Skip to main content

Advertisement

SpringerLink
Log in

Silencing of skeletal metastasis-associated genes impairs migration of breast cancer cells and reduces osteolytic bone lesions

  • Research Paper
  • Published:
Clinical & Experimental Metastasis Aims and scope Submit manuscript

Abstract

Bone sialoprotein (BSP) and osteopontin (OPN) are important factors in the metastasis of breast cancer, which were examined as targets for antineoplastic therapy by siRNA. In addition, the effect of gene silencing on their transcription factor Runx2 and their interaction partners integrin β3 and matrix metalloproteinase 2 was studied. The effect of siRNAs directed against these genes was assessed by monitoring expression levels followed by functional assays in cell culture as well as skeletal metastases caused by human MDA-MB-231luc breast cancer cells in nude rats. Upon silencing of the targets, cell migration was profoundly impaired (p < 0.001 for BSP-siRNA), but the impact on proliferation was low. Systemic administration by osmotic mini-pumps of BSP-siRNA but not OPN-siRNA decreased osteolytic lesions (p = 0.067). Extraosseous tumour growth was not affected. As an alternative approach, non-viral, polymeric based formulations of siRNAs in nanoparticles (NP) were developed. Locoregional administration of the two siRNAs targeting OPN and BSP encapsulated in these biodegradable NP reduced skeletal lesions even more efficiently (p = 0.03). Compared to systemic administration, this treatment caused not only a more pronounced anti-osteolytic effect at a 25-fold lower total siRNA dose, but also had a slight reducing effect on tumour incidence (p = 0.095). In conclusion, the siRNA treatment had a small effect on cellular proliferation but a significant efficacy against migration of and osteolysis induced by MDA-MB-231 cells. Our data underline that siRNA mediated knockdown is a powerful tool for identifying targets for pharmacological intervention. In addition, encapsulation of siRNA into biodegradable NP is a strategy, which promises well for using siRNA.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

Abbreviations

BSP:

Bone sialoprotein

ECM:

Extracellular matrix

hrBSP:

Human recombinant BSP

ITGB3:

Integrin β3

MDA-MB-231luc :

Luciferase tagged MDA-MB-231 human breast cancer cells

MMP:

Matrix metalloproteinase

MRI:

Magnetic resonance imaging

NP:

Nanoparticles

nt:

Nucleotide(s)

OPN:

Osteopontin

PEI:

Polyethyleneimine

PLGA:

Poly(d,l-lactide-co-glycolide)

siRNA:

Small interfering RNA

TGF:

Transforming growth factor

VCT:

Volume computed tomography

References

  1. Weinberg RA (2006) The biology of cancer. Garland Science, Oxford

    Google Scholar 

  2. Berenson JR (2001) Zoledronic acid in cancer patients with bone metastases: results of Phase I and II trials. Semin Oncol 28(2 Suppl 6):25–34

    Article  PubMed  CAS  Google Scholar 

  3. Hortobagyi GN (1998) Treatment of breast cancer. New Engl J Med 339(14):974–984

    Article  PubMed  CAS  Google Scholar 

  4. McArthur HL, Hudis CA (2009) Trastuzumab: a picky partner? Clin Cancer Res 15(20):6311–6313

    Article  PubMed  CAS  Google Scholar 

  5. Slamon DJ, Leyland-Jones B, Shak S et al (2001) Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. New Engl J Med 344(11):783–792

    Article  PubMed  CAS  Google Scholar 

  6. Theriault RL, Lipton A, Hortobagyi GN et al (1999) Pamidronate reduces skeletal morbidity in women with advanced breast cancer and lytic bone lesions: a randomized, placebo-controlled trial. Protocol 18 Aredia Breast Cancer Study Group. J Clin Oncol 17(3):846–854

    PubMed  CAS  Google Scholar 

  7. Bos PD, Zhang XH, Nadal C et al (2009) Genes that mediate breast cancer metastasis to the brain. Nature 459(7249):1005–1009

    Article  PubMed  CAS  Google Scholar 

  8. Kang Y, Siegel PM, Shu W et al (2003) A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 3(6):537–549

    Article  PubMed  CAS  Google Scholar 

  9. Heinegard D, Oldberg A (1989) Structure and biology of cartilage and bone matrix noncollagenous macromolecules. FASEB J 3(9):2042–2051

