Abstract
Bidirectional communication between tumours and neurons has emerged as a key facet of the tumour microenvironment that drives malignancy1,2. Another hallmark feature of cancer is epigenomic dysregulation, in which alterations in gene expression influence cell states and interactions with the tumour microenvironment3. Ependymoma (EPN) is a paediatric brain tumour that relies on epigenomic remodelling to engender malignancy4,5; however, how these epigenetic mechanisms intersect with extrinsic neuronal signalling during EPN tumour progression is unknown. Here we show that the activity of serotonergic neurons regulates EPN tumorigenesis, and that serotonin itself also serves as an activating modification on histones. We found that inhibiting histone serotonylation blocks EPN tumorigenesis and regulates the expression of a core set of developmental transcription factors. High-throughput, in vivo screening of these transcription factors revealed that ETV5 promotes EPN tumorigenesis and functions by enhancing repressive chromatin states. Neuropeptide Y (NPY) is one of the genes repressed by ETV5, and its overexpression suppresses EPN tumour progression and tumour-associated network hyperactivity through synaptic remodelling. Collectively, this study identifies histone serotonylation as a key driver of EPN tumorigenesis, and also reveals how neuronal signalling, neuro-epigenomics and developmental programs are intertwined to drive malignancy in brain cancer.
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Data availability
ChIP–seq and RNA-seq data have been deposited in the NCBI Gene Expression Omnibus under the accession number GSE246033. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048170. All other data in this article are available from the corresponding authors on reasonable request. Uncropped immunoblots associated with Figs. 2, 3 and Extended Data Fig. 7 are provided in the supplementary information. Source data are provided with this paper.
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Acknowledgements
This work was supported by US National Institutes of Health grants R35-NS132230 and R01-NS124093 to B.D., R01-CA284455 to B.D. and S.C.M., R01-CA223388 to B.D. and J.L.N., and R01NS128184, R01CA280203 and U01CA281823 to S.C.M.; the National Cancer Institute Cancer Target Discovery and Development grant U01-CA217842 to B.D.; DOD-IDEA (CA220510), and an DOD-IMPACT (CA220247) award to S.C.M.; National Cancer Institute Cancer Center Support Grant, P30 CA021765, St. Jude Children’s Research Hospital Research Collaborative on Transcription Regulation in Pediatric Cancer Grant, Alex’s Lemonade Stand Foundation ‘A’ Award to S.C.M; S.C.M is supported by funding from the National Brain Tumor Society and CERN Foundation; the ALSAC Foundation; and the Cancer Prevention Research Institute of Texas (CPRIT) (RP210027 to H.-C.C., F31-CA243382 to E.H.-H., T32-5T32HL092332-19 to B.L., 1K99-DC019668 to D.S. and AHA-23POST1019413 to M.R.W.). We are thankful for support from the David and Eula Wintermann Foundation and the Adrienne Helis Malvin Medical Research Foundation. We thank I. Maze for providing the H3.3-Q5A constructs, and we acknowledge the Optogenetics and Viral Vectors Core at the Jan and Dan Duncan Neurological Research Institute. BCM Mass Spectrometry Proteomics Core is supported by a Dan L. Duncan Comprehensive Cancer Center NIH award (P30 CA125123) and a CPRIT Core Facility Award (RP210227). Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award number P50HD103555 for use of the Microscopy Core facilities and the Animal Phenotyping and Preclinical Endpoints Core facilities. Images in schematics were created using Biorender.com.
