PMID：25822025.pdf

The Journal of Clinical Investigation R es e aRc h aRticle183 9jci.org Volume 125 Number 5 May 2015IntroductionOsteosarcoma,the most common primary malignant bone tumor,occurs most frequently in children and adolescents and has a strong tendency to metastasize.Pulmonary metastasis is the main cause of medical therapy failure and death in osteosa-rcoma patients.Despite an intensive search for new therapeutic strategies,survival has not improved over the past 2 decades(1).Given that controlling metastasis is the key to improving survival,there is an urgent need to investigate the underlying mecha-nisms of osteosarcoma metastasis and to develop more effective approaches to suppress lung metastasis.It is widely accepted that the multistep process of cancer evo-lution is driven by both genetic and epigenetic abnormalities(2).Unlike genetic alterations,epigenetic changes are potentially revers-ible,making them attractive and promising targets for therapeutic intervention.Previous studies indicated that abnormal DNA meth-ylation,a major epigenetic modification,is involved in the dysreg-ulation of the cell cycle and apoptosis as well as in the proliferation and differentiation of osteosarcoma(3,4).However,the epigenetic mechanisms of osteosarcoma metastasis remain largely unknown.Iroquois homeobox 1(IRX1),a member of the Iroquois homeobox family of transcription factors,plays a crucial role in embryonic development(5).Recently,IRX1 was identified as a potential tumor-suppressor gene in head and neck squamous cell carcinoma(HNSCC)and gastric cancer(6,7);however,further efforts are required to determine the exact function of IRX1 in the development of other cancers,including osteosarcoma.IRX1 is involved in limb development(8)and the etiology of kyphoscoli-osis(9),suggesting that aberrant IRX1 expression may contribute to abnormal bone formation.Moreover,a gain of chromosome 5p15.33(chromosomal location of IRX1)has been frequently detected in osteosarcoma cell lines(10).Therefore,the role of IRX1 in the pathogenesis of osteosarcoma is of interest.Chemokine(C-X-C motif)ligand 14(CXCL14),also known as BRAK,is a small cytokine belonging to the CXC chemokine family(11),and the gene encoding CXCL14 is commonly recognized as a tumor-suppressor gene(1214).However,recent studies indicated that CXCL14 might actually promote tumor progression in some types of cancer(1518).For example,CXCL14 was upregulated in pancreatic cancer tissues and enhanced the invasiveness of pan-creatic cancer cells(17).CXCL14 transcript levels are markedly high in papillary thyroid carcinoma and are positively correlated with lymph node metastasis(16).We previously showed that over-expression of CXCL14 predicts poor overall survival in osteosar-coma patients(19);however,its biological function in osteosar-coma metastasis requires further assessment.Osteosarcoma is a common malignant bone tumor with a propensity to metastasize to the lungs.Epigenetic abnormalities have been demonstrated to underlie osteosarcoma development;however,the epigenetic mechanisms that are involved in metastasis are not yet clear.Here,we analyzed 2 syngeneic primary human osteosarcoma cell lines that exhibit disparate metastatic potential for differences in epigenetic modifications and expression.Using methylated DNA immunoprecipitation(MeDIP)and microarray expression analysis to screen for metastasis-associated genes,we identified Iroquois homeobox 1(IRX1).In both human osteosarcoma cell lines and clinical osteosarcoma tissues,IRX1 overexpression was strongly associated with hypomethylation of its own promoter.Furthermore,experimental modulation of IRX1 in osteosarcoma cell lines profoundly altered metastatic activity,including migration,invasion,and resistance to anoikis in vitro,and influenced lung metastasis in murine models.These prometastatic effects of IRX1 were mediated by upregulation of CXCL14/NF-B signaling.In serum from osteosarcoma patients,the presence of IRX1 hypomethylation in circulating tumor DNA reduced lung metastasisfree survival.Together,these results identify IRX1 as a prometastatic gene,implicate IRX1 hypomethylation as a potential molecular marker for lung metastasis,and suggest that epigenetic reversion of IRX1 activation may be beneficial for controlling osteosarcoma metastasis.IRX1 hypomethylation promotes osteosarcoma metastasis via induction of CXCL14/NF-B signalingJinchang Lu,1 Guohui Song,1 Qinglian Tang,1 Changye Zou,1 Feng Han,2 Zhiqiang Zhao,1 Bicheng Yong,1,3 Junqiang Yin,1 Huaiyuan Xu,1 Xianbiao Xie,1 Tiebang Kang,4 YingLee Lam,2 Huiling Yang,5 Jingnan Shen,1 and Jin Wang11Department of Musculoskeletal Oncology,the First Affiliated Hospital of Sun Yat-Sen University,Guangzhou,Guangdong,China.2Division of General Orthopaedics and Oncology,Department of Orthopaedics and Traumatology,University of Hong Kong,Queen Mary Hospital,Hong Kong,China.3Department of Pediatric Orthopedics,Guangzhou Women and Childrens