Research on the mechanism of exosomes from different sources influencing the progression of lung cancer.

As a key regulator of intercellular communication, exosomes are essential for tumor cells. In our study, we will explore the mechanisms of exosomes from different sources on lung cancer. We isolated CD8+T cells and cancer-associated fibroblasts (CAFs) from venous blood and tumor tissues of lung cancer patients, and isolated exosomes. MiR-2682 was high expression in CD8+T-derived exosomes, and lncRNA-FOXD3-AS1 was upregulated in CAF-derived exosomes. Online bioinformatics database analysis showed that RNA Binding Motif Protein 39 (RBM39) was identified as the target of miR-2682, and eukaryotic translation initiation factors 3B (EIF3B) was identified as the RNA binding protein of FOXD3-AS1. CD8+T-derived exosomes inhibited the growth of A549 cells and promoted apoptosis, while miR-2682 inhibits reversed these effects of CD8+T-derived exosomes. CAF-derived exosomes promoted the growth of A549 cells and inhibited apoptosis, while FOXD3-AS1 siRNA reversed the effect of CAF-derived exosomes. Mechanism studies have found that miR-2682 inhibits the growth of lung cancer cells by inhibiting the expression of RBM39. FOXD3-AS1 promoted the growth of lung cancer cells by binding to EIF3B. In vivo experiments showed that CD8+T cell-derived exosome miR-2682 inhibited lung cancer tumor formation, while CAF-derived exosome FOXD3-AS1 promoted lung cancer tumor formation. This study provides mechanistic insights into the role of miR-2682 and FOXD3-AS1 in lung cancer progression and provides new strategies for lung cancer treatment.

Deciphering a cell death-associated signature for predicting prognosis and response to immunotherapy in lung squamous cell carcinoma.

BACKGROUND: Lung squamous cell carcinoma (LUSC) is a subtype of non-small cell carcinoma, accounting for about 30% of all lung cancers. Yet, the evaluation of prognostic outcome and therapy response of patients with LUSC remains to be resolved. This study aimed to explore the prognostic value of cell death pathways and develop a cell death-associated signature for predicting prognosis and guiding treatment in LUSC. METHODS: Transcriptome profiles and corresponding clinical information of LUSC patients were gathered from The Cancer Genome Atlas (TCGA-LUSC, n = 493) and Gene Expression Omnibus database (GSE74777, n = 107). The cell death-related genes including autophagy (n = 348), apoptosis (n = 163), and necrosis (n = 166) were retrieved from the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases. In the training cohort (TCGA-LUSC), LASSO Cox regression was used to construct four prognostic signatures of respective autophagy, apoptosis, and necrosis pathway and genes of three pathways. After comparing the four signatures, the cell death index (CDI), the signature of combined genes, was further validated in the GSE74777 dataset. We also investigated the clinical significance of the CDI signature in predicting the immunotherapeutic response of LUSC patients. RESULTS: The CDI signature was significantly associated with the overall survival of LUSC patients in the training cohort (HR, 2.13; 95% CI, 1.62‒2.82; P < 0.001) and in the validation cohort (HR, 1.94; 95% CI, 1.01‒3.72; P = 0.04). The differentially expressed genes between the high- and low-risk groups contained cell death-associated cytokines and were enriched in immune-associated pathways. We also found a higher infiltration of naive CD4+ T cells, monocytes, activated dendritic cells, neutrophils, and lower infiltration of plasma cells and resting memory CD4+ T cells in the high-risk group. Tumor stemness indices, mRNAsi and mDNAsi, were both negatively correlated with the risk score of the CDI. Moreover, LUSC patients in the low-risk group are more likely to respond to immunotherapy than those in the high-risk group (P = 0.002). CONCLUSIONS: This study revealed a reliable cell death-associated signature (CDI) that closely correlated with prognosis and the tumor microenvironment in LUSC, which may assist in predicting the prognosis and response to immunotherapy for patients with LUSC.

Exosomal lncRNA FOXD3-AS1 upregulates ELAVL1 expression and activates PI3K/Akt pathway to enhance lung cancer cell proliferation, invasion, and 5-fluorouracil resistance.

