Aspirin Suppresses the Growth and Metastasis of Osteosarcoma

Published OnlineFirst July 22, 2015; DOI: 10.1158/1078-0432.CCR-15-0198

Cancer Therapy: Preclinical Clinical Cancer Research Aspirin Suppresses the Growth and Metastasis of Osteosarcoma through the NF-kB Pathway Dan Liao, Li Zhong, Tingmei Duan, Ru-Hua Zhang, Xin Wang, Gang Wang, Kaishun Hu, Xiaobin Lv, and Tiebang Kang

Abstract

Purpose: Aspirin has recently been reported to reduce both t test or ANOVA with the Bonferroni post hoc test were used for the the incidence and the risk of metastasis in colon cancer. statistical comparisons. However, there is no evidence at the cellular levels or in the Results: Aspirin reduced cell viability in a dose- and time- animal models for such an effect of aspirin on cancer dependent manner in osteosarcoma cell lines, and aspirin syn- metastasis. ergistically sensitized osteosarcoma cells to cisplatin (DDP) Experimental Design: MTT assay, colony formation assay, and in vitro and in vivo (P < 0.001). Moreover, aspirin markedly apoptosis assay were employed to analyze the effects of aspirin on repressed the migration and invasion of osteosarcoma cells the osteosarcoma cell viability in vitro. The NF-kB activity was in vitro (P < 0.001), and dramatically diminished the occurrence measured by the NF-kB p65 luciferase reporter. Western blotting of osteosarcoma xenograft metastases to the lungs in vivo was used to analyze the proteins in cells. The migration and (P < 0.001). Mechanistically, aspirin diminishes osteosarcoma invasion abilities of osteosarcoma cells in vitro were measured migration, invasion, and metastasis through the NF-kB pathway. by the Transwell assay. Xenograft-bearing mice were used to assess Conclusion: Aspirin suppresses both the growth and metasta- the roles of aspirin in both tumor growth and metastasis of sis of osteosarcoma through the NF-kB pathway at the cellular osteosarcoma in vivo (n ¼ 5–8 mice/group). An unpaired Student level and in the animal models. Clin Cancer Res; 1–11. 2015 AACR.

Introduction level and in the animal models to argue that aspirin may be an excellent auxiliary drug for treating patients with osteosarcoma. Osteosarcoma, which has a peak incidence in adolescence, is an extremely aggressive primary malignant bone tumor of childhood Materials and Methods (1). Currently, the 5-year overall survival is 75% to 77% for the primary nonmetastatic disease, however, no more than 20% in Reagents and cell culture metastatic osteosarcoma (2, 3). Patients with metastasis or recur- U2OS and MG63 cells were cultured according to the instruc- rence present a formidable challenge despite modern multimodal tions from the ATCC. ZOS and U2OS/MTX300 cells were therapy (4, 5). Therefore, finding new strategies for the patients described previously (15). HEK-293T cells were purchased from with osteosarcoma are necessary and urgent. the ATCC, cultured in DMEM (Invitrogen) with 10% FBS Aspirin, an NSAID, was used primarily for anti-inflammation (GIBCO), 1 mmol/L glutamine, and 100 U/mL each of penicillin and analgesic (6). Interestingly, a recent population study and streptomycin. All cell lines used in this study were authen- fi reported that acetylsalicylic acid (aspirin) reduces both the inci- ticated using short-tandem repeat pro ling less than 6 months dence and the risk of metastasis in colon cancer (7). However, ago when this project was initiated, and the cells have not been in there is no evidence at the cellular level or in animal models for culture for more than 2 months. such an effect of aspirin on cancer metastasis, even though aspirin Aspirin was purchased from Sigma-Aldrich. Antibodies against is widely used for both the prevention and treatment of many p65(C22B4), XIAP(3B6), CIAP1(D5G9), CIAP2(58C7), BCL2 a diseases, including cancers in clinical settings (7–14). In this (50E3), Survivin(6E4), Livin(D61D1), and -Tublin(2148S) report, we tried to provide comprehensive evidence at the cellular were purchased from Cell Signaling Technology. An antibody against GAPDH(SC-166574) was purchased from Santa Cruz Biotechnology.

State Key Laboratory of Oncology in South China, Collaborative Plasmids Innovation Center for Cancer Medicine, Sun Yat-Sen University Cancer The full-length p65 cDNA was cloned into the pBABE vector Center, Guangzhou, China. from human 293T cells. P65 was generated by PCR ampliﬁcation, Note: Supplementary data for this article are available at Clinical Cancer subcloned into the pBABE and pCDNA3.1 vectors, and veriﬁed by Research Online (http://clincancerres.aacrjournals.org/). DNA sequencing. The p65 shRNA was purchased from Sigma- Corresponding Author: Tiebang Kang, State Key Laboratory of Oncology in Aldrich. The CMV-luciferase plasmid was a gift from Prof. Xiaom- South China, Sun Yat-Sen University Cancer Center, 651 Dongfeng East Road, ing Xie (Sun Yat-Sen University, Guangzhou, China; ref. 16). Guangzhou 510060, China. Phone: 86-20-8734-3183; Fax: 86-20-8734-3170; E-mail: [email protected] Cell viability assay doi: 10.1158/1078-0432.CCR-15-0198 U2OS, MG63, or ZOS cells were seeded in 96-well plates at a 2015 American Association for Cancer Research. density of 3,000 cells per well. They were treated with different

