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Transient RNA silencing of tissue factor pathway inhibitor-2 modulates lung cancer cell invasion

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Abstract

Tissue Factor Pathway Inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates metalloproteinases (MMPs) involved in extracellular matrix (ECM) degradation. Its secretion in ECM makes TFPI-2 a potential inhibitor to regulate tumour invasion and metastasis. Moreover, TFPI-2 is frequently downregulated, particularly in aggressive cancers. In this study, we silenced TFPI-2 in the NCI-H460 non-small cell lung cancer cell line and evaluated the role of TFPI-2 in cell invasion and its impact on MMPs expression. As the effects of siRNA are transient, the consequences of both gene silencing and restoration to normal expression could be studied kinetically in the same cells. We showed that TFPI-2 expression by NCI-H460 cells was effectively downregulated using specific small interfering RNA and this silencing was associated with an increase in the invasive potential of tumour cells while migration was not affected. We also showed that mRNA levels and protein expression of MMP-2, -3, -9, -14 were not influenced by TFPI-2 silencing. Moreover, the gelatinase activity of MMP-2 and MMP-9 was unmodified. In contrast, MMP-1 mRNA levels and protein were significantly and similarly increased in cells transfected with TFPI-2 siRNA. In conclusion, this study confirms that TFPI-2 downregulation can contribute to tumour invasion of lung cancer cells.

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References

  1. Egeblad M, Werb Z (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2(3):161–174. doi:10.1038/nrc745

    Article  PubMed  CAS  Google Scholar 

  2. Stamenkovic I (2003) Extracellular matrix remodelling: the role of matrix metalloproteinases. J Pathol 200(4):448–464. doi:10.1002/path.1400

    Article  PubMed  CAS  Google Scholar 

  3. Vihinen P, Kahari VM (2002) Matrix metalloproteinases in cancer: prognostic markers and therapeutic targets. Int J Cancer 99(2):157–166. doi:10.1002/ijc.10329

    Article  PubMed  CAS  Google Scholar 

  4. Westermarck J, Kahari VM (1999) Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J 13(8):781–792

    PubMed  CAS  Google Scholar 

  5. Noel A, Jost M, Maquoi E (2008) Matrix metalloproteinases at cancer tumor-host interface. Semin Cell Dev Biol 19(1):52–60. doi:10.1016/j.semcdb.2007.05.011

    Article  PubMed  CAS  Google Scholar 

  6. Polette M, Nawrocki-Raby B, Gilles C et al (2004) Tumour invasion and matrix metalloproteinases. Crit Rev Oncol Hematol 49(3):179–186. doi:10.1016/j.critrevonc.2003.10.008

    Article  PubMed  Google Scholar 

  7. Bjorklund M, Koivunen E (2005) Gelatinase-mediated migration and invasion of cancer cells. Biochim Biophys Acta 1755(1):37–69

    PubMed  Google Scholar 

  8. Chand HS, Foster DC, Kisiel W (2005) Structure, function and biology of tissue factor pathway inhibitor-2. Thromb Haemost 94(6):1122–1130

    PubMed  CAS  Google Scholar 

  9. Petersen LC, Sprecher CA, Foster DC et al (1996) Inhibitory properties of a novel human Kunitz-type protease inhibitor homologous to tissue factor pathway inhibitor. Biochemistry 35(1):266–272. doi:10.1021/bi951501d

    Article  PubMed  CAS  Google Scholar 

  10. Rao CN, Mohanam S, Puppala A et al (1999) Regulation of ProMMP-1 and ProMMP-3 activation by tissue factor pathway inhibitor-2/matrix-associated serine protease inhibitor. Biochem Biophys Res Commun 255(1):94–98. doi:10.1006/bbrc.1999.0153

    Article  PubMed  CAS  Google Scholar 

  11. Iino M, Foster DC, Kisiel W (1998) Quantification and characterization of human endothelial cell-derived tissue factor pathway inhibitor-2. Arterioscler Thromb Vasc Biol 18(1):40–46

    PubMed  CAS  Google Scholar 

  12. Iochmann S, Reverdiau-Moalic P, Hube F et al (2002) Demonstration of inducible TFPI-2 mRNA synthesis in BeWo and JEG-3 trophoblast cells using a competitive RT-PCR. Thromb Res 105(3):217–223. doi:10.1016/S0049-3848(02)00018-X

