Pathway In Non-small Cell Lung Cancer Research Articles

FOXP3 facilitates the invasion and metastasis of non‑small cell lung cancer cells through regulating VEGF, EMT and the Notch1/Hes1 pathway

Forkhead box P3 (FOXP3) is a specific marker of regulatory T cells (Tregs) that is also expressed in tumour cells. Previous studies have revealed that FOXP3 can promote metastasis in several types of cancer, including non-small cell lung cancer (NSCLC); however, the underlying mechanism of FOXP3 remains unclear. The aim of the present study was to investigate the effect of FOXP3 on vascular endothelial growth factor (VEGF), epithelial-to-mesenchymal transition (EMT) and the Notch1/Hes1 pathway in NSCLC. After FOXP3 small interfering RNA (siRNAs) were transfected into A549 cells, the expression of FOXP3 mRNA and protein was determined by reverse transcription-quantitative PCR and western blotting. Cell migration and invasion were analyzed by Transwell assays. The concentrations of matrix metalloproteinase (MMP)-2, MMP-9 and VEGF in the cell supernatant were evaluated by ELISA. The expression of relevant proteins involved in EMT and Notch1/Hes1 pathway were assessed via western blotting. Additionally, the expression of FOXP3, CD31 and E-cadherin was detected by immunohistochemical (IHC) staining of 55 human NSCLC tissue samples. The results demonstrated that FOXP3 knockdown significantly inhibited the cell migratory and invasive abilities, decreased the concentrations of MMP-2, MMP-9 and VEGF, downregulated the protein expression of vimentin, N-cadherin, Notch1 and Hes family BHLH transcription factor 1 (Hes1), and upregulated the protein expression of E-cadherin. Furthermore, FOXP3 expression was positively associated with CD31+ vascular endothelial cells and negatively correlated with E-cadherin in NSCLC tissues. In addition, the Notch1/Hes1 pathway inhibitor DAPT significantly downregulated the expression of FOXP3 in a dose-dependent manner. Taken together, these findings demonstrated that FOXP3 may facilitate the invasive and migratory abilities of NSCLC cells via regulating the angiogenic factor VEGF, the EMT and the Notch1/Hes1 pathway.

Targeted silencing of TEM8 suppresses non‑small cell lung cancer tumor growth via the ERK/Bcl‑2 signaling pathway.

Non-small cell lung cancer (NSCLC) is one of the most common malignancies with high rates of mortality. Although great progress has been made with the development of novel immunotherapies and targeted therapeutic strategies, the 5-year total survival rate of lung cancer has remained unchanged over the past few decades. Therefore, more effective therapeutics are urgently needed. Tumor endothelial marker 8 (TEM8) is an integrin-like cell surface transmembrane protein that has been demonstrated to be upregulated in numerous cancer types and previously showed promise for targeted cancer therapy. However, the role of TEM8 in NSCLC remains poorly understood. The present study aimed to investigate the effects of silencing TEM8 on expression and regulation of extracellular signal-regulated kinase (ERK)1/2 signaling pathways in NSCLC. In the present study, a lentiviral vector that encoded a short hairpin RNA targeting TEM8 was designed and transfected into Xuanwei Lung Cancer (XWLC)-05 lung cancer cells to silence TEM8 expression. Male BALB/c-nu/nu mice were then given subcutaneous injections in the right dorsal flank with XWLC-05 cells. Microvessel density was measured using an anti-CD34 antibody. The mRNA and protein levels of ERK1/2 and Bcl-2 in XWLC-05 cells or xenograft tumor tissues were detected by reverse transcription-quantitative polymerase chain reaction and western blotting. TEM8 knockdown was found to significantly inhibit tumor growth and conferred an anti-angiogenic ability in vivo. Furthermore, TEM8 knockdown suppressed the expression of Bcl-2 mediated by ERK1/2 activity in XWLC-05 cells or tissues from mice with NSCLC. To conclude, these results suggest that the targeted silencing of TEM8 may serve as an effective method of treating NSCLC.

