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Regulation of the JMJD3 (KDM6B) histone demethylase in glioblastoma stem cells by STAT3

View Article: PubMed Central - PubMed

ABSTRACT

The growth factor and cytokine regulated transcription factor STAT3 is required for the self-renewal of several stem cell types including tumor stem cells from glioblastoma. Here we show that STAT3 inhibition leads to the upregulation of the histone H3K27me2/3 demethylase Jmjd3 (KDM6B), which can reverse polycomb complex-mediated repression of tissue specific genes. STAT3 binds to the Jmjd3 promoter, suggesting that Jmjd3 is a direct target of STAT3. Overexpression of Jmjd3 slows glioblastoma stem cell growth and neurosphere formation, whereas knockdown of Jmjd3 rescues the STAT3 inhibitor-induced neurosphere formation defect. Consistent with this observation, STAT3 inhibition leads to histone H3K27 demethylation of neural differentiation genes, such as Myt1, FGF21, and GDF15. These results demonstrate that the regulation of Jmjd3 by STAT3 maintains repression of differentiation specific genes and is therefore important for the maintenance of self-renewal of normal neural and glioblastoma stem cells.

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STAT3 controls GBM-SC neurosphere formation and proliferation through repression of Jmjd3.A. Knockdown of Jmjd3 rescues neurosphere formation in the presence of S3I-201. Representative images of GS6-22 and GS7-2 cells infected with either control or shJmjd3 containing lentivirus treated with DMSO or S3I-201 (50 μM). Images for GS6-22 cells were taken after 6 days and GS7-2 cells were taken after 4 days, based on differences in sphere formation rate in these lines. B. Quantification of neurosphere formation in GS6-22 cells. After 6 days of inhibitor treatment, the number of spheres per 100 cells was counted. Values represent the mean of triplicates within each treatment; bars SE (**p<0.01). C. After 4 days of inhibitor treatment, GS7-2 cell sphere formation was quantified as described for GS6-22 cells. Values represent the mean of triplicates within each treatment; bars SE (**p<0.01 relative to DMSO control, upon shJMJD3 infection). D. Representative images of GS6-22 and GS7-2 cells infected with overexpression retrovirus containing either a control (empty) plasmid, Jmjd3, or a catalytic domain mutant of Jmjd3. After three days of selection, cells were dissociated and replated at 100 cells per ml. Pictures were taken at 50X seven days after replating. E. Quantification of neurosphere formation capacity in GS6-22 and GS7-2 cells infected with the Jmjd3 overexpression retrovirus. Neurosphere formation assay was performed in triplicate (**p<0.01). F. GS6-22 and G. GS7-2 cells were infected with either control, Jmjd3, or Jmjd3 mutant retroviruses as previously described. Cells were pulsed with 30 μM BrdU for 16 hours. After 24 hours, cells were fixed and stained with an anti-BrdU antibody. Cells were also stained with 7-AAD at this time. The percentage of BrdU positive cells was analyzed using flow cytometry. Values represent the mean of 3 experiments; bars SD of the mean (*p<0.05).
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pone.0174775.g002: STAT3 controls GBM-SC neurosphere formation and proliferation through repression of Jmjd3.A. Knockdown of Jmjd3 rescues neurosphere formation in the presence of S3I-201. Representative images of GS6-22 and GS7-2 cells infected with either control or shJmjd3 containing lentivirus treated with DMSO or S3I-201 (50 μM). Images for GS6-22 cells were taken after 6 days and GS7-2 cells were taken after 4 days, based on differences in sphere formation rate in these lines. B. Quantification of neurosphere formation in GS6-22 cells. After 6 days of inhibitor treatment, the number of spheres per 100 cells was counted. Values represent the mean of triplicates within each treatment; bars SE (**p<0.01). C. After 4 days of inhibitor treatment, GS7-2 cell sphere formation was quantified as described for GS6-22 cells. Values represent the mean of triplicates within each treatment; bars SE (**p<0.01 relative to DMSO control, upon shJMJD3 infection). D. Representative images of GS6-22 and GS7-2 cells infected with overexpression retrovirus containing either a control (empty) plasmid, Jmjd3, or a catalytic domain mutant of Jmjd3. After three days of selection, cells were dissociated and replated at 100 cells per ml. Pictures were taken at 50X seven days after replating. E. Quantification of neurosphere formation capacity in GS6-22 and GS7-2 cells infected with the Jmjd3 overexpression retrovirus. Neurosphere formation assay was performed in triplicate (**p<0.01). F. GS6-22 and G. GS7-2 cells were infected with either control, Jmjd3, or Jmjd3 mutant retroviruses as previously described. Cells were pulsed with 30 μM BrdU for 16 hours. After 24 hours, cells were fixed and stained with an anti-BrdU antibody. Cells were also stained with 7-AAD at this time. The percentage of BrdU positive cells was analyzed using flow cytometry. Values represent the mean of 3 experiments; bars SD of the mean (*p<0.05).

