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Mannose phosphate isomerase regulates fibroblast growth factor receptor family signaling and glioma radiosensitivity.

Cazet A, Charest J, Bennett DC, Sambrooks CL, Contessa JN - PLoS ONE (2014)

Bottom Line: However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition.In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation.Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Therapeutic Radiology, Yale School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
Asparagine-linked glycosylation is an endoplasmic reticulum co- and post-translational modification that enables the transit and function of receptor tyrosine kinase (RTK) glycoproteins. To gain insight into the regulatory role of glycosylation enzymes on RTK function, we investigated shRNA and siRNA knockdown of mannose phosphate isomerase (MPI), an enzyme required for mature glycan precursor biosynthesis. Loss of MPI activity reduced phosphorylation of FGFR family receptors in U-251 and SKMG-3 malignant glioma cell lines and also resulted in significant decreases in FRS2, Akt, and MAPK signaling. However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition. The reductions in FGFR signaling were not caused by loss of FGF ligands or receptors, but instead were caused by interference with receptor dimerization. Investigations into the cellular consequences of MPI knockdown showed that cellular programs driven by FGFR signaling, and integral to the clinical progression of malignant glioma, were impaired. In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation. Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells. Furthermore, this work identifies MPI as a potential enzymatic target for disrupting cell surface receptor-dependent survival signaling and as a novel approach for therapeutic radiosensitization.

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MPI knockdown blocks FGFR1 signaling in SKMG-3 human glioma cells.A. Efficiency of MPI-siRNA knockdown. SKMG-3 cells were transfected with increasing concentrations of MPI targeted siRNA or with a control, non-silencing siRNA sequence. MPI expression was then evaluated by western blot and data for three experiments was quantified as above. 100 pMol siRNA was used for all further experiments. Asterisks (*) indicate a significant decrease of MPI expression after siRNA experiment compared to the expression in the control cells. *p≤0.05. B. MPI siRNA inhibits FGFR1 phosphorylation and signaling. Western blot analysis of MPI-siRNA knockdown in SKMG-3 cells stimulated with 50 ng/mL FGF-1 was performed to determine induction of FGFR1, FRS2, Akt, and ERK phosphorylation. Actin expression was used as a control for protein loading. Quantification of Akt and ERK phosphorylation relative to total protein was determined for two experiments as described previously (right panel). C. MPI siRNA does not inhibit EGFR or Met activation. Western blot analysis of control or MPI-siRNA knockdown following stimulation with 10 ng/mL EGF or 30 ng/mL HGF/SF was performed to determine induction of EGFR or Met phosphorylation.
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pone-0110345-g005: MPI knockdown blocks FGFR1 signaling in SKMG-3 human glioma cells.A. Efficiency of MPI-siRNA knockdown. SKMG-3 cells were transfected with increasing concentrations of MPI targeted siRNA or with a control, non-silencing siRNA sequence. MPI expression was then evaluated by western blot and data for three experiments was quantified as above. 100 pMol siRNA was used for all further experiments. Asterisks (*) indicate a significant decrease of MPI expression after siRNA experiment compared to the expression in the control cells. *p≤0.05. B. MPI siRNA inhibits FGFR1 phosphorylation and signaling. Western blot analysis of MPI-siRNA knockdown in SKMG-3 cells stimulated with 50 ng/mL FGF-1 was performed to determine induction of FGFR1, FRS2, Akt, and ERK phosphorylation. Actin expression was used as a control for protein loading. Quantification of Akt and ERK phosphorylation relative to total protein was determined for two experiments as described previously (right panel). C. MPI siRNA does not inhibit EGFR or Met activation. Western blot analysis of control or MPI-siRNA knockdown following stimulation with 10 ng/mL EGF or 30 ng/mL HGF/SF was performed to determine induction of EGFR or Met phosphorylation.

Mentions: In order to generalize the role of MPI in modulating FGFR activation in glioma, MPI expression was targeted in SKMG-3 cells through transfection with a specific siRNA sequence designed to target MPI [19]. Transient siRNA transfection demonstrated near complete reduction of MPI expression in comparison with scramble control siRNA by western blot at 100 pmol (Fig. 5A), and this concentration was used for all further siRNA experiments. Because the SKMG-3 cell line expresses FGFR1, we analyzed receptor phosphorylation and signaling after siRNA transfection both with and without FGF1 stimulation. Our results show that transient MPI knockdown blocks FGF1-induced tyrosine phosphorylation of FGFR1 as well as downstream activation of FRS2 and MAPK (Fig. 5B). In SKMG-3 cells FGF1 does not activate Akt, and correspondingly, MPI knockdown also did not reduce Akt phosphorylation levels. The effects of MPI knockdown were also evaluated for ligand-induced activation of EGFR and Met, and consistent with the findings in U-251 cells, MPI knockdown did not diminish activation of either RTK (Fig. 5C). The results of siRNA knockdown of MPI in SKMG-3 cells therefore provide further experimental evidence for the role of MPI in regulating FGF family receptor signaling in glioma.


