Secreted phosphoprotein 24 kD (Spp24) inhibits the growth of human osteosarcoma through the BMP‐2/Smad signaling pathway

Autocrine stimulation of tumor cells is an important mechanism for the growth of skeletal tumors. In tumors that are sensitive, growth factor inhibitors can dramatically reduce tumor growth. In this study, our aim was to investigate the effects of Secreted phosphoprotein 24 kD (Spp24) on the growth of osteosarcoma (OS) cells in the presence and absence of exogenous BMP‐2 both in vitro and in vivo. Our study demonstrated that Spp24 inhibited proliferation and promoted apoptosis of OS cells as confirmed by 3‐(4,5‐dimethylthiazolyl‐2)‐2,5‐diphenyltetrazolium bromide assay and immunohistochemical staining. We found that BMP‐2 increased the mobility and invasiveness of tumor cells in vitro whereas Spp24 inhibited both of these processes alone and in the presence of exogenous BMP‐2. Phosphorylation of Smad1/5/8 and Smad8 gene expression was enhanced by treatment with BMP‐2 but inhibited by treatment with Spp24. Subcutaneous and intratibial tumor models in nude mice demonstrated that BMP‐2 promoted OS growth in vivo, while Spp24 significantly inhibited tumor growth. We conclude that the BMP‐2/Smad signaling pathway is involved in the pathogenesis of OS growth and that Spp24 inhibits the growth of human OS induced by BMP‐2 both in vitro and in vivo. Interruption of Smad signaling and increased apoptosis appear to be the primary mechanisms involved. These results confirm the potential of Spp24 as a therapeutic agent for the treatment of OS and other skeletal tumors.


| INTRODUCTION
Osteosarcoma (OS) is an aggressive malignancy that arises from osteoprogenitor cells. 1 It is the most common type of primary bone tumor and features malignant osteoblasts that produce osteoid. 2 The incidence of OS has a bimodal age distribution with the highest incidence during the second decade of life. 3,4 Surgery and chemotherapy, alone or in combination, are the mainstays in the treatment of OS. In spite of improvements in surgical techniques and the use of new chemotherapeutic regimens, the 5-year survival rate in OS patients has remained constant over the last 20 years, especially in the presence of metastatic disease. 5 Furthermore, complete surgical excision is frequently not possible and, therefore, there is a great need for efficacious adjunctive therapies. 6 Autocrine stimulation of tumor cells by growth factors is an important mechanism of tumor growth. Pertinent growth factors include platelet-derived growth factor, insulin-like growth factor, adrenomedullin, endothelin-1, parathyroid hormone-related peptide fragments, 7 and, most importantly, members of the BMP/TGF-β superfamily of cytokines and their receptors. [8][9][10][11][12][13][14][15][16] BMPs-2, -4, -6, and -7 and their receptors are upregulated in OS, breast, prostate, and lung cancer cell lines. 7,[17][18][19][20] Because of their central roles in the development, growth, and remodeling of bone, members of the TGF-β/BMP family of proteins are regarded as the most promising targets for therapeutic intervention aimed at growth factor systems in bone tumors. 21 Another concern regarding the comprehensive therapy for OS is the reconstruction of the bone defect after limb-salvage surgery.
While bone morphogenetic proteins (BMPs) have been found to be helpful additions in skeletal reconstructive surgery, a conflict exists since they have also been found, in some cases, to possess tumorenhancing properties. 22 Because of existing information supporting a role for BMPs as autocrine factors in OS cell growth, 23

| Production of Spp24
Recombinant bovine Spp24 was produced in a bacterial expression vector as described in detail previously. 24 The expression protein is engineered in such a manner as to place a (His)6-Met at the amino terminus of the recombinant form of the mature protein (lacking the leader sequence) to provide for stability and to allow purification of the protein by means of immobilized metal affinity chromatography (IMAC).

| Cell culture
The highly malignant 143B and moderately malignant MG63 cell lines used in this study were obtained from ATCC. Cells were grown in minimum essential medium (MEM) and Dulbecco's minimum essential medium (DMEM), respectively, supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 mg/mL) in a humidified atmosphere containing 5% CO 2 at 37°C. Media were changed every 3 days. Cells were passaged when cultures reached 80%-90% confluence.

| Wound-healing assay
143B and MG63 cells were seeded in 12-well microtiter plates and incubated for 24 h until 90% confluence was achieved, then starved overnight with 0.5% FBS medium. After changing back to normal culture media, a sterile 200 μL pipette tip was used to scratch wounds across the monolayer of cells and the wells were gently washed twice with phosphate-buffered saline (PBS) to remove the detached cells. 143B and MG63 cells were cultured with 2% FBS to minimize the influence of cell proliferation. Cells were treated with vehicle, BMP-2, Spp24, or BMP-2 plus Spp24 as described above.
The wounds were photographed at each time point using an inverted microscope (Leica). ImageJ software (National Institutes of Health; http://rsb.info.nih.gov/ij/) was used to quantify the migration rate by measuring the distance between the wound edges. All experiments were performed in triplicate.

