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Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway.

Rolón-Reyes K, Kucheryavykh YV, Cubano LA, Inyushin M, Skatchkov SN, Eaton MJ, Harrison JK, Kucheryavykh LY - PLoS ONE (2015)

Bottom Line: Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells.A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration.Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Universidad Central del Caribe, School of Medicine, Bayamón, Puerto Rico, United States of America.

ABSTRACT
Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells.

No MeSH data available.


Related in: MedlinePlus

FAK is not involved in microglial stimulation of migration in glioma cells.Data obtained from migration assays for glioma cells. Cells were treated with 5nM (concentration that effectively blocks FAK) and 16nM (concentration that effectively blocks Pyk2) of PF-562,271. Due to different migration abilities in all presented cell lines the Y-axis scales are adjusted for each cell line in order to demonstrate the absolute numbers of migrating cells. Results are presented as mean ± S.D. with significant differences from control in each group (+), from Mock without microglia on the bottom (#), or from Mock with microglia on the bottom (*) (p < 0.05). One-way ANOVA followed by the Tukey’s multiple comparison test was used to determine significance between groups.
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pone.0131059.g006: FAK is not involved in microglial stimulation of migration in glioma cells.Data obtained from migration assays for glioma cells. Cells were treated with 5nM (concentration that effectively blocks FAK) and 16nM (concentration that effectively blocks Pyk2) of PF-562,271. Due to different migration abilities in all presented cell lines the Y-axis scales are adjusted for each cell line in order to demonstrate the absolute numbers of migrating cells. Results are presented as mean ± S.D. with significant differences from control in each group (+), from Mock without microglia on the bottom (#), or from Mock with microglia on the bottom (*) (p < 0.05). One-way ANOVA followed by the Tukey’s multiple comparison test was used to determine significance between groups.

Mentions: Our data indicate that microglia stimulate migration of glioma cells and that treatment of glioma cells with AMCM increases their levels of phosphorylated Pyk2. To assess the involvement of microglia on activation of Pyk2 signaling in glioma cells and on glioma cell mobility, we performed migration assays for glioma cells in the presence and absence of the Pyk2/FAK inhibitor, PF-562,271. PF-562,271 readily passes the blood-brain barrier and selectively blocks FAK at 5 nM and blocks both FAK and Pyk2 at 16 nM [37], S3 and S4 Figs Therefore, we investigated both FAK and FAK/Pyk2 selective doses of PF-562,271. PF-562,271 was given two hours prior to and during the assay. The lower (FAK selective) concentration of PF-562,271 (5 nM) had no effect on glioma cell migration whether microglia were present or not (Fig 6) indicating that FAK was not involved in glioma cell migration. The higher concentration of PF-562,271 (16 nM) effectively eliminated the effect of microglia on glioma cell migration in all investigated glioma cell lines and also reduced the basal migration rates of U87 and HS683 glioma cells in the absence of microglia (Fig 6). Pyk 2 blockade (16 nM PF-562,271) did not alter the basal migration rates of A172 and C6 cells, but significantly reduced migration of these cells when stimulated by microglia (Fig 6).


Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway.

Rolón-Reyes K, Kucheryavykh YV, Cubano LA, Inyushin M, Skatchkov SN, Eaton MJ, Harrison JK, Kucheryavykh LY - PLoS ONE (2015)

FAK is not involved in microglial stimulation of migration in glioma cells.Data obtained from migration assays for glioma cells. Cells were treated with 5nM (concentration that effectively blocks FAK) and 16nM (concentration that effectively blocks Pyk2) of PF-562,271. Due to different migration abilities in all presented cell lines the Y-axis scales are adjusted for each cell line in order to demonstrate the absolute numbers of migrating cells. Results are presented as mean ± S.D. with significant differences from control in each group (+), from Mock without microglia on the bottom (#), or from Mock with microglia on the bottom (*) (p < 0.05). One-way ANOVA followed by the Tukey’s multiple comparison test was used to determine significance between groups.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476590&req=5

pone.0131059.g006: FAK is not involved in microglial stimulation of migration in glioma cells.Data obtained from migration assays for glioma cells. Cells were treated with 5nM (concentration that effectively blocks FAK) and 16nM (concentration that effectively blocks Pyk2) of PF-562,271. Due to different migration abilities in all presented cell lines the Y-axis scales are adjusted for each cell line in order to demonstrate the absolute numbers of migrating cells. Results are presented as mean ± S.D. with significant differences from control in each group (+), from Mock without microglia on the bottom (#), or from Mock with microglia on the bottom (*) (p < 0.05). One-way ANOVA followed by the Tukey’s multiple comparison test was used to determine significance between groups.
Mentions: Our data indicate that microglia stimulate migration of glioma cells and that treatment of glioma cells with AMCM increases their levels of phosphorylated Pyk2. To assess the involvement of microglia on activation of Pyk2 signaling in glioma cells and on glioma cell mobility, we performed migration assays for glioma cells in the presence and absence of the Pyk2/FAK inhibitor, PF-562,271. PF-562,271 readily passes the blood-brain barrier and selectively blocks FAK at 5 nM and blocks both FAK and Pyk2 at 16 nM [37], S3 and S4 Figs Therefore, we investigated both FAK and FAK/Pyk2 selective doses of PF-562,271. PF-562,271 was given two hours prior to and during the assay. The lower (FAK selective) concentration of PF-562,271 (5 nM) had no effect on glioma cell migration whether microglia were present or not (Fig 6) indicating that FAK was not involved in glioma cell migration. The higher concentration of PF-562,271 (16 nM) effectively eliminated the effect of microglia on glioma cell migration in all investigated glioma cell lines and also reduced the basal migration rates of U87 and HS683 glioma cells in the absence of microglia (Fig 6). Pyk 2 blockade (16 nM PF-562,271) did not alter the basal migration rates of A172 and C6 cells, but significantly reduced migration of these cells when stimulated by microglia (Fig 6).

Bottom Line: Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells.A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration.Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Universidad Central del Caribe, School of Medicine, Bayamón, Puerto Rico, United States of America.

ABSTRACT
Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells.

No MeSH data available.


Related in: MedlinePlus