Proteomics efforts for hepatocellular carcinoma drug development

Hepatocellular carcinoma (HCC) is a globally prevalent malignant tumor with high incidence and mortality rates worldwide. Data from the International Agency for Research on Cancer (IARC) indicates that HCC stands as the fifth most common cancer and the third leading cause of cancer-related deaths worldwide [1]. In addition to primary liver cancer, secondary liver cancer, resulting from metastatic spread to the liver, is more prevalent. China, where liver cancer incidence is notably high, accounts for over half of the worldwide HCC cases, posing a substantial threat to the health of its population [2, 3]. Liver cancer is exceptionally heterogeneous, displaying molecular variances in tumor cells among patients within the same clinical stage. These variances lead to diverse tumor behaviors and prognoses, rendering standard clinical treatment protocols effective for only a subset of patients. The clinical manifestations of liver cancer span from asymptomatic early stages to complex late-stage multi-organ involvement, thereby complicating early diagnosis and treatment [4].

Despite advances in liver cancer treatment in recent decades, patient prognosis remains bleak. Primary therapeutic approaches for liver cancer include surgical resection, liver transplantation, local ablation, chemotherapy, targeted therapy, and immunotherapy [5, 6]. Nevertheless, the low rate of early diagnosis leads to late-stage diagnoses for many patients, missing the optimal window for surgical intervention. Moreover, current drug treatment regimens present limitations, such as drug resistance in targeted therapies and variable response rates in immunotherapies [7]. Therefore, the development of new therapeutic strategies and drugs is crucial for enhancing the prognosis of patients with liver cancer.

Proteomics, the study of proteomes, involves the extensive analysis of protein composition and its dynamic changes. This enables the assessment of protein expression levels, post-translational modifications, and protein–protein interactions, thereby offering a comprehensive understanding of cellular activities and disease processes at the protein level [8,9,10]. Through the examination of all proteins within cells, tissues, or organisms, proteomics provides novel insights for diagnosing, treating, and developing drugs for liver cancer [11, 12]. Leveraging these technologies, researchers have discovered numerous potential biomarkers that facilitate early diagnosis, disease monitoring, and prognostic evaluation of liver cancer [13, 14]. Advancements in proteomic technologies enable researchers to carry out more precise analyses of protein expression in liver cancer tissues, such as single-cell proteomics [15, 16], which provides a deeper comprehension of variations in protein expression among different cell types within the tumor and the heterogeneous nature of liver cancer [17,18,19]. For instance, analyzing protein–protein interaction networks can unveil pivotal proteins linked to liver cancer development, potentially serving as novel therapeutic targets [20].

Post-translational protein modifications are vital for cellular signal transduction and regulation, and their irregularities are closely linked to the onset and progression of tumors, including liver cancer. Researchers are increasingly utilizing post-translational proteomics in liver cancer research to unveil alterations in post-translational modification signaling pathways, potentially identifying novel therapeutic targets [21]. Increasingly, integrative studies that combine metabolomics and proteomics are conducted, contributing to a comprehensive understanding of the molecular mechanisms driving liver cancer development [22]. Integrating omics data from various levels enables researchers to gain a better understanding of the link between metabolic irregularities and alterations in protein expression in liver cancer [23].

Proteomic technologies identify specific proteins in liver cancer for the development of targeted therapy, thereby enhancing treatment effectiveness and reducing adverse reactions. Proteomics is increasingly crucial in liver cancer research, significantly contributing to a more profound understanding of the disease’s pathogenesis, diagnosis, and treatment [24, 25]. The advancement of proteomic technologies, such as mass spectrometry and protein microarray technology, furnishes powerful tools for the development of liver cancer drugs [26]. High-throughput screening and functional analysis of proteins identify liver cancer-specific drug targets, thereby promoting targeted drug development, improving therapeutic outcomes, and reducing adverse effects [27, 28]. Concurrently, proteomics is applied to study drug mechanisms of action, providing a scientific basis for improving existing drugs and developing new medications [29].

Liver cancer presents a significant threat to human health. Challenges in treating liver cancer and the limitations of current drugs require the development of novel therapeutic strategies and medications. Proteomics has shown significant potential in diagnosing, treating, and developing drugs for liver cancer. This review summarizes the research progress of proteomics in the development of drugs for liver cancer, emphasizing the role of proteomic technologies in identifying therapeutic targets and understanding drug mechanisms of action. This review, based on comprehensive literature analysis, summarizes the applications of proteomics in identifying biomarkers and drug targets for liver cancer, studying drug mechanisms of action, and drug development, as illustrated in Scheme 1. Conducting comprehensive research into the proteomic characteristics of liver cancer aims to develop more effective treatment interventions for patients.

Schematic illustration of Proteomics Efforts for hepatocellular carcinoma drug development

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Proteomic technologies, commonly employed in research, are classified as targeted proteomics and untargeted proteomics. Targeted proteomics uses methods to detect and quantify specific pre-selected proteins or protein groups [30, 31]. These methods include multiple reaction monitoring, selected reaction monitoring, and specific antibody detection techniques [32,33,34,35]. Targeted proteomics demonstrates high sensitivity and specificity, particularly suited for analyzing the expression, modification, and quantification of specific proteins [36, 37]. In developing drugs for HCC, targeted proteomics can identify protein biomarkers related to liver cancer and monitor the effects of drugs on these markers [38]. Untargeted proteomics is a method aimed at detecting, identifying, and quantifying as many proteins as possible within the proteome [39, 40]. Common methods comprise liquid chromatography-mass spectrometry, two-dimensional gel electrophoresis combined with mass spectrometry analysis, and other related techniques [41,42,43,44]. Untargeted proteomics can comprehensively reveal the expression of all proteins in a sample, aiding in the discovery of new disease-associated biomarkers or proteins, but it generally necessitates more extensive sample preparation and poses greater technical challenges. In developing therapies for HCC, untargeted proteomics could be utilized to uncover potential therapeutic targets or biomarkers and to elucidate the mechanisms of liver cancer pathogenesis [45].

E. El-Khateeb and colleagues conducted a comprehensive quantitative analysis of microsomal samples from human cirrhotic liver tissue. They utilized four distinct proteomic methodologies, including one targeted method and three label-free approaches, to assess the consistency of these techniques in determining variations in the abundance of drug-metabolizing enzymes and transporters. The results showed that standard-based label-free methods, such as the high three ion intensity Hi3 method and the intensity-based absolute quantification iBAQ method, exhibited decreased bias and improved precision compared to data obtained from targeted proteomics. Additionally, the study introduced the disease perturbation factor (DPF), which clarifies the diverse effects of cirrhosis on the expression patterns of proteins related to drug metabolism. The study concluded that the relative changes, as indicated by the DPF, remain consistent regardless of the specific quantification methodologies employed, and the DPF aids in understanding the variations in the expression patterns of enzymes and transporters linked to cirrhosis of various etiologies [46].

Bioinformatics tools are crucial for proteomics data analysis, encompassing database search algorithms, peptide identification software, protein quantification analysis software, and protein–protein interaction network analysis software. Bioinformatics aids researchers not only in identifying and quantifying proteins from complex mass spectrometry data, but also in elucidating protein functions and their interaction networks, thereby facilitating drug target discovery and validation [47]. X. Yi et al. conducted an extensive proteome analysis of HCC and intrahepatic cholangiocarcinoma (ICC) using data-independent acquisition (DIA) mass spectrometry. The analysis uncovered unique molecular signatures, such as aberrant lipid metabolism in HCC and activation of extracellular matrix-related pathways in ICC. The researchers created a panel of three proteins as a classifier, achieving an area under the curve of 0.92 and 90% accuracy in distinguishing ICC from HCC in a validation cohort. The findings provide fresh perspectives on the tumor biology of liver cancers and are advancing the development of more efficient diagnostic approaches and targeted drug treatments [48].

Although proteomics technology has yielded valuable insights into HCC within biomedicine, it encounters numerous technical challenges and limitations. These include sample preparation, technical complexities in protein extraction, and the intricate nature of the HCC proteome, all contributing to potential data interpretation variations. Additionally, the intricacy of bioinformatics analysis, the necessity for advanced algorithms, the integration of multi-omics data, and the standardization and reproducibility of proteomics research are pivotal in ensuring findings' applicability across diverse cohorts and studies. Furthermore, the substantial cost of proteomics analysis may hinder its widespread adoption, especially in resource-constrained environments. Addressing these challenges requires technological innovation and interdisciplinary collaboration to facilitate broader clinical applications and the formulation of personalized treatment strategies.

Proteomics technology is employed to compare protein expression levels between liver cancer patients and healthy controls. This strategy aims to identify proteins uniquely expressed in tumor states that could serve as specific biomarkers for early diagnosis, treatment, and prognosis of liver cancer, thereby establishing a new foundation for therapeutic strategies against the disease. The primary targets and their potential applications in liver cancer development are listed in Table 1.

Discovery of early diagnostic biomarkers

HCC represents the primary histological subtype of liver cancer, constituting around 90% of instances. It ranks as the third leading cause of cancer-related death globally, predominantly linked to chronic cirrhosis resulting from hepatitis viruses and metabolic disorders [1, 2]. Due to the absence of early symptoms, most HCC patients receive diagnoses at an advanced clinical stage, resulting in a bleak prognosis. Existing diagnostic approaches for HCC consist of imaging examinations along with serum protein biomarkers such as alpha-fetoprotein (AFP), PIVKA-II, and histopathology. Nevertheless, these methods encounter challenges in precisely diagnosing early-stage HCC due to limitations in expertise, sensitivity, and reliance on invasive testing procedures [49].

Proteomic analysis plays a pivotal role in identifying early diagnostic biomarkers for liver cancer. HCC is an exceptionally malignant tumor, emphasizing the criticality of early diagnosis in improving patient survival and treatment efficacy. Given the frequently subtle clinical symptoms in early-stage liver cancer, the development of effective early diagnostic biomarkers is imperative for enhancing the disease prognosis. Proteomic technologies, especially mass spectrometry, are utilized to discover biomarkers for early-stage liver cancer. Through the comparison of serum or tissue samples from liver cancer patients and healthy controls, researchers can identify differentially expressed proteins that may serve as indicators of early-stage disease [50].

Serum proteomics is a major focus for exploring early diagnostic biomarkers in liver cancer. Through the analysis of serum samples from liver cancer patients, researchers can pinpoint specific proteins or protein patterns linked to liver cancer. Proteomic investigations have revealed additional potential serum protein biomarkers, highlighting improved prospects for early cancer diagnosis [51]. M. Xu and colleagues conducted comprehensive serum proteomic analysis of samples from healthy individuals and patients with hepatitis B-related liver diseases, delineating a trajectory of liver cancer development (Fig. 1A). Observed changes in biological processes associated with cancer identified potential therapeutic targets. Biomarker panels developed using machine learning demonstrated superior performance in detecting HCC compared to the traditional AFP marker [52]. X. Xing and colleagues employed a mass spectrometry-based proteomics approach to carry out a phased discovery-verification-validation workflow involving 1,002 individuals, focusing on the early diagnosis of HCC (Fig. 1B). Leveraging a machine learning model, they discovered the P4 protein biomarker panel, comprising HABP2, CD163, AFP, and PIVKA-II. The P4 panel accurately discriminates HCC patients from individuals with liver cirrhosis and healthy subjects, surpassing current clinical prediction strategies in an independent validation cohort. The P4 panel can also accurately forecast the transition from LC to HCC and HCC diagnosis with a median lead time of 11.4 months before imaging diagnosis [53]. J. Jia and colleagues identified SPINK1 as a potential biomarker for early detection, targeted therapy, and prediction of response to immune checkpoint blockade therapy in liver cancer. Elevated SPINK1 expression positively correlates with the tumor’s immune microenvironment and the expression of immune checkpoint molecules [54]. These findings suggest that employing proteomics-driven serum biomarker discovery confers substantial benefits for liquid biopsy, considerably enhancing the potential for early HCC diagnosis.

A The serum proteomes of patients with liver disease were analyzed using the comprehensive serum proteome mapping platform known as In-Depth Serum Proteome Mapping (ID-Map). Reproduced with permission [52]. Copyright 2023, Elsevier. B Large-scale DIA-based proteomics was employed to identify potential biomarkers associated with HCC, subsequently validating these candidates in an independent cohort using a PRM-based targeted proteomic approach. Utilizing machine learning, diagnostic models were developed for HCC and evaluated their effectiveness in predicting HCC risk through long-term prospective follow-up of patients with LC. Reproduced with permission [53]. Copyright 2023, Springer Nature. DIA-MS, data-independent acquisition mass spectrometry; HC, healthy control; HCC, hepatocellular carcinoma; LC, liver cirrhosis

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Tissue proteomics research focuses on analyzing alterations in protein expression in cancer tissues, facilitating the identification of specific proteins linked to the progression of cancer by comparing the protein expression variances between cancerous and normal tissues [55]. W. Naboulsi et al. conducted a quantitative analysis of tissue proteomics to investigate HCC, specifically focusing on identifying potential biomarkers for early-stage diagnosis. By examining 50 patient samples and utilizing label-free discovery analysis and targeted selected reaction monitoring, they detected the overexpression of proteins including ATP-dependent RNA helicase (DDX39), Fibulin-5, myristoylated alanine-rich C-kinase substrate, and Serpin H1 in HCC. Significantly, their study unveiled the association of versican with well-differentiated and low-stage HCC for the first time, indicating its potential as an early diagnostic biomarker. These results underscore the significance of distinct proteins in HCC development and their prospective clinical value in enhancing early detection and treatment efficacy [56].

