NEAT1_1 confers gefitinib resistance in lung adenocarcinoma through promoting AKR1C1-mediated ferroptosis defence.

Gefitinib is one of the most extensively utilized epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) for treating advanced lung adenocarcinoma (LUAD) patients harboring EGFR mutation. However, the emergence of drug resistance significantly compromised the clinical efficacy of EGFR-TKIs. Gaining further insights into the molecular mechanisms underlying gefitinib resistance holds promise for developing novel strategies to overcome the resistance and improve the prognosis in LUAD patients. Here, we identified that the inhibitory efficacy of gefitinib on EGFR-mutated LUAD cells was partially dependent on the induction of ferroptosis, and ferroptosis protection resulted in gefitinib resistance. Among the ferroptosis suppressors, aldo-keto reductase family 1 member C1 (AKR1C1) exhibited significant upregulation in gefitinib-resistant strains of LUAD cells and predicted poor progression-free survival (PFS) and overall survival (OS) of LUAD patients who received first-generation EGFR-TKI treatment. Knockdown of AKR1C1 partially reversed drug resistance by re-sensitizing the LUAD cells to gefitinib-mediated ferroptosis. The decreased expression of miR-338-3p contributed to the aberrant upregulation of AKR1C1 in gefitinib-resistant LUAD cells. Furthermore, upregulated long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1_1 (NEAT1_1) sponged miR-338-3p to neutralize its suppression on AKR1C1. Dual-luciferase reporter assay and miRNA rescue experiment confirmed the NEAT1_1/miR-338-3p/AKR1C1 axis in EGFR-mutated LUAD cells. Gain- and loss-of-function assays demonstrated that the NEAT1_1/miR-338-3p/AKR1C1 axis promoted gefitinib resistance, proliferation, migration, and invasion in LUAD cells. This study reveals the effects of NEAT1_1/miR-338-3p/AKR1C1 axis-mediated ferroptosis defence in gefitinib resistance in LUAD. Thus, targeting NEAT1_1/miR-338-3p/AKR1C1 axis might be a novel strategy for overcoming gefitinib resistance in LUAD harboring EGFR mutation.

circNOX4 activates an inflammatory fibroblast niche to promote tumor growth and metastasis in NSCLC via FAP/IL-6 axis.

BACKGROUND: Cancer-associated fibroblasts (CAFs) orchestrate a supportive niche that fuels cancer metastatic development in non-small cell lung cancer (NSCLC). Due to the heterogeneity and plasticity of CAFs, manipulating the activated phenotype of fibroblasts is a promising strategy for cancer therapy. However, the underlying mechanisms of fibroblast activation and phenotype switching that drive metastasis remain elusive. METHODS: The clinical implications of fibroblast activation protein (FAP)-positive CAFs (FAP+CAFs) were evaluated based on tumor specimens from NSCLC patients and bioinformatic analysis of online databases. CAF-specific circular RNAs (circRNAs) were screened by circRNA microarrays of primary human CAFs and matched normal fibroblasts (NFs). Survival analyses were performed to assess the prognostic value of circNOX4 in NSCLC clinical samples. The biological effects of circNOX4 were investigated by gain- and loss-of-function experiments in vitro and in vivo. Fluorescence in situ hybridization, luciferase reporter assays, RNA immunoprecipitation, and miRNA rescue experiments were conducted to elucidate the underlying mechanisms of fibroblast activation. Cytokine antibody array, transwell coculture system, and enzyme-linked immunosorbent assay (ELISA) were performed to investigate the downstream effectors that promote cancer metastasis. RESULTS: FAP+CAFs were significantly enriched in metastatic cancer samples, and their higher abundance was correlated with the worse overall survival in NSCLC patients. A novel CAF-specific circRNA, circNOX4 (hsa_circ_0023988), evoked the phenotypic transition from NFs into CAFs and promoted the migration and invasion of NSCLC in vitro and in vivo. Clinically, circNOX4 correlated with the poor prognosis of advanced NSCLC patients. Mechanistically, circNOX4 upregulated FAP by sponging miR-329-5p, which led to fibroblast activation. Furthermore, the circNOX4/miR-329-5p/FAP axis activated an inflammatory fibroblast niche by preferentially inducing interleukin-6 (IL-6) and eventually promoting NSCLC progression. Disruption of the intercellular circNOX4/IL-6 axis significantly suppressed tumor growth and metastatic colonization in vivo. CONCLUSIONS: Our study reveals a role of the circRNA-induced fibroblast niche in tumor metastasis and highlights that targeting the circNOX4/FAP/IL-6 axis is a promising strategy for the intervention of NSCLC metastasis.

