Oncogenic Role of SFRP2 in P53-Mutant Osteosarcoma Development Via Autocrine and Paracrine Mechanism

Oncogenic role of SFRP2 in p53-mutant osteosarcoma development via autocrine and paracrine mechanism

Huensuk Kima,b,c, Seungyeul Yood,e, Ruoji Zhouf,g,AnXuf, Jeffrey M. Bernitza,b,c, Ye Yuana,b,c, Andreia M. Gomesa,b,h,i, Michael G. Daniela,b,c, Jie Sua,b,c,j, Elizabeth G. Demiccok,l, Jun Zhud,e, Kateri A. Moorea,b,c, Dung-Fang Leea,b,f,g,m,n,1,2, Ihor R. Lemischkaa,b,c,o,1,3, and Christoph Schaniela,b,c,o,p,1,2

aDepartment of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029; bThe Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029; cThe Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029; dDepartment of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029; eIcahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029; fDepartment of Integrative Biology and Pharmacology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030; gThe University of Texas MD Anderson Cancer Center, UTHealth Graduate School of Biomedical Sciences, Houston, TX 77030; hCentre for Neuroscience and Cell Biology, University of Coimbra, 3030-789 Coimbra, Portugal; iInstitute for Interdisciplinary Research, University of Coimbra, 3030-789 Coimbra, Portugal; jCancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065; kDepartment of Pathology, Icahn School of Medicine at Mount Sinai, New York, NY 10029; lDepartment of Pathology, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada; mCenter for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Houston, TX 77030; nCenter for Precision Health, School of Biomedical Informatics and School of Public Health, The University of Texas Health Science Center at Houston, Houston, TX 77030; oDepartment of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029; and pMount Sinai Institute for Systems Biomedicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029

Edited by Randall T. Moon, University of Washington, Seattle, WA, and approved October 4, 2018 (received for review August 26, 2018)

Osteosarcoma (OS), the most common primary bone tumor, is highly showed that SFRP2 hypermethylation and its decreased ex- metastatic with high chemotherapeutic resistance and poor survival pression are associated with prostate, liver, colorectal, and gas- rates. Using induced pluripotent stem cells (iPSCs) generated from Li– tric cancer (22–27). Originally, SFRP2 was reported as a secreted Fraumeni syndrome (LFS) patients, we investigate an oncogenic role antiapoptosis-related protein (28, 29). Ectopic expression of of secreted frizzled-related protein 2 (SFRP2) in p53 mutation- SFRP2 promotes cell growth and has antiapoptotic properties in associated OS development. Interestingly, we find that high renal and breast cancer (30–32). The role of SFRP2 appears to SFRP2 expression in OS patient samples correlates with poor survival. be cancer-type specific and remains controversial. Thus, in- MEDICAL SCIENCES Systems-level analyses identified that expression of SFRP2 increases vestigation and understanding of the role of SFRP2 in different during LFS OS development and can induce angiogenesis. Ectopic types of cancer, including OS, is warranted. SFRP2 overexpression in normal osteoblast precursors is sufficient Using induced pluripotent stem cells (iPSCs) derived from to suppress normal osteoblast differentiation and to promote OS LFS patients, we previously recapitulated the pathophysiological phenotypes through induction of oncogenic molecules such as FOXM1 and CYR61 in a β-catenin–independent manner. Conversely, Significance inhibition of SFRP2, FOXM1, or CYR61 represses the tumorigenic potential. In summary, these findings demonstrate the oncogenic role Li–Fraumeni syndrome is a rare disorder caused by germline of SFRP2 in the development of p53 mutation-associated OS and that TP53 mutations, predisposing patients to early-onset cancers, inhibition of SFRP2 is a potential therapeutic strategy. including osteosarcoma (OS). Here we demonstrate that strong expression of SFRP2, a reported WNT antagonist, in OS patient SFRP2 | p53 | osteosarcoma | autocrine | paracrine samples correlates with poor survival and that SFRP2 overexpression suppresses normal osteoblast differentiation, pro- steosarcoma (OS) is the most common primary bone tumor. motes OS features, and facilitates angiogenesis via autocrine OIt accounts for about 60% of all primary bone tumors and and paracrine mechanisms in an induced pluripotent stem cell about 2% of all childhood cancers (1, 2). Despite significant disease model. We show that these SFRP2-mediated phenotypes advances in OS treatment modalities, the 5-y overall survival rate are canonical WNT/β-catenin independent and are mediated has remained stable over the last 20 y at 60–70% for patients through induction of oncogenes such as FOXM1 and CYR61. We with primary OS and less than 30% for patients with metastasis further demonstrate that inhibition of SFRP2, FOXM1, or CYR61 (3, 4). This stagnation of clinical outcomes underlines the urgent represses tumorigenesis. Our data suggest that inhibition of necessity for novel model systems to study the mechanism of OS SFRP2 should be explored clinically as a strategy for treatment in a patient-specific context and to identify molecular targets for patients with p53 mutation-associated OS. the development of new therapeutic strategies. The tumor suppressor p53 regulates cell cycle, apoptosis, se- Author contributions: H.K., D.-F.L., I.R.L., and C.S. designed research; H.K., R.Z., A.X., nescence, metabolism, and cell differentiation (5–7). Therefore, it J.M.B., Y.Y., A.M.G., M.G.D., J.S., E.G.D., and K.A.M. performed research; K.A.M. contributed new reagents/analytic tools; H.K., S.Y., E.G.D., J.Z., and D.-F.L. analyzed data; and is not surprising that aberrant p53 expression contributes signifi- H.K., S.Y., D.-F.L., I.R.L., and C.S. wrote the paper. cantly to cancer development (8, 9). Half of all human sporadic The authors declare no conflict of interest. bone tumors have genetic lesions in TP53 (10, 11). Patients with Li–Fraumeni syndrome (LFS), which is caused by mutations in This article is a PNAS Direct Submission. TP53, show a 500-fold higher incidence of OS relative to the Published under the PNAS license. general population (12–14). Manipulation of p53 function con- Data deposition: The RNA-sequencing data have been deposited in the Gene Expression firmed its significance in OS development (15, 16) and identified Omnibus databank (accession nos. GSE102729 and GSE102732). mesenchymal stem cells (MSCs) and preosteoblasts (pre-OBs) as 1D.-F.L., I.R.L., and C.S. contributed equally to this work. the cellular origin of OS (17, 18). Osteoblast (OB)-restricted de- 2To whom correspondence may be addressed. Email: [email protected] or letion of p53/Mdm2 or p53/Rb resulted in OS development at a [email protected]. high penetrance of about 60% and 100%, respectively (19, 20). 3Deceased December 7, 2017. The first secreted frizzled-related protein (SFRP) was identi- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. fied as a WNT antagonist (21). As a known WNT antagonist, 1073/pnas.1814044115/-/DCSupplemental. SFRP2 is considered a tumor suppressor. Indeed, several reports