The modification and interaction of proteins represent crucial elements in the early diagnosis of liver cancer. Proteomic technologies uncover the phosphorylation and acetylation statuses of proteins, and alterations in these modifications are intricately linked to the origin and advancement of liver cancer. M. Kohansal-Nodehi et al. conducted an extensive analysis of the proteomic landscapes in HCC and CCA using DIA mass spectrometry. They analyzed plasma samples from patients with liver diseases, early-stage HCC, and late-stage HCC, identifying abnormal lipid metabolism as a characteristic of HCC, while the activation of extracellular matrix-related pathways distinguished CCA. The team developed a three-protein classifier capable of distinguishing CCA from HCC, achieving 90% accuracy in an independent validation cohort, with an area under the curve of 0.92. These findings offer new insights into the molecular features of these liver cancers and suggest the potential effectiveness of glycobiomarkers for early HCC diagnosis [57]. D. Li and colleagues conducted an extensive glycoproteomic analysis of urinary EVs using mass spectrometry to identify potential biomarkers for diagnosing HCC. Their analysis of 756 intact N-glycopeptides derived from 107 N-glycoproteins revealed 344 differentially expressed intact N-glycopeptides. These included upregulated glycoproteins LG3BP, PIGR, and KNG1, as well as downregulated ASPP2 in HCC-derived EVs compared to normal controls. The results suggest that these specific glycoforms could serve as effective non-invasive biomarkers for diagnosing HCC [58].

Proteomics has shown significant potential in identifying early diagnostic markers for liver cancer. Using mass spectrometry analysis, protein chip technology, and bioinformatics tools, researchers have pinpointed specific proteins and protein patterns linked to liver cancer. These discoveries play a crucial role in improving the early diagnosis rate of liver cancer and also open up possibilities for personalized disease treatment. Future research should focus on advancing proteomic technologies to improve their accuracy and reliability in clinical settings.

Discovery of prognostic assessment biomarkers

Assessing specific clinical indicators in patients makes it possible to predict their postoperative survival duration or their response to specific drug therapies. Insights from prognostic studies furnish essential evidence for guiding clinical decisions and aiding in the discovery and evaluation of innovative treatment approaches for patients. Developments in proteomic technologies have yielded new perspectives and advanced tools for evaluating liver cancer prognosis. Mass spectrometry-based proteomic methods notably enrich the depth and range of serum and plasma protein analysis, making them superior tools for identifying biomarkers. Q. Gao and colleagues utilized an integrated proteogenomic analysis approach to investigate the association between HCC and the hepatitis B virus (HBV), using a cohort of 159 patients with paired tumor and adjacent liver tissue samples (Fig. 2). They identified distinct subgroups with unique characteristics in metabolic reprogramming, microenvironmental dysregulation, cell proliferation, and traits indicating potential therapeutic targets. The study pinpointed two significant prognostic biomarkers, PYCR2 and ADH1A, which are linked to the observed metabolic reprogramming in HCC. This includes distinct signaling and metabolic profiles associated with mutations in CTNNB1 and TP53, with a specific focus on the role of ALDOA phosphorylation in enhancing glycolysis and cell proliferation within CTNNB1-mutated cells. This research significantly contributes to the understanding of HBV-related HCC and has the potential to influence clinical practice [59]. W. Wang and colleagues uncovered aberrant expression of the centrosome and spindle pole-associated protein (CSPP1) in 27 types of cancer through bioinformatics analysis. This aberration is strongly associated with ferroptosis and the tumor microenvironment. The study has recognized CSPP1 as a potential diagnostic and prognostic biomarker and proposed it as a fresh target for ferroptosis-based drug therapy and immunotherapy. The research findings underline the multifaceted role of CSPP1 in cancer treatment across various cancer types, paving the way for novel clinical applications [60]. L. Liu and colleagues, through comprehensive sample analysis and cellular experiments, have found that the AARS2 gene is upregulated in various cancers, particularly in liver cancer. The expression of AARS2 is influenced by copy number variations and is associated with tumor growth and migration capabilities. AARS2 holds potential as a new biomarker for predicting prognosis and immune therapy responses. Additionally, the study illustrated the gene’s link to the sensitivity of several anti-cancer drugs, offering fresh insights for cancer treatment strategies [61].

Proteomic and genomic analysis was conducted on 159 patients with hepatitis B virus (HBV)-related hepatocellular carcinoma, leading to the identification of protein subgroups linked to clinical and molecular characteristics. And two prognostic biomarkers (CTNNB1 and TP53) were discovered, which are associated with metabolic reprogramming, microenvironment dysregulation, cell proliferation, and potential therapeutic applications. Reproduced with permission [59]. Copyright 2019, Elsevier

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Protein modifications and interactions are essential for the prognostic evaluation of liver cancer. Proteomics technologies enable the identification of protein phosphorylation, acetylation, and other modifications, which may be closely linked to the prognosis of liver cancer. For example, studies have demonstrated that the phosphorylation levels of specific proteins are markedly elevated in patients with liver cancer, suggesting their potential as prognostic markers. K. Wu and colleagues discovered that the phosphorylation of the ubiquitin-like with PHD and ring finger domains (UHRF2) protein promotes HBV replication and tumor malignancy in HBV-associated HCC. The Hepatitis B virus X protein (HBx) modulates the phosphorylation of UHRF2 through the miR-222-3p-ETS1-CDK2 axis, revealing a new role for UHRF2 as a potential prognostic indicator [62]. Z. Guo and colleagues identified elevated RNF149 expression in HCC and its role in promoting tumor progression via E3 ubiquitin ligase activity. This was determined through mass spectrometry analysis and clinical sample studies, suggesting RNF149’s potential as a prognostic marker and therapeutic target for HCC treatment [63]. H. Wei and colleagues conducted multi-omics research to elucidate the role of the long non-coding RNA PAARH in HCC progression and angiogenesis. They found that PAARH promotes HCC progression and angiogenesis by upregulating HOTTIP and activating the HIF-1α/VEGF signaling pathway, suggesting PAARH’s potential as a prognostic biomarker and therapeutic target [64]. J. Wang and colleagues conducted a comprehensive study revealing that the protein FZD10, when activated by METTL3-mediated mRNA methylation, enhances the self-renewal and metastatic capabilities of liver cancer stem cells through the WNT/β-catenin and Hippo pathways. The research indicates that FZD10 expression levels are associated with lenvatinib resistance and poor outcomes in HCC, suggesting its potential as a prognostic indicator and therapeutic target (Fig. 3) [65]. Z. Tang and colleagues investigated the effects of enhancer region demethylation on the expression of MCM2 and NUP37 by analyzing datasets from the Cancer Genome Atlas (TCGA) and the Network of Oncogenomics in Denmark (NODE), and by performing real-time polymerase chain reaction and immunoblotting on samples from human HCC. They observed that overexpression of MCM2 and NUP37 in HCC patients correlates with a less favorable prognosis. These findings suggest that demethylation in the enhancer region markedly increases the expression levels of MCM2 and NUP37, potentially offering novel biomarkers for predicting prognosis in HCC patients [66].

Schematic model of the mechanism underlying FZD10-drived expansion of liver CSCs and lenvatinib resistance. Reproduced with permission [65]. Copyright 2023, Elesvier

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The integration of multi-omics approaches, spanning genomics, transcriptomics, proteomics, and metabolomics, provides a more comprehensive perspective for assessing the prognosis of liver cancer. This integration enables researchers to gain a deeper understanding of the mechanisms driving the development and progression of liver cancer, leading to the identification of novel prognostic markers. These discoveries not only improve the precision of prognosis assessment in liver cancer but also open avenues for personalized therapy. Subsequent enhancement of proteomic technologies in future research is crucial for improving clinical accuracy and reliability.

Discovery of therapeutic response and drug resistance biomarkers

Proteomic studies have revealed the mechanisms governing the sensitivity and resistance of liver cancer cells to chemotherapy and targeted therapies. By examining differential protein expression before and after treatment, researchers can identify pivotal proteins influencing treatment response and propose innovative therapeutic strategies to overcome drug resistance. S. Ji and collaborators established the Liver Cancer Organoid Biobank (LICOB) comprising 65 human liver cancer organoids. They conducted comprehensive multi-omics analyses, encompassing genomic, epigenomic, transcriptomic, and proteomic profiling, alongside high-throughput drug screening to investigate liver cancer biomarkers and precision therapy. The results illustrated that LICOB accurately mirrors the molecular characteristics of the original tumor tissues and effectively predicts drug responses through multi-omics feature analysis. Moreover, integrating LICOB’s pharmaco-proteogenomics data enabled the prediction of potential drug combinations, offering personalized treatment guidance. The study validated the synergistic inhibitory effects of the mTOR inhibitor temsirolimus and the multi-targeted tyrosine kinase inhibitor lenvatinib in these organoids and their corresponding patient-derived xenograft models. This research furnishes a valuable resource for studying liver cancer biology and pharmacological dependencies, potentially advancing functional precision medicine [23]. T. Han and colleagues, employing an extensive array of experimental methodologies and patient cohort analyses, identified that miR-183-5p.1 targets MUC15, regulating the c-MET/PI3K/AKT/SOX2 signaling pathway, consequently impacting the characteristics of liver cancer stem cells and tumorigenesis. Moreover, the expression levels of MUC15 are associated with the responsiveness of liver cancer cells to lenvatinib treatment, indicating MUC15 as a potential biomarker and therapeutic target for liver cancer [67].

The realm of proteomics has shown substantial potential in uncovering biomarkers that indicate therapeutic responses and drug resistance in liver cancer. Utilizing multi-omics approaches, including genomics, transcriptomics, proteomics, and metabolomics, provides a more comprehensive and holistic perspective for prognostic assessment in liver cancer.

Identifying drug targets through signal transduction pathway analysis

Proteomic research plays a crucial role in identifying drug targets for liver cancer, particularly through the analysis of signal transduction pathways. Employing mass spectrometry and protein microarray technology, researchers can attain a comprehensive understanding of the signal transduction mechanisms within liver cancer cells. This method offers valuable insights into the molecular foundations of liver cancer, thus aiding the development of targeted therapies. Some critical targets with potential application are listed in the Table 2.

M. Fujita and colleagues conducted a proteomic investigation of around 300 proteins in 259 cases of primary liver cancer, primarily characterized by HCV and HBV positivity. Through the integration of proteomic data, the researchers illustrated correlations between TP53 and CTNNB1 mutations in liver cancer and distinct protein expression profiles. Additionally, the study identified 22 potential prognostic biomarkers, such as CDK1 and CDKN2A. These findings have the potential to contribute to precise classification and the development of targeted therapeutic strategies for liver cancer [68]. J. Peng and colleagues investigated the impact of the CAMK2N1 gene on the development of human HCC, discovering that CAMK2N1 expression is downregulated in 47% of HCC cases and is associated with a poorer prognosis. Moreover, the study revealed that CAMK2N1 inhibits tumor growth by suppressing E2F1-mediated cell cycle signaling. Comparative proteomics analysis indicated that silencing CAMK2N1 disrupts genes regulated by E2F1 at the transcriptional level. Additionally, the study identified 22 potential prognostic markers, inclusive of CDK1 and CDKN2A. These findings offer insights for precise classification and targeted therapy for liver cancer [69]. G. Chan and colleagues investigated genetic alterations activating protein kinase A (PKA) in various tumors utilizing global phosphoproteomics and kinase activity profiling. The researchers identified two PKA downstream signaling networks: RAS/MAPK and Aurora Kinase A (AURKA)/glycogen synthase kinase (GSK3), both influencing MYC oncoproteins. The study demonstrated PKA’s primary impact on MYC translation, which could be inhibited by the eIF4A inhibitor zotatifin, resulting in reduced c-MYC expression and cell growth in vitro. Targeting PKA’s influence on translation represents a potential therapeutic strategy for PKA-driven cancers [70]. L. Chibaya and colleagues discovered that Akt is frequently activated in cancer cells, demanding heightened antioxidant activity to counteract the increased oxidative stress stemming from elevated metabolic levels. Mutations at the Mdm2 Ser183 site impede Akt phosphorylation at this location, amplifying p53-mediated aging, thereby impeding tumorigenesis [71]. M. Ayesha and colleagues identified that overexpressing miR-4521 while silencing FAM129A reduces the levels of phosphorylated FAK and AKT, eliciting anti-proliferative and anti-metastatic effects on HCC cells. This strategy holds promise for liver cancer treatment [72]. A. Lepore and colleagues discovered that JNK kinase directly binds to and phosphorylates the Ser115 site of PIN1, thereby preventing its monoubiquitination and subsequent proteasomal degradation at Lys117. This mechanism fosters the growth of ICC, implying that inhibiting PIN1 to target JNK activation could offer a viable treatment strategy for ICC [73]. T. Zhang and colleagues discovered that increased levels of E-twenty-six transformation-specific variant 1 (ETV1), mediated by hepatocyte growth factor, facilitate HCC metastasis by up-regulating protein tyrosine kinase 2 and c-MET. Modulating this signaling pathway may offer a prospective therapeutic approach for HCC cases overexpressing ETV1 [74]. H. Li and colleagues assessed the expression levels of receptor-interacting serine/threonine kinase 4 (RIPK4) in samples from HCC patients, investigating the correlation between RIPK4 expression and the clinical pathological characteristics of HCC. Furthermore, they revealed a connection between RIPK4 and the prognosis of HCC patients, demonstrating that RIPK4 influences HCC invasion and metastasis through the epithelial-mesenchymal transition (EMT) and the signal transducer and activator of transcription 3 (STAT3) pathways. Consequently, targeting the RIPK4-STAT3 axis presents a potential therapeutic strategy for inhibiting postoperative recurrence and metastasis of HCC [75]. F. Cheng and colleagues investigated the correlation between chronic HBV positivity and ubiquitin-like with PHD and ring finger domains 2 (UHRF2) expression levels in HCC tissues. Their findings indicate that the X protein (HBx)-ETS1-CDK2-UHRF2 signaling pathway significantly contributes to the pathogenesis of HBV-associated HCC, potentially paving the way for a novel therapeutic target in the treatment of human HCC [76]. L. Lin and colleagues discovered that F box and WD repeat domain containing 10 promotes the phosphorylation and ubiquitination of the Ser151 site of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), thereby activating the NF-κB signaling pathway and facilitating the progression of male HCC. It was further observed that the GAPDH inhibitor, koningic acid, can impede this process, potentially presenting a novel therapeutic approach for treatment [77]. S. Wang and colleagues developed a liver-specific formimidoyltransferase cyclodeaminase (FTCD) gene knockout mouse model to investigate the role of FTCD in HCC using multi-omics analysis. They observed high expression of FTCD in normal liver but reduced levels in HCC. The absence of FTCD resulted in the upregulation of peroxisome proliferator-activated receptor (PPAR)γ and sterol regulatory element–binding protein 2 (SREBP2) through the PTEN/Akt/mTOR signaling pathway, leading to hepatic lipid accumulation and the progression of HCC. These findings advocate the potential of targeting the Akt/mTOR pathway as a therapeutic strategy for HCC patients with diminished FTCD expression [78]. Y. Yang and colleagues conducted an investigation into the role of GPR109A in liver cancer progression, utilizing both human samples and murine models. Their study revealed an association between a glutamine-deficient tumor microenvironment and the facilitation of immunosuppressive GPR109A⁺ myeloid cell infiltration, thereby allowing cancer cells to evade immune detection. The findings showcase that targeting GPR109A has the potential to disrupt this immunosuppressive mechanism, thus enhancing the effectiveness of cancer immunotherapy. This highlights the prospect of GPR109A serving as a novel immunometabolic checkpoint and therapeutic target [79]. Y. Takeba and colleagues discovered an elevation in the expression of Interleukin-16 (IL-16) in HCC tissues. This heightened expression was linked to the promotion of HCC cell proliferation via the ERK signaling pathway, shedding light on the potential role of IL-16 in HCC progression [80]. Z. Hu et al. revealed an elevated level of circular RNA circCCAR1 in the plasma exosomes of patients with HCC. The circCCAR1 molecule promotes the growth and metastasis of HCC cells by establishing a positive feedback loop of circCCAR1/miR-127-5p/WTAP. Additionally, exosomal circCCAR1 can be absorbed by CD8⁺ T cells, leading to T cell dysfunction and enhancing resistance to anti-PD-1 therapy. These findings offer new targets for HCC immunotherapy [81].