| Matrigel invasion assay
Trans-well in vitro invasion 24-well chambers (Corning) were used following the manufacturer's instructions. The membrane of each well was precoated with 500 μL BD Matrigel (BD Biosciences).
2.5 × 10 4 143B or MG63 cells in 200 μL serum-free medium were placed into the upper chamber and then vehicle, BMP-2, Spp24, or BMP-2 plus Spp24 was added into the upper chamber. Five hundred microliters of medium containing 10% FBS was placed into the lower compartment. The trans-well chambers were incubated for 24 h at 37°C in 95% air with 5% CO 2 . The membranes were fixed with methanol and cell penetration through the membrane was detected using crystal violet staining. Cell penetration was quantified by counting the numbers of cells in five microscopic fields (at ×200 magnification) per filter. The studies were conducted in triplicate.

| Apoptosis analysis by flow cytometry (FCM)
143B cells were seeded into six-well microtiter plates at a density of 8 × 10 4 cells/well and cultured overnight. When a confluence of 70%-80% was reached, wells were washed with PBS, and cells were exposed to culture media with vehicle, BMP-2 (100 ng/mL), Spp24 (20 μg/mL), or BMP-2 + Spp24 (100 ng/mL, 20 μg/mL) for 24 h. For apoptosis analysis, 5 μL of annexin V-PE and 5 μL of 7-aminoactinomycin D (7-AAD) were added to all samples, which were then incubated at room temperature for 15 min in the dark according to the manufacturer's instructions (Thermo Scientific) and the fluorescence intensity of the samples was immediately analyzed by FCM.

| Quantitative real-time polymerase chain reaction (PCR)
To examine the effects of BMP-2 and Spp24 on Smad signaling in OS cells at the gene level, 143B and MG63 cells were seeded and grouped as Control, BMP-2 (100 ng/mL), Spp24 (20 μg/mL), BMP-2 + Spp24 (100 ng/mL, 20 μg/mL) in six-well microtiter plates at a density of 1 × 10 5 cells/well and cultured for 24 h. Total RNA was isolated by E.Z.N.A. ® HP Total RNA Kit (Omega Bio-Tek). One microgram RNA was subjected to reverse transcription using the Prime-Script RT reagent kit (Takara). Real-time PCR was performed with a 7300 Real-Time PCR system with SYBR ® Premix Ex Taq™ (Takara). The primer sequences used are listed in Table 1. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was evaluated in separate tubes for each RT reaction as a standard. Relative gene expression was analyzed by the ΔΔC T method. 25

| Co-immunoprecipitation (Co-IP)
Co-IP assays were performed using a Co-IP kit (Thermo Scientific).
143B cells were lysed in SDS lysis buffer on ice for 30 min, and after centrifugation at 4°C and 14,000g for 15 min, the supernatants were collected. The protein lysates were then incubated with anti-FLAG and anti-GFP antibodies on a rotator overnight at 4°C. Then, the mixtures were incubated with immobilized protein A/G beads (Thermo Scientific) on a rotator for 2 h at 4°C. The beads were collected by centrifugation at 3000g for 2 min and washed five times with 0.5 mL of IP wash buffer. SDS loading buffer was then added to the beads, and the samples were denatured at 95°C for 10-12 min.
Finally, the supernatants were collected and immediately analyzed by western blotting.

| Spp24 inhibits in vitro OS cell growth by inducing tumor cell apoptosis
The effects of Spp24 on 143B OS apoptosis were assessed using FCM assays and immunohistochemistry. Figure 2A were higher in the Spp24 and BMP-2 + Spp24 groups ( Figure 2C).
Immunocytochemistry of in vitro cultures of the OS cells showed that the proliferation marker PCNA was slightly upregulated in the BMP-2 group and downregulated in the Spp24 and BMP-2 + Spp24 groups ( Figure 2D,F), whereas the apoptosis marker Caspase-6 was downregulated by BMP-2 and upregulated by Spp24 ( Figure 2E,F).
Altogether, this suggests that Spp24 affected tumor cell apoptosis, which inhibited tumor growth and obviated the effects of BMP-2. the natural bone environment, not only due to its ability to bind several proteins of the TGF-β/BMP family, 27,28 but also related to its (or, more precisely, truncated fragments of it) little explored ability to stimulate intracellular signaling pathways. 29 We have engineered several protein and peptide products that, while they are thought to function by a single mechanism (BMP binding), operationally have seemingly paradoxically opposite outcomes along a spectrum from the enhancement of BMP activity ("slow-release") to inhibition of BMP activity ("sequestration"). 27 For example, bone morphogenetic protein binding peptide (BBP) is a synthetic cyclic 19 amino acid peptide that is based on the BMP binding domain of Spp24 which has a similarity to the TGF-β receptor. 30 When BBP was applied to a On the other hand, full-length Spp24, when applied to a collagen sponge to which BMP-2 was subsequently applied, dramatically inhibited the activity of BMP-2 in models of ectopic bone formation 26 and spinal fusion. 34 The latter observation suggested a role for Spp24 in the suppression of BMP/TGF autocrine systems.

| Spp24 inhibits OS growth in vivo
We employed the bone matrix protein Spp24 to test the hypothesis that the interruption of a key autocrine system could be  regulation of the BMP-2/Smad signaling at both gene and the protein expression levels. We cannot state definitively that the sequestration of BMP-2 is the only mechanism of action for the effects that we observed. Spp24 could also be acting by sequestering other members of the TGF-β family of proteins or through completely different biological mechanisms such as receptor binding and inhibitory signaling activation. While more research will be required to investigate these unknown factors, the fact remains that, empirically, Spp24 has shown great potential as an anticancer therapeutic.