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Sonic hedgehog accelerates DNA replication to cause replication stress promoting cancer initiation in medulloblastoma

Nature Cancer volume 1, pages 840–854 (2020)Cite this article

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Abstract

The mechanisms generating cancer-initiating mutations are not well understood. Sonic hedgehog (SHH) pathway activation is frequent in medulloblastoma (MB), with PTCH1 mutations being a common initiating event. Here we investigated the role of the developmental mitogen SHH in initiating carcinogenesis in the cells of origin: granule cell progenitors (GCPs). We delineate a molecular mechanism for tumor initiation in MB. Exposure of GCPs to Shh causes a distinct form of DNA replication stress, increasing both origin firing and fork velocity. Shh promotes DNA helicase loading and activation, with increased Cdc7-dependent origin firing. The S-phase duration is reduced and hyper-recombination occurs, causing copy number neutral loss of heterozygosity—a frequent event at the PTCH1/ptch1 locus. Moreover, Cdc7 inhibition to attenuate origin firing reduces recombination and preneoplastic tumor formation in mice. Therefore, tissue-specific replication stress induced by Shh promotes loss of heterozygosity, which in tumor-prone Ptch1+/− GCPs results in loss of this tumor suppressor—an early cancer-initiating event.

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Fig. 1: Copy number variants and CN-LOH are the source of PTCH1 wild-type allele inactivation.
Fig. 2: Shh causes DNA replication stress.
Fig. 3: Shh alters DNA replication dynamics.
Fig. 4: Shh promotes pre-replication complex assembly.
Fig. 5: Shh induces helicase activation and origin firing.
Fig. 6: Shh-dependent replication initiation domains revealed by EdU-seq.
Fig. 7: Origin firing is required for Shh-dependent replication stress and recombination.
Fig. 8: In vivo Cdc7 inhibition reduces origin firing and hyper-recombination, preventing MB initiation.

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Data availability

Deep-sequencing data supporting the findings of this study have been deposited in the Gene Expression Omnibus under accession codes GSE147409 (EdU-seq) and GSE147410 (RNA-seq). Human LOH data in SHH-MB were derived from the dataset EGAD00001003127 obtained with authorization from the International Cancer Genome Consortium (https://icgc.org/). Mouse CNV data were downloaded from GSE19381. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank A. Helness, S. Terouz, E. Massicotte, F. Depault, J. Cardin and J. Barthe for expert assistance, A. Dumont and M. Rondeau for RNA-seq library preparation and O. Neyret for EdU-seq library preparation. We thank O. Jumanca and the Montreal Clinical Research Institute animal facility staff for animal handling. We thank E. Drobetsky for critical reading of the manuscript. We thank the International Cancer Genome Consortium for providing access to human MB data. L.T.-O. was supported by the Caldas (Colciencias) and Djavad Mowafaghian Foundation fellowships and is now supported by an EMBO Long-Term Fellowship (ALTF-739–2019). D.G. was supported by a post-graduate scholarship from the Natural Sciences and Engineering Research Council of Canada. This work was supported by the Canadian Institutes of Health Research (FDN334023 to F.C.), Fonds de Recherche du Québec–Santé (to F.C.), Canada Foundation for Innovation (33768 to F.C.), Canadian Cancer Society Research Institute (Impact grant 702310 to G.W.B. and Innovation grant 705366 to T.H.), Medical Research Council (MC_UU_00007/5 to A.P.J.) and European Union’s Horizon 2020 Research and Innovation Programme ERC Advanced Grant (number 788093 to A.P.J.). F.C. holds the Canada Research Chair in Developmental Neurobiology.

Author information

Author notes

  1. Lukas Tamayo-Orrego

    Present address: MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK

  2. These authors contributed equally: David Gallo, Frédéric Racicot.

Authors and Affiliations

  1. Montreal Clinical Research Institute, Montreal, Quebec, Canada

    Lukas Tamayo-Orrego, Frédéric Racicot, Amandine Bemmo, Sushmetha Mohan, Samer Salameh & Frédéric Charron

  2. Integrated Program in Neuroscience, McGill University, Montreal, Quebec, Canada

    Lukas Tamayo-Orrego, Sushmetha Mohan & Frédéric Charron

  3. Department of Biochemistry and Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada

    David Gallo, Brandon Ho & Grant W. Brown

  4. Department of Medicine, University of Montreal, Montreal, Quebec, Canada

    Frédéric Racicot & Frédéric Charron

  5. Institute for Research in Immunology and Cancer, University of Montreal, Montreal, Quebec, Canada

    Trang Hoang

  6. MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK

    Andrew P. Jackson

  7. Department of Anatomy and Cell Biology, Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada

    Frédéric Charron

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Contributions

L.T.-O. and F.C. conceived of the study. L.T.-O., D.G., F.R., S.M., S.S. and B.H. performed the experiments. A.B. performed the in silico analyses. T.H. provided reagents and advice. D.G. and L.T.-O. analyzed the data. A.P.J. provided critical advice on the manuscript and supported parts of the work, as well as providing support to L.T.-O., during revision. L.T.-O., G.W.B., A.P.J. and F.C. wrote the manuscript.

