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Lung cancer is the most common cause of cancer-related death worldwide, less than 7% of patients survive 10 years following diagnosis across all stages of lung cancer. Late stage of diagnosis and lack of effective and personalized medicine reflect the need for a better understanding of the mechanisms that underlie lung cancer progression. Quantitative proteomics provides the relative different protein abundance in normal and cancer patients which offers the information for molecular interactions, signaling pathways, and biomarker identification. Here we introduce both theoretical and practical applications in the use of quantitative proteomics approaches, with principles of current technologies and methodologies including gel-based, label free, stable isotope labeling as well as targeted proteomics. Predictive markers of drug resistance, candidate biomarkers for diagnosis, and prognostic markers in lung cancer have also been discovered and analyzed by quantitative proteomic analysis. Moreover, construction of protein networks enables to provide an opportunity to interpret disease pathway and improve our understanding in cancer therapeutic strategies, allowing the discovery of molecular markers and new therapeutic targets for lung cancer.

C1GALT1 controls the crucial step of GalNAc-type O-glycosylation and is associated with both physiologic and pathologic conditions, including cancers. EPH receptors comprise the largest family of receptor tyrosine kinases (RTKs) and modulate a diverse range of developmental processes and human diseases. However, the role of C1GALT1 in the signaling of EPH receptors remains largely overlooked. Here, we showed that C1GALT1 high expression in gastric adenocarcinomas correlated with adverse clinicopathologic features and is an independent prognostic factor for poor overall survival. Silencing or loss of C1GALT1 inhibited cell viability, migration, invasion, tumor growth and metastasis, as well as increased apoptosis and cytotoxicity of 5-fluorouracil in AGS and MKN45 cells. Phospho-RTK array and western blot analysis showed that C1GALT1 depletion suppressed tyrosine phosphorylation of EPHA2 induced by soluble Ephrin A1-Fc. O-glycans on EPHA2 were modified by C1GALT1 and both S277A and T429A mutants, which are O-glycosites on EPHA2, dramatically enhanced phosphorylation of Y588, suggesting that not only overall O-glycan structures but also site-specific O-glycosylation can regulate EPHA2 activity. Furthermore, depletion of C1GALT1 decreased Ephrin A1-Fc induced migration and reduced Ephrin A1 binding to cell surfaces. The effects of C1GALT1 knockdown or knockout on cell invasiveness in vitro and in vivo were phenocopied by EPHA2 knockdown in gastric cancer cells. These results suggest that C1GALT1 promotes phosphorylation of EPHA2 and enhances soluble Ephrin A1-mediated migration primarily by modifying EPHA2 O-glycosylation. Our study highlights the importance of GalNAc-type O-glycosylation in EPH receptor-regulated diseases and identifies C1GALT1 as a potential therapeutic target for gastric cancer.

Core 1 β1,3-galactosyltransferase (C1GALT1) controls the crucial step of GalNAc-type O-glycosylation and is overexpressed in various human malignancies. However, its role in head and neck squamous cell carcinoma (HNSCC) remains unclear. Here we demonstrate that C1GALT1 expression is upregulated in HNSCC tumors and is associated with adverse clinicopathologic features. Moreover, high C1GALT1 expression predicts poor disease-free and overall survivals. C1GALT1 overexpression enhances HNSCC cell viability, migration, and invasion, which can be reversed by erlotinib. Silencing of C1GALT1 suppresses the malignant behavior both in vitro and in vivo. Mass spectrometry and lectin pull-down assays demonstrate that C1GALT1 modifies O-glycans on EGFR. Blocking O-glycan elongation on EGFR by C1GALT1 knockdown decreases EGF-EGFR binding affinity and inhibits EGFR signaling, thereby suppressing malignant phenotypes. Using molecular docking simulations, we identify itraconazole as a C1GALT1 inhibitor that directly binds C1GALT1 and promotes its proteasomal degradation, leading to significant blockade of C1GALT1-mediated effects in HNSCC cells in vitro and in vivo. Collectively, our findings demonstrate a critical role of O-glycosylation in HNSCC progression and highlight the therapeutic potential of targeting C1GALT1 in HNSCC treatment.

Background Tumor-specific CD8 +  T lymphocytes play a critical role in anticancer immunity but frequently become dysfunctional and exhausted within the immunosuppressive tumor microenvironment. Although immune checkpoint inhibitors can restore T-cell activity, resistance to these treatments remains a significant challenge. Therefore, understanding the transcriptional and regulatory mechanisms underlying CD8 + T-cell exhaustion is crucial for the development of effective therapies. Methods We developed two murine models of acquired immune checkpoint inhibitor resistance through prolonged anti-PD1 treatment. To gain insight into CD8 + T-cell exhaustion, we performed single-cell multiomics analysis, including both scRNA-seq and scATAC-seq, to capture gene expression profiles and chromatin accessibility. Moreover, we collected three external datasets to validate the results in silico. We further assessed the therapeutic potential of Runx2 through marker expression and cytotoxicity assays. Results Our single-cell analysis revealed distinct T-cell subsets, including early and terminally exhausted populations, along with their exhaustion trajectories. Runx2 was identified as a key transcription factor associated with CD8 + T-cell exhaustion in both models and correlated with immunotherapy response in clinical data. Additionally, functional marker expression and cytotoxicity assays demonstrated that inhibiting Runx2 improved CD8 +  T-cell cytotoxicity. Conclusions These findings highlight the role of Runx2 as a crucial regulator of CD8 + T-cell exhaustion in the context of prolonged immune checkpoint inhibitor treatment. Targeting Runx2 may provide a novel strategy to overcome immune checkpoint inhibitor resistance and enhance therapeutic efficacy, offering promising avenues for combination therapies.