Prognostic impact of XPO1 mutations in metastatic non-small cell lung cancer (NSCLC).

e20533 Background: Nuclear protein transport is essential in guiding the organized traffic of important proteins and RNAs between the nucleus and cytoplasm of the cell. Export of proteins from the nucleus is exclusively regulated by Exportin 1(XPO1). In cancer, XPO1 is universally hyperactive and can promote the export of important tumor suppressors to the cytoplasm, leading to their functional inactivation. XPO1 is aberrantly over expressed in NSCLC and this over expression has been linked to poor overall survival. The underlying mechanisms of XPO1 over expression are not known. Currently there are no studies evaluating the impact of XPO1 mutations on NSCLC incidence and therapy resistance. Additionally, there are no studies that examined the XPO1 related pathways in NSCLC harboring co-alterations with other driver mutations such as EGFR or ALK. Methods: Tumor samples were analyzed using next-generation sequencing (NextSeq, 592 Genes), IHC, and whole transcriptome sequencing (WTS ,NovaSeq) (Caris Life Sciences, Phoenix, AZ). PD-L1 expression was tested by IHC using 22c3 (Dako) and TPS scores were reported (cutoff > 1%). TMB was measured by totaling somatic mutations (TMB-high cut-off ³ 10 mutations per Megabase). Gene fusions were detected by RNA sequencing using either the Archer FusionPlex panel or WTS. Survival was extracted from insurance claims data and calculated from time of tissue collection to last contact using Kaplan-Meier estimate. Statistical significance was determined using chi-square and Wilcoxon rank sum test and adjusted for multiple comparisons. Results: Among 18,218 NSCLC tumors sequenced, 26 harbored XPO1 mutations. XPO1 mutant tumors were more likely to be TMB High(79% vs. 52%, p = 0.007) and less likely to have high PDL1(32% vs. 68%, p = 0.03). KRAS mutations were seen in 19%(n = 5), EGFR mutation were rare (n = 2), and no targetable fusions were seen. Among the 17,449 NSCLC tumors with clinical data, there were 24 XPO1 mutant tumors with no histology imbalance observed in mutant vs. wild-type(WT). Comparison of survival in the NSCLC group between XPO1 mutant and WT showed a negative association with a hazard ratio(HR) of 1.932 (95% CI: 1.144- 3.264 p = 0.012). Comparing the survival within the subgroup with confirmed adenocarcinoma histology (9973 XPO1 WT and 14 XPO1 mutant) showed a similar negative correlation in survival with a HR of 2.156 (95% CI: 1.027- 4.525 P = 0.037). Conclusions: Presence of XPO1 pathogenic mutations was associated with a poor survival in both the entire NSCLC cohort and the adenocarcinoma subgroup. Further studies of this negative association at the molecular level along with effect of other co-existing mutations can result in development of novel treatment strategies.

Integrative RNA-Seq and H3 Trimethylation ChIP-Seq Analysis of Human Lung Cancer Cells Isolated by Laser-Microdissection.

Simple SummaryTissue heterogeneity is one of the major problems in cancer genomics. Thus, we developed and conducted an RNA-Seq and ChIP-Seq integrative analysis of clinical lung tissue samples with the isolation of specific cell populations using laser-microdissection microscopy (LMD). The transcriptomic profile was successfully captured and somatically altered regions marked by histone H3 lysine 4 trimethylation (H3K4me3) were identified in lung cancer. We also observed the differential expressions of cancer-related genes near the altered proximal H3K4me3 regions, while altered distal H3K4me3 regions were overlapped with enhancer activity annotations of cancer regulatory genes. Additionally, proximal tumor-gained promoters were associated with the core components of polycomb repressive complex 2. Our study demonstrates the practical workflow of using LMD on clinical samples for integrative analyses, which improves the overall understanding of genetic and epigenetic dysregulation of malignancy.Our previous integrative study in gastric cancer discovered cryptic promoter activation events that drive the expression of important developmental genes. However, it was unclear if such cancer-associated epigenetic changes occurred in cancer cells or other cell types in bulk tissue samples. An integrative analysis consisting of RNA-Seq and H3K4me3 ChIP-Seq was used. This workflow was applied to a set of matched normal lung tissues and non-small cell lung cancer (NSCLC) tissues, for which the stroma and tumor cell parts could be isolated by laser-microdissection microscopy (LMD). RNA-Seq analysis showed subtype-specific differential expressed genes and enriched pathways in NSCLC. ChIP-Seq analysis results suggested that the proximal altered H3K4me3 regions were located at differentially expressed genes involved in cancer-related pathways, while altered distal H3K4me3 regions were annotated with enhancer activity of cancer regulatory genes. Interestingly, integration with ENCODE data revealed that proximal tumor-gained promoters were associated with EZH2 and SUZ12 occupancies, which are the core components of polycomb repressive complex 2 (PRC2). This study used LMD on clinical samples for an integrative analysis to overcome the tissue heterogeneity problem in cancer research. The results also contribute to the overall understanding of genetic and epigenetic dysregulation of lung malignancy.