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Correspondence to Frédéric Charron.

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Extended data

Extended Data Fig. 1 Shh, but not other GCP mitogens, causes replication stress.

a, Most γ-H2AX positive GCPs are actively replicating. 90% of BrdU-positive cells are positive for γ-H2AX, but only 14% of BrdU-negative cells are γ-H2AX positive. Two-sided Fisher’s exact test, n= 530 cells from 3 experiments (428 cells were BrdU+ and 102 were BrdU-). b, Flow cytometry scatter plot showing γ-H2AX (y axis) and DNA content (x axis) of dissociated GCPs; gate shows S-phase cells, n=3844 cells analyzed. Data from 2 independent experiments. c, BrdU and 53bp1 immunostaining of GCPs actively proliferating in response to Shh in the external granule-cell layer of the cerebellum, n=2 experiments; scale bars: 20µm and 5µm (inset). d, Images of GCPs pulsed with CldU and IdU and stained for γ-H2AX, representative of quantifications shown in e–g. e, Percentage of γ-H2AX-positive and γ-H2AX-negative cells in S-phase, G2 and G0/1; two-sided Fisher’s exact test. f, Number of γ-H2AX foci/cell in S-phase, G2 and G0/1; one-way anova and Tukey post-test, median and inter-quartile range. g, Relative γ-H2AX levels in S-phase, G2 and G0/1 GCPs, mean±95% CI; n=264 S-phase cells, n= 55 G2 cells, and n=87 G0/G1 cells from 3 experiments (e–g). h, Percentage of 53bp1-positive GCPs in S- and G2-phases of the cell cycle (left) and representative images (right); cells were pulsed sequentially with 25µM CldU for 1.5hr and 125µM IdU for 20min and fixed; IdU-positive cells are in S-phase, while CldU-positive and IdU-negative cells are in G2; two-sided Fisher’s exact test; n=241 S-phase cells and 32 G2 cells from 3 experiments. Scale bar, 5µm (d,h). i, Percentage of Ki67 (left) and p-histone H3 (pH3) positive cells in GCPs treated with vehicle (Ctl), bFgf, Egf, Igf1 or Shh; mean±sem; the number of images used was: n= 9 for Ctl, n= 7 for bFgf, n= 8 for Egf, n= 9 for Igf1, and n=10 for Shh from 2 experiments . j, Relative γ-H2AX levels in GCPs treated with vehicle (Ctl), bFgf, Egf or Shh; n= 223 cells for Ctl, n= 201 cells for bFgf, n= 200 cells for Egf, and n=163 cells for Shh, from 2 experiments. k, Relative γ-H2AX levels in BrdU+ cells (S-phase) obtained by immunofluorescence of GCPs treated with vehicle, Igf1 and Shh (mean±95% CI,); n= 34 cells in Ctl-, 60 cells in Igf1-, and 115 cells in Shh-treated samples; images related to Fig. 2e; scale bar, 25µm. Data representative of 3 independent experiments. l, Example of a Rpa70-positive cytokinesis bridge in GCPs treated with 500 nM hydroxyurea; experiment performed two times; cytokinesis failure or ultrafine anaphase bridges (UFBs) were not detected in Shh-treated GCPs.

Source data

Extended Data Fig. 2 Replicative γ-H2AX levels are elevated in the wild-type EGL compared to the intestinal epithelium, and RNA sequencing of Ctl-, Shh- and Igf1-treated GCPs.

a,b, Representative IHC (a) and IF (b) images of the intestinal epithelium and EGL stained for γ-H2AX (a) or for γ-H2AX and Ki67 (b). c, Percentage of proliferating (Ki67+) cells positive for γ-H2AX in the intestinal epithelium and EGL; two-sided Fisher’s exact test. d, Number of γ-H2AX foci/cell in proliferating (Ki67+) intestinal epithelium and EGL; mean±95% CI, two-sided t-test; n=589 intestinal cells, and n= 1290 EGL cells from 3 experiments (b–d). e, Principal component analysis (PCA) of differentially-expressed genes between Ctl-, Shh- and Igf1-treated GCPs; n=3 experiments. f, Examples of Igf1 and Shh-regulated genes; mean±sem (n=3 experiments); one-way anova and Tukey post-test. *, p≤0.05; **, p≤0.01; ***, p≤0.001. Source data for the RNAseq results shown in f can be found in Supplementary Table 1.