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50 nmol/L siRNA. The transfections were performed as described Translational Relevance previously (15). Brieﬂy, asynchronously growing cells seeded at 2.5 Osteosarcoma, an extremely aggressive form of primary 105 cells per well in a 6-well plate or at 1 106 cells in a 10-cm malignant bone tumor in childhood, has a high rate of lung plate were transfected with 2 or 12 mg plasmid DNA, respectively, metastasis. A recent population study reported that aspirin using Lipofectamine 2000 (Invitrogen). Cell lines that stably reduces both the incidence and the risk of metastasis in colon expressed the NC or p65 shRNAs were established by the Sigma cancer. Our results showed that aspirin inhibits osteosarcoma shRNA system according to the manufacturer's instructions. metastasis and enhances the chemotherapy of osteosarcoma in vitro and in vivo, which is the ﬁrst evidence at the cellular level Dual-luciferase reporter assay and in an animal model for the inhibition of aspirin on cancer Transfection was performed as described previously (15). The metastasis. This strongly argues that aspirin may be an excel- transfected p65-siRNA cells incubated for 24 hours or U2OS cells lent auxiliary drug for treating patients with osteosarcoma, stably overexpressing p65 were transfected with NF-kB p65 lucif- suggesting that it is worthy to design a clinical trial using the erase reporter and pRL-TK Renilla luciferase construct (Promega) combination of aspirin with the current standard therapies for per well using Lipofectamine 2000 (Invitrogen). After 24 hours, patients with osteosarcoma. the cells were treated with aspirin (5 mmol/L). The cells were then analyzed after an additional 24 hours according to the Dual- Luciferase Assay System protocol (Promega).

concentrations of Aspirin (0, 1, 2.5, 5, 10, or 20 mmol/L) and/or Boyden chamber assays chemotherapy drugs (DDP, 10mmol/L) for the indicated times, The migration and invasion of osteosarcoma cells were exam- m and the cell viability was measured by MTT assays as described ined using 24-well Boyden chambers with 8- m inserts coated previously (15, 17). without (migration) or with Matrigel (invasion) as previously described (21). A total of 5 104 cells per well were plated on the o Clone formation assay inserts and cultured at 37 C in the upper chambers without Colony formation assays were performed as described previ- serum and supplemented with Aspirin (5 mmol/L). After 24 ously (15, 18). ZOS or U2OS cells plated in triplicate at 500 hours, the cells that crossed the inserts were stained with crystal cells per well in 6-well plates were treated with vehicle (DMSO), violet (0.005%, sigma) and counted as the number of cells per fi 10 mmol/L DDP, 5 mmol/L aspirin, or 10mmol/L DDP plus 5 eld of view under phase-contrast microscopy. mmol/L aspirin for 48 hours, and then cultured for 10 days. The Western blotting cell clones were washed with PBS, and then fixed in methanol and Western blotting was performed as previously described (18). dyed with 0.1% crystal violet, and the colonies that contained Briefly, the cells were lysed in RIPA buffer containing protease more than 50 cells were counted. inhibitor and phosphatase inhibitor cocktails (Thermo Scientific). The nuclear protein was isolated according to the protocol pro- fl Apoptosis ow-cytometry assay vided by the Nuclear Protein Extraction Kit (Thermo Scientific). Drug-induced apoptosis was detected using an Annexin V- The protein extracts were resolved using SDS-PAGE and trans- EGFP apoptosis detection kit (KeyGEN). Treated ZOS, MG63, or ferred to polyvinylidene difluoride membranes for Western blot- m U2OS cells (negative control, 10 mol/L DDP, 5 mmol/L aspirin, ting using ECL detection reagents (Beyotime Co. Haimen). or 10 mmol/L DDP plus 5 mmol/L aspirin for 48 hours) were harvested, the assay were performed according to the manufac- Mouse xenograft turer's instruction. The stained cells were analyzed using a Animal experiments were approved by the Animal Research Cytomics FC500 flow cytometer (Beckman Coulter). Committee of Sun Yat-sen University Cancer Center and were performed in accordance with established guidelines. For osteo- The effect of combination of DDP and aspirin sarcoma xenograft growth of orthotopic animal model, U2OS/ This effect of the combination of DDP and aspirin was assessed MTX300 cells were used as previously described (15, 22). Two fl by the Chou-Talalay method (19, 20). Brie y, the cells were weeks after the cells were injected, the mice were randomly treated with serial dilutions of DDP and aspirin separately or separated into four groups (n ¼ 6). The mice were treated with simultaneously for 48 hours, and the viability of the cells was DMSO, aspirin (100 mg/kg) by intragastric administration every fi quanti ed using MTT assays. A combination index (CI) was day, and/or DDP (3 mg/kg) by i.p. injection every week. The mice calculated using CalcuSyn software following the equation: were monitored every 2 days for tumor formation and sacrificed ¼ D Dx þ D D D D (CI) ( )1/( )1 ( )2/( x)2, where ( )1 and ( )2 are the when the tumors reached approximately 1.5 cm in diameter, and x doses of drugs 1 and 2 that have effect when used in combina- the tumor xenografts were harvested and weighted. The tumor Dx Dx tion; and ( )1 and ( )2 are the doses of drugs 1 and 2 that have volume was calculated using the formula V ¼ 4/3 p[1/4 x the same effect when used alone. The molar ratio of DDP:aspirin (D1þD2)2, as described previously (22). is 1:1. A drug combination is additive, synergistic and antagonistic For the spontaneous metastasis model, human osteosarcoma ¼ < > when CI 1.0, 1.0, and 1.0, respectively. cells were transplanted orthotopically into the bones of mice as previously described (15, 22). We used 143B cells stably Transfection experiments expressing luciferase (143B-luci) or U2OS/MTX300 cells stably p65 siRNAs were synthesized by GenePharma. Transfections expressing luciferase (U2OS/MTX300-luci) and U2OS/MTX300- were performed according to the manufacturer's instructions using luci–derived cells, including U2OS/MTX300-p65sh#1, -p65sh#2, the Lipofectamine RNAiMAX transfection reagent (Invitrogen) and -vector and -p65–bearing stable knockdowns of p65,