Identification of molecular biomarkers and pathways of NSCLC: insights from a systems biomedicine perspective

BackgroundWorldwide, more than 80% of identified lung cancer cases are associated to the non-small cell lung cancer (NSCLC). We used microarray gene expression dataset GSE10245 to identify key biomarkers and associated pathways in NSCLC. ResultsTo collect Differentially Expressed Genes (DEGs) from the dataset GSE10245, we applied the R statistical language. Functional analysis was completed using the Database for Annotation Visualization and Integrated Discovery (DAVID) online repository. The DifferentialNet database was used to construct Protein–protein interaction (PPI) network and visualized it with the Cytoscape software. Using the Molecular Complex Detection (MCODE) method, we identify clusters from the constructed PPI network. Finally, survival analysis was performed to acquire the overall survival (OS) values of the key genes. One thousand eighty two DEGs were unveiled after applying statistical criterion. Functional analysis showed that overexpressed DEGs were greatly involved with epidermis development and keratinocyte differentiation; the under-expressed DEGs were principally associated with the positive regulation of nitric oxide biosynthetic process and signal transduction. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway investigation explored that the overexpressed DEGs were highly involved with the cell cycle; the under-expressed DEGs were involved with cell adhesion molecules. The PPI network was constructed with 474 nodes and 2233 connections. ConclusionsUsing the connectivity method, 12 genes were considered as hub genes. Survival analysis showed worse OS value for SFN, DSP, and PHGDH. Outcomes indicate that Stratifin may play a crucial role in the development of NSCLC.

Nrf2 regulates cell motility through RhoA\u2013ROCK1 signalling in non-small-cell lung cancer cells

Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a key transcriptional regulator of several antioxidant and anti-inflammatory enzymes. It binds to its endogenous inhibitor Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm under normal conditions. Various endogenous or environmental oxidative stresses can disrupt the Nrf2/Keap1 complex, allowing Nrf2 to translocate into the nucleus, where it induces the transcription of various cytoprotective enzymes by binding to antioxidant responsive elements. These enzymes have been reported to play a role in regulating tumour growth, angiogenesis, and chemoprevention. Invasion and migration are the most harmful aspects of cancer; they directly impacts the patients’ survival. Although the roles of Keap1/Nrf2 and their downstream genes in various cancers have been widely documented, their role in regulating cell motility still remains unclear, particularly in cancer cells. We observed that Nrf2 suppression following treatment with brusatol in non-small-cell lung cancer (NSCLC) cells with either exogenously introduced Keap1 or siNrf2 resulted in the inhibition of cell migration and invasion, with shrinking cell morphology due to decreased focal adhesions via inhibition of the RhoA–ROCK1 pathway. Nrf2 overexpression showed opposite results. Thus, the Nrf2/Keap1 pathway may affect cell motility by dysregulating the RhoA–ROCK1 signalling pathway in NSCLC.

JMJD8 Promotes Malignant Progression of Lung Cancer by Maintaining EGFR Stability and EGFR/PI3K/AKT Pathway Activation

JMJD8 is a JmjC domain-containing protein that has not been widely examined, despite its potential role in malignant tumor development. The underlying biological functions and molecular mechanisms of JMJD8 in non-small-cell lung cancer (NSCLC) remain unclear. Herein, we explored the relationship between JMJD8 and the activation of malignancy pathways in NSCLC. Immunohistochemical analyses revealed that high JMJD8 expression significantly correlated with cell differentiation and advanced TNM stages of NSCLC. The overexpression of JMJD8 promoted cell proliferation and invasion in vitro. Upon JMJD8 knockdown in lung cancer cell lines, cyclin B1, RhoA, RhoC, MMP9, and N-cadherin were down-regulated, and p21 and E-cadherin were conversely up-regulated. Key factors in the PI3K/AKT signaling pathway, such as p‑AKT, showed clear decreases in expression; additionally, the expression of epidermal growth factor receptor (EGFR), which functions upstream of PI3K, was altered. Co-immunoprecipitation experiments indicated that JMJD8 interacts with EGFR, and JMJD8 knockdown accelerated EGFR degradation. Our results suggested that JMJD8 functions as an oncogenic regulator in NSCLC. We found that JMJD8 promotes carcinogenic activity in NSCLC cells by facilitating EGFR stability, thereby activating the downstream PI3K/AKT signaling pathway. JMJD8 shows potential as a prognostic marker for lung cancer patients, providing a new target for therapeutic strategies.