www.pnas.org/cgi/doi/10.1073/pnas.1814044115 PNAS Latest Articles | 1of10 Downloaded by guest on September 30, 2021 features of LFS-mediated OS development (33, 34). Taking ad- vantage of this platform, we observed increased expression of SFRP2 during LFS iPSC-derived OB differentiation. As a result of these findings and because the exact function of SFPR2 in OS is not clear, we investigated its role in LFS p53 mutation-mediated abnormal OB differentiation, tumorigenesis, and OS development. Here, we report that SFRP2 overexpression (SFRP2OE) induces OS phenotypes, increases FOXM1 expression, and promotes angiogenesis and endothelial expression of the matricellular protein CYR61. Conversely, targeting SFRP2OE in LFS and OS has therapeutic promise for OS subtypes with p53 mutations. Results SFRP2OE Is Associated with p53 Mutation-Mediated Human OS Development. To discover potential therapeutic targets for LFS- mediated OS, we compared the genome-wide transcripts of the LFS dataset (GSE58123) composed of MSCs differentiated to OBs in vitro from two LFS (P53p.G245D) patient iPSC lines, LFS1-A and LFS2-B, and one control iPSC line, WT-1 (SI Appendix,Table S1) (33), with an OS tumor-initiating cell dataset (GSE33458) (Fig. 1A) (35). We first identified differentially expressed genes (DEGs) between LFS and WT cells in the GSE58123 dataset. Because the dataset includes only one sample for each differentiation time point (D0, D7, D14, and D17), we performed a paired t test between each of the two LFS patient iPSC-derived samples with WT cells and identified DEGs common to both LFS samples with respect to WT cells (fold change >2, paired t test P < 0.01) (Dataset S1). This method enabled extraction of consistently up- or down-regulated DEGs (Fig. 1A). We identified SFRP2, which showed the greatest fold change (>30), as an overexpressed gene Fig. 1. SFRP2OE in LFS (G245D)-mediated OS. (A, Left) Global transcriptome with potential implication in OS development. Quantitative PCR analysis of LFS P53p.G245D- and WT iPSCs-derived MSCs differentiated to OBs. (Right) DEGs between LFS and WT cells were sorted by paired t test (P < 0.01) and Western blot analyses confirmed that SFRP2 expression is > significantly overexpressed in OBs derived from different LFS iPSC with a fold change 2. SFRP2 is an overexpressed gene that is also enriched in B the signature gene list of an OS gene set (GSE33458). (B) Quantitative PCR lines compared with WT controls (Fig. 1 ). We confirmed the analysis (Right) of SFRP2 expression (D4 of differentiation; data are shown as correlation between the P53p.G245D mutation and SFRP2 ex- ± = SI Appendix mean SD; n 3 independent repeats in triplicate) in LFS P53p.G245D and WT pression by introducing the mutation into WT MSCs ( , MSCs (*P < 0.05; **P <0.0001; ANOVA). The Inset depicts Western blotting Fig. S1A). SFRP2 expression was significantly increased in WT using mouse monoclonal anti-SFRP2 antibody (catalog no. sc-365524; Santa MSCs edited to harbor the P53p.G245D mutation but not in WT Cruz Biotechnology). (C) SFRP2 expression in human WT and LFS P53p.G245D MSCs with p53 depletion (shRNAs to p53) (SI Appendix,Fig. iPSC-derived OBs (D4 of differentiation) and OS cell lines (data are shown as S1B). Fibroblasts from LFS patients with the G245D mutation also mean ± SD; n = 3 independent repeats in triplicate; **P < 0.0001, ANOVA). (D, have higher SFRP2 expression (SI Appendix,Fig.S1C). Increased Upper) Expression of SFRP2 protein in LFS P53p.G245D s.c. xenograft tumors. SFRP2 expression is not unique to P53p.G245D. OBs derived from (Lower) Human prostate tissue was used as the positive staining control, and mouse muscle tissue was used as the negative staining control. (Scale bars: LFS iPSCs carrying a heterozygous P53p.Y205C mutation and μ from various p53-mutant embryonic stem cells (ESCs) (G245D, 100 m.) (E) Representative staining of SFRP2 protein expression levels in OS tissue samples from 151 OS patients. In D and E, rabbit polyclonal anti-SFRP2 G245S, R248W, and R249S) overexpress SFRP2 compared with SI Appendix D E (catalog no. PA5-29390; Thermo Fisher Scientific) was used. (F) Survival anal- their WT counterparts ( ,Fig.S1 and ). Addition- ysis of OS patients linked to the tissue microarray. The survival curves of ally, SFRP2 expression was detected in several OS cell lines har- SFRP2 high- and low-expression groups were calculated using the log-rank boring p53 mutations (HOS, MNNG, and 143B) or MDM2 test (χ2 = 11.13) (#P = 0.0009). amplification (SJSA-1) but not in the p53 WT OS cell line U2OS or the p53-null line SaOS2 (Fig. 1C). SFRP2 was also detected in s.c. tumors obtained from mouse xenograft assays with LFS cell Global Gene-Expression Analysis of SFRP2OE. To better understand lines (Fig. 1D) (33). These results demonstrate that SFRP2OE is the molecular role of SFRP2OE in LFS cells, we generated induced by LFS-associated p53 mutations. global gene-expression profiles of WT SFRP2OE cells, LFS cells Next, we analyzed the expression of SFRP2 levels in human OS with SFRP2 knockdown (SFRP2KD), parental WT cells (WT2- tissue samples using a tissue microarray spotted with 151 OS pa- C), and LFS cells (LFS2-B) at four time points (D0, D7, D14, tient tissue samples. The OS patient samples were categorized by and D17) during in vitro OB differentiation (SI Appendix, Fig. SFRP2 staining intensity: Groups 0 and 1were considered to be S2A). We used lentiviral vectors for doxycycline-inducible over- negative to low intensity, and groups 2 and 3 were high intensity expression (36) and for shRNA depletion (37) to generate stable (Fig. 1E and SI Appendix,Fig.S1F). Interestingly, high SFRP2 SFRP2OE or SFRP2KD cell lines, respectively, (SI Appendix, expression (intensity scores 2 and 3, with 3 being the highest ex- Fig. S2B). Principal component analysis (PCA) showed that WT pression) negatively correlated with patient survival (Fig. 1F and and LFS cells are clearly distinct from each other (Fig. 2A), SI Appendix,Fig.S1G and Table S2). As the patients’ p53 status consistent with our previous LFS RNA-sequencing (RNA-seq) associated with this OS tissue array is not available, a correlation data (33). Expression patterns of SFRP2OE cells were also dis- between SFRP2 expression and p53 mutation is not possible. tinct from those of WT cells and were more evident at later There was no gender difference in tumor incidence (SI Appendix, stages of differentiation (Fig. 2A). Likewise, LFS datasets di- Fig. S1H), and the area staining positive for SFRP2 is small in the verged more strongly at late stages of differentiation, while group with low SFRP2 expression (SI Appendix,Fig.S1I). These SFRP2KD data clustered together and were distinct from LFS data indicate that SFPR2OE is associated with poor prognosis for groups (Fig. 2A). This result shows that SFRP2OE significantly OS patients. alters gene expression in pre-OBs during differentiation. Linear

