Skip to main content

Advertisement

SpringerLink
Log in

Down-regulation of osteopontin mediates a novel mechanism underlying the cytostatic activity of TGF-β

  • Original Paper
  • Published:
Cellular Oncology Aims and scope Submit manuscript

Abstract

Purpose

Loss of a cytostatic response to TGF-β has been implicated in multiple hyper-proliferative disorders, including cancer. Although several key genes involved in the cytostatic activity of TGF-β have in the past been identified, its exact mode of action is yet to be elucidated. A comprehensive understanding of the mechanisms underlying the cytostatic activity of TGF-β may open up new avenues for the development of therapeutic strategies.

Methods

Quantitative real-time RT-PCR was used to assess osteopontin (OPN) gene expression in human hepatoma-derived Huh-7 and lung adenocarcinoma-derived A549 cells. Reporter assays using an OPN promoter-luciferase construct and its mutated counterparts were performed to assess its transcriptional activity. Binding of Smad4 to the OPN gene promoter was investigated using chromatin immunoprecipitation (CHIP). The putative role of Smad4 in OPN gene expression down-regulation was also assessed using a shRNA-mediated knockdown strategy. The anti-proliferative effect of TGF-β on different cancer-derived cell lines was determined using the cell proliferation reagent WST-1.

Results

We found that the OPN expression levels dose-dependently decreased in TGF-β-treated Huh-7 and A549 cells. Our reporter assays indicated that this TGF-β-induced repression occurred at the transcriptional level, and could largely be abrogated by disruption of an element (TIE2) similar to the TGF-β inhibitory element found in other TGF-β-repressed genes. Our CHIP assay revealed that the Smad protein complex specifically binds to the OPN gene promoter, and that the TGF-β-mediated inhibition of OPN was lost upon shRNA-mediated knockdown of Smad4. Moreover, we found that the deregulation of OPN gene expression by TGF-β occurred concomitantly with loss of the TGF-β anti-proliferative response, whereas a neutralizing anti-OPN antibody partially restored this response.

Conclusions

Our results indicate that the OPN gene is a direct target of Smad-mediated TGF-β signaling, implying that OPN expression inhibition serves as a novel mechanism underlying the cytostatic activity of TGF-β.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. F. Lebrin, M. Deckers, P. Bertolino, P. ten Dijke, TGF-β receptor function in the endothelium. Cardiovasc. Res. 65, 599–608 (2004)

    Article  Google Scholar 

  2. C.-H. Heldin, M. Landström, A. Moustakas, Mechanism of TGF-β signaling to growth arrest, apoptosis, and epithelial-mesenchymal transition. Curr. Opin. Cell Biol. 21, 166–176 (2009)

    Article  CAS  PubMed  Google Scholar 

  3. E. Meulmeester, P. Ten Dijke, The dynamic roles of TGF-beta in cancer. J. Pathol. 223, 205–218 (2011)

    Article  CAS  PubMed  Google Scholar 

  4. A Maier, A.L. Peille, V. Vuaroqueaux, M. Lahn, Anti-tumor activity of the TGF-β receptor kinase inhibitor galunisertib (LY2157299 monohydrate) in patient-derived tumor xenografts. Cell. Oncol. 38, 131–144 (2015)

  5. S. Ross, C.S. Hill, How the smads regulate transcription. Int. J. Biochem. Cell Biol. 40, 383–408 (2008)

    Article  CAS  PubMed  Google Scholar 

  6. A Moustakas, C.H. Heldin, The regulation of TGFβ signal transduction. Development 136, 3699–3714 (2009)

  7. P.M. Siegel, J. Massagué, Cytostatic and apoptotic actions of TGF-β in homeostasis and cancer. Nat. Rev. Cancer 3, 807–821 (2003)

    Article  CAS  PubMed  Google Scholar 

  8. J. Massagué, TGFβ in cancer. Cell 134, 215–230 (2008)

    Article  PubMed  PubMed Central  Google Scholar 

  9. J. Xu, S. Lamouille, R. Derynck, TGF-β-induced epithelial to mesenchymal transition. Cell Res. 19, 156–172 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. G.J. Hannon, D. Beach, p15INK4B is a potential effector of TGFbeta-induced cell cycle arrest. Nature 371, 257–261 (1994)

