FSTL1 Promotes Metastasis and Chemoresistance in Esophageal

Published OnlineFirst September 7, 2017; DOI: 10.1158/0008-5472.CAN-17-1411

Cancer Tumor and Stem Cell Biology Research

FSTL1 Promotes Metastasis and Chemoresistance in Esophageal Squamous Cell Carcinoma through NFkB–BMP Signaling Cross-talk Marco Chi-Chung Lau1, Kai Yu Ng1, Tin Lok Wong1, Man Tong1, Terence K. Lee2, Xiao-Yan Ming3, Simon Law4, Nikki P. Lee4, Annie L. Cheung1, Yan-Ru Qin5, Kwok Wah Chan6, Wen Ning7, Xin-Yuan Guan3, and Stephanie Ma1

Abstract

Esophageal squamous cell carcinoma (ESCC) has a generally attenuation by shRNA or neutralizing antibody elicited the poor prognosis, and molecular markers to improve early detec- opposite effects in ESCC cells. mRNA profiling analyses sug- tion and predict outcomes are greatly needed. Here, we report gested that FSTL1 drives ESCC oncogenesis and metastasis that the BMP-binding follistatin-like protein FSTL1 is over- throughvariouspathways,withderegulationofNFkB and BMP expressed in ESCCs, where it correlates with poor overall signaling figuring prominently. Cross-talk between the NFkB survival. Genetic amplification of FSTL1 or chromosome 3q, and BMP pathways was evidenced by functional rescue experi- where it is located, occurred frequently in ESCC, where FSTL1 ments using inhibitors of NFkB and TLR4. Our results establish copy number correlated positively with higher FSTL1 protein the significance of FSTL1 in driving oncogenesis and metastasis expression. Elevating FSTL1 levels by various means was suf- in ESCC by coordinating NFkB and BMP pathway control, with ficient to drive ESCC cell proliferation, clonogenicity, migra- implications for its potential use as a diagnostic or prognostic tion, invasion, self-renewal, and cisplatin resistance in vitro and biomarker and as a candidate therapeutic target in this disease tumorigenicity and distant metastasis in vivo. Conversely, FSTL1 setting. Cancer Res; 77(21); 5886–99. 2017 AACR.

Introduction understanding of the recurrently altered genomic and molecular profiles involved in ESCC development, progression, and therapy Esophageal squamous cell carcinoma (ESCC) is the most resistance should aid in the identification of novel targets and the common histologic subtype of esophageal cancer. The disease is development of new therapies for the more effective clinical ranked as the eighth most common cancer and the sixth leading management of this disease. To date, there is no effective bio- cause of cancer mortalities worldwide, with a dismal prognosis marker for the diagnosis or prognosis of ESCC, nor a good (1). The overall prognosis for ESCC is poor due to late presen- targeted therapeutic drug for the adjuvant treatment of the tation, high incidences of tumor recurrence and metastasis, as well disease. as the ability of the tumor to acquire chemoresistance. A better To identify better molecular markers for early detection and therapeutic targeting, our laboratory previously performed transcriptome sequencing on three pairs of patient-derived 1School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of ESCC samples and their adjacent nontumor tissue counterparts 2 Hong Kong, Hong Kong. Department of Applied Biology and Chemical Tech- (2) and successfully identified a number of commonly and nology, Hong Kong Polytechnic University, Hong Kong. 3Department of Clinical differentially expressed genes at a global level. On the basis of Oncology, Li Ka Shing Faculty of Medicine, Queen Mary Hospital, The University fi fi of Hong Kong, Hong Kong. 4Department of Surgery, Li Ka Shing Faculty of this pro ling data, we have identi ed PTK6 and RAB25 as novel Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong. tumor and metastasis suppressors of ESCC (2–3), CD90 to 5Department of Clinical Oncology, First Affiliated Hospital, Zhengzhou Univer- represent a novel ESCC cancer stem cell subpopulation (4), and sity, Zhengzhou, China. 6Department of Pathology, Li Ka Shing Faculty of NRP2 as a novel oncogene in ESCC (5). However, our knowl- Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong. edge of the exact cellular and molecular mechanisms leading to 7Department of Genetics and Cell Biology, College of Life Sciences, Nankai ESCC is incomplete. University, Tianjin, China Through data mining of our transcriptome sequencing data, Note: Supplementary data for this article are available at Cancer Research we found the transmembrane glycoprotein follistatin-like 1 Online (http://cancerres.aacrjournals.org/). (FSTL1), belonging to the BM-40/SPARC/osteonectin family M. Chi-Chung Lau and K.Y. Ng contributed equally to this article. (6), to be commonly overexpressed in all three ESCC samples Corresponding Author: Stephanie Ma, School of Biomedical Sciences, Li Ka compared with its corresponding nontumor counterparts Shing Faculty of Medicine, The University of Hong Kong, Room 47, 1/F, Labo- (P < 0.005). FSTL1 became an obvious candidate gene of ratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pok Fu Lam, Hong interest as it is located on chromosome 3q13.33, which has Kong. Phone: 852-3917-9238; Fax: 852-2817-0857; E-mail: [email protected]. previously been reported to be a chromosomal amplification doi: 10.1158/0008-5472.CAN-17-1411 hotspot in ESCC (7–9). FSTL1 is involved in the development 2017 American Association for Cancer Research. of different organogenesis including early development of the

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FSTL1-Mediated NFkB and BMP Pathway Deregulation in ESCC

lung, ureter, central nervous system, and skeleton (10–14). Patients and Methods FSTL1 has also been implicated to act as an autoantigen Clinical samples associated with rheumatoid arthritis and to elicit a cardiopro- For RNA-Seq, three pairs of fresh frozen ESCC and their tective role in various cardiovascular diseases (15–18). The link adjacent nontumor tissue specimens (patients 16, 18, and 19) between FSTL1 and rheumatoid arthritis is particularly strong, were randomly selected from Linzhou Cancer Hospital (Henan, where Tanaka and colleagues first cloned the gene from syno- China). Patient 16 is a 43-year-old man with a TNM grade of vial tissues of rheumatoid arthritis patients in 1998 and found T2N0M0 (moderate differentiation and no sign of lymph node FSTL1tobedetectedintheseraandsynovialfluid of patients metastasis). Patient 18 is a 60-year-old woman with a TNM suffering from the disease (19). Mechanistically, FSTL1 has grade of T2N0M0 (moderate differentiation and no lymph been shown to regulate the TGFb/BMPpathwayandasaresult node metastasis). Patient 19 is a 54-year-old male with a TNM leading to blockage of erythroid cell differentiation and sub- grade of T3N1M0 (moderate differentiation and lymph node sequent apoptosis (20), as well as abnormal skeletal and lung metastasis). A total of 73 primary human ESCC and adjacent organogenesis in mice (11–12). In addition to TGFb/BMP, nontumor esophageal tissue samples used for FSTL1 expression FSTL1 has also been shown to activate NFkB, via both canonical studies by qRT-PCR were collected from Queen Mary Hospital and noncanonical means, to trigger inflammation in osteoar- (Hong Kong). A total of 394 ESCC and 225 nontumor esoph- thritis (21) and obesity (22). Only a small number of studies ageal formalin-fixed, paraffin-embedded tissue specimens were have reported on the role of FSTL1 in cancer, and many of them obtained from a total of 619 patients undergoing esophagect- with contradictory organ-specific roles. FSTL1 has been shown omy at Linzhou Cancer Hospital and Queen Mary Hospital and to negatively regulate the migratory and invasive abilities of used for tissue microarray construction and IHC analysis. ovarian, endometrial, renal, lung, and nasopharyngeal cancers Serum samples from a total of 104 ESCC patients (75% male, (23–26). However, FSTL1 was found to be overexpressed in 25% female, age range 22–89) and 30 normal healthy indivi- astrocytic brain tumors (27) and also to enhance the metastasis duals (66.7% male, 33.3% female, age range 21–58) were of cancer cells in breast, prostate, skin, and pancreatic cancers collectedfromQueenMaryHospitalandusedforELISAanal- (28–29). A recent study have also found knockdown of FSTL1 ysis for secretory FSTL1 expression. Above patients had received to induce mitotic cell death and BIM upregulation in non– no previous local or systemic treatment prior to operation. An small cell lung carcinoma cells (30). However, to date, the additional 37 ESCC clinical samples, of which 22 received clinical significance and functional role of endogenous and preoperative chemotherapy treatment and 15 did not were also secretory FSTL1 and the molecular mechanism by which it collected from Queen Mary Hospital for IHC work. All samples drives ESCC pathogenesis has not been reported. The link used in this study were approved by the committee for ethical between FSTL1 and the cross-talk between BMP and NFkB review of research involving human subjects at Zhengzhou pathways have also not been implicated in cancer previously. University and The University of Hong Kong/Hospital Author- In this study, we found both endogenous and secretory ity Hong Kong West Cluster, with informed consent from the FSTL1 to be markedly upregulated in ESCC tissue and serum subjects. samples and that their overexpression was significantly correlated with worst survival of ESCC patients. FSTL1 copy number positively correlated with high FSTL1 expression in both ESCC Cell lines cell lines and clinical samples, suggesting that FSTL1 gene ESCC cell line EC109 was kindly provided by Professor and/or chromosome 3q amplification contributes, at least in George Tsao (School of Biomedical Sciences, The University part, to the preferential upregulation of FSTL1 in ESCC. Over- of Hong Kong). ESCC cell lines HKESC1, EC9706, and expression of FSTL1 provoked, whereas silencing FSTL1 abro- KYSE520 were kindly provided by Professor Gopesh Srivastava gated proliferation, clonogenicity, migration, invasion, self- (Department of Pathology, The University of Hong Kong, Hong renewal, and cisplatin resistance in ESCC in vitro as well as Kong). ESCC cell lines KYSE150 and KYSE510 were obtained tumorigenicity and distant metastasis in vivo. Coculture of from DSMZ, the German Resource Centre for Biological Mate- FSTL1-containing conditioned medium or recombinant FSTL1 rial (36). 293FT and 293FN cells used for lentiviral transduc- in low FSTL1-expressing ESCC cells enhanced the cells' ability tion and human umbilical vein endothelial cells (HUVEC) to migrate and invade in vitro as well as form tumors in vivo, were purchased from Invitrogen. All cell lines used in this whereas treatment of high FSTL1-expressing ESCC cells with a study were obtained between 2013 and 2016, regularly authen- FSTL1 neutralizing antibody resulted in decreased ESCC metas- ticated by morphologic observation and AuthentiFiler STR tasis. Mechanistically, FSTL1 modulated ESCC tumorigenicity (Invitrogen) and tested for absence of mycoplasma contami- and metastasis through deregulation of both NFkB and BMP nation (MycoAlert, Lonza). Cells were used within 20 passages signaling pathways cross-talk, as evidenced by mRNA profiling after thawing. of ESCC cells with or without FSTL1 stably overexpressed, coupled with functional rescue experiments involving inhibi- Reagents tors for NFkB and TLR4 (21, 31–35). Collectively, our findings Normal goat IgG control and FSTL1 neutralizing antibody provide evidence that FSTL1 functions as an important onco- (clone 22B6) used for functional studies was purchased from gene in ESCC development and metastasis through canonical R&D Systems and obtained from Professor Wen Ning (Nankai NFkB pathway activation and BMP pathway attenuation. This is University, China), respectively. Human recombinant FSTL1 pro- also the first study to report an NFkB and BMP pathways cross- tein used for functional studies was purchased from R&D Systems talk, where they both work in concert to promote ESCC. (1694-FN-050). NFkB inhibitor (IMD-0354) and TLR4 inhibitor Clinically, FSTL1 represents a potential diagnostic and prog- (C34) were purchased from Tocris. Mitomycin C was purchased nostic biomarker as well as a therapeutic target in ESCC. from Calbiochem.

