Hepatocellular carcinoma (HCC) which accounting for 80–90% of primary liver cancers is responsible for a significant proportion of cancer-related deaths all over the world (Christopher 2006). Due to the absence of early diagnostic markers and limited efficacy of conventional treatments, prognosis of HCC remains exceedingly poor. Consequently, it is imperative to expeditiously identify highly sensitive and specific biomarkers to facilitate prognosis prediction and early detection of HCC patients. Over the recent years, an increasing amount of research has highlighted the importance of the FBXO protein family in relation to HCC, including members like FBXO31 and FBXO43 Huang et al. 2010, Wu et al. 2023. However, there was still no research article has yet explored the potential connection between the FBXO protein family and the prognosis of HCC.

In our study, amid to find out the feasible relationship between FBXO family genes and HCC, the HCC original data were retrieved from the TCGA database. Subsequently, the intersecting set of FBXO protein genes and differential expressed genes was identified by an analysis. Then, a prognostic model pertaining to FBXO family genes was constructed utilizing LASSO COX regression analysis. The model encompassed six specific genes: FBXO5, FBXO6, FBXO16, FBXO30, FBXO32, and FBXO45. To evaluate the efficacy of the risk model, various techniques including univariate analysis, multivariate COX regression, KM survival curve analysis, ROC curve analysis, and nomogram construction were employed. Subsequently, IHC was performed to verify the expression of six genes (FBXO5, FBXO6, FBXO16, FBXO30, FBXO32, and FBXO45) in HCC tissues and their homologous adjacent tissues. All the experimental findings above collectively indicate that the FBXO family genes have the potential to impact the prognostic profile of HCC.

Among predicted model genes, FBXO5, FBXO6, FBXO45 and FBXO32 have been identified as oncogenes in HCC (Lin et al. 2021; Cen et al. 2023; Sahu et al. 2017; Gao et al. 2022; Xu et al. 2018a, b). Recent research findings suggest that FBXO5 contributes to the proliferation of liver cancer cells (Gao et al. 2022; Xu et al. 2018a, 2018b) and the process is regulated through the suppression of APC/C, which hinders the stability of Skp2 while facilitating the degradation of p27kip1 Zhao et al. 2013. The FBXO5 expression level correlates with the stage and unfavorable prognosis of HCC (Gao et al. 2022). Furthermore, Chen et al. reported an overexpression of FBXO5 in HCC, suggesting its potential role in controlling the proliferation of HCC cells (Xu et al. 2018a, b). In view that FBXO6 interacts with and promotes the proteasomal degradation of NLRX1, leading to the reduced effectiveness of HCC inhibition (Cen et al. 2023) and plays a role in facilitating the ubiquitination process and promoting the degradation of the protein Chk1 to enhancing the resistance of tumor cells to specific drugs (Liu et al. 2021), suggesting that FBXO6 functions as an oncogene and has detrimental effects on the prognosis of HCC. Additionally, the identification of FBXO32 as a noteworthy contributor to the upregulation of epithelial-to-mesenchymal transition (EMT) genes’ expression substantiates its role in the metastatic progression of human tumors (Sahu et al. 2017). Moreover, the upregulation of FBXO45 has been associated with poor prognostic outcomes in HCC and plays a crucial role in activating PLK1 through IGF2BD1 mediation which further substantiating its oncogenic role in HCC progression (Lin et al. 2021). In our experiment, higher expression of FBXO5, FBXO6, FBXO32 and FBXO45 in invasive tumor specimens further underscores its role as an oncogene. Nerveless, the precise role of FBXO16 in HCC is still unclear. Additionally, the recent research documented elevated levels of FBXO30 potentially improve the prognostic outcomes in HCC because it reduced levels of EG5 who participate in distinct ubiquitination processes (Tang et al. 2022). But in our research, FBXO30 exhibits oncogenic characteristics in HCC which contradicts existing literature. Our conclusions show successful risk modelling by LASSO analysis, which was further confirmed having independent prognostic significance by univariate and multivariate COX analyses. The KM survival curves indicate a statistically significant increase in overall survival (OS) within the low-risk group compared to the high-risk group. Furthermore, the ROC analysis yielded area under the curve (AUC) values of 0.744, 0.670, and 0.638 for the 1-year, 3-year, and 5-year time points, respectively. These outcomes collectively prove the remarkable prognostic effect of the six FBXO genes in HCC patients.

