Value In Non-small Cell Lung Cancer Research Articles

Comprehensive analysis and validation reveal DEPDC1 as a potential diagnostic biomarker associated with tumor immunity in non-small-cell lung cancer.

Current evidence suggests that DEP domain containing 1 (DEPDC1) has an important effect on non-small-cell lung cancer (NSCLC). However, the diagnostic value and the regulatory function within NSCLC are largely unclear. This work utilized publicly available databases and in vitro experiments for exploring, DEPDC1 expression, clinical features, diagnostic significance and latent molecular mechanism within NSCLC. According to our results, DEPDC1 was remarkably upregulated in the tissues of NSCLC patients compared with non-carcinoma tissues, linked with gender, stage, T classification and N classification based on TCGA data and associated with smoking status and stage according to GEO datasets. Meanwhile, the summary receiver operating characteristic (sROC) curve analysis result showed that DEPDC1 had a high diagnostic value in NSCLC (AUC = 0.96, 95% CI: 0.94-0.98; diagnostic odds ratio = 99.08, 95%CI: 31.91-307.65; sensitivity = 0.89, 95%CI: 0.81-0.94; specificity = 0.92, 95%CI: 0.86-0.96; positive predictive value = 0.94, 95%CI: 0.89-0.98; negative predictive value = 0.78, 95%CI: 0.67-0.90; positive likelihood ratio = 11.77, 95%CI: 6.11-22.68; and negative likelihood ratio = 0.12, 95%CI: 0.06-0.22). Subsequently, quantitative real-time PCR (qRT-PCR) and western blotting indicated that DEPDC1 was high expressed in NSCLC cells. According to the in vitro MTS and apoptotic assays, downregulated DEPDC1 expression targeting P53 signaling pathway inhibited the proliferation of NSCLC cells while promoting apoptosis of NSCLC cells. Moreover, DEPDC1 was significantly correlated with immune cell infiltrating levels in NSCLC based on TCGA data, which were primarily associated with T cells CD4 memory activated, macrophages M1, B cells memory, mast cells resting, T cells regulatory, monocytes, and T cells CD4 memory resting. Compared with the group with high expression of DEPDC1, the group with low expression level had higher scores for immune checkpoint inhibitors (ICIs) treatment. GSEA confirmed that DEPDC1 was involved in gene expression and tumor-related signaling pathways. Finally, DEPDC1 and its associated immune-related genes were shown to be enriched in 'receptor ligand activity', 'external side of plasma membrane', 'regulation of innate immune response', and 'Epstein-Barr virus infection' pathways. The present study demonstrates that DEPDC1 may contribute to NSCLC tumorigenesis and can be applied as the biomarker for diagnosis and immunology.

A circRNA-based ceRNA network shows its diagnostic value in non-small-cell lung cancer

BackgroundNumerous studies have reported the vital roles of circular RNA (circRNA)-based competitive endogenous RNA (ceRNA) regulatory networks in cancers. Here, we established a non-small-cell lung cancer (NSCLC)-related circRNA–miRNA–mRNA axis and estimated its diagnostic value in NSCLC. MethodsThe circ_0061235–miR-3180-5p–PPM1L axis was constructed by small RNA deep sequencing, bioinformatics databases, and preliminary testing. The serum levels of the selected circ_0061235, miR-3180-5p, and PPM1L were quantified using quantitative polymerase chain reaction. Receiver operating characteristic analyses were conducted to evaluate the diagnostic power. ResultsThe levels of circ_0061235, miR-3180-5p, and PPM1L showed close correlations according to the ceRNA regulation rule. They were significantly dysregulated in NSCLC and showed the diagnostic ability to discriminate between healthy and NSCLC, and remarkably, between benign lung tumors and NSCLC. Additionally, the down-regulated levels of hsa_circ_0061235, the up-regulated levels of miR-3180-5p, and the decreased levels of PPM1L were correlated to more aggressive features of NSCLC, such as lymph node metastasis, distant metastasis, and higher stages. Intriguingly, compared to the single circ_0061235, miR-3180-5p, PPM1L, and traditional tumor markers, the diverse combinations of circ_0061235, miR-3180-5p, and PPM1L showed much higher sensitivity and specificity to differentiate greater or lesser severity of NSCLC. GO annotation and KEGG pathway analyses revealed the underlying role of the circ_0061235–miR-3180-5p–PPM1L axis in NSCLC. ConclusionsWe established a specific circRNA–miRNA–mRNA network with higher sensitivity and specificity to diagnose NSCLC, particularly more aggressive NSCLC, providing a new strategy for further developing tumor biomarkers.

Identification of novel potential drugs and miRNAs biomarkers in lung cancer based on gene co-expression network analysis.

