 10.1158/1535-7163.MCT-18-1181

Cancer Biology and Translational Studies Molecular Cancer Therapeutics Glibenclamide Targets Sulfonylurea Receptor 1 to Inhibit p70S6K Activity and Upregulate KLF4 Expression to Suppress Non--Small Cell Lung Carcinoma Kexin Xu1, Geng Sun1, Min Li1, Hongling Chen1, Zuhao Zhang1, Xixi Qian1, Ping Li1, Lin Xu2, Wenbin Huang3, and Xuerong Wang1

Abstract

Sulfonylurea receptor 1 (SUR1) is the regulatory subunit of the expression of the tumor suppressor Kruppel-like€ factor 4 ATP-sensitive potassium channels (KATP channels) and the (KLF4), and silencing KLF4 partially reversed the inhibitory receptor of antidiabetic drugs, such as glibenclamide, which effect of glibenclamide on cell growth, EMT, and migration. induce insulin secretion in pancreatic b cells. However, the We found that SUR1 targeted p70S6K to downregulate KLF4 expression and role of SUR1 in cancer are unknown. In this expression by enhancing DNA-methyltransferase 1–mediated study, we found that SUR1 expression was elevated in human methylation of the KLF4 promoter. Finally, in xenograft mouse non–small cell lung carcinoma (NSCLC) tissues and cell lines. models, SUR1 expression silencing or glibenclamide treat- SUR1 silencing suppressed the growth of NSCLC cells, while ment inhibited the growth of A549 tumors, downregulated SUR1 overexpression promoted cell growth. Targeting SUR1 p70S6K activity, and upregulated KLF4 expression. These with glibenclamide suppressed cell growth, cell-cycle progres- ﬁndings suggested that SUR1 expression was elevated in some sion, epithelial–mesenchymal transition (EMT), and cell NSCLC tissues and functioned as a tumor enhancer. Targeting migration. Moreover, SUR1 directly interacted with p70S6K SUR1 with glibenclamide inhibited NSCLC through down- and upregulated p70S6K phosphorylation and activity. In regulation of p70S6K activity and subsequent upregulation of addition, glibenclamide inhibited p70S6K, and overexpres- the expression of the tumor suppressor gene KLF4. SUR1 can sion of p70S6K partially reversed the growth-inhibiting effect be developed as a new target for cancer therapy and gliben- of glibenclamide. Furthermore, glibenclamide upregulated clamide has potential anticancer effects.

Introduction at approximately 21% (3). Therefore, identifying new therapeutic targets and agents is urgently needed. Lung cancer is the most prevalent cancer and the leading cause Sulfonylurea receptor 1 (SUR1), also named ATP-binding of cancer-related deaths worldwide (1). Approximately 83% of cassette subfamily C, member 8 (ABCC8), is the regulatory lung cancers are types of non–small cell lung carcinoma (NSCLC), subunit of the ATP-sensitive potassium channels (K channels; while the others are types of small-cell lung carcinoma (2). ATP ref. 4). The K channels are metabolic sensors that couple energy Therapeutic strategies for NSCLC have been widely developed, ATP status to ion channel activity (5). In pancreatic b cells, uptake of and although combinations of molecular-targeted drugs or glucose induces an accumulation of cytoplasmic ATP, and then immune checkpoint drugs with chemotherapy have been proven induces KATP channel closing, cell membrane depolarization, to be effective first-line treatments for selected patients, the 5-year þ voltage-dependent calcium channel opening, Ca2 influx, and survival rate of patients in the middle or late stages is still very low, subsequent insulin secretion (6). SUR1 is well-known as the receptor of sulfonylurea antidiabetic drugs, such as glibenclamide, which induce insulin secretion (7). Glibenclamide has been 1Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu Province, China. 2Department of Thoracic Surgery, Jiangsu Cancer Hospital, reported to inhibit cell growth and invasion in several cancer cell Nanjing Medical University Affiliated Cancer Hospital, Nanjing, Jiangsu Province, lines, including gastric, breast, ovarian, liver, prostate, and blad- China. 3Department of Pathology, Nanjing First Hospital, Nanjing Medical der cancer (8–11). However, the expression and function of SUR1 University, Nanjing, Jiangsu Province, China. in cancer are unknown. Note: Supplementary data for this article are available at Molecular Cancer The 70-kDa ribosomal S6 kinase (p70S6K) is a serine/threo- Therapeutics Online (http://mct.aacrjournals.org/). nine kinase (12). It is one of the major downstream effectors of the K. Xu, G. Sun, and M. Li contribute equally to this article. mammalian target of rapamycin complex 1 (mTORC1) signaling pathway and plays important roles in cell proliferation, metab- Corresponding Author: Xuerong Wang, Nanjing Medical University, 1202 Xianz- olism, differentiation, and migration. p70S6K contributes to hilou, 140 Hanzhong Road, Nanjing, Jiangsu 210029, China. Phone/Fax: 8625- 8686-2884; E-mail: [email protected] some diseases such as cancer, diabetes, and obesity (13, 14). Its mechanism in regulating protein translation, mRNA splicing, and Mol Cancer Ther 2019;XX:XX–XX cytoskeletal organization is well-known, but its mechanism in doi: 10.1158/1535-7163.MCT-18-1181 regulating transcription is still unclear (15). Because mTORC1 2019 American Association for Cancer Research. signaling pathway is an important sensor of cellular energy and

