Molecular Cell Biology

Follistatin-like Protein 1 Inhibits Lung CancerMetastasis byPreventingProteolyticActivationofOsteopontinJean Chiou1, Yu-Chan Chang1, Hsing-Fang Tsai1, Yuan-Feng Lin2, Ming-Shyan Huang3,Chih-Jen Yang4,5, and Michael Hsiao1,6

Abstract

Follistatin-like protein 1 (FSTL1) plays a critical role inlung organogenesis, but is downregulated during lung can-cer development and progression. The prognostic signifi-cance and functional consequences of FSTL1 downregula-tion in lung cancer are unclear. Here, reduced levels ofFSTL1 were detected in various tumors compared withnormal tissues and were associated with poor clinical out-come in patients with non–small cell lung cancer, particu-larly those with lung adenocarcinoma. FSTL1 expressionnegatively correlated with the metastatic potential of lungcancer cells. Antibody-based neutralization of extracellularFSTL1 increased cellular migration/invasion while additionof recombinant FSTL1 protein diminished the metastaticcapacity of lung cancer cells in vitro and in vivo. Notably,treatment with FSTL1 effectively prevented the metastatic

progression of lung cancer cells in an orthotopic animalmodel. Mechanistically, FSTL1 directly bound to the pro-form of secreted phosphoprotein 1 (SPP1)/osteopontin,restraining proteolytic activation of SPP1, which led toinactivation of integrin/CD44-associated signaling and rear-rangement of the actin cytoskeleton. Combined low expres-sion of FSTL1 and high expression of SPP1 predicted apoorer prognosis for patients with lung cancer. This studyhighlights the novel interaction between FSTL1 and SPP1and new opportunities to effectively target SPP1-drivenmetastatic cancers characterized by FSTL1 downregulation.

Significance: These findings describe the novel interactionbetween FSTL1 and SPP1 and its role in the metastaticprogression of lung adenocarcinoma.

IntroductionFollistatin-like protein 1 (FSTL1) is a secreted glycoprotein that

was originally identified as a TGFb-inducible protein. Recently,FSTL1 has increasingly been recognized as a critical developmen-tal regulator of organogenesis, particularly lung development (1).FSTL1 appears to antagonize bone morphogenetic protein 4(BMP4) signaling during lung development (2). Fstl1-deficientmice have impaired alveolar maturation due to dysfunctional

distal alveolar differentiation and insufficient surfactant produc-tion (3). Because many developmental pathways involved inembryogenesis play critical roles in oncogenesis and cancer pro-gression (4, 5) and the dysregulation of BMP pathways has beendetected during tumorigenesis and the malignant evolution oflung cancer (6–9), perturbations in the FSTL1/BMP4 pathwayduring lung cancer development are worth investigating.

Follistatin (FST) has been reported to be a prognostic biomarkerfor lung adenocarcinoma (10). Similar proteins in the follistatin-like (FSTL) protein family, including FSTL1, IGFBP7, FSTL3, FSTL4,and FSTL5, play different roles in cancers (11–13). FSTL1 has beenreported to be downregulated or undetectable in various humancancer cells, including lung cancer cells, but it is expressed at highlevels innormal lung tissues (14). Similarly, FSTL1mRNAlevelsaresubstantially reduced in a panel of lung cancer cell lines comparedwith nontumor lung cell lines (15). The neutralization of extra-cellular FSTL1 by its specific antibody promotes lung cancer cellinvasionandmetastasis (16), confirming apivotal role for FSTL1 inpreventing lung cancer progression. We presented the clinicalrelevance of FSTL1 downregulation in lung cancer (17). However,the practical application of the FSTL1 protein in combating malig-nant tumors has not yet been explored.

Lung cancer is the leading cause of cancer-related deathsworldwide (18) and is classified into non–small cell lung cancer(NSCLC, �85% of all lung cancers) and small-cell lung cancer(�15%). Moreover, NSCLC is divided into threemajor histologicsubtypes: adenocarcinoma, squamous cell carcinoma, and large-cell lung cancer. Lung adenocarcinoma is currently the mostcommon type of lung cancer in patients who have neversmoked (19) and accounts for approximately 40% of all lung

1Genomic Research Center, Academia Sinica, Taipei, Taiwan. 2Graduate Instituteof Clinical Medicine, College of Medicine, Taipei Medical University, Taipei,Taiwan. 3Department of Internal Medicine, E-DA Cancer Hospital, School ofMedicine, I-Shou University, Kaohsiung, Taiwan. 4Department of Internal Med-icine, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University,Kaohsiung, Taiwan. 5Faculty of Medicine, College of Medicine, KaohsiungMedical University, Taiwan. 6Department of Biochemistry, College of Medicine,Kaohsiung Medical University, Kaohsiung, Taiwan.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

J. Chiou and Y.-C. Chang contributed equally to this article.

Corresponding Authors: Michael Hsiao, Genomics Research Center, AcademiaSinica, 128 Academia Road, Taipei 11529, Taiwan. Phone: 886-2-2787-1243;Fax: 886-2-2789-9931; E-mail: mhsiao@gate.sinica.edu.tw; and Chih-Jen Yang,Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University, No. 68Chunghwa 3rd Road, Cianjin District, Kaohsiung 80145, Taiwan. Phone: 886-7-320-8159; E-mail: chjeya@cc.kmu.edu.tw

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cancers. Here, FSTL1 downregulation strongly predicts a poorerprognosis in patients with lung adenocarcinoma, but not squa-mous cell carcinoma, and strongly correlates with an increasedmetastatic potential of lung cancer cells in vitro and in vivo. Impor-tantly, treatment with FSTL1 inhibits the metastatic progressionof lung cancer cells by directly binding to pro-SPP1 and therebyrestraining the proteolytic activation of SPP1. Because SPP1 is acritical extracellular molecule involved in eliciting cancer metasta-sis, the identificationofprotein–protein interactions (PPI) betweenFSTL1 and SPP1 might provide a new opportunity for developingnovel anticancer agents to combat metastatic cancers.

