Cell Lung Cancer Gene Research Articles

FOXP2 inhibits the aggressiveness of lung cancer cells by blocking TGFβ signaling.

Lung cancer is associated with high morbidity and mortality rates. Forkhead box P2 (FOXP2) functions as an antitumor gene in various cancers. However, its role in lung cancer remains to be elucidated. The present study explored the potential role of FOXP2 in lung cancer. mRNA levels and protein expression were determined using RT-qPCR and western blotting, respectively. Functional analysis was performed using the CCK-8, Transwell and TUNEL assays. FOXP2 expression was downregulated in lung cancer. Notably, FOXP2 suppressed the proliferative, migratory and invasive abilities of lung cancer cells and promoted tumor cell apoptosis. In addition, FOXP2 blocked TGFβ signaling. However, SRI-011381-stimulated activation of TGFβ signaling reversed the effects of overexpressed FOXP2 and promoted the aggressiveness of lung cancer cells. FOXP2 functions as an antitumor gene in lung cancer cells. FOXP2 suppressed the malignant behavior of lung cancer by inactivating TGFβ signaling.

TRIM67 interacts with ENAH to regulate the apoptosis and autophagy of lung cancer cells.

The aim of this study was to further clarify the functional mechanism of the triangular 67 (TRIM67) gene in lung cancer cells. We detected the expression of TRIM67 in lung cancer cells by RT-qPCR and Western blot, transfected si-NC, si-TRIM67, and pcDNA-ENAH into the cells. The expression of TRIM67 and ENAH was detected by Western blot and immunofluorescence localization, and CO-IP and GST pull-down experiments verified the interaction. Flow cytometry, Western blot, and transmission electron microscopy (TEM) evaluated the apoptosis and autophagy levels. TRIM67 was highly expressed in lung cancer cell lines. Knockdown of TRIM67 promoted apoptosis and autophagy of A549 and NCI-H1299 cells. TRIM67 interacted with the ENAH protein. ENAH restored the effect of knocking down TRIM67 and further inhibited apoptosis and autophagy of A549 and NCI-H1299 cells. TRIM67 inhibits apoptosis and autophagy of lung cancer cells by interacting with ENAH.

ADAM9 drives immunosuppressive environment for lung tumor progression

Abstract Cancer cells develop the immune suppressive tumor environment, but the underlying molecular mechanism for it is still elusive. Here, we demonstrated that ADAM9 contributed to lung tumor progression and promoted IFN-related gene expression in lung cancer cells that participated in immune regulation in the tumor environment. ADAM9 knockout (KO) lung cancer cells reduced lung cancer metastasis with a decrease in neutrophil infiltration and an increase in CD8+ T cell infiltration. By performing the genome-wide approach to explore ADAM9-regulated genes in lung cancer cells, we revealed that ADAM9 regulated cytokine production and lymphocyte activation. It enhanced the master transcription factor of IFN signaling, STAT1 activation in lung cancer cells. Moreover, ADAM9 promoted IL6/IL6 receptor-STAT3 axis signaling that lead to suppression of IL12p40 secretion and MHC class II expression in dendritic cells and macrophages. Notably, we found that type I IFN related-proteins and steroid biosynthesis-related proteins were related to ADAM9-mediated STAT signaling activation by LC-MS/MS analysis. We demonstrated that ADAM9 created an immune suppressive environment by regulating IFN signaling for tumor progression. Supported by grants from NHRI (NHRI-EX112-11219BI)

In vitro model of predicting metastatic ability using tumor derived extracellular vesicles; beyond seed soil hypothesis