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nutrient status, we hypothesize that there may be cross-talk UAUUCUCUCCAAUTT-30,50-GGUCAUCAGCGUCAGCAAATT-30, 0 0 between KATP channels and p70S6K. and 5 -GCCACCCACACUUGUGAUUTT-3 ), and DNMT1 Kruppel-like€ factor 4 (KLF4), a zinc finger-type transcription (50-GGAUGAGUCCAUCAAGGAA-30,50-CCAGAGCACUACC- factor, positively or negatively regulates gene expression in a GGAAAU-30,and50-GCAAGGACAUGGUUAAAUU-30)and context-dependent manner (16). It is famous for being one of control siRNA were used as described previously (12) or were the four transcription factors that induces pluripotent stem cells designed by us and synthesized by Shanghai GenePharma. from adult fibroblasts (17). It plays important roles in many siRNAs were used at a concentration of 100 nmol/L. physiologic processes, including proliferation, differentiation, SUR1 overexpression plasmids were constructed by inserting and apoptosis (18, 19), and functions both as a tumor enhancer full-length SUR1 (NM_000352.4) into pcDNA3.1 (pcDNA- and suppressor in different types of cancers (20, 21). In lung SUR1). Full-length SUR1 was also inserted into the p3 flag- cancer, KLF4 has been reported to inhibit cell growth, invasion, CMV-14 vector to generate plasmids expressing flag-tagged pro- and metastasis, and its expression is decreased in non–small cell tein. The truncated C-terminus of SUR1 (from amino acid 1187 to carcinoma but increased in small-cell carcinoma (16, 22, 23). 1581) was amplified and cloned into the same vector by homol- Currently, the regulatory context of KLF4 in lung cancer has not ogous recombination using the ClonExpress II One Step Cloning been elucidated. Kit (C112-02, Vazyme Biotech Co., Ltd). Constructs that In this study, we found that the expression of SUR1 was expressed HA-tagged p70S6K (pRK7-S6K-HA) and control vector elevated in human lung cancer tissues. SUR1 promoted cell (pRK7) were gifted by Dr. John Blenis (Harvard Medical School, growth in parallel with downregulation of the p-p70S6K signaling Boston, MA; ref. 25). pathway through a direct interaction with p70S6K in lung cancer A549 cells with stable SUR1 silencing were established by cell lines. p70S6K downregulated KLF4 expression through infection with lentivirus carrying scramble shRNA or SUR1 shRNA DNMT1-mediated DNA methylation of the KLF4 promoter. Glib- (50-GAUCUACCGUCAAAGCUCUTT-30; Shanghai GeneChem enclamide targeted SUR1 to regulate the expression of p-p70S6K Co., Ltd). A549 cells stably overexpressing SUR1 were established and KLF4 as well as growth, EMT, and migration both in NSCLC by infection with lentivirus carrying the full-length coding cells and in xenograft mouse models. sequence of SUR1 or vector as described previously (26).

Cell growth and colony formation assays Materials and Methods Surviving cells were stained with sulforhodamine B (SRB) or Reagents crystal violet. The cell growth rate and drug inhibition were Glibenclamide (G2539-5G) and 5-Azacytidine (A2385) were calculated as described previously (26). purchased from Sigma, Inc. Trichostatin A (T6270) was purchased from Target Mol Inc. Minoxidil (S24448) and nicorandil Cell-cycle analysis (S67281) were obtained from Shanghai Yuanye Biotechnology Cells were collected, and the DNA content was detected by flow Co., Ltd. 5-Azacytidine was dissolved in PBS, and the other cytometry (FACS; Cytomics FC 500, Beckman) as we described reagents were dissolved in DMSO. Lipofectamine 2000 Transfec- previously (26). tion Reagent was purchased from Life Technologies Co., Invitro- gen. The antibodies: anti-phospho-p70S6K (Thr389) (9205), Transwell migration assay anti-p70S6K (9202), anti-phospho-S6 (Ser235/236) (4858), A migration assay was performed using Matrigel Invasion anti-S6 (2217), anti-KLF4 (12173), anti-cyclin D1 (2922), and Chambers with polyethylene terephthalate membranes on the anti-Akt (9272) were obtained from Cell Signaling Technology, bottoms of the chambers (8.0-mm pore size) from BD Transduc- Inc.; anti-SUR1 (ABCC8) (sc-25683) was obtained from Santa tion Laboratories as we described previously (12). Cruz Biotechnology Inc.; anti-b-actin (BS6007), anti-GAPDH (AP0063), anti-HA (AP0005M), anti-flag (AP0007M), anti-E- RNA isolation and qRT-PCR cadherin (BS1098), and anti-N-cadherin (BS2224) were obtained Total RNA extraction, reverse transcription, qPCR, and data from Bioworld Technology Inc.; and anti-DNMT1 (A5495) was analysis were conducted as we described previously (12). The obtained from ABclonal Inc. primers for amplification of KLF4 were 50-GAAATTCGCCCGCT- CAGATGAACT-30 (forward) and 50-TTCTCTTCTGGCAGTG- Cell lines TGGGTCAT-3 (reverse), and those for the internal control The NSCLC cell lines A549, H460, H157, H1299, and Calu-1 GAPDH were 50-ATGGGGAAGGTGAAGGTCG-30 (forward) and (ATCC) were cultured in RPMI1640 Medium (Gibco) supple- 50-GGGGTCATTGATGGCAACAATA-3 (reverse). The primers mented with 5% FBS (Gibco) at 37C in a humidified atmosphere were purchased from Invitrogen. consisting of 5% CO2. All cell lines were genetically confirmed by profiling of 20 short tandem repeats (Shanghai Biowing Applied Western blot analysis and immunoprecipitation Biotechnology Co., Ltd.) and tested for Mycoplasma contamina- Whole-cell proteins were extracted using 1% Triton X-100 lysis tion using Hoechst staining periodically. buffer as we described previously (12). For immunoprecipitation, 1% Triton X-100 in the lysis buffer was substituted with 0.7% Gene knockdown and overexpression CHAPs (C5070, Sigma Inc.). In brief, 0.5–1 mg of cell lysates was siRNA pools that targeted p70S6K (50-CCAAGGUCAUGUG- subjected to immunoprecipitation and 30–50 mg of cell lysates AAACUA-30,50-CAUGGAACAUUGUGAGAAA-30, and 50-GAC- served as input. HA and flag antibodies, secondary antibodies, GGGGUCCUCAAAUGUA-30; ref. 24), SUR1 (50-GUGGUCU- and Protein A/G PLUS-Agarose Immunoprecipitation Reagent ACUAUCACAACATT-30,50-GAUCUACCGUCAAAGCUCUTT-30, (sc-2003, Santa Cruz Biotechnology, Inc.) were used for pull- and 50-GUCUAUGCCAUGGUGUUCATT-30), KLF4 (50-GGACUU- down assays as we described previously (26).