Mentions: Neurosphere formation is a hallmark of neural stem cells, and the ability to culture neurospheres from glioblastomas strongly correlates with poor patient prognosis [49]. Because STAT3 represses Jmjd3 expression in GBM-SC, we investigated whether knockdown of Jmjd3 expression could rescue the abrogation of neurosphere formation that accompanies STAT3 inhibition. GS6-22 and GS7-2 cells were infected with lentiviruses expressing shRNA to Jmjd3 or with control lentiviruses. Control infected cells exhibited decreased neurosphere formation in response to S3I-201 treatment as expected (Fig 2A, 2B and 2C). shJmjd3 infected cells, however, were able to form spheres in the presence of S3I-201 (Fig 2A, 2B and 2C). This was also true for cells infected with a second, distinct shRNA to Jmjd3, indicating that this is a specific effect of Jmjd3 knockdown (S2A and S2B Fig). This effect was more pronounced in the GS7-2 cells (Fig 2C) than the GS6-22 cells (Fig 2B) likely due to the enhanced degree of knockdown in the GS7-2 cells (78% knockdown in the GS7-2 cells versus 55% for GS6-22 cells) (S2C and S2D Fig). These data indicate that STAT3 repression of Jmjd3 is necessary for neurosphere formation.


Regulation of the JMJD3 (KDM6B) histone demethylase in glioblastoma stem cells by STAT3
STAT3 controls GBM-SC neurosphere formation and proliferation through repression of Jmjd3.A. Knockdown of Jmjd3 rescues neurosphere formation in the presence of S3I-201. Representative images of GS6-22 and GS7-2 cells infected with either control or shJmjd3 containing lentivirus treated with DMSO or S3I-201 (50 μM). Images for GS6-22 cells were taken after 6 days and GS7-2 cells were taken after 4 days, based on differences in sphere formation rate in these lines. B. Quantification of neurosphere formation in GS6-22 cells. After 6 days of inhibitor treatment, the number of spheres per 100 cells was counted. Values represent the mean of triplicates within each treatment; bars SE (**p<0.01). C. After 4 days of inhibitor treatment, GS7-2 cell sphere formation was quantified as described for GS6-22 cells. Values represent the mean of triplicates within each treatment; bars SE (**p<0.01 relative to DMSO control, upon shJMJD3 infection). D. Representative images of GS6-22 and GS7-2 cells infected with overexpression retrovirus containing either a control (empty) plasmid, Jmjd3, or a catalytic domain mutant of Jmjd3. After three days of selection, cells were dissociated and replated at 100 cells per ml. Pictures were taken at 50X seven days after replating. E. Quantification of neurosphere formation capacity in GS6-22 and GS7-2 cells infected with the Jmjd3 overexpression retrovirus. Neurosphere formation assay was performed in triplicate (**p<0.01). F. GS6-22 and G. GS7-2 cells were infected with either control, Jmjd3, or Jmjd3 mutant retroviruses as previously described. Cells were pulsed with 30 μM BrdU for 16 hours. After 24 hours, cells were fixed and stained with an anti-BrdU antibody. Cells were also stained with 7-AAD at this time. The percentage of BrdU positive cells was analyzed using flow cytometry. Values represent the mean of 3 experiments; bars SD of the mean (*p<0.05).
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Related In: Results  -  Collection

