Breast Cancer Research and Treatment

, Volume 116, Issue 1, pp 91–102 | Cite as

Expression of TNF-α leader sequence renders MCF-7 tumor cells resistant to the cytotoxicity of soluble TNF-α

  • Dan Yan
  • Nalin Qin
  • Hailong Zhang
  • Tao Liu
  • Mingxia Yu
  • Xiaodan Jiang
  • Wei Feng
  • Jing Wang
  • Bingjiao Yin
  • Tao Zhang
  • Muxiang ZhouEmail author
  • Zhuoya LiEmail author
Preclinical Study


Transmembrane TNF-α (tmTNF-α) contains a leader sequence (LS) that can be phosphorylated and cleaved at its cytoplasmic portion, inducing IL-12 production. We observed that the breast cancer cell line MDA-MB-231 expressing transmembrane TNF-α (tmTNF-α) at high level was resistant to soluble TNF-α (sTNF-α)-induced cytotoxicity, accompanied by constitutive NF-κB activation. In contrast, MCF-7 cells expressing tmTNF-α at very low level were sensitive to sTNF-α-induced cell death and had no detectable NF-κB activation. Consistently, siRNA-mediated tmTNF-α knockdown blocked NF-κB activation and rendered MDA-MB-231 sensitive. To test our hypothesis that TNF-LS may play an important role in determining the sensitivity of tumor cells to sTNF-α, we stably transfected MCF-7 cells with TNF-LS. We found that transfection of TNF-LS or wild-type TNF-α containing LS constitutively activated NF-κB and conferred the cytotoxic resistance of MCF-7 cells, while transfection of a mutant tmTNF-α lacking the cytoplasmic segment of LS neither activated NF-κB nor affected the sensitivity. However, NF-κB inhibitor PDTC suppressed NF-κB activation and reconstituted sensitivity of TNF-LS/MCF-7 cells. To check whether TNF-LS is required to be cleaved or internalized for NF-κB activation to occur, we used signal peptide peptidase inhibitor (Z-LL)2-ketone and receptor internalization inhibitor MDC to treat cells. Interestingly, both inhibitors increased TNF-LS expression on the cell surface and enhanced NF-κB activation. These results indicate that membrane-anchored TNF-LS contributes to constitutive activation of NF-κB and resistance to sTNF-α-induced cell death. Therefore, TNF-LS appears to be responsible for tmTNF-α-induced resistance in the breast cancer cells.


Transmembrane TNF-α TNF-leader sequence Cytotoxicity NF-κB Breast cancer 



We thank Dr. Malcolm Kennedy for editing the manuscript. This work was supported by the National Natural Science Foundation of China (30670421), National Key Basic Research Program of China from the Ministry of Science and Technology of the People’s Republic of China (2004AA215162).


