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Sonic Hedgehog promotes proliferation of Notch-dependent monociliated choroid plexus tumour cells

Nature Cell Biology volume 18, pages 418–430 (2016)Cite this article

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Abstract

Aberrant Notch signalling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly paediatric brain neoplasms. We developed animal models of CP tumours, by inducing sustained expression of Notch1, that recapitulate properties of human CP tumours with aberrant NOTCH signalling. Whole-transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate differentiation. A Shh-driven signalling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from monociliated progenitors in the roof plate characterized by elevated Notch signalling. Abnormal SHH signalling and distinct ciliogenesis are detected in human CP tumours, suggesting the SHH pathway and cilia differentiation as potential therapeutic avenues.

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Figure 1: Constitutive Notch1 signalling leads to the development of CP tumours.
Figure 2: Notch-induced CP tumours undergo enhanced proliferation and exhibit defects in differentiation.
Figure 3: Notch-induced CP tumours exhibit aberrant Shh signalling.
Figure 4: Shh drives the proliferation of Notch-induced CP tumours.
Figure 5: Notch-induced CP tumours possess a solitary primary cilium.
Figure 6: Monociliated CP tumour cells are uniquely capable of transducing Shh signals.
Figure 7: Notch-induced CP tumours arise from progenitors in the roof plate.
Figure 8: Notch-induced CP tumour cells retain properties of roof plate progenitors.

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Acknowledgements

We thank all members of the laboratory for helpful discussions. We are grateful to K. Millen (Seattle Children’s Hospital Research Institute, USA), J. Kim (University of Texas Southwestern Medical Center, USA) and R. Kageyama (Institute for Virus Research Kyoto University, Japan) for providing the Lmx1a–Cre transgenic mouse strain, Smo antibody, and Hes1 antibodies, respectively, and C. Eberhart (Johns Hopkins University School of Medicine, USA), M. Taylor (The Hospital for Sick Children, Canada) and S. Santagata (Boston Children’s Hospital, USA) for providing human CP tumour samples. We wish to acknowledge the Labatt Brain Tumour Research Centre Tumour and Tissue Repository, which is supported by b.r.a.i.n child and Meagan’s Walk. We are indebted to C. Evans, A. Kelsch and E. Grandprey for excellent technical assistance. We thank W.K. Miskimins and K. Surendran for helpful suggestions, and J. Tao and D. Maher for critical reading of the manuscript and helpful discussions. This project is supported by: Boston Children’s Hospital IDDRC P30 HD18655, Sanford Research, and Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under grant number 5P20GM103548 (Cancer), which also supports Cores at Sanford Research together with NIH grant 1P20GM103620-01A1 (Pediatrics). The RNA In Situ Hybridization Core facility at Baylor College of Medicine is supported by a Shared Instrumentation grant from the NIH (1S10OD016167). Additional support was provided by the National Brain Tumor Society (R.J.W.-R.).

Author information

Authors and Affiliations

  1. Children’s Health Research Center, Sanford Research, 2301 E 60th Street North, Sioux Falls, South Dakota 57104, USA

    Li Li, Katie B. Grausam & Haotian Zhao

  2. Division of Basic Biomedical Sciences, Sanford School of Medicine of the University of South Dakota, 414 E. Clark Street, Vermillion, South Dakota 57069, USA

    Katie B. Grausam & Haotian Zhao

  3. Tumor Initiation and Maintenance Programme, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, USA

    Jun Wang & Robert J. Wechsler-Reya

  4. Department of Pathology, Boston Children’s Hospital, Boston, Massachusetts 02115, USA

    Melody P. Lun, Hart G. W. Lidov, Monica L. Calicchio & Maria K. Lehtinen

  5. Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA

    Melody P. Lun

  6. Center for Neuropathology and Prion Research, Ludwig-Maximilans-University, 81377 Munich, Germany

    Jasmin Ohli & Ulrich Schüller

  7. Department of Biology, University of South Dakota, 414 E. Clark Street, Vermillion, South Dakota 57069, USA

    Erliang Zeng

  8. Department of Computer Science, University of South Dakota, 414 E. Clark Street, Vermillion, South Dakota 57069, USA

    Erliang Zeng

  9. Department of Biochemistry and Molecular Biology, Mayo Clinic (Guggenheim-14), 200 First Street SW., Rochester, Minnesota 55905, USA

    Jeffrey L. Salisbury

  10. Microscopy and Cell Analysis Core, Mayo Clinic (Guggenheim-14), 200 First Street SW., Rochester, Minnesota 55905, USA

