Mechanism In Non-small Cell Lung Cancer Research Articles

Overexpression of mir-489–3p inhibits proliferation and migration of non-small cell lung cancer cells by suppressing the HER2/PI3K/AKT/Snail signaling pathway

BackgroundLung cancer is a highly prevalent malignancy with significant morbidity and mortality rates. MiR-489–3p, a microRNA, has been identified as a regulator of tumor cell proliferation and invasion. Its expression is downregulated in non-small cell lung cancer (NSCLC). Elucidating the molecular mechanisms underlying miR-489–3p′s role in NSCLC pathogenesis is crucial for identifying potential diagnostic and therapeutic targets. MethodsTo investigate the molecular mechanism of miR-489–3p in NSCLC, this study utilized A549, a commonly used NSCLC cell line. MiR-489–3p mimics and inhibitors were transfected into A549 cells. Additionally, co-transfection experiments using wortmannin, an inhibitor of the PI3K/AKT pathway, were performed. Expression of miR-489–3p and related proteins was analyzed by Western blotting and quantitative real-time PCR (qRT-PCR). Cell migration and proliferation were assessed by wound healing and colony formation assays, respectively. ResultsOverexpression of miR-489–3p significantly inhibited the proliferation and migration of A549 cells. This inhibitory effect was further enhanced upon co-transfected with wortmannin. Analysis of human lung specimens showed increased expression of HER2, PI3K, and AKT in lung adenocarcinoma tissues compared to adjacent non-cancerous tissues. ConclusionsThese findings suggest that miR-489–3p overexpression may inhibit NSCLC cell proliferation and migration by suppressing the HER2/PI3K/AKT/Snail signaling pathway. This study elucidates miR-489–3p′s molecular mechanisms in NSCLC and provides experimental basis for identifying early diagnostic markers and novel therapeutic targets.

Emerging role of sirtuins in non‑small cell lung cancer (Review).

Non‑small cell lung cancer (NSCLC) is a highly prevalent lung malignancy characterized by insidious onset, rapid progression and advanced stage at the time of diagnosis, making radical surgery impossible. Sirtuin (SIRT) is a histone deacetylase that relies on NAD+ for its function, regulating the aging process through modifications in protein activity and stability. It is intricately linked to various processes, including glycolipid metabolism, inflammation, lifespan regulation, tumor formation and stress response. An increasing number of studies indicate that SIRTs significantly contribute to the progression of NSCLC by regulating pathophysiological processes such as energy metabolism, autophagy and apoptosis in tumor cells through the deacetylation of histones or non‑histone proteins. The present review elaborates on the roles of different SIRTs and their mechanisms in NSCLC, while also summarizing novel therapeutic agents based on SIRTs. It aims to present new ideas and a theoretical basis for NSCLC treatment.

Expression of the non-coding RNA nc886 facilitates the development of tyrosine kinase inhibitor resistance in EGFR-mutated non-small-cell lung cancer cells

Treatment of non-small-cell lung cancer (NSCLC) patients possessing EGFR-activating mutations with tyrosine kinase inhibitors (TKIs) can confer an initial promising response. However, TKI resistance inevitably arises. Numerous TKI resistance mechanisms are identified including EGFR secondary mutations, bypass receptor tyrosine kinase (RTK) signaling, and cellular transition e.g. epithelial-mesenchymal transition (EMT). To increase the knowledge of TKI resistance we performed an epigenetic screen to identify small non-coding (nc) genes with DNA methylation alterations in HCC827 NSCLC EGFR-mutated cells with acquired TKI resistance. We analyzed Infinium Methylation EPIC 850K Array data for DNA methylation changes present in both TKI-resistant HCC827 cells with EMT and MET-amplification. Hereby, we identified that the polymorphic maternal imprinted gene nc886 (vtRNA2-1) has a decrease in promoter DNA methylation in TKI-resistant cells. This epigenetic change was associated with an increase in the expression of nc886. The induction of EMT did not affect nc886 expression. CRISPR/Cas9-mediated distortion of the nc886 sequence increased the sensitivity of HCC827 cells towards TKI. Finally, nc886 sequence distortion hindered MET RTK activation and instead was EMT the endpoint TKI resistance mechanism. In conclusion, the expression of nc886 contributes to TKI resistance in the HCC827 NSCLC cell line by supporting cell survival and selection of the endpoint TKI resistance mechanism. We propose DNA methylation and expression changes for nc886 to constitute a novel TKI resistance contributing mechanism in NSCLC.