Non-small cell lung cancer (NSCLC) is an important cause of cancer-associated deaths worldwide. Therefore, the exact molecular mechanisms of NSCLC are unidentified. The present investigation aims to identify the miRNAs with predictive value in NSCLC. The two datasets were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed miRNAs (DEmiRNA) and mRNAs (DEmRNA) were selected from the normalized data. Next, miRNA-mRNA interactions were determined. Then, co-expression network analysis was completed using the WGCNA package in R software. The co-expression network between DEmiRNAs and DEmRNAs was calculated to prioritize the miRNAs. Next, the enrichment analysis was performed for DEmiRNA and DEmRNA. Finally, the drug-gene interaction network was constructed by importing the gene list to dgidb database. A total of 3,033 differentially expressed genes and 58 DE miRNA were recognized from two datasets. The co-expression network analysis was utilized to build a gene co-expression network. Next, four modules were selected based on the Zsummary score. In the next step, a bipartite miRNA-gene network was constructed and hub miRNAs (let-7a-2-3p, let-7d-5p, let-7b-5p, let-7a-5p, and let-7b-3p) were selected. Finally, a drug-gene network was constructed while SUNITINIB, MEDROXYPROGESTERONE ACETATE, DOFETILIDE, HALOPERIDOL, and CALCITRIOL drugs were recognized as a beneficial drug in NSCLC. The hub miRNAs and repurposed drugs may act a vital role in NSCLC progression and treatment, respectively; however, these results must validate in further clinical and experimental assessments.

Cell-type-specific alternative polyadenylation promotes oncogenic gene expression in non-small cell lung cancer progression

Disrupted alternative polyadenylation (APA) is frequently involved in tumorigenesis and cancer progression by regulating the gene expression of oncogenes and tumor suppressors. However, limited knowledge of tumor-type- and cell-type-specific APA events may lead to novel APA events and their functions being overlooked. Here, we compared APA events across different cell types in non-small cell lung cancer (NSCLC) and normal tissues and identified functionally related APA events in NSCLC. We found several cell-specific 3'-UTR alterations that regulate gene expression changes showed prognostic value in NSCLC. We further investigated the function of APA-mediated 3'-UTR shortening through loss of microRNA (miRNA)-binding sites, and we identified and experimentally validated several oncogene-miRNA-tumor suppressor axes. According to our analyses, we found SPARC as an APA-regulated oncogene in cancer-associated fibroblasts in NSCLC. Knockdown of SPARC attenuates lung cancer cell invasion and metastasis. Moreover, we found high SPARC expression associated with resistance to several drugs except cisplatin. NSCLC patients with high SPARC expression could benefit more compared to low-SPARC-expression patients with cisplatin treatment. Overall, our comprehensive analysis of cell-specific APA events shed light on the regulatory mechanism of cell-specificoncogenes and provided opportunities for combination of APA-regulated therapeutic target and cell-specific therapy development.

The clinical and genomic characteristics of KRAS G12D mutated cancers.

e15151 Background: KRAS mutations are relatively common in solid tumors. It is once considered to be “non-druggable” due to its high affinity to GTP/GDP and lack of other binding sites. Recently, irreversible inhibitors targeting specific KRAS mutations have been developed. We aimed to investigate the correlation of clinical and genomic factors with KRAS G12D mutation in pancreatic cancer (PC), colorectal cancer (CRC) and non-small-cell lung cancer (NSCLC). Methods: We retrospectively reviewed the clinical and genomic data of patients with PC (n = 300), CRC (n = 300), and NSCLC (n = 1800) who provided tumor tissues and matched peripheral blood samples (served as control to eliminate germline variants) for targeted sequencing of 450 cancer-related genes. Experiments were carried out in OrigiMed’s laboratory following the CAP/CLIA standards. Tumor mutation burden (TMB)-high was defined as TMB > 10 muts/Mb. Results: The prevalence of KRAS G12D mutation in PC, CRC and NSCLC was 38.3% (115/300), 15.0% (45/300) and 2.0% (36/1800), respectively. In PC and CRC, G12D was the major type of KRAS mutations, accounting for 44.9% (115/256) and 30.8% (45/146) of all the KRAS mutations, respectively. In contrast, G12C was the most common KRAS mutation in NSCLC (35.9% [79/220]), followed by G12D (16.4% [36/220]). Patients were divided into three groups according to the KRAS mutation status: the G12D (mutated in KRAS G12D), non-G12D (mutated in KRAS, but not G12D), and wild-type (WT; no KRAS mutation) groups. Clinical features including age, sex and disease stage were not associated with the KRAS grouping in PC and CRC. However, the KRAS grouping was significantly correlated with sex in NSCLC (Female vs Male: 1.9% vs 2.1% in G12D, 4.4% vs 13.9% in non-G12D, 93.7% vs 84.1% in WT, P < 0.001). In PC, KRAS G12D and BRAF mutations were mutually exclusive (P < 0.01), while in CRC, KRAS G12D mutation was found to be co-occurring with PIK3CA mutation (P < 0.05). For NSCLC, KRAS G12D mutation was mutually exclusive with EGFR mutation (P < 0.01) and co-occurred with STK11 mutation (P < 0.05). TMB was comparable between the KRAS grouping in PC and CRC. In contrast, TMB was significantly higher in the non-G12D group (median 11.7 [range 0.7-89.8] muts/Mb) compared to the G12D (median 6.9 [range 0-30.9] muts/Mb; P < 0.001) and WT (median 4.6 [range 0-215.2] muts/Mb; P < 0.001) groups in NSCLC. A higher proportion of TMB-high was also observed in the non-G12D group (58.5%) compared to the G12D (22.2%) and WT (22.6%) groups in NSCLC. Conclusions: Patients with KRAS G12D mutation possessed unique clinical and genomic characteristics. It was more prevalent in PC and CRC and was correlated with sex in NSCLC. KRAS G12D mutation co-occurred with STK11 mutation and showed a relatively low TMB value in NSCLC, suggesting that immunotherapy might be ineffective. While KRAS non-G12D mutated NSCLC patients may be favorable to ICI. Our results provide insights into the precision medicine of KRAS G12D-mutated cancers.