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Figure 1. Aspirin inhibits the proliferation and promotes apoptosis of osteosarcoma cells. A, aspirin inhibits osteosarcoma cell (U2OS, MG63, and ZOS) viability in a time- and dose-dependent manner. Osteosarcoma cells were treated with different concentrations of aspirin for the indicated times, and the viability of these cells was measured using MTT assays; , P < 0.01. A two-sided ANOVA with a Bonferroni post hoc test was used for statistical analysis. B and C, aspirin augments the chemosensitivity of osteosarcoma cells to DDP. B, the indicated cells were treated with 10 mmol/L DDP with or without 7.5 mmol/L aspirin for 48 hours as indicated, and the viability of these cells was measured by MTT assays; n ¼ 3; , P < 0.01. (Continued on the following page.)

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Figure 2. Aspirin impedes the osteosarcoma growth in vivo. A to D, U2OS/MTX300 cells were implanted in the proximal tibia and allowed to grow for 24 days (n ¼ 6/group). Starting on day 14, the mice were given 100 mg/kg aspirin every day and/or DDP (3 mg/kg) once a week i.p. The tumor volumes and mice weight were monitored on day 24 and then every 2 days, as indicated (B and D), and the xenografts were excised and weighed on day 24 (A and C); , P < 0.01; , P < 0.001. A two-sided ANOVA with a Bonferroni post hoc test was used for statistical analysis.

overexpressing vector, and p65, respectively. One week after the lungs, qRT-PCR for human hypoxanthine-guanine-phosphor- cells were injected, the mice were randomly separated into two ibosyl transferase (hHPRT) was performed on TRizol (Invitro- groups (n ¼ 6). The mice were treated with DMSO or aspirin (100 gen)-isolated total RNA using primers for hHPRT and 18S mg/kg) by intragastric administration every day. When the xeno- rRNA. The oligo nucleotide sequences of the qRT-PCR primers grafts reached 1.5 cm in diameter, the lungs of mice bearing the are as follows: osteosarcoma tumor xenografts that stably expressed luciferase were analyzed using an IVIS Lumina Imaging System (Xenogen). Gene primer (F: forward, R: reverse) The mice were then sacriﬁced, the lungs of the mice were weighed, P65 F: 50-AGCTCAAGATCTGCCGAGTG-30 and the number of metastatic nodules in the lungs was counted. R: 50-ACATCAGCTTGCGAAAAGGA-30 0 0 The harvested lungs were ﬁxed in formalin (4 mmol/L) and stained GAPDH F: 5 -ATCACCATCTTCCAGGAGCGA-3 R: 50-CCTTCTCCATGGTGGTGAAGAC-30 by hematoxylin and eosin (H&E) for histologic assessment, and 0 0 hPRT F: 5 -TTCCTTGGTCAGGCAGTATAATCC-3 total RNA was extracted for quantitative real-time reverse tran- R: 50-AGTCTGGCTTATATCCAACACTTCG-30 scription-PCR (qRT-PCR) analysis of human HPRT mRNA 18sRNA F: 50-CGGCTACCACATCCAAGGAA-30 expression. R: 50-GCTGGAATTACCGCGGCT-30

qRT-PCR qRT-PCR was performed using a LightCycler 480 instrument (Roche Diagnostics) and SYBR Premix Ex Taq (TaKaRa) accord- Statistical analysis ing to the manufacturer's instructions. All reactions were carried The data are presented as the mean SD. The error bars indicate out in a 10 mL reaction volume in triplicate. The primers for the SD. Two-sided ANOVA with the Bonferroni post hoc test, two- GAPDH were obtained from Invitrogen. Standard curves were tailed Student t test, or Mann–Whitney test was used to compare generated, and the relative amount of target gene mRNA was the differences between subgroups. , P < 0.05 was considered to normalized to that of GAPDH. The specificity was verified by be significant; , P < 0.01 and , P < 0.001 was considered to be melting curve analysis. To quantify cancer metastasis in mouse strongly significant.

(Continued.) C, ZOS or U2OS cells plated in triplicate at 500 cells per well in 6-well plates were treated with vehicle (DMSO), 10 mmol/L DDP, 5 mmol/L aspirin, or 10 mmol/L DDP plus 5 mmol/L aspirin for 48 hours, cultured for 10 days and then subjected to clone formation assays; n ¼ 3; , P < 0.01. A two-sided ANOVA with a Bonferroni post hoc test was used for statistical analysis. D, aspirin promotes DDP-induced apoptosis in osteosarcoma cell lines. The indicated cells were treated with 10 mmol/L DDP with or without 5 mmol/L aspirin for 48 hours as indicated, and then subjected to Annexin V assays, and the percentages of apoptosis in the indicated cells were shown as the histogram; n ¼ 3. Bars, SD; , P < 0.01. A two-sided ANOVA with a Bonferroni post hoc test was used for statistical analysis. E, the combination of DDP with aspirin is synergistic at low concentrations. U2OS cells were treated with different concentrations of both DDP and aspirin for 48 hours, and the viability of the cells was analyzed by MTT assays. The molar ratio of DDP:aspirin is 1:1; n ¼ 3. A drug combination is additive when CI ¼ 1.0, antagonistic when CI > 1.0 and synergistic when CI < 1.0, respectively.