    PubMed  CAS  Google Scholar 

  10. Fisher LW, Whitson SW, Avioli LV et al (1983) Matrix sialoprotein of developing bone. J Biol Chem 258(20):12723–12727

    PubMed  CAS  Google Scholar 

  11. Fisher LW, Torchia DA, Fohr B et al (2001) Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin. Biochem Biophys Res Commun 280(2):460–465

    Article  PubMed  CAS  Google Scholar 

  12. Noda M, Rodan GA (1989) Type beta transforming growth factor regulates expression of genes encoding bone matrix proteins. Connect Tissue Res 21(1–4):71–75

    Article  PubMed  CAS  Google Scholar 

  13. Ogata Y, Yamauchi M, Kim RH et al (1995) Glucocorticoid regulation of bone sialoprotein (BSP) gene expression. Identification of a glucocorticoid response element in the bone sialoprotein gene promoter. Eur J Biochem/FEBS 230(1):183–192

    Article  CAS  Google Scholar 

  14. Pratap J, Lian JB, Javed A et al (2006) Regulatory roles of Runx2 in metastatic tumor and cancer cell interactions with bone. Cancer Metastasis Rev 25(4):589–600

    Article  PubMed  CAS  Google Scholar 

  15. Byzova TV, Kim W, Midura RJ et al (2000) Activation of integrin alpha(V)beta(3) regulates cell adhesion and migration to bone sialoprotein. Exp Cell Res 254(2):299–308

    Article  PubMed  CAS  Google Scholar 

  16. Das R, Mahabeleshwar GH, Kundu GC (2003) Osteopontin stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase type plasminogen activator through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells. J Biol Chem 278(31):28593–28606

    Article  PubMed  CAS  Google Scholar 

  17. DeMali KA, Wennerberg K, Burridge K (2003) Integrin signaling to the actin cytoskeleton. Curr Opin Cell Biol 15(5):572–582

    Article  PubMed  CAS  Google Scholar 

  18. Gordon JA, Sodek J, Hunter GK et al (2009) Bone sialoprotein stimulates focal adhesion-related signaling pathways: role in migration and survival of breast and prostate cancer cells. J Cell Biochem 107(6):1118–1128

    Article  PubMed  CAS  Google Scholar 

  19. Guo W, Giancotti FG (2004) Integrin signalling during tumour progression. Nat Rev Mol Cell Biol 5(10):816–826

    Article  PubMed  CAS  Google Scholar 

  20. Kiosses WB, Shattil SJ, Pampori N et al (2001) Rac recruits high-affinity integrin alphavbeta3 to lamellipodia in endothelial cell migration. Nat Cell Biol 3(3):316–320

    Article  PubMed  CAS  Google Scholar 

  21. Rangaswami H, Bulbule A, Kundu GC (2006) Osteopontin: role in cell signaling and cancer progression. Trends Cell Biol 16(2):79–87

    Article  PubMed  CAS  Google Scholar 

  22. Sharp JA, Waltham M, Williams ED et al (2004) Transfection of MDA-MB-231 human breast carcinoma cells with bone sialoprotein (BSP) stimulates migration and invasion in vitro and growth of primary and secondary tumors in nude mice. Clin Exp Metastasis 21(1):19–29

    Article  PubMed  CAS  Google Scholar 

  23. Bellahcene A, Castronovo V, Ogbureke KU et al (2008) Small integrin-binding ligand N-linked glycoproteins (SIBLINGs): multifunctional proteins in cancer. Nat Rev Cancer 8(3):212–226

    Article  PubMed  CAS  Google Scholar 

  24. Fedarko NS, Jain A, Karadag A et al (2001) Elevated serum bone sialoprotein and osteopontin in colon, breast, prostate, and lung cancer. Clin Cancer Res 7(12):4060–4066

    PubMed  CAS  Google Scholar 

  25. Bellahcene A, Kroll M, Liebens F et al (1996) Bone sialoprotein expression in primary human breast cancer is associated with bone metastases development. J Bone Miner Res 11(5):665–670

