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STAT6 expression in glioblastoma promotes invasive growth.

Merk BC, Owens JL, Lopes MB, Silva CM, Hussaini IM - BMC Cancer (2011)

Bottom Line: STAT6-deficient GBM cells showed a reduction in (3)H-Thymidine uptake compared to the wild-type.There was some variation among the different shRNA- silenced clones, but all had a reduction in (3)H-Thymidine uptake ranging from 35%- 70% in U-1242MG and 40- 50% in U-87MG cells.Invasiveness was decreased by 25-40% and 30-75% in U-1242MG and U-87MG cells, respectively.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Virginia, Charlottesville, VA 22908, USA. bcm4u@virginia.edu

ABSTRACT

Background: Glioblastoma (GBM) is a highly aggressive malignant primary brain tumor, characterized by rapid growth, diffuse infiltration of cells into both adjacent and remote brain regions, and a generalized resistance to currently available treatment modalities. Recent reports in the literature suggest that Signal Transducers and Activators of Transcription (STATs) play important roles in the regulation of GBM pathophysiology.

Methods: STAT6 protein expression was analyzed by Western blotting in GBM cell lines and by immunohistochemistry in a tissue microarray (TMA) of glioma patient tissues. We utilized shRNA against STAT6 to investigate the effects of prolonged STAT6 depletion on the growth and invasion of two STAT6-positive GBM cell lines. Cell proliferation was assessed by measuring (3)H-Thymidine uptake over time. Invasion was measured using an in vitro transwell assay in which cells invade through a type IV collagen matrix toward a chemoattractant (Fetal Bovine Serum). Cells were then stained and counted. Kaplan-Meyer survival curves were generated to show the correlation between STAT6 gene expression and patient survival in 343 glioma patients and in a subset of patients with only GBM. Gene expression microarray and clinical data were acquired from the Rembrandt 1 public data depository (https://caintegrator.nci.nih.gov/rembrandt/). Lastly, a genome-wide expression microarray analysis was performed to compare gene expression in wild-type GBM cells to expression in stable STAT6 knockdown clones.

Results: STAT6 was expressed in 2 GBM cell lines, U-1242MG and U-87MG, and in normal astrocytes (NHA) but not in the U-251MG GBM cell line. In our TMA study, STAT6 immunostaining was visible in the majority of astrocytomas of all grades (I-IV) but not in normal brain tissue. In positive cells, STAT6 was localized exclusively in the nuclei over 95% of the time. STAT6-deficient GBM cells showed a reduction in (3)H-Thymidine uptake compared to the wild-type. There was some variation among the different shRNA- silenced clones, but all had a reduction in (3)H-Thymidine uptake ranging from 35%- 70% in U-1242MG and 40- 50% in U-87MG cells. Additionally, STAT6- depleted cells were less invasive than controls in our in vitro transmembrane invasion assay. Invasiveness was decreased by 25-40% and 30-75% in U-1242MG and U-87MG cells, respectively. The microarray analysis identified matrix metalloproteinase 1 (MMP-1) and urokinase Plasminogen activator (uPA) as potential STA6 target genes involved in the promotion of GBM cell invasion. In a Kaplan-Meier survival curve based on Rembrandt 1 gene expression microarray and clinical data, there was a significant difference in survival (P < 0.05) between glioma patients with up- and down-regulated STAT6. Decreased STAT6 expression correlated with longer survival times. In two subsets of patients with either grade IV tumors (GBM) or Grade II/III astrocytomas, there was a similar trend that however did not reach statistical significance.

Conclusions: Taken together, these findings suggest a role for STAT6 in enhancing cell proliferation and invasion in GBM, which may explain why up-regulation of STAT6 correlates with shorter survival times in glioma patients. This report thus identifies STAT6 as a new and potentially promising therapeutic target.