Long non-coding RNA (lncRNA) FOXD3-AS1 expression is upregulated in lung cancer; however, its effect and mechanism on 5-fluorouracil (5-FU) resistance remain unclear. In this study, we determined the effects of FOXD3-AS1-enriched exosomes derived from lung cancer cells on the proliferation, invasion, and 5-FU resistance of lung cancer cells. Online bioinformatics database analysis showed that FOXD3-AS1 was upregulated in lung cancer progression. Real-time quantitative PCR results confirmed that FOXD3-AS1 expression was upregulated in lung cancer tissues and cell lines, and FOXD3-AS1 was greatly enriched in lung cancer cell-derived exosomes. ELAV-like RNA-binding protein 1 (ELAVL1) was identified as an RNA-binding protein of FOXD3-AS1. The lung cancer cell-derived exosomes promoted A549 cell proliferation and invasion and inhibited apoptosis caused by 5-FU, and transfection of si-FOXD3-AS1 or si-ELAVL1 in exosome-incubated A549 cells reversed these effects. Moreover, exosome-incubated A549 cells were co-transfected with si-FOXD3-AS1 and pcDNA-ELAVL1, showing the same cell proliferation, invasion, and 5-FU resistance as those of A549 cells treated with lung cancer cell-derived exosomes alone. Mechanistic studies identified that lung cancer cell-derived exosomes activated the PI3K/Akt pathway, and transfection of si-FOXD3-AS1 or treatment with the PI3K inhibitor LY294002 reversed the activation of the PI3K/Akt axis induced by exosomes. In conclusion, our study revealed that lung cancer cell-derived exosomal FOXD3-AS1 upregulated ELAVL1 expression and activated the PI3K/Akt pathway to promote lung cancer progression. Our findings provide a new strategy for lung cancer treatment.

Exosome-derived miR-2682-5p suppresses cell viability and migration by HDAC1-silence-mediated upregulation of ADH1A in non-small cell lung cancer.

INTRODUCTION: Increasing evidence indicated that miR-2682-5p acted as a tumor suppressor in various cancers. The current study aimed to investigate the biological function of exosomal miR-2682-5p in non-small cell lung cancer (NSCLC). METHODS: The expression of miR-2682-5p in NSCLC tissues and adjacent non-tumor tissues, NSCLC cell lines and human embryonic lung fibroblast, as well as serum and serum exosomes of NSCLC patients and healthy donors was detected by RT-qPCR. The effects of miR-2682-5p on the viability, migration, and apoptosis of NSCLC cells were detected by CCK-8, Transwell, and flow cytometry assays. Dual-luciferase reporter gene and RNA immunoprecipitation assays were used to evalutate the relationship between miR-2682-5p and HDAC1. RESULTS: Low expressed miR-2682-5p was found in tumor tissues, cell lines, serum, and serum exosomes of NSCLC patients. MiR-2682-5p overexpression suppressed NSCLC cell viability and migration and promoted apoptosis, while miR-2682-5p knockdown showed the opposite results. Furthermore, exosomes from healthy donor serum inhibited NSCLC cell viability and migration and promoted apoptosis. Dual-luciferase reporter gene and RNA immunoprecipitation assays verified that HDAC1 was a target of miR-2682-5p. HDAC1 overexpression abolished the effects of miR-2682-5p mimic on NSCLC cell viability, migration, and apoptosis. Chromatin immunoprecipitation assay indicated that HDAC1 bound to the promoter region of ADH1A. Upregulation of ADH1A counteracted the effects of HDAC1 overexpression on NSCLC cell viability, migration, and apoptosis. CONCLUSION: Taken together, exosomal miR-2682-5p inhibited NSCLC cell viability and migration and promoted apoptosis by the HDAC1/ADH1A axis, and this result might provide a novel therapeutic target for NSCLC.

Tumor Cell-Derived Exosomal miR-770 Inhibits M2 Macrophage Polarization via Targeting MAP3K1 to Inhibit the Invasion of Non-small Cell Lung Cancer Cells.