Various methods are employed in these studies, encompassing proteomic research and genetic as well as animal models, yielding profound insights into the molecular intricacies of liver cancer. Consequently, they establish the groundwork for pioneering therapeutic targets and precise interventions, presenting significant potential for transforming liver cancer treatment and patient prognostication.

Identifying drug targets through protein–protein interaction networks

The analysis of protein–protein interaction networks stands as a vital application of proteomics for the identification of potential drug targets in liver cancer. By delving into the interactions among proteins, researchers can pinpoint pivotal protein complexes that play a significant role in driving the progression of liver cancer.

K. Yuan and colleagues discovered TLNC1 as a potential oncogenic long non-coding RNA in liver cancer. TLNC1 stimulates the proliferation and spread of liver cancer cells in both in vivo and in vitro settings. By interacting with TPR, TLNC1 triggers TPR-mediated translocation of p53 from the nucleus to the cytoplasm, consequently impeding the transcription of p53 target genes and fostering the advancement of liver cancer. Hence, the TLNC1-TPR-p53 axis stands as a promising target for the development of liver cancer therapies [82]. The RNA-binding protein YTHDF1, which recognizes N6-methyladenosine, is implicated in cancer-related mechanisms. In their study, L. Wang and colleagues illustrated that YTHDF1 fosters the development of NASH-HCC by engaging EZH2-IL-6 signaling. The mobilization and stimulation of myeloid-derived suppressor cells (MDSCs) by YTHDF1 result in impaired function of cytotoxic CD8⁺ T-cells. Consequently, YTHDF1 emerges as a plausible therapeutic target for addressing NASH-HCC (Fig. 4A) [83]. Z. Li and colleagues demonstrated that the antisense long non-coding RNA LINC00624 promotes the transcriptional activation of chromodomain-helicase-DNA-binding protein 1-like (CHD1L) and B-cell CLL/lymphoma 9 protein (BCL9), thus bolstering the interaction between histone deacetylase 6 (HDAC6) and tripartite motif containing 28 (TRIM28). This disrupts the HDAC6-TRIM28-ZNF354C (ZNF354C, zinc finger protein 354C) transcriptional co-repressor complex, consequently contributing to the progression of HCC. The findings advocate LINC00624 as a potential therapeutic target for managing HCC [84]. The study by D. Du and colleagues revealed that the RNA-binding motif protein 45 (RBM45) augments the stability of the alanine-serine-cysteine transporter 2 (ASCT2) through direct binding, consequently advancing the progression of HCC. Furthermore, the research demonstrated that AG-221, a drug used in the treatment of acute myeloid leukemia, disrupts the interaction between RBM45 and ASCT2, leading to reduced stability of ASCT2 protein and inhibition of HCC progression. These findings not only suggest novel therapeutic approaches for HCC but also advocate for the RBM45-ASCT2 axis as a promising target for drug development [85]. A. Konopa and colleagues’ research suggests that lysophosphatidic acid receptor 1 (LPAR1) stimulates the activation of myocardin-related transcription factor A (MRTF-A) through the promotion of Filamin A (FLNA) phosphorylation, thereby contributing to the progression of HCC. Targeting this pathway might present a new and promising therapeutic approach for treating HCC [86]. G. Carpino et al. revealed that within the tumor microenvironment of ICC, the proteins THBS1, THBS2, and PEDF play a role in inhibiting blood vessel formation and promoting lymphangiogenesis. Modulating these proteins holds promise for therapeutic benefits in treating the disease [87]. W. Tian and colleagues conducted an analysis of TCGA data aimed at identifying lysosome-related genes linked to HCC progression. Their work led to the identification of PPT1, a gene that boosts HCC cell proliferation and plays a role in macromolecular protein metabolism. The results indicate PPT1 as a promising therapeutic target for HCC and offer new prognostic biomarkers for this condition [88]. J. Wang et al. investigated the anti-tumor impact of ZCJ14 using in vitro experiments and in vivo nude mouse models, revealing its significant capacity to suppress the growth of various cancer cells with minimal toxicity. Notably, ZCJ14 exhibits anti-tumor potential through the enhancement of steroid synthesis and the inhibition of ubiquitin-mediated protein degradation, rather than via the conventional EGFR pathway [89].

A YTHDF1 promotes NASH-HCC tumorigenesis via EZH2-IL-6 signaling, which recruits and activates MDSCs to cause cytotoxic CD8⁺ T-cell dysfunction. YTHDF1 may be a novel therapeutic target to improve responses to anti-PD-1 immunotherapy in NASH-HCC. Reproduced with permission [83]. Copyright 2023, Elesvier. B Mechanism diagram of NF-κBp65 phosphorylation drivies HCC. Inflammation, such as TNF-α, induces ARRB1 and promotes the binding of ARRB1 to NF-κBp65, facilitating the phosphorylation of p65 at the ser536 site. Subsequently, the phosphorylation of p65 (Ser536) activates the Akt/mTOR signaling pathway, driving malignant hepatocyte proliferation, leading to hepatocellular carcinoma transformation. Reproduced with permission [94]. Copyright 2021, Springer Nature

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Researching protein–protein interaction networks has emerged as a critical facet of proteomics in the quest for potential therapeutic targets in liver cancer. These comprehensive studies collectively yield abundant prospects for developing precise interventions in liver cancer treatment.

Identifying drug targets through functional proteomics research

The analysis of protein expression and function via functional proteomics yields vital insights for discerning drug targets in liver cancer. HCC accounts for approximately 80% of liver cancer cases and stands as a significant contributor to cancer-related mortality. Diverse treatment modalities for liver cancer encompass local therapy, systemic chemotherapy, hormone therapy, molecular targeted therapy, immune checkpoint therapy, surgical resection, and liver transplantation. Nonetheless, the efficacy of these treatments is often compromised by drug side effects, high recurrence rates, and low response rates, ultimately reducing the likelihood of achieving a cure for liver cancer. Post-translational modifications notably influence protein function, encompassing alterations such as phosphorylation, acetylation, methylation, and ubiquitination present in nearly all proteins. Studies consistently demonstrate an association between protein post-translational modifications (PTMs) and both the onset and progression of cancer, presenting innovative pathways for pinpointing therapeutic targets in cancer treatment [90].

Identifying drug targets through phosphoproteomics

Phosphorylation represents a crucial form of protein modification, involving the reversible transfer of phosphate groups to amino acid residues by protein kinases and their removal by protein phosphatases. This modification can induce structural and functional alterations in proteins and influence their interactions with other molecules, thereby initiating specific signaling pathways. Consequently, phosphorylation impacts the behavior of subsequent molecules, ultimately playing a role in the initiation, proliferation, migration, and invasion of tumors.

The study by Y. Jiang and colleagues employed proteomic and phosphoproteomic profiling to investigate the clinical characteristics of early-stage HCC linked with HBV infection, analyzing 110 matched pairs of tumor and non-tumor tissue samples. The quantitative proteomic data identified the heterogeneity within early-stage HCC, categorizing patients into three distinct subtypes (S-I, S-II, and S-III), each demonstrating unique clinical outcomes. Notably, subtype S-III, characterized by disrupted cholesterol homeostasis, is associated with the lowest overall survival rates and poses the highest risk of an adverse prognosis post-initial surgery. The research revealed that suppression of sterol O-acyltransferase 1 (SOAT1), notably overexpressed in the S-III subtype, disrupts cellular cholesterol distribution and significantly impedes HCC proliferation and migration. Ultimately, in a patient-derived tumor xenograft mouse model, the administration of avasimibe, an SOAT1 inhibitor, led to a substantial reduction in tumor size in individuals exhibiting elevated SOAT1 expression. The proteomic classification of early-stage HCC outlined in this study yields valuable insights into the fundamental tumor biology and underscores potential approaches for personalized therapeutics [91]. D. Chen and colleagues investigated the regulatory impact of the protein phosphatase PPM1G on the phosphorylation status of the alternative splicing factor SRSF3 in HCC. The findings revealed that PPM1G influences the alternative splicing patterns of cell cycle-related genes through the dephosphorylation of SRSF3. Additionally, the research demonstrated that the expression levels of PPM1G are modulated by various transcription factors and co-activators. These discoveries highlight the crucial role of PPM1G in HCC and provide new insights into the regulation of alternative splicing in the context of malignant transformation [92]. The study by B. Wang and colleagues examined the effects of Saikosaponin-d (SSd) on autophagy and radiosensitivity in HCC cells, elucidating the underlying molecular mechanisms. The research demonstrated that SSd induces autophagy in HCC cells by inhibiting mTOR phosphorylation and enhances radiation-induced apoptosis, suggesting a potential strategy for radiosensitization therapy in liver cancer [93]. The study by X. Xu and colleagues evaluated the expression levels of NF-κBp65 and its phosphorylated form in liver tissue samples, employing liver-specific p65 knockout mice to investigate the roles of p65 and phosphorylated p65 (p-p65) in liver cancer and their ARRB1-mediated pathways. The findings indicated that the phosphorylation of NF-κBp65 drives inflammation-mediated liver cancer development, and its inhibition can mitigate liver cancer, suggesting its potential as a novel therapeutic target for HCC (Fig. 4B) [94]. C. Jiang and colleagues discovered that SUMOylation and phosphorylation significantly influence the activity of NF-κBp65, particularly in promoting HCC cell survival and invasion. This finding reveals the oncogenic mechanism of p65 in HCC [95]. C. Sun and colleagues demonstrated that Src enhances PARP1 synthesis. Moreover, the combined application of Src and PARP1 shows potent anti-tumor effects with minimal side effects. Pharmacological inhibition of Src-mediated tyrosine phosphorylation of PARP1 may provide a therapeutic strategy for PARP1-resistant HCC patients [96].

These studies collectively enhance our comprehension of the molecular landscape of HCC, highlighting the crucial roles of protein phosphorylation modifications in tumor behavior and treatment responses. The findings emphasize the significance of targeted therapies that leverage specific molecular pathways, providing promise for more effective and personalized treatment strategies for patients with HCC.

Identifying drug targets through proteomic studies of acetylation

Protein acetylation encompasses the modification of both histone and non-histone proteins. Currently, histone acetylation has been studied more extensively than non-histone acetylation in the context of liver cancer. A delicate balance between histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulates histone acetylation levels. While HDAC-mediated deacetylation of histones results in gene silencing, HAT-mediated acetylation is linked to gene activation. Abnormal acetylation patterns in proteins have been observed in HCC tissues, with these patterns correlating with clinical stage, prognosis, and overall survival.

T. Zhang et al. investigated the role of general control of amino acid synthesis 5 like 1 (GCN5L1) in glutaminolysis and HCC. The ablation of GCN5L1 in mice accelerated HCC development, whereas its mitochondrial restriction mitigated HCC progression upon exposure to carcinogens. GCN5L1 depletion promoted tumor growth through mTORC1 activation. The study highlighted the greater significance of glutaminase 2 (GLS2) over glutaminase 1 (GLS1) in promoting HCC, emphasizing the crucial role of GLS2 in HCC pathogenesis [97]. Y. Yuan et al. revealed that H2A.Z functions as an oncogene in HCC. LincZNF337-AS1 specifically binds to H2A.Z and KAT5 at distinct sites, facilitating H2A.Z acetylation via KAT5. This molecular interaction enhances the transcription of downstream genes, influencing the proliferation, metastasis, apoptosis, and cell cycle of liver cancer cells [98]. F. Gao et al. discovered that testicle-specific protease 50 (TSP50) acts as an oncogene by promoting the Warburg effect and hepatocyte proliferation through increased acetylation of pyruvate kinase M2 (PKM2), a pivotal enzyme in aerobic glycolysis. Therefore, disrupting the acetylation site of PKM2 could potentially inhibit tumor formation in vivo [99]. W. Yang et al. demonstrated that histone deacetylase 8 (HDAC8), an isoform of histone H3 lysine 27 (H3K27), is overexpressed in various human cancers. Downregulation of HDAC8 increases global acetylation of H3K27 as well as enhancer acetylation, thereby reactivating T cell-transport chemokines produced by HCC cells. This mechanism alleviates T cell rejection in immunodeficient and humanized mouse models. Consequently, selective HDAC8 inhibitors may enhance the antitumor effect in HCC [100].

These studies offer valuable insights into the role of acetylation in HCC, highlighting the significance of acetylation-related proteins and enzymes as potential therapeutic targets. The findings enhance our understanding of cancer biology and lay the groundwork for developing targeted treatments based on acetylation modulation.