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Quantitative real-time PCR CA). FISH reactions were performed as described previously (37). Total RNA was extracted using RNA-IsoPlus (Takara) and cDNA Stained sections were counterstained with DAPI anti-fade solu- was synthesized by PrimeScript RT Master Mix (Takara). qRT-PCR tion and were examined under a Zeiss Axiophot microscope was performed with EvaGreen qPCR MasterMix (Applied Biosys- equipped with a triple-band pass filter. Samples were scored as tems) and the following primers: FSTL1, forward (5'- GCCAT- normal (2 copies) and abnormal (>2 copies). GACCTGTGACGGAAA -3'); reverse (5'- CAGCGCTGAAGTGGA- GAAGA -3') and b-actin: forward (5'- CATCCACGAAACTACCTT- CAACTCC-3'); reverse (5'- GAGCCGCCGATCCACACG-3') on an Lentiviral transduction ABI Prism 7900 System with data analyzed using the ABI SDS v2.3 FSTL1 shRNA expression vector (NM_007085) and the scram- software (Applied Biosystems). Relative expression differences bled shRNA nontarget control (NTC) were purchased from Sigma- DDC Aldrich. Sequence of the shRNAs directed against FSTL1 are (clone were calculated using the 2 t method. ID 352) 5'-CCGGCCAGGTTGATTACGATGGACACTCGAGTG- Western blot TCCATCGTAATCAACCTGGTTTTTG-3' and (clone ID 1185) Protein lysates were quantified and resolved on a SDS-PAGE 5' CCGGGTCGCCAAATCACCAGTATTTCTCGAGAAATACTGGT- gel, transferred onto a PVDF membrane (Millipore), and immu- GATTTGGCGACTTTTTG – 3'; sequence of NTC is 5'-CCGG- noblotted with a primary antibody, followed by incubation with a CAACAAGATGAAGAGCACAACTCGAGTTGGTGCTCTTCATCTTG- secondary antibody. Antibody signal was detected using an TTGTTTTT-3'. Sequences were transfected into 293FT cells, packaged enhanced chemiluminescence system (GE Healthcare). The fol- using MISSION Lentiviral Packaging Mix (Sigma-Aldrich) and used lowing antibodies were used: FSTL1 (1:2,000; AF1694, R&D to infect EC109 ESCC cells to establish cells constitutively repressing Systems), p-IkBa (1:1,000, 2859, Cell Signaling Technology), FSTL1. FSTL1 lentiviral overexpression (EX-A1145-Lv105) or empty total IkBa (1:1,000, 4812, Cell Signaling Technology), p-SMAD1 vector control plasmids were purchased from GeneCopoeia. (1:1,000, 5753, Cell Signaling Technology), total SMAD1 Sequences were transfected into 293FN cells, packaged using (1:1,000, 6944, Cell Signaling Technology), p-p65 (1:1,000, Lenti-Pac HIV expression packaging mix (GeneCopoeia) and used sc101749, Santa Cruz Biotechnology), total p65 (1:1,000, sc372, to infect KYSE150-Luc ESCC cells to establish cells constitutively Santa Cruz Biotechnology), b-actin (1:10,000; Sigma-Aldrich), expressing FSTL1. Stable clones were selected with puromycin. and histone H3 (1:1,000, ab24834, Abcam). For collection of conditioned medium from ESCC cell lines, cells were first serum- Conditioned medium preparation for functional studies starved for 24 hours and conditioned medium was subsequently Stable FSTL1-expressing cells and empty vector–transfected collected. Protein in concentrated conditioned medium was cells were cultured in DMEM with 10% FBS until 70% confluent. resolved on SDS-PAGE and Silver Staining (PlusOne Silver Stain- After complete removal of the culture medium, these cells were ing Kit, GE Healthcare) was performed to normalize the amount then continually cultured in DMEM supplemented with 3% FBS of protein. ImageJ software was used for densitometric analyses of for 24 hours before medium collection. Culture medium was then Western blot bands, and the quantification results were normal- centrifuged at 1,000 g for 30 minutes and the supernatant was ized to the loading control. collected as conditioned medium for further studies.

IHC Slides were heated for antigen retrieval in 10 mmol/L sodium XTT cell proliferation assay citrate (pH 6.0). Endogenous peroxidase activity was inhibited XTT cell proliferation assay (Roche) was performed according with 3% hydrogen peroxide. Sections were subsequently incu- to manufacturer's instructions. Absorbency was measured with a bated with monoclonal FSTL1 antibody (clone 22B6; Prof. Wen spectrophotometer (Victor3 1420 Multilabel Plate, Perkin Elmer) Ning, Nankai University, Tianjin, China). Reaction was developed at 450 nm with a reference wavelength of 750 nm. with LSABþ System-HRP (Dako). Slides were counterstained with Mayer hematoxylin. Evaluation for FSTL1 expression was per- Colony formation assay formed by a pathologist who had no prior knowledge of patient Cells were seeded in a 6-well plate at a low density and cultured data. Staining intensity was divided into three scores: no/low, in complete medium for 2 weeks with medium change every 2 medium, and high. Note only expression in the squamous epi- days. Colonies were counted and stained with 2% Crystal violet thelium lining of the nontumor esophageal samples were taken (Sigma-Aldrich). into account.

