Exosomal LRG1 promotes non-small cell lung cancer proliferation and metastasis by binding FN1 protein.

Leucine-rich α2-glycoprotein-1 (LRG1) is overexpressed in various cancers, including non-small cell lung cancer (NSCLC), but its role in NSCLC cell metastasis is not well understood. In this study, NSCLC cell exosomes were analyzed using different techniques, and the impact of exosomal LRG1 on NSCLC cell behavior was investigated through various assays both in vitro and in vivo. The study revealed that LRG1, found abundantly in NSCLC cells and exosomes, enhanced cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Exosomal LRG1 was shown to promote NSCLC cell metastasis in animal models. Additionally, the interaction between LRG1 and fibronectin 1 (FN1) in the cytoplasm was identified. It was observed that FN1 could counteract the effects of LRG1 knockdown on cell regulation induced by exosomes derived from NSCLC cells. Overall, the findings suggest that targeting exosomal LRG1 or FN1 may hold therapeutic potential for treating NSCLC.

LncRNA WT1-AS downregulates lncRNA UCA1 to suppress non-small cell lung cancer and predicts poor survival.

BACKGROUND: LncRNA WT1-AS inhibits gastric cancer, while its role in other cancers is unknown. We investigated the role of WT1-AS in non-small cell lung cancer (NSCLC). METHODS: Sixty-six NSCLC patients (40 males and 26 females; 36 to 68 years old; mean age 52.7 ± 6.4 years old) were selected from the 178 NSCLC patients operated on for lung cancer between 2010 and 2013. RT-qPCR was used to analyze the expression of lncRNA. Overexpression experiments were performed to assess interactions between lncRNAs. CCK-8 assay was carried to evaluate the roles of WT1-AS and UCA1 in regulating cell proliferation. Cell invasion and migration assays were performed to assess the roles of WT1-AS and UCA1 in regulating cell invasion and migration. Western-blot was performed to illustrate the effect of WT1-AS and UCA1 in EMT. RESULTS: WT1-AS was downregulated in NSCLC and was correlated with poor survival. The expression of WT1-AS in NSCLC was not correlated with clinical stages. LncRNA UCA1 was upregulated in cancer tissues and inversely correlated with WT1-AS. Overexpression of UCA1 did not affect WT1-AS, while overexpression of WT1-AS led to inhibited expression of UCA1. Overexpression of UCA1 resulted in increased proliferation, EMT, migration and invasion of NSCLC cells, while overexpression of WT1-AS showed opposite effects. In addition, overexpression of UCA1 inhibited the role of overexpression of WT1-AS. CONCLUSIONS: Therefore, overexpression of WT1-AS may inhibit the cell proliferation and EMT to decrease cell migration and invasion of NSCLC cells by downregulating UCA1.

Prognostic value of Beclin 1, EGFR and ALK in non-squamous non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the most malignant tumors. The study was carried out to investigate the prognostic value of Beclin 1, EGFR and ALK for this cancer. Patients diagnosed with non-squamous NSCLC and admitted to our hospital from January 2011 to September 2016 were analyzed. Expression of Beclin 1 and mutation of EGFR and ALK were assessed using polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) and analyzed for their relationship with demographic and clinical characteristics of the patients. Multivariate Cox regression models were applied to analyze the risk factors associated with survival and receiver response curves (ROC) were plotted to determine the prognostic value of Beclin 1, EGFR and ALK for patients with non-squamous NSCLC. Compared with adjacent normal tissue, Beclin 1 expression was elevated in the cancer tissue significantly; assessments of EGFR and ALK mutations showed that out of the 480 patients, 233 (48.5%) and 75 (12.6%) patients had EGFR and ALK mutations. Univariate analysis revealed that Beclin 1 level, EGFR and ALK mutations were associated with lymph node metastasis, TNM stage, tumor differentiation and prognosis, but not with gender, age and smoking status. The Kaplan-Meier survival analysis indicated that low Beclin 1 expression and positive EGFR and ALK rearrangements were associated with higher survival rate and longer progress-free survival (PFS). Multivariate Cox regression analysis showed that Beclin 1, EGFR, ALK mutations, tumor differentiation grade, TNM stage and lymph node metastasis were independently associated with PFS. ROC analysis showed that Beclin 1, EGFR and ALK were significant predictors for PFS; the areas under curve (AUC) for Beclin 1, EGFR and ALK were 0.812 (P = 0.018, cut-off value: 1.2), 0.781 (P = 0.011, cut-off value: 15%) and 0.722 (P = 0.010, cut-off value: 11%), respectively, suggesting that they have significant prognostic value for lung cancer patients. Our data indicate that Beclin 1, EGFR and ALK genes are associated with the prognosis of patients with non-squamous NSCLC. High Beclin 1 expression and negative EGFR and ALK mutations predict a poor prognosis with PFS.