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Quantitative analysis of Western blot bands was performed as measurement and sample collection were performed as we we previously described using ImageJ (27). In brief, the chemi- described previously (26). luminescent signal was collected by ImageJ [integrated optical density (IOD) of each band ¼ density area]. The value of Human tissue samples IOD ratio (IOD ratio ¼ IOD of SUR1, p-p70S6K, or KLF4/IOD Paired cancer tissues and peripheral normal tissues were col- of housekeeping gene) was calculated. The fold change (fold lected from patients with lung cancer at the Affiliated Hospital of change ¼ IOD ratio of treatment/IOD ratio of control) is pre- Nanjing Medical University from 2010 to 2015. Among 16 sented under each blot band. patients, 8 had adenocarcinoma and 8 had squamous cell carcinoma. Tissue samples were stored at 80C. Total proteins were Luciferase reporter assay prepared and subjected to Western blot analysis. The study was We constructed the KLF4 promoter (from 1500 to 137) approved by the Ethics Committee of Nanjing Medical University into the pGL3-Basic Vector (Promega) using a ClonExpress II in accordance with the Declaration of Helsinki and informed One Step Cloning Kit (C112-02, Vazyme Biotech Co., Ltd) written consent was obtained from all participating subjects through homologous recombination following the manufac- (Approval No. 2014161). All experiments were performed in turer's instructions. The primers for amplification of the KLF4 accordance with the approval guidelines of Nanjing Medical promoter were 50-GGTACCGAGCTCTTACGCGTCGAAGGAAC- University. GAGTTGTC-30 (forward) and 50-CTTAGATCGCAGATCTCGAGTT- CCTTACTTATAACTTCCT-30 (reverse). Cells were transfected Statistical analysis with pGL3-KLF4-promoter (or pGL3 vector) plasmids, and a The data are presented as the mean SD from triplicate or Dual-Luciferase Reporter Assay System (E1910, Promega Co.) quadruplicate samples. The results are representative of at least was used to detect fluorescence intensity as we described three independent experiments. Statistical significance between previously (27). two groups was analyzed using two-tailed unpaired Student t tests. When multiple groups were compared, one-way ANOVA Bisulfite sequencing PCR for DNA methylation assay was used. GraphPad software was used for data analysis. The Genomic DNA was extracted from A549 cells and subjected to results were considered to be statistically significant at P < 0.05. bisulfite conversion using the TIANamp Genomic DNA Kit and the DNA Bisulfite Conversion Kit (Tiangen Biotech CO., Ltd), respectively. Selected DNA regions of KLF4 promoter (from Results 1302 to þ1041 bp, transcription start site was defined as SUR1 expression is elevated in NSCLC tissues and SUR1 þ1 bp) were amplified by PCR using primers as follows: promotes the growth of NSCLC cells 50-CACGCCTGTAATCCCAGCACTTC-30 (forward) and 50-GACA- Currently, the expression of SUR1 in cancer is unknown. We GAGTCTCGCTGTGTCGCCC-30 (reverse). The products were first analyzed SUR1 expression in human tissue samples based on purified by Thermo Scientific GeneJet PCR Purification Kit online databases. According to the Oncomine database, SUR1 (Thermo Fisher Scientific Inc.), and then cloned into pCE2 (ABCC8) mRNA expression was higher in lung cancer tissues than TA/Blunt-Zero Vector (Vazyme Biotech Co., Ltd). Ten clones of in normal lung tissues (Fig. 1A). We also analyzed the expression each sample were selected and sequenced by Tsingke Biological of the pore-forming protein of the KATP channels Kir6.1 and Technology Co. The percentage of methylation was the ratio of Kir6.2, and the results showed that Kir6.1 (KCNJ8) was decreased methylated CpG sites to total CpG sites. but that Kir6.2 (KCNJ11) was increased in cancer tissues (Supplementary Fig. S1A). Microarray analysis Then we analyzed the correlation between SUR1 expression A549 cells were treated with 50 mmol/L glibenclamide or and patient survival. A Kaplan–Meier plotter of data from the DMSO for 24 hours in triplicate. Total RNA was extracted using website www.kmplot.com showed that high expression levels of TRizol, and quality control was performed using an Agilent RNA SUR1 were correlated with poor survival (Fig. 1B). However, 6000 Nano Kit. A GeneChip PrimeView Human Gene Expression Kir6.2 expression did not show any correlation with patient Array (901838, Affymetrix) was used. RNA was processed using a survival (P ¼ 0.8). Increased Kir6.1 was correlated with good GeneChip hybridization wash and stain kit. The data were ana- survival (P ¼ 0.02), which was consistent with the low expression lyzed by GeneChem Co., Ltd.. A fold change of more than 1.5 was of Kir6.1 in NSCLC. These results indicate that SUR1 may have used as the cut-off threshold for Gene Ontology (GO) analysis. additional functions in NSCLC (Supplementary Fig. S1B). We then examined SUR1 protein expression in 16 samples of Lung cancer xenografts in nude mice cancer tissues with paired surrounding normal tissues from Animal experiments followed the institutional guidelines and patients with NSCLC, including 8 adenocarcinoma samples and were approved by the Institutional Animal Care and Use Com- 8 squamous cell carcinoma samples. The results showed that mittee of Nanjing Medical University. Four- to 6-week-old female SUR1 expression was higher in lung cancer tissue than in paired athymic (nu/nu) mice were purchased from the Model Animal normal tissue for both adenocarcinoma (6/8) and squamous cell Research Center of Nanjing University and housed under stan- carcinoma (5/8) patients. However, there were equal amounts of dard conditions at the Animal Core Facility of Nanjing Medical SUR1 expression in 3 patients (#4, #8, and #12). In 2 patients University. A549 cells (5 106 cells/mouse) were injected sub- (#14 and #15), SUR1 expression was lower in cancer tissues than cutaneously into the right flank regions of nude mice, which were in normal tissues (Fig. 1C). The clinicopathologic characteristics treated with 200 mg/kg glibenclamide by oral gavage for 14 days. of the patients are presented in Supplementary Table S1. We also A549-scramble_shRNA and A549-SUR1_shRNA cells were also detected increased SUR1 protein expression in NSCLC cells injected into mice that were then monitored for 14 days. Tumor (A549, H157, and H1299) compared with normal human

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A B 210245_at 1.0 HR = 1.28 (1.12−1.45) Log-rank P = 0.00016

0.5

0.0 0.6 0.8 1.0

0:Normal lung tissue Probability −0.5 1: Large-cell lung carcinoma

2: Lung adenocarcinoma median-centered intensity of ABCC8 median-centered intensity of 2 −1.0 Expression 0 123 3: Squamous cell lung carcinoma Low Log High 0.0 0.2 0.4 C 0 50 100 150 200 Time (months) Patients 1 23 4 567 8 Number at risk Low 978 475 125 39 7 High 948 353 78 18 0 Tissues N NNNTTTT NNNNTTTT SUR-1

β-Actin D

Patients 9 10111213141516 Cells HBE Beas 2B A549 H157 H1299 H460 Calu-1 Tissues NNNNTTTT NNN TTT N T SUR1 SUR-1

β β-Actin -Actin

E A549 30 A549 A549 30 A549 Scramble_shRNA Vector shRNA Scramble SUR1 SUR1 OE SUR1 OE Vector SUR1 20 SUR1_shRNA 20 SUR1 SUR1 1 2.96 10 1 0.49 10 Cyclin D1 Cyclin D1 Fold enrichment

