Tumor Biology

, Volume 32, Issue 3, pp 611–622 | Cite as

Stable knockdown of S100A4 suppresses cell migration and metastasis of osteosarcoma

  • Masahiko Fujiwara
  • Takeshi G. Kashima
  • Akiko Kunita
  • Isao Kii
  • Daisuke Komura
  • Agamemnon E. Grigoriadis
  • Akira Kudo
  • Hiroyuki Aburatani
  • Masashi Fukayama
Research Article


S100A4, a 10–12 kDa calcium-binding protein, plays functional roles in tumor progression and metastasis. The present study aimed to investigate the function of S100A4 in osteosarcoma (OS) metastasis, using a loss-of-function approach. Our previous expression profiling analysis revealed that S100a4 was preferentially expressed in the highly metastatic mouse OS cell line, LM8. Introducing a short hairpin ribonucleic acid (shRNA) targeting S100a4 using a newly established vector containing insulators and transposons, we established stable LM8 subclones with almost 100% silencing of endogenous S100a4 protein. These transfectants showed a significant suppression of cell migration in vitro as well as a marked reduction in their ability to colonize the lung and form pulmonary metastases in vivo following intravenous inoculation, whereas there was no significant change in cell proliferation or cell attachment to fibronectin, laminin, and type I collagen. Expression and phosphorylation of ezrin, an emerging OS metastasis-associated factor, and expression of MMPs, remained the same in S100a4-shRNA clones. In 61 human OS, immunohistochemical analysis showed that lesional cells in 85.2% samples expressed S100A4 protein, and the immunoreactivity was primarily cytoplasmic, but it also showed occasional nuclear localization. Chondroblastic and osteoblastic OS subtypes expressed more S100A4 than fibroblastic subtypes. The causative role of S100A4 in OS lung metastasis shown in the murine xenograft model, together with the high proportion of primary human OS expressing S100A4, suggest that S100A4 protein represents an important potential target for future OS therapy.


S100A4 Cell motility Osteosarcoma Metastasis shRNA Insulator Transposon 





Short hairpin RNA


Extracellular matrix




Tissue microarray


Myosin heavy chain


Quantitative reverse transcription-polymerase chain reaction





The authors would like to thank Dr. Felsenfeld and Dr. Hackett for providing chicken HS4 insulator cores and pCMV-SB, respectively, and Mrs. Ogiwara, Ms. Yamamura, and Ms. Meguro for their technical assistance.

The work described in this report was funded by a grant (no. 16790202) from the Ministry of Education, Culture, Sports, Science & Technology to TGK and a grant from the UK Bone Cancer Research Trust .

Conflicts of interest


Supplementary material

13277_2011_160_MOESM1_ESM.doc (62 kb)
ESM 1 (DOC 75 kb)
13277_2011_160_MOESM2_ESM.doc (82 kb)
Table S1 Primers sets used in this study (DOC 81 kb)
13277_2011_160_MOESM3_ESM.doc (126 kb)
Table S2 List of the highest ten genes preferentially expressed in LM8 than in Dunn by oligonucleotide arrays (DOC 137 kb)
13277_2011_160_MOESM4_ESM.doc (126 kb)
Table S3 MMP family expression profiling by oligonucleotide arrays (DOC 143 kb)
13277_2011_160_MOESM5_ESM.jpg (450 kb)
Fig. S1 The map of pInSB(R) (JPEG 449 kb)
13277_2011_160_MOESM6_ESM.jpg (538 kb)
Fig. S2 The map of pInSB(L) (JPEG 538 kb)
13277_2011_160_MOESM7_ESM.jpg (489 kb)
Fig. S3 Construction of pInSB-Neo-shS100a4 (JPEG 489 kb)


