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.

  • Original Article
  • Published:

Fibulin-3-mediated inhibition of epithelial-to-mesenchymal transition and self-renewal of ALDH+ lung cancer stem cells through IGF1R signaling

Oncogene volume 33, pages 3908–3917 (2014)Cite this article

Subjects

Abstract

Fibulins (FBLNs), a family of extracellular matrix proteins, have recently been shown to act as tumor suppressors or activators in different cancers, and the underlying molecular mechanisms of their action in cancer remain unclear. We have previously shown that the expression of FBLN3 is suppressed by promoter hypermethylation and is associated with invasiveness in aggressive non-small cell lung cancer. In this study, we evaluated the roles and signaling mechanism of FBLN3 in lung cancer stem cells (CSCs). Forced expression of FBLN3 suppressed invasion and migration of lung adenocarcinoma cells and decreased the expression of epithelial-to-mesenchymal transition (EMT) activators, including N-cadherin and Snail. Stemness activities of lung adenocarcinoma cells were also suppressed by FBLN3 as indicated by a decrease in spheroid formation and the levels of stemness markers such as Sox2 and β-catenin. These effects of FBLN3 were mediated by the glycogen synthase kinase-3β, GSK3β/β-catenin pathway, and the upstream regulators of GSK3β, including phosphoinositide 3-kinase (PI3K)/AKT and insulin-like growth factor-1 receptor (IGF1R), were inactivated by FBLN3. Moreover, IGF1R was shown to be a direct target of FBLN3, which competitively inhibited insulin-like growth factor (IGF) action. To confirm the effect of FBLN3 on lung CSCs, aldehyde dehydrogenase-positive (ALDH+) A549 lung CSCs were sorted and treated with recombinant FBLN3 protein. FBLN3 clearly suppressed EMT, stemness activity and the over-activated IGF1R/PI3K/AKT/GSK3β pathway of the ALDH+ CSC subpopulation. In addition, injection of recombinant FBLN3 protein around subcutaneous xenografts established with ALDH+ CSCs in athymic nude mice significantly suppressed tumor growth and progression. Overall, our results show that FBLN3 suppresses both EMT and self-renewal of the lung CSCs by modulating the IGF1R/PI3K/AKT/GSK3β pathway and that FBLN3 would be useful as an alternative CSC therapy.

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
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Esposito L, Conti D, Ailavajhala R, Khalil N, Giordano A . Lung cancer: are we up to the challenge? Curr Genomics 2010; 11: 513–518.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Heist RS, Christiani D . EGFR-targeted therapies in lung cancer: predictors of response and toxicity. Pharmacogenomics 2009; 10: 59–68.

    Article  CAS  PubMed  Google Scholar 

  3. Alison MR, Lin WR, Lim SM, Nicholson LJ . Cancer stem cells: in the line of fire. Cancer Treat Rev 2012; 38: 589–598.

    Article  CAS  PubMed  Google Scholar 

  4. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008; 133: 704–715.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Rivera C, Rivera S, Loriot Y, Vozenin MC, Deutsch E . Lung cancer stem cell: new insights on experimental models and preclinical data. J Oncol 2011; 2011: 549181.

    Article  PubMed  Google Scholar 

  6. Bonnet D, Dick JE . Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997; 3: 730–737.

    Article  CAS  PubMed  Google Scholar 

  7. Korkaya H, Wicha MS . Selective targeting of cancer stem cells: a new concept in cancer therapeutics. BioDrugs 2007; 21: 299–310.

    Article  CAS  PubMed  Google Scholar 

  8. Perona R, Lopez-Ayllon BD, de Castro Carpeno J, Belda-Iniesta C . A role for cancer stem cells in drug resistance and metastasis in non-small-cell lung cancer. Clin Transl Oncol 2011; 13: 289–293.

    Article  CAS  PubMed  Google Scholar 

  9. Li J, Zhou BP . Activation of beta-catenin and Akt pathways by Twist are critical for the maintenance of EMT associated cancer stem cell-like characters. BMC Cancer 2011; 11: 49.

