Dongyao Wang

Hepatocellular carcinoma, the third leading cause of cancer‐related deaths globally, presents a critical public health burden in China due to its high incidence and mortality. While targeted therapies and immunotherapies have improved survival in advanced HCC, drug resistance remains a major therapeutic challenge. Recent studies suggest that gefitinib, an EGFR inhibitor, overcomes lenvatinib resistance, yet its mechanistic underpinnings are incompletely understood. To investigate gefitinib's metabolic effects in HCC, we conducted untargeted metabolomic profiling using two separate platforms: gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) with both hydrophilic interaction liquid chromatography (HILIC) and reversed‐phase modes. Raw data were processed by Mass Hunter, normalized with internal standards, and analyzed via SIMCA for pattern recognition. Principal component analysis (PCA) of quality control samples and experimental groups ( n = 6 each) confirmed system stability and clear inter‐group separation. Orthogonal projections to latent structures discriminant analysis models were validated by 200 permutation tests. Analysis identified 42 metabolites with VIP > 1, of which 25 showed significant alterations ( p < 0.05) post‐gefitinib treatment. KEGG/RaMP‐DB enrichment revealed perturbations in four key pathways: arginine‐proline metabolism, nitrogen metabolism, branched‐chain amino acid biosynthesis, and taurine metabolism. These results delineate gefitinib‐induced metabolic reprogramming in HCC cells, providing a foundation for targeting metabolic vulnerabilities to overcome therapy resistance.

Inhibiting the malignant proliferation is crucial for managing the cancer progression. NCAPG (Non‐SMC condensin I complex subunit G) is a main component in cell proliferation, which is highly expressed in liver tumors and negatively correlated with the overall survival of hepatocellular carcinoma (HCC) patients. In this study, we identified bufalin as a molecular glue for the first time that specifically degraded NCAPG by coupling it to cathepsin V (CTSV). Distinguished from traditional PROTACs, bufalin served as a molecular glue with the distinct advantages of a smaller molecular weight and superior bioavailability. This degradation inhibited the proliferation of HCC cells by inducing G2/M phase cell cycle arrest at low doses (20–40 nM), but did not trigger the apoptosis signaling. Using co‐immunoprecipitation and confocal microscopy, we confirmed the interaction between CTSV and NCAPG by bufalin. Knockdown of CTSV or NCAPG attenuated the anti‐proliferation effects of bufalin in HCC cells. Cell cycle analysis also showed significantly reduced G2/M arrest in knockdown cells. The downstream proliferation regulators Cyclin D1 and CDK1 were also regulated by bufalin in a CTSV/NCAPG‐dependent manner. Our study not only identified the CTSV‐NCAPG complex as a novel therapeutic target but also discovered a potential molecular glue, bufalin, which exerts anti‐proliferation effects through cell cycle regulation. These findings highlight the potential clinical translational value of bufalin as a promising, bioavailable therapeutic agent for the targeted treatment of liver cancer.

Background: Tumor cells manipulate the tumor-associated antigens presentation to escape immune surveillance; however, the molecular mechanism is not exactly clear and the measure to intervene is missing. Methods: Annexin A2 was knockout by the CRISPR-Cas9 or blocked by the small-molecule matrine, PY60, and hexapeptide. Chemically and genetically induced primary liver cancer models, and the orthotopically implanted liver tumor model were used. Tumor immune environment was analyzed by single-cell sequencing. Annexin A2-interacted proteins and tumor-associated antigens were identified by co-immunoprecipitation coupled with liquid chromatography with tandem mass spectrometry. Tumor cells killing effects were evaluated by co-culture of tumor cells and CD8 ⁺ T cells. Results: Targeting Annexin A2 effectively suppressed the progression of liver cancer. The immunosuppressive microenvironment was improved by Annexin A2 inhibition in tumor tissues. The CD8 ⁺ T cells were increased and activated by targeting Annexin A2. Mechanistically, targeting Annexin A2 inhibited its combination with HSP90. The HSP90-mediated tumor-associated antigens presentation was recovered, and the major histocompatibility complex I-presented short peptides were changed, increasing the tumor cells killing by CD8 ⁺ T cells. Interestingly, Annexin A2 was increased in liver cancer tissues and the overall survival was significantly reduced in patients with high expression. However, Annexin A2 was positively correlated with immune cell infiltration in liver cancer, implying that Annexin A2 was used by tumor cells for immune escape and immunotherapy resistance. Hence, we further confirmed that blocking Annexin A2 increased the therapeutic effects of anti-programmed cell death protein-1 both in vitro and in vivo. Conclusions: Taken together, our results identified the role of Annexin A2 in the tumor-associated antigens presentation and immune evasion, which could be an actionable target in cancer immunotherapy.