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1814044115 Kim et al. Downloaded by guest on September 30, 2021 regression with controlling for time identified DEGs between LFS/SFRP2OE down-regulated genes are associated with extra- SFRP2OE and WT, between LFS and WT, and between cellular matrix and cell–cell adhesion (SI Appendix,Fig.S2D). In- SFRP2KD and LFS groups [P < 0.001, corresponding permu- terestingly, tumor-suppressor genes such as CADM1 and tation false-discovery rate (FDR) <0.01] (Fig. 2B and Datasets CADM4 [tumor-suppressor gene database (44)] were enriched S2–S4). Database for Annotation, Visualization and Integrated in the down-regulated gene set (Fisher’s exact test, P = 0.004) (SI Discovery (DAVID) functional annotation was used to un- Appendix, Fig. S2D). derstand the general biological effect of DEGs associated with Finally, we compared SFRP2OE DEGs with the human OS Gene SFRP2OE. WT signature genes (DEGs during OB differentia- Expression Omnibus (GEO) set (GSE36001) to determine the cor- tion) were generally involved in bone mineralization and bone relation with OS features through GSEA. SFRP2OE and LFS DEGs morphogenetic protein (BMP) and WNT signaling pathways were highly correlated with human OS signature genes, whereas required for normal OB differentiation (Fig. 2C). Interestingly, SFRP2KD and WT DEGs were highly concordant with OB signature SFRP2OE signature genes were linked to cell proliferation, cell genes (Fig. 2G and SI Appendix,Fig.S2E). Additionally, SFRP2OE adhesion, and cell migration, categories implicated in the tu- signature genes were significantly enriched in up-regulated genes of morigenic role of SFRP2 (31, 38, 39), whereas SFRP2KD sig- the NCI-60 panel of cell lines harboring TP53 mutations and in the nature genes restored bone mineralization, apoptosis process, Rb1-knockout mouse, suggesting that SFRP2 strongly contributes to and negative regulation of cell proliferation (Fig. 2C). Mouse the signature of well-known OS genetic lesions, RB1 deletion, and Gene Atlas analysis (40) of DEGs from WT, SFRP2OE, and TP53 mutation (Fig. 2H and SI Appendix,Fig.S2F). Collectively, our SFRP2KD signatures confirmed that SFRP2OE significantly transcriptome analyses revealed tumorigenic properties of SFRP2OE affects differentiation, while SFRP2KD recovered normal OB in LFS and OS. differentiation expression signatures (SI Appendix, Fig. S2C). To determine whether SFRP2OE is associated with oncogenic SFRP2OE Dysregulates OB Differentiation Through BMP/WNT molecular features of LFS, we compared SFRP2OE with LFS and Suppression. We reported that LFS iPSC-derived MSCs and SFRP2KD signature genes. There were 308 up-regulated and pre-OBs have defects in OB differentiation, supporting the idea 478 down-regulated genes common between SFRP2OE and LFS that compromised p53 dysregulates OB differentiation and − expression signatures (Fisher’sexacttest,P = 1.4 × 10 171 and promotes the generation of abnormal MSCs and pre-OBs, the − 2.27 × 10 160, respectively), while 277 down-regulated and 264 up- cells of origin of OS (33). Because our global transcriptome regulated genes were inversely overlapped between SFRP2KD and analyses provided evidence that SFRP2OE potentially perturbs − LFS expression signatures (Fisher’sexacttest,P = 2.13 × 10 130 OB differentiation (Fig. 2C), we wondered if SFRP2OE in LFS − and P = 2.8 × 10 127, respectively) (SI Appendix,TableS3). For suppresses normal OB differentiation as an oncogenic down- MEDICAL SCIENCES gene functional analysis, we used the gene set enrichment analysis stream modulator of mutant p53. We performed alkaline phos- (GSEA) tool Enrichr (41, 42). Gene annotation analysis showed phatase (AP) and Alizarin red S staining on LFS, SFRP2OE, and that the up-regulated genes common to SFRP2OE and LFS WT cells at different OB differentiation time points (D0–D21). compared with WT include cell-cycle and well-known oncogenic Normal OBs exert a high level of AP activity and secrete large genes frequently found in cancers such as AURKB, BUB1B, and amounts of calcium (mineral deposition) detected by Alizarin FOXM1 (Fig. 2 D and E). Interestingly, Enrichr-embedded ChIP red S staining (45–47). SFRP2OE in WT cells clearly suppressed enrichment analysis (41–43) revealed that LFS/SFRP2OE up- normal OB differentiation, similar to its effect in LFS cells (Fig. regulated genes were enriched for FOXM1 targets (P = 1.52 × 3 A and B). In contrast, SFRP2KD in LFS cells rescued osteo- − 10 16)(Fig.2F). This suggests a correlation between SFRP2OE- genic AP activity and mineral deposition (SI Appendix, Fig. S3 A mediated cell proliferation and the oncogenic function of FOXM1 and B). In agreement with this observation, LFS and SFRP2OE target genes in human OS, irrespective of a p53 mutation. Common OBs had lower levels of pre-OB markers (ALPL and COL1A1),

AB C -40 -20 0 20 40 20 Row Z−Score 0 -20 −2 2 WT OBs SFRP2OE SFRP2KD 40 Negative muscle D17 Cell adhesion Negative cell proliferation D0 755 542 proliferation 20 WT Positive cell migration Bone mineralization Negative cell migration PC3 SFRP2OED0 LFS Inflammatory 0 D0 D0 WNT Postive D14 D14 response D7 517 584 pathway bone migration D17 D7 D7 D17

D7 D14 D14

7 7 0

7 0 0

0 Positive cell

14 17

17 14

17 14 PC2 SFRP2KD 14 Apoptosis

D17 (-log10) p-value BMP pathway

0 1 2 3 4 5 6 7 6 5 4 3 2 1 0 0 1 2 3 4 5 6 7 6 5 4 3 2 1 0 0 1 2 3 4 5 6 5 4 3 2 1 0 proliferation PC1 WT SFRP2OE LFS SFRP2KD * BUB1B 4 Up-regulated D LFS_up & EFGHNCAPG ChIP * FOXM1 SFRP2OE signatures in mutated TP53 SFRP2KD PKMYT1 Enrichment SFRP2OE_up SFRP2OE_up * AURKB Mitotic cell cycle NCAPH p-value<0.001 _down 3 Log2(RPKM) Analysis p-value<0.001 -10 NUF2 203 (p=1.8×10 ) * BUB1 NES=1.23 NES=1.74 259 Mitotic nuclear division * CDC25C 45 -9 * NDC80 (p=6.5×10 ) KIF2C 2 263 SPC24 232 * FAM83D LFS_up & * BRINP1 ESPL1 FOXM1 SFRP2KD_down * CHEK1 1 0.3 0.0 -0.2

NUSAP1 0.5 0.3 0.0 -0.2 2194 Extracellular matrix GTSE1 organization: (p=8.0×10-6) * DLGAP5 EZH2 LFS_up Extracellular structure (ES) Score Enrichment -4 OS Up Down organization: (p=2.0×10 ) WT LFS OB SFRP2OE SFRP2OE SFRP2OE

Fig. 2. Global gene-expression analysis reveals that SFRP2OE induces cell proliferation and perturbs the differentiation process. (A) PCA of RNA-seq data from SFRP2OE and SFRP2KD samples [proportion of variance; principal component 1 (PC1) = 0.4020; principal component 2 (PC2) = 0.1839; principal component 3 (PC3) = 0.0903]. (B) Heatmaps of DEGs in SFRP2OE and SFRP2KD samples (DEG cutoff P value < 0.001). (C) DAVID gene ontology (GO) term biological process analysis of DEGs from WT, SFRP2OE, and SFRP2KD cells. The top 20 GO terms of DEGs from each group are presented in the panels. The cutoff at P = 0.05 is indicated by dashed lines. (D) Enrichr GO biological process analysis of common genes among SFRP2OE up-regulated genes, LFS up-regulated genes, and SFRP2KD down-regulated genes. (E) Relative expression of cell-cycle– and cell-proliferation–related DEGs up-regulated in both SFRP2OE and LFS cells. (Asterisks indicate cancer-associated genes.) (F) Common up-regulated DEGs are enriched in FOXM1 ChIP (in U2OS) data (Enrichr). (G) GSEA of the SFRP2OE signature with the OS gene set (GSE36001). (H) GSEA of the SFRP2OE signature with the TP53 mutation gene set (MSigDB C6 analysis).