    Jeffrey L. Salisbury

  11. Cancer Biology Research Center, Sanford Research, 2301 E 60th Street North, Sioux Falls, South Dakota 57104, USA

    Haotian Zhao

  12. Department of Pediatrics, Sanford School of Medicine of the University of South Dakota, 1400 W 22nd Street, Sioux Falls, South Dakota 57105, USA

    Haotian Zhao

  13. Department of Chemistry and Biochemistry, South Dakota State University, Avera Health Science Center (SAV) 131, Brookings, South Dakota 57007, USA

    Haotian Zhao

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  1. Li Li
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Contributions

L.L. and H.Z. conceived and planned the project, and wrote the manuscript. H.G.W.L. and M.L.C. reviewed diagnoses of human tissue samples. J.W., J.O., R.J.W.-R. and U.S. analysed morphological characters and gene expression patterns of human tumour samples. M.P.L. and M.K.L. performed cilia and gene expression analyses in human tissue samples. K.B.G. provided assistance with gene expression analysis. E.Z. conduced RNA-seq data processing and analysis. J.L.S. provided technical advice, support and data analysis for electron microscopy studies.

Corresponding author

Correspondence to Haotian Zhao.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Morphological and gene expression analysis of CPs in Mcre;NICD1 mice.

(a) EYFP expression (green, arrowheads) detected with a GFP antibody demarcates CP epithelial cells derived from Atoh1+ progenitors in Mcre;EYFP mice, while GFP expression (green) labels NICD1+ tumour cells in Mcre;NICD1 mice at P0, P7, P14, P21, and P90. The expression of Otx2 (red) marks CP epithelial cells. DAPI staining (blue) labels nuclei. Scale bar: 25 μm. (b) H&E staining of CPs from Mcre;NICD1 mice and wild type (WT) littermates at P0, P7, P14, and P21. Boxed regions of the abnormal growth of CP in the enlarged 4th ventricle of Mcre;NICD1 animals are shown in higher magnification. Notice that wild type CP epithelium exhibits the ‘hobnail’ configuration (arrowheads), while abnormal CP growth in Mcre;NICD1 mice displays a ‘flattened’ appearance on ventricular surfaces (arrow). Scale bars: white, 250 μm; black: 40 μm. (c) The expression of Hes1 (red) in CPs from Mcre;NICD1 mice. GFP expression (green) labels NICD1+ CP cells. Dotted lines mark the boundary between CP epithelial cells and NICD1+ cells. DAPI staining (blue) labels nuclei. Scale bar: 25 μm. (d) Western blot analysis of Hes1 expression in CP tumours from Mcre;NICD1 mice and wild type (WT) CPs at P0, P7 and P90. β-actin serves as loading control. Molecular size marker and representative image of unprocessed blots can be found in Supplementary Figure 9.

Supplementary Figure 2 Morphological analysis of CPs in Lcre;NICD1 mice.

(a) Bright field images of brain hemispheres in Lcre;NICD1mice and wild type (WT) littermates at P14. Notice that Lcre;NICD1 mice develop abnormal growth of CP (dotted red lines) and display enlarged lateral ventricle (arrows). Vesicular sacs with accumulated cerebrospinal fluid are shown (asterisks). Scale bar: 1 mm. (b) H&E staining of CPs in hindbrain and lateral ventricles of Lcre;NICD1 animals and wild type littermates at P7, P14, and P30. Notice that compared to CPs in wild type (WT) littermates (arrowheads), Lcre;NICD1 mice develop abnormal CP growth (arrows) and exhibit enlarged 4th and lateral ventricles. Vesicular sacs with accumulated cerebrospinal fluid are shown (asterisks). Scale bars: white, 100 μm; black: 300 μm.

Supplementary Figure 3 Analysis of proliferation and gene expression in CP tumours from Lcre;NICD1 mice.

(a) The expression of Ki-67 in tumour cells in the hindbrain and lateral ventricles of Lcre;NICD1 animals at P0, P7, P14, and P90, respectively. Dotted lines mark the boundary of tumours in lateral ventricles. Notice that the expression of Ki-67 in tumour cells is gradually reduced over time. Scale bar: 25 μ m. (b) The expression of Lmx1a, Otx2, Aqp1, and cytokeratins in CP tumours in the hindbrain and lateral ventricles of Lcre;NICD1 mice at P7. CPs at these sites from P7 wild type (WT) littermates serve as control. Dotted lines mark the boundary of the 4th and lateral ventricles. Notice that tumour cells display reduced expression of Aqp1 and cytokeratins. Scale bar: 25 μm.

