Abstract
The invasion of tumor cells into brain tissue is a pathologic hallmark of malignant gliomas and contributes to treatment failures. Diffuse glioblastomas contain numerous microglial cells, which enhance the progression of gliomas; however, factors responsible for invasion-promoting role of microglia are unknown. Transforming growth factor-β (TGF-β) can enhance tumor growth, invasion, angiogenesis and immunosuppression. Antagonizing TGF-β activity has been shown to inhibit tumor invasion in vitro and tumorigenicity, but a systemic inhibition or lack of TGF-β signaling results in acute inflammation and disruption of immune system homeostasis. We developed plasmid-transcribed small hairpin RNAs (shRNAs) to downregulate the TGF-β type II receptor (TβIIR) expression, which effectively inhibited cytokine-induced signaling pathways and transcriptional responses in transiently transfected human glioblastoma cells. Silencing of TβIIR abolished TGF-β-induced glioblastoma invasiveness and migratory responses in vitro. Moreover, tumorigenicity of glioblastoma cells stably expressing TβIIR shRNAs in nude mice was reduced by 50%. Microglia strongly enhanced glioma invasiveness in the co-culture system, but this invasion-promoting activity was lost in glioma cells stably expressing shTβRII, indicating a crucial role of microglia-derived TGF-β in tumor–host interactions. Our results demonstrate a successful targeting of TGF-β-dependent invasiveness and tumorigenicity of glioblastoma cells by RNAi-mediated gene silencing.
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References
Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, Kozlowski JM et al. (1987). A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 47: 3239–3245.
Attisano L, Wrana JL . (1998). Mads and Smads in TGF beta signalling. Curr Opin Cell Biol 10: 188–194.
Badie B, Schartner J . (2001). Role of microglia in glioma biology. Microsc Res Tech 54: 106–113.
Brummelkamp TR, Bernards R, Agami R . (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550–553.
Ciechomska I, Kazmierczak P, Pyrzynska B, Kaminska B . (2003). Inhibition of Akt kinase signalling and activation of Forkhead are indispensable for upregulation of FasL expression in apoptosis of glioma cells. Oncogene 22: 7617–7627.
DaCosta Byfield S, Major C, Laping NJ, Roberts AB . (2004). SB-505124 is a selective inhibitor of transforming growth factor-beta type I receptors ALK4, ALK5, and ALK7. Mol Pharmacol 65: 744–752.
Deckers M, van Dinther M, Buijs J, Que I, Lowik C, van der Pluijm G et al. (2006). The tumor suppressor Smad4 is required for transforming growth factor beta-induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells. Cancer Res 66: 2202–2209.
Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM . (1998). Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J 17: 3091–3100.
Derynck R, Zhang YE . (2003). Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 425: 577–584.
Derynck R, Zhang Y, Feng XH . (1998). Smads: transcriptional activators of TGF-beta responses. Cell 95: 737–740.
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T . (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–498.
Friese MA, Wischhusen J, Wick W, Weiler M, Eisele G, Steinle A et al. (2004). RNA interference targeting transforming growth factor-beta enhances NKG2D-mediated antiglioma immune response, inhibits glioma cell migration and invasiveness, and abrogates tumorigenicity in vivo. Cancer Res 64: 7596–7603.
Gold LI . (1999). The role for transforming growth factor-beta. (TGF-beta) in human cancer. Crit Rev Oncog 10: 303–360.
Heldin CH, Miyazono K, ten Dijke P . (1997). TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 390: 465–471.
Hjelmeland MD, Hjelmeland AB, Sathornsumetee S, Reese ED, Herbstreith MH, Laping NJ et al. (2004). SB-431542, a small molecule transforming growth factor-beta-receptor antagonist, inhibits human glioma cell line proliferation and motility. Mol Cancer Ther 3: 737–745.
Hommel JD, Sears RM, Georgescu D, Simmons DL, DiLeone RJ . (2003). Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med 9: 1539–1544.
Jazag A, Kanai F, Ijichi H, Tateishi K, Ikenoue T, Tanaka Y et al. (2005). Single small-interfering RNA expression vector for silencing multiple transforming growth factor-beta pathway components. Nucleic Acids Res 33: e131.
Jennings MT, Pietenpol JA . (1998). The role of transforming growth factor beta in glioma progression. J Neurooncol 36: 123–140.
Keeton MR, Curriden SA, van Zonneveld AJ, Loskutoff DJ . (1991). Identification of regulatory sequences in the type 1 plasminogen activator inhibitor gene responsive to transforming growth factor beta. J Biol Chem 266: 23048–23052.
Kiefer R, Supler ML, Toyka KV, Streit WJ . (1994). In situ detection of transforming growth factor-beta mRNA in experimental rat glioma and reactive glial cells. Neurosci Lett 166: 161–164.
