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

Significance

Li–Fraumeni syndrome is a rare disorder caused by germline TP53 mutations, predisposing patients to early-onset cancers, including osteosarcoma (OS). Here we demonstrate that strong expression of SFRP2, a reported WNT antagonist, in OS patient samples correlates with poor survival and that SFRP2 overexpression suppresses normal osteoblast differentiation, promotes OS features, and facilitates angiogenesis via autocrine and paracrine mechanisms in an induced pluripotent stem cell disease model. We show that these SFRP2-mediated phenotypes are canonical WNT/β-catenin independent and are mediated through induction of oncogenes such as FOXM1 and CYR61. We further demonstrate that inhibition of SFRP2, FOXM1, or CYR61 represses tumorigenesis. Our data suggest that inhibition of SFRP2 should be explored clinically as a strategy for treatment patients with p53 mutation-associated OS.

Abstract

Osteosarcoma (OS), the most common primary bone tumor, is highly metastatic with high chemotherapeutic resistance and poor survival rates. Using induced pluripotent stem cells (iPSCs) generated from Li–Fraumeni syndrome (LFS) patients, we investigate an oncogenic role of secreted frizzled-related protein 2 (SFRP2) in p53 mutation-associated OS development. Interestingly, we find that high SFRP2 expression in OS patient samples correlates with poor survival. Systems-level analyses identified that expression of SFRP2 increases during LFS OS development and can induce angiogenesis. Ectopic SFRP2 overexpression in normal osteoblast precursors is sufficient to suppress normal osteoblast differentiation and to promote OS phenotypes through induction of oncogenic molecules such as FOXM1 and CYR61 in a β-catenin–independent manner. Conversely, inhibition of SFRP2, FOXM1, or CYR61 represses the tumorigenic potential. In summary, these findings demonstrate the oncogenic role of SFRP2 in the development of p53 mutation-associated OS and that inhibition of SFRP2 is a potential therapeutic strategy.

Osteosarcoma (OS) is the most common primary bone tumor. It accounts for about 60% of all primary bone tumors and about 2% of all childhood cancers (1, 2). Despite significant advances in OS treatment modalities, the 5-y overall survival rate has remained stable over the last 20 y at 60–70% for patients with primary OS and less than 30% for patients with metastasis (3, 4). This stagnation of clinical outcomes underlines the urgent necessity for novel model systems to study the mechanism of OS in a patient-specific context and to identify molecular targets for the development of new therapeutic strategies.

The tumor suppressor p53 regulates cell cycle, apoptosis, senescence, metabolism, and cell differentiation (5–7). Therefore, it is not surprising that aberrant p53 expression contributes significantly to cancer development (8, 9). Half of all human sporadic bone tumors have genetic lesions in TP53 (10, 11). Patients with Li–Fraumeni syndrome (LFS), which is caused by mutations in TP53, show a 500-fold higher incidence of OS relative to the general population (12–14). Manipulation of p53 function confirmed its significance in OS development (15, 16) and identified mesenchymal stem cells (MSCs) and preosteoblasts (pre-OBs) as the cellular origin of OS (17, 18). Osteoblast (OB)-restricted deletion of p53/Mdm2 or p53/Rb resulted in OS development at a high penetrance of about 60% and 100%, respectively (19, 20).

The first secreted frizzled-related protein (SFRP) was identified as a WNT antagonist (21). As a known WNT antagonist, SFRP2 is considered a tumor suppressor. Indeed, several reports showed that SFRP2 hypermethylation and its decreased expression are associated with prostate, liver, colorectal, and gastric cancer (22–27). Originally, SFRP2 was reported as a secreted antiapoptosis-related protein (28, 29). Ectopic expression of SFRP2 promotes cell growth and has antiapoptotic properties in renal and breast cancer (30–32). The role of SFRP2 appears to be cancer-type specific and remains controversial. Thus, investigation and understanding of the role of SFRP2 in different types of cancer, including OS, is warranted.

Using induced pluripotent stem cells (iPSCs) derived from LFS patients, we previously recapitulated the pathophysiological features of LFS-mediated OS development (33, 34). Taking advantage 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 with potential implication in OS development. Quantitative PCR and Western blot analyses confirmed that SFRP2 expression is significantly overexpressed in OBs derived from different LFS iPSC lines compared with WT controls (Fig. 1B). We confirmed the correlation between the P53p.G245D mutation and SFRP2 expression by introducing the mutation into WT MSCs (SI Appendix, Fig. S1A). SFRP2 expression was significantly increased in WT MSCs edited to harbor the P53p.G245D mutation but not in WT MSCs with p53 depletion (shRNAs to p53) (SI Appendix, Fig. S1B). Fibroblasts from LFS patients with the G245D mutation also have higher SFRP2 expression (SI Appendix, Fig. S1C). Increased SFRP2 expression is not unique to P53p.G245D. OBs derived from LFS iPSCs carrying a heterozygous P53p.Y205C mutation and from various p53-mutant embryonic stem cells (ESCs) (G245D, G245S, R248W, and R249S) overexpress SFRP2 compared with their WT counterparts (SI Appendix, Fig. S1 D and E). Additionally, SFRP2 expression was detected in several OS cell lines harboring p53 mutations (HOS, MNNG, and 143B) or MDM2 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 lines (Fig. 1D) (33). These results demonstrate that SFRP2OE is induced by LFS-associated p53 mutations.