Increased levels of dysfunctional mitochondria within skeletal muscle are correlated with numerous age‐related physiopathological conditions. Improving our understanding of the links between mitochondrial function and muscle proteostasis, and the role played by individual genes and regulatory networks, is essential to develop treatments for these conditions. One potential player is the mitochondrial outer membrane protein Fis1, a crucial fission factor heavily involved in mitochondrial dynamics in yeast but with an unknown role in higher‐order organisms. By using Drosophila melanogaster as a model, we explored the effect of Fis1 mutations generated by transposon Minos‐mediated integration. Mutants exhibited a higher ratio of damaged mitochondria with age as well as elevated reactive oxygen species levels compared with controls. This caused an increase in oxidative stress, resulting in large accumulations of ubiquitinated proteins, accelerated muscle function decline, and mitochondrial myopathies in young mutant flies. Ectopic expression of Fis1 isoforms was sufficient to suppress this phenotype. Loss of Fis1 led to unbalanced mitochondrial proteostasis within fly muscle, decreasing both flight capabilities and lifespan. Fis1 thus clearly plays a role in fly mitochondrial dynamics. Further investigations into the detailed function of Fis1 are necessary for exploring how mitochondrial function correlates with muscle health during aging. Ubiquitinated proteins and autophagic proteins accumulate within Drosophila melanogaster muscle fibers as they age. Here we generated Drosophila mutants for the gene Fis1, which influences mitochondrial dynamics, and observed similar phenotypes in these transgenic lines. The unbalanced proteostasis resulting from Fis1 mutation led to muscle fiber degeneration and reduced health spans. Fis1‐related mitochondrial quality control is an important factor in determining Drosophila muscle, and thus overall, health.

Background Interstitial lung disease (ILD) is the primary cause of mortality in systemic sclerosis (SSc), an autoimmune disease characterized by tissue fibrosis. SSc-related ILD (SSc-ILD) occurs more frequently in females aged 30–55 years, whereas idiopathic pulmonary fibrosis (IPF) is more prevalent in males aged 60–75 years. SSc-ILD occurs earlier than IPF and progresses rapidly. FCN1, FABP4, and SPP1 macrophages are involved in the pathogenesis of lung fibrosis; SPP1 macrophages demonstrate upregulated expression in both SSc-ILD and IPF. To identify the differences between SSc-ILD and IPF using single-cell analysis, clarify their distinct pathogeneses, and propose directions for prevention and treatment. Methods We performed single-cell RNA sequencing on NCBI Gene Expression Omnibus (GEO) databases GSE159354 and GSE212109, and analyzed lung tissue samples across healthy controls, IPF, and SSc-ILD. The primary measures were the filtered genes integrated with batch correction and annotated cell types for distinguishing patients with SSc-ILD from healthy controls. We proposed an SSc-ILD pathogenesis using cell–cell interaction inferences, and predicted transcription factors regulating target genes using SCENIC. Drug target prediction of the TF gene was performed using Drug Bank Online. Results A subset of macrophages activates the MAPK signaling pathway under oxidative stress. Owing to the lack of inhibitory feedback from ANNEXIN and the autoimmune characteristics, this leads to an earlier onset of lung fibrosis compared to IPF. During initial lung injury, fibroblasts begin to activate the IL6 pathway under the influence of SPP1 alveolar macrophages, but IL6 appears unrelated to other inflammatory and immune cells. This may explain why tocilizumab (an anti-IL6-receptor antibody) only preserves lung function in patients with early SSc-ILD. Finally, we identified BCLAF1 and NFE2L2 as influencers of MAPK activation in macrophages. Metformin downregulates NFE2L2 and could serve as a repurposed drug candidate. Conclusions SPP1 alveolar macrophages play a role in the profibrotic activity of IPF and SSc-ILD. However, SSc-ILD is influenced by autoimmunity and oxidative stress, leading to the continuous activation of MAPK in macrophages. This may result in an earlier onset of lung fibrosis than in IPF. Such differences could serve as potential research directions for early prevention and treatment.