Inhibition of non-small cell lung cancer metastasis by knocking down APE1 through regulating myeloid-derived suppressor cells-induced immune disorders.

Non-small cell lung cancer (NSCLC) represents a highly immunogenic malignancy. Immunologic tolerance facilitated by myeloid-derived suppressor cells (MDSCs) is implicated in primary or secondary resistance mechanisms in NSCLC. The potential role of APE1 in regulating NSCLC metastasis by targeting MDSCs remains uncertain. This study utilized a plasmid, Plxpsp-mGM-CSF, to induce elevated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in A549 cells. Tumor transplantation experiments involved A549, A549+GM-CSF, and A549+GM-CSF-siAPE1 cell lines. Evaluation encompassed MDSCs, Treg cells, IgG, CD3, and CD8 levels. Notably, lung cancer tissues and cells displayed markedly reduced APE1 expression. siAPE1 transfection significantly curtailed tumor growth compared to the A549+GM-CSF group. APE1 knockdown orchestrated immune system modulation in lung tumor mice, characterized by diminished MDSCs but augmented Treg cells, IgG, CD3, and CD8. Additionally, APE1 knockdown led to reduced levels of pro-MDSC cytokines (HGF, CCL5, IL-6, CCL12) and a concurrent upregulation of the anti-MDSC cytokine IL-1ra. Furthermore, APE1 knockdown impeded cell viability in both A549 and H1650 cells. Transplantation of A549-GM-CSF amplified MDSC levels, fostering accelerated tumor growth, while mitigating MDSC levels through APE1 knockdown hindered tumor progression and alleviated inflammatory infiltration in lung cancer tissues. Strategies targeting the APE1/MDSC axis offer a promising approach for lung cancer prevention and treatment, presenting novel insights for NSCLC management.

PD-L1 and alternative immune checkpoints (AICs) in non-small cell lung cancer (NSCLC): Insights into the tumor immune microenvironment (TIME) and immunotherapy (IO) outcomes.

8531 Background: PD-L1 and recently identified AICs (B7x, B7-H3, and HHLA2) may function as key immune evasion mechanisms in NSCLC and influence IO efficacy. This study explores the expression patterns of PD-L1 and AICs within the TIME and their impact on IO outcomes in NSCLC, including key molecular subtypes. Methods: NSCLC samples (n=27,629) were analyzed (Caris Life Sciences) with NextGen Sequencing on DNA (592 genes or whole exome)/RNA (whole transcriptome). Tumor cell PD-L1 expression (22c3) was assessed by IHC. Gene expression profiles were analyzed for IFN-γ score and QuantiSEQ was used to assess immune cell infiltration in the TIME. Real-world overall survival (rwOS: from time of biopsy to last contact), time on treatment [TOT: from the first pembrolizumab (pembro) treatment to the last], and OS on pembro (OSpem: from pembro start to last contact) were calculated. Mann-Whitney U, X2/Fisher-Exact, and log-rank tests were applied where appropriate. Results: PD-L1 IHC+ NSCLC and cases with high PD-L1 RNA expression had higher IFN-γ score and immune cell fractions (p<0.0001 for both) vs PD-L1 IHC- and low expression, respectively. Additionally, PD-L1 IHC+ NSCLC was associated with (a/w) higher median transcript levels (TPM) of PD-L1 and B7-H3, while PD-L1 IHC- NSCLC was a/w higher TPM of HHLA2 and B7x (p<0.0001 for all). AICs expression varied by molecular subtype, e.g., higher HHLA2/B7x RNA in EGFR mutant vs wild-type (WT) NSCLC. Moreover, PD-L1 IHC+ NSCLC and cases with high PD-L1 RNA and low B7H3 RNA were a/w longer rwOS, OSpem, and TOT; high HHLA2 RNA was a/w longer rwOS and OSpem with similar TOT; and low B7x RNA expression was a/w longer OSpem and TOT with similar rwOS (Table). Conclusions: Both PD-L1 IHC+ NSCLC and cases with high PD-L1 RNA expression are a/w a more inflammatory TIME, and superior survival and IO outcomes. Moreover, consistent with our prior HHLA2 IHC work (PMIDs: 27553831, 29374053), HHLA2 RNA expression was significantly higher in PD-L1 IHC- and EGFR mutant cases. These findings support the potential utility of transcriptomics along with protein expression for assessing novel predictive biomarkers for IO. Furthermore, the distinct expression profiles of AICs identified in our study may represent key subtypes mediating IO response in WT and EGFR mutant NSCLC. [Table: see text]