Kim et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 30, 2021 mature OB markers (BGLAP and PTH1R), osteocyte markers treatment (SI Appendix, Fig. S3E). Using the TOP/FOP flash (ZNF521 and FGF23), and OB differentiation transcriptional luciferase WNT signaling reporter system, we observed low basal factors (RUNX2 and OSX) (Fig. 3C). Interestingly, ATF4, WNT activity in all groups (Fig. 3E, no treatment). We activated whose expression is high in OS tissue and OS cell lines (48), was WNT signaling using WNT3a (100 ng/mL) or LiCl (10 mM), up-regulated in SFRP2OE and LFS OBs (Fig. 3C). These re- which inhibits GSKβ. SFRP2OE showed a reduction in WNT3a- sults, together with our finding that LFS4 (P53.pY205C)-derived mediated canonical WNT activity (Fig. 3E). LFS and HOS cells OBs also showed reduced mineral deposition (SI Appendix, Fig. exhibited little or no response to either LiCl or WNT3a (Fig. 3E S3C), indicate that SFRP2OE as a consequence of mutant and SI Appendix, Fig. S3F). Ectopic expression of constitutively p53 perturbs normal osteogenic processes, which is consistent active β-catenin (DeltaN90) in LFS did not facilitate cell pro- with the RNA-seq biological process analysis (Fig. 2C). liferation or promote cell transformation (SI Appendix, Fig. The BMP/SMAD and canonical WNT pathways are required S3G). However, knockdown of β-catenin led to severe cell death for normal osteogenesis (49–51). In particular, BMP ligands fa- of LFS cells, confirming that β-catenin is necessary for homeo- cilitate OB differentiation through the BMP/SMAD pathway stasis and viability of MSCs and OBs (SI Appendix, Fig. S3H) (55, (50, 52, 53). Using a luciferase-based phospho-SMAD reporter 56). Although it is widely accepted that hyper-WNT signaling is system, we monitored BMP signaling activity throughout in vitro associated with oncogenicity in many cancers, our data support differentiation (D0–D14). While BMP signaling was relatively the idea that hyper-WNT is not always involved in osteosarco- low in MSCs and progenitors (D4), it increased in pre-OBs magenesis. This is consistent with the observation of inactive (D10–D14) (Fig. 3D). This result is consistent with published canonical WNT activity in high-grade human OS (57). in vivo data (52, 54). As expected, SFRP2OE and LFS cells showed lower BMP signaling activity than WT OBs. It supports SFRP2OE Transforms Pre-OBs into OS-Like Cells. We next investigated the idea that SFRP2OE perturbs normal OB differentiation in the oncogenic contribution of SFRP2OE by analyzing cell mor- part through inhibition of the BMP pathway. phology and cell proliferation in ectopic SFRP2OE and SFRP2KD The effects of SFRP2 on canonical WNT signaling are context conditions. SFRP2OE in WT cells resulted in the development of dependent. Generally, SFRP2 suppresses WNT signaling by aggregated structures, which is reminiscent of tumor cell aggrega- binding WNT ligands, thus preventing their binding to WNT tion, and was enhanced in ectopic SFRP2OE in LFS cells at D14– receptors (30, 55, 56). However, SFRP2 has also been shown to D17ofOBdifferentiation(Fig.4A). Overexpression also increased activate WNT signaling at low concentrations or in certain cell proliferation during MSC culture and OB differentiation (Fig. 4B types (35, 57–59). To understand the role of SFRP2 in canonical and SI Appendix,Fig.S4A). In contrast, SFRP2KD cells did not WNT signaling in OS development, we performed Western blot form aggregated structures and showed decreased cell proliferation and β-catenin immunostaining. Compared with WT cells, LFS, compared with LFS cells (Fig. 4B and SI Appendix,Fig.S4B). SFRP2OE, and HOS cells consistently had lower levels of cy- It was reported that ectopic SFRP2 expression alters the G2 toplasmic β-catenin (SI Appendix, Fig. S3D), while the amount of phase of the cell cycle and suppresses apoptosis (32). Our analysis nuclear β-catenin increased only in WT groups with WNT3a of both cell cycle and apoptosis on D4 of OB differentiation

Fig. 3. SFRP2OE suppresses OB differentiation in vitro. (A) Osteogenic AP analysis in LFS P53p.G245D-, SFRP2OE-, and reverse tetracycline-controlled transactivator (rtTA)-expressing WT-derived pre-OB cells. (B) Alizarin red S staining in LFS P53p.G245D-, SFRP2OE-, and rtTA-expressing WT-derived pre-OB cells. (C) Quantitative PCR analysis of the indicated osteogenic markers in LFS P53p.G245D, SFRP2OE, and WT cells (data are shown as mean ± SD; n = 3 independent repeats in triplicate; *P < 0.05; ANOVA). (D) BMP signaling activity in LFS P53p.G245D, SFRP2OE, and WT cells as measured by a BMP reporter (pSBE3-Luc) (data are shown as mean ± SD; n = 3 independent repeats in triplicate; **P < 0.0001; ANOVA). (E) WNT signaling activity as measured by TOP/FOP flash luciferase reporter. Activity levels were normalized using an internal control (dual luciferase assay system). Data are presented as mean ± SD (n = 3 independent repeats in triplicate; *P < 0.05; ANOVA).