Ceruloplasmin is associated with the infiltration of immune cells and acts as a prognostic biomarker in patients suffering from glioma.

Glioma is regarded as a prevalent form of cancer that affects the Central Nervous System (CNS), with an aggressive growth pattern and a low clinical cure rate. Despite the advancement of the treatment strategy of surgical resection, chemoradiotherapy and immunotherapy in the last decade, the clinical outcome is still grim, which is ascribed to the low immunogenicity and tumor microenvironment (TME) of glioma. The multifunctional molecule, called ceruloplasmin (CP) is involved in iron metabolism. Its expression pattern, prognostic significance, and association with the immune cells in gliomas have not been thoroughly investigated. Studies using a variety of databases, including Chinese Glioma Genome Atlas (CGGA), The Cancer Genome Atlas (TCGA), and Gliovis, showed that the mRNA and protein expression levels of CP in patients suffering from glioma increased significantly with an increasing glioma grade. Kaplan-Meier (KM) curves and statistical tests highlighted a significant reduction in survival time of patients with elevated CP expression levels. According to Cox regression analysis, CP can be utilized as a stand-alone predictive biomarker in patients suffering from glioma. A significant association between CP expression and numerous immune-related pathways was found after analyzing the data using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA). Tumor Immune Estimation Resource (TIMER) and CIBERSORT analyses indicated a substantial correlation between the CP expression and infiltration of immunocytes in the TME. Additionally, immune checkpoints and CP expression in gliomas showed a favorable correlation. According to these results, patients with glioma have better prognoses and levels of tumor immune cell infiltration when their CP expression is low. As a result, CP could be used as a probable therapeutic target for gliomas and potentially anticipate the effectiveness of immunotherapy.

Function of hsa_circ_0006646 as a competing endogenous RNA to promote progression in gastric cancer by regulating the miR-665-HMGB1 axis.

Background: Mounting evidences indicate that circular RNAs (circRNAs) are a novel class of non-coding RNAs and play vital roles in the tumorigenesis and aggressiveness including gastric cancer (GC). Nevertheless, the precise functions and underlying mechanisms of circRNAs in GC remain largely unknown. Methods: The Gene Expression Omnibus (GEO) data set GSE163416 was analyzed to screen the key circRNAs in GC. hsa_circ_0006646 was chosen for further study. GC tissues and matched adjacent normal gastric mucosal epithelial tissues were obtained from the Fourth Hospital of Hebei Medical University. The expressions of hsa_circ_0006646 was detected using quantitative real-time polymerase chain reaction (qRT-PCR). hsa_circ_0006646 was knocked down to identify its effects on GC cells. Bioinformatics algorithms were analyzed to predict the microRNA (miRNAs) potentially sponged by hsa_circ_0006646 and its target genes. Fluorescence in situ hybridization (FISH) was conducted to determine the subcellular location of hsa_circ_0006646 and the predicted miRNA. Then, qRT-PCR, luciferase reporter assay, radioimmunoprecipitation assay, Western blotting, and miRNA rescue experiments were used to confirm the hsa_circ_0006646-related regulatory axis in GC. Cell Counting Kit-8 (CCK-8), colony formation, wound healing, and Transwell experiments were performed to determine the effect of the hsa_circ_0006646-related regulatory axis on GC cells' malignant behaviors in vitro. The xenograft tumor mouse model was established to evaluate the effect of hsa_circ_0006646 in vivo. Results: hsa_circ_0006646 exhibited a high expression in GC tissues as compared to corresponding adjacent normal gastric mucosal epithelial tissues and its high expression was positively correlated with TNM stage, lymph node invasion and poor prognosis (P<0.05). Knockdown of hsa_circ_0006646 suppressed the proliferation, colony formation, migration, and invasion in GC cells (all P<0.05). hsa_circ_0006646 upregulated high mobility group box 1 (HMGB1) by sponging miR-665 in GC cells (P<0.05). The hsa_circ_0006646-miR-665-HMGB1 axis promoted malignant behaviors and epithelial-mesenchymal transition (EMT) in GC cells by activating the Wnt/ß-catenin pathway (P<0.05). The existence of hsa_circ_0006646-miR-665-HMGB1 axis was confirmed in GC specimens (P<0.05). Consequently, down-regulated hsa_circ_0006646 inhibited the progression and EMT of GC cells in vivo (P<0.05). Conclusions: For the first time, we demonstrated that hsa_circ_0006646-miR-665-HMGB1 axis exerted its tumor-promoting effects in GC, which suggested that hsa_circ_0006646 could be potentially targeted for GC treatment.