Identifying drug targets through proteomic studies of lactylation

Lysine lactylation (Kla) originates from intracellular lactate. Research demonstrates that Kla occurs on core histones in a p300-dependent manner and facilitates the epigenetic activation of M2-like gene expression in macrophages upon bacterial challenge, contributing to the preservation of immune homeostasis. Considering the elevated glycolysis and accumulation of intracellular lactate in cancer cells, Kla is likely to play a substantial role in cancers, including liver cancer.

Z. Yang et al. collected and analyzed tumor and adjacent liver samples from 52 patients with HBV-related HCC (HBV-HCC), conducting comprehensive lactylome and proteome analyses using mass spectrometry. They identified 9,275 lactylation sites, most of which were on non-histone proteins. The findings revealed that Kla plays a crucial role in regulating cellular metabolic pathways in HCC, particularly by inhibiting adenylate kinase 2 (AK2) activity, which facilitates tumor cell proliferation and metastasis. The lactylation level of AK2 correlates with poor prognosis in HCC patients, suggesting a novel therapeutic target [101]. L. Bi and colleagues investigated the regulatory effects of HDAC11 on the promoter region and protein expression of LKB1 using techniques such as mass spectrometry. Their results indicate that high HDAC11 expression is associated with poor prognosis in HCC, whereas HDAC11 deficiency reduces tumorigenesis and extends survival. HDAC11 inhibits glycolysis by modulating the LKB1/AMPK signaling pathway, which affects cancer stem cell properties and HCC progression. Additionally, HDAC11 overexpression reduces the sensitivity of HCC to sorafenib, suggesting that it could serve as a potential target for HCC treatment and for overcoming drug resistance [102]. J. Jin and colleagues employed SILAC quantitative proteomics and crystallographic studies to investigate the regulatory impact of SIRT3 on Kla in HCC cells. Their research identified cyclin E2 (CCNE2) as a key substrate for SIRT3-mediated delactylation and elucidated a mechanism by which CCNE2 lactylation influences HCC cell growth. The study suggests that activation of SIRT3 by Honokiol could induce cell apoptosis and inhibit HCC progression in vivo, positioning SIRT3 as a promising target for HCC treatment [103]. L. Pan et al. investigated the effects of demethylzeylasteral (DML) on liver cancer stem cells (LCSCs) using RNA sequencing, GC–MS analysis, and mouse xenograft models. The study found that DML suppresses LCSC tumorigenicity by inhibiting histone lactylation, identifying lactate as a key molecular target. DML’s interference with the glycolysis pathway and subsequent reduction in lactate levels led to decreased H3 histone lactylation, thereby reducing LCSC proliferation and migration while promoting apoptosis. These findings suggest that DML has potential as an adjunct treatment for HCC by modulating lactate and histone lactylation [104].

These studies provide valuable insights into the molecular mechanisms underlying HCC progression and offer promising avenues for developing targeted therapies to improve patient outcomes. The exploration of lactylation and its regulatory pathways opens new frontiers in understanding and treating liver cancer, underscoring the importance of targeting metabolic and epigenetic modifications in cancer therapy.

Identifying drug targets through methylation proteomics research

Histone methylation, which involves the addition of methyl groups to specific residues on histone proteins, alters their epigenetic state. This modification is implicated in modulating antiviral immune responses and promoting resistance to apoptosis, thereby potentially facilitating the progression of liver cancer. In a study conducted by J. Gao and colleagues, it was found in clinical samples and experimental mouse models that reduced expression of betaine-homocysteine methyltransferase (BHMT) correlates with the malignant phenotypes of HCC and indicates poorer patient prognosis. BHMT inhibits the activity of glucose-6-phosphate dehydrogenase (G6PD) by regulating its arginine methylation levels, which may impede the progression of HCC and suggest novel therapeutic strategies for its management [105]. A study by V. Ortiz-Barahona and colleagues revealed through experiments and data analysis that the epigenetic silencing of the RNA methyltransferase NSUN7 in liver cancer is associated with poorer patient prognosis. NSUN7 introduces 5-methylcytosine modifications that stabilize CCDC9B mRNA. Depletion of NSUN7 leads to mRNA destabilization and increased levels of the MYC-regulating protein IVNS1ABP, which enhances the sensitivity of liver cancer cells to bromodomain inhibitors. Furthermore, the study showed that elevated methylation of the NSUN7 promoter in primary liver cancer correlates with reduced overall survival times [106]. These findings underscore the importance of methylation-related modifications in liver cancer biology. Integrating methylation proteomics into cancer research provides promising insights into the molecular mechanisms underlying liver cancer. The studies presented highlight how epigenetic modifications influence the progression of liver cancer and impact patient survival rates, with implications for developing targeted therapies to improve outcomes. Further research into these epigenetic regulators could yield new strategies for predicting disease progression and enhancing treatment effectiveness.

Identifying drug targets through ubiquitination proteomics research

Protein ubiquitination, a multifunctional post-translational modification, is notably acknowledged for its involvement in guiding protein degradation using the ubiquitin–proteasome system. The ubiquitin ligase is implicated in tumorigenesis through diverse mechanisms.

F. Ji and colleagues identified, using integrated proteomics methods, that elevated expression of the E3 ubiquitin ligase SYVN1 is associated with tumor metastasis. Additionally, SYVN1 interacts with heat shock protein 90 (HSP90) and modulates the ubiquitination of eukaryotic elongation factor 2 kinase (EEF2K), positioning it as a potential target for HCC treatment [107]. H. Li and colleagues demonstrated that UBE2O is negatively correlated with IFIT3 protein expression in the interferon signaling pathway and decreases IFIT3 stability through ubiquitination, thereby diminishing the therapeutic effect of interferon-α. Furthermore, they found that arsenic trioxide inhibits UBE2O activity and upregulates IFIT3 expression, thereby enhancing the therapeutic effect of interferon-α. These findings suggest that targeting UBE2O to modulate IFIT3 expression represents a promising therapeutic strategy for improving interferon therapy response in HCC patients [108]. P. Li et al. identified, through computational analysis and experimental validation, that circMRPS35 is highly expressed in HCC. It regulates STX3 by adsorbing miR-148a, thereby affecting PTEN stability and promoting tumor progression. Furthermore, the 168-amino acid peptide encoded by circMRPS35 is upregulated by chemotherapy drugs, enhancing HCC resistance to cisplatin. This study provides new biomarkers and therapeutic targets for diagnosing and treating HCC [109]. S. Tey and colleagues, through the analysis of EVs in the serum of HCC patients, demonstrated that pIgR levels in EVs from late-stage patients are significantly elevated. These pIgR-enriched EVs enhance cancer stemness and tumorigenesis via the PDK1/Akt/GSK3β/β-catenin signaling pathway. The study suggests that combined treatment with anti-pIgR antibodies and sorafenib effectively suppresses tumor growth, presenting a new therapeutic strategy for HCC [110]. T. Wong and colleagues, through the analysis of publicly available transcriptomic data and immunohistochemical validation on tissue microarrays from over 100 HCC patients, identified that PTK7 is significantly upregulated in advanced and metastatic HCC. PTK7 facilitates HCC cell metastasis and epithelial-mesenchymal transition (EMT) via the SOX9 and TGF-β signaling pathways. The study advocates for PTK7 as a novel therapeutic target for HCC metastasis and supports the development of anti-metastatic therapies targeting PTK7 [111]. M. Qu and colleagues investigated the functional implications of the Bmal1 and Clock genes in liver cancer through gene knockdown experiments. They demonstrated that the downregulation of these genes induces cell apoptosis and cell cycle arrest by modulating the expression of Wee1 and p21. These findings reveal a novel role of circadian clock regulators in liver cancer proliferation and provide new perspectives on cancer treatment through the modulation of the circadian clock [112]. M. Archederra and colleagues, through the analysis of mouse models and human HCC patient samples, discovered that hypermethylation of the ADAMTSL5 gene correlates with tumor characteristics and drug resistance in HCC cells. The study demonstrates that downregulation of ADAMTSL5 diminishes the tumorigenic properties of HCC cells and enhances their susceptibility to clinical drugs, suggesting that ADAMTSL5 may serve as a potential therapeutic target for HCC [113]. Z. Ge and colleagues, by analyzing samples from 47 HCC patients, observed that the combined TIGIT/PD-1 blockade markedly improved the functionality of tumor-infiltrating CD8⁺ T cells compared to single PD-1 blockade. These findings suggest that this combined blockade strategy could represent a novel immunotherapeutic approach for HCC patients [114]. S. Wang and colleagues employed a chemical proteomic strategy to perform comprehensive kinomic profiling of tumors and adjacent non-tumorous liver tissues from HCC patients, identifying 124 differentially expressed kinases, 45 of which predict poor patient prognosis. Additionally, the study found that certain kinase inhibitors in combination markedly inhibit the proliferation of HCC cell lines, suggesting that targeting these kinases could be therapeutically beneficial for HCC [115]. These studies highlight the critical role of ubiquitination in HCC progression and resistance, providing new insights and potential therapeutic targets for enhanced treatment strategies. The findings underscore the value of integrating proteomics and molecular analysis in identifying and validating novel biomarkers and drug targets, which could substantially improve HCC diagnosis and therapy.

Molecular mechanism research of drug action

The application of proteomics in studying drug mechanisms of action provides crucial insights into the molecular mechanisms of drug action [116]. Proteomic analysis, through a comprehensive assessment of protein expression profiles in liver cancer cells, reveals the molecular underpinnings of drug effects. For instance, quantitative proteomics strategies have identified proteins with altered expression in response to drug treatment, which may be directly or indirectly involved in the drugs’ pharmacological effects. Additionally, this analysis has uncovered resistance characteristics of solid tumors and potential drug combinations, enhancing our understanding of how drugs impact liver cancer cells at the molecular level.

J. Zecha et al. employed a method known as decryptM proteomics to investigate the effects of 31 anticancer drugs on 13 human cancer cell models. This approach elucidated the mechanisms by which these drugs regulate post-translational modifications of proteins. Additionally, the study revealed that specific drugs, such as rituximab, induce cell death by activating B cell receptor signaling pathways, whereas trastuzumab and pertuzumab affect breast cancer cells through distinct signaling mechanisms. These findings offer new strategies for personalized cancer therapy [117]. Tianjin Medical University Cancer Institute and Hospital has initiated a clinical trial (NCT06301399) to evaluate the efficacy and safety of rituximab combined with PD-1/PD-L1 inhibitors and targeted therapy for advanced hepatocellular carcinoma. This phase II intervention study, encompassing 20 participants, aims primarily to assess the objective response rate and the time to disease progression or death. The expected results are set for publication in 2026. Pending validation of its potential and efficacy through the upcoming study results, this research might introduce new therapeutic options for advanced HCC patients. Y. Gong et al. investigated the impact of Smad3 C-terminal phosphorylation site mutations on the efficacy of salvianolic acid B (Sal B) in combating hepatocarcinogenesis. Their findings suggest that phosphorylated Smad3C (pSmad3C) plays a significant role in the effects of Sal B on HCC. The molecular mechanism likely involves the promotion of Smad3 phosphorylation isoform conversion, which is crucial for the anti-hepatocarcinogenic effects of Sal B [118]. Y. Guo et al. investigated the effects of 17β-estradiol (E2) on apoptosis in various liver cell lines, including LO2, HepG2, and HepG2.2.15 cells. They hypothesized that E2 might induce oxidative stress in HepG2 cells by increasing Foxo3a phosphorylation, potentially leading to apoptosis. This finding suggests a possible therapeutic approach for treating HCC [119]. Z. Liu et al. employed iTRAQ proteomics technology to identify that JS-K, a novel compound, modulates the expression of 182 proteins in HBV-positive liver cancer cells (HepG2.2.15), significantly affecting pathways such as endocytosis. Additionally, the study demonstrated that JS-K binds to TAGLN through molecular docking, offering a novel mechanism for JS-K’s resistance to HBV-positive liver cancer [120]. Z. Xue et al. employed a multi-omics approach integrating pharmaco-omics and proteomics to investigate the antitumor effects of Icaritin on HCC and its underlying molecular mechanisms. The findings revealed that Icaritin may exert its anti-HCC effects by targeting the FYN gene, modulating cell proliferation, inhibiting angiogenesis, and promoting apoptosis. Both in vitro and in vivo studies demonstrated significant tumor growth inhibition by Icaritin, providing a scientific basis for developing new therapeutic strategies for HCC [121]. Beijing Shenzhou Cell Biotechnology Co., Ltd. has initiated a Phase III clinical trial (NCT05594927) to evaluate the efficacy and safety of Icaritin capsules compared to Huachansu tablets in patients with unresectable and poor-prognosis HCC. With a total enrollment of 261 participants, the primary endpoint of this study is overall survival, while secondary endpoints include disease progression time, progression-free survival, and objective response rate. This study represents a significant advancement in HCC treatment and has the potential to introduce innovative therapeutic options once research outcomes are validated. Additionally, Zhejiang Cancer Hospital has initiated a clinical study (NCT05903456) to evaluate the efficacy and safety of combining Icaritin capsules with lenvatinib and TACE for the treatment of unresectable, non-metastatic HCC. This phase II study aims to enroll 20 patients, with the primary endpoint being the objective response rate, and secondary endpoints including progression-free survival and overall survival. Although recruitment has not commenced yet, the study shows promise in introducing new therapeutic approaches for HCC patients. The findings will play a crucial role in assessing the potential of combination therapy and providing essential data support for broader clinical applications in the future. Z. Li et al. investigated the antitumor mechanisms of ergosterone using an H22 tumor-bearing mouse model and employing transcriptomic and proteomic analyses. They identified Lars2, Sirpα, and Hcls1 as key regulatory factors, providing potential targets for the development of new cancer therapeutic strategies [122].

These studies underscore the critical role of proteomics in deciphering the intricate molecular landscape of liver cancer and its response to therapeutic agents. The findings not only enhance our understanding of drug effects at the molecular level but also provide essential insights into potential resistance mechanisms and the identification of novel therapeutic targets. The diversity of approaches illustrates the extensive range of techniques used to elucidate the complex interactions between drugs and liver cancer cells. Such comprehensive investigations lay the foundation for developing personalized and targeted therapeutic interventions, offering renewed hope for improved outcomes in HCC management.