  1. 1.
    Kempf-Bielack B, Bielack SS, Jurgens H, Branscheid D, Berdel WE, Exner GU, et al. Osteosarcoma relapse after combined modality therapy: an analysis of unselected patients in the Cooperative Osteosarcoma Study Group (COSS). J Clin Oncol. 2005;23:559–68.PubMedCrossRefGoogle Scholar
  2. 2.
    Asai T, Ueda T, Itoh K, Yoshioka K, Aoki Y, Mori S, et al. Establishment and characterization of a murine osteosarcoma cell line (LM8) with high metastatic potential to the lung. Int J Cancer. 1998;76:418–22.PubMedCrossRefGoogle Scholar
  3. 3.
    Kashima T, Nakamura K, Kawaguchi J, Takanashi M, Ishida T, Aburatani H, et al. Overexpression of cadherins suppresses pulmonary metastasis of osteosarcoma in vivo. Int J Cancer. 2003;104:147–54.PubMedCrossRefGoogle Scholar
  4. 4.
    Santamaria-Kisiel L, Rintala-Dempsey AC, Shaw GS. Calcium-dependent and -independent interactions of the S100 protein family. Biochem J. 2006;396:201–14.PubMedCrossRefGoogle Scholar
  5. 5.
    Boye K, Maelandsmo GM. S100A4 and metastasis: a small actor playing many roles. Am J Pathol. 2010;176:528–35.PubMedCrossRefGoogle Scholar
  6. 6.
    Davies BR, Barraclough R, Davies MP, Rudland PS. Production of the metastatic phenotype by DNA transfection in a rat mammary model. Cell Biol Int. 1993;17:871–9.PubMedCrossRefGoogle Scholar
  7. 7.
    Davies MP, Rudland PS, Robertson L, Parry EW, Jolicoeur P, Barraclough R. Expression of the calcium-binding protein S100A4 (p9Ka) in MMTV-neu transgenic mice induces metastasis of mammary tumours. Oncogene. 1996;13:1631–7.PubMedGoogle Scholar
  8. 8.
    Ambartsumian NS, Grigorian MS, Larsen IF, Karlstrom O, Sidenius N, Rygaard J, et al. Metastasis of mammary carcinomas in GRS/A hybrid mice transgenic for the mts1 gene. Oncogene. 1996;13:1621–30.PubMedGoogle Scholar
  9. 9.
    Maelandsmo GM, Hovig E, Skrede M, Engebraaten O, Florenes VA, Myklebost O, et al. Reversal of the in vivo metastatic phenotype of human tumor cells by an anti-CAPL (mts1) ribozyme. Cancer Res. 1996;56:5490–8.PubMedGoogle Scholar
  10. 10.
    Grigorian MS, Tulchinsky EM, Zain S, Ebralidze AK, Kramerov DA, Kriajevska MV, et al. The mts1 gene and control of tumor metastasis. Gene. 1993;135:229–38.PubMedCrossRefGoogle Scholar
  11. 11.
    Ismail TM, Zhang S, Fernig DG, Gross S, Martin-Fernandez ML, See V, et al. Self-association of calcium-binding protein S100A4 and metastasis. J Biol Chem. 2010;285:914–22.PubMedCrossRefGoogle Scholar
  12. 12.
    Ito Y, Yoshida H, Tomoda C, Uruno T, Miya A, Kobayashi K, et al. S100A4 expression is an early event of papillary carcinoma of the thyroid. Oncology. 2004;67:397–402.PubMedCrossRefGoogle Scholar
  13. 13.
    Garrett SC, Varney KM, Weber DJ, Bresnick AR. S100A4, a mediator of metastasis. J Biol Chem. 2006;281:677–80.PubMedCrossRefGoogle Scholar
  14. 14.
    Ma X, Yang Y, Wang Y, An G, Lv G. Small interfering RNA-directed knockdown of S100A4 decreases proliferation and invasiveness of osteosarcoma cells. Cancer Lett. 2010;28;299(2):171–81Google Scholar
  15. 15.