Materials and MethodsChemicals and antibodies

The recombinant FSTL1 protein was purchased from SinoBiological Inc., Beijing, P.R.C. (10924H08H50). Recombinantpro- andmature SPP1 proteins were obtained from R&D Systems(1433-OP-050) and Abnova (H00006696-Q01, H00006696-Q02), respectively. The FSTL1 antibody was purchased fromProteintech (20182-1-AP, 1:2,000). The SPP1 antibody was pur-chased from Abcam (ab8848, 1:1,000). Antibodies against Akt(9272, 1:1,000), p-AkT (9275S, 1:1,000), Src (2109S, 1:1,000),and p-Src (2101S, 1:1,000) were purchased from Cell SignalingTechnology. The antibody against b-actin (A2228, 1:5,000) waspurchased from Sigma-Aldrich.

Cell lines and cell culture conditionsThe lung cancer cell lines A549, H1299, PC13, and PC14 were

maintained in DMEM supplemented with 10% FBS and 1% pen-icillin–streptomycin–glutamine (PSG; Invitrogen). H1355, H928,CL1-0, and CL1-5 lung cancer cells were maintained in RPMI1640medium supplemented with 10% FBS and 1% PSG. The Beas2Bnormal lung epithelial cell line wasmaintained in keratinocyte-SFMmedium (Invitrogen, cat: 17005042) supplemented with 1% PSG.CL1-0 and CL1-5 cells were derived by Chu and colleagues andexhibit progressively increasing invasiveness (20). The PC13 andPC14 cell lines were generated by Lee and colleagues at the NationalCancer Center Hospital (Tokyo, Japan; ref. 21). Other lung cancercell lines and the Beas2B cell line were acquired from the ATCC.

Transwell migration and invasion assaysFor transwell migration assays, 5 � 104 cells were plated in the

top chamber on the noncoated membrane (Corning Costar). Forthe invasion assay, each well was freshly coated with Matrigel (BDBiosciences) before the invasion assay. Then, 5 � 104 cells wereplated in the top chamber on the Matrigel-coated membrane. Inboth assays, cells were plated in serum-free medium and mediumsupplementedwith10%FBS,whichwasusedas a chemoattractant,wasplaced in the lower chamber.The rhFSTL1proteinwasadded tothe upper layer. The cells were then incubated for another 16 or24 hours for themigration assay or 24 or 48 hours for the invasionassay. The cells that did not migrate or invade through the poreswere removed with a cotton swab. Cells on the lower surface of themembrane were fixed with methanol. After 10 minutes, mem-branes were stained with crystal violet. The number of cells migrat-ing or invading through themembrane were counted under a lightmicroscope (40�, three randomly selected fields/well).

Western blot analysisProteins (20–50mg)were electrophoretically separated on10%

SDS-polyacrylamide gels. After electrophoresis, the proteins were

transferred to a 0.45-mm polyvinylidene fluoride membrane thatwas blocked with 5% nonfat dry milk in PBS with Tween 20.Immunoblotting was performed using the primary antibodieswith overnight incubations at 4�C. Following washes and anincubation with the appropriate horseradish peroxidase–conjugated secondary antibodies, signals were visualized usingan enhanced chemiluminescence kit (Amersham ECL Plus). AllWestern blot analysis data are representative of at least threeindependent replicates.

Lentiviral infections and shRNA sequencesLentiviral FSTL1 shRNA constructs were purchased from the

National RNAi Core Facility Platform (Academia Sinica, Taiwan).Lentiviruses were produced by cotransfecting the shRNA-expressing vector with the pMDG and pD8.91 constructs into293T cells using calcium phosphate. Viral supernatants were thenharvested and used to infect CL1-0 or A549 cells in the presence of8 mg/mL polybrene (Santa Cruz Biotechnology). Cells were thenselected using 2 mg/mL puromycin (Santa Cruz Biotechnology).FSTL1-expressing cells were established by infecting cells with thepLenti6.2-FSTL1 virus, and viral supernatants were harvested andused to infect CL1-5 cells in the presence of 8 mg/mL polybrene.Cells were selected using 5 mg/mL blasticidin (Sigma-Aldrich).

Surface plasmon resonance experimentAll experiments were performed using ProteOn XPR36 instru-