Plasma versican and plasma exosomal versican as potential diagnostic markers for non-small cell lung cancer

Background and aimsThis study aimed to investigate the expression of plasma versican and plasma exosomal versican in non-small cell lung cancer (NSCLC) and its correlation with clinicopathological features, and to evaluate its diagnostic performance in NSCLC and its predictive function for NSCLC incidence and metastasis risk.Materials and methodsThere were 110 instances of NSCLC, 42 cases of benign lung disease, and 55 healthy controls from September 2018 to October 2020 at Tongji Hospital Affiliated to Tongji University. Blood was collected and plasma was separated before surgery, and plasma exosomes were extracted by ExoQuick kit. Morphological and molecular phenotype identification of exosomes was performed by transmission electron microscopy, Nanosight particle tracking analysis, and western blotting. Plasma versican and plasma exosomal versican were detected in all subjects to assess their expression levels and diagnostic value in NSCLC. Clinicopathological data were collected to explore correlations between abnormal plasma versican and plasma exosomal versican expression and clinicopathological parameters. Receiver operating characteristic (ROC) curve was used to judge its diagnostic performance in NSCLC, and binary logistic regression analysis was used to predict the risk of NSCLC incidence and metastasis.ResultsPlasma versican and plasma exosomal versican expression in NSCLC patients was significantly upregulated and was significantly higher in T3 + T4 patients compared with T1 + T2 patients (P < 0.05); the levels of plasma versican and plasma exosomal versican were positively correlated with lymph node metastasis, distant metastases (e.g., brain, bone), and mutation(e.g., EGFR,ALK)in NSCLC patients (all P < 0.05). Furthermore, ROC curve analysis showed that plasma versican and plasma exosomal versican had higher AUC values than NSE, CYFRA21-1, and SCC, and better diagnostic performance in NSCLC patients. However, the AUC and diagnostic performances of plasma versican and plasma exosomal versican in advanced-stage NSCLC patients were not shown to be significantly better than CEA. The results of binary logistic regression analysis showed that high levels of plasma exosomal versican had higher predictive value for lung cancer incidence, while high levels of plasma versican had higher predictive value for lung cancer metastasis.ConclusionOur findings showed that plasma versican and plasma exosomal versican might be potential diagnostic markers for NSCLC. High plasma exosomal versican expression can be used as a predictor of NSCLC risk and high plasma versican expression can be used as a predictor of NSCLC metastasis risk.

Abstract 4396: TROP2 expression in non-small cell lung cancer

Abstract Introduction: Non-small cell lung cancer (NSCLC) is a major cause of cancer-related deaths globally with high unmet need. A variety of novel therapeutic strategies are being explored to improve patient outcomes including the use of antibody drug conjugates (ADCs) to deliver highly potent, cytotoxic payloads to tumors. Trophoblast cell-surface antigen 2 (TROP2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), has emerged as an attractive target for ADCs. Sacituzumab govitecan-hziy is a novel ADC composed of a TROP2 antibody coupled to SN-38 and is being evaluated in NSCLC. High TROP2 mRNA expression is observed in many tumor types, however, TROP2 protein expression in NSCLC is not well-established. Here we characterized TROP2 expression across three independent datasets to explore the TROP2 relationship to baseline characteristics, molecular features of interest, and its prognostic value in NSCLC. Methods: We analyzed three independent datasets: (1) The Cancer Genome Atlas (TCGA) NSCLC adenocarcinoma (LUAD, N=660) and squamous cell carcinoma (LUSC, N=484) mRNA expression data, (2) a NSCLC adenocarcinoma FFPE tumor sample set (“Translational data set”; N=107), (3) an independent clinical NSCLC adenocarcinoma (N=103) and squamous cell carcinoma (N=37) data set. TROP2 IHC was performed on tumor sample sets (2) and (3) using the SP295 antibody (Robust Prototype Assay, Ventana) with H-Score and assessment of the percentage of membrane-positive tumor cells as readouts. Wilcoxon rank-sum and Kruskal-Wallis tests (continuous variables), Kaplan-Meier method (survival probability curves) and log-rank test (time to event outcome) were used (GraphPad Prism 8.1.2 and R version 4.0.5). Results: TROP2 mRNA was highly expressed in NSCLC and expression was similar in adenocarcinoma and squamous cell carcinoma in the TCGA data set. TROP2 expression did not vary with patient’s age, gender, race or tumor stage. Furthermore, TROP2 expression was not associated with TP53, KRAS or driver mutation status (including EGFR, ALK, ROS, RET, MET). TROP2 expression in the Translational sample set was consistent with the findings from the TCGA data analysis: TROP2 mRNA and protein expression were high and independent of patient baseline demographics, tumor stage and TP53 and KRAS mutation status. Finally, in an independent clinical NSCLC dataset, TROP2 protein was highly expressed and there was no relationship between TROP2 and baseline demographics including gender and age. TROP2 membrane H-scores and percentage of membrane-positive tumor cells also highly correlated in the two independent datasets. Conclusions: Three large independent datasets confirmed high expression of TROP2 in NSCLC both, as mRNA and protein. TROP2 expression in tumors did not differ between histological subtypes, baseline characteristics or clinically relevant driver alterations. Based on the TCGA data set, TROP2 expression was not prognostic for survival. Citation Format: Peiwen Kuo, Emon Elboudwarej, Lauri Diehl, Jilpa Patel, Sabeen Mekan, Juliane M. Jürgensmeier. TROP2 expression in non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4396.

