Clinical & Experimental Metastasis

, Volume 26, Issue 6, pp 517–526 | Cite as

Regulator of calcineurin 1 modulates cancer cell migration in vitro

  • Allan V. Espinosa
  • Motoo Shinohara
  • Leonardo M. Porchia
  • Yun Jae Chung
  • Samantha McCarty
  • Motoyasu Saji
  • Matthew D. Ringel
Research Paper

Abstract

Metastasis suppressors and other regulators of cell motility play an important role in tumor invasion and metastases. We previously identified that activation of the G protein coupled receptor 54 (GPR54) by the metastasis suppressor metastin inhibits cell migration in association with overexpression of Regulator of calcineurin 1 (RCAN1), an endogenous regulator of calcineurin. Calcineurin inhibitors also blocked cell migration in vitro and RCAN1 protein levels were reduced in nodal metastases in thyroid cancer. The purpose of the current study was to determine directly if RCAN1 functions as a motility suppressor in vitro. Several cancer cell lines derived from different cancer types with different motility rates were evaluated for RCAN1 expression levels. Using these systems we determined that reduction of endogenous RCAN1 using siRNA resulted in an increase in cancer cell motility while expression of exogenous RCAN1 reduced cell motility. In one cell line with a high migratory rate, the stability of exogenously expressed RCAN1 protein was reduced and was rescued by treatment with a proteasome inhibitor. Finally, overexpression of RCAN1 was associated with an increase in cell adhesion to collagen IV and reduced calcineurin activity. In summary, we have demonstrated that the expression of exogenous RCAN1 reduces migration and alters adhesion; and that the loss of endogenous RCAN1 leads to an increase in migration in the examined cancer cell lines. These results are consistent with a regulatory role for RCAN1 in cancer cell motility in vitro.

