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Cancer Biology and Signal Transduction

miR141–CXCL1–CXCR2 Signaling–Induced TregRecruitment Regulates Metastases and Survival ofNon–Small Cell Lung Cancer

Mingming Lv1,2, Yujun Xu1, Ruijing Tang1, Jing Ren1, Sunan Shen1,3, Yueqiu Chen1, Baorui Liu4,Yayi Hou1,3, and Tingting Wang1,3

AbstractPatients with non–small cell lung cancer (NSCLC) with malignant pleural effusion (MPE) have a short

median survival time and increased regulatory T cells (Treg). However, it is unclear whether some specific

factors in MPE are involved in Treg recruitment in the progression of NSCLC. Here, we found that Treg

population was increased in MPE and inversely correlated with patient survival (P < 0.001). Increased level

of CXCL1 in MPE was associated with recruitment of Tregs (P < 0.01). Moreover, miR141 regulated

expression of CXCL1 in lung cancer cells, whereas the luciferase test confirmed that CXCL1 is a target of

miR141. Chemotaxis assay showed that the miR141–CXCL1–CXCR2 pathway regulates migration of Tregs

into MPE. Furthermore, miR141 significantly inhibited tumor growth and metastasis in an immune-

competent mouse model. This suppressive function was mediated by the CXCL1–CXCR2 pathway and

recruitment of Tregs. Our study uncovered a causative link between microRNA and development of MPE.

Mechanistically, decreased expressions of miR141, associated with the survival of patients with NSCLC

with MPE, resulted in the increased production of CXCL1 and recruitment of Tregs to promote immune

escape of tumor. Mol Cancer Ther; 13(12); 3152–62. �2014 AACR.

IntroductionNon–small cell lung cancer (NSCLC) is the leading

cause of cancer-related death worldwide. Approximatelyhalf of patients with lung cancer developmalignant pleu-ral effusion (MPE) at some point in their life, especially inthe advanced stage of the disease (1). Currently there is nocure for MPEs and clinical treatments only palliate thesymptoms (2).Moreover, patients withNSCLCwithMPEhave a short median survival time, suggesting that pro-liferation andmetastasis of tumor cells may often occur inMPE (3). MPE is believed as a special tumor microenvi-ronment to favor the development of NSCLC, but lessinformation is available on the immune surveillance

mechanisms of MPE in patients with NSCLCwith a shortmedian survival time.

Regulatory T cells (Treg) normally function as a dom-inant inhibitory component in the immune system toactivelymaintain self-tolerance and immune homeostasisthrough suppression of various immune responses.With-in the tumor microenvironment, Tregs have been dem-onstrated to be coopted by tumor cells to escape immunesurveillance (4). The increased accumulation of Tregs intumor is associatedwith unfavorable prognosis in severalkinds of cancers (5). Foxp3 is the primary transcriptionfactor of Tregs. Foxp3þ Tregs were found in small-celllung cancer biopsies and patients with higher ratios ofFoxp3þ cells in tumor infiltrates had aworse survival rate(6). Patients with NSCLC also had an increased percent-age of Tregs than controls. The proportion of Tregsincreased in an advanced stage of NSCLC (7). Moreover,pleural fluid from benign disease containedmainly CD8þ

T cells, whereas MPEs included mainly CD4þ T cells (8).Therefore, it is reasonable to assume that Tregs could beupregulated by tumor cells in MPE and eventually affectpatient survival. It was reported that the concentration ofchemokine CCL22 in MPEs was significantly higher thanthat in the corresponding serum. CCL22 was associatedwith increased frequency of Tregs in tumor-infiltratinglymphocytes in gastric cancer (9). Interestingly, intra-pleural administration of CCL22 of patients produced amarkedprogressive influx of Tregs into pleural space (10).These data suggest that chemokine could induce Tregs

1The State Key Laboratory of Pharmaceutical Biotechnology, Division ofImmunology, Medical School, Nanjing University, Nanjing, China. 2Depart-ment of Breast Surgery, Nanjing Maternal and Child Health Care HospitalAffiliated to Nanjing Medical University, Nanjing, China. 3Jiangsu KeyLaboratory of Molecular Medicine, Nanjing, China. 4Department of Oncol-ogy,DrumTowerHospital Affiliated toMedical School ofNanjingUniversityand Clinical Cancer Institute of Nanjing University, Nanjing, China.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Authors: Tingting Wang, The State Key Laboratory ofPharmaceutical Biotechnology, Division of Immunology, Medical School,Nanjing University, 22 Hankou Road, Nanjing 210093, China. Phone: 86-25-8368-6043; Fax: 86-25-8368-6043; E-mail: [email protected]; andYayi Hou, [email protected].

doi: 10.1158/1535-7163.MCT-14-0448

�2014 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 13(12) December 20143152

on March 26, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst October 27, 2014; DOI: 10.1158/1535-7163.MCT-14-0448

