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

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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.

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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. Zimmermann H (1996) Biochemistry, localization and functional roles of ecto-nucleotidases in the nervous system. Prog Neurobiol 49(6):589–618

    Article  PubMed  CAS  Google Scholar 

  2. Resta R, Thompson LF (1997) T cell signalling through CD73. Cell Signal 9(2):131–139

    Article  PubMed  CAS  Google Scholar 

  3. Zernecke A, Bidzhekov K, Ozuyaman B et al (2006) CD73/ecto-5′-nucleotidase protects against vascular inflammation and neointima formation. Circulation 113(17):2120–2127

    Article  PubMed  CAS  Google Scholar 

  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):708–717

    Article  PubMed  CAS  Google Scholar 

  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):429–438

    Article  PubMed  CAS  Google Scholar 

  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):1355–1360

    Article  PubMed  CAS  Google Scholar 

  7. Spychala J (2000) Tumor-promoting functions of adenosine. Pharmacol Ther 87(2–3):161–173

    Article  PubMed  CAS  Google Scholar 

  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):1603–1608

    Article  PubMed  CAS  Google Scholar 

  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):2507–2511

    PubMed  CAS  Google Scholar 

  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):1747–1752

    PubMed  CAS  Google Scholar 

  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):229–238

    Article  PubMed  CAS  Google Scholar 

  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):238–245

    Article  PubMed  CAS  Google Scholar 

  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):528–534

    Article  PubMed  CAS  Google Scholar 

  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):360–364

    CAS  Google Scholar 

  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):283–289

    PubMed  CAS  Google Scholar 

  16. Bartsch JE, Staren ED, Appert HE (2003) Adhesion and migration of extracellular matrix-stimulated breast cancer. J Surg Res 110(1):287–294

    Article  PubMed  CAS  Google Scholar 

  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):612–614

    Article  PubMed  CAS  Google Scholar 

  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):99–108

    Article  PubMed  CAS  Google Scholar 

  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):107–114

    Article  PubMed  CAS  Google Scholar 

  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 79:249–258

    Article  PubMed  CAS  Google Scholar 

  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):33–40

    PubMed  CAS  Google Scholar 

  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):121–131

    Article  PubMed  CAS  Google Scholar 

  23. Bianchi V, Spychala J (2003) Mammalian 5′-nucleotidases. J Biol Chem 278(47):46195–46198

    Article  PubMed  CAS  Google Scholar 

  24. Woodhouse EC, Amanatullah DF, Schetz JA et al (1998) Adenosine receptor mediates motility in human melanoma cells. Biochem Biophys Res Commun 246(3):888–894

    Article  PubMed  CAS  Google Scholar 

  25. Othman T, Yan H, Rivkees SA (2003) Oligodendrocytes express functional A1 adenosine receptors that stimulate cellular migration. Glia 44(2):166–172

    Article  PubMed  Google Scholar 

  26. Baselga J, Arteaga CL (2005) Critical update and emerging trends in epidermal growth factor receptor targeting in cancer. J Clin Oncol 23(11):2445–2459

    Article  PubMed  CAS  Google Scholar 

  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):8221–8225

    PubMed  CAS  Google Scholar 

  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):421–431

    Article  PubMed  CAS  Google Scholar 

  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):181–188

    PubMed  CAS  Google Scholar 

  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):2362–2370

    Article  PubMed  CAS  Google Scholar 

  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):2201–2206

    PubMed  CAS  Google Scholar 

  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):1119–1123

    Article  PubMed  CAS  Google Scholar 

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Acknowledgment

We thank Dr Linda Thompson for supplying mAb 1E9.

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Authors and Affiliations

  1. Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, 138# Yixueyuan Road, Shanghai, 200032, P.R. China

    Xiuling Zhi, Sifeng Chen, Ping Zhou, Li Wang & Lianhua Yin

  2. Department of Breast Surgery, Cancer Hospital, Fudan University, Shanghai, 200032, P.R. China

    Zhimin Shao & Zhouluo Ou

Authors
  1. Xiuling Zhi
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  2. Sifeng Chen
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  3. Ping Zhou
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  4. Zhimin Shao
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  5. Li Wang
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  6. Zhouluo Ou
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  7. Lianhua Yin
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Correspondence to Ping Zhou.

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Zhi, X., Chen, S., Zhou, P. et al. RNA interference of ecto-5′-nucleotidase (CD73) inhibits human breast cancer cell growth and invasion. Clin Exp Metastasis 24, 439–448 (2007). https://doi.org/10.1007/s10585-007-9081-y

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  • DOI: https://doi.org/10.1007/s10585-007-9081-y

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