The Role of Aurora Kinase A in the Development of Hepatocellular Carcinoma and in the Regulation of Programmed Death-Ligand 1

Hepatocellular carcinoma (HCC) stands as a global health challenge and is predicted to increase in both incidence and mortality rates in the coming years. The persistent high mortality associated with HCC is primarily attributed to late diagnoses and limited therapeutic options. The recent introduction of combinatorial treatments, such as the combination of targeted therapy and immunotherapy has marginally improved outcomes in advanced-stage HCC, underscoring the imperative for identifying novel therapeutic targets. Aurora Kinase A (AURKA), a critical mitotic kinase, emerges as a significant oncogene in HCC, marked by substantial overexpression and pivotal involvement in key tumorigenic processes. Recent findings in various cancer types revealed an intriguing role of AURKA in Programmed Death-Ligand 1 (PD-L1) regulation and immune response to the tumor. However, the regulatory interplay between AURKA and PD-L1 is still unexplored in HCC. This study aims to investigate the expression patterns of AURKA and its interactors in both HCC and precancerous conditions and to evaluate the molecular and cellular effects of inhibiting and silencing AURKA in vitro, with a specific focus on the regulatory influence on PD-L1. The project employed a translational approach, by incorporating the study of human liver samples, a transgenic mouse model of HBV infection leading to HCC, and two HCC-derived cell lines. The study population included HCC patients, individuals with metabolic dysfunction-associated steatotic liver disease (MASLD), and subjects with healthy livers. The analysis of human tissues revealed a marked AURKA mRNA overexpression in 75% of HCC tissues compared to adjacent non-tumor tissues and a consistent gradual increase with the hepatocarcinogenesis progression. Corroborating these findings, the TG mouse model reflected a similar increase in Aurka expression as tumors developed when compared to pre-tumoral stages and adjacent non-neoplastic tissues. In human samples, AURKA expression positively correlated with several oncogenes, notably PD-L1, and the AURKA co-activator TPX2 Microtubule Nucleation Factor (TPX2) during the progression of the disease. AURKA protein expression exhibited a reduction in 83% of tumors compared to adjacent non-tumoral tissues. The highest AURKA expression was observed in steatotic tissues, suggesting a potential non-canonical role of AURKA in steatosis and fibrosis. However, our analysis revealed a higher percentage of phosphorylated AURKA on the Thr288 residue specifically within the tumor tissue, suggesting an increased kinase activity in the tumor. PD-L1 expression increased in HCC tissues, particularly in the non-mature forms, hinting at the initial step for subsequent membrane localization of PD-L1 or a potential non-immunological, pro-oncogenic role within cells. In vitro, the inhibition of AURKA by alisertib or AK-01 and its knockdown achieved through siRNA resulted in a notable decrease in AURKA activity and total AURKA expression, respectively. These changes were closely associated with disruptions in the mitotic process, leading to unbalanced cell division, and subsequent aneuploidy. Both the cell lines exhibited a strong dependency on AURKA expression for growth, as confirmed by the reduced viability following both AURKA inhibition and knockdown. AURKA exerted regulatory control over PD-L1 through two distinct mechanisms. The kinase-mediated activity of AURKA is crucial in influencing PD-L1 protein glycosylation and stabilization, while the presence of AURKA protein, irrespective of its kinase activity, has a notable impact on PD-L1 transcription. In conclusion, these observations suggest that AURKA is a potential therapeutic target in HCC. The combination of AURKA-targeted treatment with immune checkpoint inhibitors represents an intriguing possibility to be further explored in the identification of new effective therapeutic strategies for HCC patients.