Kjellman C, Olofsson SP, Hansson O, Von Schantz T, Lindvall M, Nilsson I et al. (2000). Expression of TGF-beta isoforms, TGF-beta receptors, and SMAD molecules at different stages of human glioma. Int J Cancer 89: 251–258.
Kulkarni AB, Huh CG, Becker D, Geiser A, Lyght M, Flanders KC et al. (1993). Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death. Proc Natl Acad Sci USA 90: 770–774.
Lakka SS, Gondi CS, Yanamandra N, Olivero WC, Dinh DH, Gujrati M et al. (2004). Inhibition of cathepsin B and MMP-9 gene expression in glioblastoma cell line via RNA interference reduces tumor cell invasion, tumor growth and angiogenesis. Oncogene 23: 4681–4689.
Lehrmann E, Kiefer R, Christensen T, Toyka KV, Zimmer J, Diemer NH et al. (1998). Microglia and macrophages are major sources of locally produced transforming growth factor-beta1 after transient middle cerebral artery occlusion in rats. Glia 24: 437–448.
Li MO, Sanjabi S, Flavell RA . (2006). Transforming growth factor-beta controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and -independent mechanisms. Immunity 25: 455–471.
Lindholm D, Castren E, Kiefer R, Zafra F, Thoenen H . (1992). Transforming growth factor-beta 1 in the rat brain: increase after injury and inhibition of astrocyte proliferation. J Cell Biol 117: 395–400.
Mantovani A, Schioppa T, Porta C, Allavena P, Sica A . (2006). Role of tumor-associated macrophages in tumor progression and invasion. Cancer Metastasis Rev 25: 315–322.
Marie JC, Liggitt D, Rudensky AY . (2006). Cellular mechanisms of fatal early-onset autoimmunity in mice with the T cell-specific targeting of transforming growth factor-beta receptor. Immunity 25: 441–454.
Markovic DS, Glass R, Synowitz M, Rooijen N, Kettenmann H . (2005). Microglia stimulate the invasiveness of glioma cells by increasing the activity of metalloprotease-2. J Neuropathol Exp Neurol 64: 754–762.
Muraoka RS, Dumont N, Ritter CA, Dugger TC, Brantley DM, Chen J et al. (2002). Blockade of TGF-beta inhibits mammary tumor cell viability, migration, and metastases. J Clin Invest 109: 1551–1559.
Nakano A, Tani E, Miyazaki K, Yamamoto Y, Furuyama J . (1995). Matrix metalloproteinases and tissue inhibitors of metalloproteinases in human gliomas. J Neurosurg 83: 298–307.
Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E et al. (1997). TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4. EMBO J 16: 5353–5362.
Oft M, Heider KH, Beug H . (1998). TGFbeta signaling is necessary for carcinoma cell invasiveness and metastasis. Curr Biol 8: 1243–1252.
Ogorelkova M, Zwaagstra J, Elahi SM, Dias C, Guilbaut C, Lo R et al. (2006). Adenovirus-delivered antisense RNA and shRNA exhibit different silencing efficiencies for the endogenous transforming growth factor-beta. (TGF-beta) type II receptor. Oligonucleotides 16: 2–14.
Ohgaki H, Kleihues P . (2005). Epidemiology and etiology of gliomas. Acta Neuropathol (Berl) 109: 93–108.
Pardridge WM . (2004). Intravenous, non-viral RNAi gene therapy of brain cancer. Expert Opin Biol Ther 4: 1103–1113.
Piek E, Westermark U, Kastemar M, Heldin CH, van Zoelen EJ, Nister M et al. (1999). Expression of transforming-growth-factor. (TGF)-beta receptors and Smad proteins in glioblastoma cell lines with distinct responses to TGF-beta1. Int J Cancer 80: 756–763.
Platten M, Wick W, Weller M . (2001). Malignant glioma biology: role for TGF-beta in growth, motility, angiogenesis, and immune escape. Microsc Res Tech 52: 401–410.
Pollard JW . (2004). Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer 4: 71–78.
Praus M, Wauterickx K, Collen D, Gerard RD . (1999). Reduction of tumor cell migration and metastasis by adenoviral gene transfer of plasminogen activator inhibitors. Gene Ther 6: 227–236.
Rao JS . (2003). Molecular mechanisms of glioma invasiveness: the role of proteases. Nat Rev Cancer 3: 489–501.
Schlingensiepen KH, Schlingensiepen R, Steinbrecher A, Hau P, Bogdahn U, Fischer-Blass B et al. (2006). Targeted tumor therapy with the TGF-beta2 antisense compound AP 12009. Cytokine Growth Factor Rev 17: 129–139.