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Figure 3. Aspirin suppresses the osteosarcoma migration, invasion, and metastasis. A, aspirin inhibits the invasion and migration abilities of osteosarcoma cells. The indicated cells were treated with or without aspirin (5 mmol/L), and cell migration and invasion were determined as described in Materials and Methods; n ¼ 3. P < 0.01. A two- tailed Student t test was used for statistical analysis. B to F, aspirin inhibits osteosarcoma metastasis to the lungs in nude mice in vivo. Both U2OS/ MTX300 cells and 143B cells stably expressing luciferase were used to analyze in vivo spontaneous metastasis to the lungs of nude mice treated with or without aspirin (100 mg/kg/d) in an orthotopic osteosarcoma model as described in Materials and Methods; n ¼ 6 for each group. B shows representative images, whereas C illustrates the statistical results for the number of metastatic nodules in the lungs of nude mice using a Mann– Whitney test. Dots, scores. Bars, SD; , P < 0.01. The expression of human HPRT mRNA relative to mouse 18S rRNA in the lungs of tumor-bearing nude mice is indicated in F. The averages and error bars correspond to the means SDs. D and E, illustrate the wet lung weights and H&E staining of lungs from the tumor-bearing nude mice, respectively. Bars, SD; , P < 0.05 and , P < 0.01. A two-tailed Student t test was used for statistical analysis.

Results viability and the clone formation ability (P < 0.05). Furthermore, Aspirin inhibits the osteosarcoma growth in vitro and in vivo Aspirin sensitized osteosarcoma cells to the DDP-induced apo- We recently showed that the inhibition of the NF-kB pathway ptosis in vitro (Fig. 1D; Supplementary Fig. S2). In addition, the impeded the growth of osteosarcoma and increased the sensitivity combination of DDP with aspirin was synergistic at low concen- of osteosarcoma to chemotherapies in vitro and in vivo (15). It is trations showed by Fa-CI plots (Fig. 1E). Collectively, these results well known that aspirin inhibits the activity of NF-kB (23, 24) and indicate that aspirin inhibits the proliferation of osteosarcoma the aspirin induces apoptosis in many cancers (25–28), we cells and promotes the apoptosis of osteosarcoma cells induced reasoned that aspirin would improve the efficiency of osteosar- by DDP in vitro. coma to chemotherapies. Using the osteosarcoma cell lines, Next, we investigated whether aspirin could inhibit the oste- including U2OS, ZOS, and MG63, we observed that aspirin osarcoma growth using the osteosarcoma orthotropic animal reduced cell viability in a dose- and time-dependent manner model in vivo. As shown in Fig. 2B, sacrificed the tumor-bearing (Fig. 1A). Because patients with osteosarcoma often develop mice at the 24th day, we found the combination of aspirin and chemoresistence in clinic, we sought to determine whether aspirin DDP showed more significant inhibition of tumor growth, com- sensitizes osteosarcoma cells to DDP, the common used chemo- pared with either aspirin or DDP alone (P < 0.001). As shown therapeutic drug in osteosarcoma patients. As shown in Fig. 1B in Fig. 2A and C, consistent with the tumor growth curve, the and C; Supplementary Fig. S1, the combination of DDP with tumor weights and volumes were more decreased in the group of aspirin was more efficiently decreasing the osteosarcoma cell combining aspirin with DDP, compared with aspirin or DDP

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Figure 4. Aspirin inhibits the NF-kB pathway. A, inhibition of the luciferase activity of NF-kB by aspirin. The indicated cells were transfected with a luciferase reporter for NF-kB, treated with or without aspirin (5 mmol/L) for 48 hours, and subjected to luciferase assays as described in Materials and Methods; , P < 0.01. A two-tailed Student t test was used for statistical analysis. B, inhibition of p65 nuclear translocation by aspirin. The indicated cells were treated with or without aspirin (5 mmol/L) for 48 hours and then subjected to cell fractionation and analyzed by Western blotting. C, downstream targets of NF-kB, such as BCL2, CIAP2, Survivin, Livin, and XIAP, were downregulated by aspirin in a dose- and time-dependent manner in both U2OS and ZOS cells. The indicated cells were treated with different concentrations of aspirin for the indicated times and then analyzed by Western blot analysis. GAPDH was used as a loading control; n ¼ 3.

alone (P < 0.01). More importantly, the combination of aspirin measured by Boyden chamber assays in vitro (P < 0.01). More and DDP did not increase their toxicities in vivo, as indicated by strikingly, using a spontaneous in vivo metastasis model of human mouse body weight (Fig. 2D). Taken together, these results reveal osteosarcoma cells (Supplementary Fig. S4), and evaluated cancer that aspirin enhances the sensitivity of osteosarcoma to che- metastasis to the lungs when xenografts reached 1.5 cm in motherapies in vitro and in vivo. diameter. Luminescence imaging for the lungs identiﬁed the severity of lung metastasis was inhibited by the aspirin Aspirin inhibits the osteosarcoma metastasis in vivo (Fig. 3B). Aspirin dramatically diminished the occurrence of Aspirin has recently been reported to reduce both the incidence osteosarcoma xenograft metastases to the lungs, as indicated by and the risk of metastasis in colon cancer (7), and osteosarcoma is the number of metastatic nodules in the lung (Fig. 3C, P < 0.05) often resistant to the conventional chemotherapy drugs because and the wet lung weight (Fig. 3E, P < 0.01). H&E staining also of high rate of lung metastasis (29), we sought to determine showed that aspirin reduced the metastasis in the lungs of the whether aspirin has inhibition on osteosarcoma metastasis in vivo. mice bearing U2OS/MTX300 or 143B xenografts compared with As shown in Fig. 3A; Supplementary Fig. S3, aspirin markedly DMSO treatment (Fig. 3D). In addition, the human HPRT mRNA suppressed the migration and invasion of osteosarcoma cells as levels detected by real-time PCR were decreased by 89% in U2OS/

