Tumor Biology

, Volume 37, Issue 7, pp 8857–8867 | Cite as

TIMP3 regulates osteosarcoma cell migration, invasion, and chemotherapeutic resistances

  • Xiu-guo Han
  • Yan Li
  • Hui-min Mo
  • Kang Li
  • Du Lin
  • Chang-qing Zhao
  • Jie Zhao
  • Ting-ting Tang
Original Article


Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases (MMPs) to limit degradation of the extracellular matrix. Low levels of TIMP3 have been demonstrated in cancer tissues at advanced clinical stages, with positive distant metastasis and chemotherapeutic resistance. We examined the role of TIMP3 in osteosarcoma (OS) cell invasiveness and chemoresistance. TIMP3 was overexpressed or knocked down in the human OS cell lines Saos2 and MG63. Cell migration and invasion capacities were then evaluated using Transwell assays, and resistance to cisplatin was assessed by CCK-8 assay and flow cytometry. Real-time PCR and western blotting were used to investigate activation of signaling pathways downstream of TIMP3. Overexpression of TIMP3 inhibited the migration and invasion of Saos2 and MG63 cells, while knockdown of TIMP3 had the opposite effect. Cell survival after exposure to cisplatin was inhibited by TIMP3 overexpression in both Saos2 and MG63 cells. Consistently, downregulation of TIMP3 gene expression significantly decreased the sensitivity of OS cells to cisplatin treatment. MMP1, MMP2, Bcl-2, and Akt1 were all downregulated following TIMP3 overexpression, while Bax and cleaved caspase-3 were upregulated. TIMP3 knockdown had opposite effects on the regulation of these genes. Taken together, our findings suggest TIMP3 as a new target for inhibition of OS progression and chemotherapeutic resistance.


TIMP3 Osteosarcoma Migration Invasion Cisplatin sensitivity 



This work was supported by grants from the National Natural Science Foundation of China (81172549, 81302341, 81370050, 81272038) and the Science and Technology Commission of Shanghai Special Experimental Animals of China (14140904002).

Authors’ contributions

XG Han and Y Li conducted the experiments. XG Han, Y Li, HM Mo, K Li, and CQ Zhao analyzed the data. XG Han helped to draft the manuscript. TT Tang and J Zhao designed the study, analyzed and interpreted the data, and drafted the manuscript. All authors read and approved the final manuscript.