Fold enrichment 0 β-Actin 0 β-Actin 012 3456 0 123456 Day Day

Figure 1. SUR1 is elevated in human NSCLC and promotes cell growth. A, ABCC8 (SUR1) mRNA expression in different subtypes of NSCLC in the Oncomine database. B, Kaplan–Meier plots showing the correlation between ABCC8 expression and patient survival from datasets at the website www.kmplot.com. C, SUR1 protein expression in human NSCLC tissues samples and paired peripheral normal tissues (8 patients with adenocarcinoma, top; 8 patients with squamous cell carcinoma, bottom) as revealed by Western blotting. D, SUR1 protein expression in NSCLC cells and normal cells as revealed by Western blotting. E, Effects of stable SUR1 silencing or overexpression on cell growth as determined by SRB assay and on SUR1 expression as determined by Western blot analysis. Bar, SD (n ¼ 4); , P < 0.05. The values under the blot bands are results of quantitative analysis of the bands. The results are representative of at least three independent experiments.

bronchial epithelial (HBE) cells and Beas 2B cells (Fig. 1D). These increased in parallel (Fig. 1E; Supplementary Fig. S2B). These ﬁndings indicate that SUR1 expression is increased in NSCLC and results suggest that SUR1 promoted cell growth in NSCLCs. indicates poor patient survival. Next, we examined the role of SUR1 in NSCLC cells. We found Glibenclamide targets SUR1 to inhibit the growth, cell-cycle that silencing of SUR1 expression in cells transiently transfected progression, EMT, and migration of NSCLC cells with SUR1 siRNAs (a pool of three sets of siRNAs) or in stable cell SUR1 is the receptor of glibenclamide in pancreatic b cells. lines (A549-SUR1_shRNA vs. A549-scramble_shRNA), inhibited Glibenclamide has been reported to inhibit the growth of several the growth of A549 and H460 cells, as determined by SRB assay. types of cancer cells; however, its effect on lung cancer is unknown. Western blot analysis conﬁrmed the successful silencing of SUR1 We found that glibenclamide inhibited the growth of the NSCLC expression and the decreased expression of cyclin D1, which is a cell lines A549, H1299, Calu-1, H157, and H460 with IC50 values cell-cycle checkpoint protein, whose expression often decreases of 95.56, 188.3, 71.89, 113.3, and 143.43 mmol/L, respectively, by when cell growth is inhibited (Fig. 1E; Supplementary Fig. S2A). SRB assay (Fig. 2A). The colony formation assay showed the same In addition, overexpression of SUR1 in cells transfected with dose-dependent inhibitory effect (Fig. 2B). We also observed cell- SUR1 plasmids or in stable cell lines (A549-SUR1 OE/A549- cycle arrest at G1-phase (Fig. 2C) and decreased cyclin D1 expres- vector) promoted cell growth. SUR1 and cyclin D1 expression sion (Supplementary Fig. S3). SUR1 total protein was not altered

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A 100 B Calu-1 Glibenclamide 0 5 50 100 80 H157 A549 A549 60 H460 H157 40 H1299

Inhibition (%) Calu-1 20

0 H460 110100 1,000 Glibenclamide (μmol/L) C A549 H460 H157 100 − 100 G −M 100 − G2 M 2 G2 M S 75 S 75 75 S − G −G − G0 G1 0 1 G0 G1 50 50 50

25 25 25 0 0

Proportion of cells (%) 0 Proportion of cells (%) DMSO Glibenclamide DMSO Glibenclamide Proportion of cells (%) DMSO Glibenclamide

DFA549 H460 100 Glibenclamide −+ −+ Scramble_shRNA E-cadherin 80 SUR1_shRNA 60 N-cadherin 40 GAPDH Inhibition (%) 20

0 1 10 100 1,000 E Control Glibenclamide Glibenclamide (μmol/L)

100

A549 80 Vector 60 SUR1 OE

Inhibition (%) 20 H460 0 1 10 100 1,000 Glibenclamide (μmol/L)

Figure 2. Glibenclamide inhibits the growth, cell-cycle progression, EMT, and migration of NSCLC cells. NSCLC cells were treated with different concentrations of glibenclamide (5–500 mmol/L) as indicated. A, Growth inhibition by a 3-day SRB assay. B, Survival cells by a 14-day colony formation assay. C, Cell-cycle analysis by ﬂow cytometry for cells treated with 100 mmol/L glibenclamide for 24 hour. Western blot analysis (D) and transwell assay for cell migration (E) in cells treated with 75 mmol/L glibenclamide for 24 hours. Scale bar, 30 mm; magniﬁcation, 100 . F, Results of a 3-day SRB assay for A549 cells with stable SUR1 overexpression or silencing treated with glibenclamide. Bars, SD. The results are representative of at least three independent experiments.

(Supplementary Fig. S3). Glibenclamide inhibited EMT (as evi- openers would promote cell growth. However, to our surprise, denced by increases in the expression of the epithelial cell marker minoxidil and nicorandil inhibited cell growth in a dose- E-cadherin and decreases in the expression of the mesenchymal dependent manner with the greatest inhibitory effect of less than cell marker N-cadherin; Fig. 2D) and cell migration (Fig. 2E). 30% of control (Supplementary Fig. S4). These results suggest that If the inhibitory effect of glibenclamide was mediated by the inhibitory effect of glibenclamide did not depend on KATP blockade of KATP channels, we speculated that KATP channel channels and that other mechanisms may exist.

