Glioblastoma multiforme (GBM) is among the most prevalent and fatal intracranial malignancies in the human brain (Wang et al. 2023). Although multiple therapies, including surgical removal, adjuvant radiation therapy and chemotherapy with temozolomide, have been accepted as the standard therapeutic treatment for GBM, the prognosis remains poor because of its diverse heterogeneity, with a five-year survival rate not exceeding 10% (Ren et al. 2022). To date, few biological risk factors and effective therapeutic targets have been identified to improve GBM treatment (Moody et al. 2021). Therefore, investigating new prognostic and promising therapeutic objectives to prolong the survival time of GBM patients is imperative.

Chemoattractant receptors are seven transmembrane G protein-coupled receptor (GPCRs) that are involved in a broad spectrum of pathophysiologic processes, including hematopoiesis, inflammation, wound healing, development (Hou et al. 2022), and malignant tumor progression (Dorsam and Gutkind 2007; Liang et al. 2020). Formyl peptide receptors (FPRs) and their variants, including FPR-like1 (FPR2) and FPR-like 2 (FPR3), are GPCRs that were originally isolated from phagocytic leucocytes and mediate the activation and chemotaxis of these cells during interactions with bacterial formylated chemotactic peptides (Busch et al. 2022). Previous studies have shown that other cell types, including astrocytoma cells, also express functional nFPRs (Le et al. 2000). Among these receptors, FPR2 is the most attractive owing to its diverse signaling pathways and a range of structurally diverse agonists, which contain high concentrations of fMLF, bacteria, viruses, and synthetic peptides (Tylek et al. 2023). In addition to formylated peptides, FPR2 also recognizes large proteins and structurally unrelated lipids as internal agonists and exhibits potent pro- or anti-inflammatory responses on the basis of the ligands that activate it. Annexin-A1 serves as an FPR2 ligand, and previous studies have confirmed that annexin-A1 activates FPR2 to promote breast cancer proliferation (Moraes et al. 2017). Our previous data demonstrated that Tat-NTS, a blocker of annexin-A1 nuclear translocation, inhibits glioblastoma cell proliferation and invasion in a dose-responsive manner (Luo et al. 2022). Agonist binding to FPR2 in leucocytes leads to the activation of phosphatidylinositol-specific phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling cascades, and mitogen-activated protein kinase (MAPK) pathways as well as the nuclear translocation of NF-κB (Li et al. 2021). More importantly, FPR2 plays a crucial role not only in many inflammatory-associated diseases but also in the progression of malignant gliomas (Tadei et al. 2018).Previous research has shown that functional FPR2 is endogenously expressed in the U87 astrocytoma cell line (Le et al. 2000), suggesting its involvement in the development of brain cancer. A previous study revealed that FPR activation in U-87 cells also enhanced the phosphorylation of Akt (Zhou et al. 2005), also known as protein kinase B. Akt is a downstream component of the PI3K pathway and has been shown to promote tumor cell survival and proliferation. Highly malignant human GBM cells express functional FPRs associated with their malignant phenotype, which is elicited by potential agonists secreted by necrotic tumor cells (Yang et al. 2011). Although FPR2 in glioblastoma tissue and glioma cells directly promotes tumor cell invasion and growth and the synthesis of matrix metalloproteinases and angiogenic vascular endothelial growth factor (Yao et al. 2008), it also transactivates EGFR, synergistically enhancing the aggressive properties of GBM cells (Huang et al. 2007). Although prior research has demonstrated that FPR2 is significantly upregulated in highly aggressive GBM cells and plays a vital role in facilitating cancer development, the clinical value of FPR2 and its role in human gliomas still need further clarification.

Autophagy is the main cytoplasmic degradation system through which cytoplasmic macromolecules and damaged organelles are transported to and digested in the lysosome by double-membrane vesicles (Lei and Klionsky 2021). Autophagy can be either constitutive or induced in response to different degrees of stress. Autophagy not only maintains cellular homeostasis but is also involved in various physiological and pathological processes, including apoptosis, aging, and tumor growth (Debnath et al. 2023). The regulation of autophagy is complicated and varies on the basis of tumor cell type. Autophagy suppresses growth and development while promoting cell death in certain tumors. Moreover, autophagy promotes the survival and proliferation of other tumors, making it a promising therapeutic target for cancer. Prior research has indicated that autophagy is capable of inhibiting GBM progression in the initial stages but facilitating GBM cell proliferation in the later stages. During autophagic flux, many dynamic membrane reorganizations are initiated with the elongation and closure of autophagosomes, resulting in the formation of autophagosomes, which ultimately effectively fuse with lysosomes to form autolysosomes. One FPR2 agonist, namely, annexin-A1, plays a crucial role in the vesiculation of multivesicular bodies (Diakonova et al. 1997) and membrane trafficking (White et al. 2006), indicating that FPR2 might also be involved in autophagy (Liu et al. 2018).