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Contributions
H.-C.C., S.C.M. and B.D. conceived the project and designed the experiments. H.-C.C., P.H., D.-J.C. and M.R.W. performed the mouse tumour experiments. H.-C.C., B.L. and E.H.-H. performed the barcoded screen. H.-C.C., D.S. and B.L. performed the RNA-seq and ChIP–seq experiments. H.-C.C. performed synaptic staining and immunostaining of tumours. A.J. performed the proteomic analysis. M.M. performed the electrophysiology studies. J.W. performed electrophysiology recording. J.L.N., S.I., G.R., T.E.M., D.W.E. and K.C.B. provided essential reagents. H.-C.C., S.V., H.S. and S.C.M. designed and performed the bioinformatics analyses. H.-C.C., S.C.M. and B.D. wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 DREADD-based activation of neurons by CNO treatment.
a, Schematic of DREADD-hM3Dq activation of ipsilateral cortical neurons in ZRFUS EPN mice. b, Low-magnification image of EPN tumours and representative BrdU staining of EPN tumours after DREADD-hM3Dq activation of ipsilateral cortical neurons via CNO (scale bar = 50 μm). c, Quantification of BrdU staining in saline versus CNO treated EPN tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, PSalinevsAAV_CNO = 0.4522, PCNOvsAAV_CNO = 0.1221, *PAAV_SalinevsAAV_CNO = 0.0337). d, Schematic of DREADD-hM3Dq activation of ipsilateral inhibitory neurons in ZRFUS EPN mice. e, Low-magnification image of EPN tumours and representative BrdU staining of EPN tumours after DREADD-based activation of ipsilateral inhibitory neurons via CNO (scale bar = 50 μm). f, Quantification of BrdU staining in saline versus CNO treated EPN tumours (Saline: n = 3, CNO: n = 3, AAV_Saline: n = 3, AAV_CNO: n = 4, mean ± s.e.m., unpaired two-sided Student’s t-test, PSalinevsAAV_CNO = 0.0770, PCNOvsAAV_CNO = 0.3896, PAAV_SalinevsAAV_CNO = 0.8727). g, Representative BrdU staining in saline and CNO (0.5 and 5 mg per kg) treated EPN tumours (scale bar = 50 μm). h, Immunofluorescence staining of FOS in the ipsilateral cortical neurons in saline versus CNO treated EPN tumours (scale bar = 50 μm). i, Quantification of FOS positive neurons in the ipsilateral cortex in saline versus CNO treated EPN tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, *P = 0.0155). j, Immunofluorescence staining of mCherry and TPH2 in the AAV injected dRN (scale bar = 30 μm). TPH2: tryptophan hydroxylase 2. k, Immunofluorescence staining of FOS in the dRN serotonergic neurons in saline versus CNO treated EPN tumours (scale bar = 50 μm). l, Quantification of FOS positive neurons in the dRN serotonergic neurons in saline versus CNO treated EPN tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, ***P = 0.0005). a and d were created using Biorender.com.
Extended Data Fig. 2 Expression of monoamine transporters in EPN.
a, Expression levels of genes under monoamine transporter GO term in human ZRFUS versus non-ZRFUS EPN tumours. b, Expression levels of genes under monoamine transporter GO term in mouse EPN tumours versus non-tumour tissues. c, Log2(FC) of serotonin transporters in human ZRFUS tumours versus non-ZRFUS tumour tissues. d, Log2(FC) of serotonin transporters in mouse EPN tumours versus non-tumour tissues. e, Normalized read counts of serotonin transporters in mouse EPN tumours versus non-tumour tissues (at least n = 10 per group, median ± upper and lower limits, box boundary states upper and lower quartiles). f, Immunofluorescence staining of SLC6A4 in mouse non-tumour cortex and EPN tumours (scale bar = 50 μm). g, Immunofluorescence staining of dopamine transporter (SLC6A3) in mouse substantia nigra, non-tumour cortex and EPN tumours (scale bar = 50 μm).