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Figure 5. Aspirin represses the osteosarcoma migration and invasion via the NF-kB pathway in vitro. A and B, aspirin has a minimal effect on the NF-kB transcriptional activity in both U2OS and ZOS cells knockdown of p65. The indicated cells were transfected with negative control or p65-siRNA for 24 hours and then transfected with an NF-kB luciferase reporter for 24 hours. The cells were treated with or without aspirin (5 mmol/L) for 24 hours, and then subjected to luciferase assays (B) as described in Materials and Methods. The efﬁciency of p65-siRNA was determined by Western blotting (A); n ¼ 3. Bars, SD; , P < 0.01. A two-sided ANOVA with a Bonferroni post hoc test was used for statistical analysis. C, aspirin has no effect on the invasion and migration abilities of osteosarcoma cells knockdown of p65. The indicated cells transfected with or without p65-siRNA (as indicated) for 24 hours were treated with or without aspirin (5 mmol/L)for 24 hours, and then the migration and invasion assays (as indicated) were subsequently performed as described in Materials and Methods; n ¼ 3; , P < 0.01. A two-tailed Student t test was used for statistical analysis. D and E, the effect of aspirin on the NF-kB transcriptional activity is rescued in both U2OS and ZOS cells stably overexpressing p65. The indicated stable transfectants (D) were transfected with an NF-kB luciferase reporter for 24 hours. The cells were treated with or without aspirin (5 mmol/L) for 24 hours, and then subjected to luciferase assays (E) as described in Materials and Methods; n ¼ 3. Bars, SD; , P < 0.01. A two-sided ANOVA with a Bonferroni post hoc test was used for statistical analysis. F, the impairment of aspirin on migration and invasion are rescued in both U2OS and ZOS cells stably overexpressing p65. The indicated stable transfectants were treated with or without aspirin (5 mmol/L) for 24 hours and then subjected to migration and invasion assays as described in Materials and Methods; n ¼ 3. Bars, SD, , P < 0.01. A two-sided ANOVA with Bonferroni post hoc test was used for statistical analysis.

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Figure 6. Aspirin suppresses osteosarcoma metastasis via the NF-kB pathway in vivo. The effect of aspirin on osteosarcoma metastasis to the lungs depends on p65 in vivo. The indicated U2OS/MTX300 cells stably expressing luciferase were used to determine the presence of in vivo spontaneous metastases to lungs of nude mice in an orthotopic model of osteosarcoma as described in Materials and Methods. A, the indicated proteins were analyzed by Western blot analysis in the indicated stable cells. B, the lungs of the mice bearing osteosarcoma tumor xenografts that stably expressed luciferase were analyzed using an IVIS Lumina Imaging System (Xenogen); n ¼ 6. C, H&E staining of lungs from the tumor-bearing nude mice in B. D, the expression of human HPRT mRNA relative to mouse 18S rRNA in the lungs of tumor-bearing nude mice in B. The averages and error bars correspond to the means SDs. E, The number of metastatic nodules in the lungs of nude mice in B using a Mann–Whitney test. Dots, scores. Bars, SD; , P < 0.01. F, the wet lung weights from the tumor- bearing nude mice in B. Bars, SD; , P < 0.05 and , P < 0.01. A two-tailed Student t test was used for statistical analysis.

MTX300 xenografts and 77% in 143B xenografts model, respec- on the osteosarcoma metastasis by modulating the NF-kB path- tively (Fig. 3E). Collectively, these results demonstrate that aspirin way. Osteosarcoma cells transfected with NF-kB luciferase plas- inhibits osteosarcoma metastasis in vivo. mid, and then incubated with aspirin for 24 hours. The p65 NF-kB luciferase reporter activity was decreased by 50% after treated with Aspirin inhibits osteosarcoma metastasis through the NF-kB aspirin in osteosarcoma cells (Fig. 4A, P < 0.01). Consistently, the pathway nuclear localization of p65 protein, the indicator of NF-kB tran- Aspirin has been shown to inhibit the activity of NF-kB, which scription activity, was also decreased in osteosarcoma cells treated plays key roles in diverse physiologic and pathologic processes, with aspirin (Fig 4B). Consequently, the NF-kB–targeted genes, including apoptosis, proliferation, migration, invasion, and such as CIAP, XIAP, BCL2, and Survivin, were inhibited by aspirin metastasis (30–32). In addition, we recently showed that the in a dose- and time-dependent manner in both ZOS and U2OS inhibition of the NF-kB pathway impaired the growth of osteo- cells (Fig. 4C). sarcoma and increased the sensitivity of osteosarcoma to che- Next, we explored whether the inhibition of aspirin on oste- motherapies in vitro and in vivo (15). Therefore, we were very osarcoma metastasis depends on the NF-kB pathway. First, as curious to determine whether aspirin also had an inhibitory effect shown in Fig. 5A–C; Supplementary Fig. S5, aspirin had no effect