Despite several studies on the role of autophagy in GBM, the pathological role of FPR2 in regulating autophagy in human GBM has not been well studied (Wan et al. 2023). In this study, we discovered that FPR2 is selectively overexpressed in glioma cells and brain tissues compared with normal astrocytes and normal brain tissues, respectively. Knockdown of FPR2 impaired the proliferation, migration and invasion of GBM cells. More importantly, the inhibition of FPR2 through the PI3K/AKT signaling pathway facilitated autophagy in GBM cells, and the process was dependent on BECN1 and ATG5. In addition, the inhibition of autophagy by FPR2 enhanced the migration and invasion of GBM cells by suppressing the degradation of Snail. Our results provide the first and most definite evidence that the inhibition of autophagy by FPR2 actively promotes the migration and invasion of GBM tumor cells.

Cell Culture and Reagents

The human glioblastoma cell lines LN-229, U87, U251, U118 and A172 were acquired from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology (China), whereas normal human astrocytes (NHA, Cat. #CC2565) were obtained from Lonza Group Ltd. (Basel, Switzerland). All these cells were cultivated in DMEM (Servicebio, Cat. #G4515; Wuhan, China) supplemented with 10% fetal bovine serum (FBS; Gibco, Cat. #16140089; USA) at 37 °C with 5% CO2/95% air in a humidified incubator. The sources of the antibodies used were as follows: FPR2 (#30989-1-AP) was purchased from Proteintech Technology (Wuhan, China). ATG5 (#2630), N-cadherin (#4061), E-cadherin (#14472), vimentin (#5741), Gsk3β (#12456), β-catenin (#8480), CDC2 (#28439), cyclin B1 (#4138), cyclin D1 (#2922),p110α (#4255), p85 (#4292), p-Akt (Ser473) (#4060), Akt (#4685), PARP (#9542), cleavedcaspase-3 (#9661), cleavedcaspase-7 (#9491), cleaved-caspase-8 (#9429), p65NF-κB (#3034), p-p65NF-κB (#3033), cPLA2 (#2832) and horseradish peroxidase-conjugated IgG antibodies (#7074 or #7076) were purchased from Cell Signaling Technology (Danvers, MA, USA).SQSTM1 (#ab207305) and BECN1 (#ab62557) were obtained from Abcam (Cambridge, UK).Cyclin B1 (#AF6000-SP) and Cdc25 (#MAB4459-SP) antibodies were purchased from the American R&D Company (R&D Systems, Minneapolis, MN). 1-Stearoyl-2-[1-14 C] arachidonoyl-sn-glycero-3-phosphocholine (2-AA-PC, 56.0 mCi/mmol) was acquired from Amersham Pharmacia Biotech (Buckinghamshire, UK) and utilized as the substrate. Unlabeled 2-AA-PC, scintillation fluid (Aquasol-2), bafilomycin A1 (BAF, B1793), 3-methyladenine (3-MA, M9281) and chloroquine (CQ, C6628) were purchased from Sigma‒Aldrich (St. Louis, MO, USA). The 0.1% crystal violet dye was purchased from Beyotime Biological Co., Ltd. (Beyotime Institute of Biotechnology, Shanghai, China), and the Transwell chambers were obtained from Corning Lifesciences (#3422, Corning, USA). Matrigel (#356234) was purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Clinical Specimens

From June 2013 to August 2019, we collected 58 paraffin-embedded glioma samples from the Neurosurgery Department at the Central Hospital of Wuhan. All the cases were confirmed by a pathologist, and 6 nontumor brain tissue samples (acquired through decompressive surgery) were utilized as normal controls. All the glioma samples included elaborate information related to clinical and pathological features. In accordance with the 2000 World Health Organization central nervous system tumor grading system, 15 cases were Grade II, 16 cases were Grade III, and 27 cases were Grade IV. Additionally, 6 Grade IV samples and 6 nontumor brain tissue samples were procured and preserved in liquid nitrogen for immunoblot analysis. The research protocol was endorsed by the Clinical Research Ethics Committee of the Central Hospital of Wuhan, and informed consent was obtained from all patients.

