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Pancreatic endoplasmic reticulum kinase activation promotes medulloblastoma cell migration and invasion through induction of vascular endothelial growth factor A.

Jamison S, Lin Y, Lin W - PLoS ONE (2015)

Bottom Line: Recent studies suggest that VEGF-A can act directly on certain tumor cell types in an autocrine manner, via binding to VEGF receptor 2 (VEGFR2), to promote tumor cell migration and invasion.Although several reports show that PERK activation increases VEGF-A expression in medulloblastoma, the most common solid malignancy of childhood, the role that either PERK or VEGF-A plays in medulloblastoma remains elusive.Moreover, using the VEGFR2 inhibitor SU5416 and the VEGF-A neutralizing antibody to block VEGF-A/VEGFR2 signaling, our results suggested that tumor cell-derived VEGF-A promoted medulloblastoma cell migration and invasion through VEGFR2 signaling, and that both VEGF-A and VEGFR2 were required for the promoting effects of PERK activation on medulloblastoma cell migration and invasion.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Minnesota, Minneapolis, United States of America; Institute for Translational Neuroscience, University of Minnesota, Minneapolis, United States of America; Masonic Cancer Center, University of Minnesota, Minneapolis, United States of America.

ABSTRACT
Evidence is accumulating that activation of the pancreatic endoplasmic reticulum kinase (PERK) in response to endoplasmic reticulum (ER) stress adapts tumor cells to the tumor microenvironment and enhances tumor angiogenesis by inducing vascular endothelial growth factor A (VEGF-A). Recent studies suggest that VEGF-A can act directly on certain tumor cell types in an autocrine manner, via binding to VEGF receptor 2 (VEGFR2), to promote tumor cell migration and invasion. Although several reports show that PERK activation increases VEGF-A expression in medulloblastoma, the most common solid malignancy of childhood, the role that either PERK or VEGF-A plays in medulloblastoma remains elusive. In this study, we mimicked the moderate enhancement of PERK activity observed in tumor patients using a genetic approach and a pharmacologic approach, and found that moderate activation of PERK signaling facilitated medulloblastoma cell migration and invasion and increased the production of VEGF-A. Moreover, using the VEGFR2 inhibitor SU5416 and the VEGF-A neutralizing antibody to block VEGF-A/VEGFR2 signaling, our results suggested that tumor cell-derived VEGF-A promoted medulloblastoma cell migration and invasion through VEGFR2 signaling, and that both VEGF-A and VEGFR2 were required for the promoting effects of PERK activation on medulloblastoma cell migration and invasion. Thus, these findings suggest that moderate PERK activation promotes medulloblastoma cell migration and invasion through enhancement of VEGF-A/VEGFR2 signaling.

No MeSH data available.


Related in: MedlinePlus

Characterization of stably transfected Daoy cell lines that allow for pharmacological controlled activation of PERK.(A) Daoy cells were transfected with the plasmid pIREs-Fv2E-PERK-ZsGreen. We obtained several stably transfected cell lines that were resistant to G418 and expressed various levels of Fv2E-PERK. (B) Western blot analysis showed that AP20187 treatment activated Fv2E-PERK and led to phosphorylation of eIF2α. The positions of activated Fv2E-PERK (p-Fv2E-PERK) and inactive Fv2E-PERK proteins were indicated. (C) Densitometry analysis of western blot results showed that AP20187 treatment increased the level of p-eIF2a in Fv2E-PERK1 cells in a dose-dependent manner. The relative protein levels are relative to actin. (D) SUnSET measurement of protein biosynthesis revealed dramatic reduction of protein biosynthesis in Fv2E-PERK1 cells treated with the high dose of AP20187 (0.1–1 nM). Nevertheless, treatment with the low dose of AP20187 (0.001–0.01 nM) only slightly reduced protein biosynthesis in the cells. (E) MTT analysis showed that treatment with the high dose of AP20187 (0. 1–1 nM) significantly inhibited Fv2E-PERK1 cell growth. Nevertheless, treatment with the low dose of AP20187 (0.001–0.01 nM) had no effect on the cell growth. The experiments were repeated at least three times. Error bars represent SD, *P < 0.05.
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pone.0120252.g001: Characterization of stably transfected Daoy cell lines that allow for pharmacological controlled activation of PERK.(A) Daoy cells were transfected with the plasmid pIREs-Fv2E-PERK-ZsGreen. We obtained several stably transfected cell lines that were resistant to G418 and expressed various levels of Fv2E-PERK. (B) Western blot analysis showed that AP20187 treatment activated Fv2E-PERK and led to phosphorylation of eIF2α. The positions of activated Fv2E-PERK (p-Fv2E-PERK) and inactive Fv2E-PERK proteins were indicated. (C) Densitometry analysis of western blot results showed that AP20187 treatment increased the level of p-eIF2a in Fv2E-PERK1 cells in a dose-dependent manner. The relative protein levels are relative to actin. (D) SUnSET measurement of protein biosynthesis revealed dramatic reduction of protein biosynthesis in Fv2E-PERK1 cells treated with the high dose of AP20187 (0.1–1 nM). Nevertheless, treatment with the low dose of AP20187 (0.001–0.01 nM) only slightly reduced protein biosynthesis in the cells. (E) MTT analysis showed that treatment with the high dose of AP20187 (0. 1–1 nM) significantly inhibited Fv2E-PERK1 cell growth. Nevertheless, treatment with the low dose of AP20187 (0.001–0.01 nM) had no effect on the cell growth. The experiments were repeated at least three times. Error bars represent SD, *P < 0.05.

