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

Aberrant Notch signaling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly pediatric 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 signaling. 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 diffferentiation. A Shh-driven signaling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from mono-ciliated progenitors in the roof plate characterized by elevated Notch signaling. Abnormal SHH signaling and distinct ciliogenesis are detected in human CP tumours, suggesting SHH pathway and cilia differentiation as potential therapeutic avenues.

Tumour cells express the CP marker Lmx1a, but not mesenchymal marker MafB 25 , and the expression of Aqp1, Transthyretin (Ttr), cytokeratins, and Otx2 is consistently reduced compared to CP epithelial cells 26 (Fig. 2d-f, Supplementary Fig. 3b, Supplementary Fig. 4e,   4f), indicating that sustained Notch signaling interferes with differentiation of tumour cells even after they become post-mitotic.

Abnormal Shh signaling in CP tumour cells
To identify signals that drive tumour cell proliferation, we compared transcriptional profiles of tumours and wild type CPs at P0 (tumour cells are proliferative) and P21 (tumour cells are post-mitotic) using RNA-seq. Tumours and CPs clustered separately in principal component analysis, indicating distinct molecular profiles (Fig. 3a). Tumour cells exhibit gene expression profiles defined by differential expression of 2,738 (P0) and 4,964 (P21) transcripts (Supplementary Table 1, Fig. 3b). Study of these differentially expressed transcripts identified 1,705 common targets, including Hes1 and Hes5, Aqp1, cytokeratins, and Otx2, all of which show significant differential expression by Q-RT-PCR and immunostaining analyses, validating RNA-seq results (Fig. 1g, Fig. 2d Table 2). We reasoned that differential genes unique to P0 tumour cells may include those involved in proliferation. To identify these genes, we excluded the 1,705 common differential targets between tumours and CPs at each time point to obtain 1,033 differential transcripts in tumours at P0 (Fig. 3b). We overlapped these 1,033 unique targets with the 4,910 differential genes between tumours of P0 and P21, further narrowing it down to 663 genes (Fig. 3b, Supplementary Table 3). Interrogating this shortened list of genes using ingenuity pathway analysis led us to a promising candidate: Shh signaling, which is also identified in analysis of larger dataset (Supplementary Tables 1,  3). Tumour cells exhibit increased expression of Gli1, Gli2, Mycn and Ccnd1 compared to wild type CP epithelium at P0 and P7. After P7, when tumour cells start to exit the cell cycle, the expression of these genes decreases to levels of those in control CPs, suggesting that decreased proliferation correlates with attenuated Shh signaling ( Fig. 3d-g, 3i, Supplementary Fig. 4b). In addition, while the expression of p27 and Cdkn2b is upregulated in non-proliferating tumour cells, Cdkn1c (p57 Kip2) is expressed at higher levels in mature CP (Fig. 2c, Supplementary Fig. 4c, Supplementary Table 1). Among hedgehog ligands, only Shh is abundantly expressed in hindbrain CP epithelial cells at birth and declines to undetectable levels after P14, resulting in lower expression levels in tumours than in wild type CPs at P0 (Fig. 3d, 3e, 3h, 3i).

Shh drives CP tumour cell proliferation
To determine the role of Shh signaling in Notch-induced CP tumour, we treated tumour cells with recombinant N-terminal fragment of Shh (ShhN). Tumour cells formed spheres under serum-free conditions and continued to express Lmx1a, indicating intact lineage characteristics under these conditions (Fig. 4a, 4c). After 96 hours, more Ki-67 + cells were detected in tumour spheres treated with ShhN than in untreated tumour spheres (Fig. 4b).
While epithelial cells formed aggregates that remained unresponsive, the size of tumour spheres and number of tumour cells were increased by ShhN, including post-mitotic tumours at P21 or later, whereas Smo inhibitor cyclopamine abolished such effects, indicating ShhN stimulates tumour cell proliferation (Fig. 4c, Supplementary Fig. 5). Together, these results indicate that Shh drives the growth of Notch-induced CP tumours.