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STAT6 expression correlates with survival in human glioma patients. The Rembrandt database was used to perform a Kaplan-Meier survival analysis of glioma patients with differential STAT6 expression. Up- and down-regulation were defined as a two-fold or greater deviation from intermediate expression. Figure 7a: Among all glioma patients (n= 343) there was a significant difference in survival (p < 0.05) between patients with increased vs. decreased STAT6 gene expression. Figure 7b: In the subset of patients with Grade IV tumors/GBM (n= 181), there was a clear trend towards a similar correlation between STAT6 expression and survival; however statistical significance was not reached. Figure 7c: In patients with Grade II/III astrocytoma (n= 105), a similar trend was observed.
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Figure 7: STAT6 expression correlates with survival in human glioma patients. The Rembrandt database was used to perform a Kaplan-Meier survival analysis of glioma patients with differential STAT6 expression. Up- and down-regulation were defined as a two-fold or greater deviation from intermediate expression. Figure 7a: Among all glioma patients (n= 343) there was a significant difference in survival (p < 0.05) between patients with increased vs. decreased STAT6 gene expression. Figure 7b: In the subset of patients with Grade IV tumors/GBM (n= 181), there was a clear trend towards a similar correlation between STAT6 expression and survival; however statistical significance was not reached. Figure 7c: In patients with Grade II/III astrocytoma (n= 105), a similar trend was observed.

Mentions: Based on our in vitro data (Figures 5 and 6) relating STAT6 expression to increased GBM growth and invasion, we hypothesized that increased STAT6 expression would also correlate with a worse prognosis in glioma patients. To test this theory, we took advantage of the publicly available patient data in the NCI Repository for Molecular Brain Neoplasia Data (REMBRANDT) database [1]. Using microarray-based gene expression data and associated clinical reports, we generated a Kaplan-Meier survival curve based on differential STAT6 expression among 343 glioma patients (Figure 7A). They included patients with GBMs (181), grade II/III astrocytomas (105), grade II/III oligodendrogliomas (50), and mixed tumors (7). Up-and down-regulation were defined as a two-fold increase or decrease in STAT6 expression, respectively, compared to the mean expression level within the given data set. Based on these criteria, STAT6 was up-regulated in ten patients, down-regulated in 72 and expressed at an intermediate level in the remaining 261 patients.


STAT6 expression in glioblastoma promotes invasive growth.

Merk BC, Owens JL, Lopes MB, Silva CM, Hussaini IM - BMC Cancer (2011)

STAT6 expression correlates with survival in human glioma patients. The Rembrandt database was used to perform a Kaplan-Meier survival analysis of glioma patients with differential STAT6 expression. Up- and down-regulation were defined as a two-fold or greater deviation from intermediate expression. Figure 7a: Among all glioma patients (n= 343) there was a significant difference in survival (p < 0.05) between patients with increased vs. decreased STAT6 gene expression. Figure 7b: In the subset of patients with Grade IV tumors/GBM (n= 181), there was a clear trend towards a similar correlation between STAT6 expression and survival; however statistical significance was not reached. Figure 7c: In patients with Grade II/III astrocytoma (n= 105), a similar trend was observed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3118945&req=5

Figure 7: STAT6 expression correlates with survival in human glioma patients. The Rembrandt database was used to perform a Kaplan-Meier survival analysis of glioma patients with differential STAT6 expression. Up- and down-regulation were defined as a two-fold or greater deviation from intermediate expression. Figure 7a: Among all glioma patients (n= 343) there was a significant difference in survival (p < 0.05) between patients with increased vs. decreased STAT6 gene expression. Figure 7b: In the subset of patients with Grade IV tumors/GBM (n= 181), there was a clear trend towards a similar correlation between STAT6 expression and survival; however statistical significance was not reached. Figure 7c: In patients with Grade II/III astrocytoma (n= 105), a similar trend was observed.
Mentions: Based on our in vitro data (Figures 5 and 6) relating STAT6 expression to increased GBM growth and invasion, we hypothesized that increased STAT6 expression would also correlate with a worse prognosis in glioma patients. To test this theory, we took advantage of the publicly available patient data in the NCI Repository for Molecular Brain Neoplasia Data (REMBRANDT) database [1]. Using microarray-based gene expression data and associated clinical reports, we generated a Kaplan-Meier survival curve based on differential STAT6 expression among 343 glioma patients (Figure 7A). They included patients with GBMs (181), grade II/III astrocytomas (105), grade II/III oligodendrogliomas (50), and mixed tumors (7). Up-and down-regulation were defined as a two-fold increase or decrease in STAT6 expression, respectively, compared to the mean expression level within the given data set. Based on these criteria, STAT6 was up-regulated in ten patients, down-regulated in 72 and expressed at an intermediate level in the remaining 261 patients.