Background A new class of regulatory elements called super-enhancers, comprised of multiple neighboring enhancers, have recently been reported to be the key transcriptional drivers of cellular, developmental, and disease states. Results Here, we defined super-enhancer RNAs as highly expressed enhancer RNAs that are transcribed from a cluster of localized genomic regions. Using the cap analysis of gene expression sequencing data from FANTOM5, we systematically explored the enhancer and messenger RNA landscapes in hundreds of different cell types in response to various environments. Applying non-negative matrix factorization (NMF) to super-enhancer RNA profiles, we found that different cell types were well classified. In addition, through the NMF of individual time-course profiles from a single cell-type, super-enhancer RNAs were clustered into several states with progressive patterns. We further investigated the enriched biological functions of the proximal genes involved in each pattern, and found that they were associated with the corresponding developmental process. Conclusions The proposed super-enhancer RNAs can act as a good alternative, without the complicated measurement of histone modifications, for identifying important regulatory elements of cell type specification and identifying dynamic cell states.

Extracellular particles (EPs) including extracellular vesicles (EVs) and exomeres play significant roles in diseases and therapeutic applications. However, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a suitable method. Therefore, a bioluminescence resonance energy transfer (BRET)‐based reporter, PalmGRET, is created to enable pan‐EP labeling ranging from exomeres (<50 nm) to small (<200 nm) and medium and large (>200 nm) EVs. PalmGRET emits robust, sustained signals and allows the visualization, tracking, and quantification of the EPs from whole animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, BRET, and fluorescence. Using PalmGRET, it is shown that EPs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. It is further demonstrated that gene knockdown of lung‐tropic membrane proteins, solute carrier organic anion transporter family member 2A1, alanine aminopeptidase/Cd13, and chloride intracellular channel 1 decreases HCC‐EP distribution to the lungs and yields distinct biodistribution profiles. It is anticipated that EP‐specific imaging, quantitative assays, and detailed in vivo characterization are a starting point for more accurate and comprehensive in vivo models of EP biology and therapeutic design. PalmGRET, a bioluminescence resonance energy transfer (BRET)‐based reporter for extracellular particles (EPs), enables pan‐EP labeling, including extracellular vesicles and exomeres. PalmGRET allows accurate visualization, tracking, and quantification of EPs from whole animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, BRET, and fluorescence. Using PalmGRET, lung‐tropic EP proteins are identified and dynamically altered biodistributions are revealed under redirected tropism.

Entry inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed to control the outbreak of coronavirus disease 2019 (COVID-19). This study developed a robust and straightforward assay that detected the molecular interaction between the receptor-binding domain (RBD) of viral spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor in just 10 min. A drug library of 1068 approved compounds was used to screen for SARS-CoV2 entry inhibition, and 9 active drugs were identified as specific pseudovirus entry inhibitors. A plaque reduction neutralization test using authentic SARS-CoV-2 virus in Vero E6 cells confirmed that 2 of these drugs (Etravirine and Dolutegravir) significantly inhibited the infection of SARS-CoV-2. With molecular docking, we showed that both Etravirine and Dolutegravir are preferentially bound to primary ACE2-interacting residues on the RBD domain, implying that these two drug blocks may prohibit the viral attachment of SARS-CoV-2. We compared the neutralizing activities of these entry inhibitors against different pseudoviruses carrying spike proteins from alpha, beta, gamma, and delta variants. Both Etravirine and Dolutegravir showed similar neutralizing activities against different variants, with EC50 values between 4.5 to 5.8 nM for Etravirine and 10.2 to 22.9 nM for Dolutegravir. These data implied that Etravirine and Dolutegravir may serve as general spike inhibitors against dominant viral variants of SARS-CoV-2.

The small nucleolar RNA host gene 1 ( SNHG1 ) is a novel oncogenic long non-coding RNA (lncRNA) aberrantly expressed in different tumor types. We previously found highly expressed SNHG1 was associated with poor prognosis and MYCN status in neuroblastoma (NB). However, the molecular mechanisms of SNHG1 in NB are still unclear. Here, we disrupted endogenous SNHG1 in the MYCN -amplified NB cell line SK-N-BE(2)C using the CRISPR/Cas9 system and demonstrated the proliferation and colony formation ability of SNHG1 -knowndown cells were suppressed. The transcriptome analysis and functional assays of SNHG1 -knockdown cells revealed SNHG1 was involved in various biological processes including cell growth, migration, apoptosis, cell cycle, and reactive oxygen species (ROS). Interestingly, the expression of core regulatory circuitry (CRC) transcription factors in MYCN -amplified NB, including PHOX2B, HAND2, GATA3, ISL1, TBX1 , and MYCN , were decreased in SNHG1 -knockdown cells. The chromatin-immunoprecipitation sequencing (ChIP-seq) and transposase-accessible chromatin using sequencing (ATAC-seq) analyses showed that chromatin status of these CRC members was altered, which might stem from interactions between SNHG1 and HDAC1/2. These findings demonstrate that SNHG1 plays a crucial role in maintaining NB identity via chromatin regulation and reveal the function of the lncRNA SNHG1 in NB.