Tumor Biology

, Volume 31, Issue 3, pp 199–207 | Cite as

Calcineurin promotes proliferation, migration, and invasion of small cell lung cancer

  • Yan Liu
  • Ye Zhang
  • Jie Min
  • Li-Li Liu
  • Ning-Qiang Ma
  • Ying-Ming Feng
  • Dong Liu
  • Ping-zhong Wang
  • De-Dong Huang
  • Yan Zhuang
  • He-Long ZhangEmail author
Research Article


A novel role for calcineurin (Cn) has been reported recently regarding the oncogenic potential in pancreatic and colorectal cancer. The aim of this study was to investigate the putative causal role calcineurin could play in the development of lung cancer with bone metastases. We found that CnAα, an isoform of calcineurin, was significantly overexpressed in lung cancer tissues with bone metastasis as compared to tumors with non-bone metastases as investigated by RT-PCR. Strong nuclear staining of tumor cells was observed in small cell lung cancer tissues with bone metastasis. Conversely, cytoplasmic staining of tumor cells was observed in small cell lung cancer tissues with non-bone metastasis. Western blots of nuclear proteins from lung cancer tissues indicated that CnAα was highly expressed in lung cancer tissues with bone metastases, but not in those with non-bone metastases. In vitro, it was demonstrated that the CnAα gene obviously promoted cell proliferation and inhibited cell apotosis. The CnAα gene affected the cell cycle and promoted G1➝S transition in SBC-3 cells. Transfection with the CnAα gene promoted cell migration and invasion. These results indicated that CnAα may affect the biological behavior of the human small cell lung cancer cell line SBC-3 in vitro and may be a candidate tumor promotor gene for developing bone metastases.


Calcineurin Small cell lung cancer Bone metastasis Proliferation Migration Invasion 



This study was supported by National Natural Science Foundation of China (No. 30873028)