    Article  PubMed  CAS  Google Scholar 

  13. Rao CN, Liu YY, Peavey CL et al (1995) Novel extracellular matrix-associated serine proteinase inhibitors from human skin fibroblasts. Arch Biochem Biophys 317(1):311–314. doi:10.1006/abbi.1995.1168

    Article  PubMed  CAS  Google Scholar 

  14. Ortego J, Escribano J, Coca-Prados M (1997) Gene expression of proteases and protease inhibitors in the human ciliary epithelium and ODM-2 cells. Exp Eye Res 65(2):289–299. doi:10.1006/exer.1997.0333

    Article  PubMed  CAS  Google Scholar 

  15. Crawley JT, Goulding DA, Ferreira V et al (2002) Expression and localization of tissue factor pathway inhibitor-2 in normal and atherosclerotic human vessels. Arterioscler Thromb Vasc Biol 22(2):218–224. doi:10.1161/hq0102.101842

    Article  PubMed  CAS  Google Scholar 

  16. Hube F, Reverdiau P, Iochmann S et al (2003) Demonstration of a tissue factor pathway inhibitor 2 messenger RNA synthesis by pure villous cytotrophoblast cells isolated from term human placentas. Biol Reprod 68(5):1888–1894. doi:10.1095/biolreprod.102.011858

    Article  PubMed  CAS  Google Scholar 

  17. Lanir N, Aharon A, Brenner B (2003) Procoagulant and anticoagulant mechanisms in human placenta. Semin Thromb Hemost 29(2):175–184. doi:10.1055/s-2003-38833

    Article  PubMed  CAS  Google Scholar 

  18. Konduri SD, Rao CN, Chandrasekar N et al (2001) A novel function of tissue factor pathway inhibitor-2 (TFPI-2) in human glioma invasion. Oncogene 20(47):6938–6945. doi:10.1038/sj.onc.1204847

    Article  PubMed  CAS  Google Scholar 

  19. Rao CN, Lakka SS, Kin Y et al (2001) Expression of tissue factor pathway inhibitor 2 inversely correlates during the progression of human gliomas. Clin Cancer Res 7(3):570–576

    PubMed  CAS  Google Scholar 

  20. Rollin J, Iochmann S, Blechet C et al (2005) Expression and methylation status of tissue factor pathway inhibitor-2 gene in non-small-cell lung cancer. Br J Cancer 92(4):775–783. doi:10.1038/sj.bjc.6602298

    Article  PubMed  CAS  Google Scholar 

  21. Wojtukiewicz MZ, Sierko E, Zimnoch L et al (2003) Immunohistochemical localization of tissue factor pathway inhibitor-2 in human tumor tissue. Thromb Haemost 90(1):140–146

    PubMed  CAS  Google Scholar 

  22. Jin M, Udagawa K, Miyagi E et al (2001) Expression of serine proteinase inhibitor PP5/TFPI-2/MSPI decreases the invasive potential of human choriocarcinoma cells in vitro and in vivo. Gynecol Oncol 83(2):325–333. doi:10.1006/gyno.2001.6394

    Article  PubMed  CAS  Google Scholar 

  23. Wong CM, Ng YL, Lee JM et al (2007) Tissue factor pathway inhibitor-2 as a frequently silenced tumor suppressor gene in hepatocellular carcinoma. Hepatology (Baltimore, Md.) 45(5):1129–1138. doi:10.1002/hep.21578

    CAS  Google Scholar 

  24. Guo H, Lin Y, Zhang H et al (2007) Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells. BMC Mol Biol 8:110. doi:10.1186/1471-2199-8-110

    Article  PubMed  Google Scholar 

  25. Hube F, Reverdiau P, Iochmann S et al (2003) Transcriptional silencing of the TFPI-2 gene by promoter hypermethylation in choriocarcinoma cells. Biol Chem 384(7):1029–1034. doi:10.1515/BC.2003.115

    Article  PubMed  CAS  Google Scholar 

  26. Konduri SD, Srivenugopal KS, Yanamandra N et al (2003) Promoter methylation and silencing of the tissue factor pathway inhibitor-2 (TFPI-2), a gene encoding an inhibitor of matrix metalloproteinases in human glioma cells. Oncogene 22(29):4509–4516. doi:10.1038/sj.onc.1206695

    Article  PubMed  CAS  Google Scholar 

  27. Nobeyama Y, Okochi-Takada E, Furuta J et al (2007) Silencing of tissue factor pathway inhibitor-2 gene in malignant melanomas. Int J Cancer 121(2):301–307. doi:10.1002/ijc.22637

