Integrative analysis of aging-related genes reveals CEBPA as a novel therapeutic target in non-small cell lung cancer.

BACKGROUND: To explore the impact of ARGs on the prognosis of NSCLC, and its correlation with clinicopathological parameters and immune microenvironment. Preliminary research on the biological functions of CEBPA in NSCLC. METHODS: Using consensus clustering analysis to identify molecular subtypes of ARGs in NSCLC patients; employing LASSO regression and multivariate Cox analysis to select 7 prognostic risk genes and construct a prognostic risk model; validating independent prognostic factors of NSCLC using forest plot analysis; analyzing immune microenvironment correlations using ESTIMATE and ssGSEA; assessing correlations between prognostic risk genes via qPCR and Western blot in NSCLC; measuring mRNA and protein expression levels of knocked down and overexpressed CEBPA in NSCLC using CCK-8 and EdU assays; evaluating the effects of knocked down and overexpressed CEBPA on cell proliferation using Transwell experiments; examining the correlation of CEBPA with T cells and B cells using mIHC analysis. RESULTS: Consensus clustering analysis identified three molecular subtypes, suggesting significant differential expression of these ARGs in NSCLC prognosis and clinical pathological parameters. There was significant differential expression between the two risk groups in the prognostic risk model, with P < 0.001. The risk score of the prognostic risk model was also P < 0.001. CEBPA exhibited higher mRNA and protein expression levels in NSCLC cell lines. Knockdown of CEBPA significantly reduced mRNA and protein expression levels of CEBPB, YWHAZ, ABL1, and CDK1 in H1650 and A549 cells. siRNA-mediated knockdown of CEBPA markedly inhibited proliferation, migration, and invasion of NSCLC cells, whereas overexpression of CEBPA showed the opposite trend. mIHC results indicated a significant increase in CD3 + CD4+, CD3 + CD8+, and CD20 + cell counts in the high CEBPA expression group. CONCLUSIONS: The risk score of the prognostic risk model can serve as an independent prognostic factor, guiding the diagnosis and treatment of NSCLC. CEBPA may serve as a potential tumor biomarker and immune target, facilitating further exploration of the biological functions and immunological relevance in NSCLC.

Cytoplasmic Accumulation of Histones Induced by BET Inhibition Protects Cells from C9orf72 Poly(PR)-Induced Cell Death.

Repeat dipeptides such as poly(proline-arginine) (polyPR) are generated from the hexanucleotide GGGGCC repeat expansions in the C9orf72 gene. These dipeptides are often considered as the genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In the study, fluorescein isothiocyanate (FITC) labeled PR20 is used to investigate PR20-induced cell death. The findings reveal that the cell death induced by PR20 is dependent on its nuclear distribution and can be blocked by a nuclear import inhibitor called importazole. Further investigation reveals that BRD4 inhibitors, such as JQ-1 and I-BET762, restrict cytoplasmic localization of PR20, thereby reducing its cytotoxic effect. Mechanistically, the inhibition of BRD4 leads to an increase in the expression of numerous histones, resulting in the accumulation of histones in the cytoplasm. These cytoplasmic histones associate with PR20 and limit its distribution within the nucleus. Notably, the ectopic expression of histones alone is enough to confer protection to cells treated with PR20. In addition, phenylephrine (PE) induces cellular hypertrophy and cytoplasmic distribution of histone, which also helps protect cells from PR20-induced cell death. The research suggests that temporarily inducing the presence of cytoplasmic histones may alleviate the neurotoxic effects of dipeptide repeat proteins.

Identifying a locus in super-enhancer and its resident NFE2L1/MAFG as transcriptional factors that drive PD-L1 expression and immune evasion.