ROS‑mediated hypomethylation of PRDX5 promotes STAT3 binding and activates the Nrf2 signaling pathway in NSCLC.

Deoxyribonucleic acid (DNA) epigenetic modification has been linked to specific sequences of CpG islands and plays roles in the progression of lung cancer. In this study, it was found that peroxiredoxin-5 (PRDX5) was highly expressed in non-small cell lung cancer (NSCLC) tissues; however, its specific regulatory mechanisms and functions in NSCLC remain unknown. The present study therefore explored the regulatory mechanism of PRDX5 under conditions of oxidative stress (OS) in NSCLC. The results revealed that 79 of 121 NSCLC patients exhibited demethylation in the PRDX5 promoter region, which was related to the tumor, node and metastasis (TNM) stage (P=0.027). PRDX5 messenger ribonucleic acid (mRNA) expression positively correlated with the demethylation status of the promoter region. The results of bisulfite sequencing polymerase chain reaction (BSP) revealed lower demethylation frequencies in H1299 cells treated with 0 µM H2O2, but maximum demethylation following treatment with 100 µM H2O2. Using chromatin immunoprecipitation (ChIP) and luciferase detection assays, the effective binding of STAT3 to the transcriptional binding sites of the PRDX5 promoter region was confirmed (2 sites confirmed: Site 1, -444 to -434 bp; and site 4, -1,417 to -1,407 bp). STAT3 knockdown significantly decreased the protein expression of PRDX5, while the overexpression of STAT3 significantly increased the protein levels of PRDX5. When PRDX5 was overexpressed in lung cancer cells under conditions of OS, the levels of the epithelial-mesenchymal transition (EMT) biomarkers, E-cadherin and vimentin, were significantly decreased and increased, respectively. By contrast, PRDX5 knockdown resulted in significantly increased E-cadherin and decreased vimentin protein expression levels. Ultimately, when PRDX5-small interfering RNA (siRNA) or pcDNA3.1-PRDX5expression vector were constructed and transfected into H1299 cells pre-treated with 100 µM H2O2, the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway was inhibited or activated. All these results suggested that the reactive oxygen species (ROS)-mediated hypomethylation of PRDX5 enhanced STAT3 binding affinity with the promoter region, and resulted in the promotion of cell migration and invasion, as well as in the activation of the Nrf2 signaling pathway in NSCLC. The demethylation status of the PRDX5 promoter may thus be used as an epigenetic biomarker in NSCLC. STAT3/PRDX5 signaling may also prove to be a potential strategy for the treatment of this type of cancer.

C-MYC-induced upregulation of LINC01503 promotes progression of non-small cell lung cancer.

The purpose of this study was to detect the expression of long intergenic non-protein-coding RNA 1503 (LINC01503) in non-small cell lung cancer (NSCLC), and to further study its biological function, as well as the regulatory relationships of c-MYC with LINC01503 and the extracellular signal regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling pathway in NSCLC. Tissue specimens were collected from 36 NSCLC patients, and the relative expression level of LINC01503 in the 36 cases of NSCLC tissue specimens and NSCLC cells was then determined using quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Then, the effects of LINC01503 on the proliferation and apoptosis of NSCLC cells were detected in vitro via Cell-Counting Kit (CCK)-8 assay, colony-forming assay and flow cytometry. Besides, the possible LINC01503 promoter-binding transcription factor was predicted using bioinformatics. After interference with c-MYC expression, the changes in the expression of LINC01503 were examined through qRT-PCR. Finally, the changes in the expressions of the molecular markers in the ERK/MAPK signaling pathway after interference with LINC01503 and c-MYC expressions were evaluated using Western blotting. According to qRT-PCR results, the expression of LINC01503 was upregulated in 30 out of 36 cases of NSCLC tissues. Compared with that in human normal bronchial epithelial cells, the expression of LINC01503 was elevated in NSCLC cells. As shown by the CCK-8 assay and colony-forming assay, the proliferation ability of NSCLC cells was weakened after interference with LINC01503 expression, and the flow cytometry results revealed the apoptosis rate of NSCLC cells was raised after interference with LINC01503 expression. Moreover, the bioinformatics prediction showed that c-MYC might be the LINC01503 promoter-binding transcription factor. Additionally, it was found through the qRT-PCR that the expression of LINC01503 declined after interference with c-MYC expression. Finally, based on Western blotting results, the expressions of phosphorylated ERK1/2 (p-ERK1/2) and p-MAPK/ERK kinase (MEK), the molecular markers in the ERK/MAPK signaling pathway, were inhibited after interference with c-MYC and LINC01503 expressions. The transcription factor c-MYC promotes the expression of LINC01503 in NSCLC and activates the ERK/MAPK signaling pathway to drive the development and progression of NSCLC.