Hospital,Guangzhou,Guangdong,China.4State Key Laboratory of Oncology in South China,Sun Yat-Sen University Cancer Center,Guangzhou,China.5Department of Pathophysiology,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou,Guangdong,China.Authorship note:Jinchang Lu,Guohui Song,and Qinglian Tang contributed equally to this work.Conflict of interest:The authors have declared that no conflict of interest exists.Submitted:August 8,2014;Accepted:February 19,2015.Reference information:J Clin Invest.2015;125(5):18391856.doi:10.1172/JCI78437.The Journal of Clinical Investigation R ese a Rch a Rti cl e1 84 0jci.org Volume 125 Number 5 May 2015Supplemental Figure 1B).We next examined IRX1 mRNA expres-sion levels in human osteosarcoma tissues and found that tissues from patients with lung metastasis showed higher IRX1 expres-sion levels than did tissues from patients without lung metastasis(Figure 1D).To further evaluate the clinical relevance of IRX1 in osteosarcoma metastasis,we collected 16 pairs of archival samples of osteosarcoma lung metastases and their corresponding primary osteosarcoma tissues.Immunohistochemical staining showed that the IRX1 expression scores were significantly higher in the lung metastases than in the primary tumors(Figure 1,E and F).These findings suggest that IRX1 plays an important role in osteosarcoma metastasis.Elevated IRX1 expression is linked to hypomethylation of its own promoter.We next examined the association between IRX1 expression and its promoter methylation status in osteosarcoma.As shown in Figure 2A,a decreased methylation level was detected by bisulfite-sequencing PCR(BSP)in the promoter region of IRX1(521 to 679 bp,which contains 5 predicted transcription factorbinding sites)in ZOSM and 143B cells compared with methylation levels detected in ZOS and MNNG-HOS cells(also see Supple-mental Figure 2,AC).Next,we analyzed IRX1 promoter methyla-tion levels in human osteosarcoma tissues.Genomic DNA isolated from 40 fresh surgical osteosarcoma specimens was treated with sodium bisulfite,amplified by PCR,and subjected to MassAR-RAY analysis.The results demonstrated that there was a signifi-cant correlation between increased IRX1 expression and reduced promoter methylation(Figure 2,B and C).Furthermore,treat-ment of ZOS and MNNG-HOS cells with the methyltransferase inhibitor 5-aza-deoxycytidine(DAC),an effective DNA demeth-ylating agent,significantly decreased the methylation level of the IRX1 promoter and resulted in activation of IRX1 gene expression(Figure 2,D and E,and Supplemental Figure 2D).Conversely,treatment of ZOSM and 143B cells with the methyl donor S-ade-nosyl-L-methionine(AdoMet),which has recently been shown to inhibit demethylase activity and induce DNA methylation(23),led to suppression of IRX1 gene expression(Figure 2,F and G,and Supplemental Figure 2E).To further explore whether DNA methylation directly reg-ulates IRX1 promoter activity,we cloned the promoter region of IRX1(521 to 679 bp)into a luciferase reporter construct.The cloned fragment was methylated in vitro by SssI methylase(meth-ylation of 12 CpGs),HpaII methylase(methylation of 2 CpGs),or HhaI methylase(methylation of 2 CpGs).IRX1 promoter activity was determined after transfection of 143B cells with methylated or mock-methylated luciferase constructs.As shown in Figure 2H,methylation repressed IRX1 promoter activity in a methylation dosedependent manner.Taken together,these results suggest that elevated IRX1 expression in osteosarcoma is associated with loss of methylation in the IRX1 promoter.IRX1 promotes the migration,invasion,and resistance to anoikis of osteosarcoma cells in vitro.Because osteosarcoma cells with a higher metastatic potential(ZOSM and 143B cells)exhib-ited elevated IRX1 expression(Figure 1,B and C),we sought to determine the role of IRX1 in osteosarcoma metastasis.It is well established that successful metastasis requires several essen-tial steps.To evaluate the contribution of IRX1 to osteosarcoma cell migration,invasion,and resistance to anoikis in vitro,we Here,we used high-throughput approaches to identify novel epigenetically regulated metastasis-related genes in osteosar-coma.We found that IRX1 is epigenetically activated in highly metastatic osteosarcoma cell lines.IRX1 expression in primary human osteosarcoma samples was positively associated with pro-moter hypomethylation.The downregulation of IRX1 in osteosa-rcoma cell lines resulted in decreased CXCL14 expression levels,inhibition of NF-B activity,and suppression of metastasis.More-over,the hypomethylation of IRX1 in serum from patients was correlated with a high risk of lung metastasis.Taken together,our findings represent a significant step forward in understanding the role of the epigenetically activated gene IRX1 in osteosarcoma metastasis and provide a potential target for epigenetic-based osteosarcoma therapy.ResultsEpigenetic screen for genes driving invasion and metastasis in osteosar-coma.We previously established 2 syngeneic human