Prognostic value of tumor-infiltrating T-lymphocytes density in the therapeutic response to initial platinum-based chemotherapy in patients with non-small cell lung cancer

Background/Aim. The fact that lung carcinomas, like other solid tumors, can be immunogenic may have a substantial prognostic value in non-small cell lung cancer (NSCLC). Specific cytotoxic T-lymphocytes (CTL) can be demonstrated in most patients with primary tumors of different histological types. Two main groups of T-lymphocytes participate in the coupled recognition of tumor-specific antigens ? CTL (CD8+) and helper T-lymphocytes (CD4+). The aim of the study was to assess the relationship between the tumor infiltration of T-lymphocytes and the therapeutic response to initial chemotherapy. Methods. Data were obtained from patients with NSCLC whose therapeutic response after four cycles of initial platinum-based chemotherapy was observed in relation to the density of tumor-infiltrating T-lymphocytes (CD4+ and CD8+) in small tumor biopsy samples. The therapeutic response was assessed in line with Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 therapeutic response evaluation system. Based on the expected therapeutic response, the patients were divided into three groups: favorable therapeutic response patients (complete and partial regression), stable disease patients, and disease progression patients. To assess the density of CD4+ and CD8+ T-lymphocytes, the number of lymphocytes was determined at ?200 magnification (1.1 mm2). Three visual fields with the densest lymphocyte infiltrate were selected for counting, and the values of all individual fields were added up. Based on the mean value, the samples were classified into the following groups: score 0, score 1, score 2, and score 3. During statistical data processing, low infiltration density combined score 0 and score 1 groups, and high infiltration density combined score 2 and score 3 groups. Based on the collected data, a database was created in SPSS 22.0 software and used for further statistical analysis. Statistical analysis of the data included descriptive and analytical statistics methods. Results. There was no significant difference in the distribution of CD4+ T-lymphocytes and CD8+ T-lymphocytes in the epithelial component of the tumor between patients with a different therapeutic response (?2 = 2.977; p = 0.226 and ?2 = 1.329; p = 0.515, respectively). There was no significant influence of the infiltration density of CD4+ T-lymphocytes and CD8+ T-lymphocytes in the stromal component of the tumor on the therapeutic response (?2 = 0.606; p = 0.739 and ?2 = 5.167; p = 0.076, respectively). Conclusion. The research did not prove that patients with a high level of tumor-infiltrating CD4+ and CD8+ T-lymphocytes in the epithelial and stromal component of the NSCLC had a better therapeutic response to standard initial chemotherapy.

TTI1 promotes non-small-cell lung cancer progression by regulating the mTOR signaling pathway.

The role of TELO2-interacting protein 1 (TTI1) in the progression of several types of cancer has been reported recently. The aim of this study was to estimate the expression and potential value of TTI1 in non-small-cell lung cancer (NSCLC) patients. The expression of TTI1 and its prognostic value in NSCLC from The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database were analyzed. To verify the bioinformatics findings, a tissue microarray containing 160 NSCLC and paired peritumoral tissues from NSCLC patients was analyzed by immunohistochemistry for TTI1. Subsequently, the roles of TTI1 in NSCLC cells were investigated in vivo by establishing xenograft models in nude mice and in vitro by transwell, CCK-8, wound healing, and colony formation assays. In addition, quantitative real-time polymerase chain reaction and western blot were applied to explore the underlying mechanism by which TTI1 promotes tumor progression. Finally, the relationship between TTI1 and Ki67 expression level in NSCLC was probed, and Kaplan-Meier and Cox analyses were performed to assess the prognostic merit of TTI1 and Ki67 in NSCLC patients. We found that the expression of TTI1 was significantly upregulated in NSCLC tissues compared to paired peritumoral tissues, which coincides with the bioinformatics findings from the TCGA and GEO databases. TTI1 was highly expressed in NSCLC patients with large tumors, advanced tumor stage, and lymphatic metastasis. In addition, the prognostic analysis identified TTI1 as an independent indication for poor prognosis of NSCLC patients. In vitro, upregulation of TTI1 in NSCLC cells could facilitate cell invasion, metastasis, viability, and proliferation. Mechanistically, our study verified that TTI1 could regulate mTOR activity, which has a pivotal role in human cancer. Consistently, the expressions of TTI1 and Ki67 had a positive relationship in NSCLC cells and tissues. Notably, patients with overexpression of TTI1 or Ki67 had a shorter overall survival rate and a higher disease-free survival rate compared to patients with low expression of TTI1 or Ki67, and the combination of TTI1 and Ki67 was an independent parameter predicting the prognosis and recurrence of NSCLC patients. We conclude that TTI1 promotes NSCLC cell proliferation, metastasis, and invasion by regulating mTOR activity, and the combination of TTI1 and Ki67 is a valuable molecular biomarker for the survival and recurrence of NSCLC patients.

