Downregulation of ankyrin 3 (ANK3) promotes malignant behaviors associated with altered adhesion dynamics and actin cytoskeleton remodeling in renal cell carcinoma

Abstract

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer, with existing therapies largely ineffective for advanced stages. Ankyrin 3 (ANK3), a protein involved in various cellular processes, has been associated with poor prognosis and immune infiltration in ccRCC. However, its role in ccRCC development and progression remains poorly understood. In the present study, ANK3-knockdown 786-O cells were generated using a shRNA approach. Cellular assays revealed that ANK3 knockdown significantly enhanced cell proliferation, migration, and invasion compared to shControl cells. ANK3-knockdown cells also displayed increased nuclear size and accumulation of G2/M phase of the cell cycle. Proteomic analysis identified 59 significantly altered proteins in ANK3-knockdown cells, primarily involved in cell adhesion and structural integrity. Among these, 10 proteins were significantly associated with overall survival in ccRCC patients. Morphological analysis showed that ANK3-knockdown cells exhibited a more spindle-like shape and increased cellular protrusions (filopodia). Western blot analysis further demonstrated altered expression levels of junctional and cytoskeleton proteins, including decreased E-cadherin and increased ZO-1, ICAM-1, vimentin, and β-actin. Immunofluorescence analysis revealed the accumulation of F-actin in the cytoplasm and filopodia in ANK3-knockdown cells. Additionally, ANK3 knockdown enhanced cell adhesion and aggregation capabilities. Finally, a decrease in ANK3 expression was confirmed in ccRCC tissues (grades I and II) compared to adjacent normal tissues. These findings suggest that ANK3 acts as a potential modulator of cell adhesion and cytoskeleton remodeling in ccRCC and may represent a promising therapeutic target for this malignancy.