Activation of HIF-1α by δ-Opioid Receptors Induces COX-2 Expression in Breast Cancer Cells and Leads to Paracrine Activation of Vascular Endothelial Cells.

Opioids promote tumor angiogenesis in mammary malignancies, but the underlying signaling mechanism is largely unknown. The current study investigated the hypothesis that stimulation of δ-opioid receptors (DOR) in breast cancer (BCa) cells activates the hypoxia-inducible factor 1α (HIF-1α), which triggers synthesis and release of diverse angiogenic factors. Immunoblotting revealed that incubation of human MCF-7 and T47D breast cancer cells with the DOR agonist d-Ala2,d-Leu5-enkephalin (DADLE) resulted in a transient accumulation and thus activation of HIF-1α DADLE-induced HIF-1α activation preceded PI3K/Akt stimulation and was blocked by the DOR antagonist naltrindole and naloxone, pertussis toxin, different phosphoinositide 3-kinase (PI3K) inhibitors, and the Akt inhibitor Akti-1/2. Whereas DADLE exposure had no effect on the expression and secretion of vascular endothelial growth factor (VEGF) in BCa cells, an increased abundance of cyclooxygenase-2 (COX-2) and release of prostaglandin E2 (PGE2) was detected. DADLE-induced COX-2 expression was also observed in three-dimensional cultured MCF-7 cells and impaired by PI3K/Akt inhibitors and the HIF-1α inhibitor echinomycin. Supernatant from DADLE-treated MCF-7 cells triggered sprouting of endothelial (END) cells, which was blocked when MCF-7 cells were pretreated with echinomycin or the COX-2 inhibitor celecoxib. Also no sprouting was observed when END cells were exposed to the PGE2 receptor antagonist PF-04418948. The findings together indicate that DOR stimulation in BCa cells leads to PI3K/Akt-dependent HIF-1α activation and COX-2 expression, which trigger END cell sprouting by paracrine activation of PGE2 receptors. These findings provide a potential mechanism of opioid-driven tumor angiogenesis and thus therapeutic targets to combat the tumor-angiogenic opioid effect. SIGNIFICANCE STATEMENT: Opioids are indispensable analgesics for treating cancer-related pain. However, opioids were found to promote tumor growth and metastasis, which questions the use of these potent pain-relieving drugs in cancer patients. Enhanced tumor vascularization after opioid treatment implies that tumor progression results from angiogenic opioid effects. Thus, understanding the signaling mechanism of opioid-driven tumor angiogenesis helps to identify therapeutic targets to combat these undesired tumor effects. The present study reveals that stimulation of δ-opioid receptors in breast cancer cells leads to an activation of HIF-1α and expression of COX-2 via PI3K/Akt stimulation, which results in a paracrine activation of vascular endothelial cells by prostaglandin E2 receptors.