Tamoxifen is an inhibitor of estrogen receptors and was originally developed for breast cancer therapy. Besides, tamoxifen is widely used for Cre‐estrogen receptor‐mediated conditional knockout in transgenic mice. However, we found that the 3‐month feeding of 0.5 g/kg tamoxifen diet dramatically lowered the body weight of mice. The liver fat content and the serum lipid indicators were all decreased. But the liver injuries were identified, as illustrated by liver/body ratio and serum ALT and AST levels. The Sirius red staining and α‐SMA staining even showed fibrosis in the liver. The increased lipid peroxidation indicators MDA and LPO, and ferroptosis markers COX‐2, GPX4, SLC7A11, and ACSL4 implied the tamoxifen‐induced ferroptosis in the liver. We further found that tamoxifen induced hepatic iron deposition. The investigation of iron transporters found that tamoxifen upregulated ferric iron reductase STEAP3, ferrous iron transporter DMT1, and iron storage molecule ferritin, which were probably the reasons for tamoxifen‐induced iron deposition. The downregulation of the transferrin receptor and upregulation of hepcidin were more likely the responses to iron deposition. In conclusion, we found that tamoxifen disturbed the iron metabolism and induced liver injuries and ferroptosis, warranting attention to the applications of tamoxifen in cancer therapy and conditional gene knockout.

Hepatocellular carcinoma (HCC) is a significant global health concern. However, there are limited effective treatments available for it. The use of natural products in the management and treatment of HCC is gaining more attention. Baicalein is a flavonoid compound that has been reported to have antitumor activities in HCC. However, the direct binding targets of baicalein are still unknown. Therefore, we used the DNA‐programmed affinity labeling method to identify the target of baicalein and validated its function in HCC cells. We set blank and competitive DNA probes as negative controls. The results showed that baicalein had 136 binding targets, of which 13 targets were differently expressed in HCC tissues. The enriched cellular process of these targets was apoptosis, which involved MAPK9. We tested the binding affinity of baicalein with MAPK9 as 89.7 nM (Kd) by surface plasmon resonance and analyzed the binding sites by virtual docking. Notably, the binding of baicalein with MAPK9 increased the protein levels of MAPK9 itself and the related downstream apoptosis signaling, triggering the apoptosis of HCC cells. However, the inhibitor of MAPK9, SP600125, blocked the baicalein‐induced apoptosis, and the amounts of MAPK9 and downstream molecules were also decreased, indicating that baicalein acted through MAPK9 to induce apoptosis of HCC cells. In conclusion, we used the DNA‐programmed affinity labeling method to identify the direct‐binding target MAPK9 of baicalein and validated its function in baicalein‐induced apoptosis of HCC cells, which would be helpful to understand and use baicalein in HCC therapy.

Identifying biologically active ligands for membrane proteins is an important task in chemical biology. We report an approach to directly identify small molecule agonists against membrane proteins by selecting DNA-encoded libraries (DELs) on live cells. This method connects extracellular ligand binding with intracellular biochemical transformation, thereby biasing the selection toward agonist identification. We have demonstrated the methodology with three membrane proteins: epidermal growth factor receptor (EGFR), thrombopoietin receptor (TPOR), and insulin receptor (INSR). A ∼30 million and a 1.033 billion-compound DEL were selected against these targets, and novel agonists with subnanomolar affinity and low micromolar cellular activities have been discovered. The INSR agonists activated the receptor by possibly binding to an allosteric site, exhibited clear synergistic effects with insulin, and activated the downstream signaling pathways. Notably, the agonists did not activate the insulin-like growth factor 1 receptor (IGF-1R), a highly homologous receptor whose activation may lead to tumor progression. Collectively, this work has developed an approach toward "functional" DEL selections on the cell surface and may provide a widely applicable method for agonist discovery for membrane proteins.

The active ingredients of Traditional Chinese Medicine are an important source of bioactive molecules and play an important role in the research and development of innovative drugs. FA-30, which is a derivative of natural product ferulic acid, inhibited cervical cancer cell proliferation and induced apoptosis as well. To understand the underlying mechanisms of FA-30, a complementary multi-omics study was conducted. Cysteine and methionine metabolism and aminoacyl-tRNA biosynthesis pathways were significantly changed both at the metabolic level and proteomic level. This may help us to get a better understanding of cervical cancer and FA-30 at the same time.