    Article  PubMed  PubMed Central  Google Scholar 

  10. de Vega S, Iwamoto T, Yamada Y . Fibulins: multiple roles in matrix structures and tissue functions. Cell Mol Life Sci 2009; 66: 1890–1902.

    Article  CAS  PubMed  Google Scholar 

  11. Schiemann WP, Blobe GC, Kalume DE, Pandey A, Lodish HF . Context-specific effects of fibulin-5 (DANCE/EVEC) on cell proliferation, motility, and invasion. Fibulin-5 is induced by transforming growth factor-beta and affects protein kinase cascades. J Biol Chem 2002; 277: 27367–27377.

    Article  CAS  PubMed  Google Scholar 

  12. Yue W, Sun Q, Landreneau R, Wu C, Siegfried JM, Yu J et al. Fibulin-5 suppresses lung cancer invasion by inhibiting matrix metalloproteinase-7 expression. Cancer Res 2009; 69: 6339–6346.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hu B, Thirtamara-Rajamani KK, Sim H, Viapiano MS . Fibulin-3 is uniquely upregulated in malignant gliomas and promotes tumor cell motility and invasion. Mol Cancer Res 2009; 7: 1756–1770.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hu Y, Pioli PD, Siegel E, Zhang Q, Nelson J, Chaturbedi A et al. EFEMP1 suppresses malignant glioma growth and exerts its action within the tumor extracellular compartment. Mol Cancer 2011; 10: 123.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kim EJ, Lee SY, Woo MK, Choi SI, Kim TR, Kim MJ et al. Fibulin-3 promoter methylation alters the invasive behavior of non-small cell lung cancer cell lines via MMP-7 and MMP-2 regulation. Int J Oncol 2012; 40: 402–408.

    Article  CAS  PubMed  Google Scholar 

  16. 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  PubMed  Google Scholar 

  17. Thiery JP, Acloque H, Huang RY, Nieto MA . Epithelial-mesenchymal transitions in development and disease. Cell 2009; 139: 871–890.

    Article  CAS  PubMed  Google Scholar 

  18. Araki K, Shimura T, Suzuki H, Tsutsumi S, Wada W, Yajima T et al. E/N-cadherin switch mediates cancer progression via TGF-beta-induced epithelial-to-mesenchymal transition in extrahepatic cholangiocarcinoma. Br J Cancer 2011; 105: 1885–1893.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lee JM, Dedhar S, Kalluri R, Thompson EW . The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol 2006; 172: 973–981.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wellner U, Schubert J, Burk UC, Schmalhofer O, Zhu F, Sonntag A et al. The EMT-activator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs. Nat Cell Biol 2009; 11: 1487–1495.

    Article  CAS  PubMed  Google Scholar 

  21. Vuoriluoto K, Haugen H, Kiviluoto S, Mpindi JP, Nevo J, Gjerdrum C et al. Vimentin regulates EMT induction by Slug and oncogenic H-Ras and migration by governing Axl expression in breast cancer. Oncogene 2011; 30: 1436–1448.

    Article  CAS  PubMed  Google Scholar 

  22. Biddle A, Mackenzie IC . Cancer stem cells and EMT in carcinoma. Cancer Metastasis Rev 2012; 31: 285–293.

    Article  Google Scholar 

  23. Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM et al. Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 2006; 9: 391–403.

    Article  CAS  PubMed  Google Scholar 

  24. Pastrana E, Silva-Vargas V, Doetsch F . Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell Stem Cell 2011; 8: 486–498.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. O'Brien CA, Kreso A, Jamieson CH . Cancer stem cells and self-renewal. Clin Cancer Res 2010; 16: 3113–3120.

    Article  CAS  PubMed  Google Scholar 

  26. Fuxe J, Vincent T, Garcia de Herreros A . Transcriptional crosstalk between TGF-beta and stem cell pathways in tumor cell invasion: role of EMT promoting Smad complexes. Cell Cycle 2010; 9: 2363–2374.

    Article  CAS  PubMed  Google Scholar 

  27. Jiang YG, Luo Y, He DL, Li X, Zhang LL, Peng T et al. Role of Wnt/beta-catenin signaling pathway in epithelial-mesenchymal transition of human prostate cancer induced by hypoxia-inducible factor-1alpha. Int J Urol 2007; 14: 1034–1039.