Shah AH, Tabayoyong WB, Kundu SD, Kim SJ, Van Parijs L, Liu VC et al. (2002). Suppression of tumor metastasis by blockade of transforming growth factor beta signaling in bone marrow cells through a retroviral-mediated gene therapy in mice. Cancer Res 62: 7135–7138.
Shull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M et al. (1992). Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature 359: 693–699.
Sliwa M, Markovic D, Gabrusiewicz K, Synowitz M, Glass R, Zawadzka M et al. (2007). The invasion promoting effect of microglia on glioblastoma cells is inhibited by cyclosporin A. Brain 130: 476–489.
Song CZ, Siok TE, Gelehrter TD . (1998). Smad4/DPC4 and Smad3 mediate transforming growth factor-beta. (TGF-beta) signaling through direct binding to a novel TGF-beta-responsive element in the human plasminogen activator inhibitor-1 promoter. J Biol Chem 273: 29287–29290.
Teicher BA . (2001). Malignant cells, directors of the malignant process: role of transforming growth factor-beta. Cancer Metastasis Rev 20: 133–143.
Uhl M, Aulwurm S, Wischhusen J, Weiler M, Ma JY, Almirez R et al. (2004). SD-208, a novel transforming growth factor beta receptor I kinase inhibitor, inhibits growth and invasiveness and enhances immunogenicity of murine and human glioma cells in vitro and in vivo. Cancer Res 64: 7954–7961.
Watters JJ, Schartner JM, Badie B . (2005). Microglia function in brain tumors. J Neurosci Res 81: 447–455.
Westerhausen Jr DR, Hopkins WE, Billadello JJ . (1991). Multiple transforming growth factor-beta-inducible elements regulate expression of the plasminogen activator inhibitor type-1 gene in Hep G2 cells. J Biol Chem 266: 1092–1100.
Wick W, Platten M, Weller M . (2001). Glioma cell invasion: regulation of metalloproteinase activity by TGF-beta. J Neurooncol 53: 177–185.
Won J, Kim H, Park EJ, Hong Y, Kim SJ, Yun Y . (1999). Tumorigenicity of mouse thymoma is suppressed by soluble type II transforming growth factor beta receptor therapy. Cancer Res 59: 1273–1277.
Yamada N, Kato M, Yamashita H, Nister M, Miyazono K, Heldin CH et al. (1995). Enhanced expression of transforming growth factor-beta and its type-I and type-II receptors in human glioblastoma. Int J Cancer 62: 386–392.
Yamamoto M, Sawaya R, Mohanam S, Loskutoff DJ, Bruner JM, Rao VH et al. (1994). Expression and cellular localization of messenger RNA for plasminogen activator inhibitor type 1 in human astrocytomas in vivo. Cancer Res 54: 3329–3332.
Yang X, Letterio JJ, Lechleider RJ, Chen L, Hayman R, Gu H et al. (1999). Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta. EMBO J 18: 1280–1291.
Ye L, Zhang H, Zhang L, Yang G, Ke Q, Guo H et al. (2006). Effects of RNAi-mediated Smad4 silencing on growth and apoptosis of human rhabdomyosarcoma cells. Int J Oncol 29: 1149–1157.
Yingling JM, Blanchard KL, Sawyer JS . (2004). Development of TGF-beta signalling inhibitors for cancer therapy. Nat Rev Drug Discov 3: 1011–1022.
Yu L, Hebert MC, Zhang YE . (2002). TGF-beta receptor-activated p38 MAP kinase mediates Smad-independent TGF-beta responses. EMBO J 21: 3749–3759.
Zamore PD, Tuschl T, Sharp PA, Bartel DP . (2000). RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101: 25–33.
Zawadzka M, Kaminska B . (2005). A novel mechanism of FK506-mediated neuroprotection: downregulation of cytokine expression in glial cells. Glia 49: 36–51.
Zupanska A, Dziembowska M, Ellert-Miklaszewska A, Gaweds-Walenych K, Kamlnska B . (2005). Cyclosporene A induces growth arrest or programmed cell death of human glioma cells. Neurochem Int 47: 430–441.
Acknowledgements
This work was supported by Grant PBZ-MIN-107-/P04/2004 from the Ministry of Science and Higher Education (BK). Aleksandra Wesolowska is a recipient of a scholarship from the Postgraduate School of Molecular Medicine. We thank Jolanta Zegarska (Department of Immunology, Medical University of Warsaw) for technical assistance with a real-time PCR and Dr Sylwia Szymczak for editorial assistance.
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Wesolowska, A., Kwiatkowska, A., Slomnicki, L. et al. Microglia-derived TGF-β as an important regulator of glioblastoma invasion—an inhibition of TGF-β-dependent effects by shRNA against human TGF-β type II receptor. Oncogene 27, 918–930 (2008). https://doi.org/10.1038/sj.onc.1210683
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DOI: https://doi.org/10.1038/sj.onc.1210683
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