Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Short Communication
  • Published:

An endogenous inhibitor of angiogenesis inversely correlates with side population phenotype and function in human lung cancer cells

Oncogene volume 33, pages 1198–1206 (2014)Cite this article

Subjects

Abstract

The side population (SP) in human lung cancer cell lines and tumors is enriched with cancer stem cells. An endogenous inhibitor of angiogenesis known as tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), characterized for its ability to inhibit matrix metalloproteinases (MMPs), has been shown by several laboratories to impede tumor progression through MMP-dependent or -independent mechanisms. We recently reported that forced expression of TIMP-2, as well as the modified form Ala+TIMP-2 (that lacks MMP inhibitory activity) significantly blocks growth of A549 human lung cancer cells in vivo. However, the mechanisms underlying TIMP-2 antitumor effects are not fully characterized. Here, we examine the hypothesis that the TIMP-2 antitumor activity may involve regulation of the SP in human lung cancer cells. Indeed, using Hoechst dye efflux assay and flow cytometry, as well as quantitative reverse transcriptase–PCR analysis, we found that endogenous TIMP-2 mRNA levels showed a significant inverse correlation with SP fraction size in six non-small cell lung cancer cell lines. In A549 cells expressing increased levels of TIMP-2, a significant decrease in SP was observed, and this decrease was associated with lowered gene expression of ABCG2, ABCB1 and AKR1C1. Functional analysis of A549 cells showed that TIMP-2 overexpression increased chemosensitivity to cytotoxic drugs. The SP isolated from TIMP-2-overexpressing A549 cells also demonstrated impaired migratory capacity compared with the SP from empty vector control. More importantly, our data provide strong evidence that these TIMP-2 functions occur independent of MMP inhibition, as A549 cells overexpressing Ala+TIMP-2 exhibited identical behavior to those overexpressing TIMP-2 alone. Our findings provide the first indication that TIMP-2 modulates SP phenotype and function, and suggests that TIMP-2 may act as an endogenous suppressor of the SP in human lung cancer cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4

Similar content being viewed by others

References

  1. Heppner GH, Miller BE . Tumor heterogeneity: biological implications and therapeutic consequences. Cancer Metastasis Rev 1983; 2: 5–23.

    Article  CAS  Google Scholar 

  2. Vermeulen L, Sprick MR, Kemper K, Stassi G, Medema JP . Cancer stem cells—old concepts, new insights. Cell Death Differ 2008; 15: 947–958.

    Article  CAS  Google Scholar 

  3. Visvader JE, Lindeman GJ . Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 2008; 8: 755–768.

    Article  CAS  Google Scholar 

  4. Wu C, Alman BA . Side population cells in human cancers. Cancer Lett 2008; 268: 1–9.

    Article  CAS  Google Scholar 

  5. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC . Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996; 183: 1797–1806.

    Article  CAS  Google Scholar 

  6. Shimano K, Satake M, Okaya A, Kitanaka J, Kitanaka N, Takemura M et al. Hepatic oval cells have the side population phenotype defined by expression of ATP-binding cassette transporter ABCG2/BCRP1. Am J Pathol 2003; 163: 3–9.

    Article  CAS  Google Scholar 

  7. Lopez J, Poitevin A, Mendoza-Martinez V, Perez-Plasencia C, Garcia-Carranca A . Cancer-initiating cells derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance. BMC Cancer 2012; 12: 48.

    Article  CAS  Google Scholar 

  8. Mitsutake N, Iwao A, Nagai K, Namba H, Ohtsuru A, Saenko V et al. Characterization of side population in thyroid cancer cell lines: cancer stem-like cells are enriched partly but not exclusively. Endocrinology 2007; 148: 1797–1803.

    Article  CAS  Google Scholar 

  9. Szotek PP, Pieretti-Vanmarcke R, Masiakos PT, Dinulescu DM, Connolly D, Foster R et al. Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness. Proc Natl Acad Sci USA 2006; 103: 11154–11159.

    Article  CAS  Google Scholar 

  10. Kondo T, Setoguchi T, Taga T . Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci USA 2004; 101: 781–786.

    Article  CAS  Google Scholar 

  11. Ho MM, Ng AV, Lam S, Hung JY . Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res 2007; 67: 4827–4833.

    Article  CAS  Google Scholar 

  12. Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM . Targeting multidrug resistance in cancer. Nat Rev Drug Discov 2006; 5: 219–234.

    Article  CAS  Google Scholar 

  13. Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001; 7: 1028–1034.

    Article  CAS  Google Scholar 

  14. Ding XW, Wu JH, Jiang CP . ABCG2: a potential marker of stem cells and novel target in stem cell and cancer therapy. Life Sci 2010; 86: 631–637.

    Article  CAS  Google Scholar 

  15. Dean M . ABC transporters, drug resistance, and cancer stem cells. J Mammary Gland Biol Neoplasia 2009; 14: 3–9.

    Article  Google Scholar 

  16. An Y, Ongkeko WM . ABCG2: the key to chemoresistance in cancer stem cells? Expert Opin Drug Metab Toxicol 2009; 5: 1529–1542.

    Article  CAS  Google Scholar 

  17. Seo DW, Li H, Guedez L, Wingfield PT, Diaz T, Salloum R et al. TIMP-2 mediated inhibition of angiogenesis: an MMP-independent mechanism. Cell 2003; 114: 171–180.

