Introduction
Hepatocellular carcinoma (HCC) is one of the most common and second most lethal liver cancer in the world (1). Unfortunately, only 18% of patients bearing HCC have a 5-year survival rate, which places it as the second most lethal cancer, preceded only pancreatic cancer (2). Therefore, a strategy that could reduce mortality from HCC is to develop an early diagnosis approach based on identifying new biomarkers. Based on a previous transcriptomic study in our lab, which revealed that annexins gene expression was modified in microdissected HCC nodules (3). Annexins A1, A2, A5, A8, and A10 belong to the A family of annexins that are Ca2+-regulated phospholipid-binding proteins. Members of this family can be peripherally coupled to the surface of negatively charged membranes when they are calcium-linked. This capability provides them with membrane-bound functions, such as organization, transport, and interaction with the actin cytoskeleton (4). The rat hepatocarcinogenesis model, induced with diethylnitrosamine (DEN), has proven to be highly effective in replicating the development of cirrhosis and heterogeneous multinodular HCC within 18 weeks. DEN's alkylating properties induce chronic liver damage, leading to inflammation and fibrosis that are associated with cirrhosis and HCC. This closely resembles the development of HCC in humans (5, 6).
Aims & Methods
We aimed to further investigate the expression profile of five annexin genes in a well-established animal model of hepatocarcinogenesis associated with cirrhosis and to identify potential serological markers for early HCC. The gene expression products of annexin were analyzed by RT-qPCR and western blots. Both mRNA and proteins were extracted from rat liver tissue at preneoplastic stages at 6 and 12 weeks and neoplasia at 18 and 22 weeks. Also, immunofluorescence was performed on rat liver samples and compared to HCC of clinical origin. Plasma samples were collected from the experimental model, and we used the ELISA technique to measure the serum levels of Annexin A1, A2, and A5.
Results
A gradual increase of annexin A1, A2, A5, and A10 was observed in liver of hepatocarcinogenesis-induced animals compared to normal liver both at mRNA and protein levels, being a statistically significant increase for annexin A1 and A5 since week 6 of the hepatocarcinogenesis model. In contrast, annexin A8 showed a significant decrease in protein levels. The histological location of annexin A1, A2, and A5 was in the tumor cells. The cellular location for annexin A1 and A5 observed in the cytoplasm and membrane of normal hepatocytes showed a re-localization to the nucleus of neoplastic cells. Notably, a significant increase in the plasma level of annexin A5 from week 12 was detected, with a gradual increase until week 22 in the hepatocarcinogenesis model. Moreover, the plasmatic level of annexin A5 significantly correlated (r2 = 0.8203) with the hepatic Annexin A5 level. Using the TCGA database, we found that the expression of ANXA2 (HR = 1.7, p = 0.0046) and ANXA5 (HR = 1.8, p = 0.00077) was associated with poor survival in HCC patients.
Conclusion
The results suggest that the expression profile of annexin A1, A5, and A10 may serve as early tumor markers, whereas annexin A2 and A8 could be used as markers of hepatocellular carcinoma progression, given their expression changes during advanced stages of hepatocarcinogenesis. Significantly, the presence of annexin A5 in the plasma of model animals suggests its potential as a biomarker for hepatocarcinogenesis.
References
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