The Expression of hnRNP A2/B1 in Benign and Malignant Lung Lesions and Its Early Diagnosis Value in NSCLC.

Lung cancer in its occurrence and development of different stages exist different biological behavior changes. This paper studies the expression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 in benign and malignant lung lesions and its early diagnosis value of nonsmall-cell lung cancer (NSCLC), aiming to provide reference for the early diagnosis and therapy of NSCLC. Some lung surgery specimens are selected from January 2021 to March 2022. All cases received no radiotherapy and chemotherapy before surgery, including 90 sufferers with benign lung lesions as the contrast set. hnRNP A2/B1 expressions are measured for comparison. The experimental results show that for lung cancer sufferers, the positive expression of hnRNP A2/B1 in their malignant lesion tissue is notoriously higher than that in their benign lesion tissue, and hnRNP A2/B1 is differently expressed in different differentiation and in different stages.

FOXP family DNA methylation correlates with immune infiltration and prognostic value in NSCLC.

Background: Forkhead box P (FOXP) family was introduced as a double-edged sword in tumorigenesis and influenced immunotherapy response by modulating host immunity. This study aimed to summarize the involvement of the FOXP family in non-small cell lung cancer (NSCLC). Methods: The UALCAN, Gene Expression Profiling Interactive Analysis (GEPIA), and Reverse transcription-quantitative polymerase chain reaction (RT‒qPCR) were used to analyse the expression levels of the FOXP family in NSCLC. The prognostic impact was evaluated using Kaplan-Meier Plotter. MethSurv, UALCAN, and cBioPortal were applied to analyse the DNA methylation and mutation status of the FOXP family respectively. COEXPEDIA, STRING, and GeneMANIA were used to explore the interaction mechanism. Finally, TISIDB was used to investigate all of the immune-related characteristics regulated by the FOXP family. Results: The expression levels of FOXP1/3/4 were dysregulated in NSCLC tissues than that in normal tissues. Groups with low expression levels of FOXP1/4 and high expression levels of FOXP2/3 were associated with poor prognosis in NSCLC. The transcriptional levels of FOXP2/3/4 were correlated with DNA methylation in NSCLC. FOXP1/3/4 DNA methylation were correlated with prognosis. Pathway enrichment analysis indicated the FOXP family was mainly related to immune-related pathways. After DNA methylation, the correlations between FOXP family and immune factors were opposite to that before alteration in NSCLC. Conclusion: This study elucidated FOXP family could serve as vital diagnostic and prognostic biomarkers in NSCLC. Our study highlighted novel potential functions of FOXP family DNA methylation in regulation of immune-related signatures in NSCLC.

Long non-coding RNA BZRAP1-AS1 functions in malignancy and prognosis for non-small-cell lung cancer.

PurposeThe function of BZRAP1-AS1 is unknown in lung cancer. We evaluated the clinicopathologic significance of BZRAP1-AS1, and its role in non-small-cell lung cancer (NSCLC) progression.Patient and methodsSixty-three NSCLC patients from Beijing Chest Hospital were included. The expression of BZRAP1-AS1 was detected by real-time quantitative polymerase chain reaction (RT-qPCR) in tumor tissues and adjacent normal tissues. Then, the clinicopathological significance and prognostic value of BZRAP1-AS1 were analyzed by using our cohort and TCGA cohort. Finally, the effect of BZRAP1-AS1 on proliferation and motility of NSCLC cell lines were evaluated by cell growth assay, colony formation assay, xenograft tumorigenesis experiment in nude mice and transwell assays respectively.ResultsCompared with adjacent normal tissues, BZRAP1-AS1 showed lower expression in NSCLC tumor tissues. As for the relationship between BZRAP1-AS1 and clinical characteristics, our results were consistent with those of TCGA data. BZRAP1-AS1 was lower in T1 than T2–T4 patients, N1-N3 than N0 patients. Low level BZRAP1-AS1 was related to shorter overall survival time (OS) in lung adenocarcinoma (LUAD), and poor first progression time (FP) in LUAD and lung squamous cell carcinoma (LUSC) patients. BZRAP1-AS1 was significantly associated with the prognosis of NSCLC patients. Overexpression of BZRAP1-AS1 inhibited proliferation and migration of H1299 and HCC827 cells.ConclusionBZRAP1-AS1 expression decreases in tumor tissues with the increase of malignancy grades in NSCLC. BZRAP1-AS1 plays an anticancer role by inhibiting cell proliferation, invasion, and metastasis, and has a potential prognostic value in NSCLC. BZRAP1-AS1 may serve as a diagnostic marker and therapeutic target for NSCLC.

P21-activated kinase 2-mediated β-catenin signaling promotes cancer stemness and osimertinib resistance in EGFR-mutant non-small-cell lung cancer.