Supplementary Figure 4 Analysis of CP tumours from Mcre;NICD1 mice and CPs of wild type (WT) mice.

(a) Results of 5-ethynyl-2’-deoxyuridine (EdU) incorporation assays are shown for tumours at P0, P7, and P14, respectively. EdU staining (magenta) labels cells in S-phase of the cell cycle. GFP expression (green) labels NICD1+ tumour cells. Dotted lines mark the boundary between GFP+ tumour cells and CP epithelium. DAPI staining (blue) labels nuclei. Scale bar: 25 μm. (b)The expression of cyclin D1 (Ccnd1) in tumours from Mcre;NICD1 mice at P0 and P14, respectively. Aqp1 expression (green) labels CP epithelium, while Ccnd1 expression (red) labels proliferating cells. Dotted lines mark the boundary between proliferative tumour cells and Aqp1+ epithelial cells. DAPI staining (blue) labels nuclei. Scale bar: 25 μm. (c) The presence of EdU-incorporating cells (magenta) and Cdkn1b+ (p27 Kip1, green) cells in tumours and WT at P7. Though tumours contain abundant cells positive for EdU or p27 expression, staining for EdU or p27 is mutually exclusive. Notice that p27+ or EdU-incorporating cells are absent in wild type CPs despite positive staining in neighboring cerebellum (Cb). Scale bar: 25 μm. (d) The expression of Cleaved Caspase-3 in CP tumours and WT CPs at P7. Cleaved Caspase-3 expression (red) is undetectable in tumour and CPs. DAPI staining (blue) marks nuclei. Scale bar: 25 μm. (e) Gene expression in WT CP epithelium at P7. The expression of Lmx1a (red), Otx2 (red), and Aqp1 (red) labels CP epithelial cells. DAPI staining (blue) marks nuclei. Scale bar: 25 μ m. (f) The expression of mesenchyme marker MafB in CPs from Mcre;NICD1 animals at P7. GFP (green) labels NICD1+ tumour cells. Dotted lines mark the boundary between GFP+ tumour cells and CP epithelium. MafB expression (red) is detected in normal CP tissue, but absent from GFP+ tumour cells. DAPI staining (blue) marks nuclei. Scale bar: 25 μm.

Supplementary Figure 5 Morphological and gene expression analysis of CPs from treated wild type animals.

H&E staining of CPs from wild type mice treated with either vismodegib or vehicle from day E15.5 for 4 days. The expression of Ki-67 (red) labels proliferating cells. Lmx1a expression (green) marks CP epithelial cells. DAPI staining (blue) labels nuclei. Notice that Ki-67+ proliferative cells are present in neighboring cerebellum (Cb); however, there is no Ki-67+ cells in CP epithelium. Scale bar: 25 μm.

Supplementary Figure 6 Transcriptome, epigenome, and genome analysis of human CP tumours.

(a) Hierarchical clustering of human CPPs and CPCs based on the expression of roof plate genes (CPP: n = 24 tumours from 24 individuals; CPC: n = 15 tumours from 15 individuals; one-way ANOVA, FDR < 0.05, fold change is shown). (b) MetaCore gene enrichment analysis of differentially methylated genes between human CPPs and CPCs (CPP: n = 5 tumours from 5 individuals; CPC: n = 15 tumours from 15 individuals). (c) Schematic illustration of results from MetaCore network analysis of differentially methylated genes between human CPPs and CPCs shown in (b). The red colored nodes indicate relatively higher expression and green nodes indicate relatively lower expression in CPPs compared to CPCs. (d) MetaCore gene enrichment analysis of copy number variations in human CP tumours (CPP: n = 37 tumours from 37 individuals; CPC: n = 38 tumours from 38 individuals). (e) Representative images show in-situ hybridization analysis of SHH expression in normal human CP (n = 5 CPs from 5 disease-free individuals) and human CP tumours (CPP: n = 11 tumour specimens from 10 individuals; CPC: n = 10 tumours from 10 individuals). Notice that SHH expression is present in normal hindbrain CP epithelium (arrowheads) and tumour tissue (arrows). Signal intensity varied among samples, likely as a result of brain regions examined, conditions under which specimens were collected and stored, and the length of time of specimens in storage. Representative images are shown. Scale bar: 50 μm.

Supplementary Figure 7 Analysis of primary cilia and gene expression.