    Article  CAS  Google Scholar 

  18. Fernandez CA, Butterfield C, Jackson G, Moses MA . Structural and functional uncoupling of the enzymatic and angiogenic inhibitory activities of tissue inhibitor of metalloproteinase-2 (TIMP-2): loop 6 is a novel angiogenesis inhibitor. J Biol Chem 2003; 278: 40989–40995.

    Article  CAS  Google Scholar 

  19. Fernandez CA, Roy R, Lee S, Yang J, Panigrahy D, Van Vliet KJ et al. The anti-angiogenic peptide, loop 6, binds insulin-like growth factor-1 receptor. J Biol Chem 2010; 285: 41886–41895.

    Article  CAS  Google Scholar 

  20. Bourboulia D, Jensen-Taubman S, Rittler MR, Han HY, Chatterjee T, Wei B et al. Endogenous angiogenesis inhibitor blocks tumor growth via direct and indirect effects on tumor microenvironment. Am J Pathol 2011; 179: 2589–2600.

    Article  CAS  Google Scholar 

  21. Seo DC, Sung JM, Cho HJ, Yi H, Seo KH, Choi IS et al. Gene expression profiling of cancer stem cell in human lung adenocarcinoma A549 cells. Mol Cancer 2007; 6: 1–8.

    Article  Google Scholar 

  22. Sung JM, Cho HJ, Yi H, Lee CH, Kim HS, Kim DK et al. Characterization of a stem cell population in lung cancer A549 cells. Biochem Biophys Res Commun 2008; 371: 163–167.

    Article  CAS  Google Scholar 

  23. Akunuru S, Palumbo J, Zhai QJ, Zheng Y . Rac1 targeting suppresses human non-small cell lung adenocarcinoma cancer stem cell activity. PloS One 2011; 6: e16951.

    Article  CAS  Google Scholar 

  24. Salcido CD, Larochelle A, Taylor BJ, Dunbar CE, Varticovski L . Molecular characterisation of side population cells with cancer stem cell-like characteristics in small-cell lung cancer. Br J Cancer 2010; 102: 1636–1644.

    Article  CAS  Google Scholar 

  25. Fletcher JI, Haber M, Henderson MJ, Norris MD . ABC transporters in cancer: more than just drug efflux pumps. Nat Rev Cancer 2010; 10: 147–156.

    Article  CAS  Google Scholar 

  26. Yoh K, Ishii G, Yokose T, Minegishi Y, Tsuta K, Goto K et al. Breast cancer resistance protein impacts clinical outcome in platinum-based chemotherapy for advanced non-small cell lung cancer. Clin Cancer Res 2004; 10: 1691–1697.

    Article  CAS  Google Scholar 

  27. Deng HB, Adikari M, Parekh HK, Simpkins H . Ubiquitous induction of resistance to platinum drugs in human ovarian, cervical, germ-cell and lung carcinoma tumor cells overexpressing isoforms 1 and 2 of dihydrodiol dehydrogenase. Cancer Chemother Pharmacol 2004; 54: 301–307.

    Article  CAS  Google Scholar 

  28. Wanichwatanadecha P, Sirisrimangkorn S, Kaewprag J, Ponglikitmongkol M . Transactivation activity of human papillomavirus type 16 E6*I on AKR1C enhances chemoresistance in cervical cancer cells. J Gen Virol 2012; 93: 1081–1092.

    Article  CAS  Google Scholar 

  29. Halon A, Nowak-Markwitz E, Donizy P, Matkowski R, Maciejczyk A, Gansukh T et al. Enhanced immunoreactivity of TIMP-2 in the stromal compartment of tumor as a marker of favorable prognosis in ovarian cancer patients. J Histochem Cytochem 2012; 60: 491–501.

    Article  CAS  Google Scholar 

  30. Stetler-Stevenson WG . The tumor microenvironment: regulation by MMP-independent effects of tissue inhibitor of metalloproteinases-2. Cancer Metastasis Rev 2008; 27: 57–66.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the Laboratory of Molecular Technology, NCI Frederick for the microarray experiment. We also thank the members of the Extracellular Matrix Pathology Section, Radiation Oncology Branch, NCI for critical discussions. This work was supported by the NCI Center for Cancer Research Intramural Research Grant # ZIA SC009179-22.

Author information

Author notes

  1. B Isaac

    Present address: 3Current Address: Bioinformatics Core Leader Room 610L, Clinical Research Building, Center for Computational Science, University of Miami, Coral Gables, FL 33124, USA.,

  2. H Han and D Bourboulia: These authors contributed equally to this work.

Authors and Affiliations

  1. Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, the National Institutes of Health, Advanced Technology Center, Bethesda, MD, USA

    H Han, D Bourboulia, S Jensen-Taubman, B Isaac, B Wei & W G Stetler-Stevenson

Authors
  1. H Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D Bourboulia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S Jensen-Taubman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. B Isaac
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. B Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. W G Stetler-Stevenson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D Bourboulia.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Han, H., Bourboulia, D., Jensen-Taubman, S. et al. An endogenous inhibitor of angiogenesis inversely correlates with side population phenotype and function in human lung cancer cells. Oncogene 33, 1198–1206 (2014). https://doi.org/10.1038/onc.2013.61

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2013.61

Keywords

This article is cited by

Search

Advanced search

Quick links