  1. 1.
    Cui R, Takahashi F, Ohashi R, Gu T, Yoshioka M, Nishio K, et al. Abrogation of the interaction between osteopontin and αvβ3 integrin reduces tumor growth of human lung cancer cells in mice. Lung Cancer. 2007;57:302–10.CrossRefPubMedGoogle Scholar
  2. 2.
    Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. Cancer statistics, CA. CA Cancer J Clin. 2008;58:71–96.CrossRefPubMedGoogle Scholar
  3. 3.
    Ogino H, Yano S, Kakiuchi S, Muguruma H, Ikuta K, Hanibuchi M, et al. follistatin suppresses the production of experimental multiple-organ metastasis by small cell lung cancer cells in natural killer cell-depleted SCID mice. Clin Cancer Res. 2008;14(3):660–7.CrossRefPubMedGoogle Scholar
  4. 4.
    Klee CB, Krinks MH. Purification of cyclic 3', 5'-nucleotide phosphodiesterase inhibitory protein by affinity chromatography on activator protein coupled to Sepharose. Biochemistry. 1978;17(1):120–6.CrossRefPubMedGoogle Scholar
  5. 5.
    Giri PR, Marietta CA, Higuchi S, Kincaid RL. Molecular and phylogenetic analysis of calmodulin-dependent protein phosphatase (calcineurin) catalytic subunit genes. DNA Cell Biol. 1992;11(5):415–24.CrossRefPubMedGoogle Scholar
  6. 6.
    Yokoyama N, Furuyama S, Wang JH. Demonstration of calmodulin-stimulated phosphatase isozymes by monoclonal antibodies. J Biol Chem. 1990;265(14):8170–5.PubMedGoogle Scholar
  7. 7.
    Muramatsu T, Giri PR, Higuchi S, Kincaid RL. Molecular cloning of a calmodulin-dependent phosphatase from murine testis: identification of a developmentally expressed nonneural isoenzyme. Proc Nati Acad Sci USA. 1992;89:529–33.CrossRefGoogle Scholar
  8. 8.
    Usuda N, Arai H, Sasaki H, Hanai T, Nagata T, Muramatsu T, et al. Differential subcellular localization of neural isoforms of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin) in central nervous system neurons: immunohistochemistry on formalin-fixed paraffin sections employing antigen retrieval by microwave irradiation. J Histochem Cytochem. 1996;44(1):13–8.PubMedGoogle Scholar
  9. 9.
    Mukai H, Chang CD, Tanaka H, Ito A, Kuno T, Tanaka C. cDNA cloning of a novel testis—specific calcineurin B-like protein. Biochem Biophys Res Commun. 1991;179(3):1325–30.CrossRefPubMedGoogle Scholar
  10. 10.
    Awumey EM, Moong BS, Sodam BR, Koval AP, Adebanjo OA, Kumegawa M, et al. Molecular and functional evidence for calcineurin-aα and β isoforms in the osteoclast; novel insights into cyclosporin A action on bone resorption. Biochem Biophys Res Commun. 1999;254:248–52.CrossRefPubMedGoogle Scholar
  11. 11.
    Sun L, Peng Y, Zaidi N, Zhu LL, Iqbal J, Yamoah K, et al. Evidence that calcineurin is required for the genesis of bone-resorbing osteoclasts. Am J Physiol Renal Physio. 2006;292:F285–91.CrossRefGoogle Scholar
  12. 12.
    Sun L, Moonga BS, Lu M, Zaidi N, Iqbal J, Blair HC, et al. Molecular cloning, expression, and function of osteo-clastic calcineurin Aα. Am J Physiol Physiol. 2003;285:F575–83.Google Scholar
  13. 13.
    Sun L, Blair HC, Peng Y, Zaidi N, Adebanjo OA, Wu XB, et al. Calcineurin regulates bone formation by the osteoblast. PNAS. 2005;102(47):17130–5.CrossRefPubMedGoogle Scholar
  14. 14.
    Yamamoto M, Suzuki Y, Kihira H, Miwa H, Kita K, Nagao M, et al. Expressions of four major protein Ser/Thr phosphatases in human primary leukemic cells. Leukemia. 1999;13(4):595–600.CrossRefPubMedGoogle Scholar
  15. 15.
    Eckstein LA, Van Quill KR, Bui SK, Uusitalo MS, O'Brien JM. Cyclosporin A inhibits calcineurin/nuclear factor of activated T-cells signaling and induces apoptosis in retinoblastoma cells. IOVS. 2005;46(3):782–90.Google Scholar
  16. 16.
    Buchholz M, Schatz A, Wagner M, Michl P, Linhart T, Adler G, et al. Overexpression of c-myc in pancreatic cancer caused by ectopic activation of NFATc1 and the Ca2+/ calcineurin signaling pathway. EMBO J. 2006;25(15):3714–24.CrossRefPubMedGoogle Scholar