ments developed by Bio-Rad Haifa (Haifa). The GLC sensor chipand amine coupling kitwere also purchased fromBio-Rad. For theimmobilization of the recombinant FSTL1 protein, FSTL1 wascoupled to the carboxymethylated alginate surface of a GLCcapacity chip (Bio-Rad) according to the protocol described inthe Bio-Rad ProteOn One-Shot Kinetics Kit Instruction Manual,with slight modifications. Direct binding experiments were per-formed on the Bio-Rad ProteOn XPR 36 protein interaction arraysystem (Bio-Rad). Briefly, the surfacewas activatedwith 0.1mol/LN-hydroxysuccinimide and 0.25 mol/L N-ethyl-N'-(3-dimethy-laminopropyl)carbodiimide at aflow rate of 25mL/minute. FSTL1was diluted in 10 mmol/L sodium acetate (pH 5.5) and immo-bilized at 25�C using a flow rate of 25 mL/minute for 288 seconds(120 mL). Activated carboxylic groups were quenched with aninjection of 1 mol/L ethanolamine (pH 8.0). Analyte solutionswere prepared at designated concentrations in filtered anddegassed PBS buffer (20 mmol/L Na-phosphate and 150mmol/L NaCl, pH 7.4). All binding experiments were conductedat 25�Cwith a constant flow rate of 100 mL/minute of PBS buffer.Sensograms for all binding interactions were recorded in real timeand analyzed after subtracting the blank channel. After eachmeasurement, the surface was regenerated with 1.0 mol/L NaCl.The equilibrium dissociation constants (KD) for evaluating theprotein-analyte binding affinity were determined with a steady-state affinity fitting analysis using the results from ProteOnManager 2.0 (Bio-Rad).

Microarray analysisTotal RNAwas extracted fromCL1-0 cells that had been treated

with the FSTL1 antibody for 0 and 6 hours using a TRIzol RNAExtraction Kit (Invitrogen). The synthesis of cDNAs from totalRNA and microarray hybridization/scanning were performedwith Affymetrix GeneChip products (HG-U133A) by the GRCMicroarray Core Facility (Academia Sinica). Datafiles (.CEL)wereconverted into probe set values (log2) by RMA normalizationusingGeneSpring (Agilent) and submitted to theGeneExpression

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Omnibus (GEO) database with accession number GSE75961.Data files (.CEL) from public microarray databases were alsoconverted into probe set values (log2) by RMA normalizationusing GeneSpring prior to performing further analyses. Normal-ized RNA data from The Cancer Genome Atlas (TCGA) weredownloaded from Cancer Browser.

Animal studiesAll animal studies were performed in accordance with a pro-

tocol approved by the Academia Sinica Institutional Animal Careand Use Committee (No. 11-12-277 to Michael Hsiao). Femaleand male age-matched NOD-SCID mice (supplied by LASCO,Taiwan) between 6 and 8 weeks old were used to assess tumorgrowth in xenograft and lung colonizationmetastasismodels. Forprimary tumor growth assays, viable cells [5� 106 cells/100 mL ofPBS]were subcutaneously injected into the backs ofmice. Primarytumor growth rates were analyzed bymeasuring the tumor length(L) and width (W) and calculating the volume with the formulaLW2/2. For experimental metastasis assays, 1 � 106 viable cellswere resuspended in 0.1 mL of PBS and introduced into thecirculation via a tail vein injection. Lungmetastasiswas quantified14 days after the injection and the metastatic tumors were tracedevery two weeks using luciferase-based, noninvasive biolumines-cent imaging. The analysiswas performedusing theXenogen IVIS-200 system (Xenogen). The orthotopic lung metastasis modelrequired 1� 106 viable cells resuspended in 0.01 mL of Matrigel:PBS at 1:1 ratio. The mixture was introduced into the left lungs ofmice via an orthotopic injection. Lung metastases were tracedwith bioluminescent imaging every 2 days after treatment withrhFSTL1 (10 mg/kg).

Patient selectionNinety-six patients diagnosed with NSCLC at the Kaohsiung

Medical University Hospital of Taiwan from 1991 to 2007(KMUH-IRB-E(I)-20160099), including 58 patients with adeno-carcinoma, 32 patients with squamous cell carcinoma, and 6patients with large cell carcinoma, were included in thisstudy (17). All patients received standard treatment protocolsaccording to hospital guidelines. Requirement for informed con-sent was waived by the Institutional Review Boards of KaohsiungMedical University Hospital of Taiwan. Patients with operablestage I-III NSCLC underwent lobectomy or pneumonectomywithmediastinal lymphadenectomy. No adjuvant chemotherapy wasadministered to patients with completely resected stage I NSCLC.Patients with resectable stage II and III NSCLC were treated withpostoperative adjuvant platinum-based chemotherapy. Patientswith unresectable locally advanced or metastatic disease receivedchemotherapywithorwithout radiotherapy. Follow-updatawereavailable for all patients, and the longest clinical follow-up timewas 190 months. All tumors were staged according to the cancerstaging manual of the American Joint Committee on Cancer andthe histologic cancer type was classified according to the WorldHealth Organization classification.

IHC stainingThree representative 1-mm diameter cores from each tumor,

which were taken from formalin-fixed paraffin embedded tissues,were selected on the basis of the morphology typical of thediagnosis. IHC stainingwas performed on serial 5-mmthick tissuesections cut from the TMA. IHC staining for FSTL1was performedusing an automated immunostainer (VentanaDiscovery XT auto-stainer). The antigens were retrieved by heat-induced antigen

retrieval in TRIS-EDTA buffer for 30 minutes. The slides werestained with a polyclonal rabbit FSTL1 antibody (1:250; Gene-Tex). After an overnight incubation at 4�C, the slides were visu-alized using the 3,30-diaminobenzidine (DAB) peroxidase sub-strate kit (Vector Laboratories). The IHC staining assessment wasindependently conducted by 2 pathologists who were blinded topatient outcomes. For FSTL1 IHC, cytoplasmic expressions intumor cells in the cores was evaluated. The intensity and percent-age of immunoreactive cells were recorded. The intensity ofstaining was scored using a four-tier scale and defined as follows:0, no staining; 1þ, weak staining; 2þ, moderate staining; and 3þ,strong staining. The extent of staining was scored by determiningthe percentage of positive cells: 0, 0%–25%; 1þ, 26%–50%; 2þ,51%–75%; and 3þ, 75%–100%. The final IHC scores wereobtained by multiplying the scores for the intensity and extentof staining. All patients were divided into two groups according tothe final IHC scores. A low IHC expression level was defined as ascore less than 4, and a score equal to or greater than 5was definedas high expression.

