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.

  • Article
  • Published:

ANO1 regulates the maintenance of stemness in glioblastoma stem cells by stabilizing EGFRvIII

Oncogene volume 40, pages 1490–1502 (2021)Cite this article

Subjects

Abstract

Glioblastoma multiforme (GBM) or glioblastoma is the most deadly malignant brain tumor in adults. GBM is difficult to treat mainly due to the presence of glioblastoma stem cells (GSCs). Epidermal growth factor receptor variant III (EGFRvIII) has been linked to stemness and malignancy of GSCs; however, the regulatory mechanism of EGFRvIII is largely unknown. Here, we demonstrated that Anoctamin-1 (ANO1), a Ca2+-activated Cl– channel, interacts with EGFRvIII, increases its protein stability, and supports the maintenance of stemness and tumor progression in GSCs. Specifically, shRNA-mediated knockdown and pharmacological inhibition of ANO1 suppressed the self-renewal, invasion activities, and expression of EGFRvIII and related stem cell factors, including NOTCH1, nestin, and SOX2 in GSCs. Conversely, ANO1 overexpression enhanced the above phenomena. Mechanistically, ANO1 protected EGFRvIII from proteasomal degradation by directly binding to it. ANO1 knockdown significantly increased survival in mice and strongly suppressed local invasion of GSCs in an in vivo intracranial mouse model. Collectively, these results suggest that ANO1 plays a crucial role in the maintenance of stemness and invasiveness of GSCs by regulating the expression of EGFRvIII and related signaling molecules, and can be considered a promising therapeutic target for GBM treatment.

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

Fig. 1: ANO1 is preferentially expressed in GSCs.
Fig. 2: Knockdown of ANO1 suppressed GSC activities.
Fig. 3: Pharmacological inhibition of ANO1 suppressed GSC activities.
Fig. 4: Effect of ANO1 on the expression of stemness factors in GSCs.
Fig. 5: Overexpression of ANO1 increases GSC activities.
Fig. 6: ANO1 regulates EGFRvIII at the protein level, but not at the RNA level.
Fig. 7: ANO1 interacts with EGFRvIII and regulates its stability in GSCs.
Fig. 8: Knockdown of ANO1 enhances survival of intracranial GSC mice models.

Similar content being viewed by others

References

  1. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl J Med. 2005;352:987–96.

    Article  CAS  PubMed  Google Scholar 

  2. Bleeker FE, Molenaar RJ, Leenstra S. Recent advances in the molecular understanding of glioblastoma. J Neurooncol. 2012;108:11–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Furnari FB, Fenton T, Bachoo RM, Mukasa A, Stommel JM, Stegh A, et al. Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev. 2007;21:2683–710.

    Article  CAS  PubMed  Google Scholar 

  4. Gallego O. Nonsurgical treatment of recurrent glioblastoma. Curr Oncol. 2015;22:e273–e281.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Murat A, Migliavacca E, Gorlia T, Lambiv WL, Shay T, Hamou MF, et al. Stem cell-related “Self-renewal” signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma. J Clin Oncol. 2008;26:3015–24.

    Article  CAS  PubMed  Google Scholar 

  6. Jordan CT, Guzman ML, Noble M. Cancer stem cells. N. Engl J Med. 2006;355:1253–61.

    Article  CAS  PubMed  Google Scholar 

  7. Hatanpaa KJ, Burma S, Zhao D, Habib AA. Epidermal growth factor receptor in glioma: signal transduction, neuropathology, imaging, and radioresistance. Neoplasia. 2010;12:675–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Mukherjee B, McEllin B, Camacho CV, Tomimatsu N, Sirasanagandala S, Nannepaga S, et al. EGFRvIII and DNA double-strand break repair: a molecular mechanism for radioresistance in glioblastoma. Cancer Res. 2009;69:4252–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. de la Iglesia N, Puram SV, Bonni A. STAT3 regulation of glioblastoma pathogenesis. Curr Mol Med. 2009;9:580–90.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Yin J, Park G, Kim TH, Hong JH, Kim YJ, Jin X, et al. Pigment epithelium-derived factor (PEDF) expression induced by EGFRvIII promotes self-renewal and tumor progression of glioma stem cells. PLoS Biol. 2015;13:e1002152.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Huang PH, Mukasa A, Bonavia R, Flynn RA, Brewer ZE, Cavenee WK, et al. Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma. Proc Natl Acad Sci USA. 2007;104:12867–72.