Downregulation of lncRNA MIR17HG reduced tumorigenicity and Treg-mediated immune escape of non-small-cell lung cancer cells through targeting the miR-17-5p/RUNX3 axis.

Long noncoding RNA MIR17HG was involved with the progression of non-small-cell lung cancer (NSCLC), but specific mechanisms of MIR17HG-mediated immune escape of NSCLC cells were still unknown. The present study investigated the function of MIR17HG on regulatory T cell (Treg)-mediated immune escape and the underlying mechanisms in NSCLC. Expression of MIR17HG and miR-17-5p in NSCLC tissue samples were detected using quantitative real-time PCR (qRT-PCR). A549 and H1299 cells were transfected with sh-MIR17HG, miR-17-5p inhibitor, or sh-MIR17HG + miR-17-5p inhibitor, followed by cocultured with Tregs. Cell proliferation was measured using 5-ethynyl-20-deoxyuridine (Edu) staining assay and cell counting kit-8 (CCK-8) assay. Flow cytometry was used for determining positive numbers of FOXP3+CD4+/CD25+/CD8+ Tregs. Through subcutaneous injection with transfected A549 cells, a xenograft nude mouse model was established. Weights and volumes of xenograft tumors were evaluated. Additionally, the expressions of immune-related factors includingtransforming growth factor beta (TGF-β), vascular endothelial growth factor A (VEGF-A), interleukin-10 (IL-10), IL-4, and interferon-gamma (IFN-γ) in cultured cells, were evaluated by enzyme-linked immunosorbent assay and western blot analysis. Then, miR-17-5p was decreased and MIR17HG was enhanced in both NSCLC tissues and cell lines. MIR17HG knockdown significantly suppressed cell proliferation, tumorigenicity, and immune capacity of Tregs in A549 and H1299 cells, whereas sh-MIR17HG significantly reduced expression levels of VEGF-A, TGF-β, IL-4, and IL-10 but promoted the IFN-γ level in vitro and in vivo. Moreover, downregulation of miR-17-5p significantly reversed the effects of sh-MIR17HG. Additionally, we identified that runt- related transcription factor 3 (RUNX3) was a target of miR-17-5p, and sh-MIR17HG and miR-17-5p mimics downregulated RUNX3 expression. In conclusion, downregulation of MIR17HG suppresses tumorigenicity and Treg-mediated immune escape in NSCLC through downregulating the miR-17-5p/RUNX3 axis, indicating that this axis contains potential biomarkers for NSCLC.

Cellular Senescence in Non-Small Cell Lung Cancer.

Lung cancer has the highest mortality rate amongst all malignancies worldwide, and is the second-highest incidence of cancer in women. Non-small cell lung cancer (NSCLC) is responsible for approximately 80% of lung cancer cases. Recent studies indicate that cellular senescence may be a promising cancer biomarker. However, the regulation of cellular senescence and its underlying mechanisms in NSCLC are not yet fully understood. Here, we present a comprehensive analysis of the genes linked to cellular senescence in NSCLC. We also describe the secretory phenotype associated with NSCLC and examine its immune profile and prognostic potential. Our findings offer novel insights into the development of effective NSCLC treatments.

Loss of Key EMT-Regulating miRNAs Highlight the Role of ZEB1 in EGFR Tyrosine Kinase Inhibitor-Resistant NSCLC.

