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

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Notch-induced CP tumours display aberrant Shh signaling. (a) Principal component analysis of CP tumours (black dots) and wild type CPs (WT, red dots) at P21 (n=3 specimens/genotype). (b) Venn diagram of differential genes (FDR < 0.05) between tumours and CPs at P21 (A, n=3 specimens/genotype), at P0 (B, n=3 specimens/genotype), and of tumour cells between P0 and P21 (C, n=3 specimens/time point). (c) Hierarchical clustering of tumours and CPs (n=3 specimens/genotype/time point) based on genes expressed in roof plate (RP) and CP (one-way ANOVA, FDR < 0.05, fold change is shown). (d) Volcano-plot analysis of gene expression of tumours and CPs at P0 (n=3 specimens/genotype), and of tumours at P0 and P21 (n=3 specimens/time point). Differential transcripts with statistical significance (FDR < 0.05, -log10 of p-value, y-axis) are shown in red (< 2 fold change) or green (> 2 fold change) dots. Non-significant genes (FDR > 0.05) are shown in black or yellow dots (2-fold cutoff). Arrows label select genes with significant differential expression. (e) Median FKPM values of differential genes between tumours and CPs (n=3 specimens/genotype/time point, mean ± SEM, two-way ANOVA, *, P< 0.05; **, P<0.01; ***, P<0.001). Q-RT-PCR analysis of the expression of Gli1 and Mycn (f), and Shh (h) in tumours (black circles) and CPs (white circles) at different time points (data from technical replicates of each specimen set in a single experiment are shown; experiment was repeated independently once with similar results. Raw data can be found in Supplementary Table 9). (g) Western blot analysis of Ccnd1 expression in tumours and CPs. β-actin serves as loading control (n=1 specimen/genotype/time point, representative blot image from one of three independent repeated experiments is shown). Molecular size marker and representative image of unprocessed blots can be found in Supplementary Figure 9. (i) In-situ hybridization analysis of Shh, Gli1, and Mycn expression in tumours and wild type CPs. Shh expression is detected in epithelium (arrowheads), but absent in tumour cells (arrows) at P0, a pattern complementary to those of Gli1 and Mycn. Scale bar: 25μm.
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Figure 3: Notch-induced CP tumours display aberrant Shh signaling. (a) Principal component analysis of CP tumours (black dots) and wild type CPs (WT, red dots) at P21 (n=3 specimens/genotype). (b) Venn diagram of differential genes (FDR < 0.05) between tumours and CPs at P21 (A, n=3 specimens/genotype), at P0 (B, n=3 specimens/genotype), and of tumour cells between P0 and P21 (C, n=3 specimens/time point). (c) Hierarchical clustering of tumours and CPs (n=3 specimens/genotype/time point) based on genes expressed in roof plate (RP) and CP (one-way ANOVA, FDR < 0.05, fold change is shown). (d) Volcano-plot analysis of gene expression of tumours and CPs at P0 (n=3 specimens/genotype), and of tumours at P0 and P21 (n=3 specimens/time point). Differential transcripts with statistical significance (FDR < 0.05, -log10 of p-value, y-axis) are shown in red (< 2 fold change) or green (> 2 fold change) dots. Non-significant genes (FDR > 0.05) are shown in black or yellow dots (2-fold cutoff). Arrows label select genes with significant differential expression. (e) Median FKPM values of differential genes between tumours and CPs (n=3 specimens/genotype/time point, mean ± SEM, two-way ANOVA, *, P< 0.05; **, P<0.01; ***, P<0.001). Q-RT-PCR analysis of the expression of Gli1 and Mycn (f), and Shh (h) in tumours (black circles) and CPs (white circles) at different time points (data from technical replicates of each specimen set in a single experiment are shown; experiment was repeated independently once with similar results. Raw data can be found in Supplementary Table 9). (g) Western blot analysis of Ccnd1 expression in tumours and CPs. β-actin serves as loading control (n=1 specimen/genotype/time point, representative blot image from one of three independent repeated experiments is shown). Molecular size marker and representative image of unprocessed blots can be found in Supplementary Figure 9. (i) In-situ hybridization analysis of Shh, Gli1, and Mycn expression in tumours and wild type CPs. Shh expression is detected in epithelium (arrowheads), but absent in tumour cells (arrows) at P0, a pattern complementary to those of Gli1 and Mycn. Scale bar: 25μm.

Mentions: 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–f, Fig. 3d, 3e, Supplementary Fig. 1c, 1d, Supplementary Fig. 3b, Supplementary Fig. 4e, Supplementary Table 1). Though tumour cells express higher mRNA levels for roof plate markers Lmx1a, Gdf7, Zic3, Zic4, and Msx2, the expression of many genes found in CP epithelium is significantly lower27–32 (Fig. 3c, 3e, Supplementary Table 1).