ELISA Cell motility and invasion assays Serum FSTL1 and secretory FSTL1 in conditioned media was Migration and invasion assays were conducted in 24-well quantified using the human FSTL1 ELISA Kit (Cloud-Clone Millicell hanging inserts (Millipore) and 24-well BioCoat Corp.). Matrigel Invasion Chambers (BD Biosciences), respectively. Cells resuspended in serum-free medium were added to the FISH top chamber and medium supplemented with 10% FBS was Dual-color FISH was undertaken using BAC clone at 3q13.33 added to the bottom chamber as a chemoattractant. After 48 covering the FSTL1 gene (RP11-343B4) labeled in Spectrum hours of incubation at 37C, cells that migrated or invaded Green (Vysis) and a reference BAC clone localized to the centro- through the membrane (migration) or Matrigel (invasion) were mere of chromosome 3 (RP11-301H7), labeled in Spectrum Red fixed and stained with 2% Crystal violet (Sigma-Aldrich). The (Vysis). BACs were obtained from the BACPAC Resource Center at number of cells was counted in three random fields under 40 the Children's Hospital Oakland Research Institute (Oakland, objective lens.

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FSTL1-Mediated NFkB and BMP Pathway Deregulation in ESCC

Spheroid formation assay genes was used for gene expression analysis. Microarray profiling Single cells were cultured in 300 mL of serum-free DMEM/F12 was performed as a service at Macrogen Inc. Statistical significance medium (Invitrogen) supplemented with 20 ng/mL human of the expression data was determined using fold change. Hier- recombinant EGF (Sigma-Aldrich), 10 ng/mL human recombi- archical cluster analysis was performed using complete linkage nant basic fibroblast growth factor (Sigma-Aldrich), 4 mg/mL and Euclidean distance as a measure of similarity. Data analysis insulin (Sigma-Aldrich), B27 (1:50; Invitrogen), 500 U/mL pen- and visualization of differentially expressed genes was conducted icillin, 500 mg/mL streptomycin (Invitrogen), and 1% methylcel- using R 3.1.2. Pathway analyses were performed using Gene Set lulose (Sigma-Aldrich). Cells were cultured in suspension in poly- Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis HEMA–coated 24-well plates. Cells were replenished with 30 mL (IPA) software. Statistical significance of the expression data was of supplemented medium every second day. To propagate spheres determined using z-score and P value. in vitro, spheres were collected by gentle centrifugation and dissociated to single cells using TrypLE Express (Invitrogen). Statistical analysis Following dissociation, trypsin inhibitor (Invitrogen) was used Statistical analyses were performed using GraphPad Prism 6.0 to neutralize the reaction, and the cells were cultured to generate (GraphPad Software, Inc.) and SPSS version 21.0 (IBM). Inde- the next generation of spheres. pendent Student t test was used for the analysis of all in vitro and in fi Annexin V apoptosis assay vivo assays. Clinicopathologic signi cance in clinical samples was x2 Cells were treated with cisplatin (1 mg/mL) for 48 hours. evaluated by test or Fisher exact test for categorical data and – Following treatment, cells were harvested and stained with pro- independent Student t test for continuous data. Kaplan Meier pidium iodide (PI) and FITC-conjugated Annexin V as provided analysis and log-rank test were used for survival analysis. Statis- fi fi by the Annexin V-FLUOS Staining Kit (Roche). Samples were tical signi cance was de ned as P 0.05 ( , P 0.05; , P 0.01; analyzed on BDFACSCanto II (BD Biosciences) with data ana- , P 0.001). Error bars represent SEM of the data. lyzed by FlowJo (Tree Star Inc.). Results In vivo subcutaneous tumorigenicity and tail vein Transcriptome sequencing profiling identifies FSTL1 to be metastasis models frequently overexpressed in ESCC The study protocol was approved by and performed in accor- Transcriptome sequencing was previously performed in our dance with the Committee of the Use of Live Animals in Teaching laboratory on three pairs of matched tumor and adjacent and Research at The University of Hong Kong. Tumorigenicity was nontumor tissues from patients of Chinese origin with ESCC determined by subcutaneous injection into the flank of 4–5 weeks (patients 16, 18, and 19) using the Illumina Genome Analyzer old female Balb/c nude mice (n ¼ 9). For conditioned medium IIx platform (Solexa; GSE29968; ref. 2). FSTL1 was a candidate treatment, ESCC cells were first pretreated with conditioned gene of interest due to increased expression in all three ESCC medium for 24 hours and then coinjected subcutaneously into samples compared with its corresponding nontumor counter- the flank of 4–5 weeks old female Balb/c nude mice (n ¼ 5), with parts (Fig. 1A, left; P < 0.005) and that the chromosomal conditioned media replenished subcutaneously directly around location (3q) on which FSTL1 is localized, has previously been the injection site every 3 days. For neutralizing antibody treat- reported to be frequently amplified in ESCC (7–9). qRT-PCR ment, ESCC cells were subcutaneously injected into the flanks of was performed to validate our RNA-Seq finding, and the results 4–5 weeks old female Balb/c nude mice. When tumor volume were concordant with the transcriptomic results (Fig. 1A, right). reaches approximately 100 mm3,25mg of FSTL1 neutralizing To determine whether the upregulation of FSTL1 was a com- antibody or IgG control was injected intratumorally every other mon event, we extended our analysis to an additional 73 paired day. Mice were sacrificed two days after the sixth treatment (n ¼ 5). nontumor and primary ESCC samples. By qRT-PCR, FSTL1 was Tumor incidence and tumor size were recorded. Tumor volumes foundtobeupregulated(defined by a fold change increase of were calculated as volume (cm3) – L W2 0.5 with L and W greater than 2) in over 57% of primary ESCCs when compared representing the largest and smallest diameters, respectively. with their corresponding nontumor esophageal tissue samples. Tumors formed were harvested for histologic analysis. For tail On average, ESCC displayed an average of 2.57-fold higher vein metastasis, luciferase-labeled cells were intravenously FSTL1 expression than nontumor esophageal tissue (Fig. 1B; injected into 6–7 weeks old Balb/c nude mice (n ¼ 10) through Supplementary Table S1). Proteomic FSTL1 levels was also the lateral tail vein. Seven weeks after implantation, mice were examined by IHC using a tissue microarray comprising of administered with 100 mg/kg D-luciferin via peritoneal injection 5 225 nontumor and 394 ESCC cases in which FSTL1 was minutes before bioluminescent imaging (IVIS 100 Imaging Sys- consistently detected in the majority of the ESCC sectors but tem, Xenogen). Lungs of the mice were collected and fixed for at reduced levels in the normal esophageal epithelia (Fig. 1C; paraffin sectioning. Sections were stained with hematoxylin and Supplementary Fig. S1). Note that only expression in the eosin (H&E) for histologic examination for sign of tumor growth squamous epithelium lining of the nontumor esophageal and metastasis. samples (highlighted in red dotted lines in Fig. 1C) was taken into account. Eighty-four percent of all nontumor samples Agilent cDNA microarray and analysis displayed low or medium FSTL1 expression levels, whereas Total RNA was harvested and purified using RNeasy Plus Mini only 56.1% of the tumor samples showed low or medium Kit (Qiagen). Quality of the RNA was analyzed on Agilent 2100 FSTL1 expression level. In contrast, only 16% of the nontumor Bioanalyzer at Centre for Genomic Sciences at the University of samples showed high FSTL1 levels as compared with 43.9% in Hong Kong. SurePrint G3 Human Gene Expression v3 8 60K ESCC samples. High FSTL1 expression correlated with "older Microarray Kit (Agilent Technologies) covering 26,083 Entrez age," as defined by age over the median in our cohort sampled

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Figure 1. FSTL1 is frequently overexpressed in ESCC. A, Left, reads per kilobase per million (RPKM) value of FSTL1 in three pairs of nontumor (N) and ESCC (T) clinical samples examined by RNA-Seq (patients 16, 18 and 19; left). Right, validation of FSTL1 expression by qRT-PCR in the same three ESCCs and their adjacent nontumor samples. B, Left, FSTL1 expression in 73 matched nontumor (NT) and primary ESCC as detected by qRT-PCR. , P < 0.001. Right, waterfall plot showing fold-change expression of FSTL1 in the 73 ESCC clinical samples as compared with their nontumor counterpart. C, Representative IHC staining of FSTL1 in matched non-neoplastic squamous epithelium (nontumor) and poorly differentiated ESCC tissue from three patient samples. Scale bar, 100 mm. Bar chart summary of the distribution of FSTL1 expression levels in nontumor versus ESCC examined by IHC on tissue microarrays (n ¼ 619 total cases; P < 0.001). D, Kaplan–Meier survival analysis comparing the cumulative survival rate of all patients with different FSTL1 expression levels (P ¼ 0.0021). E, Box plot showing expression of FSTL1 in the sera collected from 30 healthy normal individuals and 104 ESCC patients (, P < 0.001).