Investigation of drug resistance mechanisms

Drug resistance stands as a primary impediment in liver cancer treatment. Proteomic techniques, including tandem mass tag (TMT)-based quantitative proteomics, have been instrumental in scrutinizing the molecular mechanisms of drug resistance in liver cancer cells. Through the comparison of protein expression disparities between sensitive and resistant cell lines, researchers have pinpointed proteins that potentially govern tumor sensitivity to drugs, thereby offering a fresh perspective for surmounting drug resistance.

B. Hao et al. utilized TMT-based quantitative proteomics to investigate HDACi-resistant solid tumors, identifying ribosome biogenesis proteins essential for tumor sensitivity. The analysis revealed potential drug combinations to enhance HDACi sensitivity and highlighted the Rho signaling pathway and kinases PDK1 and ROCK as potential resistance markers. These findings suggest new therapeutic strategies for overcoming HDACi resistance in solid tumors [123]. Z. Pan et al. investigated the impact of NAT10 on liver cancer metastasis and drug resistance using immunohistochemistry, bioinformatics analysis, and both in vitro and in vivo experiments. The study uncovered a novel mechanism by which NAT10-mediated mRNA ac4C modification regulates tumor metastasis and highlighted the regulatory role of the NAT10-HSP90AA1 complex in mediating endoplasmic reticulum stress-induced metastasis and drug resistance in HCC cells [124]. W. Hsu et al. investigated the effects of Platycodin D (PD), a triterpenoid saponin derived from Platycodon grandiflorus, on HCC cells resistant to histone deacetylase inhibitors (HDACi). The study found that PD inhibits cell viability and reverses HDACi resistance by suppressing ERK1/2-mediated cofilin-1 phosphorylation. Additionally, combining PD with apicidin, an HDACi, significantly increased apoptosis in resistant cells, indicating that PD may serve as a potential therapeutic agent for overcoming chemotherapy resistance in HCC [125]. H. Chu et al. utilized proteomic analysis to illustrate the role of folate receptor α (FOLR1) in contributing to sorafenib resistance in HCC through the activation of autophagy. This finding offers new insights into addressing and managing sorafenib resistance [126]. S. Lauer and colleagues elucidated novel mechanisms of the DNAJ-PKAc fusion kinase in fibrolamellar carcinoma. The study identified the recruitment of the Bcl2-associated athanogene 2 (BAG2) protein and clarified its role in chemoresistance, thereby offering new biomarkers and potential therapeutic targets. This research employed proximity proteomics and live-cell imaging techniques, enhancing the understanding of the molecular underpinnings of chemoresistance in fibrolamellar carcinoma [127]. W. Han and colleagues discovered that WD repeat domain 4 (WDR4) enhances stemness and promotes drug resistance by upregulating tripartite motif protein 28 (TRIM28) through the examination of lenvatinib-resistant HCC cell lines and clinical samples. Elevated levels of WDR4 and TRIM28 are significantly associated with poor patient prognosis, indicating that WDR4 could serve as a promising therapeutic target for improving the effectiveness of lenvatinib treatment [128]. Through multi-omics analysis, C. Wu and colleagues demonstrated that AKR1C3 facilitates lipid droplet formation in HCC, thereby enhancing cellular resistance to sorafenib (Fig. 5). These findings suggest that lipid metabolism mediated by AKR1C3 is a potential target for HCC therapy [129]. Ascentawits Pharmaceuticals, Ltd is presently conducting a Phase I/II clinical trial (NCT06239155) to evaluate the safety, tolerability, pharmacokinetics, efficacy, and the correlation with AKR1C3 enzyme expression of AST-3424 in treating patients with advanced solid tumors. The study follows a non-randomized, open-label design and aims to recruit a total of 51 patients. Although the study is ongoing and definitive conclusions have not been reached, the expected results will provide crucial data for the ongoing development of AST-3424. This research has the potential to introduce new therapeutic options for patients with advanced cancer; however, a comprehensive assessment of its efficacy and safety will depend on the completion of the study and subsequent data analysis. K. Abushawish and colleagues employed UHPLC-QTOF-MS multi-omics analysis to investigate Hep3B liver cancer cells exhibiting resistance to sorafenib. They identified significant changes in 27 metabolites and 18 proteins, affecting key pathways such as amino acid and nucleotide metabolism and energy production. These changes could potentially reveal new biomarkers, which may aid in understanding and counteracting drug resistance in liver cancer [130]. B. Haberkorn and colleagues employed cell culture and protein expression analysis to investigate the Ct-OATP1B3 protein. The study revealed that this protein is localized in lysosomes and is capable of transporting specific kinase inhibitors, such as encorafenib, into these organelles. This process reduces the drug concentration in the cytoplasm, potentially enhancing the resistance of cancer cells to certain anticancer drugs. These findings offer new insights into the mechanisms underlying cancer drug resistance [131]. M. Huang and colleagues, through proteomic screening and in vitro and in vivo experiments, discovered that METTL1 and WDR4 are significantly upregulated in HCC cells resistant to lenvatinib. The knockdown of METTL1 decreases drug resistance, while its overexpression promotes it. The study reveals a new mechanism involving METTL1 in inducing drug resistance by regulating the translation of EGFR pathway genes through m7G tRNA modification. This finding offers potential intervention targets for liver cancer therapy [132].

The formation of AKR1C3-dependent lipid droplets (LD) is crucial for HCC patients’ adaptation to sorafenib by regulating lipid and energy homeostasis. AKR1C3-dependent LD accumulation protects HCC cells from sorafenib-induced mitochondrial lipotoxicity by regulating lipid autophagy. Targeting AKR1C3 may be a promising therapeutic strategy for HCC tumors. Reproduced with permission [129]. Copyright 2022, Ivyspring International Publisher

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These findings underscore the complexity of drug resistance mechanisms in liver cancer and emphasize the importance of advanced proteomic and multi-omics approaches in identifying novel therapeutic targets. This research highlights the need to understand these mechanisms at a molecular level to develop more effective treatment strategies and enhance patient outcomes in liver cancer therapy.

Drug interaction study

Proteomics is vital for studying drug interactions. By analyzing how various drugs affect protein expression, researchers have elucidated the underlying mechanisms of these interactions. Y. Xu et al. investigated the mRNA expression and genetic alterations of 36 histone acetylation regulators in 1,599 HCC samples. Their study revealed a significant correlation between histone acetylation patterns and malignant tumor pathways, as well as the tumor microenvironment. Additionally, the study demonstrated that the HAscore is strongly associated with sensitivity to antitumor drugs and identified 116 related therapeutic agents. Furthermore, the HAscore was linked to the efficacy of PD-L1 and PD-1 blockade, significantly affecting the response rate [133]. Q. Jin et al. investigated the role of miR-22-5p, derived from the long non-coding RNA MIR22HG (microRNA-22 host gene), in enhancing the radiosensitivity of HCC by suppressing histone deacetylase 2 (HDAC2) activity. Radiation was observed to upregulate MIR22HG expression while concurrently downregulating HDAC2 expression. Inhibition of HDAC2 leads to histone acetylation in the MIR22HG promoter region, resulting in increased MIR22HG expression and miR-22-5p production, thereby enhancing the sensitivity of liver cancer to radiotherapy [134]. X. Ren and colleagues employed flow cytometry and mass spectrometry to compare the effects of three multi-receptor tyrosine kinase inhibitors, sorafenib, regorafenib, and lenvatinib, on liver cancer Huh-7 cells. Their findings indicated that lenvatinib and regorafenib were more effective than sorafenib in inducing cell cycle arrest and apoptosis. These inhibitors had a more pronounced effect on downregulating key proteins in the p53 pathway and other signaling pathways. The study elucidates potential molecular mechanisms underlying the combination therapy of immune checkpoint inhibitors with these drugs [135].

The application of proteomics enhances our understanding of drug targets in liver cancer and serves as a powerful tool for developing novel therapeutic strategies. As technology advances and its application intensifies, proteomics is expected to play an increasingly significant role in liver cancer treatment.

Advances in science and technology, evolving treatment paradigms, the implementation of liver cancer prevention and screening programs, along with the development of novel diagnostic and therapeutic technologies, have significantly transformed the landscape of liver cancer diagnosis and management. Drug therapy remains a predominant and convenient method for liver cancer treatment. Recently, the emergence of various new liver cancer treatment drugs, including immune checkpoint inhibitors, traditional Chinese medicine, and combination therapies, has led to their increasingly widespread use in clinical practice.

Chemical drugs

In recent years, considerable advancements have been achieved in the development of chemical drugs for liver cancer treatment. Researchers have discovered several compounds with potential therapeutic efficacy through high-throughput screening and subsequent structural optimization. The evolution of these drugs leverages traditional drug discovery methodologies while also incorporating the latest advancements in proteomics. D. Othman et al. synthesized new benzimidazole-triazole hybrids and found that compounds 5a and 6g exhibit potent antitumor effects against various cancer cell lines. Compound 5a shows excellent inhibition of EGFR and Topo II, while compound 6g demonstrates moderate activity. The study elucidated the anticancer mechanisms and multi-target inhibition potential of these compounds through in vitro experiments and molecular simulations [136]. J. Zhou et al. discovered a novel organic selenium compound, phenyl-2-pyrimidylidene-4-allyl-3-seleno-urea (CU27), through functional screening. CU27 effectively inhibits liver cancer stem cells and reduces tumor size and metastatic potential by suppressing c-Myc transcriptional activity. In mouse models, CU27 also demonstrated the ability to sensitize sorafenib-resistant tumors to sorafenib, suggesting its potential as a candidate for treating drug-resistant liver cancer (Fig. 6) [137]. Y. Pang and colleagues investigated the effects of 5-caffeoylquinic acid (5-CQA) on HCC through cell viability testing and molecular biology methods. They found that 5-CQA significantly inhibits the proliferation of HCC cells, with its antitumor effects mediated through the abnormal activation of the HIF-1α/glucose transporters/glycolysis pathway. The findings suggest that targeting this pathway can enhance the therapeutic effects of 5-CQA and its derivatives on HCC [138]. Y. Chen et al. discovered that polyphyllin D induces G2/M phase arrest in liver cancer cells by disrupting cholesterol biosynthesis and inhibiting cell growth, thereby revealing a novel mechanism of action for liver cancer treatment [139]. L. Shi et al. investigated therapeutic strategies for liver malignancies, including ICC and HCC, using high-throughput screening, proteomics, and in vitro drug resistance models. They discovered that the small molecule 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1; lificiguat) exhibits selective activity against certain types of liver cancer cells. This selectivity is determined by the expression of the liver-specific cytosolic sulfotransferase enzyme SULT1A1, which sulfates YC-1, facilitating its covalent binding to lysine residues on protein targets and enabling the enrichment of RNA- binding factors. Additionally, the study identified structurally related small molecules activated by SULT1A1, representing a previously unexplored category of small molecules. This research lays the groundwork for the preclinical development of these drugs and underscores the broader potential of utilizing SULT1A1 activity for selective targeting strategies [140]. Z. Li et al. investigated the anti-tumor mechanism of Inonotus hispidus petroleum ether extract using an H22 tumor-bearing mouse model, employing whole transcriptome and proteome analyses. Their study identified 957 differentially expressed genes and 405 differentially expressed proteins, suggesting that five key genes/proteins (Lilrb4a, Nrp1, Gzma, Gstt1, and Pdk4) may regulate anti-tumor pathways. These findings provide molecular insights for developing new anti-tumor strategies [141]. S. Chen and colleagues used quantitative proteomics to investigate the effects of nitidine chloride (NC) on the Bel-7402 liver cancer cell line, revealing that NC inhibits cell proliferation and induces apoptosis. Their study suggests that NC targets mitochondria and activates the JNK/c-Jun signaling pathway, offering new insights into the molecular mechanisms and potential therapeutic strategies for liver cancer [142]. Researchers J. Song and Y. Li synthesized four organotin benzohydroxamate derivatives (OTBH) and demonstrated that these compounds exhibit significant antitumor activity through both in vitro and in vivo experiments. Their analysis identified 203 differentially expressed proteins in HepG2 cells and 146 differentially expressed proteins in rat liver tissues in response to OTBH treatment. The findings suggest that these proteins are associated with microtubule-related processes and apoptotic pathways. Further assays confirmed that OTBH derivatives directly target the colchicine-binding site on tubulin proteins, disrupting the microtubule network and inducing cancer cell apoptosis. These results highlight the potential of OTBH derivatives as novel microtubule-targeting anticancer agents [143]. Researchers from S. Wang et al. employed iTRAQ proteomics, cell culture, and various detection techniques to elucidate the mechanism by which compound 6c induces mitochondrial and lysosomal dysfunction, autophagic cell death, and DNA damage in liver cancer cells via increased levels of reactive oxygen species (ROS). The study underscores the critical role of ROS in cellular responses to 6c, suggesting that antioxidants such as N-acetyl-L-cysteine can partially reverse these effects. These findings provide a rationale for the application of ROS inducers in cancer therapy [144]. These studies advance our understanding of novel anticancer agents and mechanisms, revealing potential therapeutic targets and strategies for liver cancer and related malignancies. The findings underscore the importance of targeting specific pathways and molecular interactions to enhance treatment efficacy and overcome drug resistance. The advancement of chemical drugs in anti-HCC drug development has instilled new optimism for liver cancer treatment. Future studies must concentrate on addressing drug resistance, evaluating toxicity profiles, and refining personalized therapeutic approaches to enhance both the efficacy of treatments and the overall quality of life for affected patients.

