Pathogenesis Of Lung Adenocarcinoma Research Articles

Effects of hub genes on the clinicopathological and prognostic features of lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a common malignancy; however, the majority of its underlying molecular mechanisms remain unknown. In the present study, weighted gene co-expression network analysis was applied to construct gene co-expression networks for the GSE19804 dataset, in order to screen hub genes associated with the pathogenesis of LUAD. In addition, with the aid of the Database for Annotation, Visualization and Integrated Discovery, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes, pathway enrichment analyses were performed on the genes in the selected module. Using the GSE40791 dataset and The Cancer Genome Atlas database, the hub genes were identified. It was discovered that the turquoise module was the most significant module associated with the tumor stage of LUAD. After performing functional enrichment analyses, it was indicated that the turquoise module was mainly enriched in signal transduction. Additionally, at the transcriptional and translational level, nine hub genes were identified and validated: Carbonic anhydrase 4 (CA4), platelet and endothelial cell adhesion molecule 1 (PECAM1), DnaJ member B4 (DNAJB4), advanced glycosylation end-product specific receptor (AGER), GTPase, IMAP family member 6 (GIMAP6), chromosome 10 open reading frame 54 (C10orf54), dedicator of cytokinesis 4 (DOCK4), Golgi membrane protein 1 (GOLM1) and platelet activating factor acetylhydrolase 1b catalytic subunit 3 (PAFAH1B3). CA4, PECAM1, DNAJB4, AGER, GIMAP6, C10orf54 and DOCK4 were expressed at lower levels in the tumor samples, whereas GOLM1 and PAFAH1B3 were highly expressed in tumor samples. In addition, all hub genes were associated with prognosis. In conclusion, one module and nine genes were recognized to be associated with the tumor stage of LUAD. These findings may enhance the understanding of the progression and prognosis of LUAD.

Identification of a panel of mitotic spindle-related genes as a signature predicting survival in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is one of the most malignant tumor types worldwide. Our objective was to identify a genetic signature that could predict the prognosis of patients with LUAD. We extracted gene data sets from The Cancer Genome Atlas and obtained differentially expressed genes that were highly expressed at every stage. These genes were analyzed using gene set enrichment analysis to obtain four biological processes associated with LUAD. Subsequently, Cox univariate and multivariate analyses were performed to generate four optimized models (G2M checkpoint, E2F targets, mitotic spindle, and glycolysis). We identified a mitotic spindle-related signature (KIF15, BUB1, CCNB2, CDK1, KIF4A, DLGAP5, ECT2, and ANLN), which could be an independent prognostic indicator, to predict the prognosis of patients with LUAD. This new discovery should offer opportunities to explore the pathogenesis of LUAD and prove clinically useful in predicting LUAD patient prognosis.

Silencing of ARL14 Gene Induces Lung Adenocarcinoma Cells to a Dormant State.

Recently, a growing number of ADP ribosylation factor (ARF) family members has been suggested to be critical in tumorigenesis. However, the effects of most ARF members on lung adenocarcinoma pathogenesis are still not well disclosed yet. In this study, ARF-like GTPase 14 (ARL14) was screened as an important prognostic factor of lung adenocarcinoma from The Cancer Genome Atlas (TCGA) database and validated by our in vitro experiments. It was found that silencing of ARL14 gene inhibited cell proliferation and the abilities of cell migration and invasion, and it also attenuated radiation damage of lung adenocarcinoma cells but had no effect on the proliferation of normal lung cells. Notably, ARL14 siRNA blocked the extracellular signal-regulated kinase (ERK)/p38 signaling pathway and induced cell cycle arrest in G0 phase, ultimately leading to cell dormancy. Moreover, ARL14 siRNA enhanced the expression of cell death activator DFFA-like effector (CIDEC) that had opposite roles in cell proliferation and migration to ALR14. Collectively, our results suggest that ARL14 has an important role in the pathogenesis of lung adenocarcinoma through CIDEC/ERK/p38 signaling pathway, and thus it could be applied as a new candidate of prognosis indicator and/or therapeutic target of lung adenocarcinoma.

P2.03-48 Tumor-Derived Granulocyte Chemotactic Protein 2 Cooperates with Proteases to Drive Lung Adenocarcinoma

Lung adenocarcinoma (LADC) commonly arises in the lungs of smokers that are heavily affected by chronic inflammation. Inflammatory signaling from tumor to host cells is critically involved in the pathogenesis of LADC, but the exact mechanisms by which the lung epithelium interacts with the immune system during carcinogenesis are obscure. Objectives: We discovered that murine and human LADC cell lines overexpress the inflammatory and angiogenic CXC chemokine granulocyte chemotactic protein 2 (GCP2, CXCL6) compared with normal epithelial cells. GCP2 is processed by immune proteases: neutrophil elastase (NE, ELANE), proteinase 3 (PR3, PRTN3) and matrix metalloproteinase 9 (MMP9, MMP9). We suppose that GCP2 interacts with these proteases to drive LADC and aimed at investigating its function(s). GCP2, MMP9 and PR3 expressions were determined by ELISA and immunohistochemistry. Mouse and human microarray data were analyzed using Affymetrix Transcriptome Analysis Console. GCP2 silencing using dedicated shRNA pools (SantaCruz Biotechnology) and NE/PR3 or MMP9 compound knockout mice were used to study GCP2 interaction with immune proteases in LADC progression. Murine and human LADC tumors and cell lines overexpress GCP2, MMP9, NE and PR3 at the mRNA and protein levels. GCP2 is sequestered to tumor cells, whereas proteases are produced by tumor-infiltrating immune cells. LADC-secreted GCP2 is incompletely processed and require MMP9, NE or PR3 for full activation and vasoactive effects. Moreover, GCP2-silenced LADC cells injected in mice show a lack in metastasis formation. Our results indicate that tumor-originated GCP2 cooperates with immune proteases to drive LADC, providing a paradigm of how the respiratory epithelium coopts the innate immune system during carcinogenesis.

Involvement of Dual Strands of miR-143 (miR-143-5p and miR-143-3p) and Their Target Oncogenes in the Molecular Pathogenesis of Lung Adenocarcinoma.

Our analyses of tumor-suppressive microRNAs (miRNAs) and their target oncogenes have identified novel molecular networks in lung adenocarcinoma (LUAD). Moreover, our recent studies revealed that some passenger strands of miRNAs contribute to cancer cell malignant transformation. Downregulation of both strands of the miR-143 duplex was observed in LUAD clinical specimens. Ectopic expression of these miRNAs suppressed malignant phenotypes in cancer cells, suggesting that these miRNAs have tumor-suppressive activities in LUAD cells. Here, we evaluated miR-143-5p molecular networks in LUAD using genome-wide gene expression and miRNA database analyses. Twenty-two genes were identified as potential miR-143-5p-controlled genes in LUAD cells. Interestingly, the expression of 11 genes (MCM4, RAD51, FAM111B, CLGN, KRT80, GPC1, MTL5, NETO2, FANCA, MTFR1, and TTLL12) was a prognostic factor for the patients with LUAD. Furthermore, knockdown assays using siRNAs showed that downregulation of MCM4 suppressed cell growth, migration, and invasion in LUAD cells. Aberrant expression of MCM4 was confirmed in the clinical specimens of LUAD. Thus, we showed that miR-143-5p and its target genes were involved in the molecular pathogenesis of LUAD. Identification of tumor-suppressive miRNAs and their target oncogenes may be an effective strategy for elucidation of the molecular oncogenic networks of this disease.

REG4 is an indicator for KRAS mutant lung adenocarcinoma with TTF-1 low expression.

Recent research has classified lung adenocarcinoma patients with KRAS mutation into three subtypes by co-occurring genetic events in TP53 (KP subgroup), STK11/LKB1 (KL subgroup) and CDKN2A/B inactivation plus TTF-1 low expression (KC subgroup). The aim of this study was to identify valuable biomarkers by searching the candidate molecules that contribute to lung adenocarcinoma pathogenesis, especially KC subtype. We analyzed the publicly available database and identified the candidate REG4 using the E-GEOD-31210 dataset, and then confirmed by TCGA dataset. In addition, an independent cohort of 55 clinical samples was analyzed by quantitative real-time PCR analysis. Functional studies and RNA sequencing were performed after silencing the REG4 expression. REG4, an important regulator of gastro-intestinal carcinogenesis, was highly expressed in KRAS mutant lung adenocarcinoma with low expression of TTF-1 (KC subtype). The results were validated both by gene expression analysis and immunohistochemistry study in an independent 55 clinical samples from Fudan University Shanghai Cancer Center. Further in vitro and in vivo functional assays revealed silencing REG4 expression significantly reduces cancer cell proliferation and tumorigenesis. Moreover, RNA sequencing and GSEA analysis displayed that REG4 knockdown might induce cell cycle arrest by regulating G2/M checkpoint and E2F targets. Our results indicate that REG4 plays an important role in KRAS-driven lung cancer pathogenesis and is a novel biomarker of lung adenocarcinoma subtype. Future studies are required to clarify the underlying mechanisms of REG4 in the division and proliferation of KC tumors and its potential therapeutic value.

Abstract 4623: Notch3 in lung adenocarcinoma pathogenesis and heterogeneity

Abstract Lung cancer is the leading cause of cancer related death worldwide. Non-small lung cancer accounts for 85% of diagnoses and the predominant subtype is lung adenocarcinoma (LUAD). LUAD has a less than 20% response rate to conventional chemotherapy and despite identification of several therapeutic targets, the majority of diagnoses continue to lead to death, highlighting the need for better treatments. Notch signaling in mammals is complex, with multiple receptors, ligands and modifiers. Notch ligands at the surface of one cell interact with receptors on an adjacent cell, triggering cleavage and nuclear translocation of the intracellular domain which interacts with Rbpj/Csl to control the transcription of target genes. In LUAD, significant evidence suggests that Notch plays an oncogenic role. Previously, we identified a population of Tumor Propagating Cells (TPC), which had greater capacity for tumor formation and are enriched after treatment with cisplatin. The TPC population was defined by the expression of CD24, ITGB4 and was Notch high. Further functional validation showed that specifically Notch3 contributes to this propagation phenotype and is key in establishing functional intra-tumoral heterogeneity. However, the TPC population is still heterogeneous and likely only a subset of these cells are true TPCs. Furthermore, the gene expression signature of the mouse TPCs has prognostic significance in human LUAD, however the TPC population hasn’t been defined in human patients, limiting our ability to target the Notch pathway therapeutically. To assess the heterogeneity of the mouse TPC population we performed FACS on tumors from KrasLSLG12D; Tp53flox/flox; YFPLSL twelve weeks after tumor initiation via AdCre. Tumors were sorted for bulk population (YFP+Lin-) and for TPC (YFP+Lin-CD24+ITGB4+Notchhi) population and single cell sequencing was performed using the 10X genomics platform. We hypothesize that aberrant Notch3 signaling is a critical pathway for survival in at least some LUAD and that Notch activation leads to gene expression changes that are important for driving human LUAD maintenance. Patient-derived xenografts (PDX) more closely recapitulate important aspects of human cancer, particularly with regards to the role of intratumoral heterogeneity and self-renewal pathways. To characterize Notch3 expressing cells in human LUAD, we performed FACS on our cohort of LUAD PDXs and sorted the Notch3+ population. We found expression of Notch3 and expression of Notch ligand Jagged2 in all LUAD tumors regardless of driver mutation. Citation Format: Kieren D. Marini, Stanley Leung, Alex Lee, Alejandro Sweet-Cordero. Notch3 in lung adenocarcinoma pathogenesis and heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4623.

Abstract 2635: ROR1-CAVIN3 interaction required for caveolae-dependent endocytosis and pro-survival signaling in lung adenocarcinoma

Abstract TTF-1/NKX2-1, a homeobox-containing transcription factor indispensable for lung morphogenesis, was previously identified by four independent groups including us, as a “lineage-survival” oncogene involved in the pathogenesis of lung adenocarcinoma. We have further shown that TTF-1 induces expression of the receptor tyrosine kinase-like orphan receptor 1 (ROR1), which in turn sustains a favorable balance between pro-survival PI3K-AKT and pro-apoptotic ASK1-p38 signaling in both ROR1 kinase activity-dependent and -independent manner. It is interesting to note that while alterations in EGFR signaling are involved as a “driver” in lung adenocarcinoma pathogenesis, ROR1 appears to play a “sustainer role” for the EGFR-mediated signaling. Previously, we have also found an unanticipated function of this ROR1 receptor tyrosine kinase (RTK) as a scaffold of CAVIN1 and caveolin-1 (CAV1), two essential structural components of caveolae. This kinase-independent function of ROR1 facilitates the interactions of CAVIN1 and CAV1 at the plasma membrane, thereby preventing the lysosomal degradation of CAV1. Caveolae structures and pro-survival signaling towards AKT through multiple RTKs, including EGFR, MET, and IGF-IR are consequently sustained. Therefore, ROR1 is an attractive target for overcoming EGFT-TKI resistance due to various mechanisms such as EGFR T790M double mutation and bypass signaling from other RTKs. Caveolae function in vesicular and cholesterol trafficking, and have roles in internalization of cholera toxins and the SV40 virus, as well as glycosylphosphatidylinositol (GPI)-anchored proteins and various RTKs. Endocytosis supports various cellular functions, such as nutrient uptake, intracellular signaling, morphogenesis, and defense against pathogens, and is tightly regulated, while it also ensures an appropriate amplitude of growth factor signaling by recycling RTKs back to the cell surface or sorting them to lysosomes for degradation. Here we report that ROR1 possesses a novel scaffold function indispensable for efficient caveolae-dependent endocytosis. CAVIN3 was found to bind with ROR1 at a site distinct from sites for CAV1 and CAVIN1, a novel function required for proper CAVIN3 subcellular localization and caveolae-dependent endocytosis, but not caveolae formation itself. Furthermore, evidence of a mechanistic link between ROR1-CAVIN3 interaction and consequential caveolae trafficking, which was found to utilize a binding site distinct from those for ROR1 interactions with CAV1 and CAVIN1, with RTK-mediated pro-survival signaling towards AKT in early endosomes in lung adenocarcinoma cells was also obtained. The present findings warrant future study to enable development of novel therapeutic strategies for inhibiting the multifaceted scaffold functions of ROR1 in order to reduce the intolerable death toll from this devastating cancer. Citation Format: Tomoya Yamaguchi, Miyu Hayashi, Lisa Ida, Masatoshi Yamamoto, Can Lu, Taisuke Kajino, Jinglei Cheng, Masahiro Nakatochi, Hisanori Isomura, Motoshi Suzuki, Toyoshi Fujimoto, Takashi Takahashi. ROR1-CAVIN3 interaction required for caveolae-dependent endocytosis and pro-survival signaling in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2635.

POLQ Overexpression Is Associated with an Increased Somatic Mutation Load and PLK4 Overexpression in Lung Adenocarcinoma.

DNA Polymerase Theta (POLQ) is a DNA polymerase involved in error-prone translesion DNA synthesis (TLS) and error-prone repair of DNA double-strand breaks (DSBs). In the present study, we examined whether abnormal POLQ expression may be involved in the pathogenesis of lung adenocarcinoma (LAC). First, we found overexpression of POLQ at both the mRNA and protein levels in LAC, using data from the Cancer Genome Atlas (TCGA) database and by immunohistochemical analysis of our LAC series. POLQ overexpression was associated with an advanced pathologic stage and an increased total number of somatic mutations in LAC. When H1299 human lung cancer cell clones overexpressing POLQ were established and examined, the clones showed resistance to a DSB-inducing chemical in the clonogenic assay and an increased frequency of mutations in the supF forward mutation assay. Further analysis revealed that POLQ overexpression was also positively correlated with Polo Like Kinase 4 (PLK4) overexpression in LAC, and that PLK4 overexpression in the POLQ-overexpressing H1299 cells induced centrosome amplification. Finally, analysis of the TCGA data revealed that POLQ overexpression was associated with an increased somatic mutation load and PLK4 overexpression in diverse human cancers; on the other hand, overexpressions of nine TLS polymerases other than POLQ were associated with an increased somatic mutation load at a much lower frequency. Thus, POLQ overexpression is associated with advanced pathologic stage, increased somatic mutation load, and PLK4 overexpression, the last inducing centrosome amplification, in LAC, suggesting that POLQ overexpression is involved in the pathogenesis of LAC.

REG4 as a novel biomarker of lung adenocarcinoma with mutated KRAS and TTF-1 low expression.

e14541 Background: Recent research has classified lung adenocarcinoma patients with KRAS mutation into three subtypes by co-occurring genetic events in TP53 (KP subgroup), STK11/LKB1 (KL subgroup) and CDKN2A/B inactivation coupled with low expression of the TTF-1 (KC subgroup). The aim of this study was to identify novel biomarkers through searching the candidate molecules that contributing to lung adenocarcinoma pathogenesis, especially KC subtype. Methods: We analyzed the publicly available database and identified the candidate REG4 using the E-GEOD-31210 dataset, and then confirmed by TCGA dataset. In addition, an independent cohort of 55 clinical samples was analyzed by quantitative real-time PCR analysis. Functional studies and RNA sequencing were performed after the silencing REG4 expression. Results: REG4, an important regulator of gastro-intestinal carcinogenesis, was highly expressed in KRAS mutated lung adenocarcinoma with low expression of TTF-1 (KC subtype). The results were validated both by gene expression analysis and immunohistochemistry study in an independent 55 clinical samples from Fudan University Shanghai Cancer Center. Further in vitro and in vivo functional assays revealed silencing REG4 expression significantly reduce cancer cell proliferation and tumorigenesis. Moreover, RNA sequencing and GSEA analysis displayed that REG4 knockdown might induce the cell cycle arrest by regulating G2/M checkpoint and E2F targets. Conclusions: Our results indicate that REG4 plays an important role in KRAS-driven lung cancer pathogenesis and is a novel biomarker of lung adenocarcinoma subtype. Future studies are required to clarify the underlying mechanism of REG4 in the division and proliferation of KC tumors and its potential therapeutic value.

Molecular Pathogenesis of Gene Regulation by the miR-150 Duplex: miR-150-3p Regulates TNS4 in Lung Adenocarcinoma.

Based on our miRNA expression signatures, we focused on miR-150-5p (the guide strand) and miR-150-3p (the passenger strand) to investigate their functional significance in lung adenocarcinoma (LUAD). Downregulation of miR-150 duplex was confirmed in LUAD clinical specimens. In vitro assays revealed that ectopic expression of miR-150-5p and miR-150-3p inhibited cancer cell malignancy. We performed genome-wide gene expression analyses and in silico database searches to identify their oncogenic targets in LUAD cells. A total of 41 and 26 genes were identified as miR-150-5p and miR-150-3p targets, respectively, and they were closely involved in LUAD pathogenesis. Among the targets, we investigated the oncogenic roles of tensin 4 (TNS4) because high expression of TNS4 was strongly related to poorer prognosis of LUAD patients (disease-free survival: p = 0.0213 and overall survival: p = 0.0003). Expression of TNS4 was directly regulated by miR-150-3p in LUAD cells. Aberrant expression of TNS4 was detected in LUAD clinical specimens and its aberrant expression increased the aggressiveness of LUAD cells. Furthermore, we identified genes downstream from TNS4 that were associated with critical regulators of genomic stability. Our approach (discovery of anti-tumor miRNAs and their target RNAs for LUAD) will contribute to the elucidation of molecular networks involved in the malignant transformation of LUAD.

Integrated analysis reveals five potential ceRNA biomarkers in human lung adenocarcinoma.

BackgroundCompeting endogenous RNAs (ceRNAs) are a newly identified type of regulatory RNA. Accumulating evidence suggests that ceRNAs play an important role in the pathogenesis of diseases such as cancer. Thus, ceRNA dysregulation may represent an important molecular mechanism underlying cancer progression and poor prognosis. In this study, we aimed to identify ceRNAs that may serve as potential biomarkers for early diagnosis of lung adenocarcinoma (LUAD).MethodsWe performed differential gene expression analysis on TCGA-LUAD datasets to identify differentially expressed (DE) mRNAs, lncRNAs, and miRNAs at different tumor stages. Based on the ceRNA hypothesis and considering the synergistic or feedback regulation of ceRNAs, a lncRNA–miRNA–mRNA network was constructed. Functional analysis was performed using gene ontology term and KEGG pathway enrichment analysis and KOBAS 2.0 software. Transcription factor (TF) analysis was carried out to identify direct targets of the TFs associated with LUAD prognosis. Identified DE genes were validated using gene expression omnibus (GEO) datasets.ResultsBased on analysis of TCGA-LUAD datasets, we obtained 2,610 DE mRNAs, 915 lncRNAs, and 125 miRNAs that were common to different tumor stages (|log2(Fold change)| ≥ 1, false discovery rate < 0.01), respectively. Functional analysis showed that the aberrantly expressed mRNAs were closely related to tumor development. Survival analyses of the constructed ceRNA network modules demonstrated that five of them exhibit prognostic significance. The five ceRNA interaction modules contained one lncRNA (FENDRR), three mRNAs (EPAS1, FOXF1, and EDNRB), and four miRNAs (hsa-miR-148a, hsa-miR-195, hsa-miR-196b, and hsa-miR-301b). The aberrant expression of one lncRNA and three mRNAs was verified in the LUAD GEO dataset. Transcription factor analysis demonstrated that EPAS1 directly targeted 13 DE mRNAs.ConclusionOur observations indicate that lncRNA-related ceRNAs and TFs play an important role in LUAD. The present study provides novel insights into the molecular mechanisms underlying LUAD pathogenesis. Furthermore, our study facilitates the identification of potential biomarkers for the early diagnosis and prognosis of LUAD and therapeutic targets for its treatment.

TRNA-based prognostic score in predicting survival outcomes of lung adenocarcinomas.

As the most abundant noncoding RNA in cells, tRNA plays an important role in tumorigenesis and development. The report of tRNA on the pathogenesis of lung adenocarcinoma is rare. It is of great clinical significance to explore the relationship between tRNA expression and prognosis of lung adenocarcinoma. The expression level of tRNAs in lung adenocarcinoma tissues and paracarcinoma tissues was detected using a tRNA RT-qPCR array. A total of 104 lung adenocarcinomas were included in the analysis of the correlation between candidate tRNAs expression and prognosis. A tRNA-based prognostic model was constructed and validated using Cox proportional hazards regression. A nomogram was built to help clinicians develop treatment strategies. We screened a series of differentially expressed tRNAs between lung adenocarcinoma tissues and paracarcinoma tissues. Among these tRNAs, tRNAAsnATT , tRNAIleAAT , tRNALeuTAA , mt-tRNATrpTCA , mt-tRNALeuTAA , tRNAProAGG , tRNALysCTT-1 and tRNALeuAAG were associated with the clinicopathological characteristics of lung adenocarcinoma. tRNALysCTT-1 , mt-tRNASerGCT and tRNATyrATA were associated with cancer-specific survival. We constructed a prognostic model for lung adenocarcinoma using specific tRNA expression levels as reference factors. Multivariate analyses showed that tRNA-based prognostic score was a significant and important prognostic factor. The prognostic model based on the tRNAs expression signatures can help predict the prognosis of patients with lung adenocarcinoma.

Screening of important lncRNAs associated with the prognosis of lung adenocarcinoma, based on integrated bioinformatics analysis.

The study aimed to elucidate the mechanisms underlying the occurrence and development of lung adenocarcinoma, and to reveal long non-coding RNA (lncRNA) prognostic factors to identify patients at high risk of disease recurrence or metastasis. Based on extensive RNA sequencing data and clinical survival prognosis information from patients with lung adenocarcinoma, obtained from The Cancer Genome Atlas and the Gene Expression Omnibus databases, a co-expression network of lncRNAs with different expression levels was built using weighted correlation network analysis and MetaDE.ES. The prognostic lncRNAs were identified using the Cox proportional hazards model and Kaplan-Meier survival curves to construct a risk scoring system. The reliability of the system was confirmed in validation datasets. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on the genes significantly associated with the prognostic lncRNAs using gene set enrichment analysis. A total of 58 and 1,633 differentially expressed lncRNAs and mRNAs were identified, respectively. Considering the module stability, annotation, correlation between modules and clinical factors, and the differential expression levels of lncRNAs, 32 differentially expressed lncRNAs were selected from the brown, red, blue, green and yellow modules for subsequent survival analysis. A signature-based risk scoring system involving five lncRNAs [DIAPH2 antisense RNA 1, FOXN3 antisense RNA 2, long intergenic non-protein coding RNA 652, maternally expressed 3 and RHPN1 antisense RNA 1 (head to head)] was developed. The system successfully distinguished between low- and high-risk prognostic samples. System effectiveness was further verified using two independent validation datasets. Further KEGG pathway analysis indicated that the target genes of the five prognostic lncRNAs were associated with a number of cellular processes and signaling pathways, including the cell receptor-mediated signaling and cell adhesion pathways. A five-lncRNA signature predicts the prognosis of patients with lung adenocarcinoma. These prognostic lncRNAs may be potential diagnostic markers. The present results may help elucidate the pathogenesis of lung adenocarcinoma.

Elevated Notch1 enhances interleukin-22 production by CD4+ T cells via aryl hydrocarbon receptor in patients with lung adenocarcinoma.

Notch signaling induced interleukin (IL)-22 secretion by CD4+ T cells via retinoid-related orphan nuclear receptor γt (RORγt) or aryl hydrocarbon receptor (AhR). Previous studies have demonstrated that Notch-AhR-IL-22 axis took part in the pathogenesis of chronic viral infection, however, its role in cancer has not been fully elucidated. Thus, the aim of current study was to investigate the involvement of Notch-AhR-IL-22 axis in the pathogenesis of lung adenocarcinoma. A total of 37 late-stage lung adenocarcinoma patients and 17 healthy individuals were enrolled. CD4+ T cells were purified from peripheral bloods and bronchoalveolar lavage fluids (BALF), and were stimulated with γ-secretase inhibitor (GSI). mRNA corresponding to Notch receptors and transcriptional factors were measured by real-time PCR. IL-22 concentration was investigated by ELISA. The bioactivity (including cellular proliferation, cell cycle, apoptosis, and invasion) of lung adenocarcinoma cell line A549 was also assessed in response to recombinant IL-22 stimulation in vitro. Notch1 mRNA expression was significantly elevated in CD4+ T cells purified from peripheral bloods and tumor site BALF in lung adenocarcinoma patients. IL-22 expression and RORγt/AhR mRNA in BALF was also remarkably increased in tumor site. Inhibition of Notch signaling by GSI did not affect cellular proliferation, but reduced IL-22 production in CD4+ T cells from BALF, along with down-regulation of AhR, but not RORγt. Moreover, IL-22 stimulation promoted A549 cells invasion. The current data indicated that elevated Notch1 induced higher IL-22 secretion by CD4+ T cells in lung adenocarcinoma patients, and Notch-AhR-IL-22 axis took part in the pathogenesis of lung adenocarcinoma.

Transcriptomic Alterations in Lung Adenocarcinoma Unveil New Mechanisms Targeted by the TBX2 Subfamily of Tumor Suppressor Genes.

T-box (TBX) transcription factors are evolutionary conserved genes and master transcriptional regulators. In mammals, TBX2 subfamily (TBX2, TBX3, TBX4, and TBX5) genes are expressed in the developing lung bud and tracheae. Our group previously showed that the expression of TBX2 subfamily was significantly high in human normal lungs, but markedly suppressed in lung adenocarcinoma (LUAD). To further elucidate their role in LUAD pathogenesis, we first confirmed abundant expression of protein products of the four members by immunostaining in adult human normal lung tissues. We also found overall suppressed expression of these genes and their corresponding proteins in a panel of human LUAD cell lines. Transient over-expression of each of the genes in human (NCI-H1299), and mouse (MDA-F471) derived lung cancer cells was found to significantly inhibit growth and proliferation as well as induce apoptosis. Genome-wide transcriptomic analyses on NCI-H1299 cells, overexpressing TBX2 gene subfamily, unraveled novel regulatory pathways. These included, among others, inhibition of cell cycle progression but more importantly activation of the histone demethylase pathway. When using a pattern-matching algorithm, we showed that TBX's overexpression mimic molecular signatures from azacitidine treated NCI-H1299 cells which in turn are inversely correlated to expression profiles of both human and murine lung tumors relative to matched normal lung. In conclusion, we showed that the TBX2 subfamily genes play a critical tumor suppressor role in lung cancer pathogenesis through regulating its methylating pattern, making them putative candidates for epigenetic therapy in LUAD.

Comprehensive analysis of tumor immune infiltration associated with endogenous competitive RNA networks in lung adenocarcinoma

Cancer immunotherapy has achieved unprecedented success in the treatment of cancer. However, different patients have different responses to immunotherapy. More and more studies have shown that tumor immune heterogeneity has an important influence on the prognosis of cancer. Therefore, understanding the clinical impact of tumor immune infiltration and the regulatory mechanism of RNA molecules is crucial for exploring the pathogenesis of lung adenocarcinoma (LUAD) and the development of immunotherapy protocols.The endogenous competitive RNA hypothesis provides new ideas for studying immune heterogeneity. Therefore, by using the method of immune genomics, this article explores the relationship between immune infiltration and prognosis of patients with lung adenocarcinoma, and found that B-cell immune infiltration highly affects the survival of patients. Through differential analysis, differential mRNAs, lncRNAs and miRNAs were extracted, and 318 differentially expressed mRNAs related to B cell immunity were screened by correlation analysis, and prognosis of patients with COX risk regression model was predicted and analyzed. Through multiple database searches, an immune-related ceRNA regulatory network was constructed, containing 3 key mRNAs, 4 miRNAs, and 50 lncRNAs. Three mRNAs and most miRNAs, lncRNAs, are significantly associated with LUAD prognosis. Bioinformatics analysis of the network showed that LINC00337 may up-regulate the expression of PBK and KIF23 through competitive binding of has-mir-373 and has-mir-519d. The competitive binding of has-mir-373 and has-mir-372 can up-regulate the expression of SLC7A11. The interaction between these RNAs may have an important regulatory role in the immune infiltration in lung adenocarcinoma, thereby affecting the patient's prognosis and immunotherapy efficacy.

Dual strands of the miR-145 duplex (miR-145-5p and miR-145-3p) regulate oncogenes in lung adenocarcinoma pathogenesis.

Our original microRNA (miRNA) expression signatures (based on RNA sequencing) revealed that both strands of the miR-145 duplex (miR-145-5p, the guide strand, and miR-145-3p, the passenger strand) were downregulated in several types of cancer tissues. Involvement of passenger strands of miRNAs in cancer pathogenesis is a new concept in miRNA biogenesis. In our continuing analysis of lung adenocarcinoma (LUAD) pathogenesis, we aimed here to identify important oncogenes that were controlled by miR-145-5p and miR-145-3p. Downregulation of miR-145-5p and miR-145-3p was confirmed in LUAD clinical specimens. Functional assays showed that miR-145-3p significantly blocked the malignant abilities in LUAD cells, e.g., cancer cell proliferation, migration and invasion. Thus, the data showed that expression of the passenger strand of the miR-145-duplex acted as an anti-tumor miRNA. In LUAD cells, we identified four possible target genes (LMNB2, NLN, SIX4, and DDC) that might be regulated by both strands of miR-145. Among the possible targets, high expression of LMNB2 predicted a significantly poorer prognosis of LUAD patients (disease-free survival, p = 0.0353 and overall survival, p = 0.0017). Overexpression of LMNB2 was detected in LUAD clinical specimens and its aberrant expression promoted malignant transformation of LUAD cells. Genes regulated by anti-tumor miR-145-5p and miR-145-3p are closely involved in the molecular pathogenesis of LUAD. We suggest that they are promising prognostic markers for this disease. Our approach, based on the roles of anti-tumor miRNAs, will contribute to improved understanding of the molecular pathogenesis of LUAD.

Abstract 5513: Role of the evolutionarily conserved TBX2 subfamily of transcription factors in the molecular pathogenesis of human lung adenocarcinoma

Abstract T-box (TBX) transcription factors are evolutionary conserved genes and master regulators of transcription repression and activation. In mammals, 18 members were described functionally and structurally, of which the TBX2 subfamily (TBX2, TBX3, TBX4, TBX5) genes were shown to be expressed early on in the developing lung bud and tracheae. Despite these insights into the role of the TBX2 subfamily in normal lung organogenesis, little is known about the role of these genes in pathological pulmonary conditions in humans; particularly lung cancer, an aggressive malignancy that is the leading cause of cancer-deaths worldwide. To fill this void, our group previously surveyed the expression of TBX2 subfamily in various publicly available datasets and found that all four members were preferentially and highly expressed in human normal lung, but markedly and consistently suppressed in lung adenocarcinoma (LUAD) the most common histological subtype of lung cancer. We also showed that the subfamily was also suppressed in preneoplastic lesions preceding the development of LUADs. Following the above and to further elucidate the role of the TBX2 subfamily in LUAD pathogenesis, we first probed and confirmed abundant expression of protein products of the four members by immunostaining in adult human normal lung tissues. On the other hand, quantitative real-time PCR and western blotting analyses demonstrated overall suppressed expression of the genes and corresponding proteins in a panel of human LUAD cell lines. Transient over-expression of each of the four genes in human LUAD cell lines (H1299 and H1944) was found to overall significantly inhibit cancer cell growth and proliferation. Additionally, over-expression of the four genes induced apoptosis, evidenced by sub-G0/G1 accumulation following cell cycle analysis, in both cell lines (ranging from 40% to 90% compared to control). To understand genome-wide effects of TBX2 subfamily in LUAD, we interrogated global expression programs downstream of these transcription factors by RNA-Seq in H1299 cells engineered to over-express the four members separately. We unraveled novel signaling cues signifying canonical pathways found in our analysis to be directly regulated by members of the TBX2 subfamily. These included, among others, inhibition of cell cycle progression and glycolysis, suppression of pathways mediated by epidermal growth factor (EGFR) and WNT signaling and activation of the major anti-tumor immune marker interferon gamma (IFNG). All in all, our findings point to tumor suppressor roles for TBX2 subfamily in human LUAD pathogenesis and suggest “oncophenotypes” downstream of these factors as putative targets for lung cancer therapy. Citation Format: Athar Khalil, Nehme El-Hachem, Batoul Dekmak, Humam Kadara, Georges Nemer. Role of the evolutionarily conserved TBX2 subfamily of transcription factors in the molecular pathogenesis of human lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5513.

Oncogenic role of miR‑183‑5p in lung adenocarcinoma: A comprehensive study of qPCR, invitro experiments and bioinformatic analysis.

Despite the fact that previous studies have reported the aberrant expression of miR-183-5p in lung adenocarcinoma (LUAD), the oncogenic role of miR-183-5p in LUAD and its underlying mechanisms have remained elusive. Hence, we attempted to elucidate the clinicopathological significance of miR-183-5p expression in LUAD and identify the biological function of miR-183-5p in LUAD in this study. Meta-analysis of Gene Expression Omnibus (GEO) data, data mining of The Cancer Genome Atlas (TCGA) and real-time quantitative polymerase chain reaction (qPCR) were performed to evaluate the clinicopathological significance of miR-183-5p in LUAD. Then, the effect of miR-183-5p on cell growth in LUAD was assessed by in vitro experiments. Additionally, the target genes of miR-183-5p were identified via miRWalk v.2.0 and TCGA. Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Disease Ontology (DO) analysis were further carried out for the target genes. The targetability between target genes in key KEGG pathways and miR-183-5p was validated by independent samples t-test, Pearson's correlation test and immunohistochemistry results from the Human Protein Atlas (HPA). According to the results, miR-183-5p was overexpressed in LUAD and exhibited significant diagnostic value. Moreover, miR-183 expression was associated with tumor progression in the TCGA data. In vitro experiments revealed the positive influence of miR-183-5p on cell viability and proliferation as well as the negative effect of miR-183-5p on caspase-3/7 activity in LUAD, which supports the finding that target genes of miR-183-5p are mainly enriched in gene pathways containing cell adhesion molecules (CAMs) and gene pathways important in cancer. Therefore, we conclude that miR-183-5p acts as an oncogene in LUAD and participates in the pathogenesis of LUAD via the interaction networks of its target genes.