    Article  CAS  PubMed  Google Scholar 

  12. Del Vecchio CA, Giacomini CP, Vogel H, Jensen KC, Florio T, Merlo A, et al. EGFRvIII gene rearrangement is an early event in glioblastoma tumorigenesis and expression defines a hierarchy modulated by epigenetic mechanisms. Oncogene. 2013;32:2670–81.

    Article  PubMed  Google Scholar 

  13. Emlet DR, Gupta P, Holgado-Madruga M, Del Vecchio CA, Mitra SS, Han SY, et al. Targeting a glioblastoma cancer stem-cell population defined by EGF receptor variant III. Cancer Res. 2014;74:1238–49.

    Article  CAS  PubMed  Google Scholar 

  14. Schroeder BC, Cheng T, Jan YN, Jan LY. Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell. 2008;134:1019–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Caputo A, Caci E, Ferrera L, Pedemonte N, Barsanti C, Sondo E, et al. TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science. 2008;322:590–4.

    Article  CAS  PubMed  Google Scholar 

  16. Yang YD, Cho H, Koo JY, Tak MH, Cho Y, Shim WS, et al. TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature. 2008;455:1210–5.

    Article  CAS  PubMed  Google Scholar 

  17. Huang F, Zhang H, Wu M, Yang H, Kudo M, Peters CJ, et al. Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction. Proc Natl Acad Sci USA. 2012;109:16354–9.

    Article  CAS  PubMed  Google Scholar 

  18. Ma MM, Gao M, Guo KM, Wang M, Li XY, Zeng XL, et al. TMEM16A contributes to endothelial dysfunction by facilitating Nox2 NADPH oxidase-derived reactive oxygen species generation in hypertension. Hypertension. 2017;69:892–901.

    Article  CAS  PubMed  Google Scholar 

  19. Cho H, Yang YD, Lee J, Lee B, Kim T, Jang Y, et al. The calcium-activated chloride channel anoctamin 1 acts as a heat sensor in nociceptive neurons. Nat Neurosci. 2012;15:1015–21.

    Article  CAS  PubMed  Google Scholar 

  20. Oh U, Jung J. Cellular functions of TMEM16/anoctamin. Pflug Arch. 2016;468:443–53.

    Article  CAS  Google Scholar 

  21. Pedemonte N, Galietta LJ. Structure and function of TMEM16 proteins (anoctamins). Physiol Rev. 2014;94:419–59.

    Article  CAS  PubMed  Google Scholar 

  22. Duvvuri U, Shiwarski DJ, Xiao D, Bertrand C, Huang X, Edinger RS, et al. TMEM16A induces MAPK and contributes directly to tumorigenesis and cancer progression. Cancer Res. 2012;72:3270–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Liu F, Cao QH, Lu DJ, Luo B, Lu XF, Luo RC, et al. TMEM16A overexpression contributes to tumor invasion and poor prognosis of human gastric cancer through TGF-beta signaling. Oncotarget. 2015;6:11585–99.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Britschgi A, Bill A, Brinkhaus H, Rothwell C, Clay I, Duss S, et al. Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling. Proc Natl Acad Sci USA. 2013;110:E1026–E1034.

    Article  CAS  PubMed  Google Scholar 

  25. Sui Y, Sun M, Wu F, Yang L, Di W, Zhang G, et al. Inhibition of TMEM16A expression suppresses growth and invasion in human colorectal cancer cells. PLoS One. 2014;9:e115443.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Liu W, Lu M, Liu B, Huang Y, Wang K. Inhibition of Ca2+-activated Cl– channel ANO1/TMEM16A expression suppresses tumor growth and invasiveness in human prostate carcinoma. Cancer Lett. 2012;326:41–51.

