Caffeine Binding Non-Canonical Biased Adenosine A₂AR GPCR Modulates Neu1 Activation and Signaling in Breast Cancer Cells

Dysregulated receptor signaling networks contribute to breast cancer progression and metastatic plasticity, particularly in triple-negative breast cancer (TNBC), an aggressive subtype with limited therapeutic options. Among these signaling systems, the adenosine A₂A receptor (A₂AR), a G protein‑coupled receptor (GPCR) highly expressed in TNBC, contributes to immunosuppression and tumor progression. Caffeine, a consumed A₂AR antagonist, has been reported to modulate cancer-associated signaling pathways, although its non-canonical mechanisms remain poorly understood. We hypothesized that caffeine engages a biased signaling axis involving neuromedin B receptor (NMBR), matrix metalloproteinase‑9 (MMP9), and neuraminidase‑1 (Neu1) to modulate cancer‑associated phenotypes in breast cancer cells. Using sialidase assays, we found that caffeine induced Neu1 enzymatic activity in both MDA‑MB‑231 (TNBC) and MCF‑7 (luminal) cells in a dose‑dependent manner. This induction was abrogated by pharmacological inhibitors of NMBR (BIM‑23127), MMP9 (MMP9i), or Neu1 (oseltamivir phosphate), indicating dependence on the GPCR‑MMP9‑Neu1 signaling platform. Immunofluorescence co‑localization analysis revealed strong spatial proximity between A₂AR and Neu1 on the cell surface (Pearson’s r = 0.8968), supporting the feasibility of signaling crosstalk. Functionally, caffeine treatment of MDA‑MB‑231 cells significantly increased the epithelial marker E‑cadherin and decreased the mesenchymal marker N‑cadherin, while vimentin expression showed limited changes. Tunneling nanotube (TNT) density, a measure of intercellular communication, was markedly reduced at higher caffeine concentrations (≥2 mM). In RAW‑Blue reporter cells, caffeine decreased secreted embryonic alkaline phosphatase (SEAP) activity, suggesting reduced NF-κB/AP-1-dependent transcriptional activity. Cell viability was reduced by caffeine in a dose‑ and time‑dependent manner in both cell lines. Overall, these findings demonstrate that caffeine activates a non‑canonical, biased A₂AR‑NMBR‑MMP9‑Neu1 signaling axis in breast cancer cells, leading to a more epithelial phenotype, reduced TNT communication, and attenuated NF‑κB/AP‑1 activity. This work identifies a previously unrecognized mode of caffeine action and suggests that targeting this signaling platform may offer new therapeutic opportunities for aggressive breast cancer subtypes.

Department dc:contributor.department

Biomedical and Molecular Sciences

Year dc:date.issued

2026

Author dc:creator

• Li, Yunfan

Advisor dc:contributor.supervisor

• Szewczuk, Myron. R

• Breast cancer
• Triple-negative breast cancer
• Caffeine
• Neu1
• Adenosine A₂A receptor

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1974/36454

OAI identifier oai:identifier

oai:queensu.scholaris.ca:1974/36454