Fig. 1.

SFRP2OE in LFS (G245D)-mediated OS. (A, Left) Global transcriptome 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) with a fold change >2. SFRP2 is an overexpressed gene that is also enriched in the signature gene list of an OS gene set (GSE33458). (B) Quantitative PCR analysis (Right) of SFRP2 expression (D4 of differentiation; data are shown as mean ± SD; n = 3 independent repeats in triplicate) in LFS P53p.G245D and WT MSCs (*P < 0.05; **P <0.0001; ANOVA). The Inset depicts Western blotting using mouse monoclonal anti-SFRP2 antibody (catalog no. sc-365524; Santa Cruz Biotechnology). (C) SFRP2 expression in human WT and LFS P53p.G245D iPSC-derived OBs (D4 of differentiation) and OS cell lines (data are shown as mean ± SD; n = 3 independent repeats in triplicate; **P < 0.0001, ANOVA). (D, Upper) Expression of SFRP2 protein in LFS P53p.G245D s.c. xenograft tumors. (Lower) Human prostate tissue was used as the positive staining control, and mouse muscle tissue was used as the negative staining control. (Scale bars: 100 μm.) (E) Representative staining of SFRP2 protein expression levels in OS tissue samples from 151 OS patients. In D and E, rabbit polyclonal anti-SFRP2 (catalog no. PA5-29390; Thermo Fisher Scientific) was used. (F) Survival analysis of OS patients linked to the tissue microarray. The survival curves of SFRP2 high- and low-expression groups were calculated using the log-rank test (χ² = 11.13) (^#P = 0.0009).

Next, we analyzed the expression of SFRP2 levels in human OS tissue samples using a tissue microarray spotted with 151 OS patient tissue samples. The OS patient samples were categorized by SFRP2 staining intensity: Groups 0 and 1were considered to be negative to low intensity, and groups 2 and 3 were high intensity (Fig. 1E and SI Appendix, Fig. S1F). Interestingly, high SFRP2 expression (intensity scores 2 and 3, with 3 being the highest expression) negatively correlated with patient survival (Fig. 1F and SI Appendix, Fig. S1G and Table S2). As the patients’ p53 status associated with this OS tissue array is not available, a correlation between SFRP2 expression and p53 mutation is not possible. There was no gender difference in tumor incidence (SI Appendix, Fig. S1H), and the area staining positive for SFRP2 is small in the group with low SFRP2 expression (SI Appendix, Fig. S1I). These data indicate that SFPR2OE is associated with poor prognosis for OS patients.

Global Gene-Expression Analysis of SFRP2OE.

To better understand the molecular role of SFRP2OE in LFS cells, we generated global gene-expression profiles of WT SFRP2OE cells, LFS cells with SFRP2 knockdown (SFRP2KD), parental WT cells (WT2-C), and LFS cells (LFS2-B) at four time points (D0, D7, D14, and D17) during in vitro OB differentiation (SI Appendix, Fig. S2A). We used lentiviral vectors for doxycycline-inducible overexpression (36) and for shRNA depletion (37) to generate stable SFRP2OE or SFRP2KD cell lines, respectively, (SI Appendix, Fig. S2B). Principal component analysis (PCA) showed that WT and LFS cells are clearly distinct from each other (Fig. 2A), consistent with our previous LFS RNA-sequencing (RNA-seq) data (33). Expression patterns of SFRP2OE cells were also distinct from those of WT cells and were more evident at later stages of differentiation (Fig. 2A). Likewise, LFS datasets diverged more strongly at late stages of differentiation, while SFRP2KD data clustered together and were distinct from LFS groups (Fig. 2A). This result shows that SFRP2OE significantly alters gene expression in pre-OBs during differentiation. Linear regression with controlling for time identified DEGs between SFRP2OE and WT, between LFS and WT, and between SFRP2KD and LFS groups [P < 0.001, corresponding permutation false-discovery rate (FDR) <0.01] (Fig. 2B and Datasets S2–S4). Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation was used to understand the general biological effect of DEGs associated with SFRP2OE. WT signature genes (DEGs during OB differentiation) were generally involved in bone mineralization and bone morphogenetic protein (BMP) and WNT signaling pathways required for normal OB differentiation (Fig. 2C). Interestingly, SFRP2OE signature genes were linked to cell proliferation, cell adhesion, and cell migration, categories implicated in the tumorigenic role of SFRP2 (31, 38, 39), whereas SFRP2KD signature genes restored bone mineralization, apoptosis process, and negative regulation of cell proliferation (Fig. 2C). Mouse Gene Atlas analysis (40) of DEGs from WT, SFRP2OE, and SFRP2KD signatures confirmed that SFRP2OE significantly affects differentiation, while SFRP2KD recovered normal OB differentiation expression signatures (SI Appendix, Fig. S2C).

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).

