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Stable knockdown of S100A4 suppresses cell migration and metastasis of osteosarcoma

  • Research Article
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Tumor Biology

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.

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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

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Acknowledgements

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

None.

Author information

Author notes

  1. Takeshi G. Kashima

    Present address: Histopathology Department, Nuffield Orthopaedic Centre, Windmill Road, Oxford, OX3 7LD, UK

Authors and Affiliations

  1. Department of Human Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

    Masahiko Fujiwara, Takeshi G. Kashima, Akiko Kunita, Daisuke Komura & Masashi Fukayama

  2. Biken Pathology Laboratory, Kotobiken Medical Laboratories, Tokyo, Japan

    Masahiko Fujiwara

  3. Departments of Craniofacial Development and Orthodontics, King’s College London, London, UK

    Takeshi G. Kashima & Agamemnon E. Grigoriadis

  4. Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan

    Isao Kii & Akira Kudo

  5. Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan

    Daisuke Komura & Hiroyuki Aburatani

Authors
  1. Masahiko Fujiwara
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Correspondence to Takeshi G. Kashima.

Electronic supplementary material

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ESM 1

(DOC 75 kb)

Table S1

Primers sets used in this study (DOC 81 kb)

Table S2

List of the highest ten genes preferentially expressed in LM8 than in Dunn by oligonucleotide arrays (DOC 137 kb)

Table S3

MMP family expression profiling by oligonucleotide arrays (DOC 143 kb)

Fig. S1

The map of pInSB(R) (JPEG 449 kb)

Fig. S2

The map of pInSB(L) (JPEG 538 kb)

Fig. S3

Construction of pInSB-Neo-shS100a4 (JPEG 489 kb)

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Fujiwara, M., Kashima, T.G., Kunita, A. et al. Stable knockdown of S100A4 suppresses cell migration and metastasis of osteosarcoma. Tumor Biol. 32, 611–622 (2011). https://doi.org/10.1007/s13277-011-0160-y

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  • DOI: https://doi.org/10.1007/s13277-011-0160-y

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