The working hypothesis of CU27’s mechanism of action in liver cancer stem cells (L-CSCs). A The dimerization of Myc with Max through direct interaction of the basic-helix-loop-helix/leucine zipper (bHLH/LZ) domains enables them cooperative binding to the c-Myc promoter containing E-box (CACGTG or CATGTTG) and its target genes (HNRNPU, MCM7, E2F1, HNRNPA1, etc.). This process leads to the activation of c-Myc and its target gene expression, thereby promoting self-renewal of CSCs. B CU27 can bind to the c-Myc bHLH/LZ domain, promoting the dissociation of the c-Myc-Max complex and preventing its occupancy of the target gene promoters, inhibiting the expression of c-Myc and its target genes. This effect leads to the inhibition of L-CSC self-renewal and CSC-related traits (such as chemoresistance and metastasis). Reproduced with permission [137]. Copyright 2022, John Wiley and Sons

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Targeted therapy drugs

The inherent complexity and heterogeneity of liver cancer present significant challenges to the effectiveness of conventional chemotherapy agents. Targeted therapeutics, particularly those informed by proteomics data, exhibit substantial promise in addressing liver cancer. Proteomic analyses have enabled researchers to pinpoint a variety of molecular targets intricately linked to the progression of liver cancer. L. Goyal and colleagues evaluated the efficacy of futibatinib in ICC patients with FGFR2 fusions or rearrangements, demonstrating an objective response rate of 42% and a median duration of response of 9.7 months. These findings indicate the therapeutic potential of futibatinib for this patient population [145]. Mayo Clinic is presently conducting a Phase II clinical trial (NCT04828486) to evaluate the effectiveness and safety of a combined treatment comprising Futibatinib and Pembrolizumab for patients diagnosed with FGF19-positive advanced or metastatic hepatocellular carcinoma. Employing a single-arm, non-randomized design, the study aims to enroll 25 patients, with the primary endpoint focusing on progression-free survival, and secondary endpoints including overall survival and overall response rate. Since the trial is still in the recruitment phase, definitive conclusions have not been reached. While this research shows promise for introducing new therapeutic options for a specific subset of hepatocellular carcinoma patients, a comprehensive assessment of efficacy and safety depends on the completion and analysis of the study data. E. Azmy and colleagues synthesized novel CDK2 inhibitors, encompassing pyrolo[2,3-c]pyrazole and pyrolo[2,3-d]pyrimidine derivatives, along with their bioisosteres. This study revealed that these compounds possess significant cytotoxic effects, with lower IC₅₀ values than sorafenib. Molecular docking and dynamic simulations confirmed their high-affinity interaction with CDK2. Furthermore, 3D-QSAR and ADME/TOPKAT analyses substantiated their favorable pharmacokinetic profiles and therapeutic potential [146]. L. Cai and colleagues conducted research on the inhibitor B029-2, which targets the epigenetic regulators p300 and CBP in HCC. Their study indicated that p300/CBP-mediated acetylation of histones H3K18/K27 is upregulated in HCC tissues. B029-2 specifically diminishes this acetylation, resulting in substantial antitumor activity by impairing glycolytic function and nucleotide synthesis. The effects of B029-2 are partially mitigated by the overexpression of PSPH and DTYMK. This research underscores the potential of B029-2 as a therapeutic agent that modulates histone acetyltransferase activity [147]. L. Sun and colleagues have identified a novel mechanism whereby rapamycin directly targets and inhibits the STAT3 pathway. This intervention subsequently modulates c-Myc expression, leading to a reduction in tumor growth within a xenograft mouse model. The findings of this study provide novel insights into the development of STAT3 inhibitors as potential anticancer therapeutics [148]. Y. Pu and colleagues utilized mass spectrometry-based proteomics to demonstrate that cyclin-dependent kinase (CDK) inhibitors significantly lowered the IC₅₀ value of liver cancer cells resistant to 5-fluorouracil, thereby reversing their drug resistance [149].

Despite the notable advancements achieved by targeted therapy drugs in liver cancer treatment, significant challenges persist. The molecular heterogeneity inherent to liver cancer demands personalized therapeutic strategies, underscored by the need for detailed proteomic analyses to discern patient-specific molecular targets. Furthermore, drug resistance remains a perplexing issue, warranting additional investigation into the molecular underpinnings of liver cancer cells’ resistance to targeted therapies. Proteomics plays a pivotal role in the investigation of targeted therapy for liver cancer. Proteomic technologies serve as the foundation for personalized treatment by identifying distinct protein subtypes and mutations in liver cancer. They also facilitate immunotherapeutic strategies by uncovering the tumor immune microenvironment. Moreover, the analysis of protein interaction networks aids in the exploration of novel target proteins and the elucidation of drug resistance mechanisms, thereby presenting new opportunities to overcome drug resistance. Proteomic technologies find extensive application in both preclinical and clinical phases, encompassing drug target discovery, treatment monitoring, and biomarker prediction. In-depth research in these domains will foster innovation in the field of liver cancer treatment, offering patients more effective treatment alternatives.

Immune checkpoint inhibitors

Immune checkpoints, proteins integral to immune cell function, play a pivotal role in modulating immune responses. Dysregulation of these proteins can influence tumor initiation and progression. Designed to target these checkpoints, immune checkpoint inhibitors aim to counteract their suppressive activity, thereby potentiating the immune system’s capacity to identify and eradicate tumors. In a phase Ib/II clinical trial, cadonilimab, a bispecific antibody targeting PD-1 and CTLA-4, showed promising antitumor efficacy and a favorable safety profile among patients with advanced HCC. The trial observed an objective response rate of 16.7%, a median progression-free survival of 3.7 months, and the overall survival had not been reached at the time of data analysis [150]. K. Wang and colleagues executed a multicenter, open-label, randomized, controlled phase II trial to assess the efficacy and safety of sintilimab, an anti-programmed cell death protein 1 (PD-1) inhibitor, as adjuvant therapy in patients with resected high-risk HCC exhibiting microvascular invasion. The study revealed that sintilimab significantly prolonged recurrence-free survival over active surveillance and demonstrated an acceptable safety profile. These findings indicate the therapeutic potential of PD-1 inhibitors as efficacious adjuvant treatment for this patient cohort [151]. Immune checkpoint inhibitors, such as cadonilimab and sintilimab, have demonstrated efficacy in the treatment of HCC and warrant consideration as standard therapeutic agents. Continuous research is imperative for the refinement of patient selection criteria, optimization of treatment duration, and mitigation of treatment-related toxicities. The integration of these findings into clinical practice necessitates a thorough evaluation of their effects on patient quality of life and overall survival.

The emergence of immune checkpoint inhibitors signifies a notable advancement in HCC treatment. These inhibitors operate by obstructing immune inhibitory signals, such as PD-1 and CTLA-4, thus augmenting the immune system's capacity to target tumors. Consequently, they hold the potential to integrate into standard HCC treatment protocols. However, it is vital to acknowledge that the effectiveness of immune checkpoint inhibitors may differ among patients. Hence, there exists an urgent necessity to discover predictive efficacy biomarkers utilizing advanced techniques such as proteomics to enable more precise patient selection.

Proteomic techniques provide a valuable approach for analyzing changes in protein expression within the immune microenvironment. This analysis is essential for achieving a deeper understanding of the mechanism of action of immune checkpoint inhibitors and predicting their efficacy. Furthermore, proteomic analysis can lead to the discovery of novel biomarkers and therapeutic targets, propelling the advancement of personalized medicine. Moreover, the implementation of combination therapy strategies holds great promise in improving response rates and treatment efficacy. Proteomics can play a pivotal role in identifying specific targets for combination therapy, further enhancing its potential. Subsequent research endeavors should prioritize optimizing treatment regimens, including factors such as treatment duration, dose adjustments, and the mitigation of treatment-related toxicities. Long-term follow-up studies are indispensable for assessing the sustained efficacy and safety of immune checkpoint inhibitors. Equally crucial is evaluating the treatment's impact on patient quality of life.

In summary, immune checkpoint inhibitors present new and valuable treatment options for HCC patients. Nevertheless, further research is necessary to refine the application of these therapies and integrate them effectively into clinical practice. Proteomic techniques will play a crucial role in this process, facilitating the advancement of personalized medicine and ultimately resulting in improved treatment outcomes and enhanced quality of life for HCC patients.

Biologics

Proteomics, as a potent tool, has shown significant potential in the development of biopharmaceuticals for treating liver cancer. R. Carrasco-Reinado and colleagues conducted a proteomic analysis of the marine microalgae Nannochloropsis gaditana. Their research revealed that the protein UCA01 exhibits antitumor properties, selectively suppressing the proliferation of cancer cells while sparing normal cells. This discovery underscores the potential of applied proteomics in biotechnological applications [152]. X. Hou and colleagues demonstrated that extracellular microparticles, specifically apoHPC-MPs derived from apoptotic liver progenitor cells, can convey a death signal that potently inhibits the proliferation of liver cancer-initiating cells. Using rat models, the study established that these microparticles are internalized by specific liver progenitor cells, inducing cell death through the transmitted signals, which may thus prevent the onset of liver cancer (Fig. 7A). This research introduces a novel therapeutic strategy for hepatic malignancies [153]. D. Zhu et al. employed proteomics technology to identify a peptide, RR-171, with antitumor activity from human umbilical cord serum. Their findings demonstrated that RR-171 could induce apoptosis in HCC cells in vitro, activate the NF-κB signaling pathway, and effectively accumulate in solid tumors in vivo. This resulted in significant antitumor effects without apparent systemic toxicity, suggesting its potential as a new therapeutic approach for HCC treatment [154]. P. Ruenchit and colleagues conducted a comprehensive investigation using proteomic and bioinformatics methods to explore the effects of Trichinella spiralis infective larval extract on three types of human cancer cells. The findings demonstrated significant inhibitory effects, particularly on the proliferation of liver cancer cells (HepG2). The anticancer peptide, termed peptide 2, induced reactive oxygen species accumulation, thereby inhibiting HepG2 cell proliferation without inducing apoptosis or necrosis, highlighting its potential as an adjunct agent for liver cancer treatment [155]. P. Bottoni and her team used high-resolution respirometry and proteomic analysis to investigate the effects of PPAR agonists on mitochondrial respiration in HepG2 cells. The findings revealed that these drugs caused complex disruptions in mitochondrial oxidative metabolism and structural damage, highlighting the need to monitor their potential toxicity in clinical use [156].

A MPs derived from apoptotic HPCs effectively inhibit liver cancer development. HPCs preferentially uptake MPs from apoptotic HPCs compared to MPs derived from apoptotic hepatocytes and apoptotic liver tumor cells. Proteomic analysis of MPs derived from apoptotic HPCs reveals an enrichment of proteins involved in cell death pathways. Thus, HPC-derived MPs contain a death signal capable of inducing the killing of hepatic tumor-initiating cells. Reproduced with permission [153]. Copyright 2022, Springer Nature. B The synergistic mechanism between CAN and TD. COF: optimal compound composition; CAN: cantharidin; TD: CAG , Rc and Rd. promotion: ; inhibition: .Reproduced with permission [170]. Copyright 2022, Elsevier

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The integration of proteomics in developing biopharmaceuticals for liver cancer has introduced novel concepts and methodologies, offering crucial backing for research and clinical application in biomedicine. As proteomics technology continually advances and refines, it is anticipated to play a more prominent role in crafting biopharmaceuticals for liver cancer, thereby expanding treatment alternatives for patients.

Natural medicines

The application of traditional Chinese medicine (TCM) in HCC treatment offers significant advantages. TCM for liver cancer presents unique features: it enhances immunity and regulates liver qi, eliminates liver toxins, resolves blood stasis, detoxifies, softens and disperses masses, dissolves liver nodules, inhibits liver tumors, prevents tumor metastasis and recurrence, alleviates symptoms, and extends survival.