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1814044115 Kim et al. Downloaded by guest on September 30, 2021 revealed significantly more SFRP2OE cells in S phase (72.17 ± cells significantly reduced tumorigenesis (Fig. 4H). In serial 3.22%) compared with WT cells (29.82 ± 1.68%) (Fig. 4C and SI transplantation analysis, SFRP2OE cells maintained their Appendix,Fig.S4C). This implies that SFRP2OE promotes S-phase tumor-formation ability (SI Appendix, Fig. S4H), while the anti- entry, cell-cycle progression, and, thus, cell proliferation. In contrast, body in HOS consistently suppressed it over three consecutive SFRP2KD resulted in significantly less cell proliferation with more transplantations (SI Appendix, Fig. S4I). Additionally, using the SFRP2KD cells in G0/G1 (21.51 ± 2.68%) and fewer in S phase CAM assay, we checked whether FOXM1 inhibition could sup- (77.36 ± 2.86%) compared with LFS cells (G0/G1: 8.60 ± 4.32%; S press tumor formation. We used FOXM1 shRNA (FOXM1KD) phase: 90.51 ± 1.99%) (Fig. 4 B and C and SI Appendix,Fig.S4B and the small molecule FDI-6, a reported potent inhibitor of and D). No statistical difference in apoptosis was found among the FOXM1 (60). The result showed that both FOXM1KD and various groups (Fig. 4C). Collectively, we observed that SFRP2OE FDI-6 treatment effectively decreased SFRP2-mediated tumor promotes S-phase entry at the pre-OB stage. formation (SI Appendix, Fig. S4 J and K). These in ovo data Anchorage-independent growth is a hallmark of cancer cell demonstrate that SFRP2OE contributes to LFS-mediated tu- transformation. We used the soft agar colony-formation assay to morigenesis and OS development and that targeting SFRP2 with determine the transformation ability of SFRP2. SFRP2OE and an antibody suppresses OS formation. LFS cells were able to form oncogenic colonies (Fig. 4D and SI Appendix, Fig. S4 E and F). Both the number and size of colonies Secreted SFRP2 Facilitates Angiogenesis and Tumorigenesis Through formed by SFRP2OE and LFS cells were significantly greater CYR61 Induction in Endothelial Cells. Angiogenesis is a critical com- than those of WT cells. In contrast, SFRP2KD cells formed ponent of solid tumor growth. SFRP2 has been shown to function significantly fewer colonies than LFS cells (Fig. 4E). These data as an angiogenic factor in human triple-negative breast cancer, clearly demonstrate the tumorigenic ability of SFRP2. angiosarcoma, and melanoma (38, 58, 61). However, whether To determine the tumorigenicity of SFRP2OE, we performed SFRP2 has any angiogenic properties in OS remains unknown. in ovo chick chorioallantoic membrane (CAM) assays (SI Ap- Because SFRP2 is a secreted protein, we hypothesized that MSCs pendix, Fig. S4G). This assay provides a convenient and common and pre-OBs in LFS patients could secrete SFRP2 into the bone way to check the oncogenicity of sarcoma cells (58, 59). matrix where it initiates migration and sprouting of endothelial cells, SFRP2OE cells showed increased tumor formation and cell thereby facilitating neoangiogenesis. To investigate this, we con- numbers compared with WT cells (Fig. 4F). SFRP2KD cells had ducted an in vitro angiogenesis assay; we cultured human vascular suppressed tumor formation compared with LFS cells (Fig. 4G). endothelial cells (HUVECs) on Matrigel in WT-, LFS-, or Since SFRP2 is a secreted molecule, we wondered if a blocking SFRP2OE-conditioned medium (CM) for 16 h and then quantified antibody would reduce OS features. We used the well- the tube formation rate. As expected, WT CM was not able to documented p53 mutant osteosarcoma cell line HOS. As hy- significantly induce tube formation (Fig. 5 A and B). In contrast, MEDICAL SCIENCES pothesized, adding a monoclonal anti-SFRP2 antibody to HOS LFS and SFRP2OE CM dramatically increased tube formation, as

Fig. 4. SFRP2OE in pre-OBs induces cell transformation. (A) Morphological changes in WT/SFRP2OE and LFS (LFS2-B)/SFRP2OE cells (red arrow indicates aggregated cells). (Scale bars: 100 μm.) (B) Cell-proliferation analysis using the CyQUANT cell fluorescent DNA content assay of WT cells expressing rtTA and WT SFRP2OE cells (Left) and LFS (LFS2-B expressing empty shRNA) and SFRP2KD cells (Right) [data are shown as mean ± SD (n = 4; *P < 0.05; **P < 0.0001, two- way ANOVA)]. (C) Cell-cycle analysis of rtTA-expressing WT and SFRP2OE cells (Left) and LFS (LFS2-B) and SFRP2KD (SFRP2 shRNA-1) cells (Right) using BrdU/7- AAD at D4 of OB differentiation (data are shown as mean ± SD; n = 3 independent repeats in duplicate; **P < 0.0001, two-way ANOVA). (D and E, Upper) Soft agar colony formation of rtTA-expressing WT and SFRP2OE cells (Dox, doxycycline) (D) and LFS2-B (LFS), LFS2-B with empty shRNA (Empty), SFRP2 shRNA-1 (KD-1) cells (E). Colonies over 50 μm in size were included in the quantification of colony numbers. (Scale bars: 100 μm.) (Lower) Quantification. Data are expressed as mean ± SD; each symbol represents an individual colony >50 μm in size; **P < 0.0001, one-way ANOVA). (F–H) CAM assays (Upper) and analysis (Lower) of rtTA-expressing WT and SFRP2OE cells (F), LFS (LFS2-B) expressing empty shRNA (Empty) and SFRP2 shRNA-1 (SFRP2KD-1) cells (G), and HOS cells treated with SFRP2 neutralizing antibody (catalog no. sc-365524; Santa Cruz Biotechnology) (H). White ring diameter, 10 mm. White lines outline tumors. In F and G, representative images from two independent CAMs are shown for each condition. In G and H, D7 OBs were used. (F–H, Lower) Analysis (data are shown as mean ± SD; each symbol represents CAM of an individual egg; *P < 0.05; **P < 0.0001, one-way ANOVA in F and unpaired t test in G and H).

Kim et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 30, 2021 did the addition of recombinant SFRP2 protein to WT CM (Fig. 5 and CDH13 (Fig. 5F). High expression levels of CYR61, known to A and B). Moreover, SFRP2KD CM dramatically decreased tube facilitate angiogenesis, were recently associated with poor prog- formation to the levels of the negative control and WT CM. This nosis in OS (62, 63). Interestingly, we also observed increased ex- result is consistent with other reports showing the angiogenic pression of CYR61 in endothelial cells treated with SFRP2OE and function of SFRP2 in angiosarcoma and melanoma (38, 61). LFS CM (Fig. 5 F and G). Thus, we investigated if SFRP2OE To investigate how SFRP2 increases angiogenesis, we performed increases angiogenesis and tumor formation through an action of RNA-seq profiling of HUVECs after treatment with LFS, CYR61. Measuring the level of neovascularization and tumor size SFRP2OE, and WT CM. PCA with triplicate samples from each in a CAM assay, we found that CYR61 antibody treatment de- condition showed that the replicates clustered together but were creases angiogenesis and tumor formation in the presence of distinct among the conditions (Fig. 5C). To identify SFRP2- SFRP2 (Fig. 5 H–J). This supports the idea that secreted SFRP2 mediated effects on angiogenesis induction, we focused on iden- facilitates angiogenesis of endothelial cells and tumorigenesis, at tifying and analyzing DEGs between SFRP2OE and WT condi- least in part, through CYR61. tions that are also differentially expressed between LFS and WT Our RNA-seq analysis revealed the up-regulation of antiapoptosis- conditions. Using DESeq2 (Padj < 0.01), we identified 498 com- related genes in HUVECs by LFS and SFRP2OE CM (SI Appendix, mon genes (258 up-regulated and 240 down-regulated) between Fig. S5D). Indeed, the number of viable HUVECs was higher in 1,826 LFS and 969 SFRP2OE DEGs (Fig. 5D and SI Appendix, SFRP2OE CM and LFS CM than in WT CM (SI Appendix,Fig. Fig. S5 A and B). DAVID functional annotation of the 258 up- S5E). These results suggest that SFRP2 facilitates angiogenesis and regulated common genes showed that secreted SFRP2 mainly protects endothelial cells from apoptosis by inducing the expression alters angiogenesis, antiapoptosis, and inflammatory response of angiogenic/oncogenic factors and antiapoptosis regulators, re- processes of endothelial cells (Fig. 5E). Most up-regulated angio- spectively. Intriguingly, cell-cycle–relatedDEGsinHUVECstreated genic genes are either well-known angiogenic markers or procancer with LFS CM and SFRP2OE CM were mainly down-regulated genes such as VEGFC, CYR61, ANGPT2, VASP, EPAS1, JUNB, (SI Appendix,Fig.S5C and F and Datasets S5–S7). There was