    Article  PubMed  CAS  Google Scholar 

  28. Pulukuri SM, Patibandla S, Patel J et al (2007) Epigenetic inactivation of the tissue inhibitor of metalloproteinase-2 (TIMP-2) gene in human prostate tumors. Oncogene 26(36):5229–5237. doi:10.1038/sj.onc.1210329

    Article  PubMed  CAS  Google Scholar 

  29. Rao CN, Segawa T, Navari JR et al (2003) Methylation of TFPI-2 gene is not the sole cause of its silencing. Int J Oncol 22(4):843–848

    PubMed  CAS  Google Scholar 

  30. Sato N, Parker AR, Fukushima N et al (2005) Epigenetic inactivation of TFPI-2 as a common mechanism associated with growth and invasion of pancreatic ductal adenocarcinoma. Oncogene 24(5):850–858. doi:10.1038/sj.onc.1208050

    Article  PubMed  CAS  Google Scholar 

  31. Dykxhoorn DM, Novina CD, Sharp PA (2003) Killing the messenger: short RNAs that silence gene expression. Nature Reviews. Mol Cell Biol 4(6):457–467. doi:10.1038/nrm1129

    CAS  Google Scholar 

  32. Blindt R, Vogt F, Lamby D et al (2002) Characterization of differential gene expression in quiescent and invasive human arterial smooth muscle cells. J Vasc Res 39(4):340–352. doi:10.1159/000065546

    Article  PubMed  CAS  Google Scholar 

  33. Chabot V, Reverdiau P, Iochmann S et al (2006) CCL5-enhanced human immature dendritic cell migration through the basement membrane in vitro depends on matrix metalloproteinase-9. J Leukoc Biol 79(4):767–778. doi:10.1189/jlb.0804464

    Article  PubMed  CAS  Google Scholar 

  34. Pulukuri SM, Gorantla B, Rao JS (2007) Inhibition of histone deacetylase activity promotes invasion of human cancer cells through activation of urokinase plasminogen activator. J Biol Chem 282(49):35594–35603. doi:10.1074/jbc.M705867200

    Article  PubMed  CAS  Google Scholar 

  35. Kempaiah P, Chand HS, Kisiel W (2007) Identification of a human TFPI-2 splice variant that is upregulated in human tumor tissues. Mol Cancer 6:20. doi:10.1186/1476-4598-6-20

    Article  PubMed  Google Scholar 

  36. Aagaard L, Rossi JJ (2007) RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev 59(2–3):75–86. doi:10.1016/j.addr.2007.03.005

    Article  PubMed  CAS  Google Scholar 

  37. Montgomery MK, Xu S, Fire A (1998) RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc Natl Acad Sci USA 95(26):15502–15507. doi:10.1073/pnas.95.26.15502

    Article  PubMed  CAS  Google Scholar 

  38. Timmons L, Fire A (1998) Specific interference by ingested dsRNA. Nature 395(6705):854. doi:10.1038/27579

    Article  PubMed  CAS  Google Scholar 

  39. Achenbach TV, Brunner B, Heermeier K (2003) Oligonucleotide-based knockdown technologies: antisense versus RNA interference. Chem Bio Chem 4(10):928–935. doi:10.1002/cbic.200300708

    PubMed  CAS  Google Scholar 

  40. Bertrand JR, Pottier M, Vekris A et al (2002) Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem Biophys Res Commun 296(4):1000–1004. doi:10.1016/S0006-291X(02)02013-2

    Article  PubMed  CAS  Google Scholar 

  41. Bilanges B, Stokoe D (2005) Direct comparison of the specificity of gene silencing using antisense oligonucleotides and RNAi. Biochem J 388(Pt 2):573–583. doi:10.1042/BJ20041956

    PubMed  CAS  Google Scholar 

  42. Elbashir SM, Harborth J, Lendeckel W et al (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411(6836):494–498. doi:10.1038/35078107

    Article  PubMed  CAS  Google Scholar 

  43. Wun TC, Kretzmer KK, Girard TJ et al (1988) Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains. J Biol Chem 263(13):6001–6004

    PubMed  CAS  Google Scholar 

  44. Sprecher CA, Kisiel W, Mathewes S et al (1994) Molecular cloning, expression, and partial characterization of a second human tissue-factor-pathway inhibitor. Proc Natl Acad Sci USA 91(8):3353–3357. doi:10.1073/pnas.91.8.3353