Osimertinib (AZD9291) is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), used for treating patients with advanced non-small-cell lung cancer (NSCLC) harboring EGFR-activating mutations or the resistant T790M mutation. However, acquired resistance to osimertinib is inevitable in EGFR-mutant NSCLC. By employing a global mass spectrometry-based phosphoproteomics approach, we identified that the activated p21-activated kinase 2 (PAK2)/β-catenin axis acts as a driver of osimertinib resistance. We found that PAK2 directly phosphorylates β-catenin and increases the nuclear localization of β-catenin, leading to the increased expression and transcriptional activity of β-catenin, which in turn enhances cancer stem-like properties and osimertinib resistance. Moreover, we revealed that HER3 as an upstream regulator of PAK2, drives the activation of PAK2/β-catenin pathways in osimertinib-resistant cells. The clinical relevance of these findings was further confirmed by examining tissue specimens from patients with EGFR-mutant NSCLC. The results demonstrated that the levels of HER3, phospho-PAK2 (p-PAK2) and β-catenin in the tissues from patients with EGFR-mutant NSCLC, that had relapsed after treatment with osimertinib, were elevated compared to those of the corresponding untreated tissues. Additionally, the high levels of HER3, p-PAK2 and β-catenin correlated with shorter progression-free survival (PFS) in patients with EGFR-TKI-treated NSCLC. We additionally observed that the suppression of PAK2 via knockdown or pharmacological targeting with PAK inhibitors markedly restored the response of osimertinib-resistant NSCLC cells to osimertinib both in vitro and in vivo. In conclusion, these results indicated that the PAK2-mediated activation of β-catenin is important for osimertinib resistance and targeting the HER3/PAK2/β-catenin pathway has potential therapeutic value in NSCLCs with acquired resistance to osimertinib.

The diagnostic value of has_circ_0006423 in non-small cell lung cancer and its role as a tumor suppressor gene that sponges miR-492

The diagnosis and treatment of non-small cell lung cancer (NSCLC) are not ideal. We identified NSCLC-related has_circ_0006423 in database. qRT-PCR was used to measure expression levels of hsa_circ_0006423 and miR-492 in the plasma and tissue samples, and 3 NSCLC cell lines, respectively. We analyzed the relationship between expression levels of hsa_circ_0006423 and clinicopathological factors and miR-492 expression in plasma and tissue samples. Assess the diagnostic value of hsa_circ_0006423 and miR-492 in NSCLC. Cell function vitro experiment to explore the effect of has_circ_0006423 on NSCLC. We found has_circ_0006423 is lower expressed in NSCLC and miR-492 is opposite, has_circ_0006423 and miR-492 has diagnostic value in NSCLC. In A549 and NCI-H1299 cells, hsa_circ_0006423 inhibited the proliferation, migration, and invasion of NSCLC cells by sponging miR-492 and accelerating NSCLC cell apoptosis. This effect may be due to the combination of has_circ_0006423 and miR-492 affecting the progression of NSCLC.

M6A regulator expression patterns predict the immune microenvironment and prognosis of non-small cell lung cancer.

The m6A methylation modification is one of the most common mRNA modifications, and involved in a variety of biological processes, such as cell death, cancer stem cell formation and tumorigenesis. Increasing evidences have demonstrated that the expression patterns of m6A regulators are significantly correlated with PD-L1 level some solid tumors, but few study has explored the function of m6A regulators in the immune microenvironment and prognosis in non-small cell lung cancer (NSCLC). Survival analysis was independently conducted for 20 m6A regulators to explore their prognostic value in NSCLC, and then the prognostic risk model based on m6A regulator expression profiles is built to stratify NSCLC patients. Also, the correlation analysis between immune infiltrating cells and m6A regulators is used to reveal the impact of m6A on immune microenvironment of NSCLC. Furthermore, to explore the function of m6A as biomarker of anit-PD-L1 therapeutic effect, we explored the associations of tumor mutation burden (TMB) and PD-L1 levels to 20 m6A regulator expression patterns in NSCLC. First, the expressions of 20 m6A regulators in NSCLC tissues were significantly increased compared to normal tissues. Survival analysis revealed that three genes, METTL3, HNRNPC and VIRMA, were markedly correlated to the prognosis of NSCLC patients. In particular, cox regression analysis verified that METTL3 could be used as an independent prognostic factor to predict the survival rate of NSCLC patients. Second, the risk prognostic model built on seven m6A regulators can effectively stratify NSCLC patients, and the low-risk subgroup had better prognosis compared to high-risk group. Finally, a few m6A regulators showed significant associations with immune microenvironment, as well as TMB and PD-L1 level, suggesting that the m6A RNA methylation is indicative of therapeutic effect of anti-PD-L1 treatment. Our study identified some m6A regulatory factors as independent risk factors for the prognosis of NSCLC, and the expression patterns of m6A regulators are also correlated to the immune infiltration, as well as TMB and PD-L1 level in NSCLC. The m6A regulators could be used as biomarkers indicative of immunotherapy to NSCLC patients.

