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Activation of hypoxia signaling induces phenotypic transformation of glioma cells: implications for bevacizumab antiangiogenic therapy.

Xu H, Rahimpour S, Nesvick CL, Zhang X, Ma J, Zhang M, Zhang G, Wang L, Yang C, Hong CS, Germanwala AV, Elder JB, Ray-Chaudhury A, Yao Y, Gilbert MR, Lonser RR, Heiss JD, Brady RO, Mao Y, Qin J, Zhuang Z - Oncotarget (2015)

Bottom Line: Exposure of glioma cells to 1% oxygen tension increased cell proliferation, expression of EMT-associated proteins and enhanced cell migration in vitro.These phenotypic changes were significantly attenuated by pharmacologic knockdown of hypoxia-inducible Factor 1α (HIF1α) or HIF2α, indicating that HIFs represent a therapeutic target for mesenchymal GBM cells.These findings provide insights into potential development of novel therapeutic targeting of angiogenesis-specific pathways in GBM.

View Article: PubMed Central - PubMed

Affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.

ABSTRACT
Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), can attenuate tumor-associated edema and improve patient symptoms but based on magnetic resonance imaging, is associated with non-enhancing tumor progression and possibly gliosarcoma differentiation. To gain insight into these findings, we investigated the role of hypoxia and epithelial-mesenchymal transition (EMT)-associated proteins in GBM. Tumor markers of hypoxia and EMT were upregulated in bevacizumab-treated tumors from GBM patients compared to untreated counterparts. Exposure of glioma cells to 1% oxygen tension increased cell proliferation, expression of EMT-associated proteins and enhanced cell migration in vitro. These phenotypic changes were significantly attenuated by pharmacologic knockdown of hypoxia-inducible Factor 1α (HIF1α) or HIF2α, indicating that HIFs represent a therapeutic target for mesenchymal GBM cells. These findings provide insights into potential development of novel therapeutic targeting of angiogenesis-specific pathways in GBM.

No MeSH data available.


Related in: MedlinePlus

Time-course microfluidic chip migration assay of U87 glioma cells overexpressing HIF1α and HIF2αU87 glioma cells were transfected with GFP alone or GFP with a wild-type hemagglutinin-tagged (HA)-HIF1α gain-of-function construct. HIF1α gain-of-function cells were either transfected with HA-HIF1α alone or with nt-siRNA or HIF1α-siRNA. Cell cultures were then placed in the microfluidic chip assay, exposed to 21% (a) or 1% (b) oxygen saturation and imaged at 0, 6, 12, 18 and 24 hours, as indicated. The same set of experiments were performed using HA-HIF2α and HIF2α-siRNA in (c) and (d). Exposure to either hypoxia or transfection with HIF1α and HIF2α gain-of-function constructs enhanced cell migration, and migration was significantly reduced by siRNA knockdown of HIF1α and HIF2α, respectively. Scale bar = 100 μm. (e) Quantification of microfluidic chamber invasion assays shown in panels a-d. The y-axis reflects the number of invaded cells at each distance at 24 h (standard deviation [S.D.]).
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Figure 6: Time-course microfluidic chip migration assay of U87 glioma cells overexpressing HIF1α and HIF2αU87 glioma cells were transfected with GFP alone or GFP with a wild-type hemagglutinin-tagged (HA)-HIF1α gain-of-function construct. HIF1α gain-of-function cells were either transfected with HA-HIF1α alone or with nt-siRNA or HIF1α-siRNA. Cell cultures were then placed in the microfluidic chip assay, exposed to 21% (a) or 1% (b) oxygen saturation and imaged at 0, 6, 12, 18 and 24 hours, as indicated. The same set of experiments were performed using HA-HIF2α and HIF2α-siRNA in (c) and (d). Exposure to either hypoxia or transfection with HIF1α and HIF2α gain-of-function constructs enhanced cell migration, and migration was significantly reduced by siRNA knockdown of HIF1α and HIF2α, respectively. Scale bar = 100 μm. (e) Quantification of microfluidic chamber invasion assays shown in panels a-d. The y-axis reflects the number of invaded cells at each distance at 24 h (standard deviation [S.D.]).

Mentions: Hypoxia is associated with changes in gene expression and metabolism that collectively contribute to its effect on cell phenotype. To determine the relative contribution of HIF1α and HIF2α to glioma cell migration, U87 GBM cells were transfected with hemagglutinin-tagged HIF α-subunit constructs with or without siRNA to a HIF α-subunit and then placed under 1% or 21% oxygen saturation for 24 hours (Fig. 6). Under normoxic conditions, cells that overexpressed HIFα migrated further than cells transfected with green fluorescent protein (GFP) alone. There was no discernible difference between HIF1α- and HIF2α-over-expressing cells. Migration was reduced following HIF1α knockdown and was nearly abolished following HIF2α knockdown. These findings indicate that both HIF1α and HIF2α are individually sufficient to enhance GBM cell migration and that HIF2α, in particular, is necessary for hypoxia-induced cell migration.