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We further determined whether the effect of glibenclamide was To identify the role of p70S6K in glibenclamide-induced mediated by SUR1 in NSCLC. We found that overexpression of growth inhibition, we conducted a rescue experiment. Through SUR1 reduced the inhibitory effect of glibenclamide, increasing SRB assay we observed that overexpression of p70S6K by trans- the IC50 from 92.72 to 127.9 mmol/L, while silencing of SUR1 fection of the pRK7-S6K-HA plasmid promoted cell growth and enhanced the effect of glibenclamide, decreasing the IC50 from almost completely attenuated the growth-inhibiting effect of 100.3 to 69.21 mmol/L (Fig. 2F). glibenclamide (Fig. 3F). In contrast, knockdown of p70S6K These findings suggest that glibenclamide targeted SUR1 to expression by transfection with siRNAs (a pool of three sets) inhibit cell growth, the EMT, and migration in NSCLCs. enhanced glibenclamide-induced growth inhibition (Fig. 3G). Western blotting was used to confirm the successful overexpres- p70S6K plays a critical role in mediating the effects of sion or silencing of p70S6K. These findings suggest that p70S6K glibenclamide in NSCLC cells plays a critical role in mediating the effect of glibenclamide in In our previous study which aimed to identify the proteins that NSCLC cells. interact with p70S6K, we identified physical binding between SUR1 and p70S6K. In this experiment, we designed an immu- KLF4 is the downstream effector of glibenclamide noprecipitation assay to pull down p70S6K and examined its To reveal the downstream molecules that mediated the growth- binding proteins by mass spectrometry analysis. Following this inhibiting effect of SUR1, we conducted a microarray analysis of strategy, HEK293T cells were transfected with pRK7-S6K-HA A549 cells treated with glibenclamide for 24 hours using a human plasmid to overexpress p70S6K and immunoprecipitated with gene chip from Affymetrix (Fig. 4A). We identified 57 down- anti-p70S6K antibody. Then the precipitates were separated by regulated genes and 62 upregulated genes. GO biological process SDS-PAGE and silver stained. We noted a new band located analysis suggested that the downregulated genes were involved in at approximately 45 kDa in p70S6K overexpression samples cell migration, adhesion, apoptosis, and death. The upregulated that was not present in control samples, suggesting the presence genes were involved in amine, amino acid, small molecule, and of some proteins that may physically bind to p70S6K (Supple- nitrogen biosynthesis and metabolism (Supplementary mentary Fig. S5A). We then excised the band and conducted mass Table S2). Among these genes, KLF4 was the most markedly spectrometry analysis. The results showed that it was a fragment of regulated gene, with 14-fold greater expression in treated cells SUR1 (GI: 784882; Supplementary Fig. S5B). It should be noted than in control cells. We first verified that KLF4 mRNA and protein that the immunoprecipitation was performed using 0.7% CHAPs levels were increased significantly in four NSCLC cell lines (Fig. 4B lysis buffer, because when 1% Triton X-100 lysis buffer was used, and C). We found that knockdown of KLF4 expression using we did not detect any new bands in the immunoprecipitates. siRNAs promoted cell growth, the EMT, and migration (Supple- These results indicate that the binding of p70S6K with SUR1 was a mentary Fig. S4), suggesting that KLF4 is a tumor suppressor, type of protein–protein interaction rather than chemical bond. which is consistent with other reports (20). Moreover, silencing To confirm this finding, we examined the physical binding of KLF4 expression partially reversed the glibenclamide-induced p70S6K and SUR1 by immunoprecipitation. First, we cotrans- inhibition of cell growth, the EMT, and migration (Fig. 4D–F). fected HEK293T cells with HA-tagged p70S6K and flag-tagged These findings suggest that KLF4 plays an important role in SUR1 constructs, and then pulled down one tag to immunoblot mediating the effects of glibenclamide. the other (Fig. 3A). The results showed that exogenous SUR1 and p70S6K bound to each other. Second, we overexpressed HA- SUR1 activates p70S6K to reduce KLF4 expression by enhancing tagged p70S6K in A549 cells and pulled down HA to immunoblot DNMT1-mediated methylation of the KLF4 promoter SUR1. Exogenous p70S6K bound to endogenous SUR1 in cancer We then examined the effect of SUR1 and p70S6K on KLF4 cells (Fig. 3B). Third, we overexpressed the C-terminal fragment of expression in the aforementioned protein samples (in Figs. 1 SUR1 (amino acid 1187–1581; SUR1 CT-flag) and HA-tagged and 3; Supplementary Fig. S2). First, we found that silencing SUR1 p70S6K, and then pulled down flag to immunoblot HA. The C- expression transiently or stably upregulated KLF4 protein levels, terminal truncate specifically bound to p70S6K (Fig. 3C). These but overexpression of SUR1 downregulated KLF4 protein levels results suggest that SUR1 interacted with p70S6K through its C- (Fig. 5A; Supplementary Fig. S2). Consistently, p70S6K knock- terminus in NSCLCs. down increased KLF4 expression, and p70S6K overexpression We then examined the effect of SUR1 on p70S6K activity, which decreased KLF4 expression (Fig. 5B). was indicated by T389 phosphorylation of p70S6K. In the afore- Next, we examined how KLF4 was regulated by p70S6K. DNA mentioned SUR1 transient or stable knockdown cells, we found methylation and histone acetylation have been reported to that phosphorylation of p70S6K (p-p70S6K T389) was decreased, inhibit KLF4 promoter activity in NSCLC. Using 5-Azacytidine and in SUR1-overexpressing cells p-p70S6K levels were increased and Trichostatin A to inhibit global DNA methylation and histone (Fig. 3D; Supplementary Fig. S2); these results occurred without acetylation, respectively, we found that KLF4 mRNA expression changes in total p70S6K levels, suggesting that SUR1 activated that had been downregulated by p70S6K was rescued by p70S6K. 5-Azacytidine (Fig. 5C), but not Trichostatin A (Supplementary We also detected the effect of glibenclamide on p70S6K activity. Fig. S7), suggesting that DNA methylation was involved in KLF4 Glibenclamide pretreatment dramatically decreased the levels of expression. The rescue effect of 5-Azacytidine was also confirmed p-p70S6K and its downstream target p-S6 after only 5 minutes on the protein levels of KLF4 in A549 cells (Fig. 5C). Moreover, a when cells were stimulated with serum for 30 minutes, suggesting dual-luciferase reporter assay showed that p70S6K decreased and that glibenclamide inhibited serum-induced p70S6K activation that 5-Azacytidine increased KLF4 promoter (from 1500 to (Fig. 3E). Moreover, p-p70S6K decreased in a dose-dependent 137) activity. In the combination group, 5-Azacytidine partially manner under glibenclamide treatment for 24 hours (Supple- reversed the effect of p70S6K on KLF4 promoter activity (Fig. 5D). mentary Fig. S3). Furthermore, bisulfite sequencing PCR (BSP) analysis showed

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A HEK293T B A549 IP-flag IP-HA Input IP: IgG HA Input SUR1-flag − + − ++− −−+ − + + p70S6K-HA −−++ −− + + − − + + p70S6K-HA +++ flag SUR1 HA HA GAPDH

C A549 D IP-flag Input A549 A549 SUR1 CT-flag − ++ − ++ shRNA Scramble SUR1SUR1 OE Vector SUR1 p70S6K-HA −−+ −−+ p-p70S6K p-p70S6K HA 1 0.05 1 3.40 flag p70S6K p70S6K GAPDH

E A549 H460 H157 H1299

Time (min) 0 5 15 30 60 0 5 15 30 60 0 5 15 30 60 0 5 15 30 60 p-p70S6K p70S6K p-S6 S6 β-Actin