SHH and NOTCH signaling in human CP tumours
We examined NOTCH and SHH signaling in human CP tumours. First, we utilized published datasets and analyzed tumour transcriptomes, epigenomes, and genomes. Principal component analysis of human CPPs and normal CPs revealed distinct molecular profiles for CPPs 13 (Fig. 4j). MetaCore enrichment analysis of differential genes in human CPPs placed NOTCH and SHH signaling among significantly enriched pathways (Fig. 4k, Supplementary Table 4). Second, we repeated gene expression profiling of CP tumours in Mcre:NICD1 animals and wild type CPs at P0 and P21 using a similar microarray approach.
The differentially expressed genes in murine CPPs overlapped with those identified in human CPPs (Fig. 4l). MetaCore analysis of these common differential transcripts between murine and human CPPs revealed significant enrichment for genes in both pathways, indicating murine CPPs display striking resemblance to human CPPs (Fig. 4m, Supplementary Table 5). Third, we examined datasets from recently published studies of human CP tumours and found higher expression levels of roof plate markers in CPPs compared to CPCs 14 , 15 ( Supplementary Fig. 6a). MetaCore gene expression analysis showed significant enrichment for SHH and NOTCH pathways in human CPCs, an observation also supported by analysis of methylation and copy number variation data (Supplementary Fig. 6b-d, Supplementary Table 6). Q-RT-PCR analysis of human CP tumours revealed increased expression for genes of both pathways in CP tumours compared to normal human CP epithelial cells (Fig. 4n). In situ hybridization demonstrated SHH mRNA in both CPPs and CPCs (Percentage of SHH-positive cells in CPP: 24.82 ± 3.32, n=11 specimens examined from 10 individuals; CPC: 36.33 ± 5.40, n=10; Disease-free control: 100%; n=5; Of note, signal strength varied among samples, likely due to brain regions sampled, specimen age and storage) ( Supplementary Fig. 6e). Together, these results indicate that human CP tumours display aberrant NOTCH and SHH signaling, suggesting that both pathways may play a similarly important role in human CP tumourigenesis.

Notch-induced CP tumour cells are mono-ciliated
In vertebrates, Shh-driven signaling occurs via the primary cilium 16 , 17 . To understand the mechanisms by which tumour cells with sustained Notch signaling respond to Shh, we examined primary cilia in CP tumour and epithelial cells. Transmission electron microscopy and staining for cilia markers ADP-ribosylation factor-like 13b (Arl13b) 35 , γ-tubulin, and acetylated α-tubulin revealed multiple short primary cilia in wild type and EYFP + epithelial cells in Math1-Cre;Rosa26-EYFP mice, whereas a single, longer primary cilium is present in tumour cells (epithelial cells: 1.70 ± 0.11 μm, n=10; tumour cells: 3.78 ± 0.13 μm, n=12; two-tailed unpaired t test, p<0.0001; data from a single experiment are shown, raw data are available in Supplementary Table 9) (Fig. 5a-c, Supplementary Fig. 7a, 7b). Gene expression profiling showed that many genes involved in ciliogenesis are downregulated in tumour cells 36 -38

Mono-ciliated CP tumour cells are uniquely capable of transducing Shh signals
To determine whether the distinct cilia pattern of tumour cells affects Shh signaling, we characterized Shh-driven Smo ciliary translocation. Both CP epithelial and tumour cells express Lmx1a when grown with serum; however, only tumour cells can proliferate (Fig. 6a, 6b, Supplementary Fig. 7g). After serum removal, cells were treated with ShhN or SAG, a Shh pathway agonist 42