  1. 1.
    Bian ZY, Fan QM, Li G, Xu WT, Tang TT. Human mesenchymal stem cells promote growth of osteosarcoma: involvement of interleukin-6 in the interaction between human mesenchymal stem cells and Saos-2. Cancer Sci. 2010;101(12):2554–60. doi: 10.1111/j.1349-7006.2010.01731.x.CrossRefPubMedGoogle Scholar
  2. 2.
    Tu B, Du L, Fan QM, Tang Z, Tang TT. STAT3 activation by IL-6 from mesenchymal stem cells promotes the proliferation and metastasis of osteosarcoma. Cancer Lett. 2012;325(1):80–8. doi: 10.1016/j.canlet.2012.06.006.CrossRefPubMedGoogle Scholar
  3. 3.
    Du L, Fan Q, Tu B, Yan W, Tang T. Establishment and characterization of a new highly metastatic human osteosarcoma cell line derived from Saos2. International journal of clinical and experimental pathology. 2014;7(6):2871–82.PubMedPubMedCentralGoogle Scholar
  4. 4.
    Haddox CL, Han G. Osteosarcoma in pediatric patients and young adults: a single institution retrospective review of presentation, therapy, and outcome. 2014;2014:402509. doi: 10.1155/2014/402509.
  5. 5.
    Eilber F, Giuliano A, Eckardt J, Patterson K, Moseley S, Goodnight J. Adjuvant chemotherapy for osteosarcoma: a randomized prospective trial. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 1987;5(1):21–6.CrossRefGoogle Scholar
  6. 6.
    Xu JQ, Zhang WB, Wan R, Yang YQ. MicroRNA-32 inhibits osteosarcoma cell proliferation and invasion by targeting Sox9. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2014;35(10):9847–53. doi: 10.1007/s13277-014-2229-x.CrossRefGoogle Scholar
  7. 7.
    Zhang Y, Yang CQ, Gao Y, Wang C, Zhang CL, Zhou XH. Knockdown of CXCR7 inhibits proliferation and invasion of osteosarcoma cells through inhibition of the PI3K/Akt and beta-arrestin pathways. Oncol Rep. 2014;32(3):965–72. doi: 10.3892/or.2014.3290.PubMedGoogle Scholar
  8. 8.
    Kansara M, Teng MW, Smyth MJ, Thomas DM. Translational biology of osteosarcoma. Nature reviews Cancer. 2014. doi: 10.1038/nrc3838.
  9. 9.
    Arpino V, Brock M, Gill SE. The role of TIMPs in regulation of extracellular matrix proteolysis. Matrix biology : journal of the International Society for Matrix Biology. 2015;44–46:247–54. doi: 10.1016/j.matbio.2015.03.005.CrossRefGoogle Scholar
  10. 10.
    Itoh Y. Membrane-type matrix metalloproteinases: their functions and regulations. Matrix biology : journal of the International Society for Matrix Biology. 2015;44–46:207–23. doi: 10.1016/j.matbio.2015.03.004.CrossRefGoogle Scholar
  11. 11.
    Jacob A, Prekeris R. The regulation of MMP targeting to invadopodia during cancer metastasis. Frontiers in cell and developmental biology. 2015;3:4. doi: 10.3389/fcell.2015.00004.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Wu H, Wang W, Xu H. Depletion of C3orf1/TIMMDC1 inhibits migration and proliferation in 95D lung carcinoma cells. Int J Mol Sci. 2014;15(11):20555–71. doi: 10.3390/ijms151120555.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Wang CY, Liou JP, Tsai AC, Lai MJ, Liu YM, Lee HY, et al. A novel action mechanism for MPT0G013, a derivative of arylsulfonamide, inhibits tumor angiogenesis through up-regulation of TIMP3 expression. Oncotarget. 2014;5(20):9838–50.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Adissu HA, McKerlie C, Di Grappa M, Waterhouse P, Xu Q, Fang H, et al. Timp3 loss accelerates tumour invasion and increases prostate inflammation in a mouse model of prostate cancer. Prostate. 2015;75(16):1831–43. doi: 10.1002/pros.23056.CrossRefPubMedGoogle Scholar
  15. 15.
    Duan L, Perez RE, Hansen M, Gitelis S, Maki CG. Increasing cisplatin sensitivity by schedule-dependent inhibition of AKT and Chk1. Cancer biology & therapy. 2014;15(12):1600–12. doi: 10.4161/15384047.2014.961876.CrossRefGoogle Scholar
  16. 16.
    Han XG, Du L, Qiao H, Tu B, Wang YG, Qin A, et al. CXCR1 knockdown improves the sensitivity of osteosarcoma to cisplatin. Cancer Lett. 2015;369(2):405–15. doi: 10.1016/j.canlet.2015.09.002.CrossRefPubMedGoogle Scholar
  17. 17.
    Gan R, Yang Y, Yang X, Zhao L, Lu J, Meng QH. Downregulation of miR-221/222 enhances sensitivity of breast cancer cells to tamoxifen through upregulation of TIMP3. Cancer Gene Ther. 2014;21(7):290–6. doi: 10.1038/cgt.2014.29.CrossRefPubMedGoogle Scholar
  18. 18.
    Acunzo M, Visone R, Romano G, Veronese A, Lovat F, Palmieri D, et al. miR-130a targets MET and induces TRAIL-sensitivity in NSCLC by downregulating miR-221 and 222. Oncogene. 2012;31(5):634–42. doi: 10.1038/onc.2011.260.PubMedGoogle Scholar