FGA549 H460 A549 H460 120 100 100 100 120 80 80 100 80 60 60 80 60 40 40 60 40

40 Survival (%) 20 Survival (%) 20 Survival (%) 20

Survival (%) 20 0 0 0 −−+ + −−+ + −−++ 0 + Glibenclamide Glibenclamide Glibenclamide Glibenclamide −−+ siRNA Control p70S6K siRNA Control p70S6K Plasmid Vector p70S6K Plasmid Vector p70S6K siRNA Control p70S6K siRNA Control p70S6K Plasmid Vector p70S6K Plasmid Vector p70S6K p70S6K p70S6K p70S6K p70S6K β β β-Actin β-Actin -Actin -Actin

Figure 3. p70S6K plays a critical role in mediating the effects of SUR1 and glibenclamide in NSCLC cells. HEK293T cells were cotransfected with ﬂag-tagged SUR1 and HA- tagged p70S6K (A), A549 cells were transfected with HA-tagged p70S6K alone (B) or cotransfected with ﬂag-tagged C-terminal SUR1 (C) constructs for 48 hours as indicated. The cells were then subjected to immunoprecipitation (IP) and Western blotting using the indicated antibodies. D, Cells with SUR1 silencing or overexpression (the same samples as in Fig. 1) were subjected to Western blot analysis. The values under the blot bands are the results of quantitative analysis of the bands. E, Serum-starved NSCLC cells were treated with 100 mmol/L glibenclamide for different times as indicated, and cotreated with 10% serum for 30 minutes, and then subjected to Western blot analysis. The indicated cells were transfected with p70S6K plasmid (F) or siRNA (G), treated with 100 mmol/L glibenclamide, and then subjected to SRB assays and Western blotting. Bars, SD; , P < 0.05 versus control; #, P < 0.05 versus glibenclamide treatment. The results are representative of at least three independent experiments.

that p70S6K signiﬁcantly increased the methylation levels of several types of cancers (28). We found that silencing DNMT1 KLF4 promoter (Fig. 5E). Finally, we examined whether DNA- using siRNAs partially reversed the p70S6K-induced downregula- methyltransferase 1 (DNMT1) mediated the effect of p70S6K, tion of KLF4 (Fig. 5F). These ﬁndings suggest that SUR1 activated because DNMT1 has been reported to maintain the methylation p70S6K and that p70S6K downregulated KLF4 expression by of the KLF4 promoter and to suppress the expression of KLF4 in enhancing DNMT1-mediated methylation of the KLF4 promoter.

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A Replicate 1 2 3 Upregulated genes Downregulated genes expression treat 2 Upregulated genes Log 2 4 6 8 10 12 14 2 468101214 B Log2 expression control

5 Control 2 4 Glibenclamide

3 Figure 4. treat/control

Downregulated genes KLF4 mediated the effects of 2 2 glibenclamide. A, A549 cells treated 1 Log −2 with 50 mmol/L glibenclamide for 24 hours were subjected to 0 microarray analysis. A heatmap of Relative expression of KLF4 A549 H460 Calu-1 H157 differential gene expression (>1.5- fold change) is shown. Results of 120 A549 CDqRT-PCR analysis (B) and Western A549 H460 Calu-1 H157 100 blot analysis (C) of KLF4 expression Glibenclamide −+ −+ −+ −+ 80 in NSCLCs treated with 50 mmol/L 60 glibenclamide for 24 hours. Results of KLF4 40 SRB assays (D), Western blot assays

β-Actin Survival (%) 20 (E), and transwell assays (F) on cells with or without KLF4 transient 0 Glibenclamide −+− + knockdown and with or without 75 mmol/L glibenclamide treatment siRNA Control KLF4 for an additional 24 hours. Bars, SD; E A549 H460 P < P < 120 H460 , 0.05 versus control; #, 0.05 −− ++ −− ++ versus glibenclamide treatment. KLF4 siRNA 100 Glibenclamide − +−− + − ++ Scale bar, 30 mm; magniﬁcation, 80 100 . The results are representative KLF4 60 of three independent experiments. E-cadherin 40 Survival (%) N-cadherin 20 0 GAPDH Glibenclamide −+ − + siRNAControl KLF4

F siRNA Control KLF4 Glibenclamide − + − +

A549

H460

Glibenclamide treatment or SUR1 knockdown inhibits the inoculated into nude mice. The tumor growth of the SUR1 growth of A549 tumors in xenograft mouse models silencing group was slower than that of the scramble group; the We further conﬁrmed the effects of SUR1 in vivo. SUR1-silenced silencing group exhibited a signiﬁcantly lower tumor size (Fig. 6A; A549 cells (A549-SUR1_shRNA/A549-scramble_shRNA) were Supplementary Fig. S8A) and weight (Fig. 6B; P < 0.05). At the end

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Targeting SUR1 with Glibenclamide in NSCLC

A A549 A549 shRNA Scramble SUR1 SUR1 OE Vector SUR1 KLF4 KLF4 1 2.64 1 0.54 β-Actin β-Actin Figure 5. SUR1 and p70S6K downregulated B A549 H460 A549 H460 KLF4 expression by enhancing DNMT1-mediated methylation of the siRNA Control p70S6K Control p70S6K Plasmid Vector p70S6K Vector p70S6K KLF4 promoter. A, Western blot analysis for cells with SUR1 silencing KLF4 KLF4 or overexpression. The protein 1 2.37 1 16.85 1 0.52 1 0.18 lysates are the same as those GAPDH GAPDH in Fig. 1. B, Western blot analysis for cells with p70S6K silencing or C overexpression. The protein lysates 1.5 are the same as those in Fig. 3F and Plasmid Vector p70S6K G. The values under the blot bands 1.0 are the results of quantitative 5-azacytidine − +−+ analysis of the bands. C, A549 cells 0.5 were transfected with p70S6K p70S6K plasmids for 24 hours, treated with m 0.0 or without 5-azacytidine (4 mol/L) Relative expression of KLF4 KLF4 for another 24 hours, and then 5-azacytidine −−++ subjected to qRT-PCR assays and β-Actin Western blotting. D, Cells were Plasmid Vector p70S6K cotransfected with pGL3-KLF4- promoter plasmids (with the pGL3 D F vector as a control) and p70S6K 25 plasmids, treated with 5-azacytidine DNMT1 siRNA − − + + 20 4 mmol/L for another 24 hours as − + − + indicated, and then subjected to 15 p70S6K plasmid dual-luciferase reporter assays. 10 KLF4 E, BSP analysis of KLF4 promoter in A549 cells transfected with p70S6K 5 plasmids. A schematic illustration of Renilla luciferase signals DNMT1 Ratio of firefly luciferase 0

the selected region of KLF4 and − ++ ++ promoter and the methylation of KLF4-promoter p70S6K CpG sites. Filled circle, methylated; p70S6K plasmid − −−+ + β open circle, unmethylated. Each −−+ −+ -Actin column represents one CpG site, and 5-azacytidine each row represents one clone of bacteria. F, Western blot analysis of E TSS cells cotransfected with DNMT1 siRNA and p70S6K plasmids as Vector indicated. Bars, SD; , P < 0.05 versus control; #, P < 0.05 versus p70S6K −1,302 −1,041 +1 plasmid. The results are representative of three independent experiments. 100