Statistical analysisAll observations were confirmed in at least three independent

experiments, and the data are presented as means � SD. Biolu-minescence intensity data are presented as means � SE. Survivalcurves were analyzed using the Kaplan–Meier method, and theCox proportional hazards regression analysis was used to test theprognostic significance of factors included in the univariate andmultivariate models. All statistical tests were two-sided. P < 0.05was considered significant. Analyses were performed using SPSS(Statistical Package for the Social Sciences, version 13.0) software.

ResultsDecreased expression of FSTL1 is significantly correlated with apoor prognosis for patients with lung adenocarcinoma

We analyzed the public TCGA database to examine the clinicalassociation between the expression of follistatin and the follistatin-like protein family in patients with lung cancer (Fig. 1A). Theexpression of the FST RNA significantly correlated with the tumorstatus. In contrast, the RNA expression of FSTL family membersinversely correlated with the tumor status. The results of thesurvival analysis using data from public microarray databases alsoshowed significant correlations between low FSTL1 RNA expres-sion (P ¼ 0.043) and poor first-progression survival (Fig. 1B). Wefurther compared the roles of FST family in different histologicsubtypes of lung cancer (Supplementary Fig. S1). Low FSTL1expression retained its prognostic significance in lung adenocar-cinoma (P¼ 0.00021). Forest plots of FST and FSTL protein familymembers and their corresponding hazard ratios and Cox-P valueswere generated for the KM-plotter database cohort (Fig. 1C), andthe results also showed that FSTL1 was the prognostic marker withthe lowest hazard ratio in patients with lung cancer (HR ¼ 0.78,P ¼ 9 � 10�5) and lung adenocarcinoma (LUAD; HR¼ 0.64, P ¼2.1 � 10�4). Although IGFBP7 has a low HR, the IGFBP7 probedoes not have a significant Cox-P values. On the basis of thesedata, the downregulation of FSTL1 is significantly associated withdisease-related progression in patients with LUAD.

FSTL1 expression negatively regulates the migration/invasionof LUAD cells in vitro

Next, we analyzed the level of extracellular FSTL1 (Fig. 2A) andcellular migration in the nontumor cell line Beas2B and LUAD

FSTL1 Interacts with SPP1 to Inhibit Lung Cancer Metastasis

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cell lines A549, CL1-0, CL1-5, H928, H1355, PC13, and PC14.Furthermore, we examined the endogenous protein expressionlevel FSTL1 with Western blot analysis (Supplementary Fig. S2).As shown in Fig. 2B, the endogenous FSTL1 expression levelnegatively correlated with cellular migration in the detected cells.

TheCL1-0 andCL1-5 cell lineswere establishedwith lowandhighinvasive abilities, respectively (20). The profiles of their mRNAexpression, protein expression, and secretomic analysis were wellstudied (22–27). We provided additional background informa-tion, such as microarray data, NGS data, and miRNA expression

Figure 1.

FSTL1 downregulation significantly correlates with poor prognosis in patients with LUAD. A, The TCGA database heatmap indicates the correlation betweenmRNA expression of FST and FSTL family and tumor status. B, Low FSTL1 expression was correlated with a poor first-progression survival rate in patients inKaplan Meier-plotter public database. C, HRs of FST and FSTL protein family. FSTL1 was the only prognostic biomarker that had the lowest HR in both lung cancerand LUAD patients cohort of Kaplan Meier-plotter public database (n¼ 1,926 and 720).

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Figure 2.

FSTL1 downregulation correlates with the enhanced migration/invasion abilities of LUAD cells in vitro.A, FSTL1 protein expression in lung adenocarcinoma celllines. ELISA assay of the cultured medium. B, The correlation between FSTL1 expression level and migration ability of LUAD cell lines. C, FSTL1 knockdown inCL1–0 cells. ELISA analysis of FSTL1. Bar, means� SD. D,Migration activity of FSTL1 knockdown cells. comparative xCELLigence analysis of the effect ofmigration of CL1-0 cell lines. CI values for all cell lines are plotted on the graph. The experiment was repeated three times and was consistent. Bar, SD. E, Invasionactivity of FSTL1 knockdown cells. The columns represent the mean values from three independent experiments. Bars, means� SD. F, FSTL1 overexpression inCL1-5 cells. ELISA analysis of FSTL1. Bar, means� SD. G,Migration activity of FSTL1 overexpression cells. Comparative xCELLigence analysis of the effect ofmigration of CL1-5 cell lines. CI values for all cell lines are plotted on the graph. The experiment was repeated three times and was consistent. Bar, SD. H, Invasionactivity of FSTL1 overexpression cells. The columns represent the mean values from three independent experiments. Bars, means� SD. I, Knockdown of FSTL1expression promotedmetastasis in vivo. Top, representative lung images of mice injected with a nonsilencing shRNA and FSTL1 shRNA-expressing CL1- 0 cells.Arrowheads, lung metastatic nodules. Bottom, total numbers of lung metastatic nodules in individual mice 10 weeks after tail vein injection with CL1-0 cellsinfected with a nonsilencing shRNA or FSTL1 shRNA. J, FSTL1 expression inhibited metastasis in vivo. Top, representative lung images of mice injected with avector control and FSTL1-expressing CL1-5 cells. Arrowheads, lung metastatic nodules. Bottom, total numbers of lung metastatic nodules in individual mice4 weeks after tail vein injection with CL1-5 cells infected with a vector control or FSTL1. KD, knockdown; NS, non-sense control; OE, overexpressed; VC, vectorcontrol.