Extended Data Fig. 3 Synaptic staining and H3-5HT staining after dRN manipulation.
a, Low-magnification image of tumour margin and representative higher-magnification images (derived from dashed box) of peritumoral excitatory synaptic staining in saline versus CNO treated tumours from Fig. 1f (scale bar = 10 μm). b, Quantification of excitatory synaptic staining in saline versus CNO treated tumours Fig. 1f (AAV_saline: n = 4, AAV_CNO: n = 3, mean ± s.e.m., two-sided Wilcoxon rank sum test, P = 0.6286). c, Low-magnification view of tumour margin and representative higher-magnification images (derived from dashed box) of peritumoral inhibitory synaptic staining in saline versus CNO treated tumours from Fig. 1f (scale bar = 10 μm). d, Quantification of inhibitory synaptic staining in saline versus CNO treated tumours from Fig. 1f (AAV_saline: n = 4, AAV_CNO: n = 3, mean ± s.e.m., unpaired two-sided Student’s t-test, P = 0.7481). e, Low-magnification image of tumour margin and representative higher-magnification images (derived from dashed box) of peritumoral excitatory synaptic staining in saline versus CNO treated tumours from Fig. 1i (scale bar = 10 μm). f, Quantification of excitatory synaptic staining in saline versus CNO treated tumours from Fig. 1i (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, P = 0.6460). g, Low-magnification image of tumour margin and representative higher-magnification images (derived from dashed box) of peritumoral inhibitory synaptic staining in saline versus CNO treated tumours from Fig. 1i (scale bar = 10 μm). h, Quantification of inhibitory synaptic staining in saline versus CNO treated tumours from Fig. 1i (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, P = 0.9864). i, Immunofluorescence staining of H3-5HT in saline versus CNO treated tumours from Fig. 1f (scale bar = 10 μm). j, Quantification of H3-5HT intensity in saline versus CNO treated tumours from Fig. 1f (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, P = 0.2894). k, Immunofluorescence staining of H3-5HT in saline versus CNO treated tumours from Fig. 1i (scale bar = 10 μm). l, Quantification of H3-5HT intensity in saline versus CNO treated tumours from Fig. 1i (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, *P = 0.0465).
Extended Data Fig. 4 Tumour proliferation and ETV5 expression in H3.3-Q5A EPN tumours.
a, Immunofluorescence staining of H3-5HT in H3.3-WT and H3.3-Q5A EPN tumours (scale bar = 25 μm). b, Quantification of H3-5HT intensity in H3.3 control and H3.3-Q5A (n = 4 per group, mean ± s.e.m., two-sided Wilcoxon rank sum test, *P = 0.0286) c, Representative Ki67 staining of EPN control versus H3.3 wild-type and H3.3-Q5A tumours (scale bar = 50 μm). d, Quantification of Ki67 staining in EPN control versus H3.3 wild-type and H3.3-Q5A tumours (EPN control: n = 3, H3.3 wild-type: n = 5, H3.3-Q5A: n = 4, mean ± s.e.m., unpaired two-sided Student’s t-test, PEPNcontrolvsH3.3wild-type = 0.9040, **PEPNcontrolvsH3.3-Q5A = 0.0015, ****PH3.3wild-typevsH3.3-Q5A = 1.59E-05). e, Representative ETV5 staining of EPN control versus H3.3 wild-type and H3.3-Q5A tumours (scale bar = 25 μm). f, Quantification of ETV5 staining in EPN control versus H3.3 wild-type and H3.3-Q5A tumours (EPN control: n = 3, H3.3 wild-type: n = 3, H3.3-Q5A: n = 4, mean ± s.e.m., unpaired two-sided Student’s t-test, PEPNcontrolvsH3.3wild-type = 0.6180, **PEPNcontrolvsH3.3-Q5A = 0.0053, *PH3.3wild-typevsH3.3-Q5A = 0.0398).