Although the transcriptional regulation of the programmed death ligand 1 (PD-L1) promoter has been extensively studied, the transcription factor residing in the PD-L1 super-enhancer has not been comprehensively explored. Through saturated CRISPR-Cas9 screening of the core region of the PD-L1 super-enhancer, we have identified a crucial genetic locus, referred to as locus 22, which is essential for PD-L1 expression. Locus 22 is a potential binding site for NFE2:MAF transcription factors. Although genetic silencing of NRF2 (NFE2L2) did not result in a reduction of PD-L1 expression, further analysis reveals that MAFG and NFE2L1 (NRF1) play a critical role in the expression of PD-L1. Importantly, lipopolysaccharides (LPS) as the major component of intratumoral bacteria could greatly induce PD-L1 expression, which is dependent on the PD-L1 super-enhancer, locus 22, and NFE2L1/MAFG. Mechanistically, genetic modification of locus 22 and silencing of MAFG greatly reduce BRD4 binding and loop formation but have minimal effects on H3K27Ac modification. Unlike control cells, cells with genetic modification of locus 22 and silencing of NFE2L1/MAFG failed to escape T cell-mediated killing. In breast cancer, the expression of MAFG is positively correlated with the expression of PD-L1. Taken together, our findings demonstrate the critical role of locus 22 and its associated transcription factor NFE2L1/MAFG in super-enhancer- and LPS-induced PD-L1 expression. Our findings provide new insight into understanding the regulation of PD-L1 transcription and intratumoral bacteria-mediated immune evasion.

The 3' Non-Coding Sequence Negatively Regulates PD-L1 Expression, and Its Regulators Are Systematically Identified in Pan-Cancer.

The 3'-untranslated region (3'-UTR) of PD-L1 is significantly longer than the coding sequences (CDSs). However, its role and regulators have been little studied. We deleted whole 3'-UTR region by CRISPR-Cas9. Prognostic analysis was performed using online tools. Immune infiltration analysis was performed using the Timer and Xcell packages. Immunotherapy response prediction and Cox regression was performed using the R software. MicroRNA network analysis was conducted by the Cytoscape software. The level of PD-L1 was significantly and dramatically up-regulated in cells after deleting the 3'-UTR. Additionally, we discovered a panel of 43 RNA-binding proteins (RBPs) whose expression correlates with PD-L1 in the majority of cancer cell lines and tumor tissues. Among these RBPs, PARP14 is widely associated with immune checkpoints, the tumor microenvironment, and immune-infiltrating cells in various cancer types. We also identified 38 microRNAs whose individual expressions are associated with PD-L1 across different cancers. Notably, miR-3139, miR-4761, and miR-15a-5p showed significant associations with PD-L1 in most cancer types. Furthermore, we revealed 21 m6A regulators that strongly correlate with PD-L1. Importantly, by combining the identified RBP and m6A regulators, we established an immune signature consisting of RBMS1, QKI, ZC3HAV1, and RBM38. This signature can be used to predict the responsiveness of cancer patients to immune checkpoint blockade treatment. We demonstrated the critical role of the 3'-UTR in the regulation of PD-L1 and identified a significant number of potential PD-L1 regulators across various types of cancer. The biomarker signature generated from our findings shows promise in predicting patient prognosis. However, further biological investigation is necessary to explore the potential of these PD-L1 regulators.

NF-κB: a mediator that promotes or inhibits angiogenesis in human diseases?

The nuclear factor of κ-light chain of enhancer-activated B cells (NF-κB) signaling pathway, which is conserved in invertebrates, plays a significant role in human diseases such as inflammation-related diseases and carcinogenesis. Angiogenesis refers to the growth of new capillary vessels derived from already existing capillaries and postcapillary venules. Maintaining normal angiogenesis and effective vascular function is a prerequisite for the stability of organ tissue function, and abnormal angiogenesis often leads to a variety of diseases. It has been suggested that NK-κB signalling molecules under pathological conditions play an important role in vascular differentiation, proliferation, apoptosis and tumourigenesis by regulating the transcription of multiple target genes. Many NF-κB inhibitors are being tested in clinical trials for cancer treatment and their effect on angiogenesis is summarised. In this review, we will summarise the role of NF-κB signalling in various neovascular diseases, especially in tumours, and explore whether NF-κB can be used as an attack target or activation medium to inhibit tumour angiogenesis.