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EAG2 potassium channel with evolutionarily conserved function as a brain tumor target

Nature Neuroscience volume 18, pages 1236–1246 (2015)Cite this article

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Abstract

Over 20% of the drugs for treating human diseases target ion channels, but no cancer drug approved by the US Food and Drug Administration (FDA) is intended to target an ion channel. We found that the EAG2 (Ether-a-go-go 2) potassium channel has an evolutionarily conserved function for promoting brain tumor growth and metastasis, delineate downstream pathways, and uncover a mechanism for different potassium channels to functionally cooperate and regulate mitotic cell volume and tumor progression. EAG2 potassium channel was enriched at the trailing edge of migrating medulloblastoma (MB) cells to regulate local cell volume dynamics, thereby facilitating cell motility. We identified the FDA-approved antipsychotic drug thioridazine as an EAG2 channel blocker that reduces xenografted MB growth and metastasis, and present a case report of repurposing thioridazine for treating a human patient. Our findings illustrate the potential of targeting ion channels in cancer treatment.

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Figure 1: Drosophila Eag channel deficiency reduces brain tumor growth and metastasis.
Figure 2: Cross-species transcriptomic studies identified KCNT2 as a potassium channel that is enriched in SHH-MB and regulates tumor growth.
Figure 3: KCNT2 and EAG2 functionally cooperate to regulate mitotic cell volume.
Figure 4: EAG2 channel promotes MB metastasis and enriches to the trailing edge to regulate MB cell motility.
Figure 5: Thioridazine is an EAG2 blocker that reduces MB cell viability and mobility.
Figure 6: Thioridazine inhibition of EAG2 channel reduces intracranial MB growth and metastasis.
Figure 7: A subset of metastasized MB tumors display EAG2 upregulation compared with matched primary tumors.
Figure 8: A case report of thioridazine treatment of a patient with metastatic SHH-MB.

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Acknowledgements

We thank Y. Song, W.-P. Ge, S.-B. Yang, H. Yang, C. Peters, B. Piggott, S. Morrissy, D. Shih, B.K. Shoichet and all of the members of the Jan laboratory for constructive suggestions. We thank C. Chiang for critical reading of the manuscript. This work was supported by the GEMS-CTSI postdoctoral award from the Howard Hughes Medical Institute and UCSF, and the Damon Runyon Cancer Research Foundation Fellowship to X.H. (Kandis Ann Ulrich-Carleton Fellow), NIH grant P41 GM103504 to G.D.B., the Pediatric Brain Tumor Foundation and R01 grants CA133091, CA148699 and CA159859 to W.A.W. and M.D.T., the Garron Family Chair in Childhood Cancer Research at The Hospital for Sick Children and The University of Toronto, operating funds from the Canadian Institutes of Health Research, the Terry Fox Research Institute, and the Pediatric Brain Tumor Foundation to M.D.T., NIH grants R37NS040929 to Y.N.J., and R37MH065334 and R01CA185039 to L.Y.J. Y.N.J. and L.Y.J. are Howard Hughes Medical Institute investigators.

Author information

Author notes

  1. Xi Huang, Jüri Reimand, Huanghe Yang & Samantha J Stehbens

    Present address: Present address: Developmental and Stem Cell Program, Arthur and Sonia Labatt Brain Tumor Research Centre, Hospital for Sick Children, Toronto, Ontario, Canada, Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada (X.H.); Ontario Institute for Cancer Research, Toronto, Ontario, Canada, and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada (J.R.); Department of Biochemistry and Ion Channel Research Unit, Duke University Medical Center, Durham, North Carolina, USA (H.Y.); Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology, Translational Research Institute, Woolloongabba, Queensland, Australia (S.J.S.).,

  2. Xi Huang, Ye He and Adrian M Dubuc: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physiology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California, USA

    Xi Huang, Ye He, Wei Zhang, Huanghe Yang, Tongfei A Wang, Susan Younger, Suzanne Barshow, Sijun Zhu, Yuh Nung Jan & Lily Yeh Jan

  2. Department of Biophysics and Biochemistry, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California, USA

    Xi Huang, Ye He, Wei Zhang, Huanghe Yang, Tongfei A Wang, Susan Younger, Suzanne Barshow, Sijun Zhu, Yuh Nung Jan & Lily Yeh Jan

