CNIH4 promotes hepatocellular carcinoma malignant phenotype via GPCRs/ ERK mediated oxidative phosphorylation

Cornichon family AMPA receptor auxiliary protein 4 (CNIH4) is predominantly localized in the endoplasmic reticulum and aids in GPCR transport. Recent studies have found that CNIH4 is abnormally expressed and implicated in malignant tumors. However, its specific impact on hepatocellular carcinoma (HCC) and underlying mechanisms of its role in malignant progression remains elusive. Bioinformatics and clinical detection were firstly performed to assess the expression, clinical relevance, functional regulatory networks of CNIH4 in HCC. In transfected HCC cells, HCCLM3 and MHCC97H cells were assigned into Sh-NC, Sh-CNIH4 (CNIH4 knockdown), Vector and Oe-CNIH4 (CNIH4 overexpressed) groups. The effect of CNIH4 on cell proliferation, metastatic potential, and oxidative phosphorylation, including oxygen consumption rate (OCR) and ROS levels, were systematically evaluated. The antioxidant NAC, ERK1/2 inhibitor SCH772984, GPR139 agonist JNJ 63,533,054 and mGlu1 receptor antagonist BAY36-7620 were added to explore the involvement of GPCRs/ERK signaling pathway-dependent oxidative phosphorylation. In the in vivo study, an experimental HCC lung metastasis model in nude mice, established by injecting cells transfected with Oe-CNIH4 or Sh-CNIH4, was used to substantiate the pro-metastatic activity of CNIH4 and its regulatory effects on GPCRs/ERK pathway activation. Multiple public databases-based analysis elucidated that high expression of CNIH4 correlates with advanced TNM stage and poor prognosis of HCC, as well as the oxidative phosphorylation pathway. CNIH4 overexpression promoted HCC cell proliferation, migration, OCR levels and ATP production, while reducing ROS generation, through the GPCRs/ERK axis. The addition of antioxidant NAC and the ERK1/2 inhibitor SCH772984 confirmed the role of CNIH4 in malignant phenotypic progression via the ERK-dependent oxidative phosphorylation. Furthermore, the addition of GPCRs agonist and antagonist revealed that CNIH4 activates GPCRs to regulate ERK-mediated oxidative phosphorylation. In a nude mouse model of HCC lung metastasis, the pro-metastatic function of CNIH4 was further validated, together with activation of the GPCRs/ERK pathway. Our findings revealed that CNIH4 promotes HCC malignant phenotype, a process critically involving GPCR/ERK-mediated oxidative phosphorylation. These insights into CNIH4’s role in HCC may provide novel molecular targets for developing HCC therapies.