    Shi Y, Zou M, Collison K, Baitei EY, Al-Makhalafi Z, Farid NR, et al. Ribonucleic acid interference targeting S100A4 (Mts1) suppresses tumor growth and metastasis of anaplastic thyroid carcinoma in a mouse model. J Clin Endocrinol Metab. 2006;91:2373–9.PubMedCrossRefGoogle Scholar
  16. 16.
    Khanna C, Wan X, Bose S, Cassaday R, Olomu O, Mendoza A, et al. The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis. Nat Med. 2004;10:182–6.PubMedCrossRefGoogle Scholar
  17. 17.
    Hippo Y, Taniguchi H, Tsutsumi S, Machida N, Chong JM, Fukayama M, et al. Global gene expression analysis of gastric cancer by oligonucleotide microarrays. Cancer Res. 2002;62:233–40.PubMedGoogle Scholar
  18. 18.
    West AG, Gaszner M, Felsenfeld G. Insulators: many functions, many mechanisms. Genes Dev. 2002;16:271–88.PubMedCrossRefGoogle Scholar
  19. 19.
    Ivics Z, Hackett PB, Plasterk RH, Izsvak Z. Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell. 1997;91:501–10.PubMedCrossRefGoogle Scholar
  20. 20.
    Cabezon T, Celis JE, Skibshoj I, Klingelhofer J, Grigorian M, Gromov P, et al. Expression of S100A4 by a variety of cell types present in the tumor microenvironment of human breast cancer. Int J Cancer. 2007;121:1433–44.PubMedCrossRefGoogle Scholar
  21. 21.
    Shimamura T, Ito H, Shibahara J, Watanabe A, Hippo Y, Taniguchi H, et al. Overexpression of MUC13 is associated with intestinal-type gastric cancer. Cancer Sci. 2005;96:265–73.PubMedCrossRefGoogle Scholar
  22. 22.
    Kwak JM, Lee HJ, Kim SH, Kim HK, Mok YJ, Park YT, et al. Expression of protein S100A4 is a predictor of recurrence in colorectal cancer. World J Gastroenterol. 2010;16:3897–904.PubMedCrossRefGoogle Scholar
  23. 23.
    Psaila B, Lyden D. The metastatic niche: adapting the foreign soil. Nat Rev Cancer. 2009;9:285–93.PubMedCrossRefGoogle Scholar
  24. 24.
    Zreiqat H, Howlett CR, Gronthos S, Hume D, Geczy CL. S100A8/S100A9 and their association with cartilage and bone. J Mol Histol. 2007;38:381–91.PubMedCrossRefGoogle Scholar
  25. 25.
    Leonard P, Sharp T, Henderson S, Hewitt D, Pringle J, Sandison A, et al. Gene expression array profile of human osteosarcoma. Br J Cancer. 2003;89:2284–8.PubMedCrossRefGoogle Scholar
  26. 26.
    Ivics Z, Li MA, Mates L, Boeke JD, Nagy A, Bradley A, et al. Transposon-mediated genome manipulation in vertebrates. Nat Methods. 2009;6:415–22.PubMedCrossRefGoogle Scholar
  27. 27.
    Simons M, Wang M, McBride OW, Kawamoto S, Yamakawa K, Gdula D, et al. Human nonmuscle myosin heavy chains are encoded by two genes located on different chromosomes. Circ Res. 1991;69:530–9.PubMedGoogle Scholar
  28. 28.
    Conti MA, Adelstein RS. Nonmuscle myosin II moves in new directions. J Cell Sci. 2008;121:11–8.PubMedCrossRefGoogle Scholar
  29. 29.
    Dulyaninova NG, Malashkevich VN, Almo SC, Bresnick AR. Regulation of myosin-IIA assembly and Mts1 binding by heavy chain phosphorylation. Biochemistry. 2005;44:6867–76.PubMedCrossRefGoogle Scholar
  30. 30.
    Hirao M, Sato N, Kondo T, Yonemura S, Monden M, Sasaki T, et al. Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway. J Cell Biol. 1996;135:37–51.PubMedCrossRefGoogle Scholar
  31. 31.