Combination of DN604 with gemcitabine led to cell apoptosis and cell motility inhibition via p38 MAPK signaling pathway in NSCLC

Non-small-cell lung cancer (NSCLC) is the most common cancer in the world, which is still treated with Pt(II) agents as first-line drugs. As a traditional anticancer agent, gemcitabine is usually used in the combination treatment of various solid tumors with other drugs. Here, we investigate the combinatory application of gemcitabine with a Pt(II) agent (DN604, reported previously in our former research) in the treatment of NSCLC. In vitro biological assays suggested that DN604-gemcitabine treatment can effectively induce cell apoptosis and suppress cell motility, showing better anti-tumor effect than the single drug treatment or the combined treatment of cisplatin and gemcitabine. More importantly, investigation on the mechanism of the combined treatment proved that such combined treatment can suppress cell autophagy to inhibit cell motility via the activation of p38 MAPK signaling pathway. In vivo studies indicated that combination of DN604 with gemcitabine significantly inhibited the growth of tumor with nearly no influence on the normal organs and weight of mice. Our study widened the application scope of Pt(II) agents combined with gemcitabine for NSCLC treatment.

Abstract 3842: Histone methyltransferase G9a promotes migration, invasion of non-small cell lung cancer through enhancing FAK and NF-kB signal pathways

Abstract Overexpression of euchromatic histone methyltransferase 2 (EHMT2 or G9a) is associated with carcinogenesis and poor prognosis in a number of cancer types. In this study, we investigated the effect of G9a on invasion of non-small cell lung cancer (NSCLC) cells and the mechanism underlying cancer cell invasion. Knockdown of G9a was shown to drastically suppress in vitro cellular proliferation, migration, and invasion of A549 and H1299 NSCLC cells, while overexpression of G9a significantly enhanced the proliferation and invasion of these NSCLC cells. Additionally, knockdown of G9a led to significant decrease in expression of focal adhesion kinase (FAK) gene and activation of FAK pathway. UNC0638, a selective inhibitor of G9a, significantly suppressed both the proliferation, migration and invasion of these NSCLC cells, and concurrently led to a dramatic drop of global demethylation of H3K9 (H3K9me2) and suppressed the activation of FAK pathway. In addition, Defactinib, a potent FAK inhibitor, partially abolished the enhanced invasion by overexpression of G9a in these NSCLC cells. Furthermore, G9a was proved to regulate nuclear NF-κB transcriptional activity in NSCLC cells, and NF-κB inhibitor partially abolished the activated FAK signaling pathway by overexpressed G9a, which indicates that G9a-enhanced invasion and activation of FAK pathway may be mediated by NF-κB transcriptional activity. Notably, immunohistochemistry analysis further revealed that activation of FAK pathway was associated with overexpressed G9a in about 150 cases of NSCLC tissues. Altogether, these data have strongly demonstrated that G9a may promote invasion of NSCLC cells by upregulating FAK protein and pathway via enhancing NF-κB transcriptional activity, indicating a therapeutic implication of targeting these two pathways in NSCLC with overexpressed G9a. Citation Format: Ting Sun, Keqiang Zhang, Rajendra P. Pangeni, Wendong Li, Dan J. Raz. Histone methyltransferase G9a promotes migration, invasion of non-small cell lung cancer through enhancing FAK and NF-kB signal pathways [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3842.

TNNC1 Reduced Gemcitabine Sensitivity of Nonsmall-Cell Lung Cancer by Increasing Autophagy.