Despite recent advances in the treatment of non-small cell lung cancer (NSCLC), acquired drug resistance to targeted therapy remains a major obstacle. Epithelial-mesenchymal transition (EMT) has been identified as a key resistance mechanism in NSCLC. Here, we investigated the mechanistic role of key EMT-regulating small non-coding microRNAs (miRNAs) in sublines of the NSCLC cell line HCC4006 adapted to afatinib, erlotinib, gefitinib, or osimertinib. The most differentially expressed miRNAs derived from extracellular vesicles were associated with EMT, and their predicted target ZEB1 was significantly overexpressed in all resistant cell lines. Transfection of a miR-205-5p mimic partially reversed EMT by inhibiting ZEB1, restoring CDH1 expression, and inhibiting migration in erlotinib-resistant cells. Gene expression of EMT-markers, transcription factors, and miRNAs were correlated during stepwise osimertinib adaptation of HCC4006 cells. Temporally relieving cells of osimertinib reversed transition trends, suggesting that the implementation of treatment pauses could provide prolonged benefits for patients. Our results provide new insights into the contribution of miRNAs to drug-resistant NSCLC harboring EGFR-activating mutations and highlight their role as potential biomarkers and therapeutic targets.

LncRNA-LINC00472 suppresses the malignant progression of non-small cell lung cancer via modulation of the miRNA-1275/Homeobox A2 axis.

Long non-coding RNAs (lncRNAs) are increasingly observed as regulatory factors for the initiation and progression of varying kinds of cancers. However, studies on lncRNAs in non-small cell lung cancer (NSCLC) progression are currently lacking. We intended to determine the role of lncRNA LINC00472 and its downstream regulatory mechanism in NSCLC, thus providing novel ideas for targeted therapies for NSCLC. The target signaling axis comprising the lncRNA/microRNA/mRNA was identified through bioinformatics analysis. Subcellular localization of LINC00472 was assessed with fluorescence in situ hybridization (FISH). Cellular function experiments were conducted to examine the proliferation, migration, invasion, and apoptosis of NSCLC cells, and dual-luciferase and RNA binding protein immunoprecipitation assays were performed to validate the binding relationship. Quantitative real-time polymerase chain reaction (qPCR) and western blot were utilized to assess the expression levels of the investigated gene and protein, respectively. The LINC00472 expression was markedly decreased in NSCLC tissues and cells. The FISH, combined with nuclear-cytoplasm separation assay, demonstrated that LINC00472 was mainly located in the cytoplasm. The overexpression of LINC00472 restrained proliferation and metastasis of NSCLC in vitro. The LINC00472 could target and repress miR-1275 level, and overexpression of LINC00472 reduced the miR-1275-dependent malignant cell phenotype in NSCLC. Further study revealed that HOXA2 was a downstream target of miR-1275 and was negatively modulated by miR-1275. Rescue assays exhibited that the overexpression of miR-1275 or inhibition of HOXA2 reversed the impact of LINC00472 overexpression on the malignant progression of NSCLC cells. The LINC00472 repressed the epithelial-mesenchymal transition (EMT) of NSCLC cells through miR-1275/HOXA2. The LINC00472 functioned as a competing endogenous RNA to modulate HOXA2 level by sponging miR-1275 in NSCLC. Simultaneously, the LINC00472/miR-1275/HOXA2 axis may be a possible therapeutic target and biomarker for NSCLC.

Current immune therapeutic strategies in advanced or metastatic non-small-cell lung cancer.

Immune escape mechanisms in non-small cell lung cancer (NSCLC) can disrupt every step of the anti-cancer immune response. In recent years, an increased understanding of the specific mechanisms fueling immune escape has allowed for the development of numerous immunotherapeutic treatments that have been introduced into the clinical practice. The advent of immunotherapy has dramatically changed the current treatment landscape of advanced or metastatic NSCLC because of its durable efficacy and manageable toxicity. In this review, we will first present a brief overview of recent evidence on immune escape mechanisms in NSCLC. We will then discuss the current promising immunotherapeutic strategies in advanced or metastatic NSCLC tumors.

Epithelial-mesenchymal transition is associated with osteopontin-induced EGFR‑TKI resistance in EGFR mutant non-small cell lung cancer.

Resistance restricts the long-term therapeutic efficacy of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in the treatment of non-small cell lung cancer (NSCLC) with positive EGFR mutations. The present study sought to identify the potential protein osteopontin (OPN) involved in EGFR-TKI resistance and examine its therapeutic mechanism in NSCLC. The expression of OPN in NSCLC tissues was evaluated by immunohistochemistry (IHC). Western blot (WB), quantitative real‑time polymerase chain reaction (qRT-PCR), and immunofluorescence staining were used to analyze OPN and epithelial-mesenchymal transition (EMT)-related protein expression in the PC9 and PC9 gefitinib resistance (PC9GR) cells. Enzyme-linked immunosorbent assays (ELISAs) were used to detect the secreted OPN. Cell Counting Kit-8 (CCK-8) assays and flow cytometry were used to examine the effect of OPN on the gefitinib-induced growth and death of PC9 or PC9GR cells. OPN was upregulated in the human NSCLC tissues and cells resistant to EGFR-TKIs. The overexpression of OPN inhibited EGFR-TKI-induced apoptosis and was associated with the formation of EMT. By activating the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT)-EMT pathway, OPN contributed to the development of EGFR-TKI resistance. Reducing OPN expression and inhibiting PI3K/AKT signaling improved EGFR-TKI sensitivity significantly more than the use of either agent alone. This study showed that OPN increased EGFR-TKI resistance in NSCLC through the OPN-PI3K/AKT-EMT pathway. Our findings may provide a possible therapeutic target for overcoming EGFR-TKI resistance in this pathway.

A prognostic model based on ferroptosis-related long non-coding RNA signatures and immunotherapy responses for non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) ranks high in the incidence of malignant tumors, with limited treatment options and poor prognosis. Ferroptosis is a newly discovered cell death mechanism based on iron and reactive oxygen species (ROS). The role of ferroptosis-related long non-coding RNAs (lncRNAs) and associated prognostic mechanisms in NSCLC require investigation. We constructed a prognostic multi-lncRNA signature based on ferroptosis-related differentially expressed lncRNAs in NSCLC. The levels of ferroptosis-related lncRNA in normal lung cells and lung adenocarcinoma cells were verified by RT-PCR. We identified eight differentially expressed lncRNAs associated with NSCLC prognosis. The expression of AC125807.2, AL365181.3, AL606489.1, LINC02320, and AC099850.3 was upregulated, while SALRNA1, AC026355.1, and AP002360.1 were downregulated in NSCLC cell lines. Kaplan-Meier analysis showed that a high-risk patient group was associated with poor NSCLC prognosis. A risk assessment model based on ferroptosis-related lncRNAs was superior to NSCLC prognosis based on traditional clinicopathological features. Gene Set Enrichment Analysis (GSEA) identified immune- and tumor-related pathways in low-risk group patients. In addition, The Cancer Genome Atlas (TCGA) showed that T cell function during APC co-inhibition, APC co-stimulation, chemokine receptor (CCR), MHC class I, parainflammation, T cell co-inhibition, and check-point expression differed significantly between low- and high-risk groups. M6A-related mRNA comparisons between these groups also revealed significant differences in ZC3H13, RBM15, and METTL3 expression. Our new model of lncRNA-associated ferroptosis effectively predicted NSCLC prognoses.

Wnt/β-Catenin Signaling and Resistance to Immune Checkpoint Inhibitors: From Non-Small-Cell Lung Cancer to Other Cancers.