Source data

Extended Data Fig. 3 Nucleosides are sufficient to induce origin firing and γ-H2AX in GCPs.

a, GSEA (Gene-set enrichment analysis) plot of pyrimidine metabolism in Shh vs. Ctl GCPs, n=3 experiments. b, mRNA levels of representative nucleotide metabolism genes from a; mean±sem, one-way anova and Tukey post-test, n=3 experiments, *, p≤0.05; **, p≤0.01; ***, p≤0.001. c, Mass spectrometry analysis of nucleotide levels in Ctl-, Shh- and Igf1-treated GCPs, n=4 experiments; mean±sem, one-way anova and Tukey post-test, *, p≤0.05; **, p≤0.01; ***, p≤0.001. d, Flow cytometry scatter plot of Ctl- and nucleoside (AGCT)-treated GCPs; n= 63329 Ctl cells and n= 61648 AGCT cells analyzed from 2 experiments. e, BrdU fluorescence in S-phase (BrdU+) GCPs in control and nucleoside-treated GCPs; n= 36 Ctl- and n= 39 AGCT-treated cells from a representative experiment, two-tailed t-test. f, Western blot for γ-H2AX of GCPs treated with Ctl, nucleosides and Shh; bar graph shows quantification of γ-H2AX levels relative to actin (n=4 experiments; mean±sem). g, S-phase time (hrs.) of GCPs treated with nucleosides and/or Shh (n=2 experiments). h, DNA fork rate (kbp/min) of control and nucleoside-treated GCPs, two-sided Mann-Whitney test, median; n=190 forks in Ctl- and n=283 forks in nucleoside-treated GCPs. i, Average DNA fork density of Ctl- and nucleoside-treated GCPs; 300Mb of combed DNA were scored, n=2 experiments (g, i). Source data for the RNAseq results shown in b can be found in Supplementary Table 1.

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Extended Data Fig. 4 Shh regulates the pre-replication complex.

a, Relative fork density in GCPs in response to Ctl, Igf1, Shh, Shh+Cdc7i and nucleosides; mean±95% CI, two-tailed t-test; the number of experiments per condition is: n= 6 for Ctl, n= 2 for nucleosides, n= 2 for Igf1, n= 4 for Shh, and n= 2 for Shh+Cdc7i. b–e, Relative mRNA levels of origin recognition complex (Orc) subunits (b), licensing and preinitiation complex factors (c), components of the CMG (Cdc45-Mcm-Gins) helicase (d) and helicase-activating kinases (e) in Ctl-, Shh- and Igf1-stimulated GCPs; mean±sem, n=3 experiments, one-way anova and Tukey post-test, *, p≤0.05; **, p≤0.01; ***, p≤0.001. f, Flow cytometry scatter plot displaying chromatin-bound Mcm2 (y-axis) and DNA content (Dapi, x-axis) in Ctl- and Shh-treated GCPs; n=2 experiments. g, Histogram of chromatin-bound Mcm2 in the G0/G1 population (gate on f), n=2 experiments. h, Merge images of immunofluorescence shown in Fig. 4c, n=4 experiments. Source data for the RNAseq results shown in b-e can be found in Supplementary Table 1.

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Extended Data Fig. 5 Shh promotes helicase activation.

a, Chromatin-bound p-Mcm2 ser40/41 in asynchronous and S-phase (BrdU+) Ctl-, Cdc7i-, Shh- and Shh+Cdc7i-treated GCPs; mean±95%CI; n= 275 cells for Ctl-, n=130 cells for Cdc7i-, n= 263 cells for Shh-, and n= 277 cells for Shh+Cdc7i-treated samples. For BrdU+ cells: n= 44 in Ctl, n= 20 in Cdc7i, n=71 in Shh, and n=65 in Shh+Cdc7i samples. Data from 2 independent experiments. b, Chromatin-bound Gins2 in asynchronous and S-phase (BrdU+) Ctl-, Cdc7i-, Shh- and Shh+Cdc7i-treated GCPs; mean±95%CI, n= 353 cells for Ctl-, n=291 cells for Cdc7i-, n= 322 cells for Shh-, and n= 191 cells for Shh+Cdc7i-treated samples; for BrdU+ cells: n= 112 in Ctl, n= 75 in Cdc7i, n=160 in Shh, and n=90 in Shh+Cdc7i samples; data from 2 independent experiments. Scale bar, 5µm (a, b). c, DNA synthesis (BrdU fluorescence/cell) in S-phase cells in Ctl, Shh or Shh+Cdc7i-treated GCPs; mean±95% CI; one-way anova, n= 31 cells in Ctl, n= 70 in Shh and n= 28 in Shh+Cdc7i samples. Data from 2 independent experiments. d, S-phase time in Shh and Shh+Cdc7i treated GCPs; median, two-tailed t-test, n= 23 images in Shh and n=21 images in Shh+Cdc7i; data from 2 independent experiments.