50 p70S6K Percentage of methylation (%) 0 Plasmid Vector p70S6K

of the experiment, the tumors were collected and confirmed We further confirmed the growth-inhibiting effects of gliben- histologically by hematoxylin and eosin staining (Supplementary clamide in vivo. Nude mice were inoculated with A549 cells and Fig. S8B). Western blot analysis was used to examine p70S6K and treated with 200 mg/kg glibenclamide by oral gavage for 14 days. KLF4 signals as mentioned previously. We found that the levels of As expected, tumor size and weight were significantly lower in p-p70S6K were decreased and that KLF4 levels were increased, the glibenclamide-treated group than in the control group similar to what we observed in vitro (Fig. 6C). These results (P < 0.05; Fig. 6D and E; Supplementary Fig. S8A). Moreover, suggested that SUR1 upregulated p-p70S6K and downregulated glibenclamide treatment decreased p-p70S6K and increased KLF4 to inhibit the growth of A549 xenografts in the nude mouse KLF4 signals, consistent with the findings in vitro (Fig. 6F). model. Notably, the body weight of the mice showed no significant

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ABA549 1,000 0.8 ) A549 3 750 Scramble shRNA 0.6 SUR1 shRNA 500 0.4

0.2 250 Tumor weight (g) Tumor

Tumor size (mm Tumor 0.0 0 Scramble shRNA SUR1 shRNA 0 2 4 6 8 101214 Day C Scramble_shRNA SUR1_shRNA Figure 6. SUR1 silencing or glibenclamide SUR1 suppressed the growth of NSCLC tumors in nude mouse models. p-p70S6K Tumor sizes (A), tumor weights (B), and Western blot analysis results p70S6K (C) for nude mice injected with control cells or cells with KLF4 stable SUR1 knockdown (n ¼ 8). β-Actin Nude mice burdened with A549 (n ¼ 6 for each group) xenografts D E were treated with glibenclamide 0.6 A549 600 A549 (oral gavage 200 mg/kg/d) or its )

3 vehicle for 14 days. Tumor size (D), 500 Control 0.4 tumor weight (E), and protein Glibenclamide 400 expression (F) were examined. In 300 A and D: points, average tumor size; 0.2 bars, SD; , P < 0.05. In B and E: 200 points, individual tumor weight; Tumor weight (g) Tumor 100 0.0 horizontal line, mean tumor weight; Tumor size (mm Tumor 0 Control Glibenclamide bars, SD; , P < 0.05. 02468101214 Day F Control Glibenclamide

p-p70S6K

p70S6K KLF4

β-Actin

difference between the two groups, and no injury of livers was cell carcinoma cell lines) compared with the normal lung cell lines observed by pathologic examination of liver tissues, suggesting no HBE and Beas 2B. Fourth, gain-of-function and loss-of-function obvious toxicity of glibenclamide (Supplementary Fig. S8C and studies in cell lines revealed that SUR1 promoted growth in S8D). These results suggested that glibenclamide inhibited the NSCLC cells. Fifth, in a nude mouse model, knockdown of SUR1 growth of NSCLC through p70S6K-mediated KLF4 upregulation inhibited the growth of xenografts. Although 5 of 16 patients in vivo. presented decreases or no changes in SUR1 expression in cancer tissues, our results indicate that SUR1 may function as a tumor enhancer in some patients with lung cancer. Discussion SUR1 is the receptor of sulfonylurea antidiabetic drugs, such as SUR1 is well-known as the regulatory subunit of KATP channels, glibenclamide and gliclazide. We speculated that sulfonylureas which are composed of Kir6.2/SUR1 in pancreatic b cells. In this may target SUR1 to suppress lung cancer. Thus far, preclinical and study, we focused on investigating the SUR1 in lung cancer. First, clinical studies on sulfonylureas in cancer have been very limited. we found that SUR1 expression was higher in lung cancer tissues Yang and colleagues reported that in type II diabetes mellitus, the than in normal lung tissues based on the Oncomine database. use of glibenclamide and gliclazide was associated with reduced High expression levels of SUR1 were correlated with short patient cancer risk in a dose-dependent manner in a consecutive cohort survival times based on Kaplan–Meier plots for datasets from study (29). Studies in cell lines have shown that sulfonylureas website www.kmplot.com. Second, we observed increased SUR1 inhibit cell growth, induce cell-cycle arrest and apoptosis, and expression in cancer tissues of 11 of 16 patients with lung cancer. facilitate the efﬁcacy of other anticancer agents in gastric, breast, Third, SUR1 expression was also increased in NSCLC cell lines ovarian, and hepatoblastoma (8–11, 30). In this study, we found (including adenocarcinoma, squamous cell carcinoma, and large that glibenclamide targeted SUR1 to inhibit cell growth, cell-cycle

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Targeting SUR1 with Glibenclamide in NSCLC