(>59), but did not correlate with other clinicopathologic para- Gene amplification as a mechanism of FSTL1 overexpression meters like pathologic stage, tumor size, lymph node metastasis in ESCC nor differentiation (Supplementary Table S2). Importantly, In addition to clinical samples, endogenous and secretory medium to high FSTL1 expression significantly correlated with FSTL1 expression was also examined in a panel of esophageal a worst overall survival rate. Patients with low FSTL1 expression cancer cell lines by Western blot analysis. Five of the 6 ESCC cell had, on average, a longer survival time [47 months, 95% lines examined showed a range of endogenous FSTL1 expres- confidence interval (CI), 10.28] and better prognosis as com- sion, which we later used for selection of which cells to use for pared with patients with medium and high FSTL1 expression overexpression and knockdown studies (Fig. 2A). Overexpres- [27 months; 95% CI, 6.70; and 25 months, 95% CI 4.64, sion of oncogenes in cancers is often associated with gene-copy respectively; Fig. 1D). Thus, endogenous FSTL1 overexpression number or chromosomal amplification (38). Because the locus is tightly associated with adverse ESCC pathogenesis. of FSTL1 at 3q has previously been reported to be frequently amplified in ESCC (7–9), we performed FISH analysis to High serum FSTL1 expression is tightly associated with examine whether gene and/or chromosomal amplification older age and represents a sensitive and specific biomarker represents a mechanism to FSTL1 overexpression in ESCC for ESCC diagnosis (Fig.2B).FISHanalysiswascarriedoutonbothESCCcell An old study by Tanaka and colleagues has suggested FSTL1 to linesaswellasformalin-fixed paraffin-embedded sections from exist in a soluble form, where it can be detected in the sera and 45 nontumor and 51 ESCC tissue samples. Chromosomal synovial fluid of patients suffering from rheumatoid arthritis (19). region encompassing FSTL1 was not found to be amplified in To investigate whether serum FSTL1 levels were altered in ESCC the peripheral blood mononuclear cells from healthy individ- patients, serum from 30 healthy individuals and 104 ESCC ual control nor the 2 ESCC cell lines with absent or low FSTL1 patients were collected for ELISA analysis. A marked difference expression levels [luciferase-labeled KYSE150 (KYSE150-Luc) between the two groups was observed (Fig. 1E; Supplementary and HKESC1]. Most cells only displayed two signals represent- Table S3). ing chromosome region of FSTL1 in green and two control

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Figure 2. Overexpression of FSTL1 in ESCC is in part a result of gene amplification. A, Measurement of proteomic (endogenous and secretory) FSTL1 expression levels in a panel of ESCC cell lines by Western blot analysis. B, The ideogram of chromosome 3 shows localization of FSTL1 and the BAC clones used. C and D, Detection of DNA copy number change of FSTL1 by dual-color FISH in absent/low FSTL1-expressing ESCC cell lines, high FSTL1-expressing ESCC cell lines (C), and formalin-fixed paraffin-embedded nontumor and ESCC clinical samples with known FSTL1 overexpressionintheESCCcounterpart(D). BAC probe to FSTL1 and the control reference probe to the centromere of chromosome 3 are represented by green and red signals, respectively. Nuclei, blue. Scale bar, 5 mm. Peripheral blood mononuclear cells (PBMC) from healthy individual were used as control. E, Bar chart summary of the distribution of normal and abnormal copy number in nontumor versus ESCC examined by FISH (n ¼ 96 total cases; P < 0.001). F, Bar chart summary of the distribution of FSTL1 expression levels in normal versus abnormal copy number samples examined by FISH (n ¼ 96 total cases; P < 0.0001).

signals in red (Fig. 2C). In contrast, amplification was evident FSTL1 contributes to augmented tumorigenic, metastatic, and in the 2 ESCC cell lines that express high levels of FSTL1 (EC109 chemoresistance potential in ESCC and EC9706), where most cells displayed an abnormal FSTL1 To ascertain whether there is causative relationship between copy number (Fig. 2C). Of the 45 nontumor and 51 ESCC FSTL1 overexpression and altered cancer and stem cell–like phe- tissue samples tested, FSTL1 copy number abnormality (rang- notype in ESCC, we generated stably overexpressed FSTL1 ing from 3 to 6) was found in approximate;y 7% (3 of 45) KYSE150-Luc and stably repressed FSTL1 EC109 cells by lenti- nontumor epithelial tissue, while >60% (31 of 51) ESCC cases viral-based transduction. The successful overexpression and displayed FSTL1 gene amplification (Fig. 2D and E). More knockdown of FSTL1 was successfully validated at proteomic importantly, FSTL1 copy number abnormality positively cor- levels (Fig. 3A). Overexpressing FSTL1 in KYSE150-Luc resulted related with higher proteomic FSTL1 expression (Fig. 2F). These in a significant increase in the efficiency of cells to proliferate (Fig. observations provide evidence in support of the notion that 3B) and form foci (Fig. 3C) when compared with empty vector gene or chromosome 3q amplification is, at least in part, (EV) controls. Conversely, repressing FSTL1 expression in EC109 implicated in FSTL1 overexpression in human ESCC. cells yielded an opposing effect (Fig. 3B and C). Next, we also

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Figure 3. FSTL1 contributes to aggressive cancer phenotype in ESCC cells in vitro. A, Validation of FSTL1 knockdown (EC109) and overexpression (KYSE150-Luc) at proteomic (endogenous and secretory) levels by Western blot. b-Actin and silver staining were used as loading control. B, Growth curve of indicated stable cell lines by XTT cell proliferation assay. C, Quantification of colonies induced by the indicated stable cell lines. D and E, Quantification of number of cells that migrated through a membrane (D) or invaded through a Matrigel-coated membrane (E). F, Quantification of spheroids induced by the indicated stable cell lines. G, Percentage of Annexin V–positive cells in the indicated stable cell lines following cisplatin treatment. H, Representative IHC images and bar chart summary of FSTL1(þ)cellsinKYSE520 xenograft tumors treated with varying dosage of cisplatin. Scale bars in D–H,100mm. Scale bar in foci formation image, 5 mm. , P 0.05; , P 0.01; , P 0.001.

investigated whether overexpression of FSTL1 has a promoting cell–like properties of cancer cells are thought to contribute to effect on ESCC cell migration and invasion. Stable FSTL1 over- cancer relapse due to their ability to self-renew and resist chemo- expression led to a signiﬁcant increase in the ability of cells to therapy (39). Thus, we also evaluated whether overexpression of migrate and invade through an extracellular matrix coating. On FSTL1 can promote self-renewal and confer resistance to chemo- the contrary, stable knockdown of FSTL1 resulted in opposing therapy. FSTL1 overexpression enhanced the self-renewing ability effects (Figs. 3D and E). Similar results were also obtained when of ESCC cells as demonstrated by spheroid formation assays, the same experiment was performed in the presence of mitomycin while FSTL1 knockdown resulted in the contrary (Fig. 3F). To C, where cells were inhibited to proliferate, suggesting that FSTL1- examine whether FSTL1 confers chemoresistance advantage to mediated migration and invasion is not a misinterpretation of the ESCC cells, Annexin V apoptosis ﬂow cytometry analysis was cells' altered ability to proliferate (Supplementary Fig. S2). Stem performed on cisplatin-treated ESCC cells with or without FSTL1