BACKGROUND: Non-small cell lung carcinoma (NSCLC) is a type lung cancer with high malignant behaviors. MicroRNAs (miRNAs) are known to be involved in progression of NSCLC. In order to explore potential targets for the treatment of NSCLC, bioinformatics tool was used to analyze differential expressed miRNAs between NSCLC and adjacent normal tissues. METHODS: Bioinformatics tool was used to find potential targets for NSCLC. Cell proliferation was investigated by Ki67 staining. Cell apoptosis was measured by flow cytometry. mRNA and protein expression in NSCLC cells were detected by RT-qPCR and Western-blot, respectively. Transwell assay was performed to test the cell migration and invasion. In order to investigate the function of exosomal miRNA in NSCLC, in vivo model of NSCLC was constructed. RESULTS: MiR-770 was identified to be downregulated in NSCLC, and miR-770 agomir could significantly inhibit NSCLC cell proliferation through inducing the apoptosis. Additionally, the metastasis of NSCLC cells was decreased by miR-770 agomir. MAP3K1 was identified to be the target mRNA of miR-770. Meanwhile, tumor cell-derived exosomal miR-770 inhibited M2 macrophage polarization via downregulation of MAP3K1, which in turn suppressed NSCLC cell invasion. Besides, tumor cell-derived exosomal miR-770 markedly decreased NSCLC tumor growth in vivo through suppressing M2 macrophage polarization. CONCLUSION: Tumor cell-derived exosomal miR-770 inhibits M2 macrophage polarization to inhibit the invasion of NSCLC cells via targeting MAP3K1. Thus, this study provided a new strategy for the treatment of NSCLC.

Targeted deep sequencing helps distinguish independent primary tumors from intrapulmonary metastasis for lung cancer diagnosis.

PURPOSE: Multiple lung lesions found in a single patient at the time of diagnosis often pose a diagnostic dilemma: are these lesions independent primary tumors (IPT) or the result of intrapulmonary metastases (IPM)? While traditional pathological methods sometimes have difficulty distinguishing IPM from IPT, modern molecular profiling based on next-generation sequencing techniques may provide a new strategy. METHODS: Sixteen patients with multiple tumors were enrolled in this study. We performed targeted deep sequencing (~ 2000 × coverage) on a total of 40 tumors and matched blood samples. We compared mutational profiles between tumors within each patient and across patients to evaluate if they were genetically related. Computed tomographic images and histological staining were also used to validate tumor relationships. RESULTS: A total of 125 mutations were identified in 16 patients. Twelve out of fourteen patients whose histological diagnoses favored IPT did not have any shared mutations in their multiple tumors. The other two showed discrepancies: Pt01 had a shared EGFR exon19 deletion in the two lung tumors found, and Pt16 had one common mutation (BRAFD594G) in two out of five lung tumors. Pt14 with lung metastasis from salivary gland adenoid cystic carcinoma had shared mutations; and Pt15 with suspected intrapulmonary metastasis (IPM) had identical mutations between the two tumors. Visualized data can be readily accessed through the website: mlc.opengene.org. CONCLUSION: Analysis of overlapping mutations among different tumors assists physicians in distinguishing IPM from IPT. Our findings demonstrate that DNA sequencing can provide additional evidence in clinical practice when pathology is inadequate to make a conclusive diagnosis.

TW37 enhances the pro-apoptosis and anti-migration ability of gefitinib in Non-Small Cell Lung Cancer.

B cell leukemia-2 (Bcl-2) plays important roles in the development of tumor and drug resistance. The growth of tumor cells can be inhibited by downregulating the abnormal expression of Bcl-2 protein. TW37, an effective inhibitor of Bcl-2 protein, has now been widely studied in many tumors. In our study, it was found that TW37 exerted a significant effect on the proliferation, apoptosis and migration of Non-Small Cell Lung Cancer cells. Bcl-2 is also a key downstream factor of many signaling pathways such as Epidermal Growth Factor Receptor (EGFR). TW37 enhanced the inhibition of tumorigenesis by gefitinib, an EGFR-Tyrosine Kinase Inhibitor drug. Moreover, TW37 can promote apoptosis ability by inhibiting the phosphorylation level of EGFR protein in H1975 cells. Overall, TW37 enhances the pro-apoptosis and anti-migration ability of gefitinib in Non-Small Cell Lung Cancer.