To determine whether SFRP2OE is associated with oncogenic molecular features of LFS, we compared SFRP2OE with LFS and SFRP2KD signature genes. There were 308 up-regulated and 478 down-regulated genes common between SFRP2OE and LFS expression signatures (Fisher’s exact test, P = 1.4 × 10⁻¹⁷¹ and 2.27 × 10⁻¹⁶⁰, respectively), while 277 down-regulated and 264 up-regulated genes were inversely overlapped between SFRP2KD and LFS expression signatures (Fisher’s exact test, P = 2.13 × 10⁻¹³⁰ and P = 2.8 × 10⁻¹²⁷, respectively) (SI Appendix, Table S3). For gene functional analysis, we used the gene set enrichment analysis (GSEA) tool Enrichr (41, 42). Gene annotation analysis showed that the up-regulated genes common to SFRP2OE and LFS compared with WT include cell-cycle and well-known oncogenic genes frequently found in cancers such as AURKB, BUB1B, and FOXM1 (Fig. 2 D and E). Interestingly, Enrichr-embedded ChIP enrichment analysis (41–43) revealed that LFS/SFRP2OE up-regulated genes were enriched for FOXM1 targets (P = 1.52 × 10⁻¹⁶) (Fig. 2F). This suggests a correlation between SFRP2OE-mediated cell proliferation and the oncogenic function of FOXM1 target genes in human OS, irrespective of a p53 mutation. Common LFS/SFRP2OE down-regulated genes are associated with extracellular matrix and cell–cell adhesion (SI Appendix, Fig. S2D). Interestingly, tumor-suppressor genes such as CADM1 and CADM4 [tumor-suppressor gene database (44)] were enriched in the down-regulated gene set (Fisher’s exact test, P = 0.004) (SI Appendix, Fig. S2D).

Finally, we compared SFRP2OE DEGs with the human OS Gene Expression Omnibus (GEO) set (GSE36001) to determine the correlation with OS features through GSEA. SFRP2OE and LFS DEGs were highly correlated with human OS signature genes, whereas SFRP2KD and WT DEGs were highly concordant with OB signature genes (Fig. 2G and SI Appendix, Fig. S2E). Additionally, SFRP2OE signature genes were significantly enriched in up-regulated genes of the NCI-60 panel of cell lines harboring TP53 mutations and in the Rb1-knockout mouse, suggesting that SFRP2 strongly contributes to the signature of well-known OS genetic lesions, RB1 deletion, and TP53 mutation (Fig. 2H and SI Appendix, Fig. S2F). Collectively, our transcriptome analyses revealed tumorigenic properties of SFRP2OE in LFS and OS.

SFRP2OE Dysregulates OB Differentiation Through BMP/WNT Suppression.

We reported that LFS iPSC-derived MSCs and pre-OBs have defects in OB differentiation, supporting the idea that compromised p53 dysregulates OB differentiation and promotes the generation of abnormal MSCs and pre-OBs, the cells of origin of OS (33). Because our global transcriptome analyses provided evidence that SFRP2OE potentially perturbs OB differentiation (Fig. 2C), we wondered if SFRP2OE in LFS suppresses normal OB differentiation as an oncogenic downstream modulator of mutant p53. We performed alkaline phosphatase (AP) and Alizarin red S staining on LFS, SFRP2OE, and WT cells at different OB differentiation time points (D0–D21). Normal OBs exert a high level of AP activity and secrete large amounts of calcium (mineral deposition) detected by Alizarin red S staining (45–47). SFRP2OE in WT cells clearly suppressed normal OB differentiation, similar to its effect in LFS cells (Fig. 3 A and B). In contrast, SFRP2KD in LFS cells rescued osteogenic AP activity and mineral deposition (SI Appendix, Fig. S3 A and B). In agreement with this observation, LFS and SFRP2OE OBs had lower levels of pre-OB markers (ALPL and COL1A1), mature OB markers (BGLAP and PTH1R), osteocyte markers (ZNF521 and FGF23), and OB differentiation transcriptional factors (RUNX2 and OSX) (Fig. 3C). Interestingly, ATF4, whose expression is high in OS tissue and OS cell lines (48), was up-regulated in SFRP2OE and LFS OBs (Fig. 3C). These results, together with our finding that LFS4 (P53.pY205C)-derived OBs also showed reduced mineral deposition (SI Appendix, Fig. S3C), indicate that SFRP2OE as a consequence of mutant p53 perturbs normal osteogenic processes, which is consistent with the RNA-seq biological process analysis (Fig. 2C).

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).

The BMP/SMAD and canonical WNT pathways are required for normal osteogenesis (49–51). In particular, BMP ligands facilitate OB differentiation through the BMP/SMAD pathway (50, 52, 53). Using a luciferase-based phospho-SMAD reporter system, we monitored BMP signaling activity throughout in vitro differentiation (D0–D14). While BMP signaling was relatively low in MSCs and progenitors (D4), it increased in pre-OBs (D10–D14) (Fig. 3D). This result is consistent with published in vivo data (52, 54). As expected, SFRP2OE and LFS cells showed lower BMP signaling activity than WT OBs. It supports the idea that SFRP2OE perturbs normal OB differentiation in part through inhibition of the BMP pathway.