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expression modulated. The apoptotic index of FSTL1-expressing tasis models were also conducted in vivo. KYSE150-Luc cells with ESCC cells was significantly lower than that of empty vector only FSTL1 stably overexpressed formed larger tumors as compared control cells (26.98% vs. 73.33%) after a 48-hour exposure to with EV control cells when injected subcutaneously into immu- cisplatin, whereas the apoptotic index of FSTL1-repressed ESCC nodeficient mice (Fig. 4A, top; n ¼ 9). Histologic analysis of the cells was significantly higher than that of knockdown control cells xenografts revealed tumor cells to form packed nests and invasive (56.80% and 66.10% vs. 35.98%; Fig. 3G). To further obtain cords surrounded by a desmoplastic stroma, suggestive of a poorly evidence in support of this observation, we analyzed FSTL1 differentiated carcinoma (Fig. 4A, bottom). Likewise, KYSE150- expression on tissue sections obtained from our recently estab- Luc cells with FSTL1 stably overexpressed displayed a superior lished cisplatin-resistant KYSE520 ESCC xenograft model. ability to metastasize to the lung following cell injection through KYSE520 ESCC xenografts in NOD-SCID mice were treated with the tail vein as compared with EV controls, as supported by varying doses of cisplatin (0, 1, 2.5, and 5 mg/kg), with treatment stronger bioluminescence signal in the lung (Fig. 4B and C; resulting in variable tumor inhibition among the xenografts in a n ¼ 10). Histologic analysis revealed significantly more and larger dose-dependent trend (40). Interestingly, IHC results showed a metastatic foci in the harvested lung tissues of mice injected with stepwise enrichment in the frequency of FSTL1(þ) cells in xeno- ESCC cells with FSTL1 overexpressed (Fig. 4D). These observa- graft tumors treated with increasing doses of cisplatin (Fig. 3H). tions suggest a role for FSTL1 in the regulation of proliferation, To further extrapolate this to a clinical setting, we next studied metastasis, and chemoresistance in ESCC. whether chemotherapy would enrich FSTL1(þ) cells in ESCC clinical samples. To this end, 22 and 15 ESCC clinical samples Secretory FSTL1 expression promotes, while suppression of were collected from patients who had or had not undergone FSTL1 by neutralizing antibody attenuates aggressive preoperative chemotherapy treatment prior to esophagectomy, features of ESCC cells respectively. Although not statistically significant (P ¼ 0.0767), In light of the critical roles of endogenous FSTL1 in conferring possibly due to a small sample size as samples of this sort is hard multiple cancer and stem cell–like properties in ESCC and to find, a trend toward increased FSTL1(þ) cells in ESCC patients the clinical relevance of both endogenous and secretory FSTL1 who had received chemotherapy prior to surgery was indeed in ESCC, we hypothesize that secretory FSTL1 may also be crucial observed (Supplementary Fig. S3). To further substantiate the in driving these oncogenic properties. To this goal, in vitro and role of FSTL1 in driving ESCC tumorigenicity and metastasis, in vivo experiments were carried out in three different settings, (i) subcutaneous tumorigenicity and experimental tail vein metas- FSTL1-overexpressing conditioned medium, (ii) recombinant

Figure 4. FSTL1 contributes to augmented tumorigenic and metastatic potential in ESCC cells in vivo. A, Representative xenograft tumors and H&E images of resected tumors derived from mice subcutaneously injected with KYSE150-Luc cells with or without FSTL1 stably overexpressed. Graph showing the volume of tumors generated in each group (n ¼ 9). B, Representative ex vivo imaging of lungs harvested from mice that received tail vein injections of KYSE150-Luc cells with or without FSTL1 stably overexpressed (n ¼ 10; top). B, Luciferase signals shown as dot plot (bottom). C, Graph showing the number of metastatic foci in KYSE150-Luc EV control group as compared with the same cells with FSTL1 stably overexpressed (n ¼ 10). D, Representative H&E images of resected lung sections derived from mice injected via tail vein with KYSE150-Luc cells with or without FSTL1 stably overexpressed. All scale bars, 100 mm. , P 0.05.

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FSTL1, and (iii) FSTL1 neutralizing antibody (41), all aimed FSTL1 drives ESCC through cross-talk between NFkB and SMAD at to examine the functional roles of secretory FSTL1 in ESCC signaling pathways (Fig. 5A–C, top). Treatment of low FSTL1-expressing KYSE150- To better understand the molecular mechanism underlying Luc ESCC cells with FSTL1-overexpressing conditioned medium FSTL1-driven ESCC tumor progression, we compared the tran- or human recombinant FSTL1 led to a significant increase in the scriptome profiles of KYSE150-Luc ESCC cells with or without ability of cells to migrate and invade (Fig. 5A and B). In addition, FSTL1 stably overexpressed by Agilent microarray profiling. Using treatment of KYSE150-Luc cells with FSTL1 conditioned medium a fold change cutoff of 1.5, 1,399 probes corresponding to 723 also promoted tumor growth potential in vivo, as demonstrated by protein-coding genes (471 up and 252 down) were found deregu- increased proliferative rate, endpoint tumor volume, and tumor lated. Differential expression pattern between EV control and weight (Fig. 5D). Next, we then treated high FSTL1-expressing FSTL1 OE cells was visualized using a hierarchical clustering EC109 ESCC cells with a homemade monoclonal FSTL1 neutral- heatmap (Fig. 6A). Pathway analysis was also carried out using izing antibody (41) to examine its therapeutic potential in sup- GSEA, where 10 gene sets were found to be significantly enriched pressing the oncogenic and metastatic properties of ESCC. EC109 with a false discovery rate (FDR) 0.05 (Supplementary Table displayed a diminished ability to migrate and invade when treated S4). In addition to TGFb signaling [normalized enrichment score with FSTL1 neutralizing antibody, as compared with IgG control (NES) ¼ 1.61; FDR ¼ 0.036], which FSTL1 has previously been (Fig. 5C). In vivo, treatment of ESCC cells with the FSTL1 neu- shown to be involved via antagonizing BMP (11–13, 20, 41), the tralizing antibody resulted in a marked diminished ability of the other two most significantly enriched gene sets identified were cells to grow and even led to a regression in the tumor size, as TNFa signaling via NFkB (NES ¼ 1.99; FDR ¼ 0.0) and epithelial- compared with IgG control (Fig. 5E). mesenchymal transition (NES ¼ 1.87; FDR ¼ 0.002; Fig. 6B;

Figure 5. Secretory FSTL1 contributes to aggressive features in ESCC cells. A–C, Quantiﬁcation of cells that migrated through a membrane or invaded through a Matrigel- coated membrane following treatment with conditioned medium collected from KYSE150-Luc cells with FSTL1 stably overexpressed (A) or PBS control or human recombinant FSTL1 (rFSTL1; B) or normal goat IgG control or anti-FSTL1 neutralizing antibody (C). D, Relative growth curve of xenograft tumors formed following subcutaneous injection of KYSE150-Luc cells treated with conditioned medium collected from KYSE150-Luc cells with EV (EV CM) or FSTL1 stably overexpressed (FSTL1 CM). Graphs showing tumor weight and volume of tumors generated in each group (n ¼ 5). Representative H&E images of resected tumors derived from mice in each group. E, Left, graph of average tumor volumes of mice along treatment course. Treatment administered at days 0, 2, 4, 6, 8, and 10. Right, bar graph showing tumor weight generated in each group (n ¼ 5). All scale bars, 100 mm. , P 0.05; , P 0.01; , P 0.001.

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Figure 6. FSTL1 drives ESCC through deregulated NFkB and SMAD pathways cross-talk. A, Hierarchical cluster heatmap analysis of gene expression profiles in EV versus FSTL1-overexpressing KYSE150-Luc ESCC cells. Each cell in the matrix represents a particular expression level, where red and green indicates high and low gene expression, respectively. B, GSEA comparison of differentially expressed genes in EV versus FSTL1-overexpressing KYSE150-Luc ESCC cells identified enrichment of TNFa signaling via NFkB, epithelial-mesenchymal transition (EMT), and TGFb signaling gene sets. NES, normalized enrichment score; FDR q, FDR q value. C and D, IPA prediction of 33 differentially expressed genes identified in our microarray that are known to be regulated by NFkBin FSTL1-overexpressing ESCC cells. Of these 33, 10 were also consistently found to be enriched in the GSEA TNFa signaling via NFkB gene set and thus were chosen for validation by qRT-PCR. E, Western blot analysis for expression of p-IkBa, total IkBa, p-SMAD1, and total SMAD1 in total lysate, as well as p-p65 and total p65 in the nuclear subfractionated protein lysate, in the indicated cells. b-Actin and histone H3 were used as loading controls.