Fig. 5. Secreted SFRP2 facilitates angiogenesis. (A) In vitro tube formation of human endothelial cells in CM as indicated. (B) Quantification of tube formation assay (data are shown as mean ± SD, n = 3 independent experiments; *P < 0.05; **P < 0.0001, ANOVA). (C) PCA of the transcriptome of endothelial cells treated with LFS, SFRP2OE, and WT CM (proportion of variance; PC1 = 0.483, PC2 = 0.167, PC3 = 0.133). (D) Cluster analysis of the 498 common LFS P53p. G245D and SFRP2OE DEGs in the transcriptome of HUVECs exposed to LFS, SFRP2OE, and WT CM. (E) DAVID GO functional analysis of up-regulated DEGs common to LFS P53p.G245D and SFRP2OE. (F) Expression of angiogenic markers in HUVECs treated with WT, SFRP2OE, or LFS P53p.G245D CM. (G) qPCR analysis of CYR61 expression in endothelial cells after WT, SFRP2OE, or LFS P53p.G245D CM treatment (data are shown as mean ± SD; n = 2 independent repeats in triplicate; *P < 0.0001, ANOVA). (H) Representative images of a CAM inoculated with SFRP2OE pre-OBs (D7) in the presence of control rabbit IgG (Left) or CYR61 antibody (catalog no. PA1-16580; Thermo Fisher Scientific) (Right). White lines outline tumors. White ring diameter, 10 mm. (I) Tumor formation rate of LFS P53p.G245D and SFRP2OE pre-OBs in the CAM assay in the presence of control or CYR61 antibody (data are shown as mean ± SD; each symbol represents the CAM in an individual egg; *P < 0.005; **P < 0.0001, unpaired t test). (J) Quantification of neovascularization in the CAM assay after CYR61 antibody treatment (data are shown as mean ± SD; each symbol represents the CAM in an individual egg; *P < 0.005, unpaired t test).

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1814044115 Kim et al. Downloaded by guest on September 30, 2021 little overlap in cell-cycle–related DEGs between HUVECs and large areas of necrosis reminiscent of the tissue morphology activated by LFS CM and SFRP2OE CM or in LFS and seen in human osteoblastic or fibroblastic OS (Fig. 6B and SI SFRP2OE OBs (SI Appendix, Fig. S5G). For example, positive Appendix, Fig. S6B) (64). A few of the small tumors in the cell-cycle regulators such as CDC25B, CKS1B, DLGAP5, GTSE1, SFRP2KD group showed some neoplastic features, while others JAG2, and NUSAP1 were down-regulated in endothelial cells showed induction of fibrous tissues (Fig. 6B and SI Appendix,Fig. exposed to LFS and SFRP2OE CM but were up-regulated in OBs S6B). Interestingly, LFS tumors showed lower levels of AP, a exposed to LFS and SFRP2OE (SI Appendix,Fig.S5G). Only marker of mature OBs, than tumors formed from SFRP2KD cells three genes showed a common expression pattern in the two groups: (Fig. 6B). Despite higher β-catenin expression, β-catenin was lo- JUNB and MYC, two tumorigenic factors, were up-regulated, while calized to the cytosol, granules, and cell membrane in the KRT18, an intermediate filament gene, was down-regulated in both SFRP2KD group (Fig. 6B). This finding augments the idea that OBs and HUVECs affected by SFRP2 signaling (SI Appendix,Fig. SFRP2-mediated tumorigenesis is not directly linked to hyperactive S5G). We speculated that HUVECs in LFS CM and SFRP2OE CM WNT signaling in LFS. Furthermore, we found more vaso- stopped proliferating and initiated differentiation to generate neo- formation, as assessed by expression of the CD31 endothelial vasculature. This result emphasizes that the function of SFRP2 is marker, in LFS cell-induced tumors than in the SFRP2KD group context dependent. (Fig. 6 C and D). These histological analyses support the idea that SFRP2OE facilitates neovascularization and tumorigenesis. As in Targeting SFRP2OE Effectively Suppresses OS Development. To ex- the SFRP2KD group, targeting SFRP2 in malignant OS cells amine if suppression of SFRP2 expression in LFS has any ther- (MNNG)reducedOStumorgrowth(SI Appendix,Fig.S6C). Ad- apeutic effect on OS development in vivo, we s.c. injected LFS ditionally, we checked the expression of signature genes altered by and SFRP2KD cells at D7 of OB differentiation into nu/nu mice. SFRP2KD by quantitative PCR in these primary tumors (Fig. 6E Tumors formed rapidly in mice injected with LFS3-A cells (at and SI Appendix,Fig.S6D). While CADM1 and CADM4 were 2 wk postinjection) or LFS2-B cells (at 3 wk postinjection) (SI significantly up-regulated in SFRP2KD OBs and in SFRP2KD Appendix, Fig. S6A). We attribute the difference in tumor- primary tumors compared with parental LFS and OS, oncogenic formation timing to lower SFRP2 expression in LFS2-B cells CYR61 and FOXM1 were only differentially expressed (i.e., de- than in LFS3-A cells (Fig. 1B). In contrast, mice injected with creased) between SFRP2KD and empty-vector groups in primary SFRP2KD cells did not form tumors, or the tumors were of very tumors (Fig. 6E and SI Appendix,Fig.S6D). small size (Fig. 6A and SI Appendix, Fig. S6A). Histological We next performed intratibial injection in mice (65, 66) to analysis revealed that LFS tumors were composed of large, investigate the orthotopic effect of SFRP2 depletion in OS de-

poorly differentiated malignant tumor cells with mitotic features velopment. In the LFS group, two mice generated tumors in the MEDICAL SCIENCES

Fig. 6. Targeting SFRP2 in LFS pre-OBs and the OS cell line suppresses OS development in vivo. (A) Tumor formation in nu/nu mice 4 wk after injection of LFS P53p.G245D or SFRP2KD (SFRP2 shRNA-1) cells (tumor volume = 3.14/6·L·W·H). Each symbol represents an individual mouse (data are shown as mean ± SD; n = 5; *P < 0.001; **P < 0.0001, ANOVA). (B) Immunohistochemistry analysis of LFS P53p.G245D and SFRP2KD cell-induced tumors. (C) The CD31+ area in LFS P53p. G245D and SFRP2KD cell-induced tumors. (D) Quantification of CD31 expression in tumors. Each symbol represents an induced tumor in an individual mouse (data are shown as mean ± SD; *P < 0.05, unpaired t test). (E) Expression of CYR61 altered by SFRP2KD in LFS P53p.G245D OB- and MNNG-derived s.c. primary tumors (data are shown as mean ± SD; n = 4; *P <0.05, unpaired t test). (F) Intratibial tumor formation with MNNG cells with empty shRNA (Empty) or SFRP2- shRNA-1 (SFRP2KD). Red lines outline tumors. (G) Tumor size 4 wk after intratibial injection (D7 of differentiation) of MNNG with empty shRNA (Empty) or SFRP2 shRNA-1 (SFRP2KD) or WT OBs. Each symbol represents a tumor in an individual mouse (data are shown as mean ± SD; *P < 0.05, ANOVA). (H)Im- munohistochemistry analysis of representative MNNG and SFRP2KD tumors in G.

