Colorectal cancer (CRC) is a growing public health concern due to its high mortality rate. Currently, there is a lack of valid diagnostic biomarkers and few therapeutic strategies are available for CRC treatment, especially for advanced CRC whose underlying pathogenic mechanisms remain poorly understood. In the present study, we investigated the serum samples from 20 patients with stage III or IV advanced CRC using data-independent acquisition (DIA)-based proteomics and ultra-performance liquid chromatography coupled to time-of-flight tandem mass spectrometry (UPLC-TOF-MS/MS) metabolomics techniques. Overall, 551 proteins and 719 metabolites were identified. Hierarchical clustering analysis revealed that the serum proteomes of advanced CRC are more diversified than the metabolomes. Ten biochemical pathways associated with cancer cell metabolism were enriched in the detected proteins and metabolites, including glycolysis/gluconeogenesis, biosynthesis of amino acids, glutathione metabolism, and arachidonic acid metabolism, etc. A protein-protein interaction network in advanced CRC serum was constructed with 80 proteins and 21 related metabolites. Correlation analysis revealed conserved roles of lipids and lipid-like molecules in a regulatory network of advanced CRC. Three metabolites (hydroquinone, leucenol and sphingomyelin) and two proteins (coagulation factor XIII A chain and plasma kallikrein) were selected to be potential biomarkers for advanced CRC, which are positively and significantly correlated with CEA and/or CA 19–9. Altogether, the results expanded our understanding of the physiopathology of advanced CRC and discovered novel potential biomarkers for further validation and application to improve the diagnosis and monitoring of advanced CRC.

Successful viral replication entails elimination or bypass of host antiviral mechanisms. Here, we show that shRNA-mediated knockdown of murine double minute (Mdm2) and its paralog Mdm4 enhanced the expression of early and late viral gene products during adenovirus (HAdV) infection. Remarkably, whereas the expression of HAdV genes was low in p53-deficient mouse embryonic fibroblasts (p53KO MEFs), the HAdV early gene products were efficiently expressed in Mdm2/p53 double-knockout (DKO) and Mdm4/p53 DKO MEFs, and viral capsid proteins were produced in Mdm2/p53 DKO MEFs. Thus, Mdm2 and Mdm4 seem to have potent antiviral property. In cells infected with wt HAdV or a mutant virus lacking the E1B-55K gene (dl1520), both Mdm2 and Mdm4 were rapidly depleted, whereas replication-deficient mutant viruses (Ad-GFP) or ΔpTP with deletions within the coding sequence of preterminal binding protein failed to induce their downregulation. Reduced expression of Mdm2 and Mdm4 was not due to general shutoff of host protein synthesis. Additionally, expression of a dominant-negative mutant of Cul5 did not affect Mdm2/Mdm4 downregulation. Thus, viral replication but not the presence of E1B-55K is required for Mdm2/Mdm4 degradation. Surprisingly, treatment of HAdV-infected cells with proteasome inhibitor MG132 only partially restored the protein levels of Mdm2 and Mdm4, suggesting that they may also be downregulated through an additional mechanism independent of proteasome. Interestingly, cyclin D1 and p21 appear to be downregulated similarly during HAdV infection. Collectively, our work provides the first biochemical evidence for antiviral function of Mdm2 and Mdm4 and that viruses employ efficient countermeasure to ensure viral replication.

Background/Aims: Induction of oxidative stress and reactive oxygen species (ROS) mediated-apoptosis have been utilized as effective strategies in anticancer therapy. Macranthoidin B (MB) is a potent inducer of ROS-mediated apoptosis in cancer, but its mechanism of action is poorly understood. Method: Superoxide production with MB exposure in colorectal cancer (CRC) cells was measured using lucigenin chemiluminescence and real-time PCR. MB’s inhibitory effect on proliferation and viability of CRC cells was determined by proliferation assays. MB’s effect on apoptosis of CRC cells was determined by Western blotting and annexin V-FITC/PI staining. MB’s effect on the growth of CRC xenografts in mice was assessed. An established metabolomics profiling platform combining ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS) with gas chromatography-mass spectrometry (GC-MS) was performed to determine MB’s effect on total metabolite variation in CRC cells. Results: We found that MB increases ROS generation via modulating key metabolic pathways. Using metabolomics profiling platform combining LC-MS with GC-MS, a total of 236 metabolites were identified in HCT-116 cells in which 31 metabolites were determined to be significantly regulated (p ≤ 0.05) after MB exposure. A number of key metabolites revealed by metabolomics analysis include glucose, fructose, citrate, arginine, phenylalanine, and S-adenosylhomocysteine (SAH), suggesting specific modulation of metabolism on carbohydrates, amino acids and peptides, lipids, nucleotide, cofactors and vitamins in HCT-116 CRC cells with MB treatment highly associated with apoptosis triggered by enhanced ROS and activated caspase-3. Conclusion: Our results demonstrate that MB represses CRC cell proliferation by inducing ROS-mediated apoptosis.