BackgroundAs we know, chemotherapy resistance is a critical factor leading to recurrence and metastasis of nonsmall-cell lung cancer (NSCLC). To clarify the key target and potential mechanism of resistance to gemcitabine (GEM) in NSCLC, we selected Gene Expression Omnibus Data Set and statistically analyzed a parent cell group and a GEM-resistant cell group. Results showed that the expression of troponin C1, slow skeletal and cardiac type (TNNC1) in GEM-resistant cells was higher than in parent cells, which implies that TNNC1 was associated with GEM resistance in lung cancer cells.Material/MethodsTNNC1 expression level was detected by reverse transcription-quantitative polymerase chain reaction or western blot in GEM-resistant patient serum and cell lines. It could reduce or increase autophagy response and GEM resistance accordingly by inhibition of the short interfering ribonucleic acid or by forced overexpression of TNNC1 viruses in A549 cell line and GEM-resistant cell line (A549/GemR) respectively. Blocking autophagy with 3-methyladenine increased the sensitivity of chemotherapy confirmed by flow cytometry and microtubule-associated protein 1A/1B – light chain 3 punctate assay. What’s more, in a loss-of-function model, silencing of forkhead box 03 (FOXO3) in A549/GemR cells could rescue the autophagy weakened by TNNC1.ResultsTNNC1 promoted GEM chemoresistance of NSCLC by activating cytoprotective autophagy, regulated negatively by FOXO3. This research may provide a completely new strategy for NSCLC treatment.ConclusionsTargeting the TNNC1/FOXO3 signaling pathway in NSCLC may be a novel strategy to combat GEM resistance.

Long non-coding RNA SLCO4A1-AS1 drives the progression of non-small-cell lung cancer by modulating miR-223-3p/IKKα/NF-κB signaling

ABSTRACT Globally, lung cancer is known as a major cause of cancer-associated death and non-small-cell lung cancer (NSCLC) accounts for majority of all cases. Growing evidence has emerged that long non-coding RNAs (lncRNAs) act as vital regulatory molecules in various malignancies. Nevertheless, the function of SLCO4A1 antisense RNA 1(SLCO4A1-AS1) in NSCLC is vague. This study intended to investigate the biological role and probable regulatory mechanism of SLCO4A1-AS1 in NSCLC. qRT-PCR revealed that SLCO4A1-AS1 level was upregulated in NSCLC. Function assays manifested that silence of SLCO4A1-AS1 attenuated NSCLC cell proliferation, migration and invasion but promoted NSCLC cell apoptosis. Furthermore, we disclosed that SLCO4A1-AS1 activated NF-κB pathway in NSCLC, and that IKKα, an NF-κB pathway-related gene, possessed an enhanced level in NSCLC tissues and cells. Importantly, miR-223-3p bound with SLCO4A1-AS1 and IKKα. Further, SLCO4A1-AS1 competitively bound with miR-223-3p to increase IKKα expression, thereby activating NF-κB signaling pathway. In conclusion, SLCO4A1-AS1 drove NSCLC progression by activating NF-κB signaling pathway via sponging miR-223-3p to enhance IKKα expression. Thus, SLCO4A1-AS1 might be a promising biomarker for NSCLC treatment.

ZFHX3 mutation as a protective biomarker for immune checkpoint blockade in non-small cell lung cancer.

To date, immunotherapy has opened a new chapter in the treatment of lung cancer. Precise biomarkers can help to screen subpopulations of lung cancer to provide the best treatment. Multiple studies suggest that specific gene mutations may be predictive markers in guiding non-small cell lung cancer (NSCLC) immune checkpoint inhibitor (ICI) treatment. A published immunotherapy cohort with mutational and survival data for 350 NSCLC patients was used. First, the mutational data of the immunotherapy cohort were used to identify gene mutations related to the prognosis of ICI therapy. The immunotherapy cohort and TCGA-NSCLC cohort were further studied to elucidate the relationships between specific gene mutations and tumor immunogenicity, antitumor immune response capabilities, and immune cell and mutation counts in the DNA damage response (DDR) pathway. In the immunotherapy cohort (N = 350), ZFHX3 mutations were an independent predictive biomarker for NSCLC patients receiving ICI treatment. Significant differences were observed between ZFHX3-mutant (ZFHX3-MT) and ZFHX3-wild type (ZFHX3-WT) patients regarding the overall survival (OS) time (P < 0.001, HR = 0.26, 95% Cl 0.17-0.41). ZFHX3-MT is significantly associated with higher tumor mutation burden (TMB) and neoantigen load (NAL), and ZFHX3-MT positively correlates with known immunotherapy response biomarkers, including T-cell infiltration, immune-related gene expression, and mutation counts in the DDR pathway in NSCLC. ZFHX3-MT is closely related to longer OS in NSCLC patients treated with ICIs, suggesting that ZFHX3 mutations be used as a novel predictive marker in guiding NSCLC ICI treatment.

MicroRNA‑103 modulates tumor progression bytargeting KLF7 in non‑small cell lung cancer.