The effects of SFRP2 on canonical WNT signaling are context dependent. Generally, SFRP2 suppresses WNT signaling by binding WNT ligands, thus preventing their binding to WNT receptors (30, 55, 56). However, SFRP2 has also been shown to activate WNT signaling at low concentrations or in certain cell types (35, 57–59). To understand the role of SFRP2 in canonical WNT signaling in OS development, we performed Western blot and β-catenin immunostaining. Compared with WT cells, LFS, SFRP2OE, and HOS cells consistently had lower levels of cytoplasmic β-catenin (SI Appendix, Fig. S3D), while the amount of nuclear β-catenin increased only in WT groups with WNT3a treatment (SI Appendix, Fig. S3E). Using the TOP/FOP flash luciferase WNT signaling reporter system, we observed low basal WNT activity in all groups (Fig. 3E, no treatment). We activated WNT signaling using WNT3a (100 ng/mL) or LiCl (10 mM), which inhibits GSKβ. SFRP2OE showed a reduction in WNT3a-mediated canonical WNT activity (Fig. 3E). LFS and HOS cells exhibited little or no response to either LiCl or WNT3a (Fig. 3E and SI Appendix, Fig. S3F). Ectopic expression of constitutively active β-catenin (DeltaN90) in LFS did not facilitate cell proliferation or promote cell transformation (SI Appendix, Fig. S3G). However, knockdown of β-catenin led to severe cell death of LFS cells, confirming that β-catenin is necessary for homeostasis and viability of MSCs and OBs (SI Appendix, Fig. S3H) (55, 56). Although it is widely accepted that hyper-WNT signaling is associated with oncogenicity in many cancers, our data support the idea that hyper-WNT is not always involved in osteosarcomagenesis. This is consistent with the observation of inactive canonical WNT activity in high-grade human OS (57).

SFRP2OE Transforms Pre-OBs into OS-Like Cells.

We next investigated the oncogenic contribution of SFRP2OE by analyzing cell morphology and cell proliferation in ectopic SFRP2OE and SFRP2KD conditions. SFRP2OE in WT cells resulted in the development of aggregated structures, which is reminiscent of tumor cell aggregation, and was enhanced in ectopic SFRP2OE in LFS cells at D14–D17 of OB differentiation (Fig. 4A). Overexpression also increased proliferation during MSC culture and OB differentiation (Fig. 4B and SI Appendix, Fig. S4A). In contrast, SFRP2KD cells did not form aggregated structures and showed decreased cell proliferation compared with LFS cells (Fig. 4B and SI Appendix, Fig. S4B).

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).

It was reported that ectopic SFRP2 expression alters the G2 phase of the cell cycle and suppresses apoptosis (32). Our analysis of both cell cycle and apoptosis on D4 of OB differentiation revealed significantly more SFRP2OE cells in S phase (72.17 ± 3.22%) compared with WT cells (29.82 ± 1.68%) (Fig. 4C and SI Appendix, Fig. S4C). This implies that SFRP2OE promotes S-phase entry, cell-cycle progression, and, thus, cell proliferation. In contrast, SFRP2KD resulted in significantly less cell proliferation with more SFRP2KD cells in G0/G1 (21.51 ± 2.68%) and fewer in S phase (77.36 ± 2.86%) compared with LFS cells (G0/G1: 8.60 ± 4.32%; S phase: 90.51 ± 1.99%) (Fig. 4 B and C and SI Appendix, Fig. S4 B and D). No statistical difference in apoptosis was found among the various groups (Fig. 4C). Collectively, we observed that SFRP2OE promotes S-phase entry at the pre-OB stage.

Anchorage-independent growth is a hallmark of cancer cell transformation. We used the soft agar colony-formation assay to determine the transformation ability of SFRP2. SFRP2OE and 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 formed by SFRP2OE and LFS cells were significantly greater than those of WT cells. In contrast, SFRP2KD cells formed significantly fewer colonies than LFS cells (Fig. 4E). These data clearly demonstrate the tumorigenic ability of SFRP2.

To determine the tumorigenicity of SFRP2OE, we performed in ovo chick chorioallantoic membrane (CAM) assays (SI Appendix, Fig. S4G). This assay provides a convenient and common way to check the oncogenicity of sarcoma cells (58, 59). SFRP2OE cells showed increased tumor formation and cell numbers compared with WT cells (Fig. 4F). SFRP2KD cells had suppressed tumor formation compared with LFS cells (Fig. 4G). Since SFRP2 is a secreted molecule, we wondered if a blocking antibody would reduce OS features. We used the well-documented p53 mutant osteosarcoma cell line HOS. As hypothesized, adding a monoclonal anti-SFRP2 antibody to HOS cells significantly reduced tumorigenesis (Fig. 4H). In serial transplantation analysis, SFRP2OE cells maintained their tumor-formation ability (SI Appendix, Fig. S4H), while the antibody in HOS consistently suppressed it over three consecutive transplantations (SI Appendix, Fig. S4I). Additionally, using the CAM assay, we checked whether FOXM1 inhibition could suppress tumor formation. We used FOXM1 shRNA (FOXM1KD) and the small molecule FDI-6, a reported potent inhibitor of FOXM1 (60). The result showed that both FOXM1KD and FDI-6 treatment effectively decreased SFRP2-mediated tumor formation (SI Appendix, Fig. S4 J and K). These in ovo data demonstrate that SFRP2OE contributes to LFS-mediated tumorigenesis and OS development and that targeting SFRP2 with an antibody suppresses OS formation.

Secreted SFRP2 Facilitates Angiogenesis and Tumorigenesis Through CYR61 Induction in Endothelial Cells.