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on the NF-kB luciferase reporter activity, the migration and osteosarcoma is dependent on the NF-kB pathway. This is the invasion of osteosarcoma cells knockdown of p65 by siRNA in first comprehensive evidence at the cellular levels and in the these cells (P > 0.05). Conversely, as shown in Fig. 5D–F; Sup- animal models to argue that aspirin may be benefitfortreating plementary Fig. S6, the impairment of aspirin on these events were cancer patients with potential metastasis. Consistently, during completely rescued by in these osteosarcoma cells overexpressing the preparation of our article, Ogawa and colleagues reported p65. Second, we stably knocked down and overexpressed the p65 that aspirin reduced lung cancer metastasis to regional lymph in U2OS/MTX300-luci cells, which were identified by Western nodes in vivo (42). Notably, the antitumor of aspirin is indef- blotting (Fig. 6A). Using a spontaneous metastasis model in vivo inite, as aspirin is protective in some cancer types, such as and the assays mentioned above, as shown in Fig. 6B–E, the colorectal, gastric, liver, and prostate cancers (7, 10, 43), inhibition of osteosarcoma metastasis to the lungs by aspirin was whereas aspirin is useless for some other cancer types (44– not further enhanced in the cells bearing a stable knockdown of 46). For randomized trials of aspirin, low dose and high dose p65 by shRNA, but was abolished in the cells stably overexpres- is defi ned as 75 to 300 mg daily and 300 to 1,500 mg daily, sing p65. These results demonstrate that Aspirin diminishes both respectively (47). In this report, the dose of aspirin we used the growth and metastasis of osteosarcoma through the NF-kB was 100 mg/kg daily in mice, which is highly efficient to pathway. diminish the tumor growth and metastasis using the osteosarcoma orthotopic mouse model. The dose of 100 mg/kg Discussion daily in mice is equal to 8.13 mg/kg daily in humans, meaning that the aspirin level we tested is about 400 to 700 mg daily for Patients with metastatic or relapsed osteosarcoma have a poor humans weighing 50 to 80 kg. This dose of aspirin fits the prognosis and new therapies are needed. Aspirin has recently been definition of high-dose (300–1,500 mg daily) aspirin studied reported to reduce the incidence and risk of metastasis in colon in previous randomized trials (47) and should provide ade- cancer patients (7). In the present study, we demonstrated that quate systemic exposure. At the same time, it should be noted aspirin diminishes the growth and metastasis of osteosarcoma that aspirin is used at 100 or 300 mg daily in the ongoing through the NF-kB pathway in vitro and in vivo. adjuvant aspirin trials for colorectal, breast, gastrooesopha- Aspirin can inhibit the activity of NF-kB(23,24),andthe geal, and prostate cancers (47). inhibition of the NF-kB pathway repressed the growth of In summary, we herein provide comprehensive evidence for an osteosarcoma and increased the sensitivity of osteosarcoma to inhibitory effect of aspirin on osteosarcoma metastasis and dem- chemotherapies in vitro and in vivo (15). Thus, it was reasonable onstrate that aspirin enhances osteosarcoma chemotherapy. to speculate that aspirin may sensitize osteosarcoma cells to Because osteosarcoma is an extremely aggressive cancer type that DDP. Indeed, this was the case, as our results indicated that the lacks any targeted therapies, our findings strongly suggest that combination of DDP and aspirin was more efficient in killing aspirin may be an excellent auxiliary drug for treating patients osteosarcoma cells in vitro and in vivo than either DDP or aspirin with osteosarcoma. alone. This is consistent with the fact that regular and long-time intake of aspirin with a significant reduction in the prevention Disclosure of Potential Conflicts of Interest and treatment of colorectal cancer (7, 33, 34), as inflammation No potential conflicts of interest were disclosed. has key roles in tumor initiation, promotion, and metastasis (35). In fact, SDX-308, another NSAID agent, has been shown Authors' Contributions to impair the multiple myeloma cell proliferation and osteo- Conception and design: D. Liao, T. Kang clast activity by inhibiting the NF-kB activity signaling (31). On Development of methodology: D. Liao, L. Zhong the other hand, De Luna-Bertos found that therapeutic doses of Acquisition of data (provided animals, acquired and managed patients, NSAIDs (dexketoprofen, ketorolac, metamizle, and aspirin) provided facilities, etc.): D. Liao, L. Zhong, T. Duan, X. Wang, G. Wang fi Analysis and interpretation of data (e.g., statistical analysis, biostatistics, modulated differentiation and antigenic pro le activity of computational analysis): D. Liao, L. Zhong, T. Duan, X. Wang, G. Wang MG63 cells, whereas both dexketoprofen and ketorolac inhib- Writing, review, and/or revision of the manuscript: D. Liao, T. Kang ited osteoblast growth by arresting the cell-cycle and inducing Administrative, technical, or material support (i.e., reporting or organizing apoptosis, which are influenced by the dose, duration of data, constructing databases): R.-H. Zhang, K. Hu, X. Lv treatment, and cells used (36–39). These results indicate that Study supervision: T. Kang the effects of these different NSAIDs are complicated in bone Acknowledgments biology and/or osteosarcoma, and the differences in these data The authors thank the members of the laboratory for helpful comments on may be explained by the dose, treatment duration, and different the article. NSAIDs used (40). Strikingly, recent population studies support that aspirin Grant Support reduces the risk of metastasis in colon cancer (7, 41). However, This work was supported by the key project (2013ZX10002008005; to there is no evidence at the cellular levels or in the animal T. Kang), the National Nature Science Foundation in China (81125015; to models for such an effect of aspirin on cancer metastasis, even T. Kang), the 973 project (2012CB967000; to T. Kang). though aspirin is widely used for both the prevention and The costs of publication of this article were defrayed in part by the treatment of many diseases, including cancers in clinical set- payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate tings (8, 10–14, 9). In this report, we demonstrated that this fact. aspirin markedly suppressed the migration and invasion of osteosarcoma cells in vitro, and that Aspirin inhibited the metastasis of osteosarcoma in vivo. Mechanistically, this inhi- Received January 29, 2015; revised June 18, 2015; accepted July 6, 2015; bition of aspirin on migration, invasion, and metastasis of published OnlineFirst July 22, 2015.