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migration to the pleural space. However, whether tumorcells could induce migration of Tregs into MPE throughchemokine is still unknown.MicroRNAs (miRNA) are small noncoding RNAs, 18 to

25 nucleotides, that posttranscriptionally regulate geneexpression. miRNAs are involved in the regulation of lifeprocesses, including cell proliferation, apoptosis, anddifferentiation (11, 12). It was reported that changes inthe expression profiles ofmiRNAs have been linked to thedevelopment of various types of human cancers, includ-ing NSCLC (13). Let-7 and pre-miR155 could be selectedas the prognosticmarkers in lung cancer (14). In surgicallyresected NSCLC, miR34a was related to the relapse ofpatients (15). Our previous study has found that fivemiRNAs are systematically altered in MPE of patientswith NSCLC (16), which suggest that miRNA could par-ticipate in the metastasis from primary lung cancers topleural.However,whether some specificmiRNAs inMPEare involved in Treg recruitment in the progression ofNSCLC is still unclear.In the present study, we uncovered a causative link

between miRNA and development of MPE. Mechanisti-cally, the decreased expressions of miR141, associatedwith the survival of patients with NSCLC with MPE,resulted in the increased production of CXCL1, whichrecruits Tregs to promote immune escape of tumorthrough CXCR2.

Materials and MethodsPatients and sample collectionPatients (268) with NSCLC who received treatment in

Drum Tower Hospital Affiliated to Medical School ofNanjing University and Nanjing Chest Hospital in Chinabetween January 2007 and December 2012. As shown inSupplementary Table S1, among 268 patients, 72 patientswithout MPE were diagnosed with NSCLC II–III stagesand received surgical treatment. Tumor and peritumoraltissues were acquired after operation. About 196 patientswith MPE were diagnosed with NSCLC stage IV andcannot receive operation. For these patients, 50 mL effu-sionwas acquired using thoracocentesis. Supernatant andcells were separated and collected from MPE. Blood wascollected from each patient and healthy control (n ¼ 30).Tuberculous pleural effusions (n¼ 64) were also collectedas control. All research involving human participantshave been approved by the "Drum Tower Hospital ethicscommittee" and written informed consent was obtainedfrom all subjects.

Immunohistochemistry analysisThe tissue specimens were harvested, fixed in 10%

buffered formalin, dehydrated, bisected, mounted inparaffin, and sectioned. Immunohistochemical stainingwas carried out with monoclonal antibodies againsthuman CD4 (1:50; DAKO, GeneTech Lid), humanFOXP3 (1:100; BioLegend), and mouse FOXP3 (1:200;Abcam).

Flow cytometryThe following murine and human antibodies were

used: anti–hCD4-FITC, anti–hCD25-APC, anti–hFOXP3-PE, anti–mCD4-FITC, anti–mCD25-APC, and anti–mFOXP3-PE (eBiosciences), anti–hCXCR2-APC (BDPharMingen). Flow cytometry was carried out on theFACSCalibur flow cytometer (Becton Dickinson). Datawere analyzed using FlowJo software (Treestar, Inc.).

Quantitative PCR analysisTotal RNAs were extracted from tissues, MPE and

cultured cells with TRizol Reagent (Invitrogen) accordingto the recommendations of the manufacturer. miR141detection was performed described previously (16).Real-time PCR was performed on an ABI Stepone PlusDetection System (AppliedBiosystems) using SYBRgreendye (Bio-Rad). The sequences of the primers were pre-sented in Supplementary Table S2. The reaction condi-tions were 95�C for 10 minutes, followed by 40 cycles of95�C for 15 seconds, 60�C for 30 seconds, and 72�C for30 seconds. Each sample was assayed in triplicates.

Cytokine array and ELISASerum and supernatants of effusion were analyzed for

cytokines using the Proteome Profiler Array ARY005(R&D systems) according to the manufacturer’s instruc-tions. Array images are from 5-minute exposures to X-rayfilm. Concentrations of CXCL1, CCL2, and IL8 in thesupernatant of effusion or cell cultures were measuredby commercial ELISA Kits (RayBiotech and R&D Sys-tems) according to the manufacturer’s instructions. Allsamples were assessed in triplicate.

Western blottingThe method of Western blot analysis was performed as

described previously (17). The anti-human c-Jun NH2-terminal kinase (JNK), phos-JNK, p38, phosp38, ERKs,phos-ERK, and GAPDH antibodies forWestern blot anal-ysis were from Cell Signaling Technology Inc. The anti-bodies against human CXCL1 and mouse-CXCL1 forWestern blot analysis were from Abcam Company. Pro-tein bands were detected using Westar Nova 2011 andWestar Supernova (Cyanogen). All experiments wererepeated at least three times.