Lung cancer progression is often driven by metastasis, which has resulted in a considerable increase in lung cancer-related deaths. Cell-derived extracellular vesicles (EVs), particularly exosomes, serve key roles in cellular signal transmission via microenvironment, however, their biological relevance in cancer development and metastasis still needs to be clear. Here, we demonstrate that extracellular vesicles (EVs) derived from lung cancer bone metastatic patients exhibited a great capacity to promote the progression of lung cancer cells. We carried out a comprehensive meta-analysis to identify the gene expression profile of bone metastases using publicly available microarray datasets. Furthermore, mRNA expression of six identified genes was quantified by real time PCR in lung cancer with and without bone metastasis and healthy individual derived EVs. In addition, we utilized a very novel approach by to study how lung cancer cells uptake EVs by co-culturing EVs with lung cells. We observed that EVs obtained from bone metastases patients were efficiently ingested by lung cancer cells. Morevore, integration and uptake of these EVs lead to increased lung cancer cell proliferation, migration, invasion, and sphere formation. We discovered that EV uptake increase the expression of SPP1, CD44, and POSTN genes in lung cancer cells. The data obtained from this study, support to the possibility that circulating EVs play a significant role in the formation of the pre-metastatic niche, eventually leading to metastasis.

Significant Upregulation of HERV-K (HML-2) Transcription Levels in Human Lung Cancer and Cancer Cells

Lung cancer is the second most common cancer worldwide and the leading cause of cancer death in the world. Therefore, there is an urgent need to develop new and effective biomarkers for diagnosis and treatment. Under this circumstance, human endogenous retroviruses (HERVs) were recently introduced as novel biomarkers for cancer diagnosis. This study focused on the correlation between lung cancer and HERV-K (HML-2) transcription levels. At the cellular level, different types of lung cancer cells and human normal lung epithelial cells were used to analyze the transcription levels of the HERV-K (HML-2) gag, pol, and env genes by RT–qPCR. At the level of lung cancer patients, blood samples with background information from 734 lung cancer patients and 96 healthy persons were collected to analyze the transcription levels of HERV-K (HML-2) gag, pol, and env genes. The results showed that the transcriptional levels of the HERV-K (HML-2) gag, pol, and env genes in lung cancer cells and lung cancer patient blood samples were significantly higher than those in the healthy controls, which was also verified by RNAScope ISH technology. In addition, we also found that there was a correlation between the abnormal transcription levels of HERV-K (HML-2) genes in lung cancer patients and the clinicopathological parameters of lung cancer. We also identified the distribution locations of the gag, pol, and env primer sequences on each chromosome and analyzed the function of these loci. In conclusion, HERV-K (HML-2) genes may be a potential biomarker for the diagnosis of lung cancer.

TWIST2 inhibits EMT and induces oxidative stress in lung cancer cells by regulating the FGF21-mediated AMPK/mTOR pathway

Twist related protein 2 (TWIST2) plays an important role in bone development, tumorigenesis, tumour progression and epithelial mesenchymal transition (EMT). At present, there are few reports about the role of TWIST2 in lung cancer, which need to be further explored. Therefore, the purpose of this study is to explore the role and molecular mechanism of TWIST2 in the occurrence and development of lung cancer. The expression of TWIST2 in tissues of patients and cell lines was measured using RT-qPCR and western blotting. MTT and CCK8 assays were used to detect cell proliferation and viability. Western blotting was used to measure the expression of EMT-related proteins, including E-cadherin, N-cadherin, Vimentin and Slug. The results revealed that TWIST2 is lowly expressed in the tissues of lung cancer patients and cell lines. Further studies found that overexpression of TWIST2 significantly induced apoptosis and promoted the expression of E-cadherin, as well as inhibiting the expression of N-cadherin, Vimentin and Slug. More importantly, TWIST2 induced oxidative stress in lung cancer cells. In addition, TWIST2 regulated the FGF21 and AMPK/mTOR signalling pathway, which is involved in the molecular mechanism of the gene in lung cancer cells. We suggest that the mechanism of TWIST2 inhibition of the progression of lung cancer is by regulating the FGF21-mediated AMPK/mTOR signalling pathway.