    Article  CAS  PubMed  Google Scholar 

  27. Liu J, Liu Y, Ren Y, Kang L, Zhang L. Transmembrane protein with unknown function 16A overexpression promotes glioma formation through the nuclear factor-kappaB signaling pathway. Mol Med Rep. 2014;9:1068–74.

    Article  CAS  PubMed  Google Scholar 

  28. Bill A, Gutierrez A, Kulkarni S, Kemp C, Bonenfant D, Voshol H, et al. ANO1/TMEM16A interacts with EGFR and correlates with sensitivity to EGFR-targeting therapy in head and neck cancer. Oncotarget. 2015;6:9173–88.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Lee YS, Lee JK, Bae Y, Lee BS, Kim E, Cho CH, et al. Suppression of 14-3-3gamma-mediated surface expression of ANO1 inhibits cancer progression of glioblastoma cells. Sci Rep. 2016;6:26413.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lee YS, Bae Y, Park N, Yoo JC, Cho CH, Ryoo K, et al. Surface expression of the anoctamin-1 (ANO1) channel is suppressed by protein-protein interactions with β-COP. Biochem Biophys Res Commun. 2016;475:216–22.

    Article  CAS  PubMed  Google Scholar 

  31. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003;63:5821–8.

    CAS  PubMed  Google Scholar 

  32. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 2006;444:756–60.

    Article  CAS  PubMed  Google Scholar 

  33. Bill A, Hall ML, Borawski J, Hodgson C, Jenkins J, Piechon P, et al. Small molecule-facilitated degradation of ANO1 protein: a new targeting approach for anticancer therapeutics. J Biol Chem. 2014;289:11029–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Huang X, Gollin SM, Raja S, Godfrey TE. High-resolution mapping of the 11q13 amplicon and identification of a gene, TAOS1, that is amplified and overexpressed in oral cancer cells. Proc Natl Acad Sci USA. 2002;99:11369–74.

    Article  CAS  PubMed  Google Scholar 

  35. Carles A, Millon R, Cromer A, Ganguli G, Lemaire F, Young J, et al. Head and neck squamous cell carcinoma transcriptome analysis by comprehensive validated differential display. Oncogene. 2006;25:1821–31.

    Article  CAS  PubMed  Google Scholar 

  36. Katoh M, Katoh M. FLJ10261 gene, located within the CCND1-EMS1 locus on human chromosome 11q13, encodes the eight-transmembrane protein homologous to C12orf3, C11orf25 and FLJ34272 gene products. Int J Oncol. 2003;22:1375–81.

    CAS  PubMed  Google Scholar 

  37. Carneiro A, Isinger A, Karlsson A, Johansson J, Jonsson G, Bendahl PO, et al. Prognostic impact of array-based genomic profiles in esophageal squamous cell cancer. BMC Cancer. 2008;8:98.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Wang H, Zou L, Ma K, Yu J, Wu H, Wei M, et al. Cell-specific mechanisms of TMEM16A Ca2+-activated chloride channel in cancer. Mol Cancer. 2017;16:1–17.

    Article  Google Scholar 

  39. Crottes D, Jan LY. The multifaceted role of TMEM16A in cancer. Cell Calcium. 2019;82:102050.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. De La Fuente R, Namkung W, Mills A, Verkman AS. Small-molecule screen identifies inhibitors of a human intestinal calcium-activated chloride channel. Mol Pharm. 2008;73:758–68.

    Article  Google Scholar 

  41. Sauter DR, Novak I, Pedersen SF, Larsen EH, Hoffmann EK. ANO1 (TMEM16A) in pancreatic ductal adenocarcinoma (PDAC). Pflug Arch. 2015;467:1495–508.