Through cellular experiments, M. Alfwuaires et al. demonstrated that acacetin (ACN) inhibits the activation of STAT3 and its downstream signaling pathways in liver cancer cells, resulting in reduced cancer cell proliferation and migration, and the induction of apoptosis. Given its novel role as a STAT3 inhibitor, ACN holds potential for the treatment of liver cancer [157]. J. Wang et al. employed an integrated omics approach to investigate the mechanism of action of curcumol in liver cancer cells. They found that curcumol exerts anti-cancer effects by targeting PLK1 and other cell cycle-related molecules, suggesting new strategies and potential targets for liver cancer treatment [158]. J. Gao et al. employed LC–MS analysis and TMT-based quantitative proteomics to investigate the inhibitory effects of Saussurea involucrata on liver cancer cells. The study found that the cell suspension culture ESPI significantly affected pathways related to protein metabolism and cell cycle regulation, effectively suppressing the growth of HepG2 cells in vitro and tumor growth in vivo. These findings provide a scientific rationale for the anti-hepatoma potential of S. involucrata [159]. R. Huang et al. conducted a study employing biophysical proteomics and in vivo assays to explore the effects of Bruceine D (BD) on HCC. The study revealed that BD inhibits HIF-1a-mediated glucose metabolism by targeting the interaction of ICAT/b-catenin, leading to reduced HIF-1a expression and suppressed proliferation of HCC cells as well as tumor growth. These findings suggest the potential of BD as a therapeutic agent for HCC [160]. L. Yang et al. utilized network pharmacology, proteomic analysis, bioinformatics, and in vitro biochemical methods to investigate the antiproliferative activity of berberine in HepG2 cells. The study revealed that berberine induced apoptosis, caused cell cycle arrest, and targeted AKAP12, suggesting its potential as a therapeutic agent for HCC [161]. F. Tang and colleagues utilized network pharmacology and bioinformatics analysis to demonstrate that corosolic acid inhibits HCC progression by targeting the prolyl 4-hydroxylase subunit alpha 2 (P4HA2) protein. Their findings indicated that P4HA2 expression is associated with poor survival rates in HCC patients and immune cell infiltration. Furthermore, corosolic acid was shown to reduce P4HA2 protein levels, inhibit HCC cell proliferation, and induce cell cycle arrest. These results suggest that P4HA2 may serve as a novel target for HCC treatment [162]. Z. Chu et al. employed proteomic analysis to demonstrate the potential of Celastrus orbiculatus extract (COE) in inhibiting vasculogenic mimicry (VM) in HCC by targeting EphA2. The results showed that COE reduces the expression of EphA2 and VM-associated biomarkers, thereby suppressing tumor growth and VM formation. These findings suggest the potential of COE as a novel therapeutic strategy for HCC treatment [163]. J. Lin et al. employed TMT proteomics and PRM verification technology to investigate the impact of Pien Tze Huang (PZH) on the phosphorylation of tumor metabolic enzymes in a mouse model of HCC. The study demonstrated that PZH significantly suppresses tumor weight and shares an anti-tumor mechanism with sorafenib by regulating the phosphorylation levels of enzymes involved in the glycolysis and gluconeogenesis pathways. These findings provide new molecular insights into PZH as a therapeutic agent for HCC [164]. Q. Li et al. demonstrated through cellular experiments and animal models that Cucurbitacin B (CuB) effectively inhibits the progression of HCC by inducing DNA damage-dependent cell cycle arrest without directly causing cell death via necrosis or apoptosis. These findings suggest the potential of CuB as an anti-HCC drug and indicate that its mechanism of action involves the ATM-dependent p53-p21-CDK1 and CHK1-CDC25C signaling pathways [165]. J. Yao et al. employed various proteomic and cell biological techniques to discover that the cyclin-dependent kinase 9 (CDK9) inhibitor LDC067, as well as the novel CDK9 inhibitor oroxylin A, derived from Scutellaria baicalensis, can block PINK1-PRKN-mediated mitophagy, leading to mitochondrial dysfunction and cell death. Oroxylin A demonstrates potent therapeutic efficacy against HCC and can overcome drug resistance by suppressing mitophagy. These findings offer significant insights for developing novel therapeutic approaches targeting mitophagy [166]. S. Sun and colleagues developed a novel strategy for rapidly identifying direct targets of psoralen using proteomics and network pharmacology techniques. Utilizing DARTS proteomics, they identified ABL1 as a potential toxicological target of psoralen and confirmed its binding ability and site through SPR and molecular docking. By integrating DARTS techniques with network pharmacology results from the STRING database, they elucidated the downstream mechanisms of toxicity for ABL1. This approach establishes a foundation for identifying other toxicological targets of TCM [167]. H. Husain and colleagues conducted an integrated proteomic and gene expression analysis to assess the impact of pomegranate juice on NDEA-induced liver fibrosis. They identified biomarkers such as Il1rl2, Ric8a, Krt18, and Hsp90b1, which exhibit regulated expression in the context of liver fibrosis. Pomegranate juice supplementation significantly improved liver function, reduced oxidative stress, and decreased inflammation, thereby offering new biomarkers and potential therapeutic strategies for the treatment of liver fibrosis [168]. S. Zhang and colleagues employed omics and bioinformatics analysis techniques to investigate the mechanisms and material basis of hepatotoxicity induced by Sophorae tonkinensis radix et rhizome (ST). Their findings revealed that ST treatment led to the differential expression of 254 proteins and 42 metabolites in rat liver tissues, with 7 proteins significantly enriched in key pathways. Bioinformatics analysis suggested that 20 compounds might induce liver injury by modulating the expression of 7 toxic targets. These findings provide valuable guidance for the safe application, toxicity control, and development of early warning systems for ST toxicity [169]. P. An and colleagues employed an integrated omics and bioinformatics approach to investigate the antitumor effects of the compound-composed optimal formula (COF) from Aidi injection on liver and colorectal cancers (Fig. 7B). Their results demonstrated that COF significantly inhibits tumor growth and induces apoptosis by activating protein phosphatase 2A (PP2A) and inhibiting the ubiquitin–proteasome system (UPS). This research provides a new direction for the modernization of TCM formulas and suggests COF as a potential adjunct therapy for cancer treatment [170]. W. Ku and colleagues employed mass spectrometry to investigate the effects of kaempferitrin on the HepG2 liver cancer cell line. They identified 33 differentially expressed genes and observed changes in the size and quantity of EVs. Atomic force microscopy revealed larger vesicles and a reduced number of smaller ones in the medium of treated cells. Additionally, the study indicated that kaempferitrin might regulate lipid levels by modulating the expression of genes associated with lipid metabolism, providing new insights into its potential roles in liver diseases [171]. R. Begolli and colleagues, through transcriptome and proteome analysis, discovered that Hypnea musciformis seaweed extract activated p53-mediated responses in liver cancer cells, demonstrating potential anti-cancer properties [172]. W. Jiang and colleagues discovered that coconut peel polysaccharide and its derivatives exhibit significant scavenging effects on ABTS⁺ free radicals, DPPH free radicals, and superoxide anions O²⁻. The scavenging ability of these polysaccharides intensifies with increasing concentration. Additionally, coconut polysaccharide and its derivatives demonstrate notable anti-proliferative activity against HepG2 cells in vitro, suggesting potential for use in liver cancer treatment [173].

The prospects for natural anti-cancer drugs in liver cancer treatment are promising. However, due to the complexity of the chemical composition of natural products, their pharmacological mechanisms often involve the synergistic action of multiple signaling pathways and targets. Therefore, utilizing proteomics technology to deeply investigate the molecular mechanisms of natural anti-cancer drugs and reveal their action targets and pathways is crucial for optimizing drug development and personalized treatment plans. Additionally, future research should focus on chemical structure modification and pharmacokinetics optimization of natural products to enhance their feasibility and effectiveness in clinical applications.

Nanomedicines

Additionally, innovative therapeutic approaches such as innovative nano drugs and nano drug delivery system have been developed in the field of oncology [174,175,176]. Zhao et al. created a novel theranostic nanoplatform based on a EuMOF@ZIF/AP-PEG nanocomposite capable of microwave thermo-chemotherapy and fluorescence imaging. This nanocomposite demonstrated high microwave sensitization, excellent drug loading capacity, and biocompatibility, showing significant tumor growth inhibition and potential for in vivo imaging [177]. X. Chen et al. developed a novel multifunctional immune-nanomedicine, MOF-CpG-DMXAA, to enhance anticancer immunity in HCC (Fig. 8). This nanomedicine demonstrated the capacity to reprogram tumor-associated macrophages, enhance dendritic cell maturation, and simultaneously destroy tumor blood vessels, thereby improving immunotherapy efficacy in mouse models of HCC [178]. These studies provide valuable insights into the design of nanomedicines and their applications in oncology, demonstrating the potential of these technologies to revolutionize cancer treatment by enhancing efficacy, reducing toxicity, and improving patient outcomes. Continued research and clinical translation of these nanomedicines could significantly impact the future landscape of HCC therapy.

Preparation method and schematic diagram of the mechanism of action of MOF-CpG-DMXAA in enhancing anticancer immunity. By simple coordination, CpG ODNs and the vascular disrupting agent DMXAA are introduced into MOF-801 to prepare MOF-CpG-DMXAA. CpG ODNs, DMXAA, and MOF-801 individually activate the cGAS-STING-NF-κB signaling pathway, reprogramming TAMs and promoting DCs maturation. MOF-CpG-DMXAA, based on its advantages, exerts a synergistic effect by increasing the infiltration of CD4 and CD8 T lymphocytes, disrupting tumor blood vessels, triggering systemic immune activation, and stimulating potent antitumor immunity, thus demonstrating superior immunotherapeutic efficacy against HCC. Reproduced with permission [178]. Copyright 2023, John Wiley and Sons

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Combination therapy anti-liver cancer drugs

The essence of combination therapy lies in the integration of established modalities within current cancer treatment to enhance the overall effectiveness of cancer management. Transarterial chemoembolization (TACE), programmed death-ligand 1 (PD-L1) inhibitors, and molecular-targeted therapy (MTT) have exhibited significant potential in the treatment of HCC.

TACE stands as the established treatment modality for mid to late-stage HCC; nevertheless, it is associated with challenges related to a suboptimal objective response rate and treatment resistance [179]. Proteomic technologies provide a comprehensive assessment of protein expression in the tumor microenvironment, facilitating the understanding of the molecular mechanisms underlying TACE response and recurrence. This knowledge allows for the identification of more effective targets for adjunctive therapy and contributes to the refinement of TACE treatment protocols. Recent research indicates that combining TACE with immunotherapy and molecular targeted therapy leads to superior efficacy and safety outcomes in clinical practice. Remarkably, patients receiving TACE in combination with camrelizumab (a monoclonal antibody targeting programmed death-1) and apatinib demonstrated significant improvements in overall survival, progression-free survival, and objective response rate compared to those undergoing TACE monotherapy [180]. H. Zhu et al.’s retrospective cohort study demonstrates that combining TACE with PD-1 and PD-L1 inhibitors and MTT significantly improves progression-free survival, overall survival, and objective response rate in patients with advanced HCC, suggesting a potentially effective treatment strategy [181]. Moreover, proteomic technologies aid in identifying biomarkers that predict the response to TACE treatment. These biomarkers help identify patients likely to benefit from TACE treatment, thereby facilitating the implementation of personalized treatment strategies. However, TACE treatment presents challenges, such as treatment resistance and disease recurrence. Proteomic technologies contribute to understanding the underlying molecular mechanisms driving these phenomena. For instance, analyzing changes in protein expression within the tumor microenvironment can reveal crucial proteins and signaling pathways involved in treatment resistance [182]. Proteomic technologies play a crucial role in researching and clinically implementing TACE treatment for liver cancer. They contribute to enhancing treatment effectiveness, predicting treatment responses, and developing personalized treatment strategies for patients.

In recent times, the treatment approach for HCC has shifted from relying solely on targeted therapy to integrating targeted therapy with immunotherapy. This integrated strategy aims to utilize targeted therapy drugs to inhibit tumor growth and angiogenesis, while also leveraging immunotherapy to stimulate or enhance the body’s immune response against the tumor. Proteomic technology has been utilized to explore the microenvironment of HCC, with a specific focus on the TME. Understanding the cellular composition and signaling pathways within the TME allows researchers to devise innovative combined treatment strategies, including simultaneously applying immune checkpoint inhibitors and targeted therapeutic drugs. Y. Xia and colleagues conducted a single-arm, open-label phase II clinical trial to assess the efficacy and safety of camrelizumab combined with apatinib in patients with resectable HCC. The findings revealed an objective response rate of 16.7% and manageable toxicity with the combination therapy. Additionally, dendritic cell infiltration was identified as a potential biomarker for treatment response and recurrence, while circulating tumor DNA (ctDNA) emerged as a promising biomarker predictive of pathological response and relapse [183]. As shown in Fig. 9, D. Zhang and colleagues’ research revealed that high expression of the mitochondrial translocator protein (TSPO) in HCC is linked to poor patient prognosis and fosters the proliferation, migration, and invasion of HCC cells. TSPO inhibits ferroptosis by modulating the Nrf2 signaling pathway and interacts with p62 to disrupt autophagy. Furthermore, TSPO upregulates PD-L1 through Nrf2-mediated transcription, thereby enhancing immune evasion in HCC. The combined administration of the TSPO inhibitor PK11195 and anti-PD-1 antibody demonstrated synergistic antitumor effects in a mouse model, indicating that targeting TSPO could provide a novel therapeutic approach for HCC treatment [184]. In a phase III trial for unresectable HCC, S. Qin et al. compared the efficacy of camrelizumab plus rivoceranib with that of sorafenib. Their findings revealed that the combination therapy significantly improved progression-free and overall survival, thus significance, particularly in disease control and rapid disease management, thereby providingpresenting a promising new treatment strategy [185]. In the multicenter phase 3 clinical trial, COSMIC-312, the efficacy of combining cabozantinib with atezolizumab as a first-line treatment for advanced HCC was assessed. The results indicated that, in comparison with traditional sorafenib treatment, the combined medications significantly enhanced patients’ progression-free survival, although no significant difference in overall survival was observed. Safety analyses revealed that adverse events related to the combination therapy were consistent with the known safety profiles of the drugs and were manageable. Despite the absence of improvement in overall survival, the combined therapy demonstrated clinical potential new treatment options for specific patient populations [186]. In the phase II RENOBATE trial, the combination of regorafenib and nivolumab was evaluated in patients with unresectable HCC. The study reported an objective response rate of 31.0%, a median progression-free survival of 7.38 months, and a one-year overall survival rate of over 80%. Single-cell RNA sequencing (scRNA-seq) revealed the molecular features underlying the immune response in patients with durable responses, offering clinical validation for the combination therapy and pinpointing potential biomarkers of immune responsiveness [187]. Proteomics research has played a crucial role in this field by uncovering the molecular heterogeneity and immune microenvironment of HCC, providing valuable insights for advancing innovative combined treatment approaches. Through comprehensive proteomic analysis, researchers can identify protein expression patterns associated with the immune therapy response in HCC patients. These findings contribute to the development of novel predictive biomarkers that can anticipate patient responses to specific treatment regimens.