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pone.0174775.g002: STAT3 controls GBM-SC neurosphere formation and proliferation through repression of Jmjd3.A. Knockdown of Jmjd3 rescues neurosphere formation in the presence of S3I-201. Representative images of GS6-22 and GS7-2 cells infected with either control or shJmjd3 containing lentivirus treated with DMSO or S3I-201 (50 μM). Images for GS6-22 cells were taken after 6 days and GS7-2 cells were taken after 4 days, based on differences in sphere formation rate in these lines. B. Quantification of neurosphere formation in GS6-22 cells. After 6 days of inhibitor treatment, the number of spheres per 100 cells was counted. Values represent the mean of triplicates within each treatment; bars SE (**p<0.01). C. After 4 days of inhibitor treatment, GS7-2 cell sphere formation was quantified as described for GS6-22 cells. Values represent the mean of triplicates within each treatment; bars SE (**p<0.01 relative to DMSO control, upon shJMJD3 infection). D. Representative images of GS6-22 and GS7-2 cells infected with overexpression retrovirus containing either a control (empty) plasmid, Jmjd3, or a catalytic domain mutant of Jmjd3. After three days of selection, cells were dissociated and replated at 100 cells per ml. Pictures were taken at 50X seven days after replating. E. Quantification of neurosphere formation capacity in GS6-22 and GS7-2 cells infected with the Jmjd3 overexpression retrovirus. Neurosphere formation assay was performed in triplicate (**p<0.01). F. GS6-22 and G. GS7-2 cells were infected with either control, Jmjd3, or Jmjd3 mutant retroviruses as previously described. Cells were pulsed with 30 μM BrdU for 16 hours. After 24 hours, cells were fixed and stained with an anti-BrdU antibody. Cells were also stained with 7-AAD at this time. The percentage of BrdU positive cells was analyzed using flow cytometry. Values represent the mean of 3 experiments; bars SD of the mean (*p<0.05).
Mentions: Neurosphere formation is a hallmark of neural stem cells, and the ability to culture neurospheres from glioblastomas strongly correlates with poor patient prognosis [49]. Because STAT3 represses Jmjd3 expression in GBM-SC, we investigated whether knockdown of Jmjd3 expression could rescue the abrogation of neurosphere formation that accompanies STAT3 inhibition. GS6-22 and GS7-2 cells were infected with lentiviruses expressing shRNA to Jmjd3 or with control lentiviruses. Control infected cells exhibited decreased neurosphere formation in response to S3I-201 treatment as expected (Fig 2A, 2B and 2C). shJmjd3 infected cells, however, were able to form spheres in the presence of S3I-201 (Fig 2A, 2B and 2C). This was also true for cells infected with a second, distinct shRNA to Jmjd3, indicating that this is a specific effect of Jmjd3 knockdown (S2A and S2B Fig). This effect was more pronounced in the GS7-2 cells (Fig 2C) than the GS6-22 cells (Fig 2B) likely due to the enhanced degree of knockdown in the GS7-2 cells (78% knockdown in the GS7-2 cells versus 55% for GS6-22 cells) (S2C and S2D Fig). These data indicate that STAT3 repression of Jmjd3 is necessary for neurosphere formation.

View Article: PubMed Central - PubMed

ABSTRACT

The growth factor and cytokine regulated transcription factor STAT3 is required for the self-renewal of several stem cell types including tumor stem cells from glioblastoma. Here we show that STAT3 inhibition leads to the upregulation of the histone H3K27me2/3 demethylase Jmjd3 (KDM6B), which can reverse polycomb complex-mediated repression of tissue specific genes. STAT3 binds to the Jmjd3 promoter, suggesting that Jmjd3 is a direct target of STAT3. Overexpression of Jmjd3 slows glioblastoma stem cell growth and neurosphere formation, whereas knockdown of Jmjd3 rescues the STAT3 inhibitor-induced neurosphere formation defect. Consistent with this observation, STAT3 inhibition leads to histone H3K27 demethylation of neural differentiation genes, such as Myt1, FGF21, and GDF15. These results demonstrate that the regulation of Jmjd3 by STAT3 maintains repression of differentiation specific genes and is therefore important for the maintenance of self-renewal of normal neural and glioblastoma stem cells.

No MeSH data available.


Related in: MedlinePlus