Clinical & Experimental Metastasis

, Volume 24, Issue 6, pp 439–448

RNA interference of ecto-5′-nucleotidase (CD73) inhibits human breast cancer cell growth and invasion

  • Xiuling Zhi
  • Sifeng Chen
  • Ping Zhou
  • Zhimin Shao
  • Li Wang
  • Zhouluo Ou
  • Lianhua Yin
Research Paper

Abstract

Metastasis is a leading cause of mortality and morbidity in breast cancer. Recently, dramatic overexpression of ecto-5′-nucleotidase (CD73), a glycosylphosphatidylinositol-anchored cell surface protein has been found in estrogen receptor-negative [ER (−)] breast cancer cell lines and in clinical samples. In this study, CD73 small interfering RNA (siRNA) plasmid was constructed and stably transfected into breast cancer cell MB-MDA-231 to determine the role of CD73 in breast cancer metastasis and the possible mechanism. Our study demonstrates that CD73 siRNA effectively inhibits CD73 gene expression at mRNA and protein level in MB-MDA-231 cells, leading to in vivo and in vitro growth suppression, prevention of adhesion to extracellular matrix (ECM), and inhibition of invasion and migration. These properties correlate with inhibition of matrix metalloproteinase (MMP)-2 and MMP-9 expression and activity as well as reduction of epidermal growth factor receptor (EGFR) expression. Demonstration of the role of CD73 in breast cancer may lead to new targeted therapies for breast cancer.

Keywords

CD73 Breast cancer RNA interfere Invasion Adhesion Growth 

Abbreviations

CD73

Ecto-5′-nucleotidase

ER(−)