LEMONGRASS ESSENTIAL OIL SHOWS AN ANTIPROLIFERATIVE EFFECT ON LUNG CANCER CELLS AND ITS ROLE IN EXPRESSION OF P53 GENE

This study was aimed to investigate the antiproliferative effect of lemongrass essential oil (LEO) from Cymbopogon citratus on A549 cell line and their effect on the expression of p53 gene in lung cancer cells. The LEO was extracted and anlaysed using GC-MS analysis, the cytotoxicity of LEO against lung cancer cells was examined using MMT assay and finally the expression of p53 was determined via qRT-PCR. The chromatogram of GC-MS exhibited 14 compounds in ELO, the major one is citral (56.4%). The cytotoxicity of LEO was dose dependent which proliferation of the lung cells were significantly inhibited with increased concentration of essential oil (P≤ 0.01) as well as the IC50 was 29.13 ppm. Furthermore, it showed that cell treated with IC50 of LEO had a significant higher gene expression (P≤ 0.05) in the fold of p53 expression than untreated cells with change fold of 4.47±0.7. In conclusion, C. citratus essential oil have been found to possess antiproliferative activity against lung cancer cells and upregulation of p53 gene which can be considered a promising treatment for lung cancer patients.

Metformin Reduces Histone H3K4me3 at the Promoter Regions of Positive Cell Cycle Regulatory Genes in Lung Cancer Cells.

Simple SummaryTo understand the effect of metformin on epigenetic regulation, we analyzed histone H3 methylation, DNA methylation, and chromatin accessibility in lung cancer cells. Metformin showed little effect on DNA methylation or chromatin accessibility but significantly reduced H3K4me3 levels at the promoters of positive cell cycle regulatory genes. Metformin downregulated H3K4 methyltransferase MLL2 expression and knockdown of MLL2 resulted in suppression of H3K4me3 expression and lung cancer cell proliferation. We further evaluated the clinicopathological significance of MLL2 in tumor and matched normal tissues from 42 non-small cell lung cancer patients. MLL2 overexpression was significantly associated with poor recurrence-free survival in lung adenocarcinoma. Our study facilitates the understanding of the effect of metformin on the regulation of histone H3K4me3 at promoter regions of cell cycle regulatory genes in lung cancer cells, and MLL2 may be a potential therapeutic target for lung cancer therapy.This study aimed at understanding the effect of metformin on histone H3 methylation, DNA methylation, and chromatin accessibility in lung cancer cells. Metformin significantly reduced H3K4me3 level at the promoters of positive cell cycle regulatory genes such as CCNB2, CDK1, CDK6, and E2F8. Eighty-eight genes involved in cell cycle showed reduced H3K4me3 levels in response to metformin, and 27% of them showed mRNA downregulation. Metformin suppressed the expression of H3K4 methyltransferases MLL1, MLL2, and WDR82. The siRNA-mediated knockdown of MLL2 significantly downregulated global H3K4me3 level and inhibited lung cancer cell proliferation. MLL2 overexpression was found in 14 (33%) of 42 NSCLC patients, and a Cox proportional hazards analysis showed that recurrence-free survival of lung adenocarcinoma patients with MLL2 overexpression was approximately 1.32 (95% CI = 1.08–4.72; p = 0.02) times poorer than in those without it. Metformin showed little effect on DNA methylation and chromatin accessibility at the promoter regions of cell cycle regulatory genes. The present study suggests that metformin reduces H3K4me3 levels at the promoters of positive cell cycle regulatory genes through MLL2 downregulation in lung cancer cells. Additionally, MLL2 may be a potential therapeutic target for reducing the recurrence of lung adenocarcinoma.

Cellular processes involved in lung cancer cells exposed to direct current electric field

With the rapid breakthrough of electrochemical treatment of tumors, electric field (EF)-sensitive genes, previously rarely exploited, have become an emerging field recently. Here, we reported our work for the identification of EF-sensitive genes in lung cancer cells. The gene expression profile (GSE33845), in which the human lung cancer CL1-0 cells were treated with a direct current electric field (dcEF) (300 mV/mm) for 2 h, was retrieved from GEO database. Differentially expressed genes (DEGs) were acquired, followed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) and protein-protein interaction (PPI) analysis. Hub genes were acquired and analyzed by various tools including the Human Protein Atlas, Kaplan-Meier analysis, Cytoscape, FunRich, Oncomine and cBioPortal. Subsequently, three-dimensional protein models of hub genes were modeled by Modeller 9.20 and Rosetta 3.9. Finally, a 100 ns molecular dynamics simulation for each hub protein was performed with GROMACS 2018.2. A total of 257 DEGs were acquired and analyzed by GO, KEGG and PPI. Then, 10 hub genes were obtained, and the signal pathway analysis showed that two inflammatory pathways were activated: the FoxO signaling pathway and the AGE-RAGE signaling pathway. The molecular dynamic analysis including RMSD and the radius of gyration hinted that the 3D structures of hub proteins were built. Overall, our work identified EF-sensitive genes in lung cancer cells and identified that the inflammatory state of tumor cells may be involved in the feedback mechanism of lung cancer cells in response to electric field stimulation. In addition, qualified three-dimensional protein models of hub genes were also constructed, which will be helpful in understanding the complex effects of dcEF on human lung cancer CL1-0 cells.