Activation of hypoxia signaling induces phenotypic transformation of glioma cells: implications for bevacizumab antiangiogenic therapy.

Xu H, Rahimpour S, Nesvick CL, Zhang X, Ma J, Zhang M, Zhang G, Wang L, Yang C, Hong CS, Germanwala AV, Elder JB, Ray-Chaudhury A, Yao Y, Gilbert MR, Lonser RR, Heiss JD, Brady RO, Mao Y, Qin J, Zhuang Z - Oncotarget (2015)

Time-course microfluidic chip migration assay of U87 glioma cells overexpressing HIF1α and HIF2αU87 glioma cells were transfected with GFP alone or GFP with a wild-type hemagglutinin-tagged (HA)-HIF1α gain-of-function construct. HIF1α gain-of-function cells were either transfected with HA-HIF1α alone or with nt-siRNA or HIF1α-siRNA. Cell cultures were then placed in the microfluidic chip assay, exposed to 21% (a) or 1% (b) oxygen saturation and imaged at 0, 6, 12, 18 and 24 hours, as indicated. The same set of experiments were performed using HA-HIF2α and HIF2α-siRNA in (c) and (d). Exposure to either hypoxia or transfection with HIF1α and HIF2α gain-of-function constructs enhanced cell migration, and migration was significantly reduced by siRNA knockdown of HIF1α and HIF2α, respectively. Scale bar = 100 μm. (e) Quantification of microfluidic chamber invasion assays shown in panels a-d. The y-axis reflects the number of invaded cells at each distance at 24 h (standard deviation [S.D.]).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4494911&req=5

Figure 6: Time-course microfluidic chip migration assay of U87 glioma cells overexpressing HIF1α and HIF2αU87 glioma cells were transfected with GFP alone or GFP with a wild-type hemagglutinin-tagged (HA)-HIF1α gain-of-function construct. HIF1α gain-of-function cells were either transfected with HA-HIF1α alone or with nt-siRNA or HIF1α-siRNA. Cell cultures were then placed in the microfluidic chip assay, exposed to 21% (a) or 1% (b) oxygen saturation and imaged at 0, 6, 12, 18 and 24 hours, as indicated. The same set of experiments were performed using HA-HIF2α and HIF2α-siRNA in (c) and (d). Exposure to either hypoxia or transfection with HIF1α and HIF2α gain-of-function constructs enhanced cell migration, and migration was significantly reduced by siRNA knockdown of HIF1α and HIF2α, respectively. Scale bar = 100 μm. (e) Quantification of microfluidic chamber invasion assays shown in panels a-d. The y-axis reflects the number of invaded cells at each distance at 24 h (standard deviation [S.D.]).
Mentions: Hypoxia is associated with changes in gene expression and metabolism that collectively contribute to its effect on cell phenotype. To determine the relative contribution of HIF1α and HIF2α to glioma cell migration, U87 GBM cells were transfected with hemagglutinin-tagged HIF α-subunit constructs with or without siRNA to a HIF α-subunit and then placed under 1% or 21% oxygen saturation for 24 hours (Fig. 6). Under normoxic conditions, cells that overexpressed HIFα migrated further than cells transfected with green fluorescent protein (GFP) alone. There was no discernible difference between HIF1α- and HIF2α-over-expressing cells. Migration was reduced following HIF1α knockdown and was nearly abolished following HIF2α knockdown. These findings indicate that both HIF1α and HIF2α are individually sufficient to enhance GBM cell migration and that HIF2α, in particular, is necessary for hypoxia-induced cell migration.

Bottom Line: Exposure of glioma cells to 1% oxygen tension increased cell proliferation, expression of EMT-associated proteins and enhanced cell migration in vitro.These phenotypic changes were significantly attenuated by pharmacologic knockdown of hypoxia-inducible Factor 1α (HIF1α) or HIF2α, indicating that HIFs represent a therapeutic target for mesenchymal GBM cells.These findings provide insights into potential development of novel therapeutic targeting of angiogenesis-specific pathways in GBM.

View Article: PubMed Central - PubMed

Affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.

ABSTRACT
Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), can attenuate tumor-associated edema and improve patient symptoms but based on magnetic resonance imaging, is associated with non-enhancing tumor progression and possibly gliosarcoma differentiation. To gain insight into these findings, we investigated the role of hypoxia and epithelial-mesenchymal transition (EMT)-associated proteins in GBM. Tumor markers of hypoxia and EMT were upregulated in bevacizumab-treated tumors from GBM patients compared to untreated counterparts. Exposure of glioma cells to 1% oxygen tension increased cell proliferation, expression of EMT-associated proteins and enhanced cell migration in vitro. These phenotypic changes were significantly attenuated by pharmacologic knockdown of hypoxia-inducible Factor 1α (HIF1α) or HIF2α, indicating that HIFs represent a therapeutic target for mesenchymal GBM cells. These findings provide insights into potential development of novel therapeutic targeting of angiogenesis-specific pathways in GBM.

No MeSH data available.


Related in: MedlinePlus