Pertussis Toxin Is a Robust and Selective Inhibitor of High Grade Glioma Cell Migration and Invasion

Abstract

In high grade glioma (HGG), extensive tumor cell infiltration of normal brain typically precludes identifying effective margins for surgical resection or irradiation. Pertussis toxin (PT) is a multimeric complex that inactivates diverse Gi/o G-protein coupled receptors (GPCRs). Despite the broad continuum of regulatory events controlled by GPCRs, PT may be applicable as a therapeutic. We have shown that the urokinase receptor (uPAR) is a major driver of HGG cell migration. uPAR-initiated cell-signaling requires a Gi/o GPCR, N-formyl Peptide Receptor 2 (FPR2), as an essential co-receptor and is thus, PT-sensitive. Herein, we show that PT robustly inhibits migration of three separate HGG-like cell lines that express a mutated form of the EGF Receptor (EGFR), EGFRvIII, which is constitutively active. PT also almost completely blocked the ability of HGG cells to invade Matrigel. In the equivalent concentration range (0.01–1.0 μg/mL), PT had no effect on cell survival and only affected proliferation of one cell line. Neutralization of EGFRvIII expression in HGG cells, which is known to activate uPAR-initiated cell-signaling, promoted HGG cell migration. The increase in HGG cell migration, induced by EGFRvIII neutralization, was entirely blocked by silencing FPR2 gene expression or by treating the cells with PT. When U87MG HGG cells were cultured as suspended neurospheres in serum-free, growth factor-supplemented medium, uPAR expression was increased. HGG cells isolated from neurospheres migrated through Transwell membranes without loss of cell contacts; this process was inhibited by PT by >90%. PT also inhibited expression of vimentin by HGG cells; vimentin is associated with epithelial-mesenchymal transition and worsened prognosis. We conclude that PT may function as a selective inhibitor of HGG cell migration and invasion.

A long-lived peptide-conjugated iridium(iii) complex as a luminescent probe and inhibitor of the cell migration mediator, formyl peptide receptor 2

“…After treatment with negative control (NC) siRNA or FPR2 siRNA, the monolayers of HUVECs were scratched linearly with a 200 μL pipette tip, and images of wounded monolayer were taken at 12 h. The results showed that FPR2 silencing decreased the migration of HUVEC cells compared to cells treated with NC siRNA. This indicates that FPR2 is correlated with the cell migration capacity of HUVECs, which is consistent with previous reports 54,55…”

The Urokinase Receptor Induces a Mesenchymal Gene Expression Signature in Glioblastoma Cells and Promotes Tumor Cell Survival in Neurospheres

“…It is thus intriguing that uPAR protein expression was substantially increased in U87 cells when these cells were cultured in neurospheres as opposed to monolayers. Similarly, uPAR protein expression was substantially increased when U87vIII cells were cultured in neurospheres 53 . These results may be interpreted to indicate that U87 and U87vIII cells that are capable of seeding neurospheres are uPAR-enriched.…”

“…The gene that encodes the EGF Receptor ( EGFR ) is over-expressed in up to 50% of all glioblastomas and in many of these tumors, EGFR is mutated to generate a constitutively active derivative called EGFRvIII 50 , 51 . U87 cells that express EGFRvIII are previously characterized and we previously demonstrated that these cells form neurospheres 52 , 53 . To determine whether activated EGFR signaling affects glioblastoma neurosphere formation, we prepared neurospheres with U87 and U87vIII cells under equivalent conditions.…”

Pro-Resolving FPR2 Agonists Regulate NADPH Oxidase-Dependent Phosphorylation of HSP27, OSR1, and MARCKS and Activation of the Respective Upstream Kinases

“…The observation that the deletion of the Fpr1 gene reduced inflammation in an experimental mouse model of endometriosis [ 68 ] and that FPRs were mainly expressed on neutrophils and macrophages, suggests that these receptors predominantly govern a proinflammatory response which results in chemotaxis, degranulation, and oxidative burst during infection. The release of endogenous FPR ligands can influence severe diseases associated with inflammation, including systemic inflammatory response syndrome [ 69 ], Alzheimer′s disease, amyloidosis, prion disease, obesity, diabetes [ 70 ], and several types of cancer [ 71 , 72 , 73 , 74 ]. However, FPRs can also promote the resolution of inflammation.…”