The role of formyl peptide receptor 1 (FPR1) in neuroblastoma tumorigenesis

Abstract

Background: Formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor mainly expressed by the cells of myeloid origin, where it mediates the innate immune response to bacterial formylated peptides. High expression of FPR1 has been detected in various cancers but the function of FPR1 in tumorigenesis is poorly understood.

Methods: Expression of FPR1 in neuroblastoma cell lines and primary tumors was studied using RT-PCR, western blotting, immunofluorescence and immunohistochemistry. Calcium mobilization assays and western blots with phospho-specific antibodies were used to assess the functional activity of FPR1 in neuroblastoma. The tumorigenic capacity of FPR1 was assessed by xenografting of neuroblastoma cells expressing inducible FPR1 shRNA, FPR1 cDNA or control shRNA in nude mice.

Results: FPR1 is expressed in neuroblastoma primary tumors and cell lines. High expression of FPR1 corresponds with high-risk disease and poor patient survival. Stimulation of FPR1 in neuroblastoma cells using fMLP, a selective FPR1 agonist, induced intracellular calcium mobilization and activation of MAPK/Erk, PI3K/Akt and P38-MAPK signal transduction pathways that were inhibited by using Cyclosporin H, a selective receptor antagonist for FPR1. shRNA knock-down of FPR1 in neuroblastoma cells conferred a delayed xenograft tumor development in nude mice, whereas an ectopic overexpression of FPR1 promoted augmented tumorigenesis in nude mice.

Conclusion: Our data demonstrate that FPR1 is involved in neuroblastoma development and could represent a therapy option for the treatment of neuroblastoma.

Keywords: FPR1; Formyl peptide receptor 1; Inflammation; Neuroblastoma.

Fig. 1

FPR1 gene expression and NB overall survival. a High expression of FPR1 in NB is significantly associated with a worse survival. Data were obtained from “R2: Genomics analysis and visualization platform” [http://r2.amc.nl]. Two datasets provided by Versteeg (n = 88) and Seeger (n = 102) were used. b Dot plot representing higher expression of FPR1 in Versteeg cohort compared to neural crest tissue and benign neurofibroma. Data were obtained from “R2: Genomics analysis and visualization platform” [http://r2.amc.nl]

Fig. 2

FPR1 is expressed in NB cell lines and primary tumors. a Western blotting detected a protein band of approximately 38 kDa corresponding to FPR1 in all NB cells investigated. Additionally, RT-PCR analysis revealed FPR1 expression in 7 NB cell lines. b Immunohistochemistry showing specific expression of FPR1 in tumor cells of a primary human neuroblastoma. (upper panel, HE staining, 20x and 60x; middle panel, anti-human FPR1 staining, 20x and 60x; lower panel, isotype control, 20x and 60x) c Immunofluorescence staining of FPR1 (green) in SK-N-BE(2) cells

Fig. 3

FPR1 stimulation promotes intracellular Ca2+ mobilization. a Changes in intracellular calcium level were measured by confocal scanning microscope using green fluorescent calcium-binding dye. After baseline measurement, fMLP was added to the cells’ media and subsequent fluctuations of green fluorescence were monitored. b Pretreatment with EDTA. The arrow depicts time of fMLP addition

Fig. 4

Activation of signal transduction pathways in response to the stimulation of FPR1. a Phosphorylation of Erk1/2, Akt and P38-MAPK occurred after stimulation of NB cells with 10 nM of fMLF. Numbers represent densitometric values of bands. b Incubation of the cells with Cyclosporine H blocks ERK1/2 phosphorylation

Fig. 5

FPR1-dependent tumor development and survival in animal model. Cell lines with different expression level of FPR1 were subcutaneously injected into mice, and tumor growth was recorded. Kaplan-Meier curves represent the time until tumors reached a volume of 0.2 cm³ (a) and 0.5 cm³ (b). Survival plot (c) shows the difference in survival between animals with high and low expression levels of FPR1 (the criterion for endpoint was a tumor volume ≥ 1 cm³)