Quercetin suppresses the metastatic ability of lung cancer through inhibiting Snail-dependent Akt activation and Snail-independent ADAM9 expression pathways

    Quercetin suppresses the metastatic ability of lung cancer through inhibiting Snail-dependent Akt activation and Snail-independent ADAM9 expression pathways Jer-Hwa Chang, Shu-Leung Lai, Wan-Shen Chen, Wen-Yueh Hung, Jyh-Ming Chow, Michael Hsiao, Wei-Jiunn Lee, Ming-Hsien Chien PII: DOI: Reference:

S0167-4889(17)30169-6 doi:10.1016/j.bbamcr.2017.06.017 BBAMCR 18130

To appear in:

BBA - Molecular Cell Research

Received date: Revised date: Accepted date:

16 February 2017 20 June 2017 21 June 2017

Please cite this article as: Jer-Hwa Chang, Shu-Leung Lai, Wan-Shen Chen, Wen-Yueh Hung, Jyh-Ming Chow, Michael Hsiao, Wei-Jiunn Lee, Ming-Hsien Chien, Quercetin suppresses the metastatic ability of lung cancer through inhibiting Snail-dependent Akt activation and Snail-independent ADAM9 expression pathways, BBA - Molecular Cell Research (2017), doi:10.1016/j.bbamcr.2017.06.017

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ACCEPTED MANUSCRIPT Quercetin suppresses the metastatic ability of lung cancer through inhibiting Snail-dependent Akt activation and Snail-independent

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ADAM9 expression pathways Jer-Hwa Chang1,2,#, Shu-Leung Lai3,#, Wan-Shen Chen2, Wen-Yueh Hung4, Jyh-Ming

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Chow5, Michael Hsiao6, Wei-Jiunn Lee7,8,* and Ming-Hsien Chien4,7,* School of Respiratory Therapy, College of Medicine, Taipei Medical University,

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Taipei, Taiwan; 2Division of Pulmonary Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan;

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Division of

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Pulmonary Medicine, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan; 4Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; 5Division of Hematology and

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Medical Oncology, Department of Internal Medicine, Wan Fang Hospital, Taipei

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Medical University, Taipei, Taiwan; 6Genomics Research Center, Academia Sinica, Taipei, Taiwan; 7Department of Medical Education and Research, Wan Fang Hospital,

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Taipei Medical University, Taipei, Taiwan; 8Department of Urology, School of Medicine, Taipei Medical University, Taipei, Taiwan.

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*Correspondence to: Ming-Hsien Chien, PhD, Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan; Phone: +886-2-27361661 ext. 3237; Fax: +886-2-27390500; Email: [email protected] or Wei-Jiunn Lee, PhD, Department of Medical Education and Research, Wan Fang Hospital, Taipei Medical University, 111 Hsing Long Road, Section 3, Taipei 116, Taiwan. Phone: +886-2-29307930 ext. 2551; Fax: +886-2-29302448; E-mail: [email protected] #

Jer-Hwa Chang and Shu-Leung Lai contributed equally to this work.

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ACCEPTED MANUSCRIPT Abstract Metastasis is the major cause of death from lung cancer. Quercetin, a widely

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distributed bioflavonoid, is well known to induce growth inhibition in a variety of

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human cancer cells, but how it affects lung cancer cell invasion and metastasis is

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unclear. Herein, we found that quercetin inhibited the migration/invasion of non-small cell lung cancer (NSCLC) cell lines and bone metastasis in an orthotopic A549 xenograft model by suppressing the Snail-mediated epithelial-to-mesenchymal

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transition (EMT). Moreover, survival times of animals were also prolonged after

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quercetin treatment. Mechanistic investigations found that quercetin suppressed Snaildependent Akt activation by upregulating maspin and Snail-independent a disintegrin and metalloproteinase (ADAM) 9 expression pathways to modulate the invasive

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ability of NSCLC cells. In clinical samples, we observed that patients with Snailhigh/pAkthigh tumors had the shortest survival times. In addition, a lower survival rate was

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also found in ADAM9high patients than in ADAM9low patients. Overall, our results provide new insights into the role of quercetin-induced molecular regulation in

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suppressing NSCLC metastasis and suggest that quercetin has potential therapeutic applications for metastatic NSCLC.

Keywords: Non-small cell lung cancer, Invasion, Snail, Akt, A disintegrin and metalloprotease 9, Quercetin

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ACCEPTED MANUSCRIPT Abbreviations Adenocarcinoma, ADC; A disintegrin and metalloprotease 9, ADAM9; epithelial-to-

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mesenchymal transition, EMT; extracellular matrix, ECM; immunohistochemical,

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IHC; matrix metalloproteinase, MMP; phosphatase and tensin homologue, PTEN;

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glutathione, GSH; non-small cell lung cancer, NSCLC; urokinase plasminogen

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activator, uPA.

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ACCEPTED MANUSCRIPT 1. Introduction Lung cancer is the leading cause of cancer-related deaths worldwide [1]. Non-

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small cell lung cancer (NSCLC) comprises 85% of lung cancers, and

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adenocarcinomas (ADCs) are the most common histotype [2]. The prognosis of patients with metastatic, or stage IV NSCLC is generally considered poor, with a

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median survival of 8~10 months and a 2-year survival of no more than 10%~20% [3].

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Chemotherapy is an important therapeutic strategy for cancer treatment. However, most patients eventually develop acquired resistance even after combination therapy,

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although they are initially sensitive to chemotherapy. Indeed, metastasis and

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therapeutic resistance are the major causes of failure of cancer treatment.