progression, the EMT, and migration and inhibited xenografts in a KLF4 has been proven to be a tumor suppressor in lung nude mouse model. The dose of glibenclamide for diabetic cancer (16, 36). In this study, we found that KLF4 played an patients is not more than 15 mg/day, which equals approximately important role in glibenclamide-induced inhibition of cell 2 mg/kg/day for mice. The dose we used in the animal experi- growth, the EMT, and migration. We also verified that SUR1 and ments was 200 mg/kg/day, which was selected according to the p70S6K downregulated KLF4 expression. We then explored how dose of sulofenur, a diary sulfonylurea anticancer agent, used in KLF4 was downregulated by p70S6K. It has been reported that phase II clinical trials (31, 32). Although our dose was much increased DNA methylation of the KLF4 promoter is correlated higher than the clinical dose for diabetic patients, we did not with low KLF4 expression in chronic lymphocytic leukemia (37). observe any evidence of toxicity, for example, body weight loss, A histone deacetylase inhibitor has been reported to increase sickness, or liver injury in our experiments. These findings suggest KLF4 expression in lung cancer (36). In this study, we found that glibenclamide targeted SUR1 to inhibit NSCLC both in vitro that p70S6K-induced downregulation of KLF4 was reversed and in vivo. by 5-azacytidine, but not Trichostatin A, indicating that DNA To date, the mechanism of glibenclamide in cancer has not methylation was involved. Then, we showed that p70S6K- been elucidated. Qian and colleagues reported that in gastric induced inactivation of the KLF4 promoter (from 1500 to cancer, glibenclamide regulates the ROS–JNK pathway to induce 137 bp) was attenuated by 5-azacytidine treatment using a apoptosis (8). In breast cancer, glibenclamide inhibits cell growth luciferase assay. p70S6K significantly increased the methylation by inducing G0–G1 arrest (9). In ovarian cancer, it suppresses cell levels of KLF4 promoter by BSP analysis. Furthermore, DNMT1 invasion through inhibition of platelet-derived growth factor knockdown attenuated p70S6K-induced downregulation of KLF4 secretion (10). From the beginning of this study, we found that expression. These results suggested that p70S6K decreased KLF4 the expression and survival correlations for Kir6.1 and Kir6.2 in expression by enhancing DNMT1-mediated methylation of the the online database were not consistent with that for SUR1. KLF4 promoter. In addition, we searched for transcription factors Moreover, KATP channel openers, minoxidil and nicorandil, that possibly bound to the KLF4 promoter (1.0 kb upstream of did not promote cell growth but rather inhibited cell growth, the starting code) using Alibaba2.1 software, and found that SP1, indicating that the effect of glibenclamide may not depend on NF-B, C/EBPa, and NF-1 binding sequences were present. p70S6K KATP channels. Therefore, we focused our study on SUR1 but not has been reported to activate NF-B and regulate AP-1 transcription on KATP channels. Through gain-of-function and loss-of-function in breast cancer (38). Furthermore, KLF4 is also downregulated studies, we revealed that SUR1 phosphorylated and activated by some miRNAs, such as miR-7, miR-10, miR-25, and miR- p70S6K. In addition, glibenclamide inhibited p70S6K after as 3120-5p (39). Therefore, other mechanisms may exist as well. little as 5 minutes, the effect was sustained for at least 24 hours. How SUR1 interacted with p70S6K and downregulated KLF4 Overexpression of p70S6K almost completely attenuated transcription deserves further investigation. the growth-inhibiting effect of glibenclamide, indicating that In summary, this study revealed that SUR1 was overexpressed p70S6K plays an important role in mediating the effects of in lung cancer. SUR1 promoted NSCLC by interacting with glibenclamide. p70S6K, increasing its activity, and subsequently downregulating SUR1 is the regulatory subunit that directly binds around the KLF4 expression through a mechanism involving enhancement of pore protein Kir6.xs to form the KATP channel (33). Glibencla- DNMT1-mediated methylation of the KLF4 promoter. Gliben- mide directly binds to SUR1 and induces a conformational clamide targeted SUR1 to exert anticancer effects both in vitro and change in SUR1 (4). Currently, the existence and functions of in vivo. These findings suggest SUR1 as a new target for cancer SUR1 alone or as a component of complexes other than KATP therapy and suggest the potential anticancer effects of glibencla- channels are quite unknown. Seghers and colleagues reported the mide in NSCLC. existence of KATP channel–independent regulation of insulin secretion in a SUR1-knockout mouse model (34). Hambrock Disclosure of Potential Conflicts of Interest and colleagues observed that in HEK293 cells exogenous over- No potential conflicts of interest were disclosed. expression of SUR1, but not SUR2B or SUR1 mutant (M1289T), enhanced glibenclamide-induced apoptosis (35). These findings Authors' Contributions suggest that SUR1 may have other functions in addition to Conception and design: K. Xu, G. Sun, W. Huang, X. Wang regulation of KATP channels. In this study, we detected physical Development of methodology: K. Xu, G. Sun, M. Li, W. Huang, X. Wang binding of p70S6K and SUR1 through the C-terminus of SUR1. Acquisition of data (provided animals, acquired and managed patients, Although, no Kir6.1 or Kir6.2 was detected when we pulled down provided facilities, etc.): K. Xu, G. Sun, H. Chen, Z. Zhang, W. Huang, X. Wang Analysis and interpretation of data (e.g., statistical analysis, biostatistics, SUR1, whether by proteomics analysis or immunoblotting, we computational analysis): K. Xu, G. Sun, M. Li, H. Chen, Z. Zhang, X. Qian, P. Li, cannot exclude the function of SUR1 assembled in KATP channels L. Xu, W. Huang, X. Wang in lung cancer due to the preliminary nature of the data in this Writing, review, and/or revision of the manuscript: K. Xu, H. Chen, Z. Zhang, study. It is possible that some experimental conditions may miss W. Huang, X. Wang some protein interactions or fail to detect SUR1-Kir6.x complexes Administrative, technical, or material support (i.e., reporting or organizing at low abundance in lung cancer. We speculate that SUR1 alone, data, constructing databases): K. Xu, G. Sun, M. Li, H. Chen, Z. Zhang, X. Qian, – – P. Li, L. Xu, X. Wang SUR1 p70S6K complexes, SUR1 Kir6.x complexes, and Study supervision: K. Xu, H. Chen, Z. Zhang, P. Li, W. Huang, X. Wang unknown SUR1 complexes all exist in lung cancer and respond to different cell contexts. This possibility is interesting and Acknowledgments fi deserves further investigation. In this study, for the rst time we This work is supported by the National Natural Science Foundation of China found a new complex, SUR1/p70S6K, which may have new under grant no. 81473241, 81172004, and 81102458 to X. Wang; Key Labo- functions through regulation of the p70S6K signaling pathway ratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical in NSCLC cells. University, Nanjing, Jiangsu, China, 210029 (to X. Wang); and the "Six talent

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peaks project" in Jiangsu Province (to W. Huang). We thank Dr. John Blenis advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate (Harvard Medical School, Boston, MA) for providing us with the pRK7-S6K-HA this fact. plasmids and the control vector pRK7.

The costs of publication of this article were defrayed in part by the Received October 17, 2018; revised March 19, 2019; accepted July 17, 2019; payment of page charges. This article must therefore be hereby marked published ﬁrst July 24, 2019.

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Glibenclamide Targets Sulfonylurea Receptor 1 to Inhibit p70S6K Activity and Upregulate KLF4 Expression to Suppress Non--Small Cell Lung Carcinoma

Kexin Xu, Geng Sun, Min Li, et al.

Mol Cancer Ther Published OnlineFirst July 24, 2019.

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