LKB۱ dictates sensitivity to immunotherapy through Skp۲-mediated ubiquitination of immune checkpoint proteins in HCC with python analysis

Objective(s): Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with limited treatment options, particularly in advanced stages. Immune checkpoint inhibitors (ICIs) targeting PD-۱, PD-L۱, and CTLA-۴ have shown promise in cancer immunotherapy, but response rates in HCC remain variable. Materials and Methods: Hep۳B and HepG۲ HCC cells were cultured and genetically manipulated to overexpress or deplete LKB۱. Western blotting, real-time PCR, and immunofluorescence were used to assess PD-L۱ expression at the protein and mRNA levels. The role of Skp۲ in PD-L۱ regulation was evaluated through shRNA-mediated knockdown and overexpression. Additionally, kinase-dead LKB۱ mutants were expressed to determine the importance of LKB۱ kinase activity in PD-L۱ stability. ImageJ software and Python-based computational tools were employed for quantitative analysis of immunofluorescence and Western blot data.Results: LKB۱ overexpression up-regulated PD-L۱ protein levels in HCC cells, while its depletion reduced PD-L۱ expression, indicating a post-translational regulatory mechanism. Although Skp۲ expression remained unchanged upon LKB۱ modulation, Skp۲ overexpression in LKB۱-deficient cells increased PD-L۱ levels, suggesting a context-dependent role for Skp۲ in PD-L۱ stability. Furthermore, wild-type LKB۱, but not the kinase-dead mutant, restored PD-L۱ expression, highlighting the essential role of LKB۱ kinase activity in PD-L۱ regulation.Conclusion: This study identifies LKB۱ as a critical regulator of PD-L۱ stability in HCC, with implications for tumor immune evasion and immunotherapy response. While Skp۲ appears to influence PD-L۱ stability in specific contexts, LKB۱’s kinase activity is essential for PD-L۱ regulation.

کلیدواژه ها:

HCC ، Immune checkpoint - proteins ، LKB۱ ، Python analysis ، Skp۲