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The epithelial-to-mesenchymal transition (EMT) is an important event in promoting NSCLC metastasis, acquiring chemoresistance,

and

determining

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insensitivity to an epidermal growth factor receptor (EGFR) inhibitor [4-6]. Loss of epithelial markers and acquisition of mesenchymal markers are typical features of the EMT. Among these, loss of E-cadherin is considered a hallmark of EMT. A number of transcriptional factors have been identified as transcriptional repressors of Ecadherin during the EMT, such as Snail, Slug, ZEB, Twist, and FOXC2 [4]. Among these transcriptional factors, Snail (Snail1) and Slug (Snail2) are the most thoroughly investigated EMT regulators in lung cancer and were reported to be correlated with cancer invasion and resistance to targeted or chemotherapy [6-8]. Moreover, these 4

ACCEPTED MANUSCRIPT transcription factors were also reported to regulate matrix metalloproteinases (MMPs)

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in lung cancer [8]. MMPs are zinc-dependent proteases that play definitive roles in

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proteolytic degradation of extracellular matrix (ECM) components and aid in

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breaching the basement membrane, thus leading to tumor invasion and metastasis. Thus, seeking and investigating compounds with medicinal effects on the Snail

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family-mediated EMT should be a good strategy for NSCLC.

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Flavonoids have recently garnered extensive attention due to their interesting biological activities including their abilities to inhibit enzymes, their antioxidant

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properties, and their effects on immune responses [9]. These properties can explain

including

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the beneficial effects that flavonoid intake exerts on different human pathologies hypertension,

cardiovascular

diseases,

neurodegenerative

disease,

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inflammatory conditions, and cancer. Quercetin (3,3’,4’,5,7‐pentahydroxyflavone) is the most abundant bioflavonoid compound and is widely distributed in plants and foods such as apples, onions, and tea. Use of quercetin does not raise concerns regarding its toxicity due to quercetin having been used as a dietary supplement for many years [10]. The significant antioxidant and anti-inflammatory effects of quercetin have been extensively reviewed [11]. Recently, increasing numbers of studies have demonstrated that quercetin shows tumor growth inhibitory effects in a variety of in vitro and in vivo experimental models of neoplasia [12-14]. Although

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ACCEPTED MANUSCRIPT most research on quercetin focused on its growth‐inhibitory action through induction

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of apoptosis [12] or autophagy [14], enhancement of chemosensitivity [15], and

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modulation of cancer stemness [16], more recently, some work focused on the effects

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of quercetin on tumor cell invasion and metastasis. For example, quercetin was reported to suppress the motility of melanoma and breast cancer cells via inhibiting

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MMP‐9 expression and the EMT, respectively [17, 18].

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To the present, whether and how quercetin impacts the metastatic ability of NSCLC cells and the underlying mechanisms of how quercetin regulates cell motility

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are still unclear. In the present study, we investigated the antimetastatic effect of

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quercetin on two NSCLC cell lines (A549 and HCC827) and its underlying

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mechanisms in vitro and in an orthotopic xenograft model.

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ACCEPTED MANUSCRIPT 2. Materials and methods

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2.1. Materials

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Quercetin (Q4951), dimethyl sulfoxide (DMSO), and Ly294002 were purchased

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from Sigma-Aldrich (St. Louis, MO). Propidium iodide (PI) was purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS), antibiotics, molecular weight standards, trypsin-EDTA, trypan blue stain, and all medium additives were obtained

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from Life Technologies (Gaithersburg, MD). An antibody specific for E-cadherin was obtained from Abcam (Cambridge, MA). Antibodies specific for Snail, Slug, Twist,

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and unphosphorylated or phosphorylated (p-) forms of the corresponding Akt were obtained from Cell Signaling Technology (Danvers, MA). Antibodies specific for

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vimentin, N-cadherin, and maspin were purchased from BD Biosciences (San Jose,

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CA). Antibodies specific for ADAM9 and β-actin were obtained from Santa Cruz

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Biotechnology (Santa Cruz, CA). Unless otherwise specified, other chemicals used in this study were purchased from Sigma Chemical (St. Louis, MO).

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2.2. Cell culture

The NSCLC cell lines A549, H1975, and HCC827 were purchased from American Type Culture Collection (ATCC, Manassas, VA), and the CL1-3 NSCLC cell line was established in the National Health Research Institute laboratory and displayed progressively increasing invasiveness [19]. All cells were maintained in RPMI 1640 supplemented with 10% FBS and 1% penicillin‐streptomycin‐glutamine. All cells were incubated in a CO2 incubator containing 5% CO2 at 37 °C. 2.3. Cell viability detection by an MTS assay

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ACCEPTED MANUSCRIPT NSCLC cells (5×103) were plated in 96-well plates, treated with quercetin (1~100 μM) for 24 and 48 h, and then subjected to a cell-viability assay (MTS assay;

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Promega, Madison WI) according to the manufacturer’s instructions. Data were

immunosorbent

assay

(ELISA)

reader

(MQX200;

Bio-Tek

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enzyme-linked

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collected from three replicates. The absorbance (A) was read at 492 nm using an

Instruments, Winooski, VT). The cell viability rate (percentage) was determined with the formula: [A492, quercetin / A492, vehicle]  100 %.

