PKA-dependent phosphorylation of PSMG1 reduces bortezomib sensitivity in hepatocellular carcinoma
Wei-Jing Liu1, Shi-Jie Zeng2, Song-Wang Cai3
1Guangdong Provincial Key Laboratory of Pharmaceutical Preparations Research and Evaluation, Guangdong Provincial Engineering Center of Topical Precise Drug Delivery System, MOE Engineering Research Center of Small Molecule Drugs, School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, China; Cancer Minimally Invasive Therapies Centre, Guangdong Second Provincial General Hospital, Department of Cell Biology & Institute of Biomedicine, Guangdong Provincial Biotechnology & Engineering Technology Research Center, Guangdong Provincial Key Laboratory of Bioengineering Medicine, Genomic Medicine Engineering Research Center of Ministry of Education, MOE Key Laboratory of Tumor Molecular Biology, National Engineering Research Center of Genetic Medicine, State Key Laboratory of Bioactive Molecules and Druggability Assessment, College of Life Science and Technology, Jinan University, Guangzhou, Guangdong 510632, China.
Aims:
This study aimed to characterize PSMG1 expression and its phosphorylation status in hepatocellular carcinoma (HCC), and dissect its functional role in regulating bortezomib (BTZ) chemosensitivity.
Methods:
Western blotting, Flag co-immunoprecipitation and GST pull-down assays were applied to profile protein abundance, protein-protein binding and phosphorylation signaling cascades. Flow cytometry quantified apoptotic cell populations. Fluorogenic peptide substrates were utilized to measure cellular chymotrypsin-like proteasome activity. MTT, colony formation and Transwell migration assays were performed to evaluate proliferative and migratory phenotypes. In vivo anti-tumor efficacy of BTZ monotherapy and BTZ/H89 co-treatment was validated in subcutaneous HCC xenograft mouse models.
Results:
PSMG1 protein was aberrantly overexpressed in HCC tissues, and high PSMG1 abundance independently predicted dismal patient survival. Genetic ablation of PSMG1 suppressed chymotrypsin-like proteasome activity and sensitized HCC cells to BTZ, without altering intrinsic cell proliferation or migratory capacity. Protein kinase A (PKA) physically bound PSMG1 and was associated with its phosphorylation at residues T18 and T54. This post-translational modification was correlated with elevated chymotrypsin-like proteasome activity and reduced sensitivity of HCC cells to BTZ. Consistently, pharmacological blockade of PKA via H89 enhanced BTZ cytotoxicity in HCC. Immunohistochemical profiling revealed elevated PKA-pT197 levels in HCC lesions, which exhibited a modest positive correlation with PSMG1-pT18 phosphorylation signals.
Conclusions:
PKA activity is associated with PSMG1 phosphorylation, which may contribute to reduced BTZ sensitivity in HCC. Pharmacological inhibition of PKA with H89 enhanced bortezomib cytotoxicity in preclinical HCC models, providing a preliminary rationale for combination strategies to increase proteasome inhibitor sensitivity in HCC management.
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