Hepatocellular carcinoma remains a significant challenge to human health, with a high rate of recurrence even after surgical resection. In recent years, many studies have demonstrated that CTCs are closely related to the metastasis, epithelial-mesenchymal transition (EMT), and recurrence of malignant tumors, including HCC (30). Therefore, it is critical to screen molecules associated with CTCs to identify validated biomarkers for predicting HCC. Here, we construct ed a reliable prognostic signature based on CTCs related genes and explored its clinical utilization in HCC patients. Moreover, we initially investigated the biological role of CDCA8 in hepatocellular carcinoma and identified CDCA8 as a potential therapeutic target.
Firstly, we identified 258 CRGs by analyzing the DEGs in TCGA and ICGC databases and CTC expression profiles of hepatocellular carcinoma. Then, these genes were screened to construct a five-CRGs signature in the TCGA cohort. Next, we utilized the KM survival analysis and ROC analysis to confirm the prognostic value of the signature. In addition, similar results were also validated in the ICGC cohort and GEO cohort. Univariate and multifactorial Cox analyses confirmed that signature was an independent prognostic factor. Moreover, the nomogram for all three cohorts demonstrated a good predictive power of the model.
To better understand the interactions between these CRGs, we further analyzed them using the STRING website and visualized them with Cytoscape software. In addition, we identified ten genes that may play important roles in HCC development, including TOP2A, CCNB2, CDCA8, BIRC5, AURKB, CCNB1, BUB1, BUB1B, KIF20A, TTK. Many genes have been validated in HCC, such as TTK as a target for intrahepatic spread therapy in hepatocellular carcinoma; BUB1B activates mTORC1 to promote hepatocellular carcinoma progression; KIF20A may serve as an independent prognostic factor for overall and disease-free survival in hepatocellular carcinoma patients (31–33). As indicated by KEGG, CRGs promote development, metastasis, and recurrence of hepatocellular carcinoma probably through the cell cycle and p53 signaling pathway. GSEA analysis of five signature genes and high-risk groups in the prognostic model identified various oncogenesis-related features, including cell cycle, p53 signaling pathway, WNT signaling pathway, DNA replication, TGF-beta signaling pathway, and notch signaling pathway.
To evaluate the value of the signature in clinical terms, we explored correlations with clinical characteristics and found that the high-risk group was positively associated with tumor stage, tumor grade, and T stage. Immune cell infiltration analysis revealed that CDCA8 and HAVCR1 may be associated with several immune cell abundances, like B cell, CD8 + T cell, macrophage, neutrophil, and dendritic cell. In recent years, immune checkpoint inhibitors have shown a potential therapeutic role for advanced HCC (34). As mentioned previously, immune checkpoints such as PD-1, PD-L1, and CTLA4 and tumor mutation burden (TMB) were highly expressed in the high-risk subgroup, suggesting that the high-risk group patients may benefit more from immunotherapy. In addition, Pearson correlation analysis indicated that CDCA8 and HAVCR1 were also positively correlated with immune checkpoints, which further revealed the contributions of these two genes to the development of hepatocellular carcinoma in terms of immunity.