Stable FPR2 Knockdown and Overexpression and Transient Transfection

Lentiviral vectors, including LV2-pGLV constructs with either FPR2 shRNA or scramble nontarget shRNA and LV5 vectors harboring FPR2, were developed by GenePharma Co. (Shanghai, China) and validated through sequencing. The target sequence for the human FPR2 shRNA was designed as follows: 5′-UAUAAGGGCCACCACCUGAUAUGGG-3′. The efficiency of the knockdown was confirmed in previous studies (Khau et al. 2011). U87 and LN-229 cells were transfected with lentivirus particles and subsequently screened with puromycin for a two-week period. Glioma cell lines with stable knockdown or overexpression of FPR2, along with corresponding control cell lines, were established. siRNAs targeting BECN1 (sc-29797; Santa Cruz, USA), ATG5 (sc-41445; Santa Cruz, USA), control siRNA (sc-37007; Santa Cruz, USA) or SQSTM1 (sc-29679; Santa Cruz, USA) were transiently transfected into stable FPR2-knockdown and FPR2-overexpressing glioma cells using Lipofectamine 3000 (Invitrogen, USA) following the manufacturer’s protocols.

Real-Time RT‒PCR

Total RNA was extracted from glioma cells with TRIzol, and quantitative real-time PCR was subsequently performed in accordance with the manufacturer’s instructions and our previous study (Wei et al. 2021). The housekeeping gene GAPDH was used as an internal control. The sequences of primers used were as follows: FPR2, 5’-CTTGTGATCTGGGTGGCTGGA (sense) and 3’-CATTGCCTGTAACTCAGTCTC (antisense) and GAPDH, 5’-GCCAAAGGGTCATCATCTC (sense) and 3’-GTAGAGGCAGGGATGATGTTC (antisense) (Rossi et al. 2015).

Cell Counting Kit-8 (CCK-8) Assay

Cell growth was evaluated using a CCK-8 assay in accordance with the manufacturer’s instructions and as previously described (Wei et al. 2021). Briefly, stable transfected U87 cells were seeded in 96-well plates at a density of 2 × 103 cells per well. Cells from each group were cultured for 24, 48, 72 h and 96 h before CCK-8 assay kit (Beyotime, Shanghai, China) was applied and 10 µl reaction solution was added to every well and subsequently incubated for 1 h. The optical density was then detected by an EnSpire Manager spectrophotometer plate reader (Turku, Singapore) at 450 nm (excitation) and 600 nm (emission).

Flow Cytometry Analysis

During the exponential growth phase, U87 cells were plated at a density of 4 × 105 cells per well in 6-well culture plates. Glioma cells transduced with different vectors were prepared for flow cytometry analysis according to the manufacturer’s instructions. The cell cycle progression and apoptosis was assessed using a FACSCalibur flow cytometer (BD Biosciences, USA). To examine the cell cycle, a PI/RNase Staining Buffer Solution Cell Cycle Analysis Kit (#550825; BD Bioscience, USA) was used. To measure the rates of apoptosis, an apoptosis detection kit (#C1062M, Beyotime, Shanghai, China) was used. The percentage of apoptotic cells was calculated by adding together the early apoptotic cells in the lower right quadrant and the late apoptotic cells in the upper right quadrant.

Western Blot Analysis

The proteins from the cells or tissues were extracted in RIPA lysis buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail (Roche, Germany). Following centrifugation at 14,000×g for 20 min at 4 °C, proteins were combined with 5× loading buffer (#P0015, Beyotime) and heated to boiling for 10 min. Equal quantities of protein (30 µg) were loaded per lane for immunoblot analysis. The proteins were separated and then blotted onto PVDF (#IPVH00010; Millipore, Billerica, USA) membranes. After three washes with TBST, the membranes were blocked with TBST containing 5% nonfat milk, followed by overnight incubation with the appropriate primary antibodies in TBST at 4 °C. After three washes with TBST, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies, which were diluted to a 1:10000 ratio in TBST, at room temperature for 60 min. After three washes with TBST, the protein blots were visualized using an enhanced chemiluminescence kit (Pierce Biotech, Rockford, IL) and quantified using NIH ImageJ 1.4.3 (Bethesda, MD, USA) after normalization to the internal control.