  19. 19.
    Garofalo M, Di Leva G, Romano G, Nuovo G, Suh SS, Ngankeu A, et al. miR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation. Cancer Cell. 2009;16(6):498–509. doi: 10.1016/j.ccr.2009.10.014.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Qi JH, Anand-Apte B. Tissue inhibitor of metalloproteinase-3 (TIMP3) promotes endothelial apoptosis via a caspase-independent mechanism. Apoptosis : an international journal on programmed cell death. 2015;20(4):523–34. doi: 10.1007/s10495-014-1076-y.CrossRefGoogle Scholar
  21. 21.
    Yang J, Cheng D, Zhou S, Zhu B, Hu T, Yang Q. Overexpression of X-box binding protein 1 (XBP1) correlates to poor prognosis and up-regulation of PI3K/mTOR in human osteosarcoma. Int J Mol Sci. 2015;16(12):28635–46. doi: 10.3390/ijms161226123.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Tang H, Ji F, Sun J, Xie Y, Xu Y, Yue H. RBEL1 is required for osteosarcoma cell proliferation via inhibiting retinoblastoma 1. Molecular medicine reports. 2015. doi: 10.3892/mmr.2015.4670.
  23. 23.
    Tang QL, Xie XB, Wang J, Chen Q, Han AJ, Zou CY, et al. Glycogen synthase kinase-3beta, NF-kappaB signaling, and tumorigenesis of human osteosarcoma. J Natl Cancer Inst. 2012;104(10):749–63. doi: 10.1093/jnci/djs210.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Ferrari S, Serra M. An update on chemotherapy for osteosarcoma. Expert opinion on pharmacotherapy. 2015:1–10. doi: 10.1517/14656566.2015.1102226.
  25. 25.
    Saitoh Y, Setoguchi T, Nagata M, Tsuru A, Nakamura S, Nagano S et al. Combination of Hedgehog inhibitors and standard anticancer agents synergistically prevent osteosarcoma growth. International journal of oncology. 2015. doi: 10.3892/ijo.2015.3236.
  26. 26.
    Green DR, Llambi F. Cell death signaling. Cold Spring Harbor perspectives in biology. 2015;7:12. doi: 10.1101/cshperspect.a006080.CrossRefGoogle Scholar
  27. 27.
    Martelli AM, Evangelisti C, Chappell W, Abrams SL, Basecke J, Stivala F, et al. Targeting the translational apparatus to improve leukemia therapy: roles of the PI3K/PTEN/Akt/mTOR pathway. Leukemia. 2011;25(7):1064–79. doi: 10.1038/leu.2011.46.CrossRefPubMedGoogle Scholar
  28. 28.
    Yang J, Zhang W. New molecular insights into osteosarcoma targeted therapy. Curr Opin Oncol. 2013;25(4):398–406. doi: 10.1097/CCO.0b013e3283622c1b.CrossRefPubMedGoogle Scholar
  29. 29.
    Zhao Z, Tao L, Shen C, Liu B, Yang Z, Tao H. Silencing of Barkor/ATG14 sensitizes osteosarcoma cells to cisplatin-induced apoptosis. International journal of molecular medicine. 2014;33(2):271–6. doi: 10.3892/ijmm.2013.1578.PubMedGoogle Scholar
  30. 30.
    Wang G, Rong J, Zhou Z, Duo J. Novel gene P28GANK confers multidrug resistance by modulating the expression of MDR-1, Bcl-2 and Bax in osteosarcoma cells. Mol Biol. 2010;44(6):1010–7.CrossRefGoogle Scholar
  31. 31.
    Zollinger A, Stuhmer T, Chatterjee M, Gattenlohner S, Haralambieva E, Muller-Hermelink HK, et al. Combined functional and molecular analysis of tumor cell signaling defines 2 distinct myeloma subgroups: Akt-dependent and Akt-independent multiple myeloma. Blood. 2008;112(8):3403–11. doi: 10.1182/blood-2007-11-119362.CrossRefPubMedGoogle Scholar
  32. 32.
    Kimura R, Ishikawa C, Rokkaku T, Janknecht R, Mori N. Phosphorylated c-Jun and Fra-1 induce matrix metalloproteinase-1 and thereby regulate invasion activity of 143B osteosarcoma cells. Biochim Biophys Acta. 2011;1813(8):1543–53. doi: 10.1016/j.bbamcr.2011.04.008.CrossRefPubMedGoogle Scholar
  33. 33.
    Wen X, Liu H, Yu K, Liu Y. Matrix metalloproteinase 2 expression and survival of patients with osteosarcoma: a meta-analysis. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2014;35(1):845–8. doi: 10.1007/s13277-013-1116-1.CrossRefGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  1. 1.Shanghai Key Laboratory of Orthopedic Implants, Department of Orthopedic Surgery, Shanghai Ninth People’s HospitalShanghai Jiao Tong University School of MedicineShanghaiChina
  2. 2.Institution of Hematology, Xuzhou Medical College, Department of HematologyThe Affiliated Hospital of Xuzhou Medical CollegeJiangsu ProvinceChina
  3. 3.Department of Orthopedic Surgery, Shanghai First People’s HospitalShanghai Jiao Tong University School of MedicineShanghaiChina

Personalised recommendations