A Novel Neutrophil Extracellular Traps Signature for Overall Survival Prediction and Tumor Microenvironment Identification in Gastric Cancer.

Background: Neutrophil extracellular traps (NETs) released by neutrophils are crucial for cancer development, metastasis, and can indicate gastric cancer (GC) patients' prognosis. This study reveals the relevance of NETs-related genes to GC through transcriptome analysis. Methods: We obtained transcriptome sequencing data of GC from UCSC Xena and screened prognostic NETs-related genes by GEPIA2 database. The signature for NETs was subsequently created using the LASSO-Cox regression. The clinical value of model was further explored using the nomogram and was externally validated by the GEO database. After that, we employed GO, KEGG, and GSEA enrichment analyses to evaluate the bio-functional enrichment and related pathways. Additionally, ESTIMATE, MCP counter, and ssGSEA scores were used to investigate the immunological microenvironment of GC patients. Finally, in the external cohort, neutrophil elastase (NE)-DNA complexes were measured by ELISA, and the prognostic value of NE-DNA in GC was investigated using Cox analysis. Results: Seven NETs-associated genes (PDE4B, CD93, CTSG, IL6, ELANE, KCNJ15, and CRISPLD2) were filtered to establish the signature and participated in building the nomogram. In comparison to the high-risk group, the overall survival (OS) was much longer in the low-risk group (P=0.005). The validation cohort demonstrated the acceptable predictive ability of the nomogram. The signature was enriched in biological features such as extracellular matrix organization, epithelial-mesenchymal transition and inflammatory response. Moreover, there were substantial differences in immune cell infiltration across the different risk groups (p<0.001), especially the high-risk group having more immune cells that are engaged in the antigen presentation process and associated functions. Finally, in the external cohort, NE-DNA levels were shown to be an independent factor affecting OS prognosis (p=0.006). Conclusion: Overall, this research identified a novel signature based on seven NETs-associated genes to predict prognosis and identify tumor microenvironment of GC. And high NE-DNA level may be a critical factor in the poor OS associated with NETs.

Flavonoid 4,4'-dimethoxychalcone induced ferroptosis in cancer cells by synergistically activating Keap1/Nrf2/HMOX1 pathway and inhibiting FECH.

Flavonoids are widely distributed in plants as secondary metabolites and have various biological benefits such as anti-tumor, anti-oxidant, anti-inflammatory and anti-aging. We previously reported that 4,4'-dimethoxychalcone (DMC) suppressed cancer cell proliferation by aggravating oxidative stress and inducing G2/M cell cycle arrest. In the present study, we explored the underlying mechanisms by which DMC inhibited cancer cell growth. Given that ferrochelatase (FECH) is a potential target of DMC identified by thermal proteome profiling (TPP) method, herein, we confirmed that DMC inhibited the enzymatic activity of FECH. Furthermore, we proved that DMC induced Keap1 degradation via ubiquitin-proteasome system, which led to the nuclear translocation of Nrf2 and upregulated Nrf2 targeted gene HMOX1. FECH inhibition and HMOX1 upregulation resulted in iron overload and triggered ferroptosis in cancer cells. Collectively, we revealed that DMC induced ferroptosis by synergistically activating Keap1/Nrf2/HMOX1 pathway and inhibiting FECH. Our findings indicate that FECH contributes to the non-canonical ferroptosis induction, shed light on the mechanisms of DMC inhibiting cancer cell growth, and set an example for studying biological functions of flavonoids.

High VHL Expression Reverses Warburg Phenotype and Enhances Immunogenicity in Kidney Tumor Cells.

Clear cell renal cell carcinoma (ccRCC) is a frequently occurring renal cancer. The Von Hippel-Lindau disease tumor suppressor VHL, a known tumor suppressor gene, is frequently mutated in about 50% of patients with ccRCC. However, it is unclear whether VHL influences the progression of ccRCC tumors expressing wild-type VHL. In the present study, we found that higher expression of VHL was correlated with the better disease-free survival (DFS) in ccRCC patients using The Cancer Genome Atlas (TCGA) datasets. We revealed that VHL overexpression in ccRCC cells inhibited epithelial-mesenchymal transition (EMT), sterol regulatory element-binding protein 1 (SREBP1)-regulated triglyceride synthesis, and cell proliferation. Proteomic analysis provided us a global view that VHL regulated four biological processes, including metabolism, immune regulation, apoptosis, and cell movement. Importantly, we found that VHL overexpression led to up-regulated expression of proteins associated with antigen processing and interferon-responsive proteins, thus rendering ccRCC cells more sensitive to interferon treatment. We defined an interferon-responsive signature (IRS) composed of ten interferon-responsive proteins, whose mRNA expression levels were positively correlated with DFS in ccRCC patients. Taken together, our results propose that the subset of ccRCC patients with high VHL expression benefit from immunotherapy.

CA9 Silencing Promotes Mitochondrial Biogenesis, Increases Putrescine Toxicity and Decreases Cell Motility to Suppress ccRCC Progression.

Carbonic anhydrase IX (CA9), a pH-regulating transmembrane protein, is highly expressed in solid tumors, and particularly in clear cell renal cell carcinoma (ccRCC). The catalytic mechanisms of CA9 are well defined, but its roles in mediating cell migration/invasion and survival in ccRCC remain to be determined. Here, we confirmed that the mRNA expression of CA9 in ccRCC was significantly higher than that in para-carcinoma tissues from analysis of the datasets in The Cancer Genome Atlas. CA9 knockdown upregulated oxidative phosphorylation-associated proteins and increased mitochondrial biogenesis, resulting in the reversal of the Warburg phenotype and the inhibition of cell growth. Our study revealed that CA9 knockdown upregulated mitochondrial arginase 2 (ARG2), leading to the accumulation of putrescine, which suppressed ccRCC proliferation. Surfaceomics analysis revealed that CA9 knockdown downregulated proteins associated with extracellular matrix (ECM)-receptor interaction and cell adhesion, resulting in decreased cell migration. CA9 silencing also downregulated amino acid transporters, leading to reduced cellular amino acids. Collectively, our data show that CA9 knockdown suppresses proliferation via metabolic reprogramming and reduced cell migration, reaffirming that CA9 is a potential therapeutic target for ccRCC treatment.