  17. 17.
    Miki T, Yano S, Hanibuchi M, Sone S. Bone metastasis model with multiorgan dissemination of human small-cell lung cancer (SBC-5) cells in natural killer cell-depleted SCID mice. Oncol Res. 2000;12(5):209–17.PubMedGoogle Scholar
  18. 18.
    Kakiuchi S, Daigo Y, Tsunoda T, Yano S, Sone S, Nakamura Y. Genome-wide analysis of organ-preferential metastasis of human small cell lung cancer in mice. Mol Canc Res. 2003;1:485–99.Google Scholar
  19. 19.
    Jiang H, Wang PZ, Zhang Y, Xu Z, Sun L, Wang LM, et al. Hantaan virus induces toll-like receptor 4 expression, leading to enhanced production of beta interferon, interleukin-6 and tumor necrosis factor-alpha. Virology. 2008;380(1):52–9.CrossRefPubMedGoogle Scholar
  20. 20.
    Liu J, Farmer Jr JD, Lane WS, Friedman J, Weissman I, Schreiber SL. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 1991;66:807–15.CrossRefPubMedGoogle Scholar
  21. 21.
    McCaffrey PG, Perrino BA, Soderling TR. NFATp, a T lymphocyte DNA-binding protein that is a target for calcineurin and immunosuppressive drugs. J Biol Chem. 1993;268:3747–52.PubMedGoogle Scholar
  22. 22.
    Aperia A, Ibarra F, Svensson LB, Klee C, Greengard P. Calcineurin mediates alpha-adrenergic stimulation of Na+, K+-ATPase activity in renal tubule cells. Proc Natl Acad Sci. 1992;89:7394–7.CrossRefPubMedGoogle Scholar
  23. 23.
    Luan S, Li W, Rusnak F, Assmann SM, Schreiber SL. Immunosuppressants implicate protein phosphatase regulation of K+ channels in guard cells. Proc Natl Acad Sci. 1993;90:2202–6.CrossRefPubMedGoogle Scholar
  24. 24.
    Wang YL, Wang Y, Tong L. Overexpression of calcineurin B subunit (CnB) enhances the oncogenic potential of HEK293 cells. Cancer Sci. 2008;99(6):1100–8.CrossRefPubMedGoogle Scholar
  25. 25.
    Al Daraji WI, Grant KR, Ryan K, et al. Localization of calcineurin/NFAT in human skin and psoriasis and inhibition of calcineurin/NFAT activation in human keratinocytes by cyclosporin A. J Invest Dermatol. 2002;118:779–88.CrossRefGoogle Scholar
  26. 26.
    Al-Daraji WI, Grant KR, Ryan K, Saxton A, Reynolds NJ. Cyclosporine A inhibits colorectal cancer proliferation probably by regulating expression levels of c-Myc, p21WAF1/CIP1 and proliferating cell nuclear antigen. Cancer Lett. 2009;285:66–72.CrossRefGoogle Scholar
  27. 27.
    Terada N, Lucas JJ, Gelfand EW. Differential regulation of the tumor suppressor molecules, retinoblastoma susceptibility gene product (Rb) and p53, during cell cycle progression of normal human T cells. J Immunol. 1991;147:698–704.PubMedGoogle Scholar
  28. 28.
    Tomono M, Toyoshima K, Ito M, Amano H, Kiss Z. Inhibitors of calcineurin block expression of cyclins A and E induced by fibroblast growth factor in Swiss 3 T3 fibroblasts. Arch Biochem Biophys. 1998;353:374–8.CrossRefPubMedGoogle Scholar
  29. 29.
    Lipskaia L, Lompre AM. Alteration in temporal kinetics of Ca2+ signaling and control of growth and proliferation [J]. Biol Cell. 2004;96:55–68.CrossRefPubMedGoogle Scholar
  30. 30.
    Mosieniak G, Pyrzynska B, Kaminska B. Nuclear factor of activated T cells (NFAT) as a new component of the signal transduction pathway in glioma cells. J Neurochem. 1998;71:134–41.PubMedCrossRefGoogle Scholar
  31. 31.
    Amati B, Alevizopoulos K, Vlach J. Myc and the cell cycle. Front Biosci. 1998;3:d250–68.PubMedGoogle Scholar
  32. 32.
    Mizunuma M, Hirata D, Miyahara K, Tsuchiya E, Miyakawa T. Role of calcineurin and Mpk1 in regulating the onset of mitosis in budding yeast. Nature. 1998;392:303–6.CrossRefPubMedGoogle Scholar
  33. 33.
    Sugiura R, Sio SO, Shuntoh H, Kuno T. Molecular genetic analysis of the calcineurin signaling pathways. CMLS. 2001;58:278–88.CrossRefPubMedGoogle Scholar