Numerous studies have identified that microRNAs (miRs) play a crucial role in the tumorigenesis of non-small cell lung cancer (NSCLC). However, to the best of our knowledge, the physiological function of miR-103 in NSCLC is not fully understood. Experiments in the present study revealed that miR-103 expression was increased in NSCLC cell lines. In addition, a series of methods, including MTT, colony formation, 5-ethynyl-2′-deoxyuridine, Transwell, wound healing, flow cytometric, reverse transcription-quantitative PCR and western blot assays, were performed, which revealed that overexpression of miR-103 enhanced cell growth, migration, invasion and epithelial-mesenchymal transition (EMT), and suppressed apoptosis of A549 and H1299 cells. Additionally, a dual-luciferase reporter assay indicated that miR-103 directly targets the 3′-untranslated region of Kruppel-like factor 7 (KLF7), and KLF7 expression was negatively regulated by miR-103 expression. Furthermore, the present findings demonstrated that miR-103 promoted EMT via regulating the Wnt/β-catenin signaling pathway in NSCLC. Collectively, the current results demonstrated that miR-103 serves a tumorigenesis role in NSCLC development by targeting KLF7, at least partly via the Wnt/β-catenin signaling pathway. Consequently, these findings indicated that miR-103/KLF7/Wnt/β-catenin may provide a novel insight into underlying biomarkers for improving the diagnosis and treatment of NSCLC.

MicroRNA-147 targets BDNF to inhibit cell proliferation, migration and invasion in non-small cell lung cancer.

Lung cancer is one of the most common cancers that threaten human life and health. Recently, microRNAs (miRNAs) have been shown to play a unique role in many malignancies. Although the dysregulation of miR-147 has been detected in non-small cell lung cancer (NSCLC), the biological function of miR-147 is still unknown in NSCLC. The expression of miR-147 was observed by real-time quantitative polymerase chain reaction (RT-qPCR). Methyl thiazolyl tetrazolium (MTT) and Transwell assays were used to investigate the function of miR-147 in NSCLC. Target genes of miR-147 were verified using dual luciferase reporter assay. Western blot analysis was used to explore the PI3K/AKT pathway. The expression of miR-147 was decreased in NSCLC tissues, which was associated with poor prognosis in NSCLC patients. Furthermore, overexpression of miR-147 inhibited the viability and metastasis of NSCLC cells. In addition, miR-147 inhibited epithelial-mesenchymal transition (EMT) and inactivated the PI3K/AKT pathway in NSCLC. Furthermore, miR-147 directly targets brain-derived neurotrophic factor (BDNF) and negatively regulates BDNF expression in NSCLC. Upregulation of BDNF attenuated the inhibitory effect of miR-147 in NSCLC. In conclusion, miR-147 inhibits cell proliferation, migration and invasion in NSCLC through suppressing BDNF expression.

Downregulation of LINC00665 confers decreased cell proliferation and invasion via the miR-138-5p/E2F3 signaling pathway in NSCLC

Non-small cell lung cancer (NSCLC) is a type of malignant tumor which threatens human health and life. Recently, some researches on long non-coding RNAs (lncRNAs) in NSCLC has elucidated critical regulatory roles in cell proliferation, migration, and invasion, the relative clinical significance and mechanisms of action are still unclear. This study focuses on the important role of a novel lncRNA LINC00665 in the development of NSCLC. Long intergenic non-protein coding RNA 665 gene (LINC00665) was found through microarray analysis and was measured by real-time quantitative PCR (RT-qPCR). The interactions between LINC00665 and miR-138-5p as well as the interactions between miR-138-5p and E2F3 (E2F transcription factor 3) were explored by bioinformatics analysis and dual-luciferase assays. CCK-8, transwell and mouse xenograft assays were performed to investigate the effects of LINC00665 and miR-138-5p on NSCLC proliferation and invasion. As a result, LINC00665 expression was upregulated in NSCLC lung tissues and cells. Downregulated LINC00665 could arrest A549 and H1299 cell proliferation and invasion in vitro, and this finding was recapitulated in vivo. LINC00665 directly regulated the expression of miR-138-5p. Additionally, E2F3 was one of the targets of miR-138-5p; E2F3 without 3′UTR could reverse the inhibitory effects of downregulated LINC00665 on proliferation and invasion in A549 and H1299 cells. In conclusion, dysregulation of LINC00665 plays a vital role in NSCLC progression, indicating that its downregulation may confer decreased cell proliferation and invasion via the miR-138-5p/E2F3 signaling pathway.