Cell cultureCell lines (A549, LLC, 293T, Jurkat, and B16F-10) were

obtained from the Cell Bank at the China Academy ofScience and were passaged in our laboratory for less than6 months. 16HBE cells, a normal human bronchial epi-thelial cell line, were kindly provided by Dr. LongbangChen (Jinling Hospital, School of Medicine, Nanjing Uni-versity, Nanjing, China).

Plasmid construct and luciferase analysisThe 30UTRs (untranslated regions) of CXCL1, which

contain the target sites for miR141, were PCR amplifiedand then inserted into XbaI sites of the pGL3-control

miR141–CXCL1–CXCR2 Recruits Tregs in MPE

www.aacrjournals.org Mol Cancer Ther; 13(12) December 2014 3153




vector (Promega). 293T cells were seeded in 24-well plates1 day before transfection. miR141-specific precursor (pre-miR141), pre-miR control (pre–miR-NC), miR141-specificinhibitor(anti-miR141), and anti-miR negative control(anti–miR-NC), purchased from Ambion, were transient-ly transfected into the cells together with the luciferasereporter constructs described above (200 ng) and pRL-CMV (20 ng; Promega). After 48 hours, the luciferaseactivitywas determined using the dual luciferase reporterassay system (Promega). The relative reporter activitywasobtained by normalization to the Renilla luciferase activ-ity. All experiments were performed in triplicate.

Chemotaxis assayChemotaxis assay was performed in a 24-well Trans-

well chambers (Corning; Costar) with 3-mmpore polycar-bonate filters. Briefly, Transwell membranes were coatedwith fibronectin (5 mg/mL; Millipore; FC010) for 30 min-utes at 37�C before use. Human Tregs were purified fromhealthy PBMCs (peripheral blood mononuclear cells) bymagnetic cell sorting (Miltenyi Biotec). Tregs were addedto the top chamber resuspended in RPMI medium plus0.5% BSA at 1� 105 cells perwell. MPE frompatients withlung cancer or supernatant of transfected cell cultureswasplaced in thebottomchamberof theTranswell in avolumeof 600 mL. After 4 hours of incubation at 37 �C in 5% CO2

atmosphere, cells that migrated through the filters in thelower compartment were collected, fixed, and counted byflow cytometry (10, 11). To show that CXCL1 and CXCR2were responsible for Treg migration, blocking experi-ments were done by mixing the MPE with 7 mg/mL ofanti-CXCL1 or mixing the Tregs with 2 mg/mL of anti-CXCR2 (R&D Systems Inc.). The percentage of migrationwas calculated by comparing with corresponding controlvalue.

Animal modelThe following experimental protocol was approved

by the Animal Care and Use Committee of NanjingUniversity. Female C57BL/6 (4–6 weeks of age) werepurchased from the Model Animal Research Center ofNanjing University, and maintained under specificpathogen-free conditions (for more details, see Supple-mentary Information).

Two kinds of animal models were used in this study.First, 4 � 106 Lewis lung cancer cells were injected s.c.into the right flank of female C57BL/6 mice. The miR141angomir or angomir control (RiboBio Co Ltd.) togetherwith pcDNA3.1-CXCL1 or pcDNA3.1 vector wereinjected in vivo intratumorally thrice a week. Tumordiameters were monitored every 3 days, and tumorvolumes were calculated by the formula: volume ¼(width)2 � length/2. After 21 days, mice were sacri-ficed; tumors were removed and prepared for flowcytometry and quantitative PCR (qPCR) analysis . Sec-ond, the pGMLV–PE1–GFP–pre-miR141 was con-structed (pGMLV–PE1, a miRNA overexpression Vec-tor; Shanghai Genomeditech Co. Ltd.), and the lenti-

viruses were produced in 293T cells. After estimating amultiplicity of infection using a standard procedure,control viruses (LV-Ctrl) or pre-miR141–expressingviruses (LV-miR141) were used to infect B16F-10 cells.GFP-expressing B16F-10 melanoma cells were sortedafter infected with LV-miR141 or LV-Ctrl. A total of 1 �105 sorted B16F-10 cells were injected into the lateral tailvein of C57BL/6 mice. After 28 days, mice were sacri-ficed; lung tissues with metastasis were removed,photoed, and prepared for IHC and qPCR analysis.

Statistics analysisData are presented as mean � SEM. Normally distrib-

uted data were analyzed using one-way ANOVA fol-lowed by the Student Newman–Keuls post hoc test. Com-parisons between paired or unpaired two groups wereperformed using the Student t test. Spearman correlationwas used to test correlation between two continuousvariables. Statistical analysis was performed using Spssversion 15�0 for Windows software (SPSS, Inc.). A P valueof <0.05 was considered to be statistically significant.