Angiogenesis is a critical component of solid tumor growth. SFRP2 has been shown to function as an angiogenic factor in human triple-negative breast cancer, angiosarcoma, and melanoma (38, 58, 61). However, whether SFRP2 has any angiogenic properties in OS remains unknown. Because SFRP2 is a secreted protein, we hypothesized that MSCs and pre-OBs in LFS patients could secrete SFRP2 into the bone matrix where it initiates migration and sprouting of endothelial cells, thereby facilitating neoangiogenesis. To investigate this, we conducted an in vitro angiogenesis assay; we cultured human vascular endothelial cells (HUVECs) on Matrigel in WT-, LFS-, or SFRP2OE-conditioned medium (CM) for 16 h and then quantified the tube formation rate. As expected, WT CM was not able to significantly induce tube formation (Fig. 5 A and B). In contrast, LFS and SFRP2OE CM dramatically increased tube formation, as did the addition of recombinant SFRP2 protein to WT CM (Fig. 5 A and B). Moreover, SFRP2KD CM dramatically decreased tube formation to the levels of the negative control and WT CM. This result is consistent with other reports showing the angiogenic function of SFRP2 in angiosarcoma and melanoma (38, 61).

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).

To investigate how SFRP2 increases angiogenesis, we performed RNA-seq profiling of HUVECs after treatment with LFS, SFRP2OE, and WT CM. PCA with triplicate samples from each condition showed that the replicates clustered together but were distinct among the conditions (Fig. 5C). To identify SFRP2-mediated effects on angiogenesis induction, we focused on identifying and analyzing DEGs between SFRP2OE and WT conditions that are also differentially expressed between LFS and WT conditions. Using DESeq2 (Padj < 0.01), we identified 498 common genes (258 up-regulated and 240 down-regulated) between 1,826 LFS and 969 SFRP2OE DEGs (Fig. 5D and SI Appendix, Fig. S5 A and B). DAVID functional annotation of the 258 up-regulated common genes showed that secreted SFRP2 mainly alters angiogenesis, antiapoptosis, and inflammatory response processes of endothelial cells (Fig. 5E). Most up-regulated angiogenic genes are either well-known angiogenic markers or procancer genes such as VEGFC, CYR61, ANGPT2, VASP, EPAS1, JUNB, and CDH13 (Fig. 5F). High expression levels of CYR61, known to facilitate angiogenesis, were recently associated with poor prognosis in OS (62, 63). Interestingly, we also observed increased expression of CYR61 in endothelial cells treated with SFRP2OE and LFS CM (Fig. 5 F and G). Thus, we investigated if SFRP2OE increases angiogenesis and tumor formation through an action of CYR61. Measuring the level of neovascularization and tumor size in a CAM assay, we found that CYR61 antibody treatment decreases angiogenesis and tumor formation in the presence of SFRP2 (Fig. 5 H–J). This supports the idea that secreted SFRP2 facilitates angiogenesis of endothelial cells and tumorigenesis, at least in part, through CYR61.

Our RNA-seq analysis revealed the up-regulation of antiapoptosis-related genes in HUVECs by LFS and SFRP2OE CM (SI Appendix, Fig. S5D). Indeed, the number of viable HUVECs was higher in SFRP2OE CM and LFS CM than in WT CM (SI Appendix, Fig. S5E). These results suggest that SFRP2 facilitates angiogenesis and protects endothelial cells from apoptosis by inducing the expression of angiogenic/oncogenic factors and antiapoptosis regulators, respectively. Intriguingly, cell-cycle–related DEGs in HUVECs treated with LFS CM and SFRP2OE CM were mainly down-regulated (SI Appendix, Fig. S5 C and F and Datasets S5–S7). There was little overlap in cell-cycle–related DEGs between HUVECs activated by LFS CM and SFRP2OE CM or in LFS and SFRP2OE OBs (SI Appendix, Fig. S5G). For example, positive cell-cycle regulators such as CDC25B, CKS1B, DLGAP5, GTSE1, JAG2, and NUSAP1 were down-regulated in endothelial cells exposed to LFS and SFRP2OE CM but were up-regulated in OBs exposed to LFS and SFRP2OE (SI Appendix, Fig. S5G). Only three genes showed a common expression pattern in the two groups: JUNB and MYC, two tumorigenic factors, were up-regulated, while KRT18, an intermediate filament gene, was down-regulated in both OBs and HUVECs affected by SFRP2 signaling (SI Appendix, Fig. S5G). We speculated that HUVECs in LFS CM and SFRP2OE CM stopped proliferating and initiated differentiation to generate neo-vasculature. This result emphasizes that the function of SFRP2 is context dependent.

Targeting SFRP2OE Effectively Suppresses OS Development.