    Article  CAS  Google Scholar 

  42. Simon S, Grabellus F, Ferrera L, Galietta L, Schwindenhammer B, Muhlenberg T, et al. DOG1 regulates growth and IGFBP5 in gastrointestinal stromal tumors. Cancer Res. 2013;73:3661–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Shiwarski DJ, Shao C, Bill A, Kim J, Xiao D, Bertrand CA, et al. To “grow” or “go”: TMEM16A expression as a switch between tumor growth and metastasis in SCCHN. Clin Cancer Res. 2014;20:4673–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Eskilsson E, Rosland GV, Talasila KM, Knappskog S, Keunen O, Sottoriva A, et al. EGFRvIII mutations can emerge as late and heterogenous events in glioblastoma development and promote angiogenesis through Src activation. Neuro Oncol. 2016;18:1644–55.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wang H, Yao F, Luo S, Ma K, Liu M, Bai L, et al. A mutual activation loop between the Ca2+-activated chloride channel TMEM16A and EGFR/STAT3 signaling promotes breast cancer tumorigenesis. Cancer Lett. 2019;455:48–59.

    Article  CAS  PubMed  Google Scholar 

  46. Crottès D, Lin YH, Peters CJ, Gilchrist JM, Wiita AP, Jan YN, et al. TMEM16A controls EGF-induced calcium signaling implicated in pancreatic cancer prognosis. Proc Natl Acad Sci USA. 2019;116:13026–35.

    Article  PubMed  Google Scholar 

  47. Ji Q, Guo S, Wang X, Pang C, Zhan Y, Chen Y, et al. Recent advances in TMEM16A: structure, function, and disease. J Cell Physiol. 2019;234:7856–73.

    Article  CAS  PubMed  Google Scholar 

  48. Inda MM, Bonavia R, Mukasa A, Narita Y, Sah DW, Vandenberg S, et al. Tumor heterogeneity is an active process maintained by a mutant EGFR-induced cytokine circuit in glioblastoma. Genes Dev. 2010;24:1731–45.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Giese A, Bjerkvig R, Berens ME, Westphal M. Cost of migration: invasion of malignant gliomas and implications for treatment. J Clin Oncol. 2003;21:1624–36.

    Article  CAS  PubMed  Google Scholar 

  50. Cheng L, Wu Q, Guryanova OA, Huang Z, Huang Q, Rich JN, et al. Elevated invasive potential of glioblastoma stem cells. Biochem Biophys Res Commun. 2011;406:643–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the Bio-Synergy Research Project (NRF-2017M3A9C4092979 to JYP) of the National Research Foundation of Korea, and by a grant from the Korea Institute of Radiological and Medical Sciences (KIRAMS), funded by the Ministry of Science and ICT (MSIT), the Republic of Korea (No. 50531-2019 to MJP).

Author information

Author notes

  1. These authors contributed equally: Hee-Jin Kim, Jeong-Yub Kim

Authors and Affiliations

  1. Radiation Therapeutics Development Team, Division of Radiation Cancer Science, Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea

    Hee-Jin Kim, Jeong-Yub Kim, Chan-Woong Jung & Myung-Jin Park

  2. School of Biosystems and Biomedical Sciences, College of Health Sciences, Korea University, Seoul, Republic of Korea

    Hee-Jin Kim, Young-Sun Lee, Joon-Yong An & Jae-Yong Park

  3. Department of Life Sciences, Korea University, Seoul, Republic of Korea

    Chan-Woong Jung

  4. Department of Biochemistry, School of Medicine, Daegu Catholic University, Daegu, Republic of Korea

    Eun Ho Kim

  5. Department of Neurosurgery, Daegu Catholic University School of Medicine, Daegu, Republic of Korea

    Ki-Hong Kim

  6. Department of Neurosurgery, Halla General Hospital, Jeju, Republic of Korea

    Sang Pyung Lee

Authors
  1. Hee-Jin Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeong-Yub Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chan-Woong Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Young-Sun Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joon-Yong An
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eun Ho Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ki-Hong Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sang Pyung Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jae-Yong Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Myung-Jin Park
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jae-Yong Park or Myung-Jin Park.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, HJ., Kim, JY., Jung, CW. et al. ANO1 regulates the maintenance of stemness in glioblastoma stem cells by stabilizing EGFRvIII. Oncogene 40, 1490–1502 (2021). https://doi.org/10.1038/s41388-020-01612-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41388-020-01612-5

Search

Advanced search

Quick links