ResultsTreg population is increased in MPE and inverselycorrelated with patient survival

We first measured the frequencies of Tregs in lungcancers. Representative images of IHC double stainingfor CD4 and FOXP3 showed that double-positive cellswere increased in lung cancer tissues compared withperitumoral tissue (Fig. 1A). Summarized data from allindividuals showed higher levels of FOXP3 mRNAs intumor cells of patients with NSCLC, especially in patientswithMPE (Fig. 1B).We also determined the frequencies ofTregs (clarified as a percentage of CD4þCD25þFOXP3þ

cells/total CD4þ cells) in blood and MPE using flowcytometry. As shown in Fig. 1C and D, Tregs in PBMCwere increased in patients with NSCLC than those inhealthy controls. Moreover, Tregs in MPE was muchhigher than that in the PBMC from the same individual(7.65% � 0.62% vs. 5.34% � 0.50%, P < 0.05). More Tregswere found in malignant effusion than those in tubercu-lous pleural effusion (Supplementary Fig. S1A). Further-more, we found a significant correlation between FOXP3expression and patient survival time for 196 patients withNSCLCwithMPE (P < 0.001, Fig. 1E). This correlation alsoexists in the NSCLCwithout MPE patients group (n¼ 72)and the total NSCLC group (n ¼ 268, Supplementary Fig.S1B). The expression levels of FOXP3 mRNA were alsoassociated disease stages (Supplementary Fig. S1C).Together these results suggest that Tregs were recruitedin lung cancers especially in MPE, which was correlatedwith patient survival.

Increased level of CXCL1 in MPE is associated withrecruitment of Tregs

We detected cytokine concentrations in serum fromhealthy control, effusions from patients with tuberculosis

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and effusions from patients with NSCLC using cytokinearray (Fig. 2A and Supplementary Fig. S2A). As quan-tified in Fig. 2B, several cytokines were activated in theMPE of patients with NSCLC, including CXCL1, CCL2,IL8, and IL1RA. We then confirmed the mRNA andprotein expression level of these cytokines using qPCRand ELISA, respectively. As shown in Fig. 2C and Fig.2D, CXCL1 was the unique chemokine that was signif-icantly increased in the effusion of patients with NSCLCin both mRNA (0.03 � 0.006 vs. 0.006 � 0.001, P < 0.001)and protein levels (984.3 � 135.5 pg/mL vs. 528.1 � 50.8pg/mL, P < 0.01). A significant correlation was foundbetween FOXP3 expression and CXCL1 concentration(P < 0.01, R2 ¼ 0.67), whereas no correlation was foundbetween FOXP3 expression and CCL2 or IL8 concen-tration (Fig. 2E).To identify the phenotype of pleural cells expressing

CXCL1 protein, we isolated tumor cells and immune cellsby magnetic bead and identify the CXCL1 expression

using double immunofluorescence staining. As shown inSupplementary Fig. S2B, the significant expression ofCXCL1 was found in all cancer cells, which suggest thatthe malignant cancer cells are the primary cell sources ofCXCL1 in MPE.

miR141 regulates expression of CXCL1 in lungcancers

In our previous study, we found that the expressions ofsix miRNAs were increased in the longer survival groupof patients with NSCLC, namely miR134, miR141,miR106b, miR224, miR720, and miR1260 (8). We thenapplied the prediction programs TargetScan and miRan-da to identify the potential regulators of CXCL1, andfound that CXCL1 has putative miR141-binding elementsin the 30UTRs (Fig. 3A). We thus proceeded to determinewhether miR141 indeed target CXCL1 expression usingthe dual luciferase reporter assay system in 293T cells.Wefound that miR141 suppressed the expression of CXCL1

Figure 1. Increase of Tregs in tumor microenvironment is correlated with patient survival. A, IHC double staining for CD4 and FOXP3 of representativeperitumoral tissues and tumor tissues (original magnification, �400). B, peritumoral tissues and tumor tissues were acquired from patients with 72NSCLC without MPE. For 196 patients with NSCLC with MPE, effusion was acquired using thoracocentesis and effusion cells were collected. The mRNAexpressions of FOXP3 in peritumoral tissues and tumor tissues of patients with NSCLCwithoutMPE (n¼ 72) and in effusion cells of patients with NSCLCwithMPE (n ¼ 196) were measured by qRT-PCR and normalized to that of b-actin. C, representative image of Tregs in blood from healthy controls and patientswith NSCLC using flow cytometric analysis. The population of Tregs was clarified as a percentage of CD4þCD25þFOXP3þ cells/total CD4þ cells. D,summarized data of Tregs frequencies in peripheral blood from healthy controls (n ¼ 30), and in the blood (n ¼ 196) and MPE (n ¼ 196) from patients withNSCLC.E, thecorrelationbetweenFOXP3 expression andpatient survival time for each individual (196patientswithNSCLCwithMPE)was assessedby linearregression; error bars, mean � SEM; �, P < 0.05; ��, P < 0.01; and ��, P < 0.001.






protein encoded by a wild-type CXCL1 30UTR in a dose-dependent way, and no effect was observed in the muta-tion group (Fig. 3B).