To explore the specific pathological processes of the six FBXO family genes in HCC, we analyzed the role of them in the high and low-risk groups by KEGG. Moreover, the most pertinent outcome of the current investigation is the enrichment of cytochrome P450 which is associated with the processing of drugs in metabolism pathways (Patel et al. 2020). Notably, the existing literature highlights the association between CYP enzyme activity and the susceptibility to various forms of cancer development and with consistent evidence establishing a connection between CYP polymorphisms and the occurrence of liver cancer (Yeo et al. 2012). In addition, another study has presented Cytochrome P450-based cancer gene therapy as a potentially effective approach for activating prodrugs in cancer treatment, showcasing significant prospects for improving the safety and effectiveness of cancer chemotherapeutics (Agundez 2004). These studies underscore the crucial role played by cytochrome P450 enzymes in both the advancement and control of cancer, thereby drawing attention to the potential therapeutic strategies that could be developed by targeting this pathway. Hence, we examined the relationship between our six FBXOs gene-related drugs in our article. Belinostat, a histone deacetylation inhibitor which warrants consideration for enhancing its therapeutic efficacy in HCC by leveraging potential role of FBXO16 because its clinical anticancer activity and good tolerability in the treatment of HCC (Waxman et al. 1999). The drug prediction analysis presented in Fig. 5C reveals a potential negative correlation between FBXO30 and acetalax. Additionally, a prior study has provided evidence that cancer cell lines expressing higher levels of TRPM4 display heightened susceptibility to acetalax (Li et al. 2021). Furthermore, acetalax has exhibited its capacity to induce the expression of tumor necrosis factor (TNF) α and the degradation of TNFR1, thereby demonstrating its anti-tumor effects in mouse xenograft models (Yang et al. 2022). The drug predictions of FBXO32 reveal associations with allopurinol in HCC whose unique capacity to inhibit first-pass metabolism in cancer chemotherapy may enhance drug bioavailability and action in HCC treatment (Zimm et al. 1983). Regarding FBXO45, drug prediction analysis associates it with erlotinib. Encouragingly, studies have indicated that the bound of erlotinib and Lenvatinib yields significant synergistic effects both in vivo and vitro, thus offering a promising therapeutic approach for HCC (Hu et al. 2022). In the current experiment, a significant relation between the FBXO gene family and drug metabolism CYP450 was observed. Consequently, it is plausible to hypothesize that FBXO may have a potential role in drug therapy for HCC. However, additional investigation is required to validate these findings in future experiments.

The KEGG analyses also revealed a correlation between the FBXO genes and Retinol, a vital micronutrient, as known as vitamin A. This micronutrient assumes a pivotal role in the regulation of stem cell functions, cellular differentiation, and metabolism in various cell types (Gudas 2022). In various tissues, retinol ultimately transforms into retinoic acid through a sequence of biochemical reactions and leads to its immunomodulatory impacts by attaching to the retinoic acid receptor (RAR) (Mora et al. 2008). Moreover, the liver serves as a key immune-related organ situated at the center, consistently encountering blood rich in antigens from the intestines via the portal vein (Ruf et al. 2021). In the summary, our empirical inquiry has unveiled a robust association between the FBXO gene family and retinol metabolism, thereby suggesting a plausible connection between the FBXO family and immune responses. In this study, we used ssGSEA which found the tumor samples has notable disparities between low-risk and high-risk groups of different immune cell infiltrations in HCC patients. Interestingly, our analysis indicated significant variations in the levels of MHC-class I and Type II-IFN among these groups. MHC class I is a pivotal cell surface protein in the immune system (Reynisson et al. 2020) which not only restrains the evasion of the immune system by tumor cells (Lin et al. 2023) but also intensifies the engulfment of tumor cells, making it a promising target for anti-cancer immunotherapy (Barkal et al. 2018). Our findings revealed a substantially higher expression of MHC class I in the high-risk group than the low-risk group in HCC. This suggests that MHC class I could emerge as a novel potential focus for HCC immunotherapy, offering the possibility of improving the unfavorable prognosis of HCC. Type II-IFN (also known as Interferon γ) is a cytokine that plays diverse roles in enhancing protective immune responses and immunopathological processes, thereby playing a crucial role in safeguarding against tumor development (Ikeda et al. 2002). Intriguingly, Type II-IFN prompting the upregulation of MHC class I in cells and stimulating the delivery of the antigens of MHC class I are the pivotal element in anti-tumor immune responses (Ren et al. 2022). However, our results revealed that the expression of Type II-IFN was noticeably higher in the low-risk group compared with the high-risk group. This finding opens a new avenue for exploring Type II-IFN as a prospective immunotherapeutic strategy during the low-risk phase, aiming to preempt the early deterioration and migration of HCC, or as a target for immunotherapy in the context of early-stage tumors.