Sonic Hedgehog promotes proliferation of Notch-dependent monociliated choroid plexus tumour cells
Notch-induced CP tumours display aberrant Shh signaling. (a) Principal component analysis of CP tumours (black dots) and wild type CPs (WT, red dots) at P21 (n=3 specimens/genotype). (b) Venn diagram of differential genes (FDR < 0.05) between tumours and CPs at P21 (A, n=3 specimens/genotype), at P0 (B, n=3 specimens/genotype), and of tumour cells between P0 and P21 (C, n=3 specimens/time point). (c) Hierarchical clustering of tumours and CPs (n=3 specimens/genotype/time point) based on genes expressed in roof plate (RP) and CP (one-way ANOVA, FDR < 0.05, fold change is shown). (d) Volcano-plot analysis of gene expression of tumours and CPs at P0 (n=3 specimens/genotype), and of tumours at P0 and P21 (n=3 specimens/time point). Differential transcripts with statistical significance (FDR < 0.05, -log10 of p-value, y-axis) are shown in red (< 2 fold change) or green (> 2 fold change) dots. Non-significant genes (FDR > 0.05) are shown in black or yellow dots (2-fold cutoff). Arrows label select genes with significant differential expression. (e) Median FKPM values of differential genes between tumours and CPs (n=3 specimens/genotype/time point, mean ± SEM, two-way ANOVA, *, P< 0.05; **, P<0.01; ***, P<0.001). Q-RT-PCR analysis of the expression of Gli1 and Mycn (f), and Shh (h) in tumours (black circles) and CPs (white circles) at different time points (data from technical replicates of each specimen set in a single experiment are shown; experiment was repeated independently once with similar results. Raw data can be found in Supplementary Table 9). (g) Western blot analysis of Ccnd1 expression in tumours and CPs. β-actin serves as loading control (n=1 specimen/genotype/time point, representative blot image from one of three independent repeated experiments is shown). Molecular size marker and representative image of unprocessed blots can be found in Supplementary Figure 9. (i) In-situ hybridization analysis of Shh, Gli1, and Mycn expression in tumours and wild type CPs. Shh expression is detected in epithelium (arrowheads), but absent in tumour cells (arrows) at P0, a pattern complementary to those of Gli1 and Mycn. Scale bar: 25μm.
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Figure 3: Notch-induced CP tumours display aberrant Shh signaling. (a) Principal component analysis of CP tumours (black dots) and wild type CPs (WT, red dots) at P21 (n=3 specimens/genotype). (b) Venn diagram of differential genes (FDR < 0.05) between tumours and CPs at P21 (A, n=3 specimens/genotype), at P0 (B, n=3 specimens/genotype), and of tumour cells between P0 and P21 (C, n=3 specimens/time point). (c) Hierarchical clustering of tumours and CPs (n=3 specimens/genotype/time point) based on genes expressed in roof plate (RP) and CP (one-way ANOVA, FDR < 0.05, fold change is shown). (d) Volcano-plot analysis of gene expression of tumours and CPs at P0 (n=3 specimens/genotype), and of tumours at P0 and P21 (n=3 specimens/time point). Differential transcripts with statistical significance (FDR < 0.05, -log10 of p-value, y-axis) are shown in red (< 2 fold change) or green (> 2 fold change) dots. Non-significant genes (FDR > 0.05) are shown in black or yellow dots (2-fold cutoff). Arrows label select genes with significant differential expression. (e) Median FKPM values of differential genes between tumours and CPs (n=3 specimens/genotype/time point, mean ± SEM, two-way ANOVA, *, P< 0.05; **, P<0.01; ***, P<0.001). Q-RT-PCR analysis of the expression of Gli1 and Mycn (f), and Shh (h) in tumours (black circles) and CPs (white circles) at different time points (data from technical replicates of each specimen set in a single experiment are shown; experiment was repeated independently once with similar results. Raw data can be found in Supplementary Table 9). (g) Western blot analysis of Ccnd1 expression in tumours and CPs. β-actin serves as loading control (n=1 specimen/genotype/time point, representative blot image from one of three independent repeated experiments is shown). Molecular size marker and representative image of unprocessed blots can be found in Supplementary Figure 9. (i) In-situ hybridization analysis of Shh, Gli1, and Mycn expression in tumours and wild type CPs. Shh expression is detected in epithelium (arrowheads), but absent in tumour cells (arrows) at P0, a pattern complementary to those of Gli1 and Mycn. Scale bar: 25μm.
Mentions: 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–f, Fig. 3d, 3e, Supplementary Fig. 1c, 1d, Supplementary Fig. 3b, Supplementary Fig. 4e, Supplementary Table 1). Though tumour cells express higher mRNA levels for roof plate markers Lmx1a, Gdf7, Zic3, Zic4, and Msx2, the expression of many genes found in CP epithelium is significantly lower27–32 (Fig. 3c, 3e, Supplementary Table 1).

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus