Acetylcholine-induced YAP stabilization enhances HIF-1 signaling under hypoxia via the nAchR-alpha7/PI3K/PDPK1 pathway in pancreatic cancer cells

The pancreas is highly innervated by the autonomic nervous system. Recent studies have extensively addressed the potential role of the autonomic nervous system in the development of pancreatic ductal adenocarcinoma (PDAC).Hypoxia-inducible factor-1alpha (HIF-1alpha) regulates tumor adaptation to hypoxia by controlling genes linked to angiogenesis, metastasis, and therapy resistance. We previously showed that neurotransmitter acetylcholine (Ach) enhances HIF-1alpha expression in pancreatic cancer cells via the nAchR-alpha7/PDPK1/YAP pathway under hypoxia, but the underlying molecular mechanisms remained unclear. Following our previous findings, this study aims to elucidate the molecular signaling mechanism by which Ach regulates HIF-1alpha expression under hypoxic conditions in pancreatic cancer cells. Here, we treated human pancreatic cancer cells with Ach under 0.5% oxygen. Protein levels, phosphorylation, and interactions were assessed using Western blot, qRT-PCR, and co-immunoprecipitation. PI3K, PDPK1, and YAP expression were silenced by siRNA. In vivo, the effect of Ach was tested using tail-vein Ach injections in a subcutaneous xenograft mouse model. We found that Ach activated PI3K downstream of nAchR-alpha7, increasing PDPK1 phosphorylation. This reduced YAP phosphorylation at serine 397, stabilizing YAP and promoting its nuclear translocation. In the nucleus, YAP bound to HIF-1alpha, enhancing its stability and transcriptional activity. Knockdown of PI3K, PDPK1, or YAP suppressed Ach-induced HIF-1alpha upregulation. In vivo , Ach significantly promoted tumor growth in mice implanted with control pancreatic cancer cells, but had no effect in mice bearing tumors derived from nAchR-alpha7-silenced cells. Analysis of clinical datasets revealed that high nAchR-alpha7 expression correlates with poor prognosis in PDAC patients. Collectively, these findings reveal that Ach promotes pancreatic tumor progression under hypoxia via the nAchR-alpha7/PI3K/PDPK1/YAP pathway, which stabilizes HIF-1alpha. This signaling axis represents a promising therapeutic target in acetylcholine-associated pancreatic cancer.