osteosarcoma cell lines,ZOS and ZOSM,which were derived from a primary tumor and a skip metastasis,respectively,in the same patient(20).Biologically,ZOSM cells are more invasive and metastatic than are ZOS cells.To explore the underlying epigenetic mechanisms related to metastasis,we performed a methylated DNA immuno-precipitation(MeDIP)assay in combination with expression pro-filing using these 2 primary cell lines.MeDIP and expression array data were then filtered and integrated.Compared with ZOS,18 genes showed promoter hypomethylation and were upregulated in ZOSM,while 8 genes exhibited promoter hypermethylation and were downregulated in ZOSM cells(Supplemental Table 1 and Figure 1A).Some of these upregulated genes,including HOXB7,EpCAM,and CXCL6,were previously reported to be involved in cancer metastasis.Notably,high expression levels of PRAME were recently found to be associated with poor prognosis and lung metastasis of human osteosarcoma in our previous study(21).The identification of these known metastasis-related genes suggested that our high-throughput platform was effective for the discovery of genes that drive osteosarcoma metastasis.Therefore,using real-time PCR,we determined the expres-sion levels of the 26 differentially expressed genes selected(listed in Figure 1A).Among these genes,our results demonstrated that the greatest difference in expression levels of IRX1 was observed between ZOS and ZOSM cells(Supplemental Figure 1A).There-fore,we focused on IRX1 in this study,while other candidates such as SALL1 were investigated in separate studies.On the basis of our real-time PCR results,there was a greater than 15-fold increase in IRX1 expression in highly metastatic ZOSM cells com-pared with that detected in nonmetastatic ZOS cells(Figure 1B).To confirm that the IRX1 expression pattern was not specific to only the primary cell lines,we examined IRX1 expression levels in 2 other commonly used osteosarcoma cell lines,MNNG-HOS(poorly metastatic)and 143B(highly metastatic),both of which are derived from the TE85 human osteosarcoma cell line(22).As expected,higher IRX1 mRNA expression was detected in the highly metastatic 143B cells relative to the mRNA levels in MNNG-HOS cells(Figure 1B).Accordingly,IRX1 protein levels were also substantially upregulated in ZOSM and 143B cells,as indicated by Western blot and immunofluorescence analyses(Figure 1C and The Journal of Clinical Investigation R es e aRc h aRticle18 41jci.org Volume 125 Number 5 May 2015anoikis is a physiological barrier to metastasis and that resis-tance to anoikis may facilitate distant organ metastasis(24,25),we further examined the effect of IRX1 deletion on the anoikis of osteosarcoma cells by culturing cells on ultra-low attachment plates.After a 48-hour incubation,we found that ZOSM-shIRX1 and 143B-shIRX1 cells were more sensitive to detachment-in-duced apoptosis than were their scrambled controls(Figure 3D).These results suggest that IRX1 regulates the metastatic behav-ior of osteosarcoma cells.first generated 2 lentiviral constructs encoding IRX1-targeting shRNAs and established cell lines in which IRX1 was stably knocked down(Figure 3A).We found that IRX1 knockdown did not influence cell proliferation(Supplemental Figure 3A).How-ever,IRX1 downregulation inhibited the ability of ZOSM and 143B cells to fill the wound gap(Figure 3B)and migrate through Transwell membranes(Figure 3C).Suppression of IRX1 expres-sion significantly reduced the invasive ability of both cell lines,as measured by Boyden chamber assays(Figure 3C).Given that Figure 1.A high-throughput method to screen for epigenetically activated metastasis-driving genes in osteosarcoma.(A)Heatmap clustering of MeDIP and expression array data obtained from the 2 primary osteosarcoma cell lines ZOSM and ZOS.(B)Real-time PCR analysis.Data represent the mean SD of 3 separate determinations.(C)Western blot analysis.(D)IRX1 mRNA expression in human osteosarcoma tissue(n=70)was determined by real-time PCR.(E)Immunohistochemical staining of IRX1 in 16 pairs of primary osteosarcoma samples and their corresponding lung metastasis tissues.Scale bars:100 m.(F)Statistical analysis showed a significant increase in IRX1 expression in lung metastases relative to the expression in primary osteosarcoma samples.*P 0.05 by Students t test(B and D)and Wilcoxon signed-rank test(F).The Journal of Clinical Investigation R ese a Rch a Rti cl e1 84 2jci.org Volume 125 Number 5 May 2015Figure 2.Elevated IRX1 expression is associated with hypomethylation of its promoter.(A)BSP analysis of the IRX1 promoter(521 to 679)in 4 osteosarcoma cell lines.(B)Sequenom MassARRAY analysis of the IRX1 promoter in 40 human osteosarcoma tissues.Data shown are from 8 repre-sentative samples.(C)IRX1 expression was associated with a low methylation rate.P=0.001 by Spearmans rank correlation analysis.(D)BSP analysis of the IRX1 promoter in ZOS and MNNG/HOS cells after treatment with 2 M DAC for 3 days.(E)Western blot a