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2.4. Cell vitality detection by thiol measurement

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The effect of quercetin on intracellular depletion of glutathione (GSH) levels, which represents the majority of intracellular free thiols in cells, was measured using

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VitaBright-48 dye (Chemometec A/S, Allerød, Denmark). After 24-h treatment of

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quercetin, NSCLC cells were detached by trypsinization, and the cell suspension was mixed with a vitaBright-48- and propidium iodide (PI)-containing dye. Stained cells

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were immediately loaded into NCSlide A8TM (Chemometec A/S). The fluorescence intensity (k excitation = 360 nm and k emission = 485 nm) was measured with a

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NucleoCounter NC-250TM (Chemometec A/S). 2.5. In vitro wound-closure assay A549 cells or HCC827 cells (5 × 105) were plated in 6-well plates for 24 h, wounded by scratching with a pipette tip, then incubated with RPMI 1640 complete medium, and treated with or without quercetin (10~50 μM) for 24 h. Finally, the cells were stained with crystal violet and photographed using a phase-contrast microscope (100×). 2.6. Transwell migration and invasion assays

Migration and invasion assays were performed as described previously [20]. Briefly, transwell migration assays used 3 × 104 cells plated in a noncoated top 8

ACCEPTED MANUSCRIPT chamber (24-well insert; pore size, 8 μm; Corning Costar, Corning, NY) and incubated for 24 h. The invasion assay used 5 × 104 cells plated in a Matrigel (BD

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Biosciences, Bedford, MA)-coated top chamber and incubated for 24 h. In both assays,

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cells which had been pretreated for 24 h with quercetin (10~50 μM) were plated in

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medium without serum or growth factors, and medium supplemented with serum was used as a chemoattractant in the lower chamber. After 24 h of incubation, cells that had not migrated or invaded through the pores were removed with a cotton swab.

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Cells on the lower surface of the membrane were fixed with methanol and stained

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with crystal violet. The number of cells migrating through or invading through the membrane was counted under a light microscope (×100, three random fields per well).

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2.7. Immunofluorescence microscopy

NSCLC cells grown on cover slips were treated with or without quercetin and

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fixed in 4% paraformaldehyde, permeabilized, and stained with Alexa Fluor 594 Phalloidin (Thermo Fisher Scientific) to observe actin rearrangement. Slides were

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examined and photographed using a Zeiss Axiophot fluorescence microscope (Carl Zeiss Microimaging, Gottingen, Germany). Nuclei were counterstained with 4’,6diamino-2-phenylindole (DAPI).

2.8. Preparation of total cell extracts and Western blot analysis

Protein lysates were prepared as described previously [13]. A Western blot analysis was performed with primary antibodies as indicated.

2.9. DNA transfection

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ACCEPTED MANUSCRIPT The myr-Akt and pLEX-Snail plasmids were respectively provided by Dr. C. C. Chen and Dr. T. C. Kuo (National Taiwan University). To overexpress Akt or Snail,

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semiconfluent cultures of A549 cells in a 6-mm2 Petri dish were transfected with 5 μg

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of an empty or expression vector (pcDNA3.1 or pLEX-MCS) using Invitrogen

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Lipofectamine 2000 Transfection Reagent. After incubation for 24 h, cells were analyzed for the expressions of p-Akt and Snail by immunoblotting to confirm the

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2.10. Lentiviral production and infection

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overexpressed status.

Short hairpin (sh)RNAs were purchased from the National RNAi Core Facility at

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Academic Sinica (Taipei, Taiwan). The target sequence of ADAM9 shRNA was 5’-

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GCCAGAATAACAAAGCCTATT-3. The lentiviral vector and its packaging vectors were transfected into 293T packaging cells by calcium phosphate transfection. Briefly,

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106 293T cells were transfected with 10 µg of a shRNA-expressing plasmid together with 10 µg of pCMVDR8.91 (the packaging vector) and 1 µg of pMD.G (the

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envelope vector). After 5 h of incubation, transfection medium was replaced with fresh culture medium. Forty-eight hours later, lentivirus-containing medium was collected from the transfection and spun down at 1500 rpm for 5 min to pelletize the cell debris, the supernatant was filtered through a 0.45-µm filter, and target cells were infected with fresh lentivirus-containing medium (supplemented with 8 µg/mL polybrene) for 24 h. 2.11. Human protease array The protease expression profile was analyzed in cells using the Proteome Profiler Human Protease Array (R&D Systems). Cell lysate samples (200 μg) were applied per array set comprised of two nitrocellulose membranes with the spotted capture antibodies. The bound material was detected using the biotinylated antibodies 10

ACCEPTED MANUSCRIPT followed by streptavidin conjugated with horseradish peroxidase followed by chemiluminescence detection (ECL-Plus; Santa Cruz Biotechnology). Pixel density of

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the spots was quantified using Image-Pro Plus software.

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2.12. Reverse-transcriptase polymerase chain reaction (RT-PCR)

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Messenger (m)RNA was isolated and amplified as described previously [21]. Primer sequences of maspin were F: 5’-GGCACAACAAAACTCGAAACAT-3’ and R: 5’-TACATATGACAAGTACTGGCGG-3’.

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2.13. In vivo antitumor activity by quercetin treatment

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All animal experiments were carried out in accordance with a protocol approved by guidelines of the Taipei Medical University Animal Ethics Research Board.

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Female SCID mice (6~8 weeks old) were used. For experimental metastasis assays,

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A549-luc/PLE or A549-luc/Snail cells (5 × 105) were pretreated with or without 50

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μM of quercetin for 24 h and then resuspended in 100 μl of PBS and injected into the lateral tail vein of female SCID mice. Lung metastatic progression was monitored and quantified using a noninvasive bioluminescence system (Xenogen IVIS-200 system).

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For orthotopic metastasis assays, 5 × 105 A549-luc/PLE or A549-luc/Snail cells were resuspended in a 1:1 mixture of phosphate-buffered saline (PBS) and GFR-Matrigel. This mixture was then injected into the left lateral thorax of each mouse. Seven days after the injection, mice were randomized into experimental and control groups according to bioluminescence images, such that treatment began with similar mean tumor sizes in each group. Treated animals received daily intraperitoneal (i.p.) injections of 500 mg/kg quercetin dissolved in DMSO. The injection volume was 200 μl (25 μl of a stock solution and 175 μl PBS) each time. The control group was given 200 μl of vehicle (25 μl DMSO and 175 μl PBS) only. The following day after

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ACCEPTED MANUSCRIPT quercetin treatment, we used this live imaging device to monitor the tumor size and location.