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Extended Data Fig. 6 Shh induces replication initiation.

a, Percentage of BrdU+ cells in Ctl-, Cdc7i-, Shh- and Shh+Cdc7i-treated GCPs; mean±95% CI, one-way anova and Tukey post-test, n=20 images in Ctl- and n=10 in Cdc7-, Shh-, and Shh+Cdc7i-treated samples from two experiments. b, Fork speed (kbp/min) in Ctl-, Cdc7i-, Shh- and Shh+Cdc7i-treated GCPs; median, Kruskal-Wallis test; n= 285 forks in Ctl, n=120 forks in Cdc7i, n= 256 forks in Shh, and n= 224 forks in Shh+Cdc7i. Data from 2 independent experiments. c, Inter-origin distance (IOD) in Ctl-, Cdc7i-2-, Shh- and Shh+Cdc7i-2-treated GCPs; Cdc7i-2 is 200nM Tak-931 added during the last 4hr. of the experiment; Kruskal-Wallis test; median and inter-quartile range; median IOD indicated below graph; number of IOD measured is: n= 49 in Ctl-, n=108 in Cdc7i-2-, n=173 in Shh-, and n=146 in Shh+Cdc7i-2-treated samples; experiment performed one time. d, Top, composite plot of EdU peak signal (Fpkm); bottom, composite plot for nucleotide frequency (%) centered around EdU peak. e, Annotation of genomic regions containing EdU-seq peaks in Ctl- and Shh-treated GCPs according to sequence composition. f, Annotation of regions containing peaks according to sequence composition for repetitive and non-repetitive sequences; n=942 peaks for Ctl samples, and n= 4321 peaks for Shh samples from 2 independent experiments (d–f). g, Distance (kbp) between replication initiation zones in Ctl-, Shh and Shh+Cdc7i-treated GCPs; Median and inter-quartile range (IQR); one-way anova; average also indicated (+) in g; n= 2627 distances in Ctl, 4466 distances in Shh, and 3137 distances in Ctl+Cdc7i samples; data obtained from one experiment.

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Extended Data Fig. 7 Shh promotes homologous recombination.

a, GSEA plot for double strand break (DSB) repair enriched genes in Shh-treated compared to control GCPs, n=3 animals/group. b, GSEA plot for the Fanconi anemia pathway in Shh-treated vs. control GCPs, n=3 animals/group. c, mRNA levels of non-homologous end-joining genes in Ctl-, Shh- and Igf1-stimulated GCPs; mean±sem, one-way anova and Tukey post-test, n=3 animals/group; *, p≤0.05; **, p≤0.01; ***, p≤0.001. d, mRNA levels of homologous recombination genes in Ctl-, Shh- and Igf1-stimulated GCPs; mean±sem, n=3 animals/group, one-way anova; *, p≤0.05; **, p≤0.01; ***, p≤0.001. e, Rad51 total nuclear levels assessed by immunofluorescence in BrdU- or BrdU+ GCPs treated with control or Shh; mean±95% CI; n=207 Ctl and n=210 Shh (asynchronous cells); n= 61 in Ctl and n=110 Shh (BrdU+ cells). f, Chromatin-bound Rad51 levels assessed by immunofluorescence in BrdU- or BrdU+ GCPs treated with control or Shh; n= 92 Ctl and n=97 Shh (BrdU-negative cells), n=56 Ctl and n=51 Shh (BrdU+ cells); mean±95% CI, one-way anova, Tukey post-test. Data from 2 independent experiments (e, f). Representative images on the right; scale bar, 5µm. g, Number of 53bp1 foci/cell in S-phase (BrdU+) Ctl- and Shh-treated GCPs; mean±95%CI, two-sided t-test, n= 44 Ctl and n= 35 Shh S-phase cells from 2 experiments. h, SCEs/metaphase in GCPs treated with Ctl, Cdc7i, bFgF, Egf and Shh; mean±95%CI, one-way anova, Tukey post-test, n= 20 in Ctl and in Cdc7i, n= 45 in bFgf, n= 50 in Egf, and n= 29 metaphases in Shh, from 2 experiments. i, Percent RaDR-GFP-positive events indicative of recombination events in Ctl- and Shh-treated GCPs; n=4 experiments, mean, paired t-test. Source data for the RNAseq results shown in c-d can be found in Supplementary Table 1.

