Brain metastasis (BM) is common in patients with non-small cell lung cancer (NSCLC) and is associated with a poor prognosis. Ceramide synthase 1 (CERS1) participates in malignancy development, but its potential role in NSCLC BM remains unclear. This study aimed to explore the physiological effects and molecular mechanism of CERS1 in NSCLC BM. CERS1 expression was evaluated in NSCLC tissues and cell lines, and its physiological roles were subsequently explored in vivo and in vitro. Mass spectrometry and co-immunoprecipitation were performed to explore CERS1-interacting proteins. The associated signaling pathways of CERS1 in NSCLC BM were further investigated using bioinformatics analysis and molecular biotechnology. We demonstrated that CERS1 was significantly downregulated in NSCLC cell lines and BM tissues, and its upregulation was associated with better prognoses. In vitro, CERS1 overexpression inhibited cell migration, invasion, and the ability to penetrate the blood-brain barrier. Moreover, CERS1 interacted with ubiquitin-specific protease 14 (USP14) and inhibited BM progression by downregulating the PI3K/AKT/mTOR signaling pathway. Further, CERS1 expression substantially suppressed BM tumor formation in vivo. This study demonstrated that CERS1 plays a suppressor role in NSCLC BM by interacting with USP14 and downregulating the PI3K/AKT/mTOR signaling pathway, thereby serving as a novel therapeutic target for NSCLC BM.

Background: The increase in carbapenem-resistant Klebsiella pneumoniae (CRKP), especially the emergence of tigecycline-resistant K. pneumoniae (KP), is a serious public health concern. However, the underlying mechanism of tigecycline resistance is unclear. In this study, we evaluated the role of the CusS-CusR two-component system (TCS), which is associated with copper/silver resistance, in tigecycline resistance in CRKP. Methods: Following the in vitro evolution of tigecycline-resistant KP, the minimum inhibitory concentrations of tigecycline were determined using the micro-broth dilution method. RNA sequencing and data analysis were performed to identify differentially expressed genes. Quantitative PCR (qPCR) was performed to verify the genes of interest. Genes associated with tigecycline resistance, such as ramR, tex (T), and tet (A), were detected by PCR, and then mutants were confirmed by sequencing. Additionally, the efflux pump-associated genes soxS, oqxA, oqxB, acrE, and acrF were also analyzed by qPCR. CusR was deleted and complemented by the suicide vector pKO3-Km plasmid and pGEM-T-easy plasmid, respectively. Results: Nine strains of KP were evaluated in our study. Strains A2 and A3 were evolved from A1, B2, and B3 were evolved from B1, and C2 and C3 were evolved from C1. The tigecycline minimum inhibitory concentration for A1, B1, and C1 was 0.5 μg/mL; that for A2, B2, and C3 was 16.0 μg/mL; and that for A3, B3, and C3 was 32.0 μg/mL. RNA-sequencing and qPCR confirmed that the differentially expressed genes cusE, cusS, cusR, cusC, cusF, cusB, and cusA showed higher expression in C2 and C3 than in C1. Genes related to the efflux pump AcrAB-TolC showed higher expression in B2 and B3 than in B1. No mutants of ramR, tex (T), or tet (A) were detected. SoxS, oqxA, oqxB, acrE, and acrF did not show increased expression in any group. After deletion and complementation of cusR among C3, the MIC of tigecycline decreased to 4 μg/mL, and then recovered to 32 μg/mL. The expression of cusFBCA, correspondingly decreased and increased significantly. Conclusion: In addition to its primary function in resistance to copper/silver, the CusS-CusR two-component system is associated with CRKP resistance to tigecycline.