Migration and Invasion Assays

First, the Transwell chamber was immersed in complete medium for 2 h to ensure proper hydration. GBM cells were collected and quantified under a microscope and then plated into the upper wells at a density of 5 × 104 cells per well. The chamber medium was composed of DMEM enriched with 0.1% bovine serum albumin, while the lower part contained DMEM supplemented with 10% FBS. The migration pattern of glioblastoma cells was subsequently examined. After 24 h of cultivation, the chamber was removed. The cells that penetrated the chamber membrane were fixed in 4% paraformaldehyde and subsequently stained with 0.1% crystal violet (Beyotime, Shanghai, China). The cells were visualized under a microscope (Olympus, USA), and five random microscopic fields were imaged. For invasion assays, the chambers were precoated with a uniform layer of Matrigel (BD Biosciences), and the subsequent steps were similar to those used in the migration assays.

Assay of Cytosolic Phospholipase A2 (cPLA2) Activity

cPLA2 activity was evaluated via sonicated liposomes prepared with 1-stearoyl-2-[1-14 C]-arachidonyl-sn-glycero-3-phosphocholine as the substrate, in accordance with a previously established methodology(Kim et al. 2001).

Immunohistochemical Analysis

Immunohistochemical staining of FPR2 was performed on brain tissue sections that were fixed with 4% paraformaldehyde, embedded in paraffin wax, and cut into 5-µm sections. The tissue sections were deparaffinized with xylene and subsequently rehydrated through a series of graded alcohol solutions. The samples were then incubated with 3% hydrogen peroxide for 10 min to inhibit the activity of endogenous peroxidases. The slices were heated in citrate buffer for 20 min to facilitate antigen repair. Afterward, the tissue slices were incubated with an anti-FPR2 antibody (1:200, #30989-1-AP, Proteintech Technology) overnight at 4 °C. The sections were then incubated with a biotinylated secondary antibody, developed using an avidin-biotin peroxidase complex and counterstained with Harris’ modified hematoxylin. After imaging, two pathologists independently evaluated the samples. The staining intensity was categorized as follows: strong, 3; moderate, 2; weak, 1; and no staining, 0. The score was determined on the basis of the proportion of FPR2-positive cells: if the percentage of positive cells was less than 5%, a score of 0 was assigned; if it was between 5% and 10%, the score was 1; if it was between 10% and 50%, the score was 2; if it was between 50% and 80%, the score was 3; and if it was greater than 80%, the score was 4. The total score ranged between 0 and 12. The above two scores were multiplied; a score equal to or greater than 4 was considered positive, and a score less than 4 was considered negative.

Nuclear Factor NF-κB p65 Transcription Factor Activity Assay

Nuclear extracts from stably transfected U87 cells were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Rockford, USA). The activity of NF-κB was evaluated by employing 9 µg of nuclear extract along with an NF-κB (p65) Transcription Factor Assay Kit (KA1341;Abnova, USA), following the manufacturer’s protocols. The absorbance was measured at 450 nm using an EnSpire Manager spectrophotometer plate reader (Turku, Singapore).

Tumor Xenografts in Nude Mice

Following the NIH guidelines and the requirements of the “Ethical Review System for Laboratory Animal Welfare of the Myhalic Biotechnology Co., Ltd” (approval number HLK-20241115-001), mouse experiments was performed. The parental U87 cells, which had been stably transfected, were adjusted to a concentration of 1 × 107 cells/ml. A volume of 200 µl containing 2 × 106 cells with either the sh-Scramble or FPR2 knockdown sequence in PBS was subcutaneously injected into both regions. Starting from day 3, the tumor volume was measured every 3 days. The tumor volume was determined using the following formula: tumor width2 × length × 0.5. On the 30th day, tumor tissues were acquired for weighing or Western blot analysis.

Statistical Analysis

Statistical analyses were conducted using GraphPad Prism (version 8.0) software. The data are expressed as the means ± SDs. One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test was used to evaluate differences among groups. A P value of < 0.05 was considered to indicate statistical significance. Kaplan‒Meier survival curves were constructed and evaluated using the log-rank test for comparative analysis, utilizing data from the CGGA glioma cancer dataset. The correlation between FPR2 and SQSTM1 in glioma patient tissues was assessed using the Pearson correlation coefficient (http://gepia.cancer-pku.cn).