Kim et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 30, 2021 tibia 5 mo postinjection, and one mouse formed a tumor on the transcriptomic analysis of SFRP2OE and SFRP2KD cells showed flank of the body but not intratibially (SI Appendix, Fig. S6E). that SFRP2 has multiple functions during abnormal differentiation This might have been caused by a failure of proper intratibial and tumorigenesis in LFS OS development. Specifically, SFRP2OE injection. Three mice died within 3 mo of injection with no visible changes the cell-cycle profile and increases the expression of cell- exterior tumor formation but with clear signs of weight loss. The proliferation–related genes such as AURKB and FOXM1.AURKB actual cause of death was undetermined. In stark contrast, the regulates chromosomal segregation during mitosis and meiosis, and SFRP2KD group did not form any osteosarcomatous lesions (SI aberrant expression of AURKB is observed in several cancers (70, Appendix,Fig.S6E). To corroborate this finding, we depleted 71). FOXM1 is also overexpressed in many cancers (72, 73). Using SFRP2 in MNNG cells. Suppressing SFRP2 expression in MNNG our LFS OB model in the in ovo CAM assay, we found that in- cells significantly reduced tumor incidence and size (Fig. 6 F and hibition of FOXM1 impedes OS development (SI Appendix, Fig. G). Primary lesions showed characteristics of primary human OS S4 J and K). FOXM1 regulates the transcription of cell-cycle and with osteolytic, mitotically active undifferentiated cells as well as proliferation genes (74, 75), including the SKP2 and CKS1 sub- wide bone destruction (Fig. 6H and SI Appendix,Fig.S6F). Mi- units of the SCF ubiquitin ligase complex. These two proteins croscopically, MNNG tumors showed less AP activity, more pro- ubiquitinate p21 (Cip1) and p27 (Kip1), regulators of cell-cycle + + liferating (Ki67 ) cells, and more neovascularized (CD31 )areas progression into S phase, for degradation (76). Therefore, the than the SFRP2-depleted MNNG tumors (Fig. 6H). The bone observed increase in cell proliferation and in the number of cells in structure in the SFRP2KD group was relatively intact, although S phase of LFS and SFRP2OE cells may be explained by SFRP2OE- the tumors still contained undifferentiated tumorigenic cells in the induced FOXM1. One may assume that SFRP2 depletion in LFS cells proximal tibia (Fig. 6G and SI Appendix,Fig.S6F). Conclusively, should reduce FOXM1 expression. However, this is not what we ob- the in vivo xenograft study unequivocally demonstrates that de- served. We explain this by the autoregulation of FOXM1 expression pletion of SFRP2 in LFS-mediated tumorigenesis and OS devel- (77, 78). Thus, once FOXM1 expression is induced, its expression is opment has potential therapeutic benefits. maintained, and SFRP2 depletion has no apparent effect. Neverthe- less, FOXM1 inhibition by shRNA and FDI-6 small-molecule treat- Discussion ment effectively decreased tumor formation (SI Appendix,Fig.S4J We show that LFS patient-specific iPSCs and their MSCs/OBs and K). It is noteworthy that FOXM1 up-regulation was correlated provide a platform for investigating the early development of OS, with a poor prognosis in OS patients (79). This fits well with our own and we identify and evaluate a potential therapeutic target, SFRP2. observation that high SFRP2 expression in OS tissues is linked to Using molecular and functional experiments, traditional in vivo decreased patient survival (Fig. 1F and SI Appendix,Fig.S1G). studies, and bioinformatic analyses, we found that SFRP2 expres- Additionally, we found that down-regulated genes common to sion contributes significantly to LFS-mediated tumorigenesis and LFS and SFRP2OE that are up-regulated in SFRP2KD cells in- OS development by facilitating angiogenesis and cell proliferation. clude cell-adhesion genes such as CADM1 and CADM4. They are Most significantly, we demonstrated that targeting SFRP2 has a known tumor suppressors in various cancers (80–83). The signifi- potential therapeutic application in LFS and OS. cance and role of CAMD1 and CADM4 down-regulation in OS We found that SFRP2 is up-regulated in LFS patient cells and development is unknown. A possibility is that their down-regulation OS cell lines harboring TP53 mutations. We showed that is linked to anchorage-independent cell growth and cell egress from SFRP2 exerts oncogenicity in MSCs/OBs both in vitro and in vivo. the bone environment, resulting in metastasis. This warrants future These findings conflict with the hyper-WNT signaling paradigm in investigation. Taken together, the abnormal expression of these cancer biology. Generally, SFRP2 is known as a WNT pathway specific genes (AURKB, FOXM1, CADM1, and CADM4) clearly antagonist due to its ability to sequester WNT ligand from indicates the oncogenic property of SFRP2OE in OS. the frizzled receptor and is considered to be a tumor suppressor. Angiogenesis is a critical hallmark of cancer progression. In this Indeed, promoter hypermethylation of SFRP2 is found with in- study we show that secreted SFRP2 facilitates angiogenesis in vitro creased WNT signaling in many cancers (67, 68). One study found and in vivo. Global transcriptome analysis revealed up-regulation that enhanced WNT signaling as a consequence of SFRP2 meth- of angiogenesis-related and antiapoptotic genes in endothelial cells ylation is associated with OS cell invasion (69). However, in the grown in LFS, SFRP2 cell-conditioned, and SFRP2-supplemented LFS context, SFRP2 functions as an oncogenic factor. We con- media. Angiogenesis-related genes such as CDH13, EPAS1, and sistently observed that SFRP2OE suppressed WNT signaling and CYR61 are highly associated with focal adhesion and tumorigen- that the majority of β-catenin remained localized to the cytoplasm. esis. Particularly, CYR61, a secreted extracellular matrix protein, is Our data also indicate that lower β-catenin expression and inactive a proangiogenic/tumorigenic molecule validated in many cancers β-catenin are associated with the initiation of abnormal OB dif- (84, 85). Interestingly, CYR61 mediates specific functions in differentiation and are maintained to the terminal stage of osteo- ferent types of cells through binding to distinct integrins during sarcomagenesis. This finding is consistent with a report of inactive angiogenesis (86, 87) and metastasis (88). CYR61 is overexpressed β-catenin and WNT signaling in high-grade human OS (57). Our in OS compared with normal bone tissues, and its depletion causes study adds evidence of the complexity of WNT pathway regulation inhibition of neoangiogenesis in the developing tumor (62). We and its diverse outcomes in different conditions. found that CYR61 inhibition prevents SFRP2-mediated OS de- The mechanism by which SFRP2 exerts its role in OS develop- velopment in the in ovo CAM assay (Fig. 5 H and I). Collectively, ment is unknown and remains elusive, context dependent, and even the SFRP2–CYR61 axis in LFS may alter the extracellular matrix controversial in many other cancers. A recent paper described an environment, thus promoting angiogenesis, tumorigenesis, and pos- age-related increase in SFPR2 expression in fibroblasts that acti- sibly metastasis. Surprisingly, SFRP2 did not increase the expression vated a multistep signaling cascade in melanoma cells, resulting in a of cell-cycle–related genes in endothelial cells, in contrast to its ef- reduction of β-catenin and MITF expression and consequently in fects in LFS and SFRP2OE OBs. the loss of APE1. The absence of APE1 affected the DNA damage Overall, our data provide insights into the role of SFRP2 in OS response of the cells, increased metastasis, and rendered the cells initiation and development. Using the powerful tool of iPSC-based more resistant to chemotherapy (38). In our study, we observed that modeling, we show that SFRP2 has a dual function. SFRP2 increases SFRP2OE suppresses β-catenin, but MITF or APE1 expression was the proliferative capacity of pre-OBs and also can promote angio- not altered. Instead, we found that SFRP2OE in OBs up-regulates genesis through cell-adhesion/cell-matrix factors; SFRP2-FOXM1 in FOXM1 in OBs as an autocrine factor and CYR61 in endothelial LFS OBs/OS and SFRP2-CYR61 in endothelial cells contribute to cellsasaparacrinefactorinaβ-catenin–independent way. There- OS development. We did not observe a significant effect of SFRP2 fore, we concluded that SFRP2-associated osteosarcomagenesis in metastasis of LFS/OS. However, a recent report showed that develops through a mechanism different from that in melanoma. SFRP2 potentially plays a crucial role in metastatic OS, where its Oncogenesis is attributed to abnormal differentiation, aberrant ectopic expression enhanced invasiveness and metastasis of human cell adhesion, a dysregulated cell cycle, and overproliferation. Our and mouse OS cells without affecting cell proliferation (89). In