Notch-induced CP tumour arises from roof plate progenitors
The similarity in gene expression between tumour and roof plate cells, together with elevated Notch signaling and solitary primary cilium in the latter, suggests that Notchinduced CP tumour is related to the roof plate. To delineate the developmental origin of CP tumour, we first analyzed the distribution of Atoh1 + progenitors in hindbrain roof plate using Math1 M1GFP mice with enhanced green fluorescent protein (EGFP) fused to the Cterminal of Atoh1 43 . In addition to rhombic lip, Atoh1:EGFP + cells are present in the Lmx1 + /Otx2 + upper roof plate but largely absent from lower roof plate (Fig. 7a, Supplementary Fig. 8a). Second, we analyzed tumour formation in Mcre;NICD1 animals during development. At day E12.5, though many NICD1 + /GFP + cells are located in rhombic lip bordering Lmx1a + roof plate, some NICD1 + /GFP + cells are present within CP forming into papillary structures (Fig. 7b). These prospective Lmx1a + tumour cells undergo proliferation (Ki-67 + ), and remain undifferentiated (Aqp1 − ) (Fig. 7b, Supplementary Fig.  8b). At day E14.5, most NICD1 + /GFP + tumour cells are found in Lmx1a + upper roof plate and rostral half of the CP (Fig. 7c). Third, proliferative progenitors (Lmx1a + /Ki-67 + /Aqp1 − ) in hindbrain roof plate differentiate into post-mitotic epithelial cells that form the CP epithelium (Lmx1a + /Ki-67 − /Aqp1 + ) during embryogenesis 44 , 45 . The shape and size roof plate are similar between wild type and Mcre;NICD1 mice; however, tumour cells remain proliferative and undifferentiated (Ki-67 + /Aqp1 − ) even after their incorporation into CP epithelium (Fig. 8a, Supplementary Fig. 8c). Together, these results indicate that CP tumours in these animals arise from roof plate progenitors and migrate into the CP where they continue to undergo Shh-driven proliferation throughout development (Fig. 8b, 8c).

DISCUSSION
In this study, we examined Notch signaling in CP tumours, a group of rare brain neoplasms most commonly found in children. Consistent with a role for NOTCH pathway in these tumours 8 -11 , analysis of human CP tumour datasets revealed significant enrichment for NOTCH signaling. Using animal models, we showed that sustained Notch 1 signaling leads to CP tumours that, similar to human CP tumours, express CP markers and undergo increased proliferation. Cross-species molecular analysis demonstrates that these CP tumours closely resemble their human counterpart, validating our animal models as accurate representation of human disease.
CP tumours are thought to originate from CP epithelium 14 , 15

Human Samples
All human CP specimens were procured with informed consent from human subjects following the requirements by institutional review boards at Sanford Research, Sanford Burnham Prebys Medical Discovery Institute, and Ludwig-Maximilans-University. All CP specimens from Boston Children's Hospital were obtained with informed consent from human subjects under an approved institutional review board protocol (Supplementary Table  7). Normal human CP epithelial cells (ScienCell, San Diego, CA) were used as control in gene expression analysis. All tissues were handled in accordance with guidelines and regulations for the research use of human brain tissue set forth by the NIH (http:// bioethics.od.nih.gov/humantissue.html). Diagnoses of human CP specimens from Boston Children's Hospital were reviewed by two neuropathologists (H.G.W.L., S.S.) using standard WHO criteria 66 .

Isolation and culture of primary CP cells
Multiple sets of CP specimens from Mcre;NICD1 and/or wild type mice were collected. To obtain sufficient number of cells, each set of specimens included tissues pooled from multiple animals of the same genotype. Gender information is not available for animals at P0 and P7. Both male and female animals were used at other time points (see Supplementary  Table 9 for information on animals used for each experiment  Fig. 4a-4d, Fig. 6b-6d, 6f-6h, Supplementary Fig. 7g). Results from these studies confirmed their identity. Given the short time (< 8 days) during which CP cells were maintained as primary cultures supplemented with antibiotics, we did not perform test for mycoplasma contamination.