C1ql4 regulates breast cancer cell stemness and epithelial-mesenchymal transition through PI3K/AKT/NF-κB signaling pathway.

Background: The stemness characteristic of breast cancer (BC) is a crucial factor underlying cancer recurrence and metastasis after operative therapy and chemoradiotherapy. Understanding the potential mechanism of breast cancer stem cells (BCSCs) may ameliorate the prognosis of patients. Methods: We collected clinical specimens of BC patients for staining and statistical analysis to verify the expression status and clinical significance of complement C1q-like 4 (C1ql4). Western blot and qRT-PCR were employed to detect the expression of molecules. Flow cytometry was used to examine cell cycle, cell apoptosis and the portion of BCSCs. Wound healing and Transwell assays were used to detect cell metastasis. The effect of C1ql4 on breast cancer progression in vivo was examined in a nude mouse tumor bearing model. Results: Our clinical analysis showed that C1ql4 was highly expressed in BC tissues and cell lines, and the high expression of C1ql4 was significantly corelated with the malignancy of BC patients. Moreover, we also found that C1ql4 was overexpressed in BCSCs. C1ql4 knockdown suppressed the BCSC and EMT properties, promoted cell cycle progression, enhanced BC cell apoptosis, and inhibited cell migration and invasion, whereas the C1ql4 overexpression exhibited the opposite effects. Mechanistically, C1ql4 promoted the activation and nuclear location of NF-κB and the expression of downstream factors TNF-α and IL-1ß. Moreover, inhibition of PI3K/AKT signaling suppressed the C1ql4-induced stemness and EMT. Conclusions: Our findings suggest that C1ql4 promotes the BC cell stemness and EMT via modulating the PI3K/AKT/NF-κB signaling, and provides a promising target for BC treatment.

Screening of Differentially Expressed Genes Based on the ACRG Molecular Subtypes of Gastric Cancer and the Significance and Mechanism of AGTR1 Gene Expression.

BACKGROUND: The Asian Cancer Research Group (ACRG) classification is a molecular classification established based on the tissues of gastric cancer (GC) patients in Asia. Patients with different ACRG subtypes differ significantly with regard to treatment response and prognosis, which indicates that the ACRG molecular classification is more valuable than the traditional pathological classification. However, the specific differentially expressed genes (DEGs) and the value of the ACRG molecular subtypes of GC have not been studied in depth. METHODS: Through the analysis of the GEO database, the DEGs in GC tissues of different ACRG molecular subtypes were investigated. The expression and mechanism of the screened angiotensin II receptor type 1 (AGTR1) gene were bioinformatically analyzed and experimentally verified. The role of AGTR1 in GC cells was mainly investigated using CCK-8, wound-healing, transwell invasion assays, qRT-PCR, and Western blotting. RESULTS: The bioinformatics results showed the presence of multiple DEGs in GC tissues with different ACRG molecular subtypes. Certain DEGs in GC tissues of different ACRG molecular subtypes have prognostic significance. AGTR1 levels in tumor tissues were significantly higher than in paired paracancerous tissues. The prognosis of GC patients with high expression of AGTR1 was poor (p < 0.05). The AGTR1 gene in GC samples was associated with the expression of immune pathways and immune checkpoint genes. After modifying AGTR1 expression in cell lines, cells' proliferation, invasion, and migration abilities and the expression of related genes changed. CONCLUSIONS: There were significant DEGs in GC tissues with different ACGR molecular types, among which the increased expression of AGTR1 was a molecular feature of MSS/EMT type gastric cancer. Further study found that AGTR1 was closely related to tumor immune infiltration and invasion and may be a new therapeutic target gene for gastric cancer.