    Salas S, Bartoli C, Deville JL, Gaudart J, Fina F, Calisti A, et al. Ezrin and alpha-smooth muscle actin are immunohistochemical prognostic markers in conventional osteosarcomas. Virchows Arch. 2007;451:999–1007.PubMedCrossRefGoogle Scholar
  32. 32.
    Khanna C, Khan J, Nguyen P, Prehn J, Caylor J, Yeung C, et al. Metastasis-associated differences in gene expression in a murine model of osteosarcoma. Cancer Res. 2001;61:3750–9.PubMedGoogle Scholar
  33. 33.
    Fukaya Y, Ishiguro N, Senga T, Ichigotani Y, Sohara Y, Tsutsui M, et al. A role for PI3K-Akt signaling in pulmonary metastatic nodule formation of the osteosarcoma cell line, LM8. Oncol Rep. 2005;14:847–52.PubMedGoogle Scholar
  34. 34.
    Ren L, Hong SH, Cassavaugh J, Osborne T, Chou AJ, Kim SY, et al. The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC. Oncogene. 2009;28:792–802.PubMedCrossRefGoogle Scholar
  35. 35.
    Bjornland K, Winberg JO, Odegaard OT, Hovig E, Loennechen T, Aasen AO, et al. S100A4 involvement in metastasis: deregulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in osteosarcoma cells transfected with an anti-S100A4 ribozyme. Cancer Res. 1999;59:4702–8.PubMedGoogle Scholar
  36. 36.
    Mathisen B, Lindstad RI, Hansen J, El-Gewely SA, Maelandsmo GM, Hovig E, et al. S100A4 regulates membrane induced activation of matrix metalloproteinase-2 in osteosarcoma cells. Clin Exp Metastasis. 2003;20:701–11.PubMedCrossRefGoogle Scholar
  37. 37.
    Wyckoff JB, Pinner SE, Gschmeissner S, Condeelis JS, Sahai E. ROCK- and myosin-dependent matrix deformation enables protease-independent tumor-cell invasion in vivo. Curr Biol. 2006;16:1515–23.PubMedCrossRefGoogle Scholar
  38. 38.
    Yammani RR, Carlson CS, Bresnick AR, Loeser RF. Increase in production of matrix metalloproteinase 13 by human articular chondrocytes due to stimulation with S100A4: Role of the receptor for advanced glycation end products. Arthritis Rheum. 2006;54:2901–11.PubMedCrossRefGoogle Scholar
  39. 39.
    Duarte WR, Shibata T, Takenaga K, Takahashi E, Kubota K, Ohya K, et al. S100A4: a novel negative regulator of mineralization and osteoblast differentiation. J Bone Miner Res. 2003;18:493–501.PubMedCrossRefGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2011

Authors and Affiliations

  • Masahiko Fujiwara
    • 1
    • 2
  • Takeshi G. Kashima
    • 1
    • 3
    • 6
  • Akiko Kunita
    • 1
  • Isao Kii
    • 4
  • Daisuke Komura
    • 1
    • 5
  • Agamemnon E. Grigoriadis
    • 3
  • Akira Kudo
    • 4
  • Hiroyuki Aburatani
    • 5
  • Masashi Fukayama
    • 1
  1. 1.Department of Human Pathology, Graduate School of MedicineUniversity of TokyoTokyoJapan
  2. 2.Biken Pathology LaboratoryKotobiken Medical LaboratoriesTokyoJapan
  3. 3.Departments of Craniofacial Development and OrthodonticsKing’s College LondonLondonUK
  4. 4.Department of Biological Information, Graduate School of Bioscience and BiotechnologyTokyo Institute of TechnologyYokohamaJapan
  5. 5.Genome Science Division, Research Center for Advanced Science and TechnologyUniversity of TokyoTokyoJapan
  6. 6.Histopathology DepartmentNuffield Orthopaedic CentreOxfordUK

Personalised recommendations