Identification and validation of key genes with prognostic value in non-small-cell lung cancer via integrated bioinformatics analysis.

BackgroundLung cancer is the most common cause of cancer‐related death among all human cancers and the five‐year survival rates are only 23%. The precise molecular mechanisms of non‐small cell lung cancer (NSCLC) are still unknown. The aim of this study was to identify and validate the key genes with prognostic value in lung tumorigenesis.MethodsFour GEO datasets were obtained from the Gene Expression Omnibus (GEO) database. Common differentially expressed genes (DEGs) were selected for Kyoto Encyclopedia of Genes and Genomes pathway analysis and Gene Ontology enrichment analysis. Protein‐protein interaction (PPI) networks were constructed using the STRING database and visualized by Cytoscape software and Molecular Complex Detection (MCODE) were utilized to PPI network to pick out meaningful DEGs. Hub genes, filtered from the CytoHubba, were validated using the Gene Expression Profiling Interactive Analysis database. The expressions and prognostic values of hub genes were carried out through Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan‐Meier plotter. Finally, quantitative PCR and the Oncomine database were used to verify the differences in the expression of hub genes in lung cancer cells and tissues.ResultsA total of 121 DEGs (49 upregulated and 72 downregulated) were identified from four datasets. The PPI network was established with 121 nodes and 588 protein pairs. Finally, AURKA, KIAA0101, CDC20, MKI67, CHEK1, HJURP, and OIP5 were selected by Cytohubba, and they all correlated with worse overall survival (OS) in NSCLC.ConclusionThe results showed that AURKA, KIAA0101, CDC20, MKI67, CHEK1, HJURP, and OIP5 may be critical genes in the development and prognosis of NSCLC.Key pointsOur results indicated that AURKA, KIAA0101, CDC20, MKI67, CHEK1, HJURP, and OIP5 may be critical genes in the development and prognosis of NSCLC. Our methods showed a new way to explore the key genes in cancer development.

Sp1 Mediates the Constitutive Expression and Repression of the PDSS2 Gene in Lung Cancer Cells.

Prenyl diphosphate synthase subunit 2 (PDSS2) is the first key enzyme in the CoQ10 biosynthesis pathway, and contributes to various metabolic and nephritic diseases. It has been reported that PDSS2 is downregulated in several types of tumors and acts as a potential tumor suppressor gene to inhibit the proliferation and migration of cancer cells. However, the regulatory mechanism of PDSS2 expression remains elusive. In the present study, we first identified and characterized the PDSS2 promoter region. We established four different luciferase reporter constructs which mainly cover the 2 kb region upstream of the PDSS2 gene transcription initiation site. Series luciferase reporter assay demonstrated that all four constructs have prominent promoter activity, and the core promoter of PDSS2 is mainly located within the 202 bp region near its transcription initiation site. Transcription factor binding site analysis revealed that the PDSS2 promoter contains binding sites for canonical transcription factors such as Sp1 and GATA-1. Overexpression of Sp1 significantly inhibited PDSS2 promoter activity, as well as its endogenous expression, at both mRNA and protein levels in lung cancer cells. Site-directed mutagenesis assay further confirmed that the Sp1 binding sites are essential for proximal prompter activity of PDSS2. Consistently, a selective Sp1 inhibitor, mithramycin A, treatment repressed the PDSS2 promoter activity, as well as its endogenous expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binds to the PDSS2 promoter in vivo. Of note, the expression of Sp1 and PDSS2 are negatively correlated, and higher Sp1 expression with low PDSS2 expression is significantly associated with poor prognosis in lung cancer. Taken together, our results strongly suggest the essential role of Sp1 in maintaining the basic constitutive expression of PDSS2, and the pathogenic implication of Sp1-mediated PDSS2 transcriptional repression in lung cancer cells.

