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

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.

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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.

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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).

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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

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