    Article  PubMed  CAS  Google Scholar 

  45. Lakka SS, Konduri SD, Mohanam S et al (2000) In vitro modulation of human lung cancer cell line invasiveness by antisense cDNA of tissue factor pathway inhibitor-2. Clin Exp Metastasis 18(3):239–244. doi:10.1023/A:1006755223357

    Article  PubMed  CAS  Google Scholar 

  46. Konduri SD, Tasiou A, Chandrasekar N et al (2000) Role of tissue factor pathway inhibitor-2 (TFPI-2) in amelanotic melanoma (C-32) invasion. Clin Exp Metastasis 18(4):303–308. doi:10.1023/A:1011085820250

    Article  PubMed  CAS  Google Scholar 

  47. Konduri SD, Tasiou A, Chandrasekar N et al (2001) Overexpression of tissue factor pathway inhibitor-2 (TFPI-2), decreases the invasiveness of prostate cancer cells in vitro. Int J Oncol 18(1):127–131

    PubMed  CAS  Google Scholar 

  48. Yanamandra N, Kondraganti S, Gondi CS et al (2005) Recombinant adeno-associated virus (rAAV) expressing TFPI-2 inhibits invasion, angiogenesis and tumor growth in a human glioblastoma cell line. Int J Cancer 115(6):998–1005. doi:10.1002/ijc.20965

    Article  PubMed  CAS  Google Scholar 

  49. Kondraganti S, Gondi CS, Gujrati M et al (2006) Restoration of tissue factor pathway inhibitor inhibits invasion and tumor growth in vitro and in vivo in a malignant meningioma cell line. Int J Oncol 29(1):25–32

    PubMed  CAS  Google Scholar 

  50. Rao CN, Cook B, Liu Y et al (1998) HT-1080 fibrosarcoma cell matrix degradation and invasion are inhibited by the matrix-associated serine protease inhibitor TFPI-2/33 kDa MSPI. Int J Cancer 76(5):749–756. doi:10.1002/(SICI)1097-0215(19980529)76:5<749::AID-IJC21>3.0.CO;2-Y

    Article  PubMed  CAS  Google Scholar 

  51. Izumi H, Takahashi C, Oh J et al (2000) Tissue factor pathway inhibitor-2 suppresses the production of active matrix metalloproteinase-2 and is down-regulated in cells harboring activated ras oncogenes. FEBS Lett 481(1):31–36. doi:10.1016/S0014-5793(00)01902-5

    Article  PubMed  CAS  Google Scholar 

  52. Kong D, Ma D, Bai H et al (2004) Expression and characterization of the first kunitz domain of human tissue factor pathway inhibitor-2. Biochem Biophys Res Commun 324(4):1179–1185. doi:10.1016/j.bbrc.2004.09.179

    Article  PubMed  CAS  Google Scholar 

  53. Chand HS, Du X, Ma D et al (2004) The effect of human tissue factor pathway inhibitor-2 on the growth and metastasis of fibrosarcoma tumors in athymic mice. Blood 103(3):1069–1077. doi:10.1182/blood-2003-06-1930

    Article  PubMed  CAS  Google Scholar 

  54. Shibuya M, Okamoto H, Nozawa T et al (2007) Proteomic and transcriptomic analyses of retinal pigment epithelial cells exposed to REF-1/TFPI-2. Invest Ophthalmol Vis Sci 48(2):516–521. doi:10.1167/iovs.06-0434

    Article  PubMed  Google Scholar 

  55. Seiki M (2003) Membrane-type 1 matrix metalloproteinase: a key enzyme for tumor invasion. Cancer Lett 194(1):1–11. doi:10.1016/S0304-3835(02)00699-7

    Article  PubMed  CAS  Google Scholar 

  56. Sounni NE, Noel A (2005) Membrane type-matrix metalloproteinases and tumor progression. Biochimie 87(3–4):329–342. doi:10.1016/j.biochi.2004.07.012

    Article  PubMed  CAS  Google Scholar 

  57. Huntington JT, Shields JM, Der CJ et al (2004) Overexpression of collagenase 1 (MMP-1) is mediated by the ERK pathway in invasive melanoma cells: role of BRAF mutation and fibroblast growth factor signaling. J Biol Chem 279:33168–33176. doi:10.1074/jbc.M405102200

