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Knockdown of EphB1 receptor decreases medulloblastoma cell growth and migration and increases cellular radiosensitization.

Bhatia S, Baig NA, Timofeeva O, Pasquale EB, Hirsch K, MacDonald TJ, Dritschilo A, Lee YC, Henkemeyer M, Rood B, Jung M, Wang XJ, Kool M, Rodriguez O, Albanese C, Karam SD - Oncotarget (2015)

Bottom Line: EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle.In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src.Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA.

ABSTRACT
The expression of members of the Eph family of receptor tyrosine kinases and their ephrin ligands is frequently dysregulated in medulloblastomas. We assessed the expression and functional role of EphB1 in medulloblastoma cell lines and engineered mouse models. mRNA and protein expression profiling showed expression of EphB1 receptor in the human medulloblastoma cell lines DAOY and UW228. EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle. It also modulated the expression of proliferation, and cell survival markers. In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src. Furthermore, EphB1 knockdown enhanced cellular radiosensitization of medulloblastoma cells in culture and in a genetically engineered mouse medulloblastoma model. Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors. Taken together, our findings establish that EphB1 plays a key role in medulloblastoma cell growth, viability, migration, and radiation sensitivity, making EphB1 a promising therapeutic target.

No MeSH data available.


Related in: MedlinePlus

EphB1 is effectively knocked down in DAOY medulloblastoma cells(A) EphB1 mRNA level is dramatically reduced upon transfection of DAOY cells with EphB1-siRNA. (B) EphB1 expression is detected by western blotting in DAOY cells and is decreased upon transfection with EphB1-targeting siRNA vs. control non-specific siRNA (Ns-siRNA).
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Figure 1: EphB1 is effectively knocked down in DAOY medulloblastoma cells(A) EphB1 mRNA level is dramatically reduced upon transfection of DAOY cells with EphB1-siRNA. (B) EphB1 expression is detected by western blotting in DAOY cells and is decreased upon transfection with EphB1-targeting siRNA vs. control non-specific siRNA (Ns-siRNA).

Mentions: The expression of EphB1 receptor varies widely in medulloblastoma [8]. We evaluated the expression of EphB1 in a human medulloblastoma cell line, DAOY, and found EphB1 to be expressed at both the mRNA and protein level (Figure 1A, 1B). To assess the role of EphB1 in medulloblastoma, we next attempted to knockdown EphB1 expression using siRNA approach. DAOY cells were transfected with either EphB1 siRNA or a control, nonspecific siRNA (Ns-siRNA). EphB1 expression was analyzed at the mRNA level at 24, 48, and 72 h post-transfection. We found that EphB1 mRNA levels were reduced to 18% or less by 24 h in the EphB1-knockdown group compared to the control, non-specific siRNA (Ns-siRNA) transfected group, with optimal knockdown efficiency observed at 72 h post-transfection (Figure 1A). Additionally, there was a substantial reduction in the levels of EphB1 protein by western blot analysis of EphB1-knockdown DAOY cells compared to control transfectants (Figure 1B). The results were also replicated in another medulloblastoma cell line, UW228 (Supplementary Figure 1A). Since western blot analysis confirmed an appreciable reduction in EphB1 protein levels, we conducted a series of experiments to determine whether EphB1 downregulation affects cell viability, cell cycle progression, migration, and radiosensitivity.


Knockdown of EphB1 receptor decreases medulloblastoma cell growth and migration and increases cellular radiosensitization.

Bhatia S, Baig NA, Timofeeva O, Pasquale EB, Hirsch K, MacDonald TJ, Dritschilo A, Lee YC, Henkemeyer M, Rood B, Jung M, Wang XJ, Kool M, Rodriguez O, Albanese C, Karam SD - Oncotarget (2015)

EphB1 is effectively knocked down in DAOY medulloblastoma cells(A) EphB1 mRNA level is dramatically reduced upon transfection of DAOY cells with EphB1-siRNA. (B) EphB1 expression is detected by western blotting in DAOY cells and is decreased upon transfection with EphB1-targeting siRNA vs. control non-specific siRNA (Ns-siRNA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496193&req=5

Figure 1: EphB1 is effectively knocked down in DAOY medulloblastoma cells(A) EphB1 mRNA level is dramatically reduced upon transfection of DAOY cells with EphB1-siRNA. (B) EphB1 expression is detected by western blotting in DAOY cells and is decreased upon transfection with EphB1-targeting siRNA vs. control non-specific siRNA (Ns-siRNA).
Mentions: The expression of EphB1 receptor varies widely in medulloblastoma [8]. We evaluated the expression of EphB1 in a human medulloblastoma cell line, DAOY, and found EphB1 to be expressed at both the mRNA and protein level (Figure 1A, 1B). To assess the role of EphB1 in medulloblastoma, we next attempted to knockdown EphB1 expression using siRNA approach. DAOY cells were transfected with either EphB1 siRNA or a control, nonspecific siRNA (Ns-siRNA). EphB1 expression was analyzed at the mRNA level at 24, 48, and 72 h post-transfection. We found that EphB1 mRNA levels were reduced to 18% or less by 24 h in the EphB1-knockdown group compared to the control, non-specific siRNA (Ns-siRNA) transfected group, with optimal knockdown efficiency observed at 72 h post-transfection (Figure 1A). Additionally, there was a substantial reduction in the levels of EphB1 protein by western blot analysis of EphB1-knockdown DAOY cells compared to control transfectants (Figure 1B). The results were also replicated in another medulloblastoma cell line, UW228 (Supplementary Figure 1A). Since western blot analysis confirmed an appreciable reduction in EphB1 protein levels, we conducted a series of experiments to determine whether EphB1 downregulation affects cell viability, cell cycle progression, migration, and radiosensitivity.

Bottom Line: EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle.In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src.Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA.

ABSTRACT
The expression of members of the Eph family of receptor tyrosine kinases and their ephrin ligands is frequently dysregulated in medulloblastomas. We assessed the expression and functional role of EphB1 in medulloblastoma cell lines and engineered mouse models. mRNA and protein expression profiling showed expression of EphB1 receptor in the human medulloblastoma cell lines DAOY and UW228. EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle. It also modulated the expression of proliferation, and cell survival markers. In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src. Furthermore, EphB1 knockdown enhanced cellular radiosensitization of medulloblastoma cells in culture and in a genetically engineered mouse medulloblastoma model. Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors. Taken together, our findings establish that EphB1 plays a key role in medulloblastoma cell growth, viability, migration, and radiation sensitivity, making EphB1 a promising therapeutic target.

No MeSH data available.


Related in: MedlinePlus