To examine if suppression of SFRP2 expression in LFS has any therapeutic effect on OS development in vivo, we s.c. injected LFS and SFRP2KD cells at D7 of OB differentiation into nu/nu mice. Tumors formed rapidly in mice injected with LFS3-A cells (at 2 wk postinjection) or LFS2-B cells (at 3 wk postinjection) (SI Appendix, Fig. S6A). We attribute the difference in tumor-formation timing to lower SFRP2 expression in LFS2-B cells than in LFS3-A cells (Fig. 1B). In contrast, mice injected with SFRP2KD cells did not form tumors, or the tumors were of very small size (Fig. 6A and SI Appendix, Fig. S6A). Histological analysis revealed that LFS tumors were composed of large, poorly differentiated malignant tumor cells with mitotic features and large areas of necrosis reminiscent of the tissue morphology seen in human osteoblastic or fibroblastic OS (Fig. 6B and SI Appendix, Fig. S6B) (64). A few of the small tumors in the SFRP2KD group showed some neoplastic features, while others showed induction of fibrous tissues (Fig. 6B and SI Appendix, Fig. S6B). Interestingly, LFS tumors showed lower levels of AP, a marker of mature OBs, than tumors formed from SFRP2KD cells (Fig. 6B). Despite higher β-catenin expression, β-catenin was localized to the cytosol, granules, and cell membrane in the SFRP2KD group (Fig. 6B). This finding augments the idea that SFRP2-mediated tumorigenesis is not directly linked to hyperactive WNT signaling in LFS. Furthermore, we found more vasoformation, as assessed by expression of the CD31 endothelial marker, in LFS cell-induced tumors than in the SFRP2KD group (Fig. 6 C and D). These histological analyses support the idea that SFRP2OE facilitates neovascularization and tumorigenesis. As in the SFRP2KD group, targeting SFRP2 in malignant OS cells (MNNG) reduced OS tumor growth (SI Appendix, Fig. S6C). Additionally, we checked the expression of signature genes altered by SFRP2KD by quantitative PCR in these primary tumors (Fig. 6E and SI Appendix, Fig. S6D). While CADM1 and CADM4 were significantly up-regulated in SFRP2KD OBs and in SFRP2KD primary tumors compared with parental LFS and OS, oncogenic CYR61 and FOXM1 were only differentially expressed (i.e., decreased) between SFRP2KD and empty-vector groups in primary tumors (Fig. 6E and SI Appendix, Fig. S6D).

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) Immunohistochemistry analysis of representative MNNG and SFRP2KD tumors in G.

We next performed intratibial injection in mice (65, 66) to investigate the orthotopic effect of SFRP2 depletion in OS development. In the LFS group, two mice generated tumors in the tibia 5 mo postinjection, and one mouse formed a tumor on the flank of the body but not intratibially (SI Appendix, Fig. S6E). This might have been caused by a failure of proper intratibial injection. Three mice died within 3 mo of injection with no visible exterior tumor formation but with clear signs of weight loss. The actual cause of death was undetermined. In stark contrast, the SFRP2KD group did not form any osteosarcomatous lesions (SI Appendix, Fig. S6E). To corroborate this finding, we depleted SFRP2 in MNNG cells. Suppressing SFRP2 expression in MNNG cells significantly reduced tumor incidence and size (Fig. 6 F and G). Primary lesions showed characteristics of primary human OS with osteolytic, mitotically active undifferentiated cells as well as wide bone destruction (Fig. 6H and SI Appendix, Fig. S6F). Microscopically, MNNG tumors showed less AP activity, more proliferating (Ki67⁺) cells, and more neovascularized (CD31⁺) areas than the SFRP2-depleted MNNG tumors (Fig. 6H). The bone structure in the SFRP2KD group was relatively intact, although the tumors still contained undifferentiated tumorigenic cells in the proximal tibia (Fig. 6G and SI Appendix, Fig. S6F). Conclusively, the in vivo xenograft study unequivocally demonstrates that depletion of SFRP2 in LFS-mediated tumorigenesis and OS development has potential therapeutic benefits.

Discussion

We show that LFS patient-specific iPSCs and their MSCs/OBs provide a platform for investigating the early development of OS, and we identify and evaluate a potential therapeutic target, SFRP2. Using molecular and functional experiments, traditional in vivo studies, and bioinformatic analyses, we found that SFRP2 expression contributes significantly to LFS-mediated tumorigenesis and OS development by facilitating angiogenesis and cell proliferation. Most significantly, we demonstrated that targeting SFRP2 has a potential therapeutic application in LFS and OS.

We found that SFRP2 is up-regulated in LFS patient cells and OS cell lines harboring TP53 mutations. We showed that SFRP2 exerts oncogenicity in MSCs/OBs both in vitro and in vivo. These findings conflict with the hyper-WNT signaling paradigm in cancer biology. Generally, SFRP2 is known as a WNT pathway antagonist due to its ability to sequester WNT ligand from the frizzled receptor and is considered to be a tumor suppressor. Indeed, promoter hypermethylation of SFRP2 is found with increased WNT signaling in many cancers (67, 68). One study found that enhanced WNT signaling as a consequence of SFRP2 methylation is associated with OS cell invasion (69). However, in the LFS context, SFRP2 functions as an oncogenic factor. We consistently observed that SFRP2OE suppressed WNT signaling and that the majority of β-catenin remained localized to the cytoplasm. Our data also indicate that lower β-catenin expression and inactive β-catenin are associated with the initiation of abnormal OB differentiation and are maintained to the terminal stage of osteosarcomagenesis. This finding is consistent with a report of inactive β-catenin and WNT signaling in high-grade human OS (57). Our study adds evidence of the complexity of WNT pathway regulation and its diverse outcomes in different conditions.