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array data, in the revised supplementary information to ensurethat readers would clearly understand the differences in these twocell lines (Supplementary Table S1). Furthermore, we artificiallyknocked down the expression of FSTL1 using its specific shRNAsin CL1-0 cells with a high endogenous FSTL1 expression level butreducedmigratory ability. The knockdown of FSTL1 substantiallydecreased the levels of the FSTL1 protein (Fig. 2C; SupplementaryFig. S3A), but increased the migration (Fig. 2D; SupplementaryFig. S3B) and invasion (Fig. 2E) ofCL1-0 cells.We also establishedPC14 shFSTL1 clones, which presented similar phenomena withCL1-0 cells (Supplementary Fig. S3C and S3D). Conversely, weoverexpressed the exogenous FSTL1 gene in CL1-5 cells with a lowendogenous FSTL1 expression level but strong migratory ability.The overexpression of FSTL1 noticeably increased the levels of theFSTL1 protein (Fig. 2F; Supplementary Fig. S4A), but suppressedthe migration (Fig. 2G) and invasion (Fig. 2H) of CL1-5 cells. Wealso established A549 FSTL1 overexpressed cells (SupplementaryFig. S4B and S4C). We further established an animal model toevaluate the in vivometastatic potential of CL1-0 and CL1-5 cellsafter FSTL1 knockdown and overexpression, respectively. Theknockdown of FSTL1 robustly enhanced lung colonization, asshown by an increased number of lung nodules and enhancedcolony formation (Fig. 2I) of CL1-0 cells. In contrast, the over-expression of exogenous FSTL1 reduced the number of lungcolonies formed (Fig. 2J) by CL1-5 cells.

FSTL1 downregulation does not affect cell proliferation in vitrobut promotes tumor growth of LUAD cells in vivo

We next determined the proliferation rate of the less metastaticCL1-0 cells after FSTL1 knockdown to exclude the possibility thatthe enhanced migratory activity was due to increased cell prolif-eration. As shown in Supplementary Fig. S5A, FSTL1 knockdowndid not alter the proliferation rates of CL1-0 cells. Similarly, FSTL1overexpression did not alter the proliferation of CL1-5 cells(Supplementary Fig. S5B). Thus, FSTL1 downregulation pro-motes metastatic progression but does not increase the prolifer-ation of LUAD cells.

Intriguingly, FSTL1 expression negatively correlated with thegrowthof tumors composedof LUADcells in vivo (SupplementaryFig. S5C). On the basis of these findings, an FSTL1 deficiencyfosters the tumorigenesis through a cell proliferation-independent mechanism, for example, angiogenesis, in LUAD.

Reduced FSTL1 expression inhibits its antagonistic effect on theextracellular matrix and ultimately promotes the migration/invasion of LUAD cells

Because FSTL1 functions as a biological antagonist for the TGFbsuperfamily and thereby modulates cellular migration, we nextdetermined the effects of neutralizing the extracellular FSTL1withits specific antibody or replenishing it with the recombinantFSTL1 protein on the migration and invasion of LUAD cells.Treatment with an FSTL1-specific antibody dose dependentlyincreased the migration and invasion of CL1-0 cells (Fig. 3Aand B); conversely, the replenishment with the recombinantFSTL1 protein inhibited the migration and invasion of CL1-5cells in a dose-dependent manner (Fig. 3C and D). Moreover,treatment with the recombinant FSTL1 protein compromised theincrease in the migration/invasion of CL1-0 induced by FSTL1knockdown (Fig. 3E). In contrast, the addition of the FSTL1-specific antibody reversed the suppression of the migration/inva-sion of CL1-5 cells induced by FSTL1 overexpression (Fig. 3F).Therefore, FSTL1 downregulation leads to the dysregulation of

FSTL1-modulated cellular functions, likely through a protein–protein or ligand–receptor interaction in the extracellular matrix,and thereby drives metastatic evolution in LUAD.

FSTL1 treatment effectively inhibits the tumor growth andmetastatic progression of malignant LUAD cells with FSTL1downregulation in vivo

Next, we evaluated the therapeutic potential of FSTL1 ininhibiting the tumor growth and metastasis of malignant LUADcells with FSTL1 downregulation. Treatment with the recombi-nant FSTL1 protein (rhFSTL1) substantially inhibited the tumorgrowth of highly malignant CL1-5 cells that exhibit reducedendogenous FSTL1 levels (Fig. 4A). Moreover, compared withthe untreated group, the treatment with rhFSTL1 significantlysuppressed the colony-forming ability of highly invasive CL1-5cells in the lung of the animal model (Fig. 4B). Importantly, thetreatment with rhFSTL1 restrained themetastatic progression andtumor growth, of CL1-5 (Fig. 4C and F; Supplementary Fig. S6)and A549 (Supplementary Fig. S7A–S7C) cells in the orthotopiclung cancer model.

