IRX1 hypomethylation promotes osteosarcoma metastasis via induction of CXCL14/NF-κB signaling
Jinchang Lu, Guohui Song, Qinglian Tang, Changye Zou, Feng Han, Zhiqiang Zhao, Bicheng Yong, Junqiang Yin, Huaiyuan Xu, Xianbiao Xie, Tiebang Kang, YingLee Lam, Huiling Yang, Jingnan Shen, Jin Wang
Jinchang Lu, Guohui Song, Qinglian Tang, Changye Zou, Feng Han, Zhiqiang Zhao, Bicheng Yong, Junqiang Yin, Huaiyuan Xu, Xianbiao Xie, Tiebang Kang, YingLee Lam, Huiling Yang, Jingnan Shen, Jin Wang
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Research Article Oncology

IRX1 hypomethylation promotes osteosarcoma metastasis via induction of CXCL14/NF-κB signaling

Abstract

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-κB signaling. In serum from osteosarcoma patients, the presence of IRX1 hypomethylation in circulating tumor DNA reduced lung metastasis–free 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.

Authors

Jinchang Lu, Guohui Song, Qinglian Tang, Changye Zou, Feng Han, Zhiqiang Zhao, Bicheng Yong, Junqiang Yin, Huaiyuan Xu, Xianbiao Xie, Tiebang Kang, YingLee Lam, Huiling Yang, Jingnan Shen, Jin Wang

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Figure 3

IRX1 promotes migration, invasion, and resistance to anoikis in osteosarcoma cells.

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IRX1 promotes migration, invasion, and resistance to anoikis in osteosar...
(A) The effect of IRX1-targeting shRNAs was confirmed by Western blot analysis. (B) IRX1 deletion dramatically reduced the migratory ability of osteosarcoma cells in a wound-healing assay. (C) Downregulation of IRX1 resulted in a decrease in the migratory and invasive abilities of osteosarcoma cells as determined by Transwell analysis. (D) FACS analysis indicated that IRX1 suppression significantly promoted the death of nonadherent cells but had no effect on attached cells. (E) The efficiency of IRX1 overexpression in ZOS and MNNG-HOS cells was confirmed by Western blot analysis. (F) IRX1 overexpression enhanced the migratory ability of osteosarcoma cells. (G) Overexpression of IRX1 in ZOS and MNNG-HOS cells increased their migratory and invasive abilities. (H) IRX1 overexpression inhibited cell death under nonadherent culture conditions. Data represent the mean ± SD of 3 separate determinations. Scale bars: 100 μm. *P < 0.05 by Student’s t test.