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1814044115 Kim et al. Downloaded by guest on September 30, 2021 contrast, one study found that increased SFRP2 methylation result- was conducted according to federal, state, and local regulations. For further ing in reduced expression is associated with OS invasiveness (69). details see SI Appendix. These observed discrepancies in the role of SFRP2 in invasiveness and metastasis of OS might be due to differences in the progressive Human OS Tissue Microarray. The OS tissue microarray set was obtained from stageofthecellsorinthespecificOS subset studied. Detailed future E.G.D. The use of the OS microarray set and data analysis were approved by exploration of the SFRP2–FOXM1 and SFRP2–CYR61 axes during the Icahn School of Medicine’s Program for the Protection of Human Subjects OS development and metastasis will provide additional mechanistic and its Institutional Review Board. For further details see SI Appendix. insight and help develop novel therapeutic strategies to treat LFS and OS. RNA-Seq and Data Analyses. cDNA library preparation and sequencing was outsourced to Novogen. Sequence reads were aligned to human transcript Materials and Methods reference (RefSeq Genes, hg19) for expression analysis at gene levels using TopHat (97) and HTSEq (98). The raw counts of reads aligned to each gene WT and LFS iPSC and p53-Mutant ESC Culture. All iPSCs were generated using were further normalized by reads per kilobase of transcript per million reads the Sendai virus method as previously reported (33, 90). ESCs and iPSCs (SI mapped (RPKM) as the measurement of gene-expression abundance. For the Appendix, Table S1) were cultured in DMEM/F12 supplemented with 20% original dataset, we used linear regression to identify DEGs independent of knockout serum replacement, 10 ng/mL basic FGF (Invitrogen) on mouse time (D0, D7, D14, and D17) among the different conditions (WT, SFRP2OE, feeder cells (GlobalStem), or in mTeSR (STEMCELL Technologies) on Matrigel- LFS, or SFRP2KD cells) G∼time+condition. The P value cutoff (P < 0.001) was coated plates. validated by permutation test (FDR <0.01). For the HUVEC dataset, DESeq2 (Bioconductor) was used to compare gene expression among WT, LFS, and iPSC Differentiation to MSCs and OBs. iPSCs were differentiated to MSCs and SFRP2OE cells. An adjusted P value cutoff (Padj < 0.01) was applied to define OBs as previously described (33). For details see SI Appendix. DEGs. We used DAVID (version 6.8) or Enrichr (41, 42) for functional ontology and pathway analyses of DEGs depending on which method would SFRP2 Gene Expression and Knockdown. SFRP2OE and SFRP2KD, respectively, provide the best visualization for the intended experimental purpose. For were achieved by infecting iPSC-derived MSCs with lentiviral particles additional details and links to gene lists see SI Appendix. encoding for inducible SFRP2 or shRNAs against SFRP2 (SI Appendix, Table S5). For details see SI Appendix. Other Assays. Full details of quantitative real-time PCR, Western blotting, AP and Alizarin red S staining, the luciferase reporter assay, β-catenin immunos- OS Cell Lines. Human OS cell lines were obtained from Robert Maki (Icahn taining and quantification, the CyQUANT NF fluorescence proliferation assay School of Medicine, New York) and Nino Rainusso (Texas Children’s Hospital, (Thermo Fisher Scientific), and cell-cycle analysis can be found in SI Appendix. Houston). HOS, MNNG, and 143B cells were cultured in DMEM/10% FBS, and SJSA-1, U2OS, and SaOS-2 cells were cultured in RPMI/10% FBS at 37 °C and Statistical Analysis. Student’s t test (two-sided) was applied, and changes MEDICAL SCIENCES 5% CO2 in a humidified incubator. were considered statistically significant for P < 0.05. For comparison of more than two groups, a one-way or two-way (if data included different time Genome Editing. The P53p.G245D mutation was introduced into WT iPSC- points) ANOVA was applied. Survival in human subjects and mouse experi- derived MSCs using CRISPR/Cas9, appropriate donor plasmid, and guide ments was represented with Kaplan–Meier curves, and significance was es- RNAs designed on the web tool at crispr.mit.edu (91). The various hetero- timated with the log-rank test. The statistical methods used for RNA-seq zygous p53 mutant ESC lines (SI Appendix, Table S1) were generated using analysis are described under RNA-seq data analysis above. The data are TALENs designed with the ZiFiT Targeter version 4.2 (zifit.partners.org/) (92) shown as mean ± SD of at least three biological replicates. Statistical analysis as described in ref. 93. For details see SI Appendix. was conducted using Microsoft Excel or GraphPad Prism software packages.

Angiogenesis/Tube-Formation Assay. Tube formation was assayed using the In Additional Data. Histological data are available in SI Appendix, Table S6.Re- Vitro Angiogenesis Assay Kit (Abcam) as recommended by the manufacturer. sources and reagents will be provided upon proper requests and use agree- Tube formation was captured 16 h post culture initiation with an EVOS FL Cell ments. According to regulations, we do not share patient or clinical Imaging system (Thermo Fisher Scientific). Images were analyzed with the information linked to the human OS tissue microarray set except as provided ImageJ Angiogenesis Analyzer tool, which quantifies the tube-formation in this paper (SI Appendix,TableS2). images by extracting characteristic information about the formed tube meshes and branches (94). For further details see SI Appendix. ACKNOWLEDGMENTS. We thank Drs. Robert Maki and Nino Rainusso for providing OS cell lines, Dr. Marion D. Zwaka for sharing shRNAs to p53, CAM Xenograft Assay. The CAM assay with 1 × 106 differentiated OBs (D7 or D14) Drs. Zichen Wang and Avi Ma’ayan for help in RNA-seq analysis of preliminary was conducted as described previously (95). For further details see SI Appendix. data, and Dr. Avi Ma’ayan for reading and commenting on the manuscript. This work was supported by funds provided by the Graduate School of Bio- medical Sciences at the Icahn School of Medicine at Mount Sinai and by Mouse Studies. Eight-week-old male nu/nu mice (Charles River) were used for National Cancer Institute Pre- to Postdoctoral Transition Award 1F99CA212489 intratibial and s.c. xenografts. Sample size was determined based on power (to H.K.), NIH Pathway to Independence Award R00CA181496 and Cancer Pre- calculation (G*POWER software; with one-way ANOVA) and our estimation vention Research Institute of Texas Award RR160019 (to D.-F.L.), NIH Grant of surgical failure (96). All animal use was reviewed and approved by the 5R01GM078465 (to I.R.L.), and Empire State Stem Cell Fund through the New Icahn School of Medicine’s Institutional Animal Care and Use Committee and York State Department of Health Grant C024410 (to I.R.L.).

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