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Extended Data Fig. 8 High CDC7 levels characterize SHH-MB-α and are an indicator of poor prognosis in MB.

a, Overall survival analysis of 612 human medulloblastoma patients from the 4 different MB groups extracted from the Cavalli cohort and analyzed using R2; Kaplan-Meier test. b, Overall survival analysis of 172 human SHH-MB patients from the Cavalli cohort and analyzed using R2; Kaplan-Meier test. c, Dot plot of CDC7 expression in the four subtypes of SHH-MB. d, CDC7 expression in the four subtypes of SHH-MB; median and range, one-way anova test. e, GSEA plots generated from genes upregulated in SHH-MB-α versus SHH-MB-βγδ; n=612 human MBs. f, Top genes enriched in the DNA replication and DNA recombination gene sets presented in e.

Extended Data Fig. 9 Proteomics data uncovers a subtype (SHHa) of human MB characterized by DNA replication and MCM2 activation.

a, Heatmap displaying normalized enrichment scores (NES) of DNA replication and recombination gene ontologies for two subtypes of SHH-MB (SHHa and SHHb) based on proteomics data obtained from Ref. 43. b, NES of DNA recombination comparing SHHa and SHHb at the mRNA and protein level; n=9 SHHa tumors and n=5 SHHb tumors; median and range. c, d, mRNA (c) and protein (d) levels of MCM2 in SHHa and SHHb; n=10 SHHa tumors and n=5 SHHb tumors; median and range. e, Relative peptide abundance of different MCM2 phosphorylation events indicative of helicase activation (Ser40, Ser139 and Ser26/27) in SHHa and SHHb; n=10 SHHa tumors and n=5 SHHb tumors; median and range.

Extended Data Fig. 10 In vivo Cdc7 inhibition does not affect growth or cerebellum development but reduces helicase activation.

a, b, Comparison of mouse body weight at P4 and P7 in Ctl- and Cdc7i-treated pups; mean±sem, two-sided t-test. n= 16 Ctl- and n= 19 Cdc7i-treated P4 mice (a); n= 14 Ctl- and n= 18 Cdc7i-treated P7 mice (b). c, d, IGL area (c) and perimeter (d) at midline in Ctl- and Cdc7i-treated P16 mice (n=6 mice); mean±sem, two-sided t-test. e, EGL thickness in Ctl- and Cdc7i-treated P7 mice; mean±sem, two-sided t-test, n=5 mice/group. f, g Number of phospho-histone-H3 (pH3)-positive cells per 103 µm2 in Ctl- and Cdc7i-treated P7 mice (f) and representative images (g); mean±sem, two-sided t-test; n=5 animals/group. h, Percentage of BrdU+ GCPs in the EGL of the cerebellum of Ctl- and Cdc7i-treated mice; mean±sem, two-sided t-test; n=5 animals/group; scale bar, 100µm. i, Ki67 immunocytochemistry in Ctl- and Cdc7i-treated P7 mice. j, k, Quantitation of p-Mcm2 levels (j) and representative images (k) in the EGL of Ctl- and Cdc7i-treated mice; mean±sem (n=5 mice), two-sided t-test.

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Supplementary information

Reporting Summary

43018_2020_94_MOESM2_ESM.xlsx

Supplementary Table 1 Additional source data for the RNA-seq analyses shown in Extended Data Figs. 2–4 and 7. FPKM and normalized expression in control-, Igf1- and Shh-treated GCPs. The table presents data for genes involved in Igf1 and Shh signaling (Extended Data Fig. 2f), nucleotide metabolism genes (Extended Data Fig. 3b), origin recognition protein complex subunits, licensing factors, CMG helicase and helicase-activating kinases (Extended Data Fig. 4b–e) and non-homologous end-joining and homologous recombination genes (Extended Data Fig. 7c,d). Data are from three independent experiments (mean ± s.e.m.), analyzed by one-way ANOVA and Tukey’s post-hoc test.

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Tamayo-Orrego, L., Gallo, D., Racicot, F. et al. Sonic hedgehog accelerates DNA replication to cause replication stress promoting cancer initiation in medulloblastoma. Nat Cancer 1, 840–854 (2020). https://doi.org/10.1038/s43018-020-0094-7

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