Histology, Immunohistochemistry, Immunofluorescence, and Immunocytochemistry
Whole brains were fixed overnight in 4% paraformaldehyde (PFA) and processed for paraffin embedding and sectioning. Tissue sections were deparaffinized with CitriSolv (Decon Labs, King of Prussia, PA), then rehydrated with graded ethanol series. For frozen tissues, samples were further equilibrated in 20% sucrose at 4°C for 24-48 hours, embedded in TissuTek-Optimal Cutting Temperature (O.C.T.) compound (Sakura Finetek, Torrance, CA), and sectioned (15-20 μm thickness) on a cryostat. Cultured cells were fixed in 4% PFA at room temperature for 10 minutes and stored in phosphate buffered saline (PBS). For primary cilia staining, cells were fixed with cold methanol for 10 minutes and stored in PBS at 4°C.
No randomization was used to determine how samples were allocated to experimental groups and processed. Immunostaining was carried out as described previously 67 . Heatinduced eptiope retrieval was performed for paraffin-embedded tissue sections in Rodent Decloaker (Biocare Medical, Concord, CA). For immunohistochemistry, endogenous peroxidase activities were inactivated in 3% H 2 O 2 for 10 minutes at room temperature. Tissue sections were blocked with 10% normal sera in PBS-0.1% Triton X-100 for 1 hour at room temperature and incubated with primary antibodies for 1 hour. After washes with PBS, biotinylated secondary antibodies were applied for 1 hour, followed by treatment with Avidin/Biotinylated enzyme Complex and substrate/chromogen incubation (Vector laboratories, Burlingame, CA). Slides were counterstained with hematoxylin. For immunofluorescence, tissue sections or cells were sequentially probed with primary antibodies and fluorescently labeled secondary antibodies (Jackson Immunoresearch, West Grove, PA). Primary antibodies used and dilution ratios are: mouse monoclonal anti-Acetylated α-Tubulin (1:500, ab24610, clone 6-11B-1 Table 9.
For staining of primary cilia in human CP tumours and normal human CPs, the investigator was blinded to group allocation. Tissues used include: normal CP: 6 disease-free individuals; CPP: 17 tumour specimens from 16 individuals; CPC: 13 tumours from 10 individuals (Supplementary Table 9). Cilia staining was assessed by analyzing 5 distinct tissue regions.

Image Acquisition
Whole-mount images were obtained using a Nikon SMZ1000 Stereomicroscope. Light and fluorescent microscopic images were obtained by a Nikon Eclipse 90i microscope system or a Nikon confocal microscope system A1 + .

Transmission Electron Microscopy
CP specimens were collected at different time points. To ensure collection of sufficient amount of normal CP specimens, each normal CP specimen includes tissues pooled from 2 wild type animals for P7, P14 and P22. Information on animals used is available in Supplementary

Immunoblotting
Immunoblotting was carried out as described previously 67 . Multiple sets of CP specimens were collected at each time point. To obtain sufficient quantity of protein, each set of specimens may contain tissues pooled from several animals of the same genotype. Gender information is not available for animals at P0 and P7 (see Supplementary Table 9 for information on animals used

RNA preparation, Quantitative RT-PCR, In-situ hybridization, microarray, and RNA-seq
For murine tissues, three independent sets of tumour specimens from Mcre;NICD1 mice and three sets CP specimens from wild type mice were collected at each time point. To ensure that sufficient amount of RNA can be extracted, each set of specimens included tissues pooled from several animals of the same genotype. Gender information was not available for animals at P0 and P7 (see Supplementary Table 9 for information on animals used for each set of specimens). For human tissues, seven CPPs from seven individuals, two CPCs from two individuals, and normal human CP epithelial cells (ScienCell) were used (Supplementary Table 9 Table 8). The following Taqman assays for human genes were used (Life Technologies): GLI1 (Hs01110766_m1), PTCH1 (Hs00181117_m1), MYCN (Hs00232074_m1), HES5 (Hs01387463_g1), HES1 (Hs00172878_m1), HEY1 (Hs01114113_m1), ACTB (Hs99999903_m1). Data were analyzed using ABI Fast System SDS software. Transcript levels were determined as the number of transcripts for each gene relative to those of Actb (mouse) or ACTB (human). The results for each set of specimens were obtained by averaging transcript levels of technical triplicates and used for subsequent analyses. Data from Q-RT-PCR experiments can be found in Supplementary Table 9.
Exclusion was applied only on rare occasions when one of the three wells of the triplicate was a significant outlier; however, for each sample in question, Q-RT-PCRs were repeated in independent experiments, which validated the exclusion.
In-situ hybridization was performed by RNA In Situ Hybridization Core facility at Baylor College of Medicine as described previously 69 . For analysis of Shh, Mycn, and Gli1 expression, CP specimens from multiple animals were collected at P0 and P14. For analysis of Mycn expression in Mcre;NICD1 animals treated with vismodegib or vehicle, three tumours from three vismodegib-treated mice, and five tumours from five vehicle-treated mice were examined at P0. For Hes1 and Hes5 expression analysis, two Mcre;NICD1 mice and one wild type mouse were collected at day E14.5. Gender information was not available for embryos, P0 animals, and the P8 animal for sense probe controls (see Supplementary  Table 9 for information animals used for each experiment). For human tissue samples, SHH (probe #600951), PPIB (positive control, probe #313901), dapB (negative control, probe #310043) RNA were visualized using RNAscope 2.0 HD detection kit-BROWN (Advanced Cell Diagnostics, Hayward, CA) according to manufacturer's protocol. Human tissues used include: normal CP: 5 disease-free individuals; CPP: 11 tumour specimens from 10 individuals; CPC: 10 tumours from 10 individuals (Supplementary Tables 7, 9 For RNA-seq experiments, total RNA samples were ribo-depleted using the Ribominus Eukaryote System (Life Technologies), and used to generate sequencing libraries of barcoded fragment using the Ion Total RNA-Seq Kit V2 (Life Technologies). Libraries were sequenced on the Ion Proton sequencer, three libraries per Ion Proton PI Chip, using 200 bp sequencing reagents. Reads were aligned to the mouse genome (mm10) using a combination of Tophat2, and Bowtie2 (http://tophat.cbcb.umd.edu/). Differentially expressed transcripts were detected using the Cufflinks Cuffdiff package (http://cufflinks.cbcb.umd.edu/) and transcripts with a q-value <0.05 (Cuffdiff) were analyzed by ingenuity pathway analysis (Ingenuity Systems, Redwood City, CA). Principal component analysis was performed using R package rgl (http://cran.r-project.org/web/packages/rgl/index.html), a 3D visualization system based on OpenGL. Hierarchical clustering was performed using Genesis (http:// genome.tugraz.at/genesisclient/genesisclient_description.shtml).