  1. 1.
    Kriegler M, Perez C, DeFay K, Albert I, Lu SD (1988) A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: ramifications for the complex physiology of TNF. Cell 53(1):45–53. doi: 10.1016/0092-8674(88)90486-2 PubMedCrossRefGoogle Scholar
  2. 2.
    Friedmann E, Hauben E, Maylandt K et al (2006) SPPL2a and SPPL2b promote intramembrane proteolysis of TNFalpha in activated dendritic cells to trigger IL-12 production. Nat Cell Biol 8(8):843–848. doi: 10.1038/ncb1440 PubMedCrossRefGoogle Scholar
  3. 3.
    Fluhrer R, Grammer G, Israel L et al (2006) A gamma-secretase-like intramembrane cleavage of TNFalpha by the GxGD aspartyl protease SPPL2b. Nat Cell Biol 8(8):894–896. doi: 10.1038/ncb1450 PubMedCrossRefGoogle Scholar
  4. 4.
    Yamaguchi M, Murakami T, Tomimatsu T et al (1998) Autocrine inhibition of leptin production by tumor necrosis factor-alpha (TNF-alpha) through TNF-alpha type-I receptor in vitro. Biochem Biophys Res Commun 244(1):30–34. doi: 10.1006/bbrc.1998.8199 PubMedCrossRefGoogle Scholar
  5. 5.
    Haas E, Grell M, Wajant H, Scheurich P (1999) Continuous autotropic signaling by membrane-expressed tumor necrosis factor. J Biol Chem 274(25):18107–18112. doi: 10.1074/jbc.274.25.18107 PubMedCrossRefGoogle Scholar
  6. 6.
    Domonkos A, Udvardy A, Laszlo L, Nagy T, Duda E (2001) Receptor-like properties of the 26 kDa transmembrane form of TNF. Eur Cytokine Netw 12(3):411–419PubMedGoogle Scholar
  7. 7.
    Cayabyab M, Phillips JH, Lanier LL (1994) CD40 preferentially costimulates activation of CD4 + T lymphocytes. J Immunol 152(4):1523–1531PubMedGoogle Scholar
  8. 8.
    Pollok KE, Kim YJ, Hurtado J, Zhou Z, Kim KK, Kwon BS (1994) 4-1BB T-cell antigen binds to mature B cells and macrophages, and costimulates anti-mu-primed splenic B cells. Eur J Immunol 24(2):367–374. doi: 10.1002/eji.1830240215 PubMedCrossRefGoogle Scholar
  9. 9.
    Stuber E, Neurath M, Calderhead D, Fell HP, Strober W (1995) Cross-linking of OX40 ligand, a member of the TNF/NGF cytokine family, induces proliferation and differentiation in murine splenic B cells. Immunity 2(5):507–521. doi: 10.1016/1074-7613(95)90031-4 PubMedCrossRefGoogle Scholar
  10. 10.
    van Essen D, Kikutani H, Gray D (1995) CD40 ligand-transduced co-stimulation of T cells in the development of helper function. Nature 378(6557):620–623. doi: 10.1038/378620a0 PubMedCrossRefGoogle Scholar
  11. 11.
    Wiley SR, Goodwin RG, Smith CA (1996) Reverse signaling via CD30 ligand. J Immunol 157(8):3635–3639PubMedGoogle Scholar
  12. 12.
    Harashima S, Horiuchi T, Hatta N et al (2001) Outside-to-inside signal through the membrane TNF-alpha induces E-selectin (CD62E) expression on activated human CD4 + T cells. J Immunol 166(1):130–136PubMedGoogle Scholar
  13. 13.
    Mitoma H, Horiuchi T, Hatta N et al (2005) Infliximab induces potent anti-inflammatory responses by outside-to-inside signals through transmembrane TNF-alpha. Gastroenterology 128(2):376–392. doi: 10.1053/j.gastro.2004.11.060 PubMedCrossRefGoogle Scholar
  14. 14.
    Higuchi M, Nagasawa K, Horiuchi T et al (1997) Membrane tumor necrosis factor-alpha (TNF-alpha) expressed on HTLV-I-infected T cells mediates a costimulatory signal for B cell activation—characterization of membrane TNF-alpha. Clin Immunol Immunopathol 82(2):133–140. doi: 10.1006/clin.1996.4291 PubMedCrossRefGoogle Scholar
  15. 15.
    Rossol M, Meusch U, Pierer M et al (2007) Interaction between transmembrane TNF and TNFR1/2 mediates the activation of monocytes by contact with T cells. J Immunol 179(6):4239–4248PubMedGoogle Scholar