Keywords

Adhesion Calcineurin GPR54 KiSS1 Metastases Metastin Motility Thyroid Cancer 

Abbreviations

RCAN1

Regulator of calcineurin 1

NFAT

Nuclear factor of activated T-cells

FTC

Follicular thyroid cancer

GPR54

G protein coupled receptor 54

PBS

Phosphate-buffered saline

TBS

Tris-buffered saline

References

  1. 1.
    Chiang AC, Massague J (2008) Molecular basis of metastasis. N Engl J Med 359(26):2814–2823. doi:10.1056/NEJMra0805239 PubMedCrossRefGoogle Scholar
  2. 2.
    Chambers AF, Groom AC, MacDonald IC (2002) Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2(8):563–572. doi:10.1038/nrc865 PubMedCrossRefGoogle Scholar
  3. 3.
    Ramaswamy S, Ross KN, Lander ES et al (2003) A molecular signature of metastasis in primary solid tumors. Nat Genet 33(1):49–54. doi:10.1038/ng1060 PubMedCrossRefGoogle Scholar
  4. 4.
    Podsypanina K, Du YC, Jechlinger M et al (2008) Seeding and propagation of untransformed mouse mammary cells in the lung. Science 321(5897):1841–1844. doi:10.1126/science.1161621 PubMedCrossRefGoogle Scholar
  5. 5.
    Shevde LA, Welch DR (2003) Metastasis suppressor pathways-an evolving paradigm. Cancer Lett 198(1):1–20. doi:10.1016/S0304-3835(03)00304-5 PubMedCrossRefGoogle Scholar
  6. 6.
    Ohtake T, Shintani Y, Honda S et al (2001) Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 411(6787):613–617. doi:10.1038/35079135 CrossRefGoogle Scholar
  7. 7.
    Kotani M, Detheux M, Vandenbogaerde A et al (2001) The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 276(37):34631–34636. doi:10.1074/jbc.M104847200 PubMedCrossRefGoogle Scholar
  8. 8.
    Muir AI, Chamberlain L, Elshourbagy NA et al (2001) AXOR12: a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 276(31):28969–28975. doi:10.1074/jbc.M102743200 PubMedCrossRefGoogle Scholar
  9. 9.
    Lee JH, Miele ME, Hicks DJ et al (1996) KiSS-1, a novel human malignant melanoma metastasis-suppressor gene. J Natl Cancer Inst 88(23):1731–1737. doi:10.1093/jnci/88.23.1731 PubMedCrossRefGoogle Scholar
  10. 10.
    Ringel MD, Hardy E, Bernet VJ et al (2002) Metastin receptor is overexpressed in papillary thyroid cancer and activates MAP kinase in thyroid cancer cells. J Clin Endocrinol Metab 87(5):2399–2402. doi:10.1210/jc.87.5.2399 PubMedCrossRefGoogle Scholar
  11. 11.
    Ikeguchi M, Yamaguchi K, Kaibara N (2004) Clinical significance of the loss of KiSS-1 and orphan G-Protein-coupled receptor (hOT7T175) gene expression in esophageal squamous cell carcinoma. Clin Cancer Res 10(4):1379–1383. doi:10.1158/1078-0432.CCR-1519-02 PubMedCrossRefGoogle Scholar
  12. 12.
    Dhar DK, Naora H, Kubota H et al (2004) Downregulation of KiSS-1 expression is responsible for tumor invasion and worse prognosis in gastric carcinoma. Int J Cancer 111(6):868–872. doi:10.1002/ijc.20357 PubMedCrossRefGoogle Scholar
  13. 13.
    Hori A, Honda S, Asada M et al (2001) Metastin suppresses the motility and growth of CHO cells transfected with its receptor. Biochem Biophys Res Commun 286(5):958–963. doi:10.1006/bbrc.2001.5470 PubMedCrossRefGoogle Scholar
  14. 14.
    Shirasaki F, Takata M, Hatta N et al (2001) Loss of expression of the metastasis suppressor gene KiSS1 during melanoma progression and its association with LOH of chromosome 6q16.3–q23. Cancer Res 61(20):7422–7425PubMedGoogle Scholar
  15. 15.
    Jiang Y, Berk M, Singh LS et al (2005) KiSS1 suppresses metastasis in human ovarian cancer via inhibition of protein Kinase C Alpha. Clin Exp Metastasis 22(5):369–376. doi:10.1007/s10585-005-8186-4 PubMedCrossRefGoogle Scholar
  16. 16.
    Nash KT, Phadke PA, Navenot J-M et al (2007) Requirement of KISS1 secretion for multiple organ metastasis suppression and maintenance of tumor dormancy. J Natl Cancer Inst 99(4):309–321. doi:10.1093/jnci/djk053 PubMedCrossRefGoogle Scholar
  17. 17.
    Stathatos N, Bourdeau I, Espinosa AV et al (2005) KiSS-1/G protein-coupled receptor 54 metastasis suppressor pathway increases myocyte-enriched calcineurin interacting protein 1 expression and chronically inhibits calcineurin activity. J Clin Endocrinol Metab 90(9):5432–5440. doi:10.1210/jc.2005-0963 PubMedCrossRefGoogle Scholar
  18. 18.
    Fuentes JJ, Pritchard MA, Estivill X (1997) Genomic organization, alternative splicing, and expression patterns of the DSCR1 (Down syndrome candidate region 1) gene. Genomics 44(3):358–361. doi:10.1006/geno.1997.4866 PubMedCrossRefGoogle Scholar
  19. 19.