Ectopic expression of miR141 in A549 cells reducedCXCL1 mRNA level, whereas introduction of antisensemiR141 oligos induced an increase of CXCL1 (Fig. 3C).Corresponding to the changes of mRNA, the expressionand secretion of CXCL1 were also decreased with over-expressingmiR141 but increasedwith blocking ofmiR141

(Fig. 3D and E). Because the basal level of miR141 wasdifferent in tumor cells, pre-miR141 was transfected intoNCI-H460 (relatively lower basal level) and anti-miR141was transfected into SPC-A1 (relatively higher basal lev-el). As we expected, the level of CXCL1 was decreased inH460 cells and increased in SPC-A1 cells (SupplementaryFig. S3Aand S3B). These data indicated thatmiR141 couldregulate the expression and production of CXCL1 in lungcancer cells.

Figure 2. Upregulation of CXCL1 in lung cancers is associated with increase of Tregs. A, cytokine profile array from serum of healthy control and effusion ofpatientswith tuberculosis andNSCLCwere detected by capture antibodies spotted in duplicate on nitrocellulosemembranes . The high-intensity spots in thethree corners are positive controls (PC). Cytokines with visible spots were labeled. B, quantitative analysis of cytokine profiles in A. C, total cells werecollected from effusions. mRNA expression of CXCL1, CCL2, IL8, and IL1RA genes in effusion cells of patients with tuberculosis (n ¼ 64) and patients withNSCLC (n¼ 196) using qPCR and normalized to that of b-actin. D, concentration of CXCL1, CCL2, and IL8 in the pleural effusion of patients with tuberculosis(n¼ 64) and patients with NSCLC (n¼ 196) using ELISA. E, the correlation between FOXP3 expression and cytokine concentration for patients with NSCLCwas assessed by linear regression; error bars, mean � SEM; �, P < 0.05; ��, P < 0.01; and ��, P < 0.001.

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Manipulation of miR141 could not affect the cell pro-liferation, cell cycle, and cell apoptosis in A549 cells andNCI-H460 cells (Supplementary Fig. S3C–S3E). Thesenegative data suggest that miR141 cannot directly inhibittumor progression, which perhaps acts through a cellnonautonomousmanner. Thesedata alsoprovide supportto our hypothesis that miR141 may play an essential rolein cancer-suppressive function through regulating thesecreted protein.

miR141 level is inversely correlated with CXCL1level in NSCLCWe compared the expression levels of miR141 in A549

cells and a normal human bronchial epithelial cell line(16HBE). miR141 expression was lower in A549 cellsthan that in 16HBE cells, whereas the mRNA andprotein levels of CXCL1 were higher in A549 cells thanthose in 16HBE cells (Fig. 4A). As shown in Fig. 4B, wefurther compared the expression levels of miR141 in thesame pleural effusions displayed in Fig. 2 and foundthat the levels of miR141 were notably decreased in theMPE group than that in the tuberculosis group (0.64 �0.04 vs. 1.18 � 0.08, P < 0.001). More fundamentally, theconcentration of CXCL1 protein was inversely correlat-

ed with miR141 in patients with NSCLC with MPE(Fig. 4C). Moreover, there was a significant correlationbetween the miR141 expression and survival time inpatients with NSCLC with MPE (Fig. 4D). These clinicalfindings strongly support our hypothesis that theincrease of CXCL1 may be the result of reduced expres-sion of miR141 with a close association with survival inpatients with NSCLC with MPE.

The miR141–CXCL1–CXCR2 pathway regulatesmigration of Tregs to lung cancer microenvironment

To test whether miR141 may exert the cell nonautono-mous cancer-suppressive affect through inhibiting CXCL1and Treg migration, Tregs from healthy donors werefreshly isolated (Supplementary Fig. S4A) and added tothe top chambers of Transwell plates. As shown in Fig. 5A,CXCL1 significantly increased themigratory activity of theTregs. Furthermore, MPE in patients with NSCLC, whichhave a high concentration of CXCL1 could induce Tregsmigration, whereas downregulation of CXCL1 in MPEsignificantly inhibited the migration of Tregs (Fig. 5B).

To study the effect ofmiR141 onTregs accumulation, wedetected the chemo-attractive abilities of the supernatantsof A549 transfected with pre-miR141 and anti-miR141.