Extended Data Fig. 5 SLC6A4-LOF EPN analyses, expression of serotonin synthetase and SSRI treatment in EPN.
a, Kaplan–Meier survival curve of EPN control (n = 19, median = 74 days) and SLC6A4-LOF (n = 14, median = 95 days, log-rank test, P = 0.1177). b, Representative Ki67 staining of EPN control versus SLC6A4-LOF tumours (scale bar = 50 μm). c, Quantification of Ki67 staining in EPN control versus SLC6A4-LOF tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, **P = 0.0055). d, Representative H3-5HT staining of EPN control versus SLC6A4-LOF tumours (scale bar = 25 μm). e, Quantification of H3-5HT intensity in EPN control versus Slc6a4-LOF tumours (n = 4 per group, mean ± s.e.m., two-sided Wilcoxon rank sum test, *P = 0.0286). f, Immunofluorescent staining of TPH2 in mouse dRN (positive control), non-tumour cortex, and EPN tumour (scale bar = 50 μm) g, Quantification of TPH2 intensity in mouse dRN, non-tumour cortex, and EPN tumour (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, ****PdRNvsnon-tumourcortex = 3.76E-05, **PdRNvsEPNtumour = 3.73E-05, Pnon-tumourcortexvsEPNtumour = 0.7262). h, Schematic of DMSO or SSRI treatment in EPN i, Representative H3-5HT staining of DMSO versus sertraline-HCl treated tumours (scale bar = 25 μm). j, Quantification of H3-5HT intensity in DMSO versus sertraline-HCl treated tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, P = 0.0743). h was created using Biorender.com.
Extended Data Fig. 6 H3-5HT ChIP–seq and ETV5 ChIP–seq analyses in EPN.
a, Venn diagram depicting core TFs with H3-5HT peaks in mouse EPN tumours. b, ETV5 ChIP–seq heat map profiles in mouse EPN tumours and Venn diagram depicting genes overlapped between downregulated DEGs acquiring H3K27me3 peaks in ETV5-GOF tumours and ETV5 annotated genes.
Extended Data Fig. 7 Validation of candidates from functional screen.
a, Immunoblots of LHX2, LHX4, ETV5, and KLF12 in mouse non-tumour cortex versus EPN tumours (n = 3 per group). b, Kaplan–Meier survival curves of EPN control (n = 51, median = 70 days), LHX2-GOF (n = 23, median = 82 days, log-rank test, P = 0.9640), LHX4-GOF (n = 9, median=114 days, log-rank test, P = 0.3532), and KLF12-GOF (n = 23, median = 77 days, log-rank test, P = 0.7588). c, Immunoblots of LHX2, LHX4, and KLF12 in control versus corresponding GOF tumours. d, Immunoblots of ETV5 in control versus ETV5-GOF and ETV5-LOF tumours. e, RT–qPCR fold enrichment of ETV5 and Etv5 transcript (ddCt) in control versus ETV5-GOF and ETV5-LOF tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, GOF: **P = 0.0068, LOF: ***P = 0.0007). f, Surveyor Nuclease Digestion assay of Etv5 gRNA efficiency. Mouse non-tumour cortex: negative control. Two primer sets were used, and gel images are presented in left and right panel. Asterisks label the nuclease digested bands. g, Representative BrdU staining of control versus ETV5-GOF and ETV5-LOF tumours (scale bar = 50 μm). h, Quantification of BrdU staining in control versus ETV5-GOF and ETV5-LOF tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, GOF: **P = 0.00258, LOF: ***P = 0.000976). i, Immunoblots of H3K27ac and H3K27me3 in control versus ETV5-GOF tumours. Total H3: loading control. j, Ring chart showing percentage of H3K27me3 sites in ETV5-GOF tumours carrying ETS motif allowing 0 mismatch at 1,000 bp from peak centre. k, GO-terms analysis of ETV5 binding partners in mouse EPN tumours performed using Enrichr (two-sided Fisher’s exact test). l, RT–qPCR fold enrichment of gene transcript (ddCt) in control versus ETV5-GOF tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, Npy: ***P = 0.00024, Gabra5: P = 0.4342, Chrm4: *P = 0.0239, Kcnmb4: *P = 0.0109, Nptx2: P = 0.2549). m, H3K27me3 ChIP–seq peaks at Npy and Chrm4 locus in control and ETV5-GOF tumours.