Inhibition of LncRNAH19 has the effect of anti-tumour and enhancing sensitivity to Gefitinib and Chemotherapy in Non-small-cell lung cancer in vivo.

Lung cancer is one of the most common malignant diseases, which ranked first in both men and women malignancies worldwide. The survival rate of non‐small‐cell lung cancer (NSCLC) has been limited with distant metastasis and shortage of effective chemotherapeutics in recent years. Thus, novel therapeutic strategies for NSCLC are urgently explored. Here, we showed that inhibition of H19 effectively inhibited the progression of NSCLC. Moreover, down‐regulation of H19 treatment significantly enhanced the levels of PTEN and PDCD4, while suppressed the expressions of NFIB in NSCLC. Furthermore, down‐regulation of H19 combined with Gefitinib treatment significantly increased the levels of PTEN and PDCD4, while decreased the expression levels of NFIB. Moreover, the results showed that Gefitinib treatment significantly reduced the shH19‐mediated miR‐21 expression levels. Our results showed that down‐regulation of H19 combined with Gefitinib administration significantly improved the effect of shH19 treatment alone on the progression of NSCLC, which was involved in the activation of PTEN signalling pathway in NSCLC in vivo. Therefore, these findings might indicate a novel molecular mechanism, which could provide a new potential combination of therapeutic method in NSCLC.

EGFR inhibition triggers an adaptive response by co-opting antiviral signaling pathways in lung cancer.

EGFR inhibition is an effective treatment in the minority of non-small cell lung cancer (NSCLC) cases harboring EGFR-activating mutations, but not in EGFR wild type (EGFRwt) tumors. Here, we demonstrate that EGFR inhibition triggers an antiviral defense pathway in NSCLC. Inhibiting mutant EGFR triggers Type I IFN-I upregulation via a RIG-I-TBK1-IRF3 pathway. The ubiquitin ligase TRIM32 associates with TBK1 upon EGFR inhibition, and is required for K63-linked ubiquitination and TBK1 activation. Inhibiting EGFRwt upregulates interferons via an NF-κB-dependent pathway. Inhibition of IFN signaling enhances EGFR-TKI sensitivity in EGFR mutant NSCLC and renders EGFRwt/KRAS mutant NSCLC sensitive to EGFR inhibition in xenograft and immunocompetent mouse models. Furthermore, NSCLC tumors with decreased IFN-I expression are more responsive to EGFR TKI treatment. We propose that IFN-I signaling is a major determinant of EGFR-TKI sensitivity in NSCLC and that a combination of EGFR TKI plus IFN-neutralizing antibody could be useful in most NSCLC patients.

Gene microarray expression profile analysis of differentially expressed genes of potential alternative pathways in non–small cell lung cancer: In search of biomarkers

In this study, we aimed to identify genes that could be involved in alternative pathways linked to the pathogenesis of non–small cell lung cancer (NSCLC), which could provide novel molecular biomarkers. Owing to NSCLC subtypes, three individual NSCLC data sets were downloaded and analyzed. Gene–subsetting subsequent meta–analysis was performed to screen differentially expressed genes (DEGs). The functional enrichment and pathway analyses were then executed using comprehensive bioinformatics analysis. Accordingly, the three hub genes of alternative pathways and their strong correlation with NSCLC pathway were initially identified as potential candidates (common DEGs) for the progression of NSCLC. The clinicopathological functions of common DEGs in NSCLC were validated by analyzing transcription factors targeting common DEGs, and their effectiveness was validated by analysis of protein/gene interactions and biological processes annotations. Finally, elucidated genes, such as protein phosphatase 3 catalytic subunit alpha (PPP3CA) of calcium signaling pathway, ubiquitin conjugating enzyme E2–Q1 (UBE2Q1) of β–catenin–EGFR–PI3K–Akt–mTOR signaling pathway and TATA–Box binding protein like–1 (TBPL1) of RNA polymerase–II transcription initiation and promoter clearance and PI3K–Akt signaling pathway were recognized as alternative networks mostly associated with the progression of NSCLC by co–occurred with oncogenes of the extracellular–signal–regulated kinase (ERK) pathway; and these genes can confer resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in NSCLC therapy through various mechanisms. Together, these results indicate that mutation–activated alternative pathway genes and related checkpoints can confer progression to NSCLC and drug resistance; therefore, which could provide as promising biomarkers for early detection and treatment of NSCLC.