    Article  CAS  PubMed  Google Scholar 

  11. M.B. Datto, Y. Li, J.F. Panus, D.J. Howe, Y. Xiong, X.F. Wang, Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 throughp53-independent mechanism. Proc. Natl. Acad. Sci. U. S. A. 92, 5545–5549 (1995)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. J.M. Scandura, P. Boccuni, J. Massague, S.D. Nimer, Transforming growth factor beta-induced cell cycle arrest of human hematopoietic cells requires p57KIP2 upregulation. Proc. Natl. Acad. Sci. U. S. A. 101, 15231–15236 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. J. Massagué, S.W. Blain, R.S. Lo, TGFβ signaling in growth control, cancer, and heritable disorders. Cell 103, 295–309 (2000)

    Article  PubMed  Google Scholar 

  14. R. Derynck, R.J. Akhurst, A. Balmain, TGF-β signaling in tumor suppression and cancer progression. Nat. Genet. 29, 117–129 (2001)

    Article  CAS  PubMed  Google Scholar 

  15. L.M. Wakefield, A.B. Roberts, TGF-β signaling: positive and negative effects on tumorigenesis. Curr. Opin. Genet. Dev. 12, 22–29 (2002)

    Article  CAS  PubMed  Google Scholar 

  16. B. Kleuser, D. Malek, R. Gust, H.H. Pertz, H. Potteck, 17-beta-estradiol inhibits transforming growth factor-beta signaling and function in breast cancer cells via activation of extracellular signal-regulated kinase through the G protein-coupled receptor 30. Mol. Pharmacol. 74, 1533–1543 (2008)

    Article  CAS  PubMed  Google Scholar 

  17. S. Gizzo, C. Saccardi, T.S. Patrelli, R. Berretta, G. Capobianco, S. Di Gangi, A. Vacilotto, A. Bertocco, M. Noventa, E. Ancona, D. D’Antona, G.B. Nardelli, Update on raloxifene: mechanism of action, clinical efficacy, adverse effects, and contraindications. Obstet. Gynecol. Surv. 68, 467–481 (2013)

    Article  PubMed  Google Scholar 

  18. S. Gizzo, M. Noventa, C. Saccardi, P. Litta, D. D’Antona, G.B. Nardelli, Proposal on raloxifene use after prophylactic salpingo-oophorectomy in BRCA1-2: hypothesis and rationale. Eur. J. Cancer Prev. 23, 514–515 (2014)

    Article  PubMed  Google Scholar 

  19. S. Gizzo, M. Noventa, S. Di Gangi, P. Litta, C. Saccardi, D. D’Antona, G.B. Nardelli, Could in-vitro studies on Ishikawa cell lines explain the endometrial safety of raloxifene? Systematic literature review and starting points for future oncological research. Eur. J. Cancer Prev 24, 497–507 (2015)

    Article  CAS  PubMed  Google Scholar 

  20. L. Levy, C.S. Hill, Alterations in components of the TGF-β superfamily signaling pathways in human cancer. Cytokine Growth Factor Rev. 17, 41–58 (2006)

    Article  CAS  PubMed  Google Scholar 

  21. A.B. Roberts, L.M. Wakefield, The two faces of transforming growth factor beta in carcinogenesis. Proc. Natl. Acad. Sci. U. S. A. 100, 8621–8623 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. C.H. Heldin, M. Vanlandewijck, A. Moustakas, Regulation of EMT by TGFβ in cancer. FEBS Lett. 586, 1959–1970 (2012)

    Article  CAS  PubMed  Google Scholar 

  23. G.F. Weber, The metastasis gene osteopontin: a candidate target for cancer therapy. Biochim. Biophys. Acta 1552, 61–85 (2001)

    Article  CAS  PubMed  Google Scholar 

  24. H. Rangaswami, A. Bulbule, G.C. Kundu, Osteopontin: role in cell signaling and cancer progression. Trends Cell Biol. 16, 79–87 (2006)

    Article  CAS  PubMed  Google Scholar 

  25. N.I. Johnston, V.K. Gunasekharan, A. Ravindranath, C. O’Connell, P.G. Johnston, P.G. El-Tanani, Osteopontin as a target for cancer therapy. Front. Biosci. 13, 4361–4372 (2008)

    Article  CAS  PubMed  Google Scholar 

  26. J.L. Lee, M.J. Wang, P.R. Sudhir, G.D. Chen, C.W. Chi, J.Y. Chen, Osteopontin promotes integrin activation through outside-in and inside-out mechanisms: OPN-CD44V interaction enhances survival in gastrointestinal cancer cells. Cancer Res. 67, 2089–2097 (2007)

