LKB1 dictates sensitivity to immunotherapy through Skp2-mediated ubiquitination of immune checkpoint proteins in HCC with python analysis

Articles in Press

Document Type : Original Article

Authors

1 School of Medicine,Tehran University of Medical Sciences,Tehran, Iran

2 Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

3 School of Pharmacy ,Tehran University of Medical Sciences,Tehran, Iran

4 Immunology Research Center,Tabriz University of Medical Sciences,Tabriz,Iran

10.22038/ijbms.2026.91840.19829

Abstract

Objective(s): Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with limited treatment options, particularly in advanced stages. Immune checkpoint inhibitors (ICIs) targeting PD-1, PD-L1, and CTLA-4 have shown promise in cancer immunotherapy, but response rates in HCC remain variable. 
Materials and Methods: Hep3B and HepG2 HCC cells were cultured and genetically manipulated to overexpress or deplete LKB1. Western blotting, real-time PCR, and immunofluorescence were used to assess PD-L1 expression at the protein and mRNA levels. The role of Skp2 in PD-L1 regulation was evaluated through shRNA-mediated knockdown and overexpression. Additionally, kinase-dead LKB1 mutants were expressed to determine the importance of LKB1 kinase activity in PD-L1 stability. ImageJ software and Python-based computational tools were employed for quantitative analysis of immunofluorescence and Western blot data.
Results: LKB1 overexpression up-regulated PD-L1 protein levels in HCC cells, while its depletion reduced PD-L1 expression, indicating a post-translational regulatory mechanism. Although Skp2 expression remained unchanged upon LKB1 modulation, Skp2 overexpression in LKB1-deficient cells increased PD-L1 levels, suggesting a context-dependent role for Skp2 in PD-L1 stability. Furthermore, wild-type LKB1, but not the kinase-dead mutant, restored PD-L1 expression, highlighting the essential role of LKB1 kinase activity in PD-L1 regulation.
Conclusion: This study identifies LKB1 as a critical regulator of PD-L1 stability in HCC, with implications for tumor immune evasion and immunotherapy response. While Skp2 appears to influence PD-L1 stability in specific contexts, LKB1’s kinase activity is essential for PD-L1 regulation.

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Main Subjects

References

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Iranian Journal of Basic Medical Sciences

Articles in Press, Accepted Manuscript
Available Online from 16 May 2026

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