Figure 3. miR141 regulates expression of CXCL1 in lung cancers. A, putative miR141-binding sites in the 30UTR of human and mouse CXCL1. B,HEK293T cells were transiently transfected with pre-miR141 or negative control together with the pGL3 control plasmid, a modified pGL3 controlplasmid containing the wild-type CXCL1 30UTR, or a mutant CXCL1 30UTR with target sequence mutated. Pre-miR141 or negative control was added atthe indicated concentrations and the luciferase activity was analyzed 24 hours later. Data, relative firefly luciferase units. C, A549 cells weretransfected with pre-miR141, anti-miR141, and respective negative control. The relative expression of CXCL1 mRNA was determined by qPCR andnormalized to that of b-actin 48 hours later. D, A549 cells were treated as in C, and CXCL1 protein level in the surface of cells was determinedby Western blot analysis. GAPDH was used as a loading control. E, A549 cells were treated as in C, and CXCL1 protein level in culture media wasdetermined by ELISA; error bars, mean � SEM; �, P < 0.05 and ��, P < 0.01.






As shown in Fig. 5C, upregulation of miR141 inhibitedthe migration of Tregs, whereas downregulation ofmiR141 increased the migration of Treg. Moreover,neutralization of CXCL1 significantly suppressedanti-miR141–induced migration of Tregs. These dataproved that miR141 can recruit Tregs into MPE byCXCL1. Furthermore, a negative correlation betweenthe expression of miR141 and FOXP3 levels in patientswith NSCLC with MPE (Fig. 5D) was observed. How-ever, in the all NSCLC patients group and the patientswith NSCLC without MPE group, no significant cor-relation was found (Supplementary Fig. S4B).

To explore mechanisms of Tregs enrichment byCXCL1, the receptor CXCR2 expression was examinedin Tregs using flow cytometry. CXCL1 treatment couldincrease protein levels of the CXCR2 in a time- andconcentration-dependent manner (Fig. 5E and Supple-mentary Fig. S5A). In the migration test, preblockingof CXCR2 in Tregs could strongly inhibit CXCL1-and MPE-induced migration of Tregs (Fig. 5F andG). We also investigated the signaling pathwayinvolved in the downstream of CXCL1 and CXCR2.The level of phosphorylated ERK was significantlyincreased in Jurkat T cells after stimulated withCXCL1 (Fig. 5H), whereas the phosphorylated p38and JNK did not changed (Supplementary Fig. S5B).The results suggest that the induction of CXCR2expression by CXCX1 was responsible for the migra-tion of Tregs.

miR141 suppresses tumor progression throughCXCL1–CXCR2 pathway and Tregs

We wondered whether there is a beneficial effect ofmiR141 on tumor progression in vivo. We first adopted animmune-competent model with fully functional T-celllineages by injection of Lewis lung cancer cells intoC57BL/6J mouse. Mice were intratumorally injected withmiR141 agomir or/and pcDNA3.1–CXCL1, respectively.As shown in Fig. 6A, miR141 significantly suppressedtumor growth, whereas overexpression CXCL1 appearedto have a positive effect on tumor growth. Theprotein leveland mRNA expression level of CXCL1 in tumor tissueswere significantly decreased in themiR141 overexpressiongroup (Fig. 6BandC). Inaddition, the expressionofCXCR2was also increased in the CXCL1 group (SupplementaryFig. S6A). Moreover, the frequency of Tregs and theexpression of Foxp3 mRNAs in tumor tissues wereincreased in the CXCL1 overexpression group, suggestingan increased proportion of Tregs in tumor (Fig. 6D–F).Notably, overexpression of the nontargetable CXCL1could mostly eliminate miR141-reduced accumulation ofTregs. These results above strongly demonstrated thatmiR141 could suppress tumor progression through inhi-biting CXCL1 and infiltration of Tregs.

We further use B16F-10 melanoma cells in theC57BL/6 mouse model to demonstrate the role of themiR141–CXCL1–Treg pathway in promoting tumormetastasis. B16F-10 melanoma cells with lentiviral vec-tors encoding pre-miR141 (LV-miR141) or pre–miR-Ctrl

Figure 4. An inverse relationship between miR141 level and CXCL1 level in NSCLC. A, relative expression of miR141 and CXCL1 in lung cancer cells andnormal cells was determined by qPCR and normalized to that of b-actin. Protein level of CXCL1 in lung cancer cells and normal cells was determined byWestern blot analysis and ELISA. GAPDH was used as a loading control. B, expression of miR141 in total effusion cells of patients with tuberculosis (n¼ 64)and patients with NSCLC (n ¼ 196) was detected using qPCR. C, the correlation between miR141 expression and CXCL1 concentration for eachpatient with NSCLCwas assessed by linear regression. D, the correlation between miR141 expression and patient survival time for each patient with NSCLCwas assessed by linear regression; error bars, mean � SEM; ��, P < 0.001.