Simultaneous Detection of the T790M and L858R Mutations in the EGFR Gene by Oligoribonucleotide Interference-PCR.

A de novo single-nucleotide mutation in the EGFR gene can cause the development of lung cancer. EGFR tyrosine kinase inhibitors (EGFR-TKIs) are used for clinical treatment of such lung cancers, but acquired resistance often mitigates their efficacy. Accordingly, monitoring of de novo and acquired nucleotide mutations is essential for clinical treatment of lung cancers with EGFR-TKIs. Previously, we reported that oligoribonucleotide interference-PCR (ORNi-PCR) can accurately and cost-effectively detect single-nucleotide mutations. In this study, we applied ORNi-PCR to simultaneous detection of the de novo L858R and acquired T790M mutations in the EGFR gene in lung cancer cells. First, we established optimal experimental conditions for ORNi-PCR to simultaneously detect the two single-nucleotide mutations in genomic DNA from lung cancer cells. The conditions we established could also be used for ORNi-PCR using complementary DNA reverse-transcribed from extracted RNA. We found that ORNi-PCR could detect lung cancer cells possessing both single-nucleotide mutations among a large number of cells harboring wild-type sequences, even when the cancer cells constituted less than ~0.2% of all cells. Our findings demonstrate that ORNi-PCR can simultaneously detect multiple single-nucleotide mutations in a gene of interest and might therefore be useful for simultaneous detection of EGFR mutations in clinical examinations.

CA916798 gene expression is associated with multidrug resistance and predicts progression-free survival in patients with lung cancer.

CA916798 has been identified as a novel multidrug resistance gene in lung cancer cells. However, the expression patterns of CA916798 in tumor tissues prior and subsequent to chemotherapy remain unclear. In the present study, CA916798 expression levels in tumor tissues prior and subsequent to chemotherapy were detected by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry analysis. The prognostic significance of CA916798 expression in tumor tissues was explored by Kaplan-Meier survival analysis and Cox proportional hazards regression analysis. The messenger RNA (mRNA) and protein expression levels of CA916798 in tumor tissues were downregulated post-chemotherapy in chemotherapy-sensitive patients with lung cancer, but not in chemotherapy-resistant patients. Downregulation of CA916798 mRNA and protein expression post-chemotherapy was significantly associated with improved progression-free survival time. The findings from the present study suggest that platinum-based chemotherapy may induce the expression of CA916798, and CA916798 may be a promising biomarker to predict chemotherapy resistance and improve therapies for patients with lung cancer.

NDR2 kinase contributes to cell invasion and cytokinesis defects induced by the inactivation of RASSF1A tumor-suppressor gene in lung cancer cells

BackgroundRASSF1A, a tumor suppressor gene, is frequently inactivated in lung cancer leading to a YAP-dependent epithelial-mesenchymal transition (EMT). Such effects are partly due to the inactivation of the anti-migratory RhoB GTPase via the inhibitory phosphorylation of GEF-H1, the GDP/GTP exchange factor for RhoB. However, the kinase responsible for RhoB/GEF-H1 inactivation in RASSF1A-depleted cells remained unknown.MethodsNDR1/2 inactivation by siRNA or shRNA effects on epithelial-mesenchymal transition, invasion, xenograft formation and growth in SCID−/− Beige mice, apoptosis, proliferation, cytokinesis, YAP/TAZ activation were investigated upon RASSF1A loss in human bronchial epithelial cells (HBEC).ResultsWe demonstrate here that depletion of the YAP-kinases NDR1/2 reverts migration and metastatic properties upon RASSF1A loss in HBEC. We show that NDR2 interacts directly with GEF-H1 (which contains the NDR phosphorylation consensus motif HXRXXS/T), leading to GEF-H1 phosphorylation. We further report that the RASSF1A/NDR2/GEF-H1/RhoB/YAP axis is involved in proper cytokinesis in human bronchial cells, since chromosome proper segregation are NDR-dependent upon RASSF1A or GEF-H1 loss in HBEC.ConclusionTo summarize, our data support a model in which, upon RASSF1A silencing, NDR2 gets activated, phosphorylates and inactivates GEF-H1, leading to RhoB inactivation. This cascade induced by RASSF1A loss in bronchial cells is responsible for metastasis properties, YAP activation and cytokinesis defects.