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2.14. Statistical analysis

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Data points represent the mean ± standard error of the mean (SEM). Data were analyzed using Student's t-test when two groups were compared. A one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was used to analyze three or

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more groups. Differences were considered significant at the 95% confidence interval

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(p < 0.05).

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ACCEPTED MANUSCRIPT 3. Results 3.1. Quercetin exhibits anti-migratory and anti-invasive activities against human

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NSCLC cells

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Recent reports indicated that long-term treatment (3 days) with quercetin can inhibit

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the growth of human NSCLC cell lines [12]. To further investigate the pharmacological potential of quercetin against NSCLC, we examined the effects of short-term treatment (24 h) with quercetin on cell migration and invasion of NSCLC

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cells. First of all, the cytotoxic effects of quercetin treatment for 24 or 48 h at various

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concentrations (0~100 μM) on four NSCLC cell lines (HCC827, CL1-3, H1975, and A549) are shown in Fig. 1A. Treatment with quercetin (0~50 μM) for 24 h did not

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alter the viability of any tested NSCLC cells, compared to that of the controls. The

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decrease in the intracellular glutathione (GSH) concentration was demonstrated in response to very different apoptotic stimuli, including quercetin-induced cell death in

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lung cancer [22]. The effect of quercetin on the intracellular content of GSH was assessed using the Vita-Bright-48 dye, a probe which forms a fluorescent compound

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when conjugated with free thiols such as GSH. Treatment of A549 or HCC827 cells with 50 μM quercetin for 24 h did not significantly change the VitaBright-48 fluorescence intensity in either cells (Fig. 1B). These findings indicated that 24-h treatment with this quercetin concentration range (0~50 μM) exhibited no cytotoxicity in human NSCLC cells, and all subsequent experiments used this quercetin concentration range. To further study the effect of quercetin on NSCLC cell migration and invasion abilities, we respectively performed wound-closure, transwell migration, and Matrigel invasion assays on two metastatic NSCLC cell lines, A549 and HCC827. After treating A549 or HCC827 cells with various concentrations of quercetin for 24 h, results showed that quercetin concentration-dependently suppressed the woundclosure, migratory, and invasive abilities at concentrations of 10~50 μM (Fig. 1C-E). 13

ACCEPTED MANUSCRIPT 3.2. Quercetin suppresses Snail family-mediated EMT progression in NSCLC To determine whether quercetin suppresses cell motility accompanied by acquisition

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of cell morphological changes. A549 and HCC 827 cells had a spindle- or fibroblast-

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like morphology prior to treatment with quercetin, but most cells exhibited a round or

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oval shape after treatment with quercetin for 24 h (Suppl. Fig. S1). The morphological transformation correlated with major changes in the actin cytoskeleton was revealed by phalloidin staining. A549 or HCC827 control cells displayed well-formed F-actin-

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containing microfilament bundles within the cytoplasm, whereas cells treated with

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quercetin contained few microfilament bundles (Fig. 2A, B), suggesting that F-actincontaining microfilament bundle rearrangements may be involved in quercetin-

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inhibited cell motility. In addition, the EMT plays a crucial role in promoting NSCLC

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metastasis [4]. The effects of quercetin on the EMT of NSCLC cells were examined by treating A549 or HCC827 cells with various concentrations of quercetin for 24 h

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and observing EMT-related proteins. Results from Western blotting (Fig. 2C, D) showed that treatment of NSCLC cells with quercetin induced the expression of the

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epithelial marker, E-cadherin, and suppressed expressions of the mesenchymal markers, N-cadherin and vimentin. The molecular signals involved in quercetinmediated suppression of the EMT were elucidated by investigating several EMTrelated repressive transcriptional factors (Snail, Slug, and Twist) which target Ecadherin [23]. Treatment with quercetin significantly decreased expressions of Snail family members (Snail and Slug) in A549 and HCC827 cells, but had less effect on Twist (Fig. 2C, D). These data suggest that downregulating Snail family members might be important for the quercetin-mediated inhibition of the EMT in NSCLC cells. 3.3. Snail plays a critical role in quercetin-mediated suppression of cell motility Next, to determine whether Snail modulates tumor cell migration and invasion, we overexpressed Snail in A549 cells (Fig. 3A). The migratory and invasive abilities 14

ACCEPTED MANUSCRIPT were enhanced in A549 cells transiently transfected with Snail compared to control A549 cells (A549/Neo) (Fig. 3C, D). Moreover, the quercetin-mediated suppression

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of Snail expression (Fig. 3A), EMT progression (Fig. 3B), and migratory/invasive

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abilities (Fig. 3C, D) in A549 cells were all significantly reversed. To validate the

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clinical significance of Snail in patients with lung cancer, a cohort of 426 lung cancer cases from the The Cancer Genome Atlas (TCGA) was analyzed. The resulting Kaplan-Meier plot showed that patients with high Snail expression had poor overall

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survival (p = 0.0008; Fig. 3E). These data suggest that downregulating Snail is crucial

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for the quercetin-induced suppression of the EMT and cell motility, and that high Snail levels predict a poor prognosis in patients with lung cancer.

