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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.


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Mono-ciliated CP tumour cells are uniquely capable of transducing Shh signals. (a) Dissociated CP tumour cells from Mcre;NICD1 animals were initially cultured in the presence of 10% fetal bovine serum (FBS). Cultured CP tumour cells were then switched to serum-free conditions for 2 days followed by treatment with ShhN or SAG for 1.5 or 3 days for signaling and proliferation analysis, respectively. (b) The expression of Lmx1a (red) and Ki-67 (red) is shown in CP tumour cells cultured with FBS. GFP (green) labels NICD1+ CP tumour cells, while Aqp1 labels CP epithelial cells. DAPI staining (blue) labels nuclei. Scale bar: 25μm. After treatment with ShhN, SAG, or vehicle, Smo (red) localization is shown in CP tumour (c) or epithelial cells (d). Arl13b expression (green) labels primary cilia, while DAPI staining (blue) labels nuclei. Scale bar: 10μm. (e) Q-RT-PCR analysis of Ptch1 and Smo expression in CP tumours from Mcre;NICD1 animals (black circles) and wild type CPs (WT, while circles) at P7 (data from technical replicates of each set of specimen in a single experiment are shown; experiment was not repeated. Raw data can be found in Supplementary Table 9). (f) The expression of Ki-67 (green) in CP tumour cells from Mcre;NICD1 animals treated with ShhN for 48 hours treatment is shown. Arl13b expression (red) labels primary cilia, DAPI staining (blue) labels nuclei. Notice that Ki-67 expression is present in mono-ciliated CP tumour cell, while multi-ciliated CP epithelial cell lacks Ki-67 expression. Scale bar: 10μm. (g) The expression of Ki-67 (red) in CP tumour cells from Mcre;NICD1 animals treated as indicated is shown. GFP (green) labels NICD1+ tumour cells. DAPI staining (blue) labels nuclei. Scale bar: 25μm. (h) Quantitation of the percentage of Ki-67+ cells in NICD1+/GFP+ tumour cells after 72-hour treatment as indicated (n=4 specimens/treatment, data from a single experiment are shown, raw data are available in Supplementary Table 9; mean ± SEM, two-way ANOVA, ***, P< 0.001; ****, P<0.0001).
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Figure 6: Mono-ciliated CP tumour cells are uniquely capable of transducing Shh signals. (a) Dissociated CP tumour cells from Mcre;NICD1 animals were initially cultured in the presence of 10% fetal bovine serum (FBS). Cultured CP tumour cells were then switched to serum-free conditions for 2 days followed by treatment with ShhN or SAG for 1.5 or 3 days for signaling and proliferation analysis, respectively. (b) The expression of Lmx1a (red) and Ki-67 (red) is shown in CP tumour cells cultured with FBS. GFP (green) labels NICD1+ CP tumour cells, while Aqp1 labels CP epithelial cells. DAPI staining (blue) labels nuclei. Scale bar: 25μm. After treatment with ShhN, SAG, or vehicle, Smo (red) localization is shown in CP tumour (c) or epithelial cells (d). Arl13b expression (green) labels primary cilia, while DAPI staining (blue) labels nuclei. Scale bar: 10μm. (e) Q-RT-PCR analysis of Ptch1 and Smo expression in CP tumours from Mcre;NICD1 animals (black circles) and wild type CPs (WT, while circles) at P7 (data from technical replicates of each set of specimen in a single experiment are shown; experiment was not repeated. Raw data can be found in Supplementary Table 9). (f) The expression of Ki-67 (green) in CP tumour cells from Mcre;NICD1 animals treated with ShhN for 48 hours treatment is shown. Arl13b expression (red) labels primary cilia, DAPI staining (blue) labels nuclei. Notice that Ki-67 expression is present in mono-ciliated CP tumour cell, while multi-ciliated CP epithelial cell lacks Ki-67 expression. Scale bar: 10μm. (g) The expression of Ki-67 (red) in CP tumour cells from Mcre;NICD1 animals treated as indicated is shown. GFP (green) labels NICD1+ tumour cells. DAPI staining (blue) labels nuclei. Scale bar: 25μm. (h) Quantitation of the percentage of Ki-67+ cells in NICD1+/GFP+ tumour cells after 72-hour treatment as indicated (n=4 specimens/treatment, data from a single experiment are shown, raw data are available in Supplementary Table 9; mean ± SEM, two-way ANOVA, ***, P< 0.001; ****, P<0.0001).

Mentions: 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 agonist42 (Fig. 6a). While such treatment failed to promote ciliary accumulation of Smo in epithelial cells, it led to translocation of Smo into the solitary primary cilium of tumour cells (Shh: n=3, 71.78±6.93%, p<0.0001; SAG: n=3, 74.99±3.03%, p<0.0001; two-way ANOVA) (Fig. 6c, 6d), even though both cell types express similar levels of Ptch1 and Smo (Fig. 6e), indicating that Shh signaling in the primary cilium is preserved in tumour cells, but lost in epithelial cells despite their multiple primary cilia. Indeed, ShhN or SAG restored the percentage of Ki-67+ tumour cells after serum removal, an effect that can be reversed by cyclopamine, indicating that tumour cells are uniquely capable of responding to Shh through proliferation (Fig. 6f–h).


