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. 2009 Apr;119(4):837-51.
doi: 10.1172/JCI37175. Epub 2009 Mar 23.

Wnt Inhibitory Factor 1 Is Epigenetically Silenced in Human Osteosarcoma, and Targeted Disruption Accelerates Osteosarcomagenesis in Mice

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Wnt Inhibitory Factor 1 Is Epigenetically Silenced in Human Osteosarcoma, and Targeted Disruption Accelerates Osteosarcomagenesis in Mice

Maya Kansara et al. J Clin Invest. .
Free PMC article


Wnt signaling increases bone mass by stimulating osteoblast lineage commitment and expansion and forms the basis for novel anabolic therapeutic strategies being developed for osteoporosis. These strategies include derepression of Wnt signaling by targeting secreted Wnt pathway antagonists, such as sclerostin. However, such therapies are associated with safety concerns regarding an increased risk of osteosarcoma, the most common primary malignancy of bone. Here, we analyzed 5 human osteosarcoma cell lines in a high-throughput screen for epigenetically silenced tumor suppressor genes and identified Wnt inhibitory factor 1 (WIF1), which encodes an endogenous secreted Wnt pathway antagonist, as a candidate tumor suppressor gene. In vitro, WIF1 suppressed beta-catenin levels in human osteosarcoma cell lines, induced differentiation of human and mouse primary osteoblasts, and suppressed the growth of mouse and human osteosarcoma cell lines. Wif1 was highly expressed in the developing and mature mouse skeleton, and, although it was dispensable for normal development, targeted deletion of mouse Wif1 accelerated development of radiation-induced osteosarcomas in vivo. In primary human osteosarcomas, silencing of WIF1 by promoter hypermethylation was associated with loss of differentiation, increased beta-catenin levels, and increased proliferation. These data lead us to suggest that derepression of Wnt signaling by targeting secreted Wnt antagonists in osteoblasts may increase susceptibility to osteosarcoma.


Figure 1
Figure 1. A high-throughput screen for epigenetically silenced tumor suppressor genes linking transformation with loss of differentiation in osteosarcoma.
(A) Design of high-throughput screen and filtering strategy for identification of epigenetically silenced tumor suppressor genes linking differentiation and transformation in osteosarcoma. (B) Dose-dependent induction of ALP activity in G292 cells treated with dAC for 5 d. Values are mean ± SEM of 3 separate determinations. (C) Effect of demethylation on expression of osteoblast- and muscle-specific genes, as well as a set of known oncogenes. Data are from transcriptional profiling, and are the median fold change across all 6 cell lines after treatment for 3 d with 5–10 μM dAC. The experiment was performed independently twice. (D) RT-PCR confirmation of changes in gene expression in SJSA and G292 cells. OSX, osterix.
Figure 2
Figure 2. Epigenetic silencing of Wif1 derepresses Wnt signaling.
(A) Western blot showing marked suppression of β-catenin levels after treatment of osteosarcoma cell lines with 5–10 μM dAC for 3 d. (B) RT-PCR showing induction of WIF1 transcript after demethylation of B143 cells for 72 h. (C) B143 cells were treated with 3 μg/ml recombinant GST-WIF1 or GST alone (control) for 24 h, then fixed and stained for β-catenin (green) and counterstained with propidium iodide (red). Shown are brightfield (left), coregistered (middle), and β-catenin intensity heatmap (right) images. Scale bar: 100 μm. The experiment was performed independently 3 times with similar results. (D) Effect of WIF1 expression on TCF/LEF reporter activity. TOP-flash and FOP-flash luciferase reporter constructs were cotransfected with constant amounts of LacZ vector control (10:1 ratio) into B143 cells with varying amounts of WIF1 expression vector. Cells were harvested within 24 h, and luciferase activity was measured. Data are normalized to β-galactosidase expression. Values represent independent transfections done in triplicate and are presented as fold change relative to FOP-flash (mean ± SEM). *P < 0.01 versus control, Tukey test with 1-way ANOVA.
Figure 3
Figure 3. WIF1 regulates osteoblast differentiation and suppresses growth of osteosarcoma cell lines in vitro.
(A) WIF1 induced markers of bone differentiation. Primary human bone–derived NHB osteoblasts were flow sorted using antibodies to STRO1 and ALP. Cultures were treated with GST-WIF1 or equimolar amounts of GST for 24 h. The percentage of cells in each quadrant is indicated. (B) Quantitative RT-PCR analysis of osteoblast gene expression in flow-sorted ALP-positive NHB cells after treatment for 3 d with GST-WIF1. Values show fold change relative to GST treated control. The experiment was performed in triplicate. ON, osteonectin. (CE) MC3T3E1 cells were infected with empty vector control or with shRNAmir hairpin to Wif1 (462) or nonsense hairpin (848) and selected using puromycin. (C) Quantitative RT-PCR analysis and (D) Western blot of Wif1 and Runx2 in control and Wif1 knockdown cells. (E) Mineralization of control and Wif1 knockdown cells after treatment with β-glycerophosphate and ascorbic acid for 2 wk. Cells were stained with alizarin red. (F) NHB cells were immortalized with LXSN-HPV16-E6/E7 (E6/E7) or control LXSN vector, and stably transfected cells were analyzed by flow cytometry for STRO1 and ALP expression. The percentage of cells in each quadrant is indicated. (G) RT-PCR for Wif1 and OC and (H) Western blot for WIF1 and Runx2 in immortalized cells compared with control. (I) Effect of WIF1 and control GST on B143 cells. Cells were stained with Crystal violet. (J) Cell lines and primary NHB cells were plated at 30% confluence and treated with 3 μg/ml GST-WIF1 or GST alone for 3 d followed by an MTT assay. Data (mean ± SEM for triplicate wells) are expressed as percentages relative to GST control.
Figure 4
Figure 4. Gene targeting Wif1 in vivo.
(A) Gene targeting strategy. Mice were generated containing a knockin allele of tau/LacZ in the first exon of Wif1, resulting in loss of expression of the Wif1 gene. (B) Southern blot of derived mice tails, demonstrating WT, heterozygous, and homozygous alleles. (CG) Expression of Wif1 during mouse embryogenesis was detected by in situ hybridization. (C) Wif1 expression was initiated at E9.0 within the fore limb and weakly in the developing somites. (D) At E11.5, Wif1 expression was detected in the branchial arches, limb buds, and somites. (E) Wif1 was expressed in the apical ectodermal ridge (AER) and in the dorsal (D) and ventral (V) limb buds at E11.5. (F and G) Expression within the fore-limb bud at E11.5 was stronger on the dorsal (F) than the ventral (G) side. A, anterior; P, posterior; Pr, proximal; Di, distal. (HJ) LacZ expression under the control of the native Wif1 promoter in the developing embryo at E14.5. Patterns of β-galactosidase expression reflect endogenous patterns of Wif1 expression. (H) LacZ activity was detected in the craniofacial region, hair cells, limbs, and retina. (I) Expression of LacZ within the eyelid and retina. (J) Expression of LacZ within the digits of the hind limb.
Figure 5
Figure 5. Mice lacking expression of Wif1 undergo normal skeletal development.
(AE) Patterns of LacZ expression in the adult mouse. Mice were sectioned and stained for β-galactosidase activity. (A) Sternum. (B) Cross section through rib. (C) Cartilage surface of hip joint. (D) Salivary gland. (E) Hair follicles in dermis. Scale bars: 100 μm. C, cartilage; B, bone. (F) The knockin allele disrupted expression of endogenous Wif1. Real-time RT-PCR for Wif1 expression in tissues from WT and Wif1–/– mice. Data (mean ± SEM for 3 primer sets spanning exons 1–2, 3–4, and 7–8) were repeated twice. (G) Femoral cortical bone mineral density (BMD), measured at 25% of bone length distal to the growth plate, in female (F) and male (M) WT and WIF1 knockout mice. (H) Trabecular bone mineral density, measured at 5% of bone length distal to the growth plate. n = 4–11 for all groups, except 52-wk-old females of both genotypes and 16-wk-old WT females (n = 2). No statistically significant differences were detected.
Figure 6
Figure 6. Wif1–/– mice are susceptible to spontaneous and radiation-induced osteosarcoma.
(A) Left: Sarcoma from the hind left limb of an 89-wk-old male Wif1–/– mouse, arising adjacent to periosteum, and infiltrating skeletal muscle. The adjacent cortical bone (black arrow) and skeletal muscle fiber surrounded by sarcoma cells (red arrow) are indicated. Right: Osteosarcoma from the lumbar spine of a 56-wk-old female Wif1–/– mouse, showing a malignant osteoid (yellow arrow). Scale bars: 100 μm. (B) Radiation-induced osteosarcoma model schematic. (C) Typical imaging appearance of osteosarcomas. Top: 18F-PET scan demonstrating intense avidity of osteoblastic tumors from the same mouse. Bottom: Histology of mouse radiation-induced osteosarcoma. Original magnification, ×20. (D and E) Kaplan-Meier plots of tumor onset (D; P = 0.0032, Mantel-Cox) and survival (E; P = 0.0382, Mantel-Cox) of Wif1–/– and WT mice after exposure to 45Ca (n = 23 [WT]; 22 [Wif1–/–]). (E) Data points represent individual mice; horizontal bars and error bars denote mean ± SEM.
Figure 7
Figure 7. Expression patterns and epigenetic silencing of WIF1 in human osteosarcoma.
(A) RT-PCR showing expression of WIF1 transcript in primary human osteoblasts, and lost in primary human osteosarcoma samples. hGAPDH, human GAPDH. (B) Immunohistochemical staining for WIF1 and β-catenin in normal bone and osteosarcomas. A tissue microarray was probed with antibodies to WIF1. Arrow indicates normal osteoblast. Original magnification, ×20. (C) Heatmap clustering of 30 primary osteosarcomas using differentiation and proliferation gene cassettes. Cluster A is a gene cassette of intermediate osteoblastic lineage, cluster B contains proliferation markers, and cluster C is a gene cassette of an osteoblastic phenotype. (D) Heatmap demonstrating unsupervised hierarchical clustering of CpG methylation in primary human osteoblast cultures (green), osteosarcoma cell lines (red), and primary osteosarcoma samples (blue). Individual CpGs are identified by numbers above, and the approximate relationship between CpG location and the WIF1 coding region is identified below. Cluster A represents the tumor-associated methylation group, and cluster B represents the normal osteoblast methylation group. The region underlined below the heatmap, from 63,801,793 to 63,802,086, appeared to be differentially methylated. Green denotes no methylation; red denotes 100% methylation. Asterisks indicate individual CpGs whose degree of methylation significantly correlated with expression of WIF1 transcript (all P < 0.05, ANOVA). The schematic below shows the position of Wif1 exon 1 and the untranslated region (UTR) in relation to regions of methylation.
Figure 8
Figure 8. Role of Wnt signaling in osteoblast development and osteosarcoma.
(A) Physiologic control by Wnts of osteoblast lineage commitment and expansion is terminated by a range of secreted antagonists, including WIF1, SOST, and DKKs. (B) Therapeutic targeting of Wnt antagonists such as SOST increases bone mass by increasing the commitment and number of osteoblasts. Epigenetic silencing of WIF1 leads to unopposed activity of Wnts, resulting in failure to undergo terminal cell cycle exit and increasing the likelihood of malignant transformation.

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