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inhibition of cell motility

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3.4. Crosstalk between Akt activation and Snail expression in quercetin-mediated

Previous studies showed that the Akt signaling pathway plays an important role in the

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Snail-mediated EMT through increasing Snail stability [24]. Therefore, we determined whether Akt activation was affected in quercetin-treated A549 cells, and

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found that quercetin inhibited activation of Akt at indicated time points (10 min, 30 min, 6 h, and 24 h) (Fig. 4A). Next, the constitutively activated Akt, myr-Akt, was ectopically expressed in A549 cells to further investigate whether Akt plays an important role in quercetin-mediated suppression of cell motility. Data showed that overexpressing activated Akt significantly reversed quercetin-induced inhibition of invasion in A549 cells (Fig. 4B). Moreover, overexpressing activated Akt also increased the Snail expression level compared to control cells (Fig. 4C). Previous reports also indicated that Snail showed a transcriptional repressive effect on the tumor suppressor, maspin, which was reported to suppress lung cancer by targeting the Akt pathway [25, 26]. Our results showed that maspin mRNA and protein were upregulated time dependently after quercetin treatment in A549 cells (Fig. 4D, upper 15

ACCEPTED MANUSCRIPT panel and Suppl. Fig. S2). Moreover, compared to control cells, Snail overexpression in A549 cells decreased maspin expression, increased Akt activation, and significantly

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reversed the quercetin-induced upregulation of maspin and downregulation of p-Akt

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(Fig. 4D, lower panel; E). Taken together, these data suggest that the ability of

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quercetin to inhibit cell motility is attributable to its capacity to suppress Snail expression followed by upregulating maspin to inhibit Akt activation. We also found that Akt and Snail regulate each other to respectively maintain their activity and

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stability in A549 cells. From the same TCGA database described above, patients with

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lung tumors with Snailhigh/p-Akthigh had the shortest survival times compared to the Snailhigh/p-Aktlow, Snaillow/p-Akthigh, or Snaillow/p-Aktlow groups. The clinical data

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indicate that upregulations of Snail and p-Akt are critical events in promoting lung

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cancer progression.

3.5. Quercetin suppresses cell motility via inhibition of the Snail-independent ADAM9

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pathway

In addition to the Snail-mediated EMT, Snail was reported to upregulate several

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members of the MMP family [27, 28]. The human protease array data showed the effect of quercetin on 34 proteases and several proteases, including ADAM9, cathepsin C, cathepsin V, MMP-1, MMP-2, MMP-7, MMP-12, and the urokinase plasminogen activator (uPA), were considerably downregulated in A549 cells after quercetin treatment (Fig. 5A). Of these proteases, ADAM9 was one of the most downregulated enzymes after quercetin treatment and was reported to play a crucial role in promoting distal metastasis of lung cancer [29]. To confirm the expression of ADAM9, we performed a Western blot analysis and observed that AMAM9 expression was indeed downregulated in HCC827 and A549 cells after quercetin treatment (Fig. 5B). Next, to determine whether ADAM9 modulates tumor cell invasion, we performed knockdown of ADAM9 in A549 cells. Fig. 5C showed 16

ACCEPTED MANUSCRIPT knockdown of ADAM9 by an ADAM9-specific shRNA significantly decreased the cell invasive ability of A549 cells (Fig. 5C, lower panel), and the knockdown

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efficiency of ADAM9-specific shRNA was detected by a Western blot analysis (Fig.

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5C, upper panel). To further determine whether Snail is involved in quercetin-

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mediated downregulation of ADAM9, Western blot data showed that overexpressing Snail did not affect ADAM9 expression or reverse the quercetin-mediated inhibition of ADAM9 (Fig. 5D). Moreover, data from an RT-PCR (Fig. 5E, upper panel) and

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qPCR (Fig. 5E, lower panel) all showed that quercetin did not change the ADAM9

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mRNA level in A549 cells, indicating that Snail was not involved in the quercetinmediated posttranscriptional regulation of ADAM9 expression. Furthermore, we

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analyzed the clinical relevance of ADAM9 in lung cancer patients using gene

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expression data obtained from the publicly available Gene Expression Omnibus database (GSE13213), which revealed that ADAM9 expression was inversely

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correlated with overall survival of lung cancer patients (Fig.5F). 3.6. Significant antimetastatic effects of quercetin in an A549 orthotopic graft model

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To investigate the in vivo effects of Snail expression on tumor metastasis and the in vivo antimetastatic activity of quercetin, we established an orthotopic lung tumorbearing model by transplanting luciferase-tagged cells, A549-luc or A549-luc/Snail, into SCID mice. A549-luc or A549-luc/Snail orthotopic graft mice were treated with quercetin or the vehicle control every day by IP administration, and tumor growth and metastasis were monitored by bioluminescence imaging. A schematic timeline of this experimental design and setup was shown in Suppl. Fig. S3. Five weeks after inoculation with cancer cells, we found the distal metastatic ability (red arrow indicated) had increased in Snail-overexpressed A549 cells compared to control A549 cells (Fig. 6A). After the mice were sacrificed at the end of the experiment, ex vivo imaging of the lungs of mice showed a lower photon intensity in quercetin-treated 17

ACCEPTED MANUSCRIPT mice compared to vehicle-treated mice, and the growth inhibitory effect of quercetin was reversed in cancer cells overexpressing Snail (Fig. 6B). Moreover, from the ex

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vivo imaging of the bones, we also observed that quercetin treatment significantly

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abated bone metastasis from lung cancer, and this phenomenon was reversed in

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cancer cells overexpressing Snail (Fig. 6C). Most importantly, a parallel study in a different animal cohort was also carried out and showed that mice with A549-luc tumors and quercetin treatment had significantly longer survival times compared to

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mice with tumors and vehicle treatment (Fig. 6D). To rule out the possibility that the

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antimetastatic effect of quercetin in the orthotopic metastatic mouse model was because of the quercetin-mediated growth inhibition of primary tumors, we further investigated the antimetastatic effect of quercetin in an experimental metastasis model

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using a bioluminescence system. A549 cells were pretreated with 50 μM quercetin or the vehicle for 24 h and then injected into the lateral tail vein of SCID mice. Five

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weeks postinjection, quercetin-treated cells exhibited lower lung colony formation, as revealed by a bioluminescence imaging analysis (Fig. 6E). Taken together, these

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results revealed that quercetin treatment suppressed in vitro invasion and in vivo bone metastasis of NSCLC.