The mechanism by which SFRP2 exerts its role in OS development is unknown and remains elusive, context dependent, and even controversial in many other cancers. A recent paper described an age-related increase in SFPR2 expression in fibroblasts that activated a multistep signaling cascade in melanoma cells, resulting in a reduction of β-catenin and MITF expression and consequently in the loss of APE1. The absence of APE1 affected the DNA damage response of the cells, increased metastasis, and rendered the cells more resistant to chemotherapy (38). In our study, we observed that SFRP2OE suppresses β-catenin, but MITF or APE1 expression was not altered. Instead, we found that SFRP2OE in OBs up-regulates FOXM1 in OBs as an autocrine factor and CYR61 in endothelial cells as a paracrine factor in a β-catenin–independent way. Therefore, we concluded that SFRP2-associated osteosarcomagenesis develops through a mechanism different from that in melanoma.

Oncogenesis is attributed to abnormal differentiation, aberrant cell adhesion, a dysregulated cell cycle, and overproliferation. Our transcriptomic analysis of SFRP2OE and SFRP2KD cells showed that SFRP2 has multiple functions during abnormal differentiation and tumorigenesis in LFS OS development. Specifically, SFRP2OE changes the cell-cycle profile and increases the expression of cell-proliferation–related genes such as AURKB and FOXM1. AURKB regulates chromosomal segregation during mitosis and meiosis, and aberrant expression of AURKB is observed in several cancers (70, 71). FOXM1 is also overexpressed in many cancers (72, 73). Using our LFS OB model in the in ovo CAM assay, we found that inhibition of FOXM1 impedes OS development (SI Appendix, Fig. S4 J and K). FOXM1 regulates the transcription of cell-cycle and proliferation genes (74, 75), including the SKP2 and CKS1 subunits of the SCF ubiquitin ligase complex. These two proteins ubiquitinate p21 (Cip1) and p27 (Kip1), regulators of cell-cycle progression into S phase, for degradation (76). Therefore, the observed increase in cell proliferation and in the number of cells in S phase of LFS and SFRP2OE cells may be explained by SFRP2OE-induced FOXM1. One may assume that SFRP2 depletion in LFS cells should reduce FOXM1 expression. However, this is not what we observed. We explain this by the autoregulation of FOXM1 expression (77, 78). Thus, once FOXM1 expression is induced, its expression is maintained, and SFRP2 depletion has no apparent effect. Nevertheless, FOXM1 inhibition by shRNA and FDI-6 small-molecule treatment effectively decreased tumor formation (SI Appendix, Fig. S4 J and K). It is noteworthy that FOXM1 up-regulation was correlated with a poor prognosis in OS patients (79). This fits well with our own observation that high SFRP2 expression in OS tissues is linked to decreased patient survival (Fig. 1F and SI Appendix, Fig. S1G).

Additionally, we found that down-regulated genes common to LFS and SFRP2OE that are up-regulated in SFRP2KD cells include cell-adhesion genes such as CADM1 and CADM4. They are known tumor suppressors in various cancers (80–83). The significance and role of CAMD1 and CADM4 down-regulation in OS development is unknown. A possibility is that their down-regulation is linked to anchorage-independent cell growth and cell egress from the bone environment, resulting in metastasis. This warrants future investigation. Taken together, the abnormal expression of these specific genes (AURKB, FOXM1, CADM1, and CADM4) clearly indicates the oncogenic property of SFRP2OE in OS.

Angiogenesis is a critical hallmark of cancer progression. In this study we show that secreted SFRP2 facilitates angiogenesis in vitro and in vivo. Global transcriptome analysis revealed up-regulation of angiogenesis-related and antiapoptotic genes in endothelial cells grown in LFS, SFRP2 cell-conditioned, and SFRP2-supplemented media. Angiogenesis-related genes such as CDH13, EPAS1, and CYR61 are highly associated with focal adhesion and tumorigenesis. Particularly, CYR61, a secreted extracellular matrix protein, is a proangiogenic/tumorigenic molecule validated in many cancers (84, 85). Interestingly, CYR61 mediates specific functions in different types of cells through binding to distinct integrins during angiogenesis (86, 87) and metastasis (88). CYR61 is overexpressed in OS compared with normal bone tissues, and its depletion causes inhibition of neoangiogenesis in the developing tumor (62). We found that CYR61 inhibition prevents SFRP2-mediated OS development in the in ovo CAM assay (Fig. 5 H and I). Collectively, the SFRP2–CYR61 axis in LFS may alter the extracellular matrix environment, thus promoting angiogenesis, tumorigenesis, and possibly metastasis. Surprisingly, SFRP2 did not increase the expression of cell-cycle–related genes in endothelial cells, in contrast to its effects in LFS and SFRP2OE OBs.

Overall, our data provide insights into the role of SFRP2 in OS initiation and development. Using the powerful tool of iPSC-based modeling, we show that SFRP2 has a dual function. SFRP2 increases the proliferative capacity of pre-OBs and also can promote angiogenesis through cell-adhesion/cell-matrix factors; SFRP2-FOXM1 in LFS OBs/OS and SFRP2-CYR61 in endothelial cells contribute to OS development. We did not observe a significant effect of SFRP2 in metastasis of LFS/OS. However, a recent report showed that SFRP2 potentially plays a crucial role in metastatic OS, where its ectopic expression enhanced invasiveness and metastasis of human and mouse OS cells without affecting cell proliferation (89). In contrast, one study found that increased SFRP2 methylation resulting in reduced expression is associated with OS invasiveness (69). These observed discrepancies in the role of SFRP2 in invasiveness and metastasis of OS might be due to differences in the progressive stage of the cells or in the specific OS subset studied. Detailed future exploration of the SFRP2–FOXM1 and SFRP2–CYR61 axes during OS development and metastasis will provide additional mechanistic insight and help develop novel therapeutic strategies to treat LFS and OS.