Mentions: The three functional domains of PERK are: a stress-sensing ER luminal domain, an ER transmembrane domain, and a cytosolic eIF2α kinase domain [34]. During ER stress, the stress-sensing ER lumenal domain senses the stress signal and initiates PERK oligomerization and autophosphorylation, resulting in activation of PERK signaling. Fv2E-PERK, an artificial PERK derivative, is generated by fusing the eIF2α kinase effector domain of PERK to a polypeptide containing tandem modified FK506 binding domains (Fv2E). It has been demonstrated that Fv2E-PERK can be induced to oligomerize and activate by an otherwise inert ligand, AP20187, in the absence of any additional stress [26]. We transfected human medulloblastoma cell line Daoy with plasmid pIREs-Fv2E-PERK-ZsGreen and obtained several stably transfected cell lines that were resistant to G418 and expressed various levels of Fv2E-PERK (Fig. 1A). The effects of overexpression of Fv2E-PERK alone on Daoy cell growth, migration, and invasion were determined. We found that stably transfected Daoy cell line # 1 (Fv2E-PERK1) expressed a high level of Fv2E-PERK and that overexpression of Fv2E-PERK alone did not significantly affect the cell growth, migration, and invasion as compared to control Daoy cells (Figs. 1, 2). Thus, Fv2E-PERK1 cell line was selected for all subsequent studies.


Pancreatic endoplasmic reticulum kinase activation promotes medulloblastoma cell migration and invasion through induction of vascular endothelial growth factor A.

Jamison S, Lin Y, Lin W - PLoS ONE (2015)