Bottom Line: STAT6-deficient GBM cells showed a reduction in (3)H-Thymidine uptake compared to the wild-type.There was some variation among the different shRNA- silenced clones, but all had a reduction in (3)H-Thymidine uptake ranging from 35%- 70% in U-1242MG and 40- 50% in U-87MG cells.Invasiveness was decreased by 25-40% and 30-75% in U-1242MG and U-87MG cells, respectively.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Virginia, Charlottesville, VA 22908, USA. bcm4u@virginia.edu

ABSTRACT

Background: Glioblastoma (GBM) is a highly aggressive malignant primary brain tumor, characterized by rapid growth, diffuse infiltration of cells into both adjacent and remote brain regions, and a generalized resistance to currently available treatment modalities. Recent reports in the literature suggest that Signal Transducers and Activators of Transcription (STATs) play important roles in the regulation of GBM pathophysiology.

Methods: STAT6 protein expression was analyzed by Western blotting in GBM cell lines and by immunohistochemistry in a tissue microarray (TMA) of glioma patient tissues. We utilized shRNA against STAT6 to investigate the effects of prolonged STAT6 depletion on the growth and invasion of two STAT6-positive GBM cell lines. Cell proliferation was assessed by measuring (3)H-Thymidine uptake over time. Invasion was measured using an in vitro transwell assay in which cells invade through a type IV collagen matrix toward a chemoattractant (Fetal Bovine Serum). Cells were then stained and counted. Kaplan-Meyer survival curves were generated to show the correlation between STAT6 gene expression and patient survival in 343 glioma patients and in a subset of patients with only GBM. Gene expression microarray and clinical data were acquired from the Rembrandt 1 public data depository (https://caintegrator.nci.nih.gov/rembrandt/). Lastly, a genome-wide expression microarray analysis was performed to compare gene expression in wild-type GBM cells to expression in stable STAT6 knockdown clones.

Results: STAT6 was expressed in 2 GBM cell lines, U-1242MG and U-87MG, and in normal astrocytes (NHA) but not in the U-251MG GBM cell line. In our TMA study, STAT6 immunostaining was visible in the majority of astrocytomas of all grades (I-IV) but not in normal brain tissue. In positive cells, STAT6 was localized exclusively in the nuclei over 95% of the time. STAT6-deficient GBM cells showed a reduction in (3)H-Thymidine uptake compared to the wild-type. There was some variation among the different shRNA- silenced clones, but all had a reduction in (3)H-Thymidine uptake ranging from 35%- 70% in U-1242MG and 40- 50% in U-87MG cells. Additionally, STAT6- depleted cells were less invasive than controls in our in vitro transmembrane invasion assay. Invasiveness was decreased by 25-40% and 30-75% in U-1242MG and U-87MG cells, respectively. The microarray analysis identified matrix metalloproteinase 1 (MMP-1) and urokinase Plasminogen activator (uPA) as potential STA6 target genes involved in the promotion of GBM cell invasion. In a Kaplan-Meier survival curve based on Rembrandt 1 gene expression microarray and clinical data, there was a significant difference in survival (P < 0.05) between glioma patients with up- and down-regulated STAT6. Decreased STAT6 expression correlated with longer survival times. In two subsets of patients with either grade IV tumors (GBM) or Grade II/III astrocytomas, there was a similar trend that however did not reach statistical significance.

Conclusions: Taken together, these findings suggest a role for STAT6 in enhancing cell proliferation and invasion in GBM, which may explain why up-regulation of STAT6 correlates with shorter survival times in glioma patients. This report thus identifies STAT6 as a new and potentially promising therapeutic target.

Show MeSH
Related in: MedlinePlus