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(LV-Ctrl) were injected into the lateral tail vein ofC57BL/6 mice. The overexpression of miR141 in theLV-miR141 group was confirmed (SupplementaryFig. S6B). As shown in Fig. 6G, overexpression miR141thoroughly prevented the lung metastasis by B16F-10

cells. IHC staining of FOXP3 showed reduced accumu-lation of Tregs in lung tumors of the LV-miR141 group(Fig. 6H). In consist with the protein level, lower levelsof FOXP3 mRNAs were detected in the LV-miR141group (Supplementary Fig. S6C). Cxcl1 mRNAs were

Figure 5. ThemiR141–CXCL1–CXCR2 pathway regulatesmigration of Tregs. A, CD4þCD25þ T cells were isolated fromperipheral blood of healthy adults andwere tested by Transwell assay for their migration toward Hanks with and without CXCL1. After plating Tregs in the top chamber for 4 hours, migratedTregs were quantified by cell counter analysis. B, pleural effusions from patients with NSCLC (n ¼ 3) with or without anti-CXCL1 were used tostimulate chemotaxis of Tregs as described in A. C, Tregs were tested by Transwell assay for their migration toward culture media of A549 cells harvested 48hours after the cells were transfected with different amounts of pre-miR141, pre–miR-NC, anti-miR141 or anti–miR-NC. CXCL1-neutralizing antibody wasadded to the culture media of anti-miR141 transfected A549 cells to determine the effect on Treg migration. D, the correlation between miR141 andFOXP3 expression in total effusion cells for each individual was assessed by linear regression. E, Tregs were stimulated with different concentrations ofCXCL1. Expression of CXCR2 in the surface of Tregs was examined using flow cytometry. F, Tregs with or without pretreatment of anti-CXCR2 weretested for their migration toward Hanks with CXCL1 using Transwell assay. G, pleural effusions from patients with NSCLC (n ¼ 3) were used to stimulatechemotaxis of Tregs as described in F. Tregs were pretreated with anti-CXCR2 or control antibody. H, the expression of pERK, ERK, in Jurkat T cellsafter stimulated with CXCL1 (4 ng/mL) was determined byWestern blot analysis. GAPDHwas used as a loading control; error bars, mean� SEM; �, P < 0.05;��, P < 0.01; and, ��, P < 0.001.






also decreased in the LV-miR141 group (Supplementa-ry Fig. S6D). The percentage of CD4þ and CD8þ cellswere not changed in these groups (SupplementaryFig. S6F).

The miR141–CXCL1–Treg link was further demon-strated using nude mice. By injection of H460 cells innude mice, we found that miR141 has no effect on tumorformation in a mouse xenograft model (Supplementary

Figure 6. Impact of miR141 and CXCL1 on tumor growth and metastasis associates with Tregs recruitment to the lung cancer microenvironment. A, a total of4� 106 Lewis lung cancer cells were injected s.c. into the right flank of femaleC57BL/6mice.Micewere injected intratumorallywith vector, miR141 agomir or/and pcDNA3.1-CXCL1, respectively, three times a week. Tumor growth curves for these four groups were measured; n ¼ 4 per experimental group;experiment repeated three times. B, the expression of miR141 in tumors as described in A was detected using qPCR. C, protein levels and mRNA levels ofCXCL1 in tumors as described in A were determined by Western blot analysis and qPCR, respectively. D, mRNA expression of Foxp3 in tumors asdescribed in A was detected using qPCR. E, frequency of Tregs in tumors as described in A was detected using flow cytometry. F, representative image ofTregs in tumors using flow cytometric analysis. G, B16F-10 melanoma cells were sorted after infected with lentiviral vectors encoding pre-miR141(LV-miR141) or control (LV-Ctrl). A total of 1 � 105 sorted B16F-10 cells were injected into the lateral tail vein of C57BL/6 mice. After 28 days, mice weresacrificed. Representative images showed the formation of tumormetastases in themouse lung by injection of different B16F-10 cells; n¼ 4 per experimentalgroup; experiment repeated three times. H, IHC staining of FOXP3 in lung tumors; error bars, mean � SEM; �, P < 0.05 and ��, P < 0.01.

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Fig. S6E). Taken together, these results of tumor modelsabove demonstrated that miR141 has a potency toinhibit tumor progression through restraining therecruitment of Tregs by inhibiting the production ofCXCL1.