Estrogen receptor-negative

siRNA

Small interfering RNA

G418

Geneticin

EGFR

Epidermal growth factor receptor

MMP

Matrix metalloproteinase

ECM

Extracellular matrix

VCAM

Vascular cell adhesion molecule

shRNA

Short hairpin RNA

RNAi

RNA interference

ATCC

American type culture collection

DMEM

Dulbecco’s modified Eagle’s medium

FCS

Fetal calf serum

FACS

Fluorescence activated cell sorter

ECL

Enhanced chemiluminescence

FN

Fibronectin

LN

Laminin

APCP

α, β-methylene ADP

dsRNA

Double-stranded RNA

CNS

Central nervous system

References

  1. 1.
    Zimmermann H (1996) Biochemistry, localization and functional roles of ecto-nucleotidases in the nervous system. Prog Neurobiol 49(6):589–618PubMedCrossRefGoogle Scholar
  2. 2.
    Resta R, Thompson LF (1997) T cell signalling through CD73. Cell Signal 9(2):131–139PubMedCrossRefGoogle Scholar
  3. 3.
    Zernecke A, Bidzhekov K, Ozuyaman B et al (2006) CD73/ecto-5′-nucleotidase protects against vascular inflammation and neointima formation. Circulation 113(17):2120–2127PubMedCrossRefGoogle Scholar
  4. 4.
    Spychala J, Lazarowski E, Ostapkowicz A et al (2004) Role of estrogen receptor in the regulation of ecto-5′-nucleotidase and adenosine in breast cancer. Clin Cancer Res 10(2):708–717PubMedCrossRefGoogle Scholar
  5. 5.
    Mujoomdar M, Bennett A, Hoskin D et al (2004) Adenosine stimulation of proliferation of breast carcinoma cell lines: evaluation of the [3H]thymidine assay system and modulatory effects of the cellular microenvironment in vitro. J Cell Physiol 201(3):429–438PubMedCrossRefGoogle Scholar
  6. 6.
    Mirza A, Basso A, Black S et al (2005) RNA interference targeting of A1 receptor-overexpressing breast carcinoma cells leads to diminished rates of cell proliferation and induction of apoptosis. Cancer Biol Ther 4(12):1355–1360PubMedCrossRefGoogle Scholar
  7. 7.
    Spychala J (2000) Tumor-promoting functions of adenosine. Pharmacol Ther 87(2–3):161–173PubMedCrossRefGoogle Scholar
  8. 8.
    Airas L, Hellman J, Salmi M et al (1995) CD73 is involved in lymphocyte binding to the endothelium: characterization of lymphocyte-vascular adhesion protein 2 identifies it as CD73. J Exp Med 182(5):1603–1608PubMedCrossRefGoogle Scholar
  9. 9.
    Fenoglio C, Necchi D, Civallero M et al (1997) Cytochemical demonstration of nitric oxide synthase and 5′ nucleotidase in human glioblastoma. Anticancer Res 17(4A):2507–2511PubMedGoogle Scholar
  10. 10.
    Ludwig HC, Rausch S, Schallock K et al (1999) Expression of CD 73 (ecto-5′-nucleotidase) in 165 glioblastomas by immunohistochemistry and electronmicroscopic histochemistry. Anticancer Res 19(3A):1747–1752PubMedGoogle Scholar
  11. 11.
    Caceres M, Guerrero J, Martinez J (2005) Overexpression of RhoA-GTP induces activation of the epidermal growth factor receptor, dephosphorylation of focal adhesion kinase and increased motility in breast cancer cells. Exp Cell Res 309(1):229–238PubMedCrossRefGoogle Scholar
  12. 12.
    Ostapkowicz A, Inai K, Smith L et al (2006) Lipid rafts remodeling in estrogen receptor-negative breast cancer is reversed by histone deacetylase inhibitor. Mol Cancer Ther 5(2):238–245PubMedCrossRefGoogle Scholar
  13. 13.
    Lee H, Lin EC, Liu L et al (2003) Gene expression profiling of tumor xenografts: in vivo analysis of organ-specific metastasis. Int J Cancer 107(4):528–534PubMedCrossRefGoogle Scholar
  14. 14.
    Zhou TT, Zhou P, Yin LH (2006) Crucial role for ecto-5′-nucleotidase (CD73) in invasion and migration of human breast cancer cell lines. Chinese J Pathophysiol 22(2):360–364Google Scholar
  15. 15.
    Saad S, Gottlieb DJ, Bradstock KF et al (2002) Cancer cell-associated fibronectin induces release of matrix metalloproteinase-2 from normal fibroblasts. Cancer Res 62(1):283–289PubMedGoogle Scholar
  16. 16.
    Bartsch JE, Staren ED, Appert HE (2003) Adhesion and migration of extracellular matrix-stimulated breast cancer. J Surg Res 110(1):287–294PubMedCrossRefGoogle Scholar
  17. 17.
    Kondo T, Nakazawa T, Murata SI et al (2006) Expression of CD73 and its ecto-5′-nucleotidase activity are elevated in papillary thyroid carcinomas. Histopathology 48(5):612–614PubMedCrossRefGoogle Scholar
  18. 18.
    Spychala J, Kitajewski J (2004) Wnt and beta-catenin signaling target the expression of ecto-5′-nucleotidase and increase extracellular adenosine generation. Exp Cell Res 296(2):99–108PubMedCrossRefGoogle Scholar
  19. 19.
    Wink MR, Tamajusuku AS, Braganhol E et al (2003) Thyroid hormone upregulates ecto-5′-nucleotidase/CD73 in C6 rat glioma cells. Mol Cell Endocrinol 205(1–2):107–114PubMedCrossRefGoogle Scholar
  20. 20.
    El-Darahali A, Fawcett H, Mader JS et al (2005) Adenosine-induced apoptosis in EL-4 thymoma cells is caspase-independent and mediated through a non-classical adenosine receptor. Exp Mol Pathol 79:249–258PubMedCrossRefGoogle Scholar
  21. 21.
    Stochaj U, Flocke K, Mathes W et al (1989) 5′-Nucleotidases of chicken gizzard and human pancreatic adenocarcinoma cells are anchored to the plasma membrane via a phosphatidylinositol-glycan. Biochem J 262(1):33–40PubMedGoogle Scholar
  22. 22.
    Navarro JM, Olmo N, Turnay J et al (1998) Ecto-5′-nucleotidase from a human colon adenocarcinoma cell line. Correlation between enzyme activity and levels in intact cells. Mol Cell Biochem 187(1–2):121–131PubMedCrossRefGoogle Scholar
  23. 23.
    Bianchi V, Spychala J (2003) Mammalian 5′-nucleotidases. J Biol Chem 278(47):46195–46198PubMedCrossRefGoogle Scholar
  24. 24.
    Woodhouse EC, Amanatullah DF, Schetz JA et al (1998) Adenosine receptor mediates motility in human melanoma cells. Biochem Biophys Res Commun 246(3):888–894PubMedCrossRefGoogle Scholar
  25. 25.
    Othman T, Yan H, Rivkees SA (2003) Oligodendrocytes express functional A1 adenosine receptors that stimulate cellular migration. Glia 44(2):166–172PubMedCrossRefGoogle Scholar
  26. 26.
    Baselga J, Arteaga CL (2005) Critical update and emerging trends in epidermal growth factor receptor targeting in cancer. J Clin Oncol 23(11):2445–2459PubMedCrossRefGoogle Scholar
  27. 27.
    Hofmann UB, Eggert AA, Blass K et al (2003) Expression of matrix metalloproteinases in the microenvironment of spontaneous and experimental melanoma metastases reflects the requirements for tumor formation. Cancer Res 63(23):8221–8225PubMedGoogle Scholar
  28. 28.
    Airas L, Niemelä J, Salmi M et al (1997) Differential regulation and function of CD73, a glycosyl-phosphatidylinositol-linked 70-kD adhesion molecule, on lymphocytes and endothelial. J Cell Biol 136(2):421–431PubMedCrossRefGoogle Scholar
  29. 29.
    Olmo N, Turnay J, Risse G et al (1992) Modulation of 5′-nucleotidase activity in plasma membranes and intact cells by the extracellular matrix proteins laminin and fibronectin. Biochem J 282(Pt 1):181–188PubMedGoogle Scholar
  30. 30.
    Ioachim E, Charchanti A, Briasoulis E et al (2002) Immunohistochemical expression of extracellular matrix components tenascin, fibronectin, collagen type IV and laminin in breast cancer: their prognostic value and role in tumour invasion and progression. Eur J Cancer 38(18):2362–2370PubMedCrossRefGoogle Scholar
  31. 31.
    Cronstein BN, Levin RI, Philips M et al (1992) Neutrophil adherence to endothelium is enhanced via adenosine A1 receptors and inhibited via adenosine A2 receptors. J Immunol 148(7):2201–2206PubMedGoogle Scholar
  32. 32.
    Sadej R, Spychala J, Skladanowski AC (2006) Ecto-5′-nucleotidase (eN, CD73) is coexpressed with metastasis promoting antigens in human melanoma cells. Nucleosides Nucleotides Nucleic Acids 25(9–11):1119–1123PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media B.V. 2007

Authors and Affiliations

  • Xiuling Zhi
    • 1
  • Sifeng Chen
    • 1
  • Ping Zhou
    • 1
  • Zhimin Shao
    • 2
  • Li Wang
    • 1
  • Zhouluo Ou
    • 2
  • Lianhua Yin
    • 1
  1. 1.Department of Physiology and PathophysiologyShanghai Medical College, Fudan UniversityShanghaiP.R. China
  2. 2.Department of Breast Surgery, Cancer HospitalFudan UniversityShanghaiP.R. China

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