    Article  PubMed  CAS  Google Scholar 

  26. Bellahcene A, Menard S, Bufalino R et al (1996) Expression of bone sialoprotein in primary human breast cancer is associated with poor survival. Int J Cancer 69(4):350–353

    Article  PubMed  CAS  Google Scholar 

  27. Bramwell VH, Doig GS, Tuck AB et al (2006) Serial plasma osteopontin levels have prognostic value in metastatic breast cancer. Clin Cancer Res 12(11 Pt 1):3337–3343

    Article  PubMed  CAS  Google Scholar 

  28. Diel IJ, Solomayer EF, Seibel MJ et al (1999) Serum bone sialoprotein in patients with primary breast cancer is a prognostic marker for subsequent bone metastasis. Clin Cancer Res 5(12):3914–3919

    PubMed  CAS  Google Scholar 

  29. Rudland PS, Platt-Higgins A, El-Tanani M et al (2002) Prognostic significance of the metastasis-associated protein osteopontin in human breast cancer. Cancer Res 62(12):3417–3427

    PubMed  CAS  Google Scholar 

  30. Shevde LA, Das S, Clark DW et al (2010) Osteopontin: an effector and an effect of tumor metastasis. Curr Mol Med 10(1):71–81

    Article  PubMed  CAS  Google Scholar 

  31. Hedley BD, Welch DR, Allan AL et al (2008) Downregulation of osteopontin contributes to metastasis suppression by breast cancer metastasis suppressor 1. Int J Cancer 123(3):526–534

    Article  PubMed  CAS  Google Scholar 

  32. Adwan H, Bauerle T, Najajreh Y et al (2004) Decreased levels of osteopontin and bone sialoprotein II are correlated with reduced proliferation, colony formation, and migration of GFP-MDA-MB-231 cells. Int J Oncol 24(5):1235–1244

    PubMed  CAS  Google Scholar 

  33. Adwan H, Bauerle TJ, Berger MR (2004) Downregulation of osteopontin and bone sialoprotein II is related to reduced colony formation and metastasis formation of MDA-MB-231 human breast cancer cells. Cancer Gene Ther 11(2):109–120

    Article  PubMed  CAS  Google Scholar 

  34. Uccello M, Malaguarnera G, Vacante M et al (2011) Serum bone sialoprotein levels and bone metastases. J Cancer Res Ther 7(2):115–119

    Article  PubMed  CAS  Google Scholar 

  35. Bauerle T, Adwan H, Kiessling F et al (2005) Characterization of a rat model with site-specific bone metastasis induced by MDA-MB-231 breast cancer cells and its application to the effects of an antibody against bone sialoprotein. Int J Cancer 115(2):177–186

    Article  PubMed  Google Scholar 

  36. Weber GF (2001) The metastasis gene osteopontin: a candidate target for cancer therapy. Biochim Biophys Acta 1552(2):61–85

    PubMed  CAS  Google Scholar 

  37. Elazar V, Adwan H, Bauerle T et al (2010) Sustained delivery and efficacy of polymeric nanoparticles containing osteopontin and bone sialoprotein antisenses in rats with breast cancer bone metastasis. Int J Cancer 126(7):1749–1760

    PubMed  CAS  Google Scholar 

  38. Dias N, Stein CA (2002) Antisense oligonucleotides: basic concepts and mechanisms. Mol Cancer Ther 1(5):347–355

    PubMed  CAS  Google Scholar 

  39. Cohen-Sacks H, Najajreh Y, Tchaikovski V et al (2002) Novel PDGFbetaR antisense encapsulated in polymeric nanospheres for the treatment of restenosis. Gene Ther 9(23):1607–1616

    Article  PubMed  CAS  Google Scholar 

  40. Elazar V, Adwan H, Rohekar K et al (2010) Biodistribution of antisense nanoparticles in mammary carcinoma rat model. Drug Deliv 17(6):408–418

    Article  PubMed  CAS  Google Scholar 

  41. Park JS, Na K, Woo DG et al (2010) Non-viral gene delivery of DNA polyplexed with nanoparticles transfected into human mesenchymal stem cells. Biomaterials 31(1):124–132