  3. Developmental and Stem Cell Program, Arthur and Sonia Labatt Brain Tumor Research Centre, Hospital for Sick Children, Toronto, Ontario, Canada

    Adrian M Dubuc, John Peacock, Vijay Ramaswamy, Livia Garzia, Xiaochong Wu, Marc Remke & Michael D Taylor

  4. Department of Neurological Surgery, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA

    Rintaro Hashizume & C David James

  5. The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada

    Jüri Reimand & Gary D Bader

  6. Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, California, USA

    Samantha J Stehbens

  7. Department of Neurology, Vanderbilt University Medical Center, Nashville, Tennessee, USA

    Michael K Cooper

  8. Department of Pediatrics, University of California, San Francisco, San Francisco, California, USA

    Craig M Forester & William A Weiss

  9. Division of Experimental Medicine, Department of Medicine, University of California, San Francisco, San Francisco, California, USA

    Charles C Kim

  10. Department of Neurological Surgery and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA

    William A Weiss

  11. Department of Neurology and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA

    William A Weiss & Sabine Mueller

  12. Department of Medicine and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA

    Marc A Shuman

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  1. Xi Huang
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Contributions

X.H. and L.Y.J. conceived and designed the study. X.H., Y.H., A.M.D., R.H., W.Z., J.R., H.Y., T.A.W., S.J.S., S.Y., S.B., S.Z., M.K.C., J.P., V.R., L.G., X.W., M.R., C.M.F., C.C.K., G.D.B. and S.M. contributed to methodology and data acquisition. X.H., Y.H., A.M.D., W.Z., J.R., S.Y., J.P., V.R., C.M.F., C.C.K., W.A.W., C.D.J., M.A.S., G.D.B., S.M., M.D.T., Y.N.J. and L.Y.J. analyzed and interpreted the data. X.H., Y.N.J. and L.Y.J. wrote and revised the manuscript.

Corresponding authors

Correspondence to Yuh Nung Jan or Lily Yeh Jan.

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Integrated supplementary information

Supplementary Figure 1 eag loss-of-function mutation reduces the growth of brain tumors induced by reduction of tumor suppressor Brat activity, and transplantation assay to study Drosophila brain tumor metastasis

a, External view of the control brains, insc-Gal4-driven bratRNAi-induced brain tumors and bratRNAi-induced brain tumors with eag loss-of-function (eag1) at 25oC (n = 9 and 8 for bratRNAi and bratRNAi; eag1, respectively, P = 0.0002, two-tailed Student’s t-test) and 29oC (n = 10 and 10 for bratRNAi and bratRNAi; eag1, respectively, P < 0.0001, two-tailed Student’s t-test). b, GFP-labeled, dpn-overexpressing primary brain lobe tumors were retrieved from 3rd instar larvae. The tumors were micro-dissected into small pieces with equal volumes. Young female adult hosts were anesthetized with CO2 and a piece of larval brain was picked up with the tip of a glass needle and injected tangentially into the mid-ventral part of the abdomen. Implanted hosts were kept at 25°C for 10 days and monitored for tumor growth in the abdomen and metastasis into the brain. Tumor formation or metastasis was detected in hosts transplanted with dpn-overexpressing tumor cells, and the tumor-transplanted hosts displayed significantly shortened survival compared to the hosts transplanted with normal brain cells retrieved from w- control larvae (n = 12 and 20 for control and dpnO/E, respectively, log-rank Kaplan-Meier test).

Supplementary Figure 2 Mutation status of KCNT2 across different cancer types and analysis of KCNT2 knockdown in MB cells

a, Analysis of large-scale cancer genomics datasets shows frequent KCNT2 gene amplification and mutations in different cancer types. Data analysis and visualization are performed using the cBioPortal Cancer Genomics website (http://www.cbioportal.org). b, Lentivirus-mediated RNAi knockdown of KCNT2 reduces the clonogenic growth of MB cells. c, KCNT2 RNAi treatment in MB cells significantly reduces KCNT2 transcript level (n = 3 independent cultures and qPCR experiments, two-tailed Student’s t-test). Error bars show ± SEM.