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References 1. Mirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival osarcoma transplanted orthotopically into bone of athymic mice. Cancer rates from 1973 to 2004: data from the Surveillance, Epidemiology, and Res 1993;53:4890–5. End Results Program. Cancer 2009;115:1531–43. 23. Kopp E, Ghosh S. Inhibition of NF-kappa B by sodium salicylate and 2. Ferrari S, Smeland S, Mercuri M, Bertoni F, Longhi A, Ruggieri P, et al. aspirin. Science 1994;265:956–9. Neoadjuvant chemotherapy with high-dose Ifosfamide, high-dose meth- 24. Yin MJ, Yamamoto Y, Gaynor RB. The anti-inflammatory agents aspirin otrexate, cisplatin, and doxorubicin for patients with localized osteosar- and salicylate inhibit the activity of I(kappa)B kinase-beta. Nature coma of the extremity: a joint study by the Italian and Scandinavian 1998;396:77–80. Sarcoma Groups. J Clin Oncol 2005;23:8845–52. 25. Lu M, Strohecker A, Chen F, Kwan T, Bosman J, Jordan VC, et al. Aspirin 3. Mialou V, Philip T, Kalifa C, Perol D, Gentet JC, Marec-Berard P, et al. sensitizes cancer cells to TRAIL-induced apoptosis by reducing survivin Metastatic osteosarcoma at diagnosis: prognostic factors and long- levels. Clin Cancer Res 2008;14:3168–76. term outcome—the French pediatric experience. Cancer 2005;104: 26. Dikshit P, Chatterjee M, Goswami A, Mishra A, Jana NR. Aspirin induces 1100–9. apoptosis through the inhibition of proteasome function. J Biol Chem 4. Gill J, Ahluwalia MK, Geller D, Gorlick R. New targets and approaches in 2006;281:29228–35. osteosarcoma. Pharmacol Ther 2013;137:89–99. 27. Gu Q, Wang JD, Xia HH, Lin MC, He H, Zou B, et al. Activation of the 5. Kager L, Zoubek A, Potschger U, Kastner U, Flege S, Kempf-Bielack B, et al. caspase-8/Bid and Bax pathways in aspirin-induced apoptosis in gastric Primary metastatic osteosarcoma: presentation and outcome of patients cancer. Carcinogenesis 2005;26:541–6. treated on neoadjuvant Cooperative Osteosarcoma Study Group proto- 28. Shiff SJ, Qiao L, Tsai LL, Rigas B. Sulindac sulfide, an aspirin-like com- cols. J Clin Oncol 2003;21:2011–8. pound, inhibits proliferation, causes cell-cycle quiescence, and induces 6. Sneader W. The discovery of aspirin: a reappraisal. BMJ 2000;321: apoptosis in HT-29 colon adenocarcinoma cells. J Clin Invest 1995; 1591–4. 96:491–503. 7. RothwellPM,WilsonM,PriceJF,BelchJF,MeadeTW,MehtaZ.Effect 29. Kager L, Zoubek A, Potschger U, Kastner U, Flege S, Kempf-Bielack B, et al. of daily aspirin on risk of cancer metastasis: a study of incident cancers Primary metastatic osteosarcoma: presentation and outcome of patients during randomised controlled trials. Lancet 2012;379:1591–601. treated on neoadjuvant Cooperative Osteosarcoma Study Group proto- 8. Fink SP, Yamauchi M, Nishihara R, Jung S, Kuchiba A, Wu K, et al. Aspirin cols. J Clin Oncol 2003;21:2011–8. and the risk of colorectal cancer in relation to the expression of 15- 30. Bharti AC, Donato N, Singh S, Aggarwal BB. Curcumin (diferuloyl- hydroxyprostaglandin dehydrogenase (HPGD). Sci Transl Med 2014;6: methane) down-regulates the constitutive activation of nuclear factor- 233re2. kappa B and IkappaBalpha kinase in human multiple myeloma cells, 9. Goodman JR, Grossman D. Aspirin and other NSAIDs as chemoprevention leading to suppression of proliferation and induction of apoptosis. Blood agents in melanoma. Cancer Pre Res 2014;7:557–64. 2003;101:1053–62. 10. Rothwell PM, Wilson M, Elwin CE, Norrving B, Algra A, Warlow CP, 31. Feng R, Anderson G, Xiao G, Elliott G, Leoni L, Mapara MY, et al. SDX-308, a et al. Long-term effect of aspirin on colorectal cancer incidence and nonsteroidal anti-inflammatory agent, inhibits NF-kappaB activity, result- mortality: 20-year follow-up of five randomised trials. Lancet 2010; ing in strong inhibition of osteoclast formation/activity and multiple 376:1741–50. myeloma cell growth. Blood 2007;109:2130–8. 11. Holmes MD, Chen WY, Li L, Hertzmark E, Spiegelman D, Hankinson 32. Fujioka S, Sclabas GM, Schmidt C, Frederick WA, Dong QG, Abbruzzese JL, SE. Aspirin intake and survival after breast cancer. J Clin Oncol 2010; et al. Function of nuclear factor kappaB in pancreatic cancer metastasis. 28:1467–72. Clin Cancer Res 2003;9:346–54. 12. Vidal AC, Howard LE, Moreira DM, Castro-Santamaria R, Andriole GL, 33. Cole BF, Logan RF, Halabi S, Benamouzig R, Sandler RS, Grainge MJ, et al. Freedland SJ. Aspirin, NSAIDs, and risk of prostate cancer: results from the Aspirin for the chemoprevention of colorectal adenomas: meta-analysis of REDUCE Study. Clin Cancer Res 2015;21:756–62. the randomized trials. J Natl Cancer Inst 2009;101:256–66. 13. Baandrup L, Kjaer SK, Olsen JH, Dehlendorff C, Friis S. Low-dose aspirin 34. Kaiser J. Will an aspirin a day keep cancer away? Science 2012;337:1471–3. use and the risk of ovarian cancer in Denmark. Ann Oncol 2015;26: 35. Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. 787–92. Cell 2010;140:883–99. 14. Muranushi C, Olsen CM, Pandeya N, Green AC. Aspirin and nonsteroidal 36. Garcia-Martinez O, Diaz-Rodriguez L, Rodriguez-Perez L, De Luna-Bertos anti-inflammatory drugs can prevent cutaneous squamous cell carcinoma: E, Reyes Botella C, Ruiz CC. Effect of acetaminophen, ibuprofen and a systematic review and meta-analysis. J Invest Dermatol 2015;135: methylprednisolone on different parameters of human osteoblast-like 975–83. cells. Arch Oral Biol 2011;56:317–23. 15. Tang QL, Xie XB, Wang J, Chen Q, Han AJ, Zou CY, et al. Glycogen synthase 37. De Luna-Bertos E, Ramos-Torrecillas J, Garcia-Martinez O, Guildford A, kinase-3beta, NF-kappaB signaling, and tumorigenesis of human osteo- Santin M, Ruiz C. Therapeutic doses of nonsteroidal anti-inflammatory sarcoma. J Natl Cancer Inst 2012;104:749–63. drugs inhibit osteosarcoma MG-63 osteoblast-like cells maturation, via- 16. Xie X, Xia W, Li Z, Kuo HP, Liu Y, Ding Q, et al. Targeted expression of bility, and biomineralization potential. ScientificWorldJournal 2013; BikDD eradicates pancreatic tumors in noninvasive imaging models. 2013:809891. Cancer Cell 2007;12:52–65. 38. De Luna-Bertos E, Ramos-Torrecillas J, Garcia-Martinez O, Diaz-Rodriguez 17. Yin JQ, Shen JN, Su WW, Wang J, Huang G, Jin S, et al. Bufalin induces L, Ruiz C. Effect of aspirin on cell growth of human MG-63 osteosarcoma apoptosis in human osteosarcoma U-2OS and U-2OS methotrexate300- line. ScientificWorldJournal 2012;2012:834246. resistant cell lines. Acta Pharmacol Sin 2007;28:712–20. 39. De Luna-Bertos E, Ramos-Torrecillas J, Manzano-Moreno FJ, Garcia-Mar- 18. Liang Y, Zhong Z, Huang Y, Deng W, Cao J, Tsao G, et al. Stem-like cancer tinez O, Ruiz C. Effects on growth of human osteoblast-like cells of three cells are inducible by increasing genomic instability in cancer cells. J Biol nonsteroidal anti-inflammatory drugs: metamizole, dexketoprofen, and Chem 2010;285:4931–40. ketorolac. Biol Res Nurs 2015;17:62–7. 19. Chou TC, Talalay P. Quantitative analysis of dose–effect relationships: the 40. Salari P, Abdollahi M. Controversial effects of non-steroidal anti-inflam- combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme matory drugs on bone: a review. Inflamm Allergy Drug Targets 2009;8: Regul 1984;22:27–55. 169–75. 20. Hong J, Hu K, Yuan Y, Sang Y, Bu Q, Chen G, et al. CHK1 targets spleen 41. Algra AM, Rothwell PM. Effects of regular aspirin on long-term cancer tyrosine kinase (L) for proteolysis in hepatocellular carcinoma. J Clin Invest incidence and metastasis: a systematic comparison of evidence from 2012;122:2165–75. observational studies versus randomised trials. Lancet Oncol 2012;13: 21. Cheng GZ, Chan J, Wang Q, Zhang W, Sun CD, Wang LH. Twist transcrip- 518–27. tionally up-regulates AKT2 in breast cancer cells leading to increased 42. Ogawa F, Amano H, Ito Y, Matsui Y, Hosono K, Kitasato H, et al. Aspirin migration, invasion, and resistance to paclitaxel. Cancer Res 2007;67: reduces lung cancer metastasis to regional lymph nodes. Biomed Pharmac- 1979–87. other 2014;68:79–86. 22. Berlin O, Samid D, Donthineni-Rao R, Akeson W, Amiel D, Woods VL Jr. 43. Protective effect of aspirin associated with SNP. Cancer Discov 2014;4: Development of a novel spontaneous metastasis model of human oste- OF5.