Materials and Methods

WT and LFS iPSC and p53-Mutant ESC Culture.

All iPSCs were generated using the Sendai virus method as previously reported (33, 90). ESCs and iPSCs (SI Appendix, Table S1) were cultured in DMEM/F12 supplemented with 20% knockout serum replacement, 10 ng/mL basic FGF (Invitrogen) on mouse feeder cells (GlobalStem), or in mTeSR (STEMCELL Technologies) on Matrigel-coated plates.

iPSC Differentiation to MSCs and OBs.

iPSCs were differentiated to MSCs and OBs as previously described (33). For details see SI Appendix.

SFRP2 Gene Expression and Knockdown.

SFRP2OE and SFRP2KD, respectively, were achieved by infecting iPSC-derived MSCs with lentiviral particles encoding for inducible SFRP2 or shRNAs against SFRP2 (SI Appendix, Table S5). For details see SI Appendix.

OS Cell Lines.

Human OS cell lines were obtained from Robert Maki (Icahn School of Medicine, New York) and Nino Rainusso (Texas Children’s Hospital, 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 5% CO₂ in a humidified incubator.

Genome Editing.

The P53p.G245D mutation was introduced into WT iPSC-derived MSCs using CRISPR/Cas9, appropriate donor plasmid, and guide RNAs designed on the web tool at crispr.mit.edu (91). The various heterozygous p53 mutant ESC lines (SI Appendix, Table S1) were generated using TALENs designed with the ZiFiT Targeter version 4.2 (zifit.partners.org/) (92) as described in ref. 93. For details see SI Appendix.

Angiogenesis/Tube-Formation Assay.

Tube formation was assayed using the In Vitro Angiogenesis Assay Kit (Abcam) as recommended by the manufacturer. Tube formation was captured 16 h post culture initiation with an EVOS FL Cell Imaging system (Thermo Fisher Scientific). Images were analyzed with the ImageJ Angiogenesis Analyzer tool, which quantifies the tube-formation images by extracting characteristic information about the formed tube meshes and branches (94). For further details see SI Appendix.

CAM Xenograft Assay.

The CAM assay with 1 × 10⁶ differentiated OBs (D7 or D14) was conducted as described previously (95). For further details see SI Appendix.

Mouse Studies.

Eight-week-old male nu/nu mice (Charles River) were used for intratibial and s.c. xenografts. Sample size was determined based on power calculation (G*POWER software; with one-way ANOVA) and our estimation of surgical failure (96). All animal use was reviewed and approved by the Icahn School of Medicine’s Institutional Animal Care and Use Committee and was conducted according to federal, state, and local regulations. For further details see SI Appendix.

Human OS Tissue Microarray.

The OS tissue microarray set was obtained from E.G.D. The use of the OS microarray set and data analysis were approved by the Icahn School of Medicine’s Program for the Protection of Human Subjects and its Institutional Review Board. For further details see SI Appendix.

RNA-Seq and Data Analyses.

cDNA library preparation and sequencing was outsourced to Novogen. Sequence reads were aligned to human transcript 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 were further normalized by reads per kilobase of transcript per million reads mapped (RPKM) as the measurement of gene-expression abundance. For the original dataset, we used linear regression to identify DEGs independent of time (D0, D7, D14, and D17) among the different conditions (WT, SFRP2OE, LFS, or SFRP2KD cells) G∼time+condition. The P value cutoff (P < 0.001) was validated by permutation test (FDR <0.01). For the HUVEC dataset, DESeq2 (Bioconductor) was used to compare gene expression among WT, LFS, and SFRP2OE cells. An adjusted P value cutoff (Padj < 0.01) was applied to define DEGs. We used DAVID (version 6.8) or Enrichr (41, 42) for functional ontology and pathway analyses of DEGs depending on which method would provide the best visualization for the intended experimental purpose. For additional details and links to gene lists 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 immunostaining and quantification, the CyQUANT NF fluorescence proliferation assay (Thermo Fisher Scientific), and cell-cycle analysis can be found in SI Appendix.

Statistical Analysis.

Student’s t test (two-sided) was applied, and changes 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 points) ANOVA was applied. Survival in human subjects and mouse experiments was represented with Kaplan–Meier curves, and significance was estimated with the log-rank test. The statistical methods used for RNA-seq analysis are described under RNA-seq data analysis above. The data are shown as mean ± SD of at least three biological replicates. Statistical analysis was conducted using Microsoft Excel or GraphPad Prism software packages.

Additional Data.

Histological data are available in SI Appendix, Table S6. Resources and reagents will be provided upon proper requests and use agreements. According to regulations, we do not share patient or clinical information linked to the human OS tissue microarray set except as provided in this paper (SI Appendix, Table S2).