(a) Transmission electron micrograph of primary cilia on CP tumour cells (arrow) or CP epithelium (arrowheads). Scale bar: 0.5 μm. (b) The expression of Arl13b (red) and γ-tubulin (red) in EYFP+ CP epithelial cells of Mcre;EYFP mice. EYFP (green) detected with a GFP antibody demarcates epithelial cells derived from Atoh1+ progenitors in Mcre;EYFP mice, while Aqp1 (yellow) marks differentiated epithelial cells. DAPI staining (blue) labels nuclei. Inset pictures show high magnification images of EYFP+ (arrows, upper) or EYFP− epithelial cells (arrowheads, lower). Scale bar: 10 μm. (c) The expression of Foxj1 (red) in CP tumour cells from Mcre;NICD1 mice at P14. Dotted lines mark boundary between tumour cells and Aqp1+ (green) CP epithelium. DAPI staining (blue) labels nuclei. Notice that Foxj1 expression is reduced in tumour cells compared to epithelial cells. Scale bar: 25 μm. (d) Schematic illustration of hindbrain roof plate and CP in mouse embryo at day E14.5. Solid line marks the transverse plane across the cerebellum, roof plate, and CP. H&E staining and the diagram show structures in this plane. LRL: lower rhombic lip; URL: upper rhombic lip; LRP: lower roof plate; URP: upper roof plate; CB: cerebellum. Scale bar: 1 mm. (e) Cilia pattern in cells within the roof plate and CP epithelium of wild type animals is shown at day E14.5. Arl13b (red) and γ-tubulin expression (red) marks primary cilia and basal bodies, respectively. DAPI staining (blue) labels nuclei. Scale bar: 10 μm. (f) Representative images show the expression of acetylated α-tubulin (act-α-tubulin, red) and AQP1 (red) in normal human CP and human CP tumours. solitary cilium (arrows) or multiple cilia (arrowheads) are magnified in inset pictures. Hoechst staining (blue) labels nuclei. Scale bar: 10 μm. (g) The expression Lmx1a (red) and Ki-67 (red) in CP cells from Mcre;NICD1 mice cultured in the presence of serum. GFP (green) labels NICD1+ tumour cells, while Aqp1 marks epithelial cells. DAPI staining (blue) labels nuclei. Scale bar: 25 μm.

Supplementary Figure 8 Notch-induced CP tumours arise from progenitors in the roof plate.

(a) Atoh1:EGFP expression in Math1M1GFP mice at day E14.5 is detected with a GFP antibody and shown in green signals. Otx2 expression (red) labels the roof plate (marked by dotted lines), while DAPI staining (blue) marks nuclei. Notice that Atoh1:EGFP+ progenitors (arrows) are present in Otx2+ upper roof plate, but absent in lower roof plate. Scale bar: 50 μm. (b) Gene expression in CPs from Mcre:NICD1 mice at day E14.5. GFP (green) marks tumour cells Ki-67 expression (red) labels proliferating cells, and Aqp1 expression (red) marks differentiated epithelial cells. DAPI staining (blue) labels nuclei. Scale bar: 30 μm. (c) Analysis of gene expression and proliferation of tumour cells and progenitors within hindbrain roof plate in Mcre;NICD1and wild type (WT) mice, respectively, at day E14.5. The expression of Lmx1a (red) marks the roof plate and CP lineage. While Ki-67 expression (green) labels proliferating cells, Aqp1 expression (green) marks differentiated epithelial cells White dotted lines mark domain of progenitors (Lmx1a+/Ki-67+/Aqp1−) in hindbrain roof plate. Boxed regions in upper roof plate are shown in higher magnification. Red bracket lines mark differentiated epithelial cells (Lmx1a+/Ki-67−/Aqp1+). Stars demarcate tumour cells (Lmx1a+/Ki-67+/Aqp1−). Scale bar: 25 μm.

Supplementary Figure 9 Representative unprocessed scanned images of blots.

Western blot analysis of Hes1 (a), Aqp1 and Otx2 (b), and Ccnd1 (c) expression in CP tumours from Mcre;NICD1 mice and CPs from wild type (WT) mice at different time points. β-actin serves as loading control. N/A: protein samples for unrelated study run on the same gel (c).

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Li, L., Grausam, K., Wang, J. et al. Sonic Hedgehog promotes proliferation of Notch-dependent monociliated choroid plexus tumour cells. Nat Cell Biol 18, 418–430 (2016). https://doi.org/10.1038/ncb3327

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