Sonic Hedgehog promotes proliferation of Notch-dependent monociliated choroid plexus tumour cells
Mono-ciliated CP tumour cells are uniquely capable of transducing Shh signals. (a) Dissociated CP tumour cells from Mcre;NICD1 animals were initially cultured in the presence of 10% fetal bovine serum (FBS). Cultured CP tumour cells were then switched to serum-free conditions for 2 days followed by treatment with ShhN or SAG for 1.5 or 3 days for signaling and proliferation analysis, respectively. (b) The expression of Lmx1a (red) and Ki-67 (red) is shown in CP tumour cells cultured with FBS. GFP (green) labels NICD1+ CP tumour cells, while Aqp1 labels CP epithelial cells. DAPI staining (blue) labels nuclei. Scale bar: 25μm. After treatment with ShhN, SAG, or vehicle, Smo (red) localization is shown in CP tumour (c) or epithelial cells (d). Arl13b expression (green) labels primary cilia, while DAPI staining (blue) labels nuclei. Scale bar: 10μm. (e) Q-RT-PCR analysis of Ptch1 and Smo expression in CP tumours from Mcre;NICD1 animals (black circles) and wild type CPs (WT, while circles) at P7 (data from technical replicates of each set of specimen in a single experiment are shown; experiment was not repeated. Raw data can be found in Supplementary Table 9). (f) The expression of Ki-67 (green) in CP tumour cells from Mcre;NICD1 animals treated with ShhN for 48 hours treatment is shown. Arl13b expression (red) labels primary cilia, DAPI staining (blue) labels nuclei. Notice that Ki-67 expression is present in mono-ciliated CP tumour cell, while multi-ciliated CP epithelial cell lacks Ki-67 expression. Scale bar: 10μm. (g) The expression of Ki-67 (red) in CP tumour cells from Mcre;NICD1 animals treated as indicated is shown. GFP (green) labels NICD1+ tumour cells. DAPI staining (blue) labels nuclei. Scale bar: 25μm. (h) Quantitation of the percentage of Ki-67+ cells in NICD1+/GFP+ tumour cells after 72-hour treatment as indicated (n=4 specimens/treatment, data from a single experiment are shown, raw data are available in Supplementary Table 9; mean ± SEM, two-way ANOVA, ***, P< 0.001; ****, P<0.0001).
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Figure 6: Mono-ciliated CP tumour cells are uniquely capable of transducing Shh signals. (a) Dissociated CP tumour cells from Mcre;NICD1 animals were initially cultured in the presence of 10% fetal bovine serum (FBS). Cultured CP tumour cells were then switched to serum-free conditions for 2 days followed by treatment with ShhN or SAG for 1.5 or 3 days for signaling and proliferation analysis, respectively. (b) The expression of Lmx1a (red) and Ki-67 (red) is shown in CP tumour cells cultured with FBS. GFP (green) labels NICD1+ CP tumour cells, while Aqp1 labels CP epithelial cells. DAPI staining (blue) labels nuclei. Scale bar: 25μm. After treatment with ShhN, SAG, or vehicle, Smo (red) localization is shown in CP tumour (c) or epithelial cells (d). Arl13b expression (green) labels primary cilia, while DAPI staining (blue) labels nuclei. Scale bar: 10μm. (e) Q-RT-PCR analysis of Ptch1 and Smo expression in CP tumours from Mcre;NICD1 animals (black circles) and wild type CPs (WT, while circles) at P7 (data from technical replicates of each set of specimen in a single experiment are shown; experiment was not repeated. Raw data can be found in Supplementary Table 9). (f) The expression of Ki-67 (green) in CP tumour cells from Mcre;NICD1 animals treated with ShhN for 48 hours treatment is shown. Arl13b expression (red) labels primary cilia, DAPI staining (blue) labels nuclei. Notice that Ki-67 expression is present in mono-ciliated CP tumour cell, while multi-ciliated CP epithelial cell lacks Ki-67 expression. Scale bar: 10μm. (g) The expression of Ki-67 (red) in CP tumour cells from Mcre;NICD1 animals treated as indicated is shown. GFP (green) labels NICD1+ tumour cells. DAPI staining (blue) labels nuclei. Scale bar: 25μm. (h) Quantitation of the percentage of Ki-67+ cells in NICD1+/GFP+ tumour cells after 72-hour treatment as indicated (n=4 specimens/treatment, data from a single experiment are shown, raw data are available in Supplementary Table 9; mean ± SEM, two-way ANOVA, ***, P< 0.001; ****, P<0.0001).
Mentions: 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 agonist42 (Fig. 6a). While such treatment failed to promote ciliary accumulation of Smo in epithelial cells, it led to translocation of Smo into the solitary primary cilium of tumour cells (Shh: n=3, 71.78±6.93%, p<0.0001; SAG: n=3, 74.99±3.03%, p<0.0001; two-way ANOVA) (Fig. 6c, 6d), even though both cell types express similar levels of Ptch1 and Smo (Fig. 6e), indicating that Shh signaling in the primary cilium is preserved in tumour cells, but lost in epithelial cells despite their multiple primary cilia. Indeed, ShhN or SAG restored the percentage of Ki-67+ tumour cells after serum removal, an effect that can be reversed by cyclopamine, indicating that tumour cells are uniquely capable of responding to Shh through proliferation (Fig. 6f–h).

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