  34. 34.
    Ryeom S, Baek KH, Rioth MJ, Lynch RC, Zaslavsky A, Birsner A, et al. Targeted deletion of the calcineurin inhibitor DSCR1 suppresses tumor growth. Cancer Cell. 2008;13:420–31.CrossRefPubMedGoogle Scholar
  35. 35.
    Wakabayashi K, Kambe F, Cao X, Murakami R, Mitsuyama H, Nagaya T, et al. Inhibitory effects of cyclosporin A on calcium mobilization-dependent interleukin-8 expression and invasive potential of human glioblastoma U251MG cells. Oncogene. 2004;23(41):6924–32.CrossRefPubMedGoogle Scholar
  36. 36.
    Du MR, Zhou WH, Dong L, Zhu XY, He YY, Yang JY, et al. Cyclosporin a promotes growth and invasiveness in vitro of human first-trimester trophoblast cells via MAPK3/MAPK1-mediated AP1 and Ca2t/Calcineurin/NFAT signaling pathways. Biol Reprod. 2008;78:1102–10.CrossRefPubMedGoogle Scholar
  37. 37.
    Miki T, Yano S, Hanibuchi M, Kanematsu T, Muguruma H, Sone S. Parathyroid hormone-related protein (PTHrP) is responsible for production of bone metastasis, but not visceral metastasis, by human small cell lung cancer SBC-5 cells in natural killer cell-depleted SCID mice. Int J Cancer. 2004;108:511–5.CrossRefPubMedGoogle Scholar
  38. 38.
    Roodman GD. Biology of osteoclast activation in cancer. J Clin Oncol. 2001;19(15):3562–71.PubMedGoogle Scholar
  39. 39.
    Johnson RA, Boyce BF, Mundy GR, Roodman GD. Tumors producing human tumor necrosis factor induced hypercalcemia and osteoclastic bone resorption in nude mice. Endocrinology. 1989;124:1424–7.CrossRefPubMedGoogle Scholar
  40. 40.
    Pfeilschifter J, Chenu C, Bird A, Mundy GR, Roodman GD. Interleukin-1 and tumor necrosis factor stimulate the formation of human osteoclast-like cells in vitro. J Bone Miner Res. 1989;4:113–8.CrossRefPubMedGoogle Scholar
  41. 41.
    Cozzolino F, Torcia M, Aldinucci D, Rubartelli A, Miliani A, Shaw AR, et al. Production of interleukin-1 by bone marrow myeloma cells. Blood. 1989;74:380–7.PubMedGoogle Scholar
  42. 42.
    Kurihara N, Bertolini D, Suda T, Akiyama Y, Roodman GD. IL-6 stimulates osteoclast-like multinucleated cell formation in long-term human marrow cultures by inducing IL-1 release. J Immunol. 1990;144:4226–30.PubMedGoogle Scholar
  43. 43.
    Black K, Garrett IR, Mundy GR. Chinese hamster ovarian cells transfected with the murine interleukin-6 gene cause hypercalcemia as well as cachexia, leukocytosis and thrombocytosis in tumor-bearing nude mice. Endocrinology. 1991;28:2657–9.CrossRefGoogle Scholar
  44. 44.
    Uy HL, Mundy GR, Boyce BF, Story BM, Dunstan CR, Yin JJ, et al. Tumor necrosis factor enhances parathyroid hormone-related protein- induced hypercalcemia and bone resorption without inhibiting bone formation in vivo. Cancer Res. 1997;57:3194–9.PubMedGoogle Scholar
  45. 45.
    Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 1998;93:165–76.CrossRefPubMedGoogle Scholar
  46. 46.
    Han JH, Choi SJ, Kurihara N, Koide M, Oba Y, Roodman GD. Macrophage inflammatory protein-1{alpha} is an osteoclastogenic factor in myeloma that is independent of receptor activator of nuclear factor {kappa}B ligand. Blood. 2001;97:3349–53.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2010

Authors and Affiliations

  • Yan Liu
    • 1
  • Ye Zhang
    • 2
  • Jie Min
    • 1
  • Li-Li Liu
    • 1
  • Ning-Qiang Ma
    • 1
  • Ying-Ming Feng
    • 1
  • Dong Liu
    • 1
  • Ping-zhong Wang
    • 2
  • De-Dong Huang
    • 2
  • Yan Zhuang
    • 2
  • He-Long Zhang
    • 1
    Email author
  1. 1.Department of Oncology, Tangdu HospitalFourth Military Medical UniversityXi’anChina
  2. 2.Center of Diagnosis and Treatment for Infectious Diseases, Tangdu HospitalThe Fourth Military Medical UniversityXi’anChina

Personalised recommendations