DiscussionMPEs are thought to be a specific tumor microenviron-

ment that facilitates NSCLC progression. The immunemechanisms ofMPE, especially how tumor cells evade theimmune system are still unclear. In the present study, werevealed the relationship between miRNA and MPEdevelopment. Mechanistically, decreased expression ofmiR141, causing increased production of CXCL1, recruitsTregs into MPE through CXCR2. This regulatory effectwas associated with the survival of patients with NSCLCwith MPE.Tregs play a pivotal role in the maintenance of the

homeostatic balance of the immune system (18, 19).Previous studies suggested that Tregs accumulate intumor microenvironment and suppress antitumorimmune responses (4, 20). In our study, we alsoobserved that Tregs were increased in lung cancers,both in peripheral blood and MPE. Some studiesreported that Tregs could be induced from naive T-cellby TGFb (21). However, the mechanism of Tregs devel-opment can not sufficiently explain our obvious accu-mulation of Treg in MPE. Therefore, we thought that theincrease of Tregs is not only through lineage develop-ment but also through migration.It has been proved that Tregs can be recruited to tumor

microenvironment by CCL22 in ovary (22), prostate (23),gastric (9), esophageal (24), hepatocellular (25), and breastcarcinomas (26–28). Qin and colleagues (10) found thatCCL22 can recruit Tregs intoMPE.However, their furtherstudy indicated that the recruitment of Tregs by CCL22also exists inpleural effusionof tuberculosis (29).Ourdatashowed that CXCL1 was increased specifically in MPE oflung cancers, but not in plural effusion of tuberculosis.More importantly, a positive correlation was foundbetween CXCL1 and Tregs. On the basis of these findings,we hypothesize and prove that CXCL1 can recruit Tregsinto MPE of NSCLC, promoting lung tumor growth andmetastasis.Studies have shown that miRNAs are involved in can-

cer pathogenesis by regulating tumor growth and metas-tasis (30, 31). Therefore, more and more researchers focuson their potential applications in cancer therapeutics (32,33). Our previous research discovered different expres-sion of miRNAs between the poor and good survivalgroups (16). miRNA array analysis showed that thesemiRNAs are involved in pathways regulating tumor cellsproliferation, cell cycles, and cell apoptosis. Although theexpression of miR141 was higher in the longer survivalgroup, gain and loss ofmiR141 had no effect on cancer cellproliferation, cell cycles, and cell apoptosis. These nega-tive data suggest miR141 cannot directly suppress tumor

progression, which perhaps acts through a cell nonauton-omous manner.

Chemokine receptor has been reported to play animportant role in Tregs recruitment. It has been reportedthat the recruitment of Tregs is often associated withCCR4 and CCR5 chemokine receptors. CCR4 canmediatemigration of Tregs to airway and to inflammation in skin,arthritic joints, and lymph nodes (34). CCR5 signalingsuppresses inflammation and reduces adverse remodel-ing of the infarcted heart–mediating recruitment of Tregs(35). In our study, we first confirm that CXCR2 couldmediate the recruitment of Tregs into MPE. Chemokinesthat activate CXCR2 are expressed by a wide variety ofestablished human cancer types. The chemokinesCXCL1–8 can all activate human CXCR2. CXCR2 is a keymediator of neutrophil migration that plays an importantrole in tumor development. CXCR2 deficiency couldinhibit inflammation-driven tumorigenesis in skin andintestine as well as spontaneous adenocarcinoma forma-tion (36). CXCR2 has been proved to be a potent protu-morigenic chemokine receptor that directs recruitment oftumor-promoting leukocytes into tissues in both tumorinitiation stage and tumor promotion stage (37). There-fore, CXCR2 receptor antagonists are a potential pharma-cologic approach in cancer and chronic inflammatorydiseases (38, 39).

In conclusion, during the development of MPE, tumorcells with decreased expression of miR141 occurred inthe lung microenvironment, causing increased produc-tion of CXCL1, which recruited Tregs into MPE. Ampli-fied immunesuppressive effects of Treg promotedimmune escape of tumor cells, causing the progressionof pleural metastasis, which affected the survival timeof patients with NSCLC. These data indicate thatmiR141–CXCL1–CXCR2 signaling in MPE may be apotent factor to decrease survival of the patients withNSCLC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Lv, B. Liu, Y. Hou, T. WangDevelopment of methodology: Y. Xu, S. Shen, T. WangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Xu, R. Tang, J. Ren, S. Shen, Y. Hou, T. WangAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): M. Lv, S. Shen, Y. ChenWriting, review, and/or revision of the manuscript: M. Lv, T. WangAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R. Tang, Y. Chen, B. Liu, Y. Hou

Grant SupportThis work was supported by the National Natural Science Foundation

of China (81101552), and the Natural Science Foundation of JiangsuProvince (BK2011571).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 4, 2014; revised September 9, 2014; accepted September30, 2014; published OnlineFirst October 27, 2014.






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Lv et al.




2014;13:3152-3162. Published OnlineFirst October 27, 2014.Mol Cancer Ther Mingming Lv, Yujun Xu, Ruijing Tang, et al.

Small Cell Lung Cancer−Regulates Metastases and Survival of Non Induced Treg Recruitment−CXCR2 Signaling−CXCL1−miR141

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