Supplementary Table S4). Similar results were also obtained recombinant FSTL1. Conversely, p-SMAD1 was upregulated in through IPA analysis where both the NFkB complex and TNF ESCC cells with FSTL1 stably repressed or treated with FSTL1 family were identified to be significantly activated downstream neutralizing antibody (Fig. 6E). This observation coincides with effectors in FSTL1 overexpressing ESCC cells (Fig. 6C; Supple- our previous pathway enrichment analysis, where we found TGFb mentary Table S5). IPA analysis predicted 33 deregulated genes signaling to be positively enriched in ESCC cells with FSTL1 identified in our microarray to be regulated by NFkB and of these, overexpressed (Fig. 6B) and together, suggests that TGFb/BMP/ 10 of them were also found in the list predicted by GSEA (Fig. 6D), SMAD is also a critical effector of FSTL1-mediated ESCC. including BIRC3, CCL2, CCL3, CD69, GFPT2, IL1A, IL1B, IL23A, To further validate the role of NFkB activation in FSTL1- IL6, and TNFRSF9, and for this reason, were selected for qRT-PCR induced tumorigenicity, metastasis, and stemenss, we analyzed validation. Correlation between qRT-PCR and microarray data the impact of introducing IMD-0354, an inhibitor of NFkB, into indicates a high level of concordance between the differential ESCC cells with FSTL1 stably overexpressed on these altered expression measurements from both platforms (Fig. 6D). More phenotypes. Successful suppression of the NFkB pathway follow- importantly, phosphorylation of key members of the NFkB ing addition of IMD-0354 in ESCC cells with FSTL1 stably over- pathway, namely IkBa and p65, were also found to be signifi- expressed was confirmed by a diminished p-IkBa and p-p65 cantly altered at the proteomic level, not only in FSTL1-over- protein expression (Fig. 7A). IMD-0354 suppressed the cancer expressing KYSE150-Luc cells, but also in FSTL1 repressed EC109 and stem cell–like properties conferred by FSTL1 overexpression, cells, KYSE150-Luc cells treated with EV or FSTL1 OE conditioned as evidenced by the diminished abilities of ESCC cells to migrate, medium, KYSE150-Luc cells with control or recombinant FSTL1 invade, form spheroids, and proliferate (Fig. 7B). Similar func- and EC109 cells treated with IgG control or FSTL1 neutralizing tional observations were also noted when IMD-0354 was used antibody, suggesting NFkB to be a critical downstream effector of against ESCC cells treated with FSTL1-overexpressing conditioned FSTL1-mediated ESCC (Fig. 6E). A number of studies in the past medium (Supplementary Fig. S4A) and ESCC cells treated with have reported FSTL1 to act as a BMP signaling antagonist in recombinant FSTL1 (Supplementary Fig. S4B). Previous studies development (11–13, 20, 41). Here, we also found altered phos- have demonstrated the existence of a cross-talk between TLR4/ phorylated SMAD1 expression in ESCC cells with or without MyD88/NFkB and BMP/SMAD signaling in osteoblastic differ- FSTL1 modulated. More specifically, p-SMAD1 was found to be entiation (42). Given that we observed both NFkB and SMAD to downregulated in ESCC cells with FSTL1 stably overexpressed or be deregulated in FSTL1-mediated ESCC, we went on to examine treated with FSTL1-overexpressing conditioned medium or the link between these two pathways and noted that addition of

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Figure 7. A, Western blot analysis for FSTL1, p-IkBa,totalIkBa, p-SMAD1, and total SMAD1 in total lysate, as well as p-p65 and total p65 in the nuclear subfractionated protein lysate, in KYSE150-Luc cells with or without FSTL1 stably overexpressed, in the presence or absence of IMD-0354. DMSO was used as a control for IMD-0354. b-Actin and histone H3 were used as loading controls. B, Quantiﬁcation of number of cells that migrated through a membrane, invaded through a Matrigel-coated membrane, formed spheroids or formed colonies in KYSE150-Luc cells with or without FSTL1 stably overexpressed in the presence or absence of IMD-0354. C, Western blot analysis for FSTL1, p-IkBa,totalIkBa, p-SMAD1, and total SMAD1 in total lysate, as well as p-p65 and total p65 in the nuclear subfractionated protein lysate, in KYSE150-Luc cells with or without FSTL1 stably overexpressed in the presence or absence of C34. b-Actin and histone H3 were used as loading controls. D, Quantiﬁcation of number of cells that migrated through a membrane, invaded through a Matrigel-coated membrane, formed spheroids or formed colonies in KYSE150-Luc cells with or without FSTL1 stably overexpressed in the presence or absence of C34. Scale bars in migration, invasion, and spheroid images at 25 mm. Scale bars in foci formation image, 5 mm. E, Cartoon diagram illustrating the proposed FSTL1-regulated oncogenic and metastatic mechanism in ESCC tumorigenesis. RE, responsive element. , P 0.05; , P 0.01; , P 0.001.

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IMD-0354 led to a rescue in p-SMAD1 signaling (Fig. 7A), that did not have chromosome 3q amplified but had FSTL1 suggesting that NFkB acts upstream of the BMP pathway and overexpressed. A recent study in prostate cancer has suggested cross-talk between the two pathways do exist and act in concert to the involvement of androgen receptor transcriptional activity in promote ESCC. driving FSTL1 upregulation (29). FSTL1 is also identified as a Previous studies have demonstrated effects of FSTL1 on TLR4/ TGFb-inducible gene (43). Studies in rheumatoid arthritis, sclero- MyD88/NFkB pathway (21, 31–35). To examine the role of TLR4 derma endothelial cells, and nasopharyngeal carcinoma have also in mediating FSTL1 induced NFkB activation in ESCC, we per- identified other potential regulatory mechanisms leading to formed rescue experiments utilizing an inhibitor of TLR4 (C34). FSTL1 inactivation, including miR-27a (44), histone deacetylase Inhibition of TLR4 attenuated expression of p-IkBa and p-p65, 5 (45), deregulations, and hypermethylation in the FSTL1 pro- promoted p-SMAD1 expression, and a concomitant diminished moter (25). abilities of ESCC cells to migrate, invade, form spheroids and Through functional studies using overexpression and sup- proliferate (Fig. 7C and D). Again, similar functional observations pression lentiviral-based expression systems as well as recom- were also noted when C34 was used against ESCC cells treated binant FSTL1, FSTL1-expressing conditioned medium and a with FSTL1-overexpressing conditioned medium (Supplementary homemade FSTL1 neutralizing antibody (kindly provided by Fig. S5A) and ESCC cells treated with recombinant FSTL1 (Sup- Prof. Wen Ning, Nankai University; ref. 41), we found the plementary Fig. S5B). Taken together, our results suggest that upregulation of FSTL1 to promote tumorigenicity, metastasis, FSTL1 promotes ESCC through stimulating NFkB activation, self-renewal, and resistance to cisplatin. Note that since FSTL1 attenuating BMP signaling and modulating the cross-talk between can also lead to increased cell proliferation, as demonstrated by these two pathways (Fig. 7E). foci formation and XTT cell proliferation assays, we must take caution when we interpret our metastasis findings, such that we must ensure that the metastasis effect is not a byproduct of the Discussion cells' altered proliferation potential. To address this, we repeat- FSTL1 has previously been reported to play both oncogenic ed our migration and invasion assays again, in the presence of and tumor-suppressive roles in various cancer types (23–30). mitomycin C, a drug used to inhibit cell proliferation. Previ- Yet, to the best of our knowledge, this is the first study to report ously, our collaborator Ning and colleagues have demonstrated its significance in ESCC. FSTL1 was first systematically identi- blockade of FSTL1 with this neutralizing antibody in mice to fied to be frequently upregulated in ESCC as compared with reduce bleomycin-induced fibrosis in vivo,suggestingthat adjacent nontumor esophageal tissues by RNA-Seq profiling. FSTL1 may serve as a novel therapeutic target for treatment of Among the other genes that were also found to be deregulated progressive lung fibrosis (41). However, use of this neutralizing in ESCC, we chose FSTL1 to focus on as first,thegeneislocated antibody for treatment of cancer in general has not been on chromosome 3q, which is a known chromosomal ampli- applied nor tested. FSTL1 encodes a 308 amino acid secretory fication hotspot in ESCC (7–9) and second, because the gene protein with a 20 amino acid signaling peptide located at the N- encodes for a secretory protein. The late clinical presentation of terminus. In this work, we have demonstrated FSTL1 to be ESCC often limits the success of treatment at the stage of secreted from ESCC cells and that it will act, via an autocrine discovery. Thus far, no serum screening methods have been manner, to promote aggressive cancer features in ESCC devel- employed in a clinical setting for neither ESCC diagnosis nor opment and progression. Past literature have found, via IHC prognosis. Thus, identification of a functionally important staining of murine joint sections, expression of FSTL1 in all cell secretory protein as a biomarker is urgently needed. Data from types of the mesenchymal lineage including osteocytes, chon- our study found FSTL1 to have obvious potential to be devel- drocytes, adipocytes, and fibroblasts. More specifically, FSTL1 oped into a diagnostic and prognostic biomarker, especially for could be induced in osteoblasts, adipocytes, and human fibro- noninvasive screening in high-risk areas, for instance China. blast-like synoviocytes (46). In colorectal cancer, FSTL1 was This is the first study to identify the significance of secretory alsofoundtobesignificantly increased in cancer-associated FSTL1 in ESCC and any tumor type. The markedly lower FSTL1 fibroblasts, without significant expression in the cancer epithe- levels in healthy individual patient sera in our study allows for lial cells (47–48). Further studies on whether FSTL1 is also a good prediction of ESCC by FSTL1. However, as the number expressed in the cancer-associated fibroblasts in ESCC stroma of healthy individuals samples included in our current study is would be worthwhile to further delineate origin of FSTL1 in limited (n ¼ 30), a study with a larger cohort of samples is addition to ESCC cells and whether the secretory protein required for further validation. As our functional studies iden- functions in both autocrine and paracrine manners. tified a role of FSTL1 in conferring resistance of ESCC cells to For a systematic identification of the underlying molecular the chemo-drug cisplatin, it would also be worthwhile to mechanism mediating FSTL1-induced ESCC, we compared the explore whether FSTL1 can be used to predict response to expression profiles of KYSE510 ESCC cells with or without FSTL1 cisplatin treatment and/or tumor recurrence following cisplat- overexpressed using Agilent microarray profiling. By pathway in therapy. enrichment analyses, we found the NFkB pathway to be activated Gene amplification is one of the most common mechanisms of and the BMP pathway to be silenced in FSTL1-overexpressing gene upregulation in cancer. Our results here suggest gene ampli- ESCC cells. Deregulation in both of these pathways have previ- fication of FSTL1 and/or chromosome 3q to be tightly correlated ously been implicated in FSTL1-mediated ESCC (11–13, 20–21, with high FSTL1 protein expression, suggesting gene amplifica- 31–35, 41). The two pathways have also been found to interlink tion may at least in part be implicated in FSTL1 activation in (41), yet our study is the first to demonstrate the cross-talk human ESCC. Yet with that said, it is unlikely that gene ampli- between NFkB and BMP signaling where they both work in fication represents the sole mechanism leading to FSTL1 upregu- concert to promote ESCC. More specifically, there is data to show lation in ESCC as we also found a small portion of clinical samples TLR signaling pathways to culminate in activation of NFkB that