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ACCEPTED MANUSCRIPT 4. Discussion Cancer metastasis and resistance to treatment (including radiotherapy, chemotherapy,

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and targeted therapy) are two major causes for the poor survival of lung cancer

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patients. The EMT is involved in cancer cell invasion, resistance to apoptosis, and

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stem cell features [7]. E-cadherin acts as a tumor suppressor inhibiting invasion and metastasis, and it is frequently repressed during tumor progression. Previous reports suggested that E-cadherin expression is downregulated in several cancer types such as

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breast cancer [30] and NSCLC [31]. In recent years, the polyphenolic flavonoid

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compound, quercetin, has attracted a great deal of attention due to its wide-ranging biological activities and low toxicity [32]. Among these activities, the effect of

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quercetin on carcinogenesis is a highly promising research area. It was demonstrated

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that quercetin induces apoptosis [12] or autophagy [14] of cancer cells, enhances chemosensitivity [15], and modulates cancer stemness [16]. However, no information

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on the effects of quercetin on the migration and invasiveness of NSCLC cells is available. Our present results exhibited that quercetin inhibited the migratory and

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invasive abilities of NSCLC cells. Moreover, quercetin treatment upregulated the epithelial maker, E-cadherin, and downregulated the mesenchymal markers, Ncadherin and vimentin, in NSCLC cells. These results imply reduced migratory and invasive properties upon quercetin treatment, which is likely due to modulation of EMT-related proteins. Consistent results from a previous study also showed that quercetin inhibits metastatic ability of colorectal cancer through suppressing EMT of cancer cells [33]. The bone is a common site of distant metastasis in cases of lung cancer. Our in vivo studies showed that quercetin treatment attenuated bone metastasis and prolonged survival in the A549 orthotopic graft model, suggesting that quercetin has potential value in clinical applications for NSCLC.

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ACCEPTED MANUSCRIPT Transcription factors involved in the EMT such as Snail, Slug, Twist, and the ZEB family mainly repress expression of E-cadherin during the EMT [34]. Our results

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showed that protein levels of Snail and Slug exhibited rapid decreases after quercetin

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treatment of A549 cells. However, the protein level of Twist only slightly decreased

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after quercetin treatment. Recent studies indicated that E-cadherin expression was negatively regulated by Snail-mediated bone metastasis of human lung cancer [35]. Actually, our study also demonstrated that NSCLC cells overexpressing Snail

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promoted the bone metastatic ability of cancer cells and reversed the quercetin-

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mediated inhibition of bone metastasis in the A549 orthotopic graft model and invasive ability of A549 cells, indicating that Snail is a critical target of quercetin in

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preventing the metastatic ability of lung cancer cells. In addition, the Snail-mediated

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metastasis of lung cancer was reported to be induced by Wnt/β-catenin signaling, and the effect of quercetin on Wnt/β-catenin signaling should be further investigated in

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the future. In addition to Wnt/β-catenin signaling, Akt/GSK-3β signaling is another key pathway regulating Snail expression in cancers [36]. The present study

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demonstrated that treatment of A549 cells with quercetin suppressed activation of Akt. Moreover, overexpressing activated Akt in A549 cells induced upregulation of Snail and reversed quercetin-mediated inhibition of the invasive ability. Based on these results, we suggest that Akt inhibition by quercetin may be one of the causes of the suppression of Snail-induced cell motility. In contrast to Akt-regulated Snail expression, Snail was also reported to modulate Akt activation in several cancer types including NSCLC [37]. Mechanistic investigations showed that Snail induces Akt activation via transcriptional repression of several tumor suppressors which target Akt such as phosphatase and tensin homologue (PTEN) [38] and maspin [25, 26]. Previous studies identified that maspin suppresses tumor growth and metastasis in vivo, inhibits basement membrane 20

ACCEPTED MANUSCRIPT invasion in vitro [38], and is functionally linked to apoptosis of human prostate and lung cancer cells by targeting Akt [26, 39]. Moreover, maspin was reported to exert a

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potent inhibitory effect on osteolysis occurring in prostate cancer bone metastasis and

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to enhance the chemosensitivity (to doxorubicin and etoposide) in lung cancer [26,

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40]. Maspin expression is transactivated by p63 and was reported to be correlated with a lower tumor stage and less lymph node involvement in lung cancer [41]. Importantly, our current study showed that quercetin significantly induced

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upregulation of maspin in A549 cells. Moreover, Snail overexpression in A549 cells

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decreased maspin expression, increased Akt activation, and significantly reversed the quercetin-induced upregulation of maspin and downregulation of p-Akt. Taken

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together, these data suggest that the ability of quercetin to inhibit cell motility might

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be attributable to its capacity to suppress Snail expression followed by upregulating maspin to inhibit Akt activation. In the future, we will investigate the effects of

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quercetin on PTEN and p63 expressions and osteolysis to further elucidate the underlying mechanisms of quercetin on p-Akt inhibition, maspin induction, and bone suppression

in

NSCLC.

Moreover,

combination

therapy

of

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metastasis

chemotherapeutic drugs with quercetin for lung cancer warrants further study in our future work.