Furthermore, the utilization of immune checkpoint inhibitors has demonstrated considerable promise in the field of HCC tumor immunotherapy (Huang et al. 2021). Our findings revealed a significant positive correlation between our predictive model and immune checkpoints, specifically encompassing CTLA-4, PDCD1, and LAG3. PDCD1 (as known as PD-1) not only plays a key role in down-regulating immune system function and proving tolerance (Li et al. 2022) but also has surfaced as a potential effective immune treatment strategy for patients dealing with advanced HCC (Shi et al. 2022). Besides, CTLA-4 functions as an inhibitory co-receptor which exerting its influence by impeding the activation and proliferation of T cells (Sangro et al. 2013). In recent research, it has been established that CTLA-4 has the capability to dampen immune responses against tumors by impeding T cell reactions and PD-1 with CD8 + T cells interacting with PD-L1 + Kupffer cells contributes to the malfunction of effector T cells within HCC, which both are associated with less favorable prognosis in individuals with HCC (Guo et al. 2021). In addition, LAG-3 belongs to the group of immune checkpoint receptors that diminish the multiplication and stimulation of T-cells by binding with its ligand (Guo et al. 2021). Additionally, LAG-3 could also potentially serve as a marker for exhaustion alongside PD-1, arising from repetitive stimulation by antigens in cancer contexts (Wang et al. 2019). Nowadays, immunotherapy has the capability to enhance the immune system by inhibiting immune checkpoints and this advancement is widely seen as a significant breakthrough in how malignancies are treated (Guo et al. 2021). Therefore, these findings in our research underscore the potential significance of these immune checkpoints in influencing the advancement of HCC.

Since FBXOs serve as E3 ubiquitin ligases, in order to further explore the role of the FBXO family in HCC, we used the UbiBrowser database to search for their substrate target proteins. The database results indicated that TP53 is the substrate target protein for all six FBXO members. The TP53 gene (also referred to as p53) encodes the p53 protein which represents one of the most frequently mutated gene and is related in over 50% of all human cancers (Juan Liu et al. 2023). In addition, the progression of cancer can be attributed to the proteolytic degradation of p53 facilitated by activated E3 ubiquitination enzymes (Ming Zhou et al. 2021). Notably, there are investigations revealing that the upregulation of the ubiquitination process targeting the tumor suppressor gene TP53 is associated with enhanced growth in ovarian cancer (Ming Zhou et al. 2021), while the ubiquitination of P53 is implicated in the pathological progression of colorectal cancer (Wang et al. 2020). FBXO proteins serve as integral components of E3 ubiquitin ligase, playing crucial regulatory roles in various cellular processes (Liu et al. 2021). Moreover, the functional prediction revealed that the FBXOs were involved in ubiquitination-related pathways such as TP5352which is as the same as ours. Based on the reported literature it is known that FBXO45 expression was related to P53 mutation in PDAC (Zhang et al. 2021). In addition, FBXO45 selectively ubiquitinates and degrades p73 which is a member of the p53 family, resulting in diminished cellular apoptosis in cancers (Wang et al. 2020). However, there was no significant difference in the expression of FBXO32 between the two groups (Jing et al. 2023). Additionally, in p53-deficient cells, up-regulation of FBXO5 could facilitate genomic instability, tetra ploidy, and cell proliferation in breast cancer (Zhang et al. 2022). Interestedly, there were rare evidence to improve that FBXO6, FBXO16, and FBXO30 had relationship with the P53-mediated ubiquitination in cancers, and our research may demonstrate them. To sum up, it is reasonably to suppose that FBXO family genes could ubiquitinate TP53 which results in p53 being more prone to mutation and promotes HCC progression.

This study holds great potential for the future. By identifying the overlap between FBXO genes and DEGs in HCC, our research provides potential novel biomarkers that can be used for the early detection, prognosis assessment and management of HCC. Moreover, KEGG analysis offered strong directions for subsequent drug development research. Additionally, the discovery of different immune infiltrating statuses can inform personalized immunotherapy, advancing precision medicine. However, our study also has certain limitations. We did not elucidate the specific molecular mechanisms of FBXO proteins in HCC, nor did we specify how these proteins influence P53 and TP53 mutations through ubiquitination. The specific impact of different immune infiltrating statuses on HCC progression and treatment response also requires further investigation. In future research, functional experiments such as cell-based assays and animal models could be conducted to explore the precise mechanisms of FBXO proteins, particularly their roles in P53 ubiquitination. Combining techniques like immunohistochemistry (IHC) and flow cytometry can provide deeper insights into the effects of different immune infiltrating statuses on HCC. Our study aims to contribute to optimized personalized treatment strategies for HCC, thereby enhancing treatment efficiency and patient survival rates. As research on drug metabolism pathways progresses, new targeted drugs and therapeutic approaches are expected to emerge. Nonetheless, our study has limitations, including the constraint of sample size, potential biases in the TCGA dataset, and the fact that our conclusions are primarily based on bioinformatics analyses. These findings require further experimental data for validation and support.