Adenosine interaction with adenosine receptor A2a promotes gastric cancer metastasis by enhancing PI3K-AKT-mTOR signaling

Abstract

The accumulation of adenosine in the tumor microenvironment is associated with tumor progression in many cancers. However, whether adenosine is involved in gastric cancer (GC) metastasis and progression, and the underlying molecular mechanism, is largely unclear. In this study, we find that GC tissues and cell lines had higher A2aR levels than nontumor gastric tissues and cell lines. A2aR expression correlated positively with TNMstage, and associated with poor outcomes. Adenosine enhanced the expression of the stemness and epithelial-mesenchymal transition-associated genes by binding to A2aR. A2aR expression on GC cells promoted metastasis in vivo. The PI3K-AKT-mTOR signaling pathway was involved in adenosine-stimulated GC cell migration and invasion. Our results indicate that adenosine promotes GC cell invasion and metastasis by interacting with A2aR to enhance PI3K-AKT-mTOR pathway signaling.

FIGURE 1:

High A2aR expression in GC is associated with poor outcomes. Immunoblotting (A) and immunohistochemistry (B) assays of A2aR expression in tissue arrays of tumor (T) and para-tumor (N) normal tissues from 97 patients with GC. Black arrow: A2aR. Scale bar = 50 µm. *, P < 0.05, t test. (C) Kaplan–Meier curves for cumulative survival show a significant association between high A2aR expression in GC and worse prognosis (P = 0.008, log-rank test). Immunoblotting (D) and immunofluorescence (E) assays both show higher A2aR expression in GC cell lines than in the nontumorigenic gastric epithelial cell line. Scale bar = 50 µm.

FIGURE 2:

Adenosine promotes GC cell invasion and metastasis through the A2aR pathway in vitro. (A) Representative scanning electron microscopy images showing significantly more pseudofoot and cilia growth. (B) Migration (top) and invasion (bottom) Transwell assays showing increased invasive capability of NECA-treated cells compared with control (Ctrl) or NECA + SCH 58261–treated cells. Scale bar = 100 µm. Bars represent mean ± SEM of at least three independent quadruplicate experiments. (C) Wound-healing assay in MKN45 cells: the scratch was measured 24 h after the treatments. Bars represent mean ± SEM of at least three independent quadruplicate experiments. (D) Immunoblot detection of tumor metastasis–related MMPs. Data are the mean ± SE of triplicate measurements. *, P < 0.05, ANOVA.

FIGURE 3:

Adenosine (NECA) regulates the expression of the stemness and EMT-associated genes through the A2aR pathway in vitro. Epithelial and mesenchymal marker expression was analyzed by immunoblotting (A) and immunofluorescence staining (B). Scale bar = 50 µm. (C) Immunoblotting detection of the stemness markers. The adenosine-induced increased stemness was confirmed by flow cytometry (D) and colony formation assay (E), but the specific antagonist of A2aR (SCH 58261) could reverse this effect. **, P < 0.01, ANOVA. All results are from three independent experiments.

FIGURE 4:

Adenosine promotes GC cell metastasis based on PI3K–AKT–mTOR pathway activation. (A) NECA-treated MKN45 cells had higher PI3K, AKT, and mTOR expression than vehicle- or NECA + SCH 58261–treated cells. (B) Immunoblotting detection of MMPs, and epithelial, mesenchymal, and stemness markers after treatment with OSI-027 (2 μM). Colony formation (C) and Transwell (D) assays confirmed the decreased invasive capability and stemness of GC cells after OSI-027 treatment. Data are the mean ± SEM, *, P < 0.05, **, P < 0.01, ANOVA. All results are from three independent experiments.

FIGURE 5:

Blocking of A2aR on GC cells suppresses lung metastasis in vivo. Short hairpin (Sh) control (negative control [NC]) or A2aR KD MKN45 cells (5 × 10⁵ cells) were injected intravenously in a 200-μl volume into male athymic BALB/c nude mice. In the group of Ctrl or A2aRi, the mice were treated intraperitoneally with vehicle or A2aR inhibitor (SCH58261, 10 mg/kg) two times every week. (A) The photon fluxes were monitored at week 4 after tumor cell injection. (B, C) The lungs in each treatment group were collected and the metastatic burden was quantified by counting colonies on the lung surface 4 wk after tumor cell injection. Results are the mean ± SEM; n = eight mice per group. (D) Representative hematoxylin–eosin staining images of lung tissue sections from each group. Scale bar = 100 µm. ***, P < 0.001, ANOVA.