The binding of FSTL1 to pro-SPP1 prevents the proteolyticactivation of SPP1 and thereby inhibits the metastaticprogression of LUAD cells

We next overexpressed exogenous His-tagged FSTL1 and per-formed Ni-NTA-based affinity chromatography (Fig. 5A) to iso-late proteins that bind to His-tagged FSTL1 in the extracellularmatrix of CL1-5 cells and to identify the PPI network of FSTL1 inthe highly metastatic CL1-5 cells. As shown in Fig. 5B, severalproteins from the extracellular matrix or exosomal compartmentwere identified by a mass spectrometry analysis of the FSTL1-interacting protein mixture (Supplementary Table 2). BecauseSPP1 has been considered an oncogenic driver that tumorigenesisand metastasis via regulating avb3 integrin and CD44-mediatedcellular functions in various cancer types, the next experimentswere thus designed to validate the PPI between FSTL1 and SPP1and the functional consequence of this PPI in governing meta-static evolution in LUAD cells. Surface plasmon resonance anal-ysis clearly revealed an interaction between FSTL1 and the pro-form, but not the cleaved forms, of SPP1, with a strong bindingaffinity (KD¼ 3.48� 10�8 mol/L; Fig. 5C). The addition of activeSPP1 fragment, but not inactive pro-SPP1 protein, robustlyrestored the FSTL1-induced decrease in the migration/invasionof CL1-5 cells (Fig. 5D). In contrast, the inclusion ofavb3 integrinand CD44-specific antibodies dramatically inhibited the increasein the migration/invasion of CL1-0 cells after FSLT1 neutraliza-tion using its specific antibody (Fig. 5E). In addition, treatmentwith the FSTL1 antibody induced the phosphorylation of Src andAkt (Fig. 5F),which are downstreameffectors ofavb3 integrin andCD44. These findings confirm that the direct binding of FSTL1 topro-SPP1, thereby restraining SPP1 proteolytic activation, is a keystep in preventing metastatic progression in LUAD.

The ILK signaling pathway is involved in FSTL1-mediatedLUAD cell migration

We performed 3 microarray analyses using CL1-0 cells trans-fectedwith orwithout shFSTL1, CL1-0 cells treatedwith orwithoutthe FSTL1 antibody (GSE79682), and CL1-5 cells treated with orwithout rhFSTL1 to determine whether another novel pathwayregulated FSTL1-mediated LUAD cell migration. The genes dis-playing 1.5-fold changes in expression levels compared with thecontrol group were subsequently subjected to the computational

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simulation with Ingenuity Pathway Analysis software to identifycanonical pathways that were altered upon FSTL1 modulation(Fig. 6A). The simulation of canonical pathway activity showedthat HMGB1 signaling, the SUMOylation pathway, and ILK sig-naling were strongly predicted to be activated (Fig. 6A, right) afterFSTL1 is blocked with its specific antibody in CL1-0 cells and inCL1-0/shFSTL1 cells.On thebasis of these results,wecompared thedataobtained fromrhFSTL1-treatedCL1-5 cells.HMGB1signaling,the SUMOylation pathway, and ILK signaling pathway were pre-dicted to be downregulated. In Fig. 6B, the IPA results revealed theactivation of most genes in the ILK signaling pathway after theantibody treatment. Furthermore, F-actin, G-actin, and fibronectin

mRNAs, which are related to cell migration, were upregulated afterFSTL1 blockade. Compared with the microarray data obtainedfrom cells treated with the antibody and recombinant protein, theexpression of genes in the ILK signaling pathway was in significantcontrast (Fig. 6C). In our microarray data, FSTL1 did not affect theexpressionof the integrinb3mRNA, the trigger of ILK signaling.Wenext examined whether FSTL1 affected the integrin b3 promoter(Fig. 6D), but did not identify any activated transcription factorsafter the FSTL1 antibody treatment (Fig. 6E). Furthermore, wepresented migration assay with 2D08 (SUMOylation pathwayinhibitor), BAY117082 (NF-kB pathway inhibitor), and quercetin(HMGB1 signaling pathway inhibitor) in FSTL1 antibody-treated

Figure 3.

FSTL1 stimuli inversely promotes the migration/invasion abilities of LUAD cells in vitro.A, FSTL1 antibody treatment induced CL1-0 cell migration. Cells withFSTL1 antibodies (1 and 10 mg/mL) were seeded on Transwell CIM-Plate 16 plates and subjected to real-time migration assays (xCELLigence) over a timeperiod of 70 hours. Bars, means� SD. B, FSTL1 recombinant protein treatment inhibited CL1-5 cell migration. Cells with FSTL1 recombinant protein (10, 30, and100 ng/mL) were seeded on Transwell CIM-Plate 16 plates and subjected to real-timemigration assays (xCELLigence) over a time period of 25 hours. Bars, means� SD. C, FSTL1 antibody treatment induced CL1-0 cell invasion ability. Cells with FSTL1 antibodies (1 and 10 mg/mL) were seeded on Transwell precoated withMatrigel in the upper well and subjected to invasion assays for 36 hours. The columns represent the mean values from three independent experiments. Bars,means� SD. D, FSTL1 recombinant protein treatment inhibited CL1-5 cell invasion. Cells with FSTL1 recombinant protein (10, 30, and 100 ng/mL) were seeded onTranswell precoated with Matrigel in the upper well and subjected to invasion assays for 16 hours. The columns represent the mean values from threeindependent experiments. Bars, means� SD. E, Recombinant FSTL1 protein restored shFSTL1-induced cell migration and invasion. Cell migration/invasiveabilities were measured by Transwell assay in CL1-0/KD (knockdown) cells. The columns represent the mean values from three independent experiments. Bars,means� SD. F, FSTL1 antibody restored FSTL1-inhibited cell migration and invasion. Cell migration/invasive abilities were measured by Transwell assay inCL1-5/OE (overexpressed) cells. The columns represent the mean values from three independent experiments. Bars, means� SD. n.s., nonsignificant.

