Annexin A5 Regulates Store-Operated Calcium Entry via Glyceraldehyde-3-Phosphate Dehydrogenase in Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is a major contributor to cancer-related mortality worldwide. Store-operated calcium (CA²⁺) entry (SOCE), the principal CA²⁺ influx pathway in non-excitable cells, has been implicated in regulating tumor cell proliferation, migration, and survival. Although annexin A5 (ANXA5) has been implicated in several malignancies, its mechanistic contribution to CA²⁺ signaling in HCC remains unclear.

Methods: SOCE-related differentially expressed genes were identified through integrated bioinformatics analyses of The Cancer Genome Atlas and the Gene Expression Omnibus datasets. Huh-7 and HepG2 cells with stable ANXA5 knockdown were established using lentiviral transduction. Molecular interactions and functional alterations were examined by co-immunoprecipitation, enzymatic activity assays, inositol 1,4,5-trisphosphate (IP₃) quantification, CA²⁺ imaging, western blotting, quantitative polymerase chain reaction (PCR), and flow cytometry. Cellular phenotypes were assessed using proliferation and migration assays, whereas tumor growth was evaluated in subcutaneous xenograft models using nude mice. Notably, all in vitro experiments in this study were validated using both Huh-7 and HepG2 cells, whereas only Huh-7 cells were employed for in vivo experiments.

Results: ANXA5 was identified as an SOCE-associated gene whose elevated expression correlated with poor prognosis in HCC. Functional assays demonstrated that ANXA5 depletion significantly suppressed HCC cell proliferation and migration. Co-immunoprecipitation assays showed reduced levels of GAPDH co-precipitating with ANXA5 in ANXA5-deficient cells, suggesting impaired association between ANXA5 and GAPDH. Although ANXA5 knockdown did not alter GAPDH expression, it markedly reduced GAPDH enzymatic activity, leading to decreased IP₃ production, impaired endoplasmic reticulum CA²⁺ release, and attenuated SOCE-mediated CA²⁺ influx. Importantly, pharmacological modulation of Phospholipase C (PLC) activity with U73122 and its inactive analog U73443 further supported the involvement of PLC-IP₃ signaling in SOCE impairment and malignant phenotypes following ANXA5 depletion. In vivo, ANXA5 silencing significantly inhibited tumor growth and was accompanied by reduced expression of Ki-67, vimentin, and the M2 macrophage marker cluster of differentiation 206 (CD206).

Conclusion: These findings support a working model in which ANXA5 interacts with GAPDH and is associated with altered IP₃ production and SOCE-dependent CA²⁺ signaling, potentially contributing to HCC progression and immune modulation. Collectively, this ANXA5/GAPDH/IP₃/SOCE axis may provide a mechanistic framework for understanding HCC development and suggests ANXA5 as a potential therapeutic target.