A novel tRNA-derived fragment AS-tDR-007333 promotes the malignancy of NSCLC via the HSPB1/MED29 and ELK4/MED29 axes.

BACKGROUND: Transfer RNA-derived fragments (tRFs) are a new class of small non-coding RNAs. Recent studies suggest that tRFs participate in some pathological processes. However, the biological functions and mechanisms of tRFs in non-small cell lung cancer (NSCLC) are largely unknown. METHODS: Differentially expressed tRFs were identified by tRF and tiRNA sequencing using 9 pairs of pre- and post-operation plasma from patients with NSCLC. Quantitative real-time PCR (qRT-PCR) and fluorescence in situ hybridization (FISH) were used to determine the levels of tRF in tissues, plasma, and cells. Gain- and loss-of-function experiments were implemented to investigate the oncogenic effects of tRF on NSCLC cells in vitro and in vivo. Chromatin immunoprecipitation (ChIP), luciferase reporter, RNA pulldown, mass spectrum, RNA immunoprecipitation (RIP), Western blot, co-immunoprecipitation (Co-IP) assays, and rescue experiments were performed to explore the regulatory mechanisms of tRF in NSCLC. RESULTS: AS-tDR-007333 was an uncharacterized tRF and significantly up-regulated in NSCLC tissues, plasma, and cells. Clinically, AS-tDR-007333 overexpression could distinguish NSCLC patients from healthy controls and associated with poorer prognosis of NSCLC patients. Functionally, overexpression of AS-tDR-007333 enhanced proliferation and migration of NSCLC cells, whereas knockdown of AS-tDR-007333 resulted in opposite effects. Mechanistically, AS-tDR-007333 promoted the malignancy of NSCLC cells by activating MED29 through two distinct mechanisms. First, AS-tDR-007333 bound to and interacted with HSPB1, which activated MED29 expression by enhancing H3K4me1 and H3K27ac in MED29 promoter. Second, AS-tDR-007333 stimulated the expression of transcription factor ELK4, which bound to MED29 promoter and increased its transcription. Therapeutically, inhibition of AS-tDR-007333 suppressed NSCLC cell growth in vivo. CONCLUSIONS: Our study identifies a new oncogenic tRF and uncovers a novel mechanism that AS-tDR-007333 promotes NSCLC malignancy through the HSPB1-MED29 and ELK4-MED29 axes. AS-tDR-007333 is a potential diagnostic or prognostic marker and therapeutic target for NSCLC.

Rapid identification of tumor-reactive T-cell receptors by RNA preamplification-based single-cell sequencing.

T-cell receptor (TCR)-transduced T (TCR-T) cell therapy has shown promising efficacy in the clinical treatment of malignant cancers. However, the populations covered by reported TCRs are still limited. Tumor infiltrating lymphocytes (TILs) are natural reservoirs of tumor-reactive T cells and TCRs. Approaches are required for the fast and cost-effective identification of tumor-reactive TCRs from TILs. The widely employed TCR identification approaches by the clonal expansion of TILs involve a TCR singularization process for the direct pairing of TCR Vα and the Vß chain. However, the clonal expansion of T cells is well known to require extensive time and effort due to the involvement of T cell cultures. Several single-cell multiplexing PCR methods followed by Sanger sequencing have been developed, representing a cost-effective and fast approach for single-cell TCR identification. In this study, an RNA-based preamplification step was included in the single-cell TCR sequencing, which can reduce the multiplexing PCR amplification to one round. Moreover, the cDNA product of RNA preamplification is derived from the whole genome mRNA, instead of TCR mRNA only by multiplexing primers-based DNA preamplification, which is valuable for many other analyses (e.g., phenotypic analysis) of the tumor-reactive T cells that can be correlated with the identified TCRs. The feasibility for both single α chain and dual α chain TILs of this approach highlights its potential value as a rapid and cost-effective sequencing strategy for the development of TCR-T therapies for solid cancers.

Human Lung Adenocarcinoma-Derived Organoid Models for Drug Screening.

Lung cancer is an extremely heterogeneous disease, and its treatment remains one of the most challenging tasks in medicine. Few existing laboratory lung cancer models can faithfully recapitulate the diversity of the disease and predict therapy response. Here, we establish 12 patient-derived organoids from the most common lung cancer subtype, lung adenocarcinoma (LADC). Extensive gene and histopathology profiling show that the tumor organoids retain the histological architectures, genomic landscapes, and gene expression profiles of their parental tumors. Patient-derived lung cancer organoids are amenable for biomarker identification and high-throughput drug screening in vitro. This study should enable the generation of patient-derived lung cancer organoid lines, which can be used to further the understanding of lung cancer pathophysiology and to assess drug response in personalized medicine.