    Article  PubMed  CAS  Google Scholar 

  58. Pulukuri SM, Rao JS (2008) Matrix metalloproteinase-1 promotes prostate tumor growth and metastasis. Int J Oncol 32(4):757–765

    PubMed  CAS  Google Scholar 

  59. Sossey-Alaoui K, Ranalli TA, Li X et al (2005) WAVE3 promotes cell motility and invasion through the regulation of MMP-1, MMP-3, and MMP-9 expression. Exp Cell Res 308(1):135–145. doi:10.1016/j.yexcr.2005.04.011

    Article  PubMed  CAS  Google Scholar 

  60. Brinckerhoff CE, Rutter JL, Benbow U (2000) Interstitial collagenases as markers of tumor progression. Clin Cancer Res 6(12):4823–4830

    PubMed  CAS  Google Scholar 

  61. Cheng S, Tada M, Hida Y et al (2008) High MMP-1 mRNA Expression is a Risk Factor for Disease-Free and Overall Survivals in Patients with Invasive Breast Carcinoma. J Surg Res 146(1):104–109. doi:10.1016/j.jss.2007.05.032

    Article  PubMed  CAS  Google Scholar 

  62. Blackburn JS, Rhodes CH, Coon CI et al (2007) RNA interference inhibition of matrix metalloproteinase-1 prevents melanoma metastasis by reducing tumor collagenase activity and angiogenesis. Cancer Res 67(22):10849–10858. doi:10.1158/0008-5472.CAN-07-1791

    Article  PubMed  CAS  Google Scholar 

  63. Jiang X, Dutton CM, Qi WN et al (2005) siRNA mediated inhibition of MMP-1 reduces invasive potential of a human chondrosarcoma cell line. J Cell Physiol 202(3):723–730. doi:10.1002/jcp.20162

    Article  PubMed  CAS  Google Scholar 

  64. George J, Gondi CS, Dinh DH et al (2007) Restoration of tissue factor pathway inhibitor-2 in a human glioblastoma cell line triggers caspase-mediated pathway and apoptosis. Clin Cancer Res 13(12):3507–3517. doi:10.1158/1078-0432.CCR-06-3023

    Article  PubMed  CAS  Google Scholar 

  65. Tasiou A, Konduri SD, Yanamandra N et al (2001) A novel role of tissue factor pathway inhibitor-2 in apoptosis of malignant human gliomas. Int J Oncol 19(3):591–597

    PubMed  CAS  Google Scholar 

  66. Ivanciu L, Gerard RD, Tang H et al (2007) Adenovirus-mediated expression of tissue factor pathway inhibitor-2 inhibits endothelial cell migration and angiogenesis. Arterioscler Thromb Vasc Biol 27(2):310–316. doi:10.1161/01.ATV.0000254147.89321.cf

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We are grateful to Walter Kisiel (Department of Pathology, University of New Mexico, Health Sciences Centre, Albuquerque, NM, USA) for kindly providing the anti-TFPI-2 rabbit polyclonal antibody. We also thank Mrs Doreen Raine for editing the English text. This study was supported by the «Ligue Contre le Cancer», the «Association pour la Recherche sur le Cancer», and the «Institut pour la Recherche sur la Thrombose et l’Hémostase».

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Authors and Affiliations

  1. Inserm U618, Protéases et Vectorisation Pulmonaires, Faculté de Médecine, Université François Rabelais, IFR 135, 10 Boulevard Tonnellé, 37032, Tours Cedex, France

    Sophie Iochmann, Claire Bléchet, Annabelle Saulnier, Aniça Amini, Guillaume Gaud, Yves Gruel & Pascale Reverdiau

  2. Service d’Hématologie-Hémostase, CHRU Trousseau, 37044, Tours, France

    Sophie Iochmann, Yves Gruel & Pascale Reverdiau

  3. Service d’Anatomie Pathologique, CHRU Bretonneau, 37032, Tours, France

    Claire Bléchet

  4. Etablissement Français du Sang Centre-Atlantique, 37032, Tours, France

    Valérie Chabot

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  1. Sophie Iochmann
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Iochmann, S., Bléchet, C., Chabot, V. et al. Transient RNA silencing of tissue factor pathway inhibitor-2 modulates lung cancer cell invasion. Clin Exp Metastasis 26, 457–467 (2009). https://doi.org/10.1007/s10585-009-9245-z

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