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Aspirin Suppresses Osteosarcoma through the NF-kB Pathway

44. Cook NR, Lee IM, Gaziano JM, Gordon D, Ridker PM, Manson JE, et al. 46. Baandrup L, Faber MT, Christensen J, Jensen A, Andersen KK, Friis S, et al. Low-dose aspirin in the primary prevention of cancer: the Women's Health Nonsteroidal anti-inﬂammatory drugs and risk of ovarian cancer: system- Study: a randomized controlled trial. JAMA 2005;294:47–55. atic review and meta-analysis of observational studies. Acta Obstet Gynecol 45. Ni X, Ma J, Zhao Y, Wang Y, Wang S. Meta-analysis on the association Scand 2013;92:245–55. between non-steroidal anti-inﬂammatory drug use and ovarian cancer. Br J 47. Langley RE, Rothwell PM. Aspirin in gastrointestinal oncology: new data on Clin Pharmacol 2013;75:26–35. an old friend. Curr Opin Oncol 2014;26:441–7.

www.aacrjournals.org Clin Cancer Res; 2015 OF11

Aspirin Suppresses the Growth and Metastasis of Osteosarcoma through the NF- κB Pathway

Dan Liao, Li Zhong, Tingmei Duan, et al.

Clin Cancer Res Published OnlineFirst July 22, 2015.

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