Characterization of stably transfected Daoy cell lines that allow for pharmacological controlled activation of PERK.(A) Daoy cells were transfected with the plasmid pIREs-Fv2E-PERK-ZsGreen. We obtained several stably transfected cell lines that were resistant to G418 and expressed various levels of Fv2E-PERK. (B) Western blot analysis showed that AP20187 treatment activated Fv2E-PERK and led to phosphorylation of eIF2α. The positions of activated Fv2E-PERK (p-Fv2E-PERK) and inactive Fv2E-PERK proteins were indicated. (C) Densitometry analysis of western blot results showed that AP20187 treatment increased the level of p-eIF2a in Fv2E-PERK1 cells in a dose-dependent manner. The relative protein levels are relative to actin. (D) SUnSET measurement of protein biosynthesis revealed dramatic reduction of protein biosynthesis in Fv2E-PERK1 cells treated with the high dose of AP20187 (0.1–1 nM). Nevertheless, treatment with the low dose of AP20187 (0.001–0.01 nM) only slightly reduced protein biosynthesis in the cells. (E) MTT analysis showed that treatment with the high dose of AP20187 (0. 1–1 nM) significantly inhibited Fv2E-PERK1 cell growth. Nevertheless, treatment with the low dose of AP20187 (0.001–0.01 nM) had no effect on the cell growth. The experiments were repeated at least three times. Error bars represent SD, *P < 0.05.
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pone.0120252.g001: Characterization of stably transfected Daoy cell lines that allow for pharmacological controlled activation of PERK.(A) Daoy cells were transfected with the plasmid pIREs-Fv2E-PERK-ZsGreen. We obtained several stably transfected cell lines that were resistant to G418 and expressed various levels of Fv2E-PERK. (B) Western blot analysis showed that AP20187 treatment activated Fv2E-PERK and led to phosphorylation of eIF2α. The positions of activated Fv2E-PERK (p-Fv2E-PERK) and inactive Fv2E-PERK proteins were indicated. (C) Densitometry analysis of western blot results showed that AP20187 treatment increased the level of p-eIF2a in Fv2E-PERK1 cells in a dose-dependent manner. The relative protein levels are relative to actin. (D) SUnSET measurement of protein biosynthesis revealed dramatic reduction of protein biosynthesis in Fv2E-PERK1 cells treated with the high dose of AP20187 (0.1–1 nM). Nevertheless, treatment with the low dose of AP20187 (0.001–0.01 nM) only slightly reduced protein biosynthesis in the cells. (E) MTT analysis showed that treatment with the high dose of AP20187 (0. 1–1 nM) significantly inhibited Fv2E-PERK1 cell growth. Nevertheless, treatment with the low dose of AP20187 (0.001–0.01 nM) had no effect on the cell growth. The experiments were repeated at least three times. Error bars represent SD, *P < 0.05.
Mentions: The three functional domains of PERK are: a stress-sensing ER luminal domain, an ER transmembrane domain, and a cytosolic eIF2α kinase domain [34]. During ER stress, the stress-sensing ER lumenal domain senses the stress signal and initiates PERK oligomerization and autophosphorylation, resulting in activation of PERK signaling. Fv2E-PERK, an artificial PERK derivative, is generated by fusing the eIF2α kinase effector domain of PERK to a polypeptide containing tandem modified FK506 binding domains (Fv2E). It has been demonstrated that Fv2E-PERK can be induced to oligomerize and activate by an otherwise inert ligand, AP20187, in the absence of any additional stress [26]. We transfected human medulloblastoma cell line Daoy with plasmid pIREs-Fv2E-PERK-ZsGreen and obtained several stably transfected cell lines that were resistant to G418 and expressed various levels of Fv2E-PERK (Fig. 1A). The effects of overexpression of Fv2E-PERK alone on Daoy cell growth, migration, and invasion were determined. We found that stably transfected Daoy cell line # 1 (Fv2E-PERK1) expressed a high level of Fv2E-PERK and that overexpression of Fv2E-PERK alone did not significantly affect the cell growth, migration, and invasion as compared to control Daoy cells (Figs. 1, 2). Thus, Fv2E-PERK1 cell line was selected for all subsequent studies.

Bottom Line: Recent studies suggest that VEGF-A can act directly on certain tumor cell types in an autocrine manner, via binding to VEGF receptor 2 (VEGFR2), to promote tumor cell migration and invasion.Although several reports show that PERK activation increases VEGF-A expression in medulloblastoma, the most common solid malignancy of childhood, the role that either PERK or VEGF-A plays in medulloblastoma remains elusive.Moreover, using the VEGFR2 inhibitor SU5416 and the VEGF-A neutralizing antibody to block VEGF-A/VEGFR2 signaling, our results suggested that tumor cell-derived VEGF-A promoted medulloblastoma cell migration and invasion through VEGFR2 signaling, and that both VEGF-A and VEGFR2 were required for the promoting effects of PERK activation on medulloblastoma cell migration and invasion.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Minnesota, Minneapolis, United States of America; Institute for Translational Neuroscience, University of Minnesota, Minneapolis, United States of America; Masonic Cancer Center, University of Minnesota, Minneapolis, United States of America.

ABSTRACT
Evidence is accumulating that activation of the pancreatic endoplasmic reticulum kinase (PERK) in response to endoplasmic reticulum (ER) stress adapts tumor cells to the tumor microenvironment and enhances tumor angiogenesis by inducing vascular endothelial growth factor A (VEGF-A). Recent studies suggest that VEGF-A can act directly on certain tumor cell types in an autocrine manner, via binding to VEGF receptor 2 (VEGFR2), to promote tumor cell migration and invasion. Although several reports show that PERK activation increases VEGF-A expression in medulloblastoma, the most common solid malignancy of childhood, the role that either PERK or VEGF-A plays in medulloblastoma remains elusive. In this study, we mimicked the moderate enhancement of PERK activity observed in tumor patients using a genetic approach and a pharmacologic approach, and found that moderate activation of PERK signaling facilitated medulloblastoma cell migration and invasion and increased the production of VEGF-A. Moreover, using the VEGFR2 inhibitor SU5416 and the VEGF-A neutralizing antibody to block VEGF-A/VEGFR2 signaling, our results suggested that tumor cell-derived VEGF-A promoted medulloblastoma cell migration and invasion through VEGFR2 signaling, and that both VEGF-A and VEGFR2 were required for the promoting effects of PERK activation on medulloblastoma cell migration and invasion. Thus, these findings suggest that moderate PERK activation promotes medulloblastoma cell migration and invasion through enhancement of VEGF-A/VEGFR2 signaling.

No MeSH data available.


Related in: MedlinePlus