Paired immunoglobin-like type 2 receptor beta (PILRB) mainly plays a crucial role in regulating innate immunity, but whether PILRB is involved in cancer is poorly understood. Here, we report that PILRB potentiates the PI3K/AKT pathway to drive gastric tumorigenesis by binding and stabilizing IRS4, which could hyperactivate the PI3K/AKT pathway. Firstly, the levels of PILRB are upregulated in human gastric cancer (GC) specimens and associated with poor prognosis in patients with GC. In addition, our data show that PILRB promotes cell proliferation, colony formation, cell migration and invasion in GC cells in vitro and in vivo. Mechanistically, PILRB recruits the deubiquitination enzymes OTUB1 to IRS4 and relieves K48-linked ubiquitination of IRS4, protecting IRS4 protein from proteasomal-mediated degradation and subsequent activation of the PI3K/AKT pathway. Importantly, the levels of PILRB are positively correlated with IRS4 in GC specimens. Meanwhile, we also found that PILRB reprogrammed cholesterol metabolism by altering ABCA1 and SCARB1 expression levels, and PILRB-expression confers GC cell resistance to statin treatment. Taken together, our findings illustrate that the oncogenic role of PILRB in gastric tumorigenesis, providing new insights into the regulation of PI3K/AKT signaling in GC and establishing PILRB as a biomarker for simvastatin therapy resistance in GC.

Alterations in copy number are crucial genetic events in the development of esophageal squamous cell carcinoma (ESCC). Here, we show that Tocopherol alpha transfer protein-like (TTPAL) is highly amplified and frequently overexpressed in human ESCC. Using Ttpal-KO mouse mode, we demonstrate that TTPAL promotes ESCC cell proliferation and accelerates tumor development by driving cholesterol biosynthesis. Mechanistically, TTPAL upregulates a key transcription factor in cholesterol biosynthesis-sterol regulatory element-binding transcription factor (SREBP2) in ESCC cells. TTPAL interacts with the RNA methyltransferase NSUN2 and relieves the ubiquitination of NSUN2, protecting NSUN2 from proteasome-mediated degradation. In turn, NSUN2 catalyzes the m5C modification of SREBP2 mRNA, and then the m5C modified SREBP2 mRNA binds to the m5C reader protein-ALYREF to enhance its stability, thereby increasing SREBP2 expression. Moreover, we validate the efficacy of cholesterol biosynthesis inhibitor simvastatin in ESCC with high TTPAL expression. Overall, our results uncover a novel function of TTPAL in regulating SREBP2 expression, revealed a previously unknown TTPAL/NSUN2/SREBP2 pathway that promotes cholesterol biosynthesis in ESCC cells, and identified sensitively to cholesterol biosynthesis inhibitor simvastatin.

Background: Hedysari Radix (HR), commonly known as Hong-Qi in Chinese, is a traditional Chinese herbal medicine recognized for possessing anti-inflammatory and anti-tumor properties. While the polysaccharides in HR have been extensively studied, other HR metabolites and their potential anti-tumor properties remain largely unknown. Methods: We employed a multi-omics strategy integrating metabolomics, network analysis, proteomics, phosphoproteomics, and molecular docking to identify HR metabolites with anti-colorectal cancer (CRC) property and investigate underlying mechanisms. Results: Using mass spectrometry-based metabolomics, we identified 1,292 metabolites across eight processed HR products. Key metabolites including medicarpin, formononetin, naringenin, and quercetin were validated via the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Notably, formononetin-derived metabolites were significantly enriched during HR processing. The metabolite-metabolite correlation analysis revealed key compounds such as flavonoids and formononetin. Subsequent network analysis combined with label-free data-independent acquisition (DIA) proteomics and phosphoproteomics in colon cancer cells identified 194 potential targets, 291 differentially expressed proteins, and 1,535 phosphorylated proteins that were regulated by formononetin. Cell-surface enzymes carbonic anhydrase IX (CA9) and membrane metalloendopeptidase (MME) were consistently identified in different analyses as key targets, and molecular docking results confirmed their strong binding to formononetin. Bioinformatics analyses further revealed significant enrichment of cancer-associated pathways, including PI3K-Akt, Hippo, HIF-1 signaling, and cholesterol metabolism upon formononetin treatment. Conclusion: The findings provide novel insights into the HR metabolome and reveal the multi-targeting roles of formononetin in CRC development, laying the foundation for developing new CRC therapeutic strategies.