Prohibitin 1 orchestrates mitochondrial-ER crosstalk via the VDAC1-GRP75-IP3R axis to drive malignancy in gastric cancer

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Abstract Background : Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), serving as critical hubs for mitochondrial- ER interactions, play a pivotal role in cancer progression. Prohibitin 1 (PHB1), an inner mitochondrial membrane protein, contributes to tumorigenesis by regulating energy metabolism and structural integrity. This study aimed to elucidate the molecular mechanism by which PHB1 regulates MAMs structural integrity and its cascade signaling in driving gastric cancer (GC) metastasis. Methods : We analyzed the expression profiles of PHB1/PHB2 in GC using the TCGA database. Immunohistochemistry was performed to detect PHB1/PHB2 protein expression in GC tissues, adjacent non-tumor tissues, and omental metastasis tissues, and to assess their correlation with clinicopathological features. Immunofluorescence was used to observe the co-localization of PHB1 with the IP3R-GRP75-VDAC1 complex. Live-cell Ca2+ imaging, ATP/ROS detection, and mitochondrial functional assays (mPTP opening/MMP) were employed to evaluate calcium flux and energy metabolism changes. Transmission electron microscopy and Western blot were utilized to analyze MAMs structural integrity. A nude mouse xenograft model was established to validate in vivo functions. Results : TCGA analysis confirmed that both PHB1 and PHB2 were overexpressed in GC. PHB1 expression significantly correlated with tumor stage, while PHB2 expression correlated with demographic characteristics. Mitochondrial PHB1 expression was significantly higher than PHB2 in omental metastasis tissues. Furthermore, PHB1 co-localized and interacted with the IP3R-GRP75-VDAC1 complex, regulating ER-to-mitochondria Ca2+ flux. PHB1 overexpression enhanced MAMs-mediated Ca2+ signaling, increased ATP production, and drove cytoskeletal remodeling, thereby promoting malignant progression. Conversely, PHB1 knockdown disrupted the IP3R-GRP75-VDAC1 axis, leading to MAMs structural dissociation and mitochondrial dysfunction. Notably, treatment with the VDAC1-specific inhibitor VBIT-12 in PHB1- overexpressing cells significantly reversed the pro-tumorigenic effects of PHB1, reduced PHB1 protein levels, and diminished its association with the GRP75-IP3R complex, demonstrating that PHB1 function depends on VDAC1 activity. In summary, PHB1 regulates the VDAC1-GRP75-IP3R complex in a VDAC1-dependent manner to maintain MAMs structural integrity. This subsequently modulates calcium homeostasis, mitochondrial energy metabolism, and cytoskeletal remodeling, ultimately driving GC progression. Conclusion : Our findings reveal a novel mechanism by which PHB1 promotes GC progression via regulating MAMs function through the VDAC1 complex, providing a theoretical basis for therapeutic targeting.

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