In addition to the EMT regulated by Snail, Snail was also reported to play another key role in regulating cell migration and invasion by affecting the so-called MMP family, such as MMP-1, MMP-2, MMP-7, MMP-9, and MMP-14, in ovarian and liver cancers [27, 28]. Another previous study also indicated that overexpression of Snail increases the expression of MMP-9 through the PI3K/Akt signaling pathway in MDCK cells [42]. Our protease array results showed that treatment of A549 cells with quercetin significantly suppressed ADAM9, cathepsin C, cathepsin V, MMP-1, MMP-2, MMP-7, MMP-12, and uPA, and we actually found that overexpressing 21

ACCEPTED MANUSCRIPT Snail in A549 cells induced upregulation of MMP-2 and MMP-7 mRNA (Suppl. Fig. S4). These results suggested that quercetin's suppression of MMP-2, and MMP-7

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might be through inhibiting Snail expression and this issue should be further

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confirmed in our future work. In addition to MMPs, we found that ADAM9 is one of

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the most downregulated enzymes after quercetin treatment, and it is involved in modulating the invasive ability of NSCLC cells. Actually, ADAM9 was reported to play a crucial role in promoting distal metastasis of lung cancer by facilitating the

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tissue plasminogen activator (tPA)-mediated cleavage of the promigratory protein,

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CDCP1 [29]. Our present study first determined whether Snail is involved in regulating of ADAM9, and found that ADAM9 expression is Snail independent in cells,

and

quercetin

suppressed

ADAM9

expression

through

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NSCLC

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posttranscriptional regulation. The effect of quercetin on tPA-mediated cleavage of CDCP1 in NSCLC cells will be further investigated. In addition, ADAM9 knockdown

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was also reported to induce epithelial phenotypic alterations through upregulating Ecadherin and sensitizing prostate cancer cells to radiation and chemotherapy [43]. We

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suggested that ADAM9 suppression by quercetin might contribute to the quercetinmediated inhibition of the EMT in NSCLC cells and warrants further study in our future work.

5. Conclusions We report here that NSCLC cells were subjected to a strategy of using quercetin to suppress migration and invasion of cells. Our data supported the conclusion of EMT suppression in NSCLC cells during quercetin treatment, which inhibits Snaildependent Akt activation by upregulating maspin and Snail-independent ADAM9 expressions; the mechanism is schematically illustrated in Fig. 7. Most importantly, our study also demonstrated that quercetin can significantly inhibit tumor bone metastasis and prolong the lifespan in a human A549 xenograft model. Our present 22

ACCEPTED MANUSCRIPT findings strongly support the development of clinical trials to determine whether quercetin or quercetin combined with other chemotherapeutic drug regimens would be

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useful in managing human NSCLC.

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Acknowledgments

This work was supported by a grant from the Ministry of Science and Technology of Taiwan, MOST 105-2320-B-038-058-MY3 (to M.-H. Chien). This study was also

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supported by grant 105TMU-TMUH-08 from Taipei Medical University Hospital (to

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S.-L. Lai).

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Conflict of interest statement

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The authors declare that no conflict of interest exists.

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ACCEPTED MANUSCRIPT Figure legends Fig. 1. Quercetin at non-cytotoxic concentrations inhibits the wound closure, cell

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migration, and invasive abilities of human non-small cell lung cancer (NSCLC) cells.

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(A) Four NSCLC cell lines were treated with the indicated concentrations of quercetin for 24 or 48 h, and cell viability was determined using a MTS assay. (B) Two NSCLC cell lines were incubated for 24 h with vehicle or with 50 μM quercetin, and cellular

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glutathione was quantified by measurement of VitaBright-48 fluorescence. (C) Effect of quercetin on wound closure in NSCLC cells. Two NSCLC cell lines were wounded

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and then treated with vehicle or quercetin (10~50 μM) for 24 h, and phase-contrast pictures of the wounds were taken (left panel). Cells migrating into the wound area

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were counted using the dashed line as time zero (right panel). (D, E) Two NSCLC cell

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lines were treated by the indicated concentrations of quercetin for the transwell

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migration and Matrigel invasion assays. The results are expressed as percentages of the control. Data are expressed as the mean ± SEM of at three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control

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group. NS, not statistically different versus the control.

Fig. 2. Effect of quercetin on cellular morphology and epithelial-to-mesenchymal transition (EMT)-related regulators in non-small cell lung cancer (NSCLC) cells. (A, B) Cellular morphology was altered in NSCLC cells treated with quercetin. Cells were treated with 50 μM of quercetin or its control for 24 h. Cells were fixed and stained for F-actin by Alexa Fluor 594 Phalloidin (red). Nuclei were counterstained with DAPI (blue). Original magnification, 400. Scare bar, 50 μm (C, D) An epithelial marker, E-cadherin, and mesenchymal markers, N-cadherin and vimentin, 30

ACCEPTED MANUSCRIPT and EMT-related transcriptional factors, Snail, Slug, and Twist, were expressed in quercetin-treated NSCLC cells. Lysates were collected from cells cultured with or

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without different concentrations of quercetin (0~50 μM) for 24 h and subjected to a

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Western blot analysis. The extents of EMT-related proteins and β-actin were

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determined using a densitometer with Image-Pro Plus image analysis software. Quantitative results of EMT-related protein levels, which were adjusted to the β-actin

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protein level.

Fig. 3. Snail plays a critical role in quercetin-mediated suppression of cell motility in

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A549 non-small cell lung cancer (NSCLC) cells. (A, B) Western blot analysis of Snail

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(A), E-cadherin, and vimentin expressions (B) in A549 cells which were transiently transfected with a vector control (A549/Neo) or pLEX-Snail (A549/Snail) followed

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by quercetin or vehicle treatment for an additional 24 h. Quantitative results of epithelial-to-mesenchymal transition (EMT)-related protein levels, which were

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adjusted to the β-actin protein level. (C, D) Migratory and Invasive abilities of A549/Snail or A549/Neo which was treated with the vehicle or 50 μM quercetin. Multiples of differences are presented as the mean ± SEM of three independent experiments. Data were analyzed using a one-way ANOVA with Tukey’s post-hoc tests at 95% confidence intervals; different letters represent different levels of significance. (E) Kaplan-Meier curves for overall lung cancer patient survival, grouped by high and low Snail expression. The p value indicates a comparison between patients with Snailhigh and Snaillow. The lung cancer dataset was retrieved from TCGA.

