Ochratoxin A drives hepatocellular carcinoma progression via GRPR-mediated activation of PLCβ/IP3R/Ca²⁺ and RPS15A/p53 pathways by regulating cancer cell fate

Ochratoxin A (OTA), a group 2B human carcinogen, has been implicated in hepatocellular carcinoma (HCC), yet its precise molecular mechanisms remain elusive. In this study, we investigated the oncogenic role of OTA using both in vitro (Huh7 and Hep-3B cell lines) and in vivo (C57BL/6 mouse models) approaches. Molecular docking, surface plasmon resonance, co-immunoprecipitation coupled with mass spectrometry (Co-IP/MS), and functional rescue experiments were employed to elucidate the underlying pathways. Our results demonstrated that OTA exposure (50 nM) significantly promoted HCC cell proliferation, migration, and invasion. Molecular docking combined with surface plasmon resonance revealed a high-affinity binding interaction between OTA and the gastrin-releasing peptide receptor (GRPR), which was subsequently validated by the observed upregulation of GRPR expression upon OTA treatment. We further demonstrated that OTA activates the canonical GRPR downstream pathway, PLCβ/IP3R/Ca 2 + , leading to increased intracellular calcium levels. Furthermore, Co-IP/MS identified ribosomal protein S15A (RPS15A) as a novel GRPR-interacting protein. RPS15A overexpression facilitated HCC progression by recruiting MDM2 to accelerate the degradation of p53, thereby upregulating the SLC7A11/GPX4 axis-a key regulator of ferroptosis. Crucially, GRPR knockdown effectively attenuated OTA-induced tumor growth both in vitro and in vivo. Collectively, our findings reveal a novel mechanism whereby OTA promotes HCC progression by binding to GRPR and activating both the PLCβ/IP3R/Ca 2+ signaling cascade and the RPS15A/p53/SLC7A11/GPX4 axis, suggesting that targeting GRPR may represent a promising therapeutic strategy for OTA-associated HCC. A hypothesis diagram of OTA promoted hepatocellular carcinoma progression by targeting GRPR and then regulating PLCβ/IP3R/Ca 2+ and RPS15A/p53 signaling. In HCC cells, OTA exerted its carcinogenic effects by binding to and activating GRPR. Specifically, the upregulated GRPR facilitated the expression of PLCβ and IP3R, which resulted in the increase of Ca 2+ concentration. In addition, GRPR upregulated RPS15A, which accelerated the degradation of p53 protein by recruiting MDM2, leading to upregulated the expression of SLC7A11/GPX4. The above molecules eventually promoted the proliferation and migration and may inhibit the ferroptosis of HCC cells. • Ochratoxin A exposure promoted the hepatocellular carcinoma progression both in vivo and in vitro. • Ochratoxin A promoted hepatocellular carcinoma progression by binding and activating GRPR. • The up-regulation of GRPR activated PLCβ/IP3R/Ca 2+ and RPS15A/p53 pathways, and resulted in HCC.