  16. 16.
    Xin L, Wang J, Zhang H et al (2006) Dual regulation of soluble tumor necrosis factor-alpha induced activation of human monocytic cells via modulating transmembrane TNF-alpha-mediated ‘reverse signaling’. Int J Mol Med 18(5):885–892PubMedGoogle Scholar
  17. 17.
    Lugering A, Schmidt M, Lugering N, Pauels HG, Domschke W, Kucharzik T (2001) Infliximab induces apoptosis in monocytes from patients with chronic active Crohn’s disease by using a caspase-dependent pathway. Gastroenterology 121(5):1145–1157. doi: 10.1053/gast.2001.28702 PubMedCrossRefGoogle Scholar
  18. 18.
    ten Hove T, van Montfrans C, Peppelenbosch MP, van Deventer SJ (2002) Infliximab treatment induces apoptosis of lamina propria T lymphocytes in Crohn’s disease. Gut 50(2):206–211. doi: 10.1136/gut.50.2.206 PubMedCrossRefGoogle Scholar
  19. 19.
    Waetzig GH, Rosenstiel P, Arlt A et al (2005) Soluble tumor necrosis factor (TNF) receptor-1 induces apoptosis via reverse TNF signaling and autocrine transforming growth factor-beta1. FASEB J 19(1):91–93PubMedGoogle Scholar
  20. 20.
    Stevenson FT, Bursten SL, Locksley RM, Lovett DH (1992) Myristyl acylation of the tumor necrosis factor alpha precursor on specific lysine residues. J Exp Med 176(4):1053–1062. doi: 10.1084/jem.176.4.1053 PubMedCrossRefGoogle Scholar
  21. 21.
    Utsumi T, Takeshige T, Tanaka K et al (2001) Transmembrane TNF (pro-TNF) is palmitoylated. FEBS Lett 500(1–2):1–6. doi: 10.1016/S0014-5793(01)02576-5 PubMedCrossRefGoogle Scholar
  22. 22.
    Pocsik E, Duda E, Wallach D (1995) Phosphorylation of the 26 kDa TNF precursor in monocytic cells and in transfected HeLa cells. J Inflamm 45(3):152–160PubMedGoogle Scholar
  23. 23.
    Watts AD, Hunt NH, Wanigasekara Y et al (1999) A casein kinase I motif present in the cytoplasmic domain of members of the tumour necrosis factor ligand family is implicated in ‘reverse signalling’. EMBO J 18(8):2119–2126. doi: 10.1093/emboj/18.8.2119 PubMedCrossRefGoogle Scholar
  24. 24.
    Jin S, Lu D, Ye S et al (2005) A simplified probe preparation for ELISA-based NF-kappaB activity assay. J Biochem Biophys Methods 65(1):20–29. doi: 10.1016/j.jbbm.2005.08.006 PubMedCrossRefGoogle Scholar
  25. 25.
    Hasegawa T, Suzuki K, Sakamoto C et al (2003) Expression of the inhibitor of apoptosis (IAP) family members in human neutrophils: up-regulation of cIAP2 by granulocyte colony-stimulating factor and overexpression of cIAP2 in chronic neutrophilic leukemia. Blood 101(3):1164–1171. doi: 10.1182/blood-2002-05-1505 PubMedCrossRefGoogle Scholar
  26. 26.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif) 25(4):402–408Google Scholar
  27. 27.
    Himeno T, Watanabe N, Yamauchi N et al (1990) Expression of endogenous tumor necrosis factor as a protective protein against the cytotoxicity of exogenous tumor necrosis factor. Cancer Res 50(16):4941–4945PubMedGoogle Scholar
  28. 28.
    Dollbaum C, Creasey AA, Dairkee SH et al (1988) Specificity of tumor necrosis factor toxicity for human mammary carcinomas relative to normal mammary epithelium and correlation with response to doxorubicin. Proc Natl Acad Sci USA 85(13):4740–4744. doi: 10.1073/pnas.85.13.4740 PubMedCrossRefGoogle Scholar
  29. 29.
    Cai Z, Bettaieb A, Mahdani NE et al (1997) Alteration of the sphingomyelin/ceramide pathway is associated with resistance of human breast carcinoma MCF7 cells to tumor necrosis factor-alpha-mediated cytotoxicity. J Biol Chem 272(11):6918–6926. doi: 10.1074/jbc.272.11.6918 PubMedCrossRefGoogle Scholar