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ACCEPTED MANUSCRIPT Fig. 4. Crosstalk between Akt activation and Snail expression in quercetin-mediated inhibition of A549 cell motility. (A) Phosphorylation levels of Akt were assessed by a

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Western blot analysis after treatment with quercetin (50 µM) for 10 min, 30 min, 6 h,

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and 24 h. Quantitative results of p-Akt protein levels were adjusted to total Akt

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protein levels. Values are presented as the mean ± SEM of three independent experiments. * p < 0.05, compared to the vehicle groups. (B, C) Invasive ability (B) and Western blot analysis of p-Akt and Snail expressions (C) in A549 cells which

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were transiently transfected with a vector control (A549/Neo) or myr-Akt (A549/myr-

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Akt) followed by quercetin or vehicle treatment for an additional 24 h. Multiples of differences are presented as the mean ± SEM of three independent experiments. Data

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were analyzed using a one-way ANOVA with Tukey’s post-hoc tests at 95%

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confidence intervals; different letters represent different levels of significance. (D, upper panel) A549 cells were treated with quercetin (50 μM) for the indicated time

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points and then subjected to an RT-PCR to analyze maspin mRNA expression. (D, lower panel, E) Western blot analysis of maspin (D, lower panel) expression and Akt

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phosphorylation (E) in A549 cells which were transiently transfected with a vector control (A549/Neo) or pLEX-Snail (A549/Snail) followed by quercetin or vehicle treatment for an additional 24 h. Quantitative results of maspin and p-Akt were respectively adjusted to the β-actin and total Akt protein levels. Multiples of differences are presented as the mean ± SEM of three independent experiments. Data were analyzed using a one-way ANOVA with Tukey’s post-hoc tests at 95% confidence intervals; different letters represent different levels of significance. (F) Combined expressions of high p-Akt and high Snail proteins were correlated with the worst overall survival of patients with lung cancer. The p value refers to a comparison between Snailhigh/p-Akthigh and other groups (Snailhigh/p-Aktlow, Snaillow/p-Akthigh, or Snaillow/p-Aktlow). 32

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Fig. 5. Quercetin suppresses cell motility via inhibition of the Snail-independent a

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disintegrin and metalloprotease (ADAM) 9 pathway. (A) Changes in expressions of

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proteases in A549 cell lysates following 24-h treatment with quercetin. Protein

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expressions were determined using an antibody array (R&D Systems) containing 34 different antibodies against proteases. The representative array blots are shown in the left panel; n = 2 trials for the experiment. Quantitative analysis of the protease array

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using a densitometer is shown in the right panel. Values are presented as the mean ±

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SEM * p < 0.05, compared to the control group. (B) HCC827 (left panel) and A549 (right panel) cells were treated with the vehicle or quercetin (50 μM) for 24 h, and a Western blot analysis was performed. Quantitative ADAM9 levels were adjusted to

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the β-actin levels. (C) A549 cells were infected with a lentivirus carrying ADAM9 shRNA or shGFP and subjected to invasion assays. Upper panel, Western blot

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analysis of ADAM9 expression. Lower panel, invasive abilities of cells expressing ADAM9 shRNA or shGFP. Multiples of differences are presented as the mean ± SEM

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of three independent experiments. * p < 0.05, compared to the control group. (D) Western blot analysis of Snail and ADAM9 protein expressions in Snailoverexpressing A549 cells. Quantitative Snail and ADAM9 levels were adjusted to βactin levels. (E) A549 cells were treated with quercetin (50 μM) for 24 h and then subjected to an RT-PCR (upper panel) or qPCR (lower panel) to analyze ADAM9 mRNA expression. (F) Kaplan-Meier analysis of ADAM9 gene expression in lung cancer tissues (GSE13213).

Fig. 6. Quercetin attenuates the snail-mediated metastatic ability in an A549 orthotopic graft model. Luciferase-tagged A549 cells were injected into the left lateral thorax of SCID mice. Seven days after tumor cell injection, mice were treated with 33

ACCEPTED MANUSCRIPT vehicle or quercetin by an i.p. injection every day for 4 consecutive weeks. The tumor size and location were monitored by bioluminescence imaging every week. (A)

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Xenogen IVIS spectrum bioluminescence images of orthotopic lung tumor growth

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and location of a metastatic tumor (red arrow) are shown. A quantitative analysis of

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Xenogen imaging signal intensity from lung tissues at the end of the study is shown in (B). (C) Bone metastasis was bioluminescently imaged at the end of the study (upper panel) with the mean signal for each group indicated (lower panel). p values from

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different compared groups are shown. (D) An overall survival curve was produced for

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tumor-bearing mice after treatment without or with quercetin using the Kaplan-Meier method. The p values were determined using a log-rank test. (E) Vehicle-treated

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A549-luc and quercetin-treated A549-luc cells were injected into the tail vein of SCID

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mice, and lung colonization was bioluminescently imaged as shown in the upper panel, with the mean signal for each group indicated (lower panel). * p < 0.05 and **

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p < 0.01 vs. the vehicle control group.

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Fig. 7. A working model shows the molecular mechanism underlying the ability of quercetin to suppress the metastasis of non-small cell lung cancer (NSCLC) cells. The anti-metastatic activity of quercetin on NSCLC cells was attributed to inhibition of Snail-dependent Akt activation via upregulating the maspin and Snail-independent ADAM9 expression pathways. Moreover, Akt activation also shows the positive feedback regulation on Snail expression in NSCLC cells. Bold solid lines indicate pathways regulated by quercetin. Bold dashed lines indicate hypothetical pathways which might be regulated by quercetin.

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ACCEPTED MANUSCRIPT

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Highlights  Quercetin inhibits the migration/invasion of NSCLC cells through suppressing the EMT.  Quercetin attenuates bone metastasis and prolongs lifespan in an in vivo model.  Quercetin suppresses snail-dependent Akt activation by upregulating maspin.  Quercetin suppresses snail-independent ADAM9 expression.  Snail or ADAM9 expression is inversely correlated with survival of patients.

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