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 po- tential. 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 over- expression 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. contrib- uted 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 develop- ment. Here, we report that SFRP2 overexpression (SFRP2OE) induces OS phenotypes, increases FOXM1 expression, and pro- motes 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 be- tween 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).