Lung cancer is the leading cause of cancer-related deaths worldwide. The standard of care for advanced non-small-cell lung cancer (NSCLC) without driver-gene mutations is a combination of an anti-PD-1/PD-L1 antibody and chemotherapy, or an anti-PD-1/PD-L1 antibody and an anti-CTLA-4 antibody with or without chemotherapy. Although there were fewer cases of disease progression in the early stages of combination treatment than with anti-PD-1/PD-L1 antibodies alone, only approximately half of the patients had a long-term response. Therefore, it is necessary to elucidate the mechanisms of resistance to immune checkpoint inhibitors. Recent reports of such mechanisms include reduced cancer-cell immunogenicity, loss of major histocompatibility complex, dysfunctional tumor-intrinsic interferon-γ signaling, and oncogenic signaling leading to immunoediting. Among these, the Wnt/β-catenin pathway is a notable potential mechanism of immune escape and resistance to immune checkpoint inhibitors. In this review, we will summarize findings on these resistance mechanisms in NSCLC and other cancers, focusing on Wnt/β-catenin signaling. First, we will review the molecular biology of Wnt/β-catenin signaling, then discuss how it can induce immunoediting and resistance to immune checkpoint inhibitors. We will also describe other various mechanisms of immune-checkpoint-inhibitor resistance. Finally, we will propose therapeutic approaches to overcome these mechanisms.

Thrombospondin-1 mimic peptide PKHB1 induced endoplasmic reticulum stress-mediated but CD47-independent apoptosis in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the most common malignancies with high morbidity and mortality. PKHB1, a serum-stable Thrombospondin-1 (TSP-1) mimic peptide, has shown some effective ability in triggering cell death against several cancers. Here, we aimed to study the potential biological function of PKHB1 and its molecular mechanism in NSCLC. Our results revealed that PKHB1 significantly suppressed NSCLC cell proliferation, cell migration, and induced apoptosis in a dose-dependent manner. Additionally, we found that PKHB1 treatment resulted in mitochondrial transmembrane potential depolarization, Ca2+ overloading as well as the upregulation of proapoptotic proteins. Mechanistically, PKHB1 induced NSCLC cells apoptosis in a CD47-independent manner. Further study revealed that PKHB1 provoked endoplasmic reticulum (ER) stress principally through the activation of CHOP and JNK signaling, which could be alleviated in the presence of 4-PBA, an ER stress inhibitor. Furthermore, xenograft tumor models showed that PKHB1 treatment could notably inhibit NSCLC tumor growth in vivo. In conclusion, these findings suggested that PKHB1 exerted antitumor efficacy in NSCLC via triggering ER stress-mediated but CD47-independent apoptosis, potentially functioned as a promising peptide-based therapeutic agent for NSCLC.

CircSCN8A suppresses malignant progression and induces ferroptosis in non-small cell lung cancer by regulating miR-1290/ACSL4 axis

ABSTRACT Circular RNAs (CircRNAs) are reported to exert vital regulatory roles in the occurrence and development of various human malignancies, including non-small cell lung cancer (NSCLC). Bioinformatics methods identified the down-regulation of circSCN8A (circBase ID: hsa_circ_0026337) in NSCLC tissues. However, its biological functions and molecular mechanisms in NSCLC remain unknown. In this study, we found that circSCN8A expression was down-regulated in NSCLC tissues and cells. Low circSCN8A expression was positively associated with aggressive clinicopathological characteristics and poor prognosis in NSCLC patients. CircSCN8A suppressed cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro and blocked tumor growth in vivo. Moreover, circSCN8A promoted cell ferroptosis in NSCLC. Mechanistically, circSCN8A acted as a competing endogenous RNA (ceRNA) by sponging miR-1290 to enhance the expression of long-chain acyl-CoA synthetase-4 (ACSL4). Furthermore, the knockdown of ACSL4 or overexpression of miR-1290 reversed the effect of circSCN8A on facilitating ferroptosis and inhibiting cell proliferation and metastasis. In summary, circSCN8A represses cell proliferation and metastasis in NSCLC by regulating the miR-1290/ACSL4 axis to induce ferroptosis. Thus, circSCN8A may represent a promising therapeutic target against NSCLC.