FSTL1 Interacts with SPP1 to Inhibit Lung Cancer Metastasis

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CL1-0 cells. The results showed that only BAY117082 reversed themigration ability induced by FSTL1 antibody (SupplementaryFig. S8). Thus, FSTL1 may mediate LUAD cell migration by acti-vating the ILK signaling pathway through different mechanismssuch as PPIs.

Blocking peptides mimic the interaction between FSTL1 andpro-SPP1 to inhibit LUAD cell migration

On thebasis of the sequence covered by FSTL1binding to SPP1,we designed 4 short peptides (15 a.a.) to mimic the behaviors ofFSTL1 (Fig. 7A). When CL1-0 cells were treated with pro-SPP1,

Figure 4.

FSTL1 Treatment suppresses tumor growth and lung metastatic ability of LUAD cells in vivo. A, CL1-5-GL cells were subcutaneously injected into NOD/SCID micethat were treated over an interval of 2 dayswith saline only (PBS) and 10 mg/kg rhFSTL1. Left, photo of tumors after 3 weeks of treatment; right, quantification oftumor weight. B, CL1-5-GL cells were intravenously injected into NOD/SCID mice that were treated over an interval of 2 dayswith saline only (PBS) and 10 mg/kgrhFSTL1. Top, luminescence image measured using a noninvasive, bioluminescence system (IVIS spectrum) at day 20. The tumor growth is expressed as thebioluminescence intensity change (5 mice/group). Left, quantification of bioluminescence intensity in whole mice at day 20. Bottom, total numbers of lungmetastatic nodules in individual mice 20 days after tail vein injection with CL1-5 cells and treated over an interval of 2 dayswith saline only (PBS) and 10 mg/kgrhFSTL1. C, CL1-5-GL cells were orthotopically injected into NOD/SCIDmice that were treated over an interval of 2 dayswith saline only (PBS) and 10 mg/kgrhFSTL1. Luminescence image measured using a noninvasive, bioluminescence system (IVIS spectrum) at days 0 (left) and 16 (right). D, Luminescence in the lungof mice treated with PBS or 10 mg/kg rhFSTL1 at day 16. E, rhFSTL1 inhibited metastasis of lung cancer in orthotopic model. Green fluorescence in the lungs ofmice treated with PBS or 10 mg/kg rhFSTL1 at day 16 after intravenous injection. (field-40�). Columns, the activity of GFP in the fields. Bars, means� SD.F, Hematoxylin and eosin staining of mice lung. Top, 12.5�; red square, 200� area; bottom, 200�.

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migration was induced. Migration was reduced after treatmentwith the combination of the P1 and P2 peptides, but not the P3and P4 peptides. Thus, blocking the exact binding site of SPP1potentially interrupted the subsequent activation of the integrin-triggered ILK signaling pathway (Fig. 7B). Furthermore, followingphalloidin staining of FSTL1 knockdown CL1-0 cells and FSTL1-overexpressing CL1-5 cells, FSTL1 inhibited intracellular F-actinpolymerization (Fig. 7C). On the other hand, the signature ofcombining a low level of FSTL1 and high level a SPP1 predicted asubstantially worse prognosis for patients with LUAD (Fig. 7D;Supplementary Table S3). These findings implicate a broad ther-apeutic valueof FSTL1 in combatingmalignancieswith a low ratioof FSTL1/SPP1 levels. We thus provide a novel model of FSTL1-mediated pro-SPP1 maturation to inhibit LUAD progression andmetastasis.

DiscussionAs shown in the current study, FSTL1 plays an important role in

preventing the metastatic progression of LUAD, but not squa-

mous cell carcinoma. Bymining data from the TCGAdatabase, weemphasize the role of FSTL1 downregulation in lung adenocar-cinoma. Higher serum FST levels have been observed in patientswith LUAD than in healthy people (10). The results convinced usbecause we observed that the overexpression of the FST mRNAcorrelated with a poor prognosis for patients with lung cancer. Inour previous studies, low FSTL1 expression significantly correlat-edwith poor prognosis in LUADbut not in SCC (17). In addition,FSTL1was reported to suppress tumor cell proliferation, invasion,and survival in NSCLC (28). Thus, the role of FSTL1 appeared todiffer from the role of FST in specific subtypes of lung adenocar-cinoma. Although a loss of FSTL1 expression predicted a poorprognosis for patients with lung adenocarcinoma in the currentstudy, it has been reported to exert the opposite effects on patientswith different types of tumors. FSTL1 expression inhibits thegrowth and invasion of lung cancer cells, and an FSTL1 antibodytreatment blocks invasion. In endometrial and ovarian cancer,FSTL1 also possesses a tumor suppressor function that regulatescell proliferation, apoptosis, invasion, and migration (29).Downregulation of FSTL1 expression has also been reported in

Figure 5.

FSTL1 inhibits cellular migration/invasion abilities via preventing theproteolytic activation of SPP1 in LUADcells. A, FPLC elution of CL1-5/FSTL1conditionedmedium binding withHis-Trap column. B, Surface plasmonresonance experiment for FSTL1 andSPP1 binding affinity. C, IngenuityPathway Analysis of microarrayresults of CL1-0 cells treated withFSTL1 antibody for 6 hours.D,Migration assay of CL1-5 treatedwith recombinant FSTL1 and SPP1protein. Bars, means� SD.E,Migration assay of CL1-0 treatedwith FSTL1 and integrin av, integrinb3, and CD44 antibody. Bars, means� SD. F,Western blot of phosphor-Src and PKB/Akt in CL1-0 cellstreated with FSTL1 and SPP1antibody. n.d., not detected;n.s., nonsignificant; �� , P < 0.01;��� , P < 0.001.