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goes on to control the expression of an array of inflammatory FSTL1 could hold potential as a novel therapy for the treatment of cytokine genes (34). Recently, FSTL1 was found to bind to TLR4 ESCC. (32) and its coreceptor CD14 (36). Recombinant FSTL1 was also found to induce IL6 and IL8 production from 293 cells in a CD14- Disclosure of Potential Conflicts of Interest fi and TLR4-dependent manner (32). In culture broblast-like No potential conflicts of interest were disclosed. synoviocytes, FSTL1 was found to activate NFkB signaling, as evident by Western blot and chromatin immunoprecipitation assays at p65-binding sites (35). Furthermore, two recent reports Authors' Contributions have also demonstrated a link between FSTL1, TLR4, and NFkB Conception and design: M. Chi-Chung Lau, K.-Y. Ng, S. Ma Development of methodology: M. Chi-Chung Lau, T.K. Lee (31, 33). However, none of the reports stated are related to cancer Acquisition of data (provided animals, acquired and managed patients, and ESCC. Taking into account the literature and in combination provided facilities, etc.): M. Chi-Chung Lau, K.-Y. Ng, T.L. Wong, M. Tong, with the fact that our profiling data, comparing empty vector and X.-Y. Ming, S. Law, N.P. Lee, Y.-R. Qin, K.W. Chan, S. Ma FSTL1-overexpressing ESCC cells, enriched for an altered NFkB Analysis and interpretation of data (e.g., statistical analysis, biostatistics, and TGFb signaling pathway, we hypothesized that FSTL1 will computational analysis): M. Chi-Chung Lau, K.-Y. Ng, T.L. Wong, X.Y. Guan, drive ESCC via modulating TLR4/NFkB/BMP signaling. S. Ma Writing, review, and/or revision of the manuscript: M. Chi-Chung Lau, In addition, there is a handful of studies where FSTL1 has been K.-Y. Ng, S. Ma found to modulate tumor immune response. For instance, Mur- Administrative, technical, or material support (i.e., reporting or organizing akami and colleagues have demonstrated FSTL1 to evoke an innate data, constructing databases): M. Chi-Chung Lau, S. Law, A.L.M. Cheung, immune response via CD14 and TLR4, thereby modulating organ Y.-R. Qin, W. Ning, S. Ma development (32). Kudo-Saito and colleagues have shown FSTL1 Study supervision: S. Ma to promote bone metastasis by causing immune dysfunction (28), and a separate study has also found epigenetic inactivation of Acknowledgments FSTL1 to mediate tumor immune evasion in nasopharyngeal We thank the Faculty Core Facility at the Faculty of Medicine, The University carcinoma (25). Thus, it would also be of interest to further explore of Hong Kong, for providing and maintaining the equipment needed for flow whether FSTL1 secreted from ESCC cells would mediate alterations cytometry, confocal microscopy, and animal imaging work. in immune cells like macrophages, T cells, etc., to modulate tumor immune response. Grant Support Findings from our current study suggest the novel oncogenic, This study was supported by funding from the Research Grants Council metastatic, and cisplatin resistance role of FSTL1 in ESCC and its of Hong Kong - Collaborative Research Fund (C7038-14G to X.Y. Guan and potential use as a diagnostic and prognostic ESCC biomarker. S. Ma). The costs of publication of this article were defrayed in part by the payment of Both endogenous and secretory FSTL1 plays important roles in page charges. This article must therefore be hereby marked advertisement in mediating aggressive cancer properties in ESCC via canonical accordance with 18 U.S.C. Section 1734 solely to indicate this fact. NFkB pathway activation and BMP pathway attenuation. BMP and NFkB pathways cross-talk to drive these aggressive cancer Received May 15, 2017; revised July 27, 2017; accepted August 30, 2017; features in FSTL1-mediated ESCC. With more studies, targeting published OnlineFirst September 7, 2017.

References 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer 10. Adams D, Larman B, Oxburgh L. Developmental expression of mouse statistics. CA Cancer J Clin 2011;61:69–90. follistatin-like 1 (Fstl1): dynamic regulation during organogenesis of the 2. Ma S, Bao JY, Kwan PS, Chan YP, Tong CM, Fu L, et al. Identification of kidney and lung. Gene Expr Patterns 2007;7:491–500. PTK6, via RNA sequencing analysis, as a suppressor of esophageal squa- 11. Geng Y, Dong Y, Yu M, Zhang L, Yan X, Sun J, et al. Follistatin-like 1 (Fstl1) mous cell carcinoma. Gastroenterology 2012;143:675–86. is a bone morphogenetic protein (BMP)4 signaling antagonist in control- 3. Tong M, Chan KW, Bao JY, Wong KY, Chen JN, Kwan PS, et al. Rab25 is a ling mouse lung development. Proc Natl Acad Sci USA 2011;108:7058–63. tumor suppressor gene with antiangiogenic and anti-invasive activities in 12. Sylva M, Li VS, Buffing AA, van Es JH, van den Born M, van der Velden S, esophageal squamous cell carcinoma. Cancer Res 2012;72:6024–35. et al. The BMP antagonist follistatin-like 1 is required for skeletal and lung 4. Tang KH, Dai YD, Tong M, Chan YP, Kwan PS, Fu L, et al. A CD90(þ) organogenesis. PLoS One 2011;6:e22616. tumor-initiating cell population with an aggressive signature and meta- 13. Xu J, Qi X, Gong J, Yu M, Zhang F, Sha H, et al. Fstl1 antagonizes BMP static capacity in esophageal cancer. Cancer Res 2013;73:2322–32. signaling and regulates ureter development. PLoS One 2012;7:e32554. 5. Fung TM, Ng KY, Tong M, Chen JN, Chai S, Chan KT, et al. Neuropilin-2 14. Yang Y, Liu J, Mao H, Hu YA, Yan Y, Zhao C. The expression pattern of promotes tumorigenicity and metastasis in oesophageal squamous cell follistatin-like 1 in mouse central nervous system development. Gene Expr carcinoma through ERK-MAPK-ETV4-MMP-E-cadherin deregulation. Patterns 2009;9:532–40. J Pathol 2016;239:309–19. 15. Shimano M, Ouchi N, Nakamura K, van Wijk B, Ohashi K, Asaumi Y, et al. 6. Miyamae T, Marinov AD, Sowders D, Wilson DC, Devlin J, Boudrea R, et al. Cardiac myocyte follistatin-like 1 functions to attenuate hypertrophy Follistatin-like protein-1 is a novel proinflammatory molecule. J Immunol following pressure overload. Proc Natl Acad Sci USA 2011;108:899–906. 2006;177:4758–62. 16. Ogura Y, Ouchi N, Ohashi K, Shibata R, Kataoka Y, Kambara T, et al. 7. Bandla S, Pennathur A, Luketich JD, Beer DG, Lin L, Bass AJ, et al. Therapeutic impact of follistatin-like 1 on myocardial ischemic injury in Comparative genomics of esophageal adenocarcinoma and squamous cell preclinical models. Circulation 2012;126:1728–38. carcinoma. Ann Thorac Surg 2012;93:1101–6. 17. Oshima Y, Ouchi N, Sato K, Izumiya Y, Pimentel DR, Walsh K. Follistatin- 8. Bellinin MF, Silva AE, Varella-Garcia M. Genomic imbalances in esoph- like 1 is an Akt-regulated cardioprotective factor that is secreted by the ageal squamous cell carcinoma identified by molecular cytogenetic tech- heart. Circulation 2008;117:3099–108. niques. Genet Mol Biol 2010;33:205–13. 18. Wei K, Serpooshan V, Hurtado C, Diez-Cunado M, Zhao M, Maruyama S, 9. Song Y, Li L, Ou Y, Gao Z, Li E, Li X, et al. Identification of genomic et al. Epicardial FSTL1 reconstitution regenerates the adult mammalian alterations in oesophageal squamous cell cancer. Nature 2014;509:91–5. heart. Nature 2015;525:479–85.

