FRI-516-YI Aurora kinase A and programmed death-ligand 1: expression dynamics in hepatocellular carcinoma development and the regulatory role of the kinase in immune checkpoint modulation

Background and aims: Aurora Kinase A (AURKA) is a pivotal mitotic kinase implicated in tumorigenic processes and overexpressed in most cancer types. Recent evidence suggests a new regulatory role for AURKA in modulating Programmed Death-Ligand 1 (PD-L1) expres- sion in breast cancer. Despite its critical role, limited information exists on AURKA’s involvement in hepatocellular carcinoma (HCC), and the regulatory interplay with PD-L1 remains unexplored. This study aims to explore AURKA and PD-L1 expression in HCC and precancerous conditions and the impact of AURKA inhibition and knockdown on the regulation of PD-L1 in vitro. Method: AURKA and PD-L1 (CD274) mRNA and protein expression were assessed using qRT-PCR and Western blot, respectively. Human samples included healthy (n = 14), metabolic dysfunction-associated steatotic liver disease (MASLD) (n = 17), tumor, and paired adjacent non-tumoral (NT) tissues from HCC patients (n = 56). Mouse samples were collected from transgenic (TG) mice with chronic hepatitis B, progressing to HCC by 12 months, and wildtype (WT) mice sacrificed at 3, 6, 9, 12, and 15 months (n = 11 per condition). AURKA was inhibited by alisertib or AK-01 for 72 h, and knockdown was achieved through siRNA for 72 h or 144 h in HCC-derived JHH6 and Huh7 cell lines. The effects were evaluated in terms of cell viability, cell cycle arrest (flow cytometry), and PD-L1 protein expression (Western blot). Results: AURKA and PD-L1 gradually increased from healthy to MASLD ( p < 0.05) and from MASLD to HCC-adjacent NT tissues ( p ≤0 .05). AURKA overexpression was observed in 75% of HCC tissues compared to NT tissues ( p < 0.001), aligning with the results from the TG mouse model, where the gene was increased in early tumor stages compared to pre-tumoral stages ( p < 0.001), NT ( p < 0.05), and WT tissues ( p < 0.001). Cd274 exhibited a marked increase during neoplastic progression in mice ( p = 0.01). AURKA positively correlated with PD-L1 in HCC, NT, and MASLD tissues (all p < 0.001). Despite the decrease of AURKA protein expression in HCC tissues ( p < 0.001), the percentage of phosphorylated AURKA (Thr288) was increased in HCC, suggesting augmented kinase activity. AURKA inhibition or knock- down increased aneuploidy (+19–48%, all p<0.01) and reduced viability (p < 0.001) in both cell lines. AK-01 treatment decreased PD- L1 glycosylated mature forms ( p < 0.01), while 144-hour knockdown reduced unglycosylated PD-L1 ( p < 0.05) in both cell lines. Conclusion: AURKA and PD-L1 expression positively correlate in liver disease, both increasing with the progression of the disease. AURKA exhibits higher kinase activity in tumors than in NT tissues. In vitro, AURKA inhibition or silencing decreases PD-L1 expression through two mechanisms, implying multiple roles of AURKA in the regulation of PD-L1. This suggests a potential use of AURKA-targeted therapy in combination with immune checkpoint inhibitors in cancer therapy.