Non‑small cell lung cancer carrying PBRM1 mutation suggests an immunologically cold phenotype leading to immunotherapy failure even with high TMB

High tumor mutation load (TMB-H, or TMB ≥ 10) has been approved by the U.S. FDA as a biomarker for pembrolizumab treatment of solid tumors, including non‑small cell lung cancer (NSCLC). Patients with cancer who have immunotherapy-resistant gene mutations cannot achieve clinical benefits even in TMB-H. In this study, we aimed to identify gene mutations associated with immunotherapy resistance and further informed mechanisms in NSCLC. A combined cohort of 350 immune checkpoint blockade-treated patients from Memorial Sloan Kettering Cancer Center (MSKCC) was used to identify genes whose mutations could negatively influence immunotherapy efficacy. An external NSCLC cohort for which profession-free survival (PFS) data were available was used for independent validation. CIBERSORT algorithms were used to characterize tumor immune infiltrating patterns. Immunogenomic features were analysed in the TCGA NSCLC cohort. We observed that PBRM1 mutations independently and negatively influence immunotherapy efficacy. Survival analysis showed that the overall survival (OS) and PFS of patients with PBRM1 mutations (MT) were significantly shorter than the wild type (WT). Moreover, compared with PBRM1-WT/TMB-H group, OS was worse in the PBRM1-MT/TMB-H group. Notably, in patients with TMB-H/PBRM1-MT, it was equal to that in the low-TMB group. The CIBERSORT algorithm further confirmed that the immune infiltration abundance of CD8+ T cells and activated CD4+ memory T was significantly lower in the MT group. Immunogenomic differences were observed in terms of immune signatures, T-cell receptor repertoire, and immune-related genes between WT and MT groups. Nevertheless, we noticed an inverse relationship, given that MT tumors had a higher TMB than the WT group in MSKCC and TCGA cohort. In conclusion, our study revealed that NSCLC with PBRM1 mutation might be an immunologically cold phenotype and exhibited immunotherapy resistance. NSCLC with PBRM1 mutation might be misclassified as immunoresponsive based on TMB.

ADGRD1 as a Potential Prognostic and Immunological Biomarker in Non-Small-Cell Lung Cancer.

ADGRD1 (GPR133), an adhesion G protein-coupled receptor (GPCR), has been linked to cancer. However, the prognostic value and regulatory function within non-small-cell lung cancer (NSCLC) is still unclear. This work adopted various bioinformatics methods, including publicly available databases as well as real-time PCR (RT-PCR), for detecting ADGRD1 expression level and investigating the correlation between ADGRD1 expression level and prognosis, tumor mutational burden (TMB), microsatellite instability (MSI), immune infiltrating cells, immune-related genes, and targeted regulation mechanisms in NSCLC. According to the results, ADGRD1 expression decreased within NSCLC, which might be the factor predicting prognosis of NSCLC. Meanwhile, ADGRD1 showed significant correlation with TMB and MSI, respectively, as well as immune cell infiltrating levels in lung adenocarcinoma (LUAD), which were primarily linked to macrophage M1, mast cell resting, T cell CD4 memory activated, and T cell CD4 memory resting and were associated with mast cell activated and mast cell resting in lung squamous cell carcinoma (LUSC). The most promising upstream regulation pathways of ADGRD1 were likely miR-142-5p, miR-93-5p, and miR-17-5p, which were overexpressed and associated with poor prognosis in NSCLC. ADGRD1 and immune-related genes correlated with ADGRD1 were shown to be enriched in "positive regulation of leukocyte activation," "external side of plasma membrane," "receptor ligand activity," and "cytokine-cytokine receptor interaction" pathways. ADGRD1 expression and regulation may be critical in determining NSCLC prognosis.

CircRNA-mediated upregulation of HOXC9 is correlated with poor outcome and immune microenvironment infiltrates in LUAD

PurposeTo assess HOXC9 expression in non-small cell lung cancer (NSCLC) using bioinformatics analysis with experimental validation and to investigate its role and potential mechanisms in NSCLC. MethodsPublic databases were explored to investigate the gene expression, clinical outcomes, immune infiltration, and potential molecular mechanisms of HOXC9 in NSCLC. Next, HOXC9 expression levels in NSCLC were verified by RT-PCR and nucleic acid electrophoresis. In vitro experiments were performed to assess the effects of HOXC9 on proliferation, migration, and invasion of NSCLC cells. ResultsHOXC9 expression was increased in NSCLC and correlated with poor prognosis in lung adenocarcinoma. In addition, HOXC9 expression levels were significantly correlated with immune cell infiltration of tumors and biomarkers of immune cells. Overexpression of HOXC9 promoted NSCLC cell proliferation, migration, and invasion in vitro. Finally, the circCENPF/hsa-miR-184 axis was identified as the most promising upstream regulatory pathway of HOXC9 in lung adenocarcinoma. ConclusionHOXC9 expression may reflect the prognosis and immune microenvironment infiltration in NSCLC, emerging as a potential oncogene and a new target for immunotherapy.