Statistical analysis & Reproducibility
Statistical analyses were performed with GraphPadPrism 6.0 (GraphPad Software Inc., La Jolla, CA). All pooled data were expressed as the mean ± SEM. Differences between two groups were compared using an unpaired two-tailed t-test. Differences between multiple groups were analyzed with one-way or two-way ANOVA followed by Tukey's multiple comparisons test.
Results were considered significant at p < 0.05 (*); p<0.01 (**); p<0.001 (***); p<0.0001 (****). For western blot analysis, the expression of Aqp1 and Otx2 at P14, and Hes1 expression at P0 and P7, were repeated in one independent experiment. Analyses of the expression of Hes1 at P90, and Ccnd1 expression were repeated in two independent experiments with similar results. RNA-seq and microarray studies were performed in single experiments. For in-situ hybridization, analyses of Mycn expression in visomodegib-treated animals, and Mycn and Gli1 expression at P0 and P14, were repeated in one independent experiment with similar results. Analysis of Shh, Hes1, and Hes5 expression was performed in single experiments (Supplementary Table 9

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.   Table 9. Experiment was not repeated for Ttr expression; Aqp1 expression analysis was repeated in one independent experiment). (f) Western blot analysis of Aqp1 and Otx2 expression in CP tumours and wild type (WT) CPs at P14. β-actin serves as loading control. Molecular size marker and representative image of unprocessed blots can be found in Supplementary Figure 9.      marks NICD1 + tumour cells in the roof plate and CP. Lmx1a expression (red) labels the roof plate and CP lineage, while DAPI staining (blue) marks nuclei. Scale bar: 300μm. Shh pathways during the roof plate/CP morphogenesis and tumour formation. Epithelial progenitors in the roof plate (RP, yellow) next to the rhombic lip (RL) exhibits active Notch signaling, possess a solitary primary cilium, and proliferate in response to Shh (green dots) secreted from multiciliated epithelial cells (grey). The self-renewal will cease as Notch pathway activity in progenitor cells is attenuated to allow for multiciliate differentiation, thereby abolishing the response of the progenitors to Shh that drives their expansion during development. (c) Epithelial progenitors with constitutive Notch pathway activity (red) remain mono-ciliated and undergo aberrant proliferation to become tumour cells that retain the ability to respond to Shh signals in the local environment and undergo Shh-driven proliferation.