    Article  CAS  PubMed  Google Scholar 

  28. Hwang CF, Chien CY, Huang SC, Yin YF, Huang CC, Fang FM et al. Fibulin-3 is associated with tumour progression and a poor prognosis in nasopharyngeal carcinomas and inhibits cell migration and invasion via suppressed AKT activity. J Pathol 2010; 222: 367–379.

    Article  CAS  PubMed  Google Scholar 

  29. Mitsiades CS, Mitsiades N, Koutsilieris M . The Akt pathway: molecular targets for anti-cancer drug development. Curr Cancer Drug Targets 2004; 4: 235–256.

    Article  CAS  PubMed  Google Scholar 

  30. Desbois-Mouthon C, Cadoret A, Blivet-Van Eggelpoel MJ, Bertrand F, Cherqui G, Perret C et al. Insulin and IGF-1 stimulate the beta-catenin pathway through two signalling cascades involving GSK-3beta inhibition and Ras activation. Oncogene 2001; 20: 252–259.

    Article  CAS  PubMed  Google Scholar 

  31. Doble BW, Woodgett JR . Role of glycogen synthase kinase-3 in cell fate and epithelial-mesenchymal transitions. Cells Tissues Organs 2007; 185: 73–84.

    Article  CAS  PubMed  Google Scholar 

  32. Cappuzzo F, Tallini G, Finocchiaro G, Wilson RS, Ligorio C, Giordano L et al. Insulin-like growth factor receptor 1 (IGF1R) expression and survival in surgically resected non-small-cell lung cancer (NSCLC) patients. Ann Oncol 2010; 21: 562–567.

    Article  CAS  PubMed  Google Scholar 

  33. Engelman JA, Janne PA, Mermel C, Pearlberg J, Mukohara T, Fleet C et al. ErbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines. Proc Natl Acad Sci USA 2005; 102: 3788–3793.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Brognard J, Clark AS, Ni Y, Dennis PA . Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res 2001; 61: 3986–3997.

    CAS  PubMed  Google Scholar 

  35. Morrison R, Schleicher SM, Sun Y, Niermann KJ, Kim S, Spratt DE et al. Targeting the mechanisms of resistance to chemotherapy and radiotherapy with the cancer stem cell hypothesis. J Oncol 2011; 2011: 941876.

    Article  PubMed  Google Scholar 

  36. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 2007; 1: 555–567.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ucar D, Cogle CR, Zucali JR, Ostmark B, Scott EW, Zori R et al. Aldehyde dehydrogenase activity as a functional marker for lung cancer. Chem Biol Interact 2009; 178: 48–55.

    Article  CAS  PubMed  Google Scholar 

  38. Jiang T, Collins BJ, Jin N, Watkins DN, Brock MV, Matsui W et al. Achaete-scute complex homologue 1 regulates tumor-initiating capacity in human small cell lung cancer. Cancer Res 2009; 69: 845–854.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ischenko I, Seeliger H, Schaffer M, Jauch KW, Bruns CJ . Cancer stem cells: how can we target them? Curr Med Chem 2008; 15: 3171–3184.

    Article  CAS  PubMed  Google Scholar 

  40. Gorelik E, Lokshin A, Levina V . Lung cancer stem cells as a target for therapy. Anticancer Agents Med Chem 2010; 10: 164–171.

    Article  CAS  PubMed  Google Scholar 

  41. McCubrey JA, Steelman LS, Abrams SL, Misaghian N, Chappell WH, Basecke J et al. Targeting the cancer initiating cell: the ultimate target for cancer therapy. Curr Pharm Des 2012; 18: 1784–1795.