    Rothermel B, Vega RB, Yang J et al (2000) A protein encoded within the Down syndrome critical region is enriched in striated muscles and inhibits calcineurin signaling. J Biol Chem 275(12):8719–8725. doi:10.1074/jbc.275.12.8719 PubMedCrossRefGoogle Scholar
  20. 20.
    Vega RB, Rothermel BA, Weinheimer CJ et al (2003) Dual roles of modulatory calcineurin-interacting protein 1 in cardiac hypertrophy. Proc Natl Acad Sci USA 100(2):669–674. doi:10.1073/pnas.0237225100 PubMedCrossRefGoogle Scholar
  21. 21.
    van Rooij E, Doevendans PA, Crijns HJ et al (2004) MCIP1 overexpression suppresses left ventricular remodeling and sustains cardiac function after myocardial infarction. Circ Res 94(3):e18–e26. doi:10.1161/01.RES.0000118597.54416.00 PubMedCrossRefGoogle Scholar
  22. 22.
    Vega RB, Yang J, Rothermel BA et al (2002) Multiple domains of MCIP1 contribute to inhibition of calcineurin activity. J Biol Chem 277(33):30401–30407. doi:10.1074/jbc.M200123200 PubMedCrossRefGoogle Scholar
  23. 23.
    Abbasi S, Lee J-D, Su B et al (2006) Protein kinase-mediated regulation of calcineurin through the phosphorylation of modulatory calcineurin-interacting protein 1. J Biol Chem 281(12):7717–7726. doi:10.1074/jbc.M510775200 PubMedCrossRefGoogle Scholar
  24. 24.
    Seo SR, Chung KC (2008) CREB activates proteasomal degradation of DSCR1/RCAN1. FEBS Lett 582(13):1889–1893. doi:10.1016/j.febslet.2008.04.059 PubMedCrossRefGoogle Scholar
  25. 25.
    Bush CR, Havens JM, Necela BM et al (2007) Functional genomic analysis reveals cross-talk between peroxisome proliferator-activated receptor gamma and calcium signaling in human colorectal cancer cells. J Biol Chem 282(32):23387–23401. doi:10.1074/jbc.M702708200 PubMedCrossRefGoogle Scholar
  26. 26.
    Iizuka M, Abe M, Shiiba K et al (2004) Down syndrome candidate region 1, a downstream target of VEGF, participates in endothelial cell migration and angiogenesis. J Vasc Res 41(4):334–344. doi:10.1159/000079832 PubMedCrossRefGoogle Scholar
  27. 27.
    Minami T, Horiuchi K, Miura M et al (2004) Vascular endothelial growth factor- and thrombin-induced termination factor, down syndrome critical region-1, attenuates endothelial cell proliferation and angiogenesis. J Biol Chem 279(48):50537–50554. doi:10.1074/jbc.M406454200 PubMedCrossRefGoogle Scholar
  28. 28.
    Schweppe RE, Klopper JP, Korch C et al (2008) Deoxyribonucleic acid profiling analysis of 40 human thyroid cancer cell lines reveals cross-contamination resulting in cell line redundancy and misidentification. J Clin Endocrinol Metab 83(11):4331–4341. doi:10.1210/jc.2008-1102 CrossRefGoogle Scholar
  29. 29.
    Ringel MD (2008) “Thyroid cancer” cell line misidentification: a time for proactive change. J Clin Endocrinol Metab 93(11):4226–4227. doi:10.1210/jc.2008-2008 PubMedCrossRefGoogle Scholar
  30. 30.
    Vasko V, Saji M, Hardy E et al (2004) Akt activation and localization correlate with tumor invasion and oncogene expression in thyroid cancer. J Med Genet 41(3):161–170. doi:10.1136/jmg.2003.015339 PubMedCrossRefGoogle Scholar
  31. 31.
    Hoffmann S, Maschuw K, Hassan I et al (2005) Differential pattern of integrin receptor expression in differentiated and anaplastic thyroid cancer cell lines. Thyroid 15(9):1011–1020. doi:10.1089/thy.2005.15.1011 PubMedCrossRefGoogle Scholar
  32. 32.
    Mulero MC, Aubareda A, Orzaez M, et al. (2009) Inhibiting the calcineurin-NFAT signaling pathway with a regulator of calcineurin-derived peptide without affecting general calcineurin phosphatase activity. J biol chem [Epub ahead of print]Google Scholar
  33. 33.
    Rinker-Schaeffer CW, O’Keefe JP, Welch DR et al (2006) Metastasis suppressor proteins: discovery, molecular mechanisms, and clinical application. Clinical Cancer Research 12(13):3882–3889. doi:10.1158/1078-0432.CCR-06-1014 PubMedCrossRefGoogle Scholar
  34. 34.
    Ryeom S, Baek K-H, Rioth MJ et al (2008) Targeted deletion of the calcineurin inhibitor DSCR1 suppresses tumor growth. Cancer Cell 13(5):420–431. doi:10.1016/j.ccr.2008.02.018 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Allan V. Espinosa
    • 1
  • Motoo Shinohara
    • 1
  • Leonardo M. Porchia
    • 1
  • Yun Jae Chung
    • 1
    • 2
  • Samantha McCarty
    • 1
  • Motoyasu Saji
    • 1
  • Matthew D. Ringel
    • 1
  1. 1.Division of Endocrinology, Diabetes, and MetabolismThe Ohio State University College of Medicine and Arthur G. James Comprehensive Cancer CenterColumbusUSA
  2. 2.Division of Endocrinology and MetabolismDepartment of Internal Medicine, Chung-Ang University College of MedicineSeoulKorea

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