Circular RNA hsa_circ_0002938 (circCRIM1) promotes the progression of esophageal squamous cell carcinoma by upregulating transcription factor 12.

Growing evidence has indicated that circular RNAs (circRNAs) play crucial roles in the tumorigenesis and progression of diverse malignancies. However, the majority of circRNAs involved in esophageal squamous cell carcinoma (ESCC) remain undefined and the exact functions and underlying mechanisms of circRNAs in ESCC still need further exploration. In this study, we identified a novel onco-circRNA hsa_circ_0002938, derived from the exons of cysteine-rich transmembrane BMP regulator 1 (CRIM1) pre-mRNA, referred to as circCRIM1. We found that the expression of circCRIM1 was higher in ESCC tissues, compared to para-carcinoma tissues. Increased expression of circCRIM1 was positively correlated with clinical parameters of ESCC patients including tumor-node-metastasis (TNM) stage, tumor invasion range, and lymph node metastasis. Functionally, the results from the experiments in vitro showed that the knockdown of circCRIM1 suppressed proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in ESCC cells. By conducting bioinformatics algorithms analyses and microRNA (miRNA) rescue experiments, we found that circCRIM1 could act as a competing endogenous RNA (ceRNA) to sponge miR-342-3p in ESCC cells, and thereby upregulated the expression of transcription factor 12 (TCF12), a key regulator promoting the EMT process. Taken together, circCRIM1 facilitates the progression of ESCC by sponging miR-342-3p to regulate TCF12 and promote EMT, and the circCRIM1/miR-342-3p/TCF12 axis may be regarded as a potential predictive biomarker and therapeutic target for treating ESCC.

Fibroblast activation protein in the tumor microenvironment predicts outcomes of PD-1 blockade therapy in advanced non-small cell lung cancer.

PURPOSE: The identification of robust predictive biomarkers of the response to programmed cell death-1 (PD-1) blockade remains a critical concern. Here, we investigated on fibroblast activation protein (FAP) as a microenvironment-derived biomarker of clinical outcomes of PD-1 blockade therapy, and the correlation between FAP expression and T cell infiltration in advanced non-small cell lung cancer (NSCLC). METHODS: A total of 135 patients with advanced NSCLC who received PD-1 blockade therapy were retrospectively analyzed. The potential associations among FAP expression, CD3 + T cell and CD8 + T cell infiltration, and clinical outcomes of immunotherapy were validated by immunohistochemistry, bioinformatic analyses, and statistical measurements. RESULTS: FAP was widely expressed in advanced NSCLC tissues. FAP was correlated with decreased density of CD8 + T cells (Spearman's rho - 0.32, p < 0.001) and immunosuppressive tumor microenvironment (TME) status. No correlations were detected between FAP and PD-L1 expression or with the density of CD3 + T cells. The patients with higher expression of FAP showed worse response rate (16.4% vs. 38.7%, p < 0.001) and worse progression-free survival (HR = 2.56, 95% CI 1.69-3.87, p < 0.001). In addition, FAP contributed to shortened overall survival in subgroups of the patients with squamous cell lung cancer (p = 0.020), PD-1 blockade monotherapy (p = 0.017), and first-line therapy (p = 0.028). CONCLUSION: FAP is a potential predictive biomarker of resistance to PD-1 blockade. Further investigation is warranted to identify a strategy for targeting FAP to alleviate the immunosuppressive TME and broaden the clinical effectiveness of PD-1 blockade therapy.