    Article  PubMed  CAS  Google Scholar 

  42. Senger DR, Perruzzi CA (1996) Cell migration promoted by a potent GRGDS-containing thrombin-cleavage fragment of osteopontin. Biochim Biophys Acta 1314(1–2):13–24

    Article  PubMed  CAS  Google Scholar 

  43. Dahlgren C, Zhang HY, Du Q et al (2008) Analysis of siRNA specificity on targets with double-nucleotide mismatches. Nucleic Acids Res 36(9):e53

    Article  PubMed  Google Scholar 

  44. He J, Zhang JF, Yi C et al (2010) miRNA-mediated functional changes through co-regulating function related genes. PLoS ONE 5(10):e13558

    Article  PubMed  Google Scholar 

  45. Soldani C, Scovassi AI (2002) Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update. Apoptosis 7(4):321–328

    Article  PubMed  CAS  Google Scholar 

  46. Gross A, McDonnell JM, Korsmeyer SJ (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13(15):1899–1911

    Article  PubMed  CAS  Google Scholar 

  47. Duffy MJ, Maguire TM, Hill A et al (2000) Metalloproteinases: role in breast carcinogenesis, invasion and metastasis. Breast Cancer Res 2(4):252–257

    Article  PubMed  CAS  Google Scholar 

  48. Bauerle T, Peterschmitt J, Hilbig H et al (2006) Treatment of bone metastasis induced by MDA-MB-231 breast cancer cells with an antibody against bone sialoprotein. Int J Oncol 28(3):573–583

    PubMed  Google Scholar 

  49. Peterschmitt J, Bauerle T, Berger MR (2007) Effect of zoledronic acid and an antibody against bone sialoprotein II on MDA-MB-231(GFP) breast cancer cells in vitro and on osteolytic lesions induced in vivo by this cell line in nude rats. Clin Exp Metastasis 24(6):449–459

    Article  PubMed  CAS  Google Scholar 

  50. Bauerle T, Hilbig H, Bartling S et al (2008) Bevacizumab inhibits breast cancer-induced osteolysis, surrounding soft tissue metastasis, and angiogenesis in rats as visualized by VCT and MRI. Neoplasia 10(5):511–520

    PubMed  Google Scholar 

  51. Shevde LA, Samant RS, Paik JC et al (2006) Osteopontin knockdown suppresses tumorigenicity of human metastatic breast carcinoma, MDA-MB-435. Clin Exp Metastasis 23(2):123–133

    Article  PubMed  CAS  Google Scholar 

  52. Davis ME, Zuckerman JE, Choi CH et al (2010) Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464(7291):1067–1070

    Article  PubMed  CAS  Google Scholar 

  53. Lundberg BB (1997) Ether lipids enhance the cytotoxic effect of teniposide and paclitaxel in liposomes against leukaemic cells in culture. Anticancer Drug Des 12(6):503–513

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported in part by the BIO-DISC project, the Federal Ministry of Education and Research (Germany) and the Ministry of Science and Technology (Israel). In addition, CR and RLS were funded by the DKFZ-MOST program in cancer research.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Toxicology and Chemotherapy Unit, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120, Heidelberg, Germany

    Christina Reufsteck, Michael Zepp & Martin R. Berger

  2. Department of Pharmaceutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, POB 12065, 91120, Jerusalem, Israel

    Rinat Lifshitz-Shovali & Gershon Golomb

  3. Department of Medical Physics in Radiology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany

    Tobias Bäuerle

  4. Mechanisms of Biomolecular Interactions, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120, Heidelberg, Germany

    Dieter Kübler

Authors
  1. Christina Reufsteck
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rinat Lifshitz-Shovali
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael Zepp
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tobias Bäuerle
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dieter Kübler
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gershon Golomb
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Martin R. Berger
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Martin R. Berger.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 367 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Reufsteck, C., Lifshitz-Shovali, R., Zepp, M. et al. Silencing of skeletal metastasis-associated genes impairs migration of breast cancer cells and reduces osteolytic bone lesions. Clin Exp Metastasis 29, 441–456 (2012). https://doi.org/10.1007/s10585-012-9462-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10585-012-9462-8

Keywords

Search

Navigation