S-3′-hydroxy-7′, 2′, 4′-trimethoxyisoxane, a novel ferroptosis inducer, promotes NSCLC cell death through inhibiting Nrf2/HO-1 signaling pathway

Background: Ferroptosis is a newly discovered and promising non-apoptotic programmed cell death (PCD), and inducing ferroptosis in cancer cells could open up a novel avenue for drug screening and cancer therapy. S-3′-hydroxy-7′, 2′, 4′-trimethoxyisoxane (ShtIX), a new isoflavane compound, has been reported to possess cytotoxicity in non-small cell lung cancer (NSCLC). The aim of this research is to explore the ShtIX-induced cell death form and its underlying molecular mechanism in NSCLC cells. Methods: Cell proliferation, cell cycle arrest, and cell death tests were used to assess the ability of ShtIX to kill NSCLC cells. Iron metabolism, Fe2+ content, reactive oxygen species (ROS) production, lipid peroxide (MDA) level, glutathione (GSH) level, and glutathione peroxidase 4 (GPX4) level were used to determine ferroptosis caused by ShtIX. We employed western blot, quantitative real-time PCR, and Nrf2 interference in NSCLC cells to investigate the roles of Nrf2/HO-1 in ShtIX-induced ferroptosis. In a xenograft nude mouse model, the anticancer efficacy of ShtIX and the function of ferroptosis were studied. Results: Our research shows that ShtIX can selectively kill NSCLC cells while sparing normal cells and that ShtIX-induced cell death can be efficiently reversed by the ferroptosis inhibitors and the iron chelator, but not by other cell death inhibitors. After cells were treated with ShtIX, there was an increase in Fe2+ content and lipid peroxidation accumulation, as well as a drop in GSH and GPX4 levels, all of which are indicators of ferroptosis. ShtIX also reduced the expression of Nrf2 and HO-1, and genetic Nrf2 silencing in NSCLC enhanced the effect of ShtIX-induced ferroptosis. Additionally, ShtIX retards tumor growth and induced ferroptosis through Nrf2/HO-1 signal pathway in the A549 xenograft model, whereas Fer-1 lessens the anticancer effect. Conclusion: This work provided the evidence that ShtIX caused ferroptosis in NSCLC cells, and inhibiting the Nrf2/HO-1 pathway can considerably exacerbate the effect of ShtIX-induced ferroptosis. The study establishes ShtIX as a promising natural ferroptosis inducer for the treatment of NSCLC.

TRIM66 Promotes Malignant Progression of Non-Small-Cell Lung Cancer Cells via Targeting MMP9.

Lung cancer has a higher incidence and mortality rate than other cancers, and over 80% of lung cancer cases were classified as non-small-cell lung cancer (NSCLC). TRIM66 is one of the crucial members of TRIM, which has a deep connection with the behavior of various malignant tumors. But it remains uncertain regarding its exact function and underlying mechanism in NSCLC. In our study, qRT-PCR and Western blot were employed to validate that TRIM66 was overexpressed in NSCLC. The migration, invasion, and epithelial-mesenchymal transformation (EMT) progression of NSCLC cells were determined by Western blotting and Transwell experiments after knocking down TRIM66, and it was found that knockdown TRIM66 inhibited the migration, invasion, and EMT processes of NSCLC cells. Next, the binding relationship between TRIM66 and MMP9 was verified by Co-IP assay. After determining the interaction between them, rescue assays showed that overexpression of MMP9 was capable to promote the migration, invasion, and EMT of NSCLC cells. However, the transfection of si-TRIM66 could reverse this facilitating effectiveness. To sum up, we concluded that by targeting MMP9, TRIM66 could exert a cancer-promoting role in the progression of NSCLC cells.