A model illustrating the role of TSPO in regulating ferroptosis and anti-tumor immunity in HCC cells. TSPO interacts with p62 and stabilizes the Nrf2 protein in HCC cells, thereby promoting the expression of antioxidant genes and PD-L1, leading to the inhibition of ferroptosis and immune escape (up). However, PK11195 inhibition of TSPO can promote the ubiquitination and proteasomal degradation of Nrf2, thereby promoting ferroptosis, sensitizing to anti-PD-1 immunotherapy and enhancing CD8T cell-mediated cytotoxicity (down). Reproduced with permission [187]. Copyright 2023, John Wiley and Sons

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Proteomics research has provided us with a valuable tool to gain a profound understanding of tumor biology and the complexities of the microenvironment, thereby driving the progress of innovative therapeutic approaches in the field of HCC treatment. Specifically, proteomic techniques have revealed numerous potential therapeutic targets and biomarkers that are crucial for designing and optimizing combination therapies. In this regard, researchers have achieved a series of compelling outcomes. For instance, Y. Li et al. revealed that fatty acid synthase (FASN), through its interaction with hypoxia-inducible factor 1-alpha (HIF1α), facilitates the nuclear translocation of HIF1α, inhibits its ubiquitination and proteasomal degradation, and subsequently enhances the transcription of SLC7A11. This process increases resistance to sorafenib-induced iron-dependent apoptosis. Orlistat, a FASN inhibitor, and sorafenib show significant anti-tumor effects both in vitro and in vivo, demonstrating a synergistic anti-cancer effect when used in combination [188]. B. Zuo and colleagues developed a novel vaccine, DEXP&A2&N, by combining dendritic cell-derived exosomes with HCC-targeting peptides and immunoadjuvants. The vaccine effectively promoted the recruitment and activation of dendritic cells in mouse models, enhanced the cross-presentation of tumor neoantigens, and activated T-cell responses, resulting in significantly slowed tumor growth and tumor eradication. This development offers a new strategy for personalized immunotherapy of HCC [189]. Y. Wang et al. developed a multifunctional nanoplatform using oxygen-saturated perfluorohexane-cored liposomes modified with CXCR4 antagonist LFC131 peptides. The nanoplatform co-delivered sorafenib and the CSF1/CSF1R inhibitor PLX3397 to address sorafenib-resistant HCC. This nanoplatform alleviated hypoxia, blocked the SDF-1α/CXCR4 axis, and activated immune responses, demonstrating synergistic antitumor effects in H22 tumor-bearing mice and HCC patient-derived xenograft models [190]. B. Li et al. reveals that rhamnetin potentially enhances the sensitivity of HCC cells to sorafenib by targeting the miR-148a/PXR axis and interacting with Sirtuin 1 (SIRT1), which could improve therapeutic outcomes. The findings suggest that rhamnetin might serve as a promising therapeutic strategy for HCC by modulating the expression of key proteins and miRNAs involved in drug resistance. Clinical data also indicate that high SIRT1 and low miR-148a levels are associated with poorer prognoses in HCC patients treated with sorafenib, highlighting the clinical relevance of these molecular interactions [191]. C. Hu et al. discovered that, in a HCC mouse model, the reduction of Lactobacillus reuteri and short-chain fatty acids (SCFAs), particularly acetate, was linked to increased IL-17A-producing type 3 innate lymphoid cells (ILC3s). They found that acetate could inhibit IL-17A production by ILC3s through histone deacetylase inhibition and Sox13-mediated acetylation. The combination of acetate with PD-1/PD-L1 blockade enhanced antitumor immunity, suggesting a potential therapeutic strategy for HCC by modulating gut microbiota and SCFA levels [192]. C. Wu et al. found that HDM201 (Siremadlin), an MDM2-p53 binding antagonist, activates p53 in the RBE and SK-Hep-1 HCC cell lines, both alone and in combination with the WIP1 phosphatase inhibitor GSK2830371 [193]. Y. Jeon et al. found that combining cabozantinib with cannabidiol (CBD) enhances the therapeutic effect on HCC cells by increasing apoptosis. This enhanced apoptosis is primarily mediated through the induction of endoplasmic reticulum (ER) stress and activation of the p53 pathway. These results suggest that the combination of cabozantinib and CBD may offer a promising therapeutic strategy for HCC [194]. J. Ru et al. discovered that IRGM promotes the interaction between YBX1 and the kinase S6K1, enhancing YBX1 phosphorylation and nuclear localization. This mechanism boosts PD-L1 transcription, inhibits CD8⁺ cytotoxic T lymphocyte (CTL) infiltration and function in HCC, and drives cancer progression. A novel therapeutic combination of immune checkpoint inhibitors (ICIs) is proposed for HCC treatment [195]. Y. Zhang et al. found that focal adhesion kinase (FAK)-mediated phosphorylation at the tyrosine 464 residue of p85β, a regulatory subunit of the PI3K complex, promotes its nuclear translocation in renal cells and drives the tumorigenesis of clear cell renal cell carcinoma (ccRCC) by repressing RB1 expression through the stabilization of EZH1/EZH2. The combination of the FAK inhibitor defactinib and sunitinib offers a new therapeutic strategy for the treatment of ccRCC [196]. Y. Kim et al. developed chemical inhibitors of Sirtuin 7 (SIRT7) and investigated its role in sorafenib-acquired resistance and the underlying molecular mechanisms in HCC. They found that SIRT7 inhibition, mediated by the DDX3X-associated NLRP3 inflammasome, suppresses the phosphorylation of ERK1/2, thereby overcoming sorafenib-acquired resistance. Combining SIRT7 inhibition with sorafenib may enhance therapeutic efficacy [197]. J. Lu et al. demonstrated using forward and reverse stable isotope labeling with amino acids in cell culture-based proteomics that shikonin promotes HCC survival through the PKM2-Nrf2-BAG3 pathway. Furthermore, they found that BAG3 knockdown may enhance the anti-cancer effects of shikonin, suggesting a novel strategy for combination therapy [198]. H. Suzuki and colleagues, through proteomic analysis and clinical studies, demonstrated that among patients with HCC treated with anti-angiogenic tyrosine kinase inhibitors (TKIs), elevated serum levels of IGFBP-1 were significantly associated with poor prognosis. Their findings indicate that IGFBP-1 promotes tumor angiogenesis by activating the integrin α5β1-focal adhesion kinase pathway, resulting in TKI resistance. Experiments confirmed that reducing IGFBP-1 expression can increase HCC sensitivity to TKIs, suggesting a combined therapy of TKIs and IGFBP-1 inhibitors as a promising treatment strategy for HCC [199]. In the phase II RENOBATE trial, the combination of bavituximab and pembrolizumab was assessed as a first-line treatment for unresectable HCC. The study reported an objective response rate of 32.1%, a median progression-free survival of 6.3 months, and a favorable safety profile. Additionally, it identified a correlation between B-cell depletion and the expression of immune checkpoints within the stroma, which may indicate that the combined therapy modulates the immune response by altering the tumor microenvironment [200].

The integration of multiple treatment modalities in HCC has increasingly become a pivotal research focus. Through the integration of various treatment modalities, combination therapy seeks to improve therapeutic efficacy, overcome drug resistance, and enhance patient prognosis. Recent studies have unveiled the potential of diverse drug combinations, thereby presenting new prospects for HCC treatment. With the ongoing deepening of our understanding of the molecular mechanisms of HCC, proteomics will continue to play a pivotal role in developing innovative therapeutic approaches, offering patients enhanced and personalized treatment options.

The complexity and heterogeneity of HCC require innovative approaches to improve therapeutic efficacy and patient outcomes. The potential future directions and emerging trends of proteomics in drug development for HCC can be strategically prioritized and encompass the following aspects:

1. (1)

   Proteomic-guided precision medicine framework. Establishing a comprehensive framework that integrates proteomic data with genomic, transcriptomic, and metabolomic information is fundamental for advancing precision medicine in the context of HCC. This integrated omics approach offers a comprehensive view of HCC biology, essential for shaping tailored precision medicine strategies. Understanding the multidimensional molecular landscape of HCC is crucial for effective treatment tailoring; however, the proteomics-based precision medicine framework faces significant challenges, including complex and heterogeneous data, tool development, and the need for clinical validation. Overcoming these challenges is vital for successfully implementing the precision medicine framework in HCC treatment. To further bolster this framework's impact, future research should focus on developing methods to extract valuable information from extensive omics data and translate it into practical clinical applications, thereby integrating the precision medicine framework into routine patient care and ultimately benefiting HCC patients.

2. (2)

   Proteomic signature-guided personalized therapy. This crucial advancement follows the establishment of a comprehensive framework. Developing proteomic signatures capable of predicting responses to various HCC therapies facilitates the creation of personalized treatment plans tailored to individual patients. Utilizing machine learning algorithms to analyze proteomic data allows the identification of patterns correlating with treatment outcomes, thereby enhancing the efficiency and effectiveness of HCC treatment. Moreover, this approach aids in uncovering novel therapeutic targets and insights into new pathways involved in HCC progression and treatment resistance. It also presents opportunities for drug repurposing as our understanding of the molecular underpinnings of HCC expands. However, several challenges must be addressed for the successful implementation of this approach, including establishing a robust bioinformatics infrastructure, obtaining large, well-annotated clinical samples for model training and validation, and integrating proteomic data with other omics data types to achieve a comprehensive understanding of the disease state. With technological advancements and deepening knowledge of the proteome, proteomic signature-guided personalized therapy is anticipated to become an increasingly integral part of the clinical toolkit for managing HCC and other complex diseases.

3. (3)

   Proteomic biomarker-driven clinical trials. Employing personalized therapy signatures, designing clinical trials based on proteomic biomarkers enhances the likelihood of participant response to experimental therapy. Stratifying patients into subgroups for tailored treatment approaches using proteomic data is essential for developing more effective treatment strategies, ultimately leading to improved patient outcomes. Clinical trials based on proteomic biomarkers play a pivotal role in promoting the personalization and precision of clinical research. With the continuous advancement of proteomic technology and the application of artificial intelligence in data analysis, this approach is anticipated to significantly influence future clinical research and treatment. However, despite the substantial potential of clinical trials based on proteomic biomarkers, significant challenges exist. The process of discovering and validating biomarkers can be exceedingly complex and time-consuming, demanding highly specialized technology and resources. Additionally, integrating these biomarkers into clinical practice requires interdisciplinary collaboration and support from regulatory agencies. Addressing these challenges is crucial for the successful implementation of proteomic biomarker-driven clinical trials, as they hold promise for advancing personalized medicine and improving patient care.

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   Dynamic proteome-pharmacodynamic modeling. Dynamic proteome-pharmacodynamic modeling plays a pivotal role in advancing our understanding of HCC biology and treatment responses. Integrating proteomic changes and pharmacodynamic responses within these models is essential for enhancing our knowledge of HCC and its treatment. By elucidating the role of drugs in HCC treatment, these models enable the prediction of treatment efficacy and resistance, leading to the development of more effective treatment plans for patients. While dynamic models hold significant potential, they also face practical challenges that require attention. These challenges encompass managing the complexity of data, establishing and validating the models, and translating research findings into clinical practice. Overcoming these hurdles is crucial for the successful implementation of dynamic modeling in HCC treatment. To further advance the field, future research should concentrate on developing methods to extract valuable information from large-scale proteomic data and translate it into practical applications in clinical practice. This will facilitate the integration of dynamic modeling approaches into routine patient care, ultimately benefiting HCC patients.

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   Proteomic drug target discovery and validation in HCC. The process of identifying novel drug targets in HCC, with a specific focus on proteins showing differential expression or modification in tumor versus normal tissues, is an ongoing and parallel endeavor. Validating these targets using functional proteomic approaches, such as RNA interference or CRISPR/Cas9 technology, is crucial for uncovering new therapeutic options. The discovery and validation of drug targets guided by proteomics have the potential to significantly enhance the success rate of drug development and advance the field of personalized medicine. However, several challenges need to be addressed in this domain, including the complexity of proteomic data, optimization of high-throughput screening technologies, and the translation of basic research findings into clinically applicable strategies. To overcome these challenges, future research should prioritize improving the sensitivity and specificity of these techniques, as well as validating the efficacy of these targets in clinical settings.

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   Proteomic analysis of the tumor microenvironment. Investigating the complex proteomic landscape of various components, including immune cells, stromal cells, and extracellular matrix components, is a crucial endeavor in understanding the tumor microenvironment. This research holds immense importance as it aids in identifying potential targets for immunotherapy and stromal-targeting drugs. Moreover, analyzing the proteomic changes induced by immune checkpoint inhibitors provides valuable insights into their mechanisms of action and resistance. The field of proteomic analysis in the tumor microenvironment is multidimensional and multilevel, unraveling the intricate nature of the tumor microenvironment and providing vital information for developing innovative therapeutic targets and strategies. Advancements in technology and continued in-depth research hold the promise of revolutionizing cancer treatment through proteomic analysis. However, it is crucial to acknowledge the challenges associated with this analysis, including sample complexity, heterogeneity, and data analysis intricacies. Additionally, effectively translating basic research findings into clinical applications is necessary. To address these challenges, future research should prioritize enhancing the sensitivity and specificity of proteomic techniques and validating the effectiveness of identified targets in clinical settings. Proteomic analysis plays a pivotal role in comprehending the tumor microenvironment and has the potential to significantly advance cancer treatment. Nonetheless, addressing challenges and refining techniques are essential to fully realize the potential of proteomic analysis in this field.

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   Proteomic-guided development of nanomedicine. Utilizing proteomic insights to design nanocarriers that specifically target HCC cells or the tumor microenvironment shows great promise in enhancing drug delivery efficacy while minimizing systemic toxicity. This emerging field of nanomedicine development, guided by proteomic biomarkers, builds upon the knowledge gained from previous research. However, protein-based nanomedicine development relying on proteomics also presents several challenges that require attention. These challenges encompass the design and optimization of nanocarriers for effective targeting, the complexity of proteomics data analysis, and the translation of basic research findings into practical clinical applications. To advance the field, future research should prioritize improving the sensitivity and specificity of proteomic techniques used in nanomedicine development. Furthermore, it is crucial to validate the therapeutic relevance of these targets in a clinical setting. By addressing these challenges and refining the proteomic-guided nanomedicine approach, we can unlock its full potential in revolutionizing targeted drug delivery for HCC and other diseases.

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   Clinical translational significance. It is crucial to emphasize the translational significance of proteomic biomarkers in clinical practice. This encompasses the vital need for clinically validating these biomarkers and addressing the challenges associated with their transition from research to clinical application. Proteomic data significantly contributes to advancing personalized medicine for HCC, offering the potential for tailored treatment plans and patient stratification. Additionally, there is a critical necessity to explore integrating proteomic technologies with existing diagnostic and prognostic tools, while recognizing the regulatory obstacles and the need for accompanying clinical guidelines. Finally, ethical considerations, such as informed consent and data privacy, are paramount when managing large-scale omics data. These factors collectively shape the implementation and impact of proteomic technologies in clinical settings.

Proteomics has emerged as a powerful tool in the biomedical field, particularly in the context of HCC, offering exciting prospects for improving therapeutic outcomes and patient prognosis. However, several challenges need to be addressed, including data complexity, technological advancements, validation, and translation to clinical applications. Overcoming these obstacles is crucial to realizing the full potential of proteomics in HCC research and integrating it into routine patient care. Future research should focus on refining analytical tools, enhancing sensitivity and specificity, strengthening bioinformatics infrastructure, and developing robust data analysis models. Additionally, interdisciplinary collaboration, the availability of fully annotated clinical samples, and regulatory support are essential for successful implementation. By addressing these challenges, proteomics has the potential to revolutionize targeted drug delivery, enable personalized treatment approaches, discover new therapeutic targets, and enhance our understanding of HCC biology. The promise of proteomics in HCC research is significant, and continued advancements in techniques and methods will further propel the field, ultimately benefiting HCC patients and paving the way for advancements in personalized medicine.