Copper homeostasis dysregulation promotes malignant transformation of renal tubular epithelial cells via the ROS/NF-κB/AID signaling axis.

Background: Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma and is characterized by aggressive biological behavior and poor clinical outcomes. However, the molecular mechanisms underlying the malignant transformation of renal tubular epithelial cells are not yet fully understood. This study aimed to investigate the role of copper homeostasis dysregulation in regulating the reactive oxygen species/nuclear factor-kappa B/activation-induced cytidine deaminase (ROS/NF-κB/AID) signaling axis during ccRCC progression. Methods: Copper ion levels, ROS accumulation, and the expression of copper transport proteins, NF-κB pathway-associated molecules, AID, and epithelial-mesenchymal transition (EMT)-related proteins were examined in ccRCC tissues, chronic nephritis tissues, adjacent non-tumor tissues, renal cancer cell lines (786-O and 769-P), and human renal tubular epithelial cells (HK-2). The copper chelator triethylenetetramine (TETA), copper (II) chloride (CuCl₂), ROS scavenger N-acetyl-L-cysteine (NAC), hydrogen peroxide (H₂O₂), NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC), and NF-κB activator lipopolysaccharide (LPS) were used to modulate signaling pathways. Cell proliferation, migration, and invasion were evaluated by 5-Ethynyl-2'-deoxyuridine (EdU) assay, colony formation, wound-healing, and Transwell invasion assays. Results: Copper levels were significantly elevated in the ccRCC tissues and renal cancer cells, accompanied by increased ROS accumulation, NF-κB signaling activation, aberrant AID overexpression, and enhanced EMT progression. Exogenous copper treatment promoted the proliferation, migration, and invasion of HK-2 cells, whereas copper chelation markedly suppressed these malignant phenotypes. Mechanistically, copper accumulation was associated with increased ROS production and activation of NF-κB signaling, accompanied by increased AID expression and EMT progression. ROS or NF-κB inhibition significantly attenuated AID expression and EMT-associated alterations. Further, TETA, NAC, and PDTC effectively reduced intracellular copper accumulation, suppressed ROS/NF-κB/AID signaling, and inhibited malignant biological behaviors in renal cancer cells. Conclusions: Copper homeostasis dysregulation promotes the malignant transformation of renal tubular epithelial cells through activation of the ROS/NF-κB/AID signaling axis. These findings suggest that targeting copper metabolism and the ROS/NF-κB/AID pathway represents a promising therapeutic strategy for ccRCC.