Mannose phosphate isomerase regulates fibroblast growth factor receptor family signaling and glioma radiosensitivity.

Cazet A, Charest J, Bennett DC, Sambrooks CL, Contessa JN - PLoS ONE (2014)

MPI knockdown blocks FGFR1 signaling in SKMG-3 human glioma cells.A. Efficiency of MPI-siRNA knockdown. SKMG-3 cells were transfected with increasing concentrations of MPI targeted siRNA or with a control, non-silencing siRNA sequence. MPI expression was then evaluated by western blot and data for three experiments was quantified as above. 100 pMol siRNA was used for all further experiments. Asterisks (*) indicate a significant decrease of MPI expression after siRNA experiment compared to the expression in the control cells. *p≤0.05. B. MPI siRNA inhibits FGFR1 phosphorylation and signaling. Western blot analysis of MPI-siRNA knockdown in SKMG-3 cells stimulated with 50 ng/mL FGF-1 was performed to determine induction of FGFR1, FRS2, Akt, and ERK phosphorylation. Actin expression was used as a control for protein loading. Quantification of Akt and ERK phosphorylation relative to total protein was determined for two experiments as described previously (right panel). C. MPI siRNA does not inhibit EGFR or Met activation. Western blot analysis of control or MPI-siRNA knockdown following stimulation with 10 ng/mL EGF or 30 ng/mL HGF/SF was performed to determine induction of EGFR or Met phosphorylation.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4196966&req=5

pone-0110345-g005: MPI knockdown blocks FGFR1 signaling in SKMG-3 human glioma cells.A. Efficiency of MPI-siRNA knockdown. SKMG-3 cells were transfected with increasing concentrations of MPI targeted siRNA or with a control, non-silencing siRNA sequence. MPI expression was then evaluated by western blot and data for three experiments was quantified as above. 100 pMol siRNA was used for all further experiments. Asterisks (*) indicate a significant decrease of MPI expression after siRNA experiment compared to the expression in the control cells. *p≤0.05. B. MPI siRNA inhibits FGFR1 phosphorylation and signaling. Western blot analysis of MPI-siRNA knockdown in SKMG-3 cells stimulated with 50 ng/mL FGF-1 was performed to determine induction of FGFR1, FRS2, Akt, and ERK phosphorylation. Actin expression was used as a control for protein loading. Quantification of Akt and ERK phosphorylation relative to total protein was determined for two experiments as described previously (right panel). C. MPI siRNA does not inhibit EGFR or Met activation. Western blot analysis of control or MPI-siRNA knockdown following stimulation with 10 ng/mL EGF or 30 ng/mL HGF/SF was performed to determine induction of EGFR or Met phosphorylation.
Mentions: In order to generalize the role of MPI in modulating FGFR activation in glioma, MPI expression was targeted in SKMG-3 cells through transfection with a specific siRNA sequence designed to target MPI [19]. Transient siRNA transfection demonstrated near complete reduction of MPI expression in comparison with scramble control siRNA by western blot at 100 pmol (Fig. 5A), and this concentration was used for all further siRNA experiments. Because the SKMG-3 cell line expresses FGFR1, we analyzed receptor phosphorylation and signaling after siRNA transfection both with and without FGF1 stimulation. Our results show that transient MPI knockdown blocks FGF1-induced tyrosine phosphorylation of FGFR1 as well as downstream activation of FRS2 and MAPK (Fig. 5B). In SKMG-3 cells FGF1 does not activate Akt, and correspondingly, MPI knockdown also did not reduce Akt phosphorylation levels. The effects of MPI knockdown were also evaluated for ligand-induced activation of EGFR and Met, and consistent with the findings in U-251 cells, MPI knockdown did not diminish activation of either RTK (Fig. 5C). The results of siRNA knockdown of MPI in SKMG-3 cells therefore provide further experimental evidence for the role of MPI in regulating FGF family receptor signaling in glioma.

Bottom Line: However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition.In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation.Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Therapeutic Radiology, Yale School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
Asparagine-linked glycosylation is an endoplasmic reticulum co- and post-translational modification that enables the transit and function of receptor tyrosine kinase (RTK) glycoproteins. To gain insight into the regulatory role of glycosylation enzymes on RTK function, we investigated shRNA and siRNA knockdown of mannose phosphate isomerase (MPI), an enzyme required for mature glycan precursor biosynthesis. Loss of MPI activity reduced phosphorylation of FGFR family receptors in U-251 and SKMG-3 malignant glioma cell lines and also resulted in significant decreases in FRS2, Akt, and MAPK signaling. However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition. The reductions in FGFR signaling were not caused by loss of FGF ligands or receptors, but instead were caused by interference with receptor dimerization. Investigations into the cellular consequences of MPI knockdown showed that cellular programs driven by FGFR signaling, and integral to the clinical progression of malignant glioma, were impaired. In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation. Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells. Furthermore, this work identifies MPI as a potential enzymatic target for disrupting cell surface receptor-dependent survival signaling and as a novel approach for therapeutic radiosensitization.

Show MeSH
Related in: MedlinePlus