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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.

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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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Tissue Microarray (TMA), representative images of staining categories. All images were probed for total STAT6 protein by IHC. Upper left: tissue sample of a Grade III (anaplastic) astrocytoma showing high staining intensity in the nuclei of the majority of cells. Upper right: GBM tissue sample demonstrating an example of low staining: some nuclei exhibit dark staining, but less than half the cells are positive for STAT6. Lower left: GBM sample showing weak staining intensity in the majority of nuclei. This was also called low staining. Lower right: Tissue sample of normal brain, showing no STAT6 staining in the nucleus or elsewhere.
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Figure 2: Tissue Microarray (TMA), representative images of staining categories. All images were probed for total STAT6 protein by IHC. Upper left: tissue sample of a Grade III (anaplastic) astrocytoma showing high staining intensity in the nuclei of the majority of cells. Upper right: GBM tissue sample demonstrating an example of low staining: some nuclei exhibit dark staining, but less than half the cells are positive for STAT6. Lower left: GBM sample showing weak staining intensity in the majority of nuclei. This was also called low staining. Lower right: Tissue sample of normal brain, showing no STAT6 staining in the nucleus or elsewhere.

Mentions: In order to relate our in vitro findings to actual human patient tumor specimens, we utilized a tissue microarray (TMA) to evaluate STAT6 expression in GBM, healthy brain, and lower grade gliomas by immunohistochemistry (IHC). Two independent investigators examined 8 sections each of normal cortex, Grade I (pilocytic) astrocytoma, and Grade IV astrocytoma (GBM), as well as 5 sections of Grade III (anaplastic) astrocytoma and 17 sections of Grade II (diffuse) astrocytoma, and evaluated the extent and intensity of STAT6 staining in each sample. Figure 2 shows examples of images from the TMA, and the numerical results of all TMA sections are summarized in Table 1. Tumor-associated endothelial cells, which frequently displayed high intensity staining of STAT6, were disregarded when describing a sample as STAT6 positive or negative.


STAT6 expression in glioblastoma promotes invasive growth.

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

Tissue Microarray (TMA), representative images of staining categories. All images were probed for total STAT6 protein by IHC. Upper left: tissue sample of a Grade III (anaplastic) astrocytoma showing high staining intensity in the nuclei of the majority of cells. Upper right: GBM tissue sample demonstrating an example of low staining: some nuclei exhibit dark staining, but less than half the cells are positive for STAT6. Lower left: GBM sample showing weak staining intensity in the majority of nuclei. This was also called low staining. Lower right: Tissue sample of normal brain, showing no STAT6 staining in the nucleus or elsewhere.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Tissue Microarray (TMA), representative images of staining categories. All images were probed for total STAT6 protein by IHC. Upper left: tissue sample of a Grade III (anaplastic) astrocytoma showing high staining intensity in the nuclei of the majority of cells. Upper right: GBM tissue sample demonstrating an example of low staining: some nuclei exhibit dark staining, but less than half the cells are positive for STAT6. Lower left: GBM sample showing weak staining intensity in the majority of nuclei. This was also called low staining. Lower right: Tissue sample of normal brain, showing no STAT6 staining in the nucleus or elsewhere.
Mentions: In order to relate our in vitro findings to actual human patient tumor specimens, we utilized a tissue microarray (TMA) to evaluate STAT6 expression in GBM, healthy brain, and lower grade gliomas by immunohistochemistry (IHC). Two independent investigators examined 8 sections each of normal cortex, Grade I (pilocytic) astrocytoma, and Grade IV astrocytoma (GBM), as well as 5 sections of Grade III (anaplastic) astrocytoma and 17 sections of Grade II (diffuse) astrocytoma, and evaluated the extent and intensity of STAT6 staining in each sample. Figure 2 shows examples of images from the TMA, and the numerical results of all TMA sections are summarized in Table 1. Tumor-associated endothelial cells, which frequently displayed high intensity staining of STAT6, were disregarded when describing a sample as STAT6 positive or negative.

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