Abstract 2131: m6A demethylase ALKBH5 promote tumor growth through IGF2BPs' recognition of m6A modified CDKN1A in non-small lung cancer

Abstract Background: N6-methyladenosine (m6A), the most prevalent and abundant RNA modification on eukaryote mRNA, is regulated by the methylation complex (writer) and demethylase (eraser), subsequently being recognized by RNA-binding protein (reader). As an m6A eraser, ALKBH5 has been shown to promote the development of breast cancer and glioblastoma. In lung cancer, two experiments described contradictory findings that one is a cancer-promoting effect and the other is a cancer-suppressing effect. Accordingly, the carcinogenic mechanisms of ALKBH5 are not completely elucidated in lung cancer. The purpose of this study is to investigate the role of m6A demethylase ALKBH5 in lung cancer. Method: We investigated the relationship between eraser protein expression level by immunostaining (IHC) and prognosis in 629 patients with non-small cell lung cancer (NSCLC). Small interfering RNA transfection was used to down-regulate ALKBH5 in PC9 and A549 cells. Then, cell function assays (cell proliferation, migration ability, cell cycle, apoptosis) were conducted in cells. In addition, mRNA and m6A expression under the ALKBH5 knockdown were comprehensively analyzed by microarray. The m6A target genes were identified by Methylated RNA immunoprecipitation (MeRIP) assay using m6A antibody. Furthermore, the mRNA stability and protein expression of the m6A target gene was examined. Result: High expression of ALKBH5 showed a poor prognosis (p&amp;lt;0.001). ALKBH5 knockdown suppressed cell proliferation ability in PC9 and A549. G1 arrest and increased apoptotic cells were observed by ALKBH5 knockdown in cells. In addition, overexpression of ALKBH5 increased the cell proliferation ability in HEK293 and BEAS2B. Comprehensive analysis of microarray revealed up-regulation of CDKN1A, E2F1, GADD45A, TIMP3, and DNMT3B, and down-regulation of CASP14 and CCNG2 by ALKBH5 knockdown. m6A microarray showed up-regulation of m6A modification in 22 genes by ALKBH5 knockdown. MeRIP qPCR with fragmented mRNA showed m6A level in 3'UTR regions of CDKN1A, TIMP3, E2F1, DNMT3B, and CCNG2 were increased by ALKBH5 knockdown. The CDKN1A mRNA was stabilized by ALKBH5 knockdown, and the increased expression of CDKN1A was down-regulated by IGF2BPs knockdown. The protein expression of p21, MFAP5, and TIMP3 was up-regulated by ALKBH5 knockdown. The up-regulated expression of p21 was p53-independent. Conclusion: High expression of ALKBH5 has an unfavorable prognostic value in NSCLC. ALKBH5 knockdown increases m6A of 3'UTR of CDKN1A, which is recognized by IGF2BPs and enhances mRNA stability. The increased expression of p21 leads to the regulation of the cell cycle, regulating cell proliferation ability. Taken together, ALKBH5 plays a cancer-promoting role in non-small cell lung cancer. Citation Format: Kazuo Tsuchiya, Katsuhiro Yoshimura, Yusuke Inoue, Yuji Iwashita, Tsutomu Ohta, Hidetaka Yamada, Hirofumi Watanabe, Hong Tao, Akikazu Kawase, Masayuki Tanahashi, Hiroshi Ogawa, Kazuhito Funai, Kazuya Shinmura, Takafumi Suda, Haruhiko Sugimura. m6A demethylase ALKBH5 promote tumor growth through IGF2BPs' recognition of m6A modified CDKN1A in non-small lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2131.

Identification of downstream signaling cascades of ACK1 and prognostic classifiers in non-small cell lung cancer.

Activated Cdc42-associated kinase 1 (ACK1) is an oncogene in multiple cancers, but the underlying mechanisms of its oncogenic role remain unclear in non-small cell lung cancer (NSCLC). Herein, we comprehensively investigated the ACK1-regulated cell processes and downstream signaling pathways, as well as its prognostic value in NSCLC. We found that ACK1 gene amplification was associated with mRNA levels in The Cancer Genome Atlas (TCGA) lung cancer cohort. The Oncomine databases showed significantly elevated ACK1 levels in lung cancer. In vitro, an ACK1 inhibitor (dasatinib) increased the sensitivity of NSCLC cell lines to AKT or MEK inhibitors. RNA-sequencing results demonstrated that an ACK1 deficiency in A549 cells affected the MAPK, PI3K/AKT, and Wnt pathways. These results were validated by gene set enrichment analysis (GSEA) of data from 188 lung cancer cell lines. Using Cytoscape, we dissected 14 critical ACK1-regulated genes. The signature with the 14 genes and ACK1 could significantly dichotomize the TCGA lung cohort regarding overall survival. The prognostic accuracy of this signature was confirmed in five independent lung cancer cohorts and was further validated by a prognostic nomogram. Our study unveiled several downstream signaling pathways for ACK1, and the proposed signature may be a promising prognostic predictor for NSCLC.

LncRNA-ATB Can Be a Biomarker for Diagnosis and Prognosis Evaluation of Non-Small Cell Lung Cancer