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FSTL1-Mediated NFkB and BMP Pathway Deregulation in ESCC

19. Tanaka M, Ozaki S, Osakada F, Mori K, Okubo M, Nakao K. Cloning of 34. Kawai T, Akira S. Signaling to NFkB by toll-like receptors. Trends Mol Med follistatin-related protein as a novel autoantigen in systemic rheumatic 2007;13:460–9. diseases. Int Immunol 1998;10:1305–14. 35. Tanaka M, Murakami K, Ozaki S, Imura Y, Tong XP, Watanabe T, 20. Wu J, Dong Y, Teng X, Cheng M, Shen Z, Chen W. Follistatin-like 1 et al. DIP2 disco-interacting protein 2 homolog A (Drosophila) is a attenuates differentiation and survival of erythroid cells through candidate receptor for follistatin-related protein/follistatin-like 1 – Smad2/3 signaling. Biochem Biophys Res Commun 2015;466:711–6. analysis of their binding with TGF-b superfamily proteins. FEBS J 21. Ni S, Miao K, Zhou X, Xu N, Li C, Zhu R, et al. The involvement of follistatin- 2010;277:4278–89. like protein 1 in osteoarthritis by elevating NF-kB-mediated inflammatory 36. Shimada Y, Imamura M, Wagata T, Yamaguchi N, Tobe T. Character- cytokines and enhancing fibroblast like synoviocyte proliferation. ization of 21 newly established esophageal cancer cell lines. Cancer 2015;17:91. 1992;69:277–84. 22. Fan N, Shu H, Wang Y, Wang Y, Zhang L, Xia Z, et al. Follistatin-like 1: a 37. Guan XY, Sham JST, Tang TC, Fang Y, Huo KK, Yang JM. Isolation of a potential mediator of inflammation in obesity. Mediators Inflamm novel candidate oncogene within a frequently amplified region at 3q26 2013;2013:752519. in ovarian cancer. Cancer Res 2001;61:3806–9. 23. Chan QK, Ngan HY, Ip PP, Liu VW, Xue WC, Cheung AN. Tumor 38. Schwab M. Amplification of oncogenes in human cancer cells. Bioessays suppressor effect of follistatin-like 1 in ovarian and endometrial carcino- 1998;20:473–9. genesis: a differential expression and functional analysis. Carcinogenesis 39. Flemming A. Cancer stem cells: targeting the root of cancer relapse. Nat 2009;30:114–21. Rev Drug Discov 2015;14:165. 24. Zhao W, Han HB, Zhang ZQ. Suppression of lung cancer cell invasion and 40. Ming XY, Fu L, Zhang LY, Qin YR, Cao TT, Chan KW, et al. Integrin a7isa metastasis by connexin43 involves the secretion of follistatin-like 1 medi- functional cancer stem cell marker in oesophageal squamous cell carci- ated via histone acetylation. Int J Biochem Cell Biol 2011;43:1459–68. noma. Nat Commun 2016;7:13568. 25. Zhou X, Xiao X, Huang T, Du C, Wang S, Mo Y, et al. Epigenetic inactivation 41. Dong Y, Geng Y, Li L, Li X, Yan X, Fang Y, et al. Blocking follistatin-like 1 of follistatin-like 1 mediates tumor immune evasion in nasopharyngeal attenuates bleomycin-induced pulmonary fibrosis in mice. J Exp Med carcinoma. Oncotarget 2016;7:16433–44. 2015;212:235–52. 26.TanX,ZhaiY,ChangW,HouJ,HeS,LinL,etal.Globalanalysisof 42. Huang RL, Yuan Y, Zou GM, Liu G, Tu J, Li Q. LPS-stimulated inflammatory metastasis-associated gene expression in primary cultures from clinical environment inhibits BMP-2-induced osteoblastic differentiation through specimens of clear-cell renal-cell carcinoma. Int J Cancer 2008;123: crosstalk between TLR4/MyD88/NF-kB and BMP/Smad signaling. Stem 1080–8. Cells Dev 2014;23:277–89. 27. Reddy SP, Britto R, Vinnakota K, Aparna H, Sreepathi HK, Thota B, et al. 43. Shibanuma M, Mashimo J, Mita A, Kuroki T, Nose K. Cloning from a mouse Novel glioblastoma markers with diagnostic and prognostic value iden- osteoblastic cell line of a set of transforming-growth-factor-beta 1-regu- tified through transcriptome analysis. Clin Cancer Res 2008;14:2978–87. lated genes, one of which seems to encode a follistatin-related polypeptide. 28. Kudo-Saito C, Fuwa T, Murakami K, Kawakami Y. Targeting FSTL1 prevents Eur J Biochem 1993;217:13–9. tumor bone metastasis and consequent immune dysfunction. Cancer Res 44. Shi DL, Shi GR, Xie J, Du XZ, Yang H. MicroRNA-27a inhibits cell 103;73:6185–93. migration and invasion of fibroblast-like synoviocytes by targeting 29. Su S, Parris AB, Grossman G, Mohler JL, Wang Z, Wilson EM. Up-regulation follistatin-like protein 1 in rheumatoid arthritis. Mol Cells 2016; of follistatin-like 1 by the androgen receptor and melanoma antigen-A11 in 39:611–8. prostate cancer. Prostate 2017;77:505–16. 45. Tsou PS, Wren JD, Amin MA, Schiopu E, Fox DA, Khanna D, et al. Histone 30. Bae K, Park KE, Han J, Kim J, Kim K, Yoon KA. Mitotic cell death caused by deacetylase 5 is overexpressed in scleroderma endothelial cells and impairs follistatin-like 1 inhibition is associated with up-regulated Bim by inacti- angiogenesis via repression of proangiogenic factors. Arthritis Rheum vated Erk1/2 in human lung cancer cells. Oncotarget 2016;7:18076–84. 2016;68:2975–85. 31. Guo J, Liang W, Li J, Long J. Knockdown of FSTL1 inhibits oxLDL-induced 46. Wilson DC, Marinov AD, Blair HC, Bushnell DS, Thompson SD, Chaly Y, inflammation responses through the TLR4/MyD88/NF-kB and MAPK et al. Follistatin-like protein 1 is a mesenchyme-derived inflammatory pathway. Biochem Biophys Res Commun 2016;478:1528–33. protein and may represent a biomarker for systemic-onset juvenile rheu- 32. Murakami K, Tanaka M, Usui T, Kawabata D, Shiomi A, Iguchi-Hashi- matoid arthritis. Arthritis Rheum 2010;62:2510–6. moto M, et al. Follistatin-related protein/follistatin-like 1 evokes an 47. Torres S, Bartolome RA, Mendes M, Barderas R, Fernandez-Acenero MJ, innate immune response via CD14 and toll-like receptor 4. FEBS Lett Pelaez-Garcia A, et al. Proteome profiling of cancer-associated fibroblasts 2012;586:319–24. identifies novel proinflammatory signatures and prognostic markers for 33. Zhang ZM, Zhang AR, Xu M, Lou J, Qiu WQ. TLR-4/miRNA-32-5p/FSTL1 colorectal cancer. Clin Cancer Res 2013;19:6006–19. signaling regulates mycobacterial survival and inflammatory responses in 48. Chen SX, Xu XE, Wang XQ, Cui SJ, Xu LL, Jiang YH, et al. Identification of Mycobacterium tuberculosis-infected macrophages. Exp Cell Res 2017; colonic fibroblast secretomes reveals secretory factors regulating colon 352:313–21. cancer cell proliferation. J Proteomics 2014;110:155–71.

www.aacrjournals.org Cancer Res; 77(21) November 1, 2017 5899

FSTL1 Promotes Metastasis and Chemoresistance in Esophageal Squamous Cell Carcinoma through NF κB−BMP Signaling Cross-talk

Marco Chi-Chung Lau, Kai Yu Ng, Tin Lok Wong, et al.

Cancer Res 2017;77:5886-5899. Published OnlineFirst September 7, 2017.

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