Extended Data Fig. 8 Comparison of Kaplan–Meier survival curves between sexes.
a–c, All Kaplan–Meier survival curves (a), table of median survival (b) and comparison of log-rank test results (c) between groups divided by sex.
Extended Data Fig. 9 Analysis of NPY-GOF EPN tumours.
a, RT–qPCR fold enrichment of NPY transcript (ddCt) in control versus NPY-GOF tumours (n = 3 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, *P = 0.0255). b, RT–qPCR fold enrichment of Npy2r transcript (ddCt) in mouse non-tumour cortex and EPN tumours (n = 3 per group, as mean ± s.e.m., unpaired two-sided Student’s t-test, *P = 0.0215). c, Immunofluorescence staining of NPY2R in mouse cortex. NeuN: neuronal marker, Aldh1l1: astrocytic marker (scale bar = 50 μm). d, Low-magnification image of the tumour margin and representative images of NPY2R staining in mouse non-tumour and EPN tumours (scale bar = 50 μm). Top right panel: Quantification of NPY2R intensity normalized to DAPI in mouse non-tumour cortex versus EPN tumours. (n = 4 per group, mean ± s.e.m., unpaired two-sided Student’s t-test, **P = 0.0014). e, GO-terms analysis of the downregulated DEGs in NPY-GOF tumours versus control performed using Enrichr (two-sided Fisher’s exact test). f, Zoom-out EEG traces from mice bearing control and NPY-GOF tumours.
Supplementary information
Supplementary information
Uncropped immunoblots associated with Figs. 2, 3 and Extended Data Fig. 7
Supplementary Tables 1-17
Supplementary Table 1. DEGs from RNA-seq data of patients with ZRFUS versus non-ZRFUS EPN; associated with Fig. 1. Supplementary Table 2. Motif analysis of genes with ZRFUS-HA and H3-5HT overlapped genes; associated with Fig. 2. Supplementary Table 3. H3-5HT annotated genes and core TF lists; associated with Fig. 2. Supplementary Table 4. TF lists in barcode screen; associated with Fig. 3. Supplementary Table 5. EPNCtrl_H3K27ac annotated genes; associated with Fig. 3. Supplementary Table 6. EPNETV5GOF_H3K27ac annotated genes; associated with Fig. 3. Supplementary Table 7. EPNCtrl_H3K27me3 annotated genes; associated with Fig. 3. Supplementary Table 8. EPNETV5GOF_H3K27me3 annotated genes; associated with Fig. 3. Supplementary Table 9. ETV5 IP–MS list; associated with Fig. 3 Supplementary Table 10. GO analysis of ETV5 interacting proteins in EPN tumour exclusively; analysed and acquired table from Enrichr datasets, associated with Extended Data Fig. 7. Supplementary Table 11. DEGs from RNA-seq data of mouse ETV5-GOF versus EPN Ctrl tumours; associated with Fig.3. Supplementary Table 12. EPN_ETV5 annotated genes; associated with Extended Data Fig. 6. Supplementary Table 13. Overlapped genes between downregulated DEGs which gain H3K27me3 peaks in mouse ETV5-GOF tumours; associated with Fig. 3. Supplementary Table 14. GO analysis of downregulated DEGs which gain H3K27me3 peaks in mouse ETV5-GOF tumours; analysed and acquired table from Enrichr datasets, associated with Fig. 3. Supplementary Table 15. DEGs from RNA-seq data of mouse NPY-GOF versus EPN Ctrl tumours; associated with Fig.4. Supplementary Table 16. GO analysis of downregulated DEGs in NPY-GOF versus EPN Ctrl tumours; analysed and acquired table from Enrichr datasets, associated with Extended Data Fig. Supplementary Table 17. Primer, antibody, and plasmid tables
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Chen, HC., He, P., McDonald, M. et al. Histone serotonylation regulates ependymoma tumorigenesis. Nature 632, 903–910 (2024). https://doi.org/10.1038/s41586-024-07751-z
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DOI: https://doi.org/10.1038/s41586-024-07751-z