    Article  CAS  PubMed  Google Scholar 

  27. A Bellahcène, V. Castronovo, K.U. Ogbureke, L.W. Fisher, N.S. Fedarko, Small integrin-binding ligand N-linked glycoproteins (SIBLINGs): multifunctional proteins in cancer. Nat. Rev. Cancer 8, 212–226 (2008)

  28. D. Coppola, M. Szabo, D. Boulware, P. Muraca, M. Alsarraj, A.F. Chambers, T.J. Yeatman, Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies. Clin. Cancer Res. 10, 184–190 (2004)

    Article  CAS  PubMed  Google Scholar 

  29. M. Higashiyama, T. Ito, E. Tanaka, Y. Shimada, Prognostic significance of osteopon- tin expression in human gastric carcinoma. Ann. Surg. Oncol. 14, 3419–3427 (2007)

    Article  PubMed  Google Scholar 

  30. P.V. Korita, T. Wakai, Y. Shirai, Y. Matsuda, J. Sakata, X. Cui, Y. Ajioka, K. Hatakeyama, Overexpression of osteopontin independently correlates with vascular invasion and poor prognosis in patients with hepatocellular carcinoma. Hum. Pathol. 39, 1777–1783 (2008)

    Article  CAS  PubMed  Google Scholar 

  31. N. Patani, F. Jouhra, W. Jiang, K. Mokbel, Osteopontin expression profiles predict pathological and clinical outcome in breast cancer. Anticancer Res. 28, 4105–4110 (2008)

    CAS  PubMed  Google Scholar 

  32. G.F. Weber, G.S. Lett, N.C. Haubein, Osteopontin is a marker for cancer aggressiveness and patient survival. Br. J. Cancer 103, 861–869 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. J. Zhang, O. Yamada, Y. Matsushita, H. Chagan-Yasutan, T. Hattori, Transactivation of human osteopontin promoter by human T-cell leukemia virus type 1-encoded tax protein. Leuk. Res. 34, 763–768 (2010)

    Article  CAS  PubMed  Google Scholar 

  34. J. Zhang, O. Yamada, T. Sakamoto, H. Yoshida, T. Iwai, Y. Matsushita, H. Shimamura, H. Araki, K. Shimotohno, Down-regulation of viral replication by adenoviral-mediated expression of siRAN against cellular cofactors for hepatitis C virus. Virology 320, 135–143 (2004)

    Article  CAS  PubMed  Google Scholar 

  35. J. Zhang, O. Yamada, S. Kida, Y. Matsushita, S. Yamaoka, H. Chagan-Yasutan, T. Hattori, Identification of CD44 as a downstream target of noncanonical NF-κB pathway activated by human T-cell leukemia virus type 1-encoded tax protein. Virology 413, 244–252 (2011)

    Article  CAS  PubMed  Google Scholar 

  36. T. Standal, M. Borset, A. Sundan, Role of osteopontin in adhesion, migration, cell survival and bone remodeling. Exp. Oncol. 26, 179–184 (2004)

    CAS  PubMed  Google Scholar 

  37. C.R. Chen, Y. Kang, P.M. Siegel, J. Massagué, E2F4/5 and p107 as smad cofactors linking the TGFbeta receptor to c-myc repression. Cell 110, 19–32 (2002)

    Article  CAS  PubMed  Google Scholar 

  38. Y. Kang, C.R. Chen, J. Massagué, A self-enabling TGFbeta response coupled to stress signaling: smad engages stress response factor ATF3 for Id1 repression in epithelial cells. Mol. Cell 11, 915–926 (2003)

    Article  CAS  PubMed  Google Scholar 

  39. N.G. Denissova, C. Pouponnot, J. Long, D. He, F. Liu, Transforming growth factor -inducible independent binding of SMAD to the Smad7 promoter. Proc. Natl. Acad. Sci. 97, 6397–6402 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. N.T. Liberati, M. Moniwa, A.J. Borton, J.R. Davie, X.F. Wang, An essential role for mad homology domain 1 in the association of Smad3 with histone deacetylase activity. J. Biol. Chem. 276, 22595–22603 (2001)

    Article  CAS  PubMed  Google Scholar 

  41. T. Alliston, L. Choy, P. Ducy, G. Karsenty, R. Derynck, TGF-β-induced repression of CBFA1 by Smad3 decreases cbfa1 and osteocalcin expression and inhibits osteoblast differentiation. EMBO J. 20, 2254–2272 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. J.S. Kang, T. Alliston, R. Delston, R. Derynck, Repression of Runx2 function by TGF-β through recruitment of class II histone deacetylases by Smad3. EMBO J. 24, 2543–2555 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. R. Sakata, S. Minami, Y. Sowa, M. Yoshida, T. Tamaki, Trichostatin a activates the osteopontin gene promoter through AP1 site. Biochem. Biophys. Res. Commun. 315, 959–963 (2004)