Objective: This study was set out to inquire into the expression and clinical significance of lncRNA activated by transforming growth factor β (LncRNA-ATB) and in cancer tissues of patients with non-small cell lung cancer (NSCLC). Methods: LncRNA-ATB in cancer tissues and adjacent tissues of 89 NSCLC patients was detected by quantitative real-time polymerase chain reaction (qRT-PCR), and its clinical diagnostic value in NSCLC was determined by receiver operating characteristic (ROC) curves. Based on the median expression of LncRNAATB in NSCLC tissues, 89 patients were allocated into high- and low-expression groups. The 3-year survival rate was calculated using Kaplan-Meier method and logrank test, and compared between lncRNA-ATB high and low expression groups by Log-rank test. Cox regression quantitatively analyzed the correlation between lncRNA-ATB and the clinical prognosis of NSCLC patients. Results: LncRNAATB presented notably higher expression in NSCLC tissues as compared to adjacent tissues (P &lt; 0.05), and was linked to smoking history, TNM stage and greatest tumor diameter (GTD) (P &lt; 0.05). The cut-off value, sensitivity and specificity, as well as the area under the curve (AUC) of NSCLC diagnosed by LncRNA-ATB were 2.103, 73.77%, 83.15% and 0.836, respectively. Patients with high lncRNA-ATB expression (40.00%) showed statistically lower than those with low lncRNA-ATB expression (63.36%). TNM stage, differentiation degree and lncRNA-ATB expression were independent variables affecting patient prognosis. Conclusions: LncRNA-ATB, with increased expression profiles in NSCLC tissues, is involved in NSCLC progression and associated with unfavourable prognosis of patients, which can be a plausible tumor marker for NSCLC patients.

Circulating Long Noncoding RNAs Act as Diagnostic Biomarkers in Non-Small Cell Lung Cancer.

Identification of novel effective early diagnostic biomarkers may provide alternative strategies to reduce the mortality for non-small cell lung cancer (NSCLC) patients. Circulating long non-coding RNAs (lncRNAs) have emerged as a new class of promising cancer biomarkers. Our study aimed to identify circulating lncRNAs for diagnosing NSCLC. A total 528 plasma samples were continuously collected and allocated to four progressive phases: discovery, training, verification, and expansion phases. The expression of candidate lung cancer related lncRNAs were detected using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). We identified a 4-lncRNA panel (RMRP, NEAT1, TUG1, and MALAT1) that provided a high diagnostic value in NSCLC (AUC = 0.86 and 0.89 for training and verification phase, respectively). Subgroup analyses showed that the 4-lncRNA panel had a sensitivity of 78.95% [95% confidence interval (CI) = 62.22%–89.86%] in stage I-II patients and 75.00% (95% CI = 52.95%–89.40%) in patients with small tumor size (≤3cm). Notably, the sensitivity of 4-lncRNA panel was significantly higher than that of routine protein panels in adenocarcinoma (CEA, CA125, and CYFRA21-1, 86.30% vs. 73.96%). Adding 4-lncRNA to protein markers significantly improved the diagnostic capacity in both adenocarcinoma (AUC=0.85, 95% CI = 0.78–0.91) and squamous cell carcinoma (AUC=0.93, 95% CI = 0.86–0.97). In conclusion, we identified a plasma 4-lncRNA panel that has considerable clinical value in diagnosing NSCLC. The 4-lncRNA panel could improve the diagnostic values of routine tumor protein markers in diagnosing NSCLC. Circulating lncRNAs could be used as promising candidates for NSCLC diagnosis.

The Immune Cell Composition in Primary Lung Squamous Cell Carcinomas Influences Patterns of Recurrence and Survival

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with squamous cell carcinoma (SCCA) being a prominent histology. Emerging data suggests that immunological parameters have predictive and prognostic value in NSCLC. In this study we determine if the immune cell infiltrate (ICI) in primary lung SCCA tumors influences patterns of recurrence and survival. We retrospectively collected data from an IRB approved prospective tissue collection protocol, which profiled all tumors for gene expression with Affymetrix Hu-RSTA microarray chips (Affymetrix; Santa Clara, CA). We identified tumor samples from patients diagnosed with lung SCCA who underwent surgery between 1997 and 2010. The CIBERSORT deconvolution algorithm was employed to infer the composition of the immune cell infiltrate (22 distinct immune cell types) in tumors via gene expression. ICI presence was dichotomized at the median, where high refers to counts above the median. Time to event analyses were assessed with Kaplan-Meier methods and compared with log rank test. Cox regression was performed to evaluate the effect of predictors on defined outcomes. We identified 258 patients for this analysis. The median age was 70 years (range, 72-88) and the majority (67%) were male and had an ECOG performance status of 1 (65%). The median tobacco pack-years was 50 (range, 0-180), 33% were current and 64% were former smokers. Stages were IA (24%), IB (27%), IIA (8%), IIB (27%) and IIIA/B (14%). Nineteen (7 %) patients received NACT, 17% received adjuvant CT and 9% received adjuvant radiotherapy. The median follow-up time was 19 months with a 2-year OS of 76% and a 2-yr freedom from recurrence of 77%. ICI analysis identified that a high presence of activated CD4+ memory T cells predicts for recurrence in the lung vs other site (77% vs 23%, p = 0.01). Similarly, a high presence of activated NK cells predicts for locoregional recurrence (62% vs 38%, p = 0.03) and high resting NK cells predict for distant recurrence (66% vs 35%, p = 0.02). Predictors for OS were male gender (HR: 1.6, 95% CI 1.1-2.5, p = 0.03), and high presence of resting dendritic cells (HR: 1.5, 95% CI 1.0-2.2, p = 0.04) and eosinophils (HR: 1.6, 95% CI 1.0-2.4, p = 0.03) on multivariable analysis. Our results suggest that the immune cell composition of primary lung SCCA tumors may influence patterns of recurrence. This data may assist with novel means to optimize treatment selection and sequencing.