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various tumor cell lines, including clear cell renal cell carcinoma,colon cancer and gastric cancer (14, 30). In contrast, a recent studyof breast cancer showed that FSTL1 increases chemoresistance byregulating tumor stemness (31). Upregulation of FSTL1 is also

observed in glioblastoma and is associated with a poor prognosisfor patients with glioblastoma (32). These findings confirm thehypothesis that the function of FSTL1 varies among differentcancer types. In addition, treatment with the recombinant FSTL1

Figure 6.

ILK signaling involved in FSTL1-inhibited LUAD cell migration. A, Left, 1.5-fold changing gene numbers are shown in the circle. Right, the tables show the rankingof the predicted canonical pathways in three microarray analyses. B, The ILK signaling network was predicted based on the common signature from theIngenuity Pathway Analysis database overlaid with microarray data from FSTL1 antibody–treated CL1-0 cells, with a 1.5 fold-change cutoff compared with CL1-0control cells. The intensity of the color indicates the degree of activating (red and pink). C, The heatmap indicates the intensity of mRNA expression in CL1-0þAnti-FSTL1 cells and CL1-5þ rhFSTL1 cells. Red, activated; blue, downregulated; white, no significant difference.D, Prediction of integrin b3 promoter bindingsite. E, Ingenuity Pathway Analysis prediction of transcription factor of integrin b3 promoter.

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protein significantly inhibited tumor growth in highly malignantCL1-5 cells with lower endogenous FSTL1 expression. Moreover,treatment with FSTL1 significantly inhibited the formation oflung cancer nodules by highly invasive CL1-5 cells in animalmodels compared with the untreated group. Importantly, treat-ment with the recombinant FSTL1 protein inhibited the meta-static progression of CL1-5 and A549 cells in an orthotopic lungtumor metastasis model.

SPP1 is a pleiotropic chemokine that is overexpressed invarious types of cancer. Elevated serum SPP1 levels are frequentlydetected in patients with metastatic cancer (33–35). Therefore,therapeutic strategies targeting osteopontin by repressing geneexpression or blocking its binding to avb3 integrin and CD44have recently been considered useful approaches to overcomecancer metastasis (36–38). Here, FSTL1 directly bound to nascent

SPP1 and thereby inhibited its proteolytic processing by matrixmetalloproteinases-3/7 or thrombin protease into active SPP1 toprevent the SPP1-induced activation of avb3 integrin and CD44inmetastatic cancer cells. Importantly, the signature of a high levelof SPP1 and low level of FSTL1 predominantly correlates with apoor distant metastasis-free survival rate in clinical patients withbasal-like breast cancer, a highly metastatic breast cancer (Sup-plementary Fig. S9A). These findings imply a broad therapeuticvalue of FSTL1 in combating SPP1-driven cancer metastasis.

In addition to SPP1, a mass spectrometry analysis of an FSTL1-interacting protein mixture obtained from highly metastatic can-cer cells showed that FSTL1 may also interact with other extra-cellular proteins. We also designed 4 short peptides (15 a.a.) tomimic the behaviors of FSTL1 and block SPP1-mediated migra-tion. However, further studies are required to delineate these PPIs

Figure 7.

Peptides interrupt the SPP1maturation to mimic FSTL1-inhibitedLUAD cell migration. A, Briefdiagram of the peptides. B, Thepeptides interrupted the CL1-0invasion induced by SPP1. Cellswith SPP1 recombinant protein(500 ng/mL) and combinedpeptides treatment (500 ng/mL)were seeded on Transwell precoatedwith Matrigel in the upper well andsubjected to invasion assays for16 hours. The columns represent themean values from three independentexperiments. Bars, means� SD.C, Left, actin polymerizationincreased after downregulation ofFSTL1 in CL1-0 cells; right, FSTL1overexpression inhibitedintracellular tubular forming in CL1-5cells. D, Kaplan–Meier survival curveanalysis of patients with lung cancerand LUADwith FSTL1 and SPP1 levelsas determined by Kaplan Meier-plotter database analysis at theendpoint of overall survival.

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of FSTL1 in metastatic cancers. On the other hand, the molecularmechanism underlying FSTL1 downregulation during metastaticprogression in cancer remains to be elucidated. In conclusion,this study is the first to document the PPI between FSTL1 andSPP1, as well as the potential usefulness of this PPI in preventingcancer metastasis in the clinic.

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

Authors' ContributionsConception and design: J. Chiou, Y.-C. Chang, Y.-F. Lin, M. HsiaoDevelopment of methodology: J. Chiou, Y.-C. ChangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Chiou, Y.-C. Chang, M.-S. Huang, M. HsiaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Chiou, Y.-C. Chang, Y.-F. Lin, C.-J. Yang, M. Hsiao

Writing, review, and/or revision of the manuscript: J. Chiou, Y.-C. Chang,M.-S. Huang, C.-J. Yang, M. HsiaoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H.-F. Tsai, M.-S. Huang, M. HsiaoStudy supervision: M.-S. Huang, C.-J. Yang, M. Hsiao

AcknowledgmentsThis study was supported by Academia Sinica and Ministry of Science and

Technology grants (AS-SUMMIT-108) awarded to Michael Hsiao.

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 March 17, 2019; revised September 23, 2019; accepted October 21,2019; published first October 25, 2019.

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