Tumor Biology

, Volume 32, Issue 3, pp 611–622

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

DOI: 10.1007/s13277-011-0160-y

Cite this article as:
Fujiwara, M., Kashima, T.G., Kunita, A. et al. Tumor Biol. (2011) 32: 611. doi:10.1007/s13277-011-0160-y
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Abstract

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.

Keywords

S100A4 Cell motility Osteosarcoma Metastasis shRNA Insulator Transposon 

Abbreviations

OS

Osteosarcoma

shRNA

Short hairpin RNA

ECM

Extracellular matrix

MMP

Metalloproteinase

TMA

Tissue microarray

MHC

Myosin heavy chain

qRT-PCR

Quantitative reverse transcription-polymerase chain reaction

IHC

Immunohistochemistry

Supplementary material

13277_2011_160_MOESM1_ESM.doc (62 kb)
ESM 1(DOC 75 kb)
13277_2011_160_MOESM2_ESM.doc (82 kb)
Table S1Primers sets used in this study (DOC 81 kb)
13277_2011_160_MOESM3_ESM.doc (126 kb)
Table S2List 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 S3MMP family expression profiling by oligonucleotide arrays (DOC 143 kb)
13277_2011_160_MOESM5_ESM.jpg (450 kb)
Fig. S1The map of pInSB(R) (JPEG 449 kb)
13277_2011_160_MOESM6_ESM.jpg (538 kb)
Fig. S2The map of pInSB(L) (JPEG 538 kb)
13277_2011_160_MOESM7_ESM.jpg (489 kb)
Fig. S3Construction of pInSB-Neo-shS100a4 (JPEG 489 kb)

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

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