Notably, we screened five small molecule drugs that were highly relevant to CRGs by using cMAP, including apigenin, sulfadimethoxine, guanabenz, nadolol, and ginkgolide A. Apigenin, an edible plant flavonoid, is an antioxidant and can also be used as a therapeutic agent to overcome several diseases and even cancer. Previous studies found that apigenin inhibits EMT and metastasis in hepatocellular carcinoma through NF-κB and snail signaling pathways and also enhances the cytotoxicity of sorafenib on HepG2 cells (35, 36). Several reports have shown that sulfadimethoxine has antitumor effects in in vitro cellular assays, but the biological contribution in hepatocellular carcinoma needs to be further investigated (37, 38). Guanabenz (GBZ), an α2-adrenergic agonist, is an FDA-approved antihypertensive drug with a good safety profile. Kang et al. demonstrated the antitumor value of GBZ in human HCC cell lines (39). In addition, GBZ not only inhibits the invasion and migration of chondrosarcoma but also serves as a potential therapeutic agent for triple-negative breast cancer (40, 41). Ginkgolide A is a platelet-activating factor antagonist extracted from Ginkgo biloba, and a literature review mentioned its many biological effects, such as anti-inflammatory, anti-cancer, anti-anxiety, anti-atherosclerotic, neurological, and hepatoprotective effects (42). However, a recent report highlighted that another derivative from Ginkgo biloba, ginkgolide C, exhibited its anti-hepatocarcinoma effection by modulating c-Met phosphorylation (43). In conclusion, our analysis is undoubtedly helpful, but its application in future studies is necessary.
In the prognostic signature, we screened five CRGs, including HAVCR1, TP53I3, MYCN, TXNRD1, and CDCA8. HAVCR1 is highly expressed in a variety of tumors, including colorectal cancer, non-small-cell lung cancer, clear cell renal cell carcinoma, and hepatocellular carcinoma; and can be an independent prognostic factor (44–47). Moreover, Ye et al. found that TIM-1+ (HAVCR1+) regulatory B cell infiltration was significantly higher in tumor tissues of HCC patients compared with paraneoplastic tissues and could promote the immune escape of hepatocellular carcinoma cells, implying that it could be used as a new immune therapeutic target (47). The p53-inducible gene 3 (PIG3), also known as TP53I3, is involved in the process of apoptosis and DNA damage response. Previous studies have revealed that TP53I3 promotes invasion and metastasis of lung cancer cells and that silencing TP53I3 enhances the chemosensitivity of non-small cell lung cancer to docetaxel (48, 49). These findings may provide us with a new strategy for the treatment of liver cancer. MYCN, a member of the MYC proto-oncogene family, Qin et al. highlighted that MYCN may be a stem cell-like marker for HCC and could be used as a therapeutic target of acyclic retinoid (ACR) for HCC (50, 51). TXNRD1 is an antioxidant enzyme that has been reported to be overexpressed in hepatocellular carcinoma. Lee et al. observed that inhibition of TXNRD1 suppressed HCC cell proliferation, promoted apoptosis, and could induce oxidative stress, which implies that it could be used as a therapeutic target for HCC (52). In addition, CDCA8 is a protein associated with the human cell division cycle and has been reported to be involved in a variety of tumorigenesis. Recent results have suggested that overexpression of CDCA8 enhances the proliferation of breast cancer, bladder cancer, and cutaneous melanoma cells and leads to poor prognosis. Interestingly, our study found that downregulation of CDCA8 inhibited proliferation, invasion, and metastasis of HCC cells and could lead to cell cycle arrest and increasing apoptosis. Therefore, targeting CDCA8 may be the next molecular strategy for the treatment and prevention of metastasis from HCC.
In our study, we focused on the analysis of CTC-related genes in HCC and constructed a signature of five CRGs. Through the validation of several databases, we found that this signature has a good predictive and clinical application profile. It is easier to apply in clinical practice as it reduces the necessity and medical expenses of whole-genome sequencing for patients. And, we explored the correlation with immune checkpoints and observed that high-risk patients may be better suited for immunotherapy. In addition, we established nomograms and screened candidate small molecule drugs, which may provide clinical benefits. However, our paper also inevitably has some limitations. Firstly, our study is still a prospective study, and future multicenter studies are needed to further validate the accuracy of the model. Secondly, since the database lacks tumor progression variables, such as vascular invasion status and tumor size, it may affect the statistical power of the nomogram and influence the predictive abilities. Finally, although we have initially explored the function of CDCA8 in HCC development, the specific molecular mechanisms are not clear; therefore, it is necessary to further elucidate the molecular mechanisms linking CDCA8 to circulating tumor cells.