Background: Lung cancer has the highest case fatality rate among cancers because of uncontrolled proliferation and early metastasis of cancer cells in the lung tissue. This study aimed to clarify the role of the non-SMC condensin I complex, subunit G (NCAPG) in lung adenocarcinoma (LUAD), explore the mechanisms of its progression, and lay the foundation for the search for new biological markers. Methods: We analyzed overlapping differentially expressed genes (DEGs) from three datasets; a protein–protein interaction (PPI) network was subsequently constructed and analyzed using Cytoscape. We then selected NCAPG for validation because of its poor prognosis and because it has not been sufficiently studied in the context of LUAD. Immunohistochemical analysis was used to detect the expression of NCAPG in LUAD tissues, and the relationships between NCAPG and clinical parameters were analyzed. In vitro and in vivo experiments were conducted to verify the role of NCAPG in LUAD. Finally, we studied the specific mechanism of action of NCAPG in LUAD. Results: Through comprehensive analysis of the GSE43458, GSE75037, and The Cancer Genome Atlas databases, we identified 517 overlapping DEGs. Among them, NCAPG was identified as a hub gene. Immunohistochemical analysis revealed that NCAPG was strongly associated with the clinical stage, M-classification, and N-classification. Univariate and multivariate Cox regression analyses indicated that NCAPG was a prognostic risk factor for LUAD, while the in vitro experiments showed that NCAPG overexpression promoted proliferation, migration, invasion, and epithelial-mesenchymal transition. Furthermore, knockdown of NCAPG inhibited LUAD progression, both in vitro and in vivo. Mechanistically, NCAPG overexpression increased p-Smad2 and p-Smad3 expressions in the transforming growth factor β (TGF-β) signaling pathway. Additionally, rescue experiments indicated that TGF-β signaling pathway inhibitors could restore the effect of NCAPG overexpression in LUAD cells. Conclusions: NCAPG may promote proliferation and migration via the TGF-β signaling pathway in LUAD.

Background: Ceramide metabolism is crucial in the progress of brain metastasis (BM). However, it remains unexplored whether targeting ceramide metabolism may arrest BM. Methods: RNA sequencing was applied to screen different genes in primary and metastatic foci and whole-exome sequencing (WES) to seek crucial abnormal pathway in BM + and BM-patients. Cellular arrays were applied to analyze the permeability of blood–brain barrier (BBB) and the activation or inhibition of pathway. Database and Co-Immunoprecipitation (Co-IP) assay were adopted to verify the protein–protein interaction. Xenograft and zebrafish model were further employed to verify the cellular results. Results: RNA sequencing and WES reported the involvement of RPTOR and ceramide metabolism in BM progress. RPTOR was significantly upregulated in BM foci and increased the permeability of BBB, while RPTOR deficiency attenuated the cell invasiveness and protected extracellular matrix. Exogenous RPTOR boosted the SPHK2/S1P/STAT3 cascades by binding YY1, in which YY1 bound to the regions of SPHK2 promoter (at -353 ~ -365 nt), further promoting the expression of SPHK2. The latter was rescued by YY1 RNAi. Xenograft and zebrafish model showed that RPTOR blockade suppressed BM of non-small cell lung cancer (NSCLC) and impaired the SPHK2/S1P/STAT3 pathway. Conclusion: RPTOR is a key driver gene in the brain metastasis of lung cancer, which signifies that RPTOR blockade may serve as a promising therapeutic candidate for clinical application.

Background: Lung squamous cell carcinoma (LUSC) is a major subtype of non-small cell lung cancer. The tumor immune microenvironment (TIME) affects the anti-tumor immune response and the patient’s prognosis, although the TIME in LUSC patients is incompletely understood.Methods: We retrospectively collected surgical specimens from patients with previously untreated primary LUSC. Histopathological examination was used to identify tumor regions and adjacent regions, and imaging mass cytometry was used to characterize the immune cells in those regions. The results were compared between regions and between patients.Results: We identified heterogeneity in the TIME on comparing different patients with LUSC, although the tumor region and adjacent region both exhibited an immune response to the tumor. The TIME typically included a large number of infiltrating and activated T-cells, especially CD8⁺ T-cells, which closely interacted with the tumor cells in the tumor region. There was limited infiltration of B-cells, NK cells, and NKT cells, while the major immune suppressor cells were CD33⁺ myeloid-derived cells. We also identified a novel population of CD3⁻CD4⁺ cells with high expression of Foxp3 and TNFα, which might modulate the tumor microenvironment and play a proinflammatory role in the TIME.Conclusions: The TIME of LUSC appears to be immunogenic and heterogenous, with predominant infiltration of activated CD8⁺ T-cells. The interactions between the tumor cells and T-cells facilitate the anti-tumor activity. A novel subpopulation of CD3⁻CD4⁺ cells with high TNFα and Foxp3 expression may modulate the tumor microenvironment and play a proinflammatory role.