Background
Epidermal growth factor receptor (EGFR) is co-activated by the μ-opioid receptor (MOR), expressed on non-small cell lung cancer (NSCLC) cells and human lung cancer. We hypothesized that clinically used opioid analgesics that are MOR agonists co-activate EGFR, resulting in growth- and survival-promoting signaling.
Methods
We used H2009, a human adenocarcinoma NSCLC cell line, with constitutive EGFR phosphorylation, which showed increased expression of MOR and delta opioid receptor (DOR) by reverse transcriptase polymerase chain reaction. We used Western immunoblotting, magnetic bead-based Bio-plex cytokine assay, immunofluorescent staining, BrdU incorporation ELISA and BioCoat™ Matrigel™ invasion assay, to examine cell signaling, cytokine expression, co-localization of MOR and EGFR in human lung cancer, cell proliferation and invasion, respectively.
Results
Like epidermal growth factor (EGF), morphine stimulated phosphorylation of EGFR, Akt/protein kinase B (Akt), and mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) signaling in H2009 cells. Opioid receptor (OR) antagonist, naloxone, EGFR tyrosine kinase inhibitor, erlotinib, and silencing of MOR and DOR abrogated morphine- and EGF-induced phosphrylation of signaling, suggestive of OR mediated co-activation of EGFR. H2009 cells secreted significantly higher levels of cytokines as compared to control Beas2B epithelial cells. H2009 conditioned medium stimulated MOR expression in Beas2B cells, suggesting that cytokines secreted by H2009 may be associated with increased OR expression in H2009. We observed co-localization of EGFR and MOR, in human NSCLC tissue. Functionally, morphine and EGF-induced proliferation and invasion of H2009 cells was ameliorated by naloxone as well as erlotinib.
Conclusion
Morphine-induced phosphorylation of EGFR occurs via ORs, leading to downstream MAPK/ERK, Akt phosphorylation, cell proliferation and increased invasion. Notably, ORs are also associated with EGF-induced phosphorylation of EGFR. Increased co-expression of MOR and EGFR in human lung cancer suggests that morphine may have a growth-promoting effect in lung cancer.