    Article  CAS  PubMed  Google Scholar 

  44. D.T. Denhardt, D. Mistretta, A.F. Chambers, S. Krishna, J.F. Porter, S. Raghuram, S.R. Rittling, Transcriptional regulation of osteopontin and the metastatic phenotype evidence for a ras-activated enhancer in the human OPN promoter. Clin. Exp. Metastasis 20, 77–84 (2003)

    Article  CAS  PubMed  Google Scholar 

  45. X. Shi, S. Bai, L. Li, X. Cao, Hoxa-9 represses transforming growth factor-beta- induced osteopontin gene transcription. J. Biol. Chem. 276, 850–855 (2001)

    Article  CAS  PubMed  Google Scholar 

  46. T.G. Hullinger, Q. Pan, H.L. Viswanathan, M.J. Somerman, TGFbeta and BMP-2 activation of the OPN promoter: roles of smad- and hox-binding elements. Exp. Cell Res. 262, 69–74 (2001)

    Article  CAS  PubMed  Google Scholar 

  47. C.H. Heldin, A. Moustakas, Role of smads in TGFβ signaling. Cell Tissue Res. 347, 21–36 (2012)

    Article  CAS  PubMed  Google Scholar 

  48. J. Massagué, TGFβ signalling in context. Nat. Rev. Mol. Cell Biol. 13, 616–630 (2012)

    Article  PubMed  PubMed Central  Google Scholar 

  49. K. Matsuzaki, Smad phospho-isoforms direct context-dependent TGF-β signaling. Cytokine Growth Factor Rev. 24, 385–399 (2013)

    Article  CAS  PubMed  Google Scholar 

  50. D. Padua, X.H. Zhang, Q. Wang, C. Nadal, W.L. Gerald, R.R. Gomis, J. Massagué, TGFbeta primes breast tumors for lung metastasis seeding through angiopoietin-like 4. Cell 133, 66–77 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. S. Gizzo, M. Quaranta, G.B. Nardelli, M. Noventa. Lipophilic Statins as Anticancer Agents: Molecular Targeted Actions and Proposal in Advanced Gynaecological Malignancies. Curr. Drug Targets 16, 1142--1159 (2015)

  52. A. Vitagliano, M. Noventa, S. Gizzo. Emerging evidence regarding statins use as novel endometriosis targeted treatment: real "magic pills" or "trendy" drugs? Some considerations. Eur. J. Obstet. Gynecol. Reprod. Biol. 184, 125-126 (2015)

  53. A. Vitagliano, M. Noventa, M. Quaranta, S. Gizzo. Statins as Targeted "Magical Pills" for the Conservative Treatment of Endometriosis: May Potential Adverse Effects on Female Fertility Represent the "Dark Side of the Same Coin"? A Systematic Review of Literature. Reprod. Sci. (2015). doi:10.1177/1933719115584446

  54. M. Matsuura, T. Suzuki, M. Suzuki, R. Tanaka, E. Ito, T. Saito, Statin-mediated reduction of osteopontin expression induces apoptosis and cell growth arrest in ovarian clear cell carcinoma. Oncol. Rep. 25, 41–47 (2011)

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Research and Development Center, FUSO Pharmaceutical Industries, LTD., 2-3-30 Morinomiya, Joto-ku, Osaka, 536-8523, Japan

    Jing Zhang, Osamu Yamada, Shinya Kida & Yoshihisa Matsushita

  2. Division of Emerging Infectious Diseases, Department of Internal Medicine, Tohoku University, Sendai, Japan

    Toshio Hattori

Authors
  1. Jing Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Osamu Yamada
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shinya Kida
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yoshihisa Matsushita
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Toshio Hattori
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jing Zhang.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal studies

This article does not contain any studies with human participants or animals performed by any of the authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, J., Yamada, O., Kida, S. et al. Down-regulation of osteopontin mediates a novel mechanism underlying the cytostatic activity of TGF-β. Cell Oncol. 39, 119–128 (2016). https://doi.org/10.1007/s13402-015-0257-1

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13402-015-0257-1

Keywords

Search

Navigation