Supplementary Figure 3 Working model: co-option of potassium channel activity regulates mitotic cell volume in MB

Left: Potassium channel activity is increased in MB cells during late G2 and mitotic phases, which regulates cell volume reduction. Right: EAG2, which localizes to the plasma membrane at late G2 and during mitosis, regulates KCNT2 mRNA expression. KCNT2 channel enriches to the plasma membrane beginning at metaphase to cooperate with EAG2 and regulate mitotic volume dynamics. EAG2 channel block by astemizole or thioridazine, or indirect inhibition of KCNT2 channel using riluzole, represents viable strategy to inhibit MB tumor growth by targeting potassium channels.

Supplementary Figure 4 EAG2 channel localizes to the trailing membrane during MB cell migration and regulates migratory polarity and cell motility

Immunofluorescence staining using EAG2 antibodies obtained from different commercial sources shows cell rear and trailing membrane enrichment during migration of multiple human MB cell lines, including SF8953-MB, DAOY, Vandy-MB-11, Vandy-MB-6 (at passage 3) and Vandy-MB-10 (at passage 3) cells. White arrows indicate migratory direction.

Supplementary Figure 5 KCNT2 channel does not enrich to the trailing edge during MB cell migration and does not regulate migratory polarity or cell motility

a, EAG2, but not KCNT2, displays cell rear and trailing edge enrichment during MB cell migration. b, KCNT2 knockdown does not considerably affect MB cell migratory polarization or the formation of lamellipodia (n = 482 and 391 randomly selected cells for Scr. shRNA and KCNT2 shRNA, respectively). c, 3-D reconstruction shows rear cell body contraction in control Vandy-MB-11 cells and cells with KCNT2 knockdown. d, Control Vandy-MB-11 cells and cells with KCNT2 knockdown show comparable migratory velocity (n = 20 for each group, two-tailed Student’s t-test). Each colored trace represents the migratory route of an individual Vandy-MB-11 cell over 5 hours. e, 5 μM thioridazine treatment for 3 days results in significantly reduced KCNT2 expression in Vandy-MB-11 cells (n = 4 independent cultures and qPCR experiments, two-tailed Student’s t-test). Error bars show ± SEM.

Supplementary Figure 6 Promazine blocks EAG2 channel and inhibits MB cell growth

a, Chemical structure of promazine. b, Promazine treatment markedly inhibits MB cell growth at 10 μM. c, Patch clamp recording shows that promazine significantly blocks EAG2 channel at 10 μM (n = 3, two-tailed Student’s t-test). Error bars show ± SEM.

Supplementary Figure 7 Working model: localized EAG2 potassium channel activity at the trailing edge regulates MB cell motility

EAG2 channel enriched at the trailing edge regulates local potassium efflux, which accompanies water loss to promote cell rear contraction and cell motility.

Supplementary Figure 8 Astemizole treatment of mice bearing subcutaneously allografted Math1-Cre; SmoM2 MB tumors

a, Astemizole treatment reduces MB cell clonal growth as neurospheres (Math1-Cre; SmoM2 cells) or monolayer colonies (Vandy-MB-11, DAOY and Ptch1+/-; p53-/- cells). b, Experimental design for acute allografting of Math1-Cre; SmoM2 MB tumors into the flanks of nu/nu BALB/C mice and astemizole treatment by oral gavage. Once palpable tumors are detected one-week post implantation, daily oral gavage of 50 mg/kg astemizole to the mice was performed for two weeks. c, 14 days of daily oral gavage of astemizole reduces proliferation and increases apoptosis in subcutaneously allografted Math1-Cre; SmoM2 tumors. d, Quantifications of tumor volumes show significantly inhibited tumor growth with astemizole treatment (n = 9 for vehicle and astemizole group, Two-Way ANOVA). Error bars show ± SEM.

Supplementary Figure 9 Full-length Western blotting images for cropped images shown in Fig. 6b

The full-length Western blotting images for the cropped images in Fig. 6b are shown with molecular weights indicated by protein ladder. The same membrane was first blotted with EAG2 antibody, followed by antibody stripping and re-blotting with α tubulin antibody. Chemiluminescence was imaged using Molecular Imager@VersaDoc MP4000 system (Bio-Rad).

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Huang, X., He, Y., Dubuc, A. et al. EAG2 potassium channel with evolutionarily conserved function as a brain tumor target. Nat Neurosci 18, 1236–1246 (2015). https://doi.org/10.1038/nn.4088

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