    Article  CAS  PubMed  Google Scholar 

  42. Prud'homme GJ . Cancer stem cells and novel targets for antitumor strategies. Curr Pharm Des 2012; 18: 2838–2849.

    Article  CAS  PubMed  Google Scholar 

  43. Hu B, Nandhu MS, Sim H, Agudelo-Garcia PA, Saldivar JC, Dolan CE et al. Fibulin-3 promotes glioma growth and resistance through a novel paracrine regulation of Notch signaling. Cancer Res 2012; 72: 3873–3885.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Camaj P, Seeliger H, Ischenko I, Krebs S, Blum H, De Toni EN et al. EFEMP1 binds the EGF receptor and activates MAPK and Akt pathways in pancreatic carcinoma cells. Biol Chem 2009; 390: 1293–1302.

    Article  CAS  PubMed  Google Scholar 

  45. Wang R, Zhang YW, Chen LB . Aberrant promoter methylation of FBLN3 gene and clinicopathological significance in non-small cell lung carcinoma. Lung Cancer 2010; 69: 239–244.

    Article  PubMed  Google Scholar 

  46. Pollak M . Insulin and insulin-like growth factor signalling in neoplasia. Nat Rev Cancer 2008; 8: 915–928.

    Article  CAS  PubMed  Google Scholar 

  47. Yang J, Ylipaa A, Sun Y, Zheng H, Chen K, Nykter M et al. Genomic and molecular characterization of malignant peripheral nerve sheath tumor identifies the IGF1R pathway as a primary target for treatment. Clin Cancer Res 2011; 17: 7563–7573.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ivanova NB, Dimos JT, Schaniel C, Hackney JA, Moore KA, Lemischka IR . A stem cell molecular signature. Science 2002; 298: 601–604.

    Article  CAS  PubMed  Google Scholar 

  49. Bendall SC, Stewart MH, Menendez P, George D, Vijayaragavan K, Werbowetski-Ogilvie T et al. IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature 2007; 448: 1015–1021.

    Article  CAS  PubMed  Google Scholar 

  50. Jones RA, Campbell CI, Wood GA, Petrik JJ, Moorehead RA . Reversibility and recurrence of IGF-IR-induced mammary tumors. Oncogene 2009; 28: 2152–2162.

    Article  CAS  PubMed  Google Scholar 

  51. Shan J, Shen J, Liu L, Xia F, Xu C, Duan G et al. Nanog regulates self-renewal of cancer stem cells through the insulin-like growth factor pathway in human hepatocellular carcinoma. Hepatology 2012; 56: 1004–1014.

    Article  CAS  PubMed  Google Scholar 

  52. Bill A, Schmitz A, Konig K, Heukamp LC, Hannam JS, Famulok M . Anti-proliferative effect of cytohesin inhibition in gefitinib-resistant lung cancer cells. PloS One 2012; 7: e41179.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Desbois-Mouthon C, Baron A, Blivet-Van Eggelpoel MJ, Fartoux L, Venot C, Bladt F et al. Insulin-like growth factor-1 receptor inhibition induces a resistance mechanism via the epidermal growth factor receptor/HER3/AKT signaling pathway: rational basis for cotargeting insulin-like growth factor-1 receptor and epidermal growth factor receptor in hepatocellular carcinoma. Clin Cancer Res 2009; 15: 5445–5456.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We acknowledge the financial supports from the Ministry of Science, ICT & Future Planning (Nuclear Research and Development Program) of the Republic of Korea and basic program of Korea Atomic Energy Research Institute.

Author information

Authors and Affiliations

  1. Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, (KAERI), Daejeon, Korea

    I G Kim, S Y Kim, S I Choi, J H Lee & K C Kim

  2. Department of Radiation Biotechnology and Applied Radioisotope, University of Science and technology (UST), Daejeon, Korea

    I G Kim, S I Choi & J H Lee

  3. Cancer Biomarkers Development Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea

    E W Cho

Authors
  1. I G Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S Y Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S I Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J H Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. K C Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. E W Cho
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to I G Kim.

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

Kim, I., Kim, S., Choi, S. et al. Fibulin-3-mediated inhibition of epithelial-to-mesenchymal transition and self-renewal of ALDH+ lung cancer stem cells through IGF1R signaling. Oncogene 33, 3908–3917 (2014). https://doi.org/10.1038/onc.2013.373

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2013.373

Keywords

This article is cited by

Search

Advanced search

Quick links