  30. 30.
    Luberto C, Hassler DF, Signorelli P et al (2002) Inhibition of tumor necrosis factor-induced cell death in MCF7 by a novel inhibitor of neutral sphingomyelinase. J Biol Chem 277(43):41128–41139. doi: 10.1074/jbc.M206747200 PubMedCrossRefGoogle Scholar
  31. 31.
    Cai Z, Capoulade C, Moyret-Lalle C et al (1997) Resistance of MCF7 human breast carcinoma cells to TNF-induced cell death is associated with loss of p53 function. Oncogene 15(23):2817–2826. doi: 10.1038/sj.onc.1201445 PubMedCrossRefGoogle Scholar
  32. 32.
    Stall AM, Kroese FG, Gadus FT, Sieckmann DG, Herzenberg LA, Herzenberg LA (1988) Rearrangement and expression of endogenous immunoglobulin genes occur in many murine B cells expressing transgenic membrane IgM. Proc Natl Acad Sci USA 85(10):3546–3550. doi: 10.1073/pnas.85.10.3546 PubMedCrossRefGoogle Scholar
  33. 33.
    Roy N, Deveraux QL, Takahashi R, Salvesen GS, Reed JC (1997) The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases. EMBO J 16(23):6914–6925. doi: 10.1093/emboj/16.23.6914 PubMedCrossRefGoogle Scholar
  34. 34.
    Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS Jr (1998) NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281(5383):1680–1683. doi: 10.1126/science.281.5383.1680 PubMedCrossRefGoogle Scholar
  35. 35.
    Benayoun B, Baghdiguian S, Lajmanovich A et al (2007) NF-{kappa}B-dependent expression of the antiapoptotic factor c-FLIP is regulated by calpain 3, the protein involved in limb-girdle muscular dystrophy type 2A. FASEB J 22(5):1521–1529PubMedCrossRefGoogle Scholar
  36. 36.
    Lwin T, Hazlehurst LA, Li Z et al (2007) Bone marrow stromal cells prevent apoptosis of lymphoma cells by upregulation of anti-apoptotic proteins associated with activation of NF-kappaB (RelB/p52) in non-Hodgkin’s lymphoma cells. Leukemia 21(7):1521–1531. doi: 10.1038/sj.leu.2404723 PubMedCrossRefGoogle Scholar
  37. 37.
    Schutze S, Machleidt T, Adam D et al (1999) Inhibition of receptor internalization by monodansylcadaverine selectively blocks p55 tumor necrosis factor receptor death domain signaling. J Biol Chem 274(15):10203–10212. doi: 10.1074/jbc.274.15.10203 PubMedCrossRefGoogle Scholar
  38. 38.
    Woo CH, Kim TH, Choi JA et al (2006) Inhibition of receptor internalization attenuates the TNFalpha-induced ROS generation in non-phagocytic cells. Biochem Biophys Res Commun 351(4):972–978. doi: 10.1016/j.bbrc.2006.10.154 PubMedCrossRefGoogle Scholar
  39. 39.
    Forster R, Kremmer E, Schubel A et al (1998) Intracellular and surface expression of the HIV-1 coreceptor CXCR4/fusin on various leukocyte subsets: rapid internalization and recycling upon activation. J Immunol 160(3):1522–1531PubMedGoogle Scholar
  40. 40.
    Micheau O, Tschopp J (2003) Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 114(2):181–190. doi: 10.1016/S0092-8674(03)00521-X PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2008

Authors and Affiliations

  • Dan Yan
    • 1
  • Nalin Qin
    • 1
  • Hailong Zhang
    • 1
  • Tao Liu
    • 1
  • Mingxia Yu
    • 1
  • Xiaodan Jiang
    • 1
  • Wei Feng
    • 1
  • Jing Wang
    • 1
  • Bingjiao Yin
    • 1
  • Tao Zhang
    • 1
  • Muxiang Zhou
    • 2
    Email author
  • Zhuoya Li
    • 1
    Email author
  1. 1.Department of Immunology, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanPeople’s Republic of China
  2. 2.Division of Pediatric Hematology/Oncology/BMTEmory University School of MedicineAtlantaUSA

Personalised recommendations