MCP-1/CCL2 Mediated by Autocrine Loop of PDGF-BB Promotes Invasion of Lung Cancer Cell by Recruitment of Macrophages Via CCL2–CCR2 Axis

Platelet-derived growth factor-BB (PDGF-BB) is recognized as a potential player in a paracrine manner in tumor stroma development. PDGF-BB has an autocrine growth function in lung cancer cells; however, the mechanism in nonsmall-cell lung cancer is not fully understood. In this study, we report that PDGF-BB increased monocyte chemoattractant protein-1 (MCP-1)-dependent macrophage recruitment and that expression of metastatic genes increased in A549 cells cocultured with RAW 264.7 macrophages. Similar to exogenous PDGF-BB, PDGF-BB might have a self-stimulation in invasion of cancer cells during reciprocal activation of cancer cells and tumor-associated macrophages through secretion of soluble proteins. Also, we found that PDGF-BB upregulates both mRNA and protein level of MCP-1 expression in human A549 cells through mitogen-activated protein kinases and phosphatidylinositol 3-kinase/Akt cell signaling pathways, binding NFκB to MCP-1 promoter site and PDGF-Rβ as critical receptor. These results suggested that MCP-1/chemokine (C-C motif) ligand 2 (CCL2) expression mediated by the autocrine loop of PDGF-BB enhanced recruitment of macrophages through CCL2-CCR2 axis, which could ultimately increase expression of metastatic genes in lung cancer cells, finally promoting invasive potential of cancer cells.

Functional Gene Knockout of NRF2 Increases Chemosensitivity of Human Lung Cancer A549 Cells In Vitro and in a Xenograft Mouse Model

Recent studies point to the evolution of drug resistance in lung cancer as being centered, at least in part, on the upregulation of various genes involved in controlling efflux or drug inactivation. Among the most important of these genes is Nuclear Factor Erythroid 2-Related Factor (NRF2), considered the master regulator of 100–200 target genes involved in cellular responses to oxidative and/or electrophilic stress. With increased focus on the development of combinatorial approaches for cancer treatment, we utilized CRISPR/Cas9 to disable the NRF2 gene in lung cancer cells by disrupting the NRF2 nuclear export signal (NES) domain; phenotypically, the protein is largely blocked from transiting into the nucleus after translation. In tissue culture, cells with this gene knockout were found to have a reduced proliferation phenotype and are more sensitive to chemotherapeutic agents, such as cisplatin and carboplatin. These observations were confirmed in xenograft mouse models wherein the homozygous knockout cells proliferate at a slower rate than the wild-type cells, even in the absence of drug treatment. Tumor growth was arrested for a period of 16 days, with a dramatic decrease in tumor volume being observed in samples receiving the combined action of CRISPR-directed gene editing and chemotherapy.

Altered expression of cellular proliferation, apoptosis and the cell cycle-related genes in lung cancer cells with acquired resistance to EGFR tyrosine kinase inhibitors.

Non-small cell lung cancers harboring somatic gain-of-function mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain respond well to treatment with EGFR tyrosine kinase inhibitors (TKIs) including gefitinib and erlotinib. However, all patients who experience a marked improvement with these drugs eventually develop disease progression due to the acquisition of drug resistance. Approximately half of the cases with acquired resistance to EGFR TKIs can be accounted for by a second-site mutation in exon 20 of the EGFR kinase domain (T790M). However, the changes of gene expression involved in EGFR TKI resistance due to the T790M mutation remain poorly defined. The present study established lung cancer cell lines that were resistant to gefitinib or erlotinib, and these cell lines were verified to contain the EGFR T790M mutation. The differential expression of genes associated with acquired resistance was verified in the present study by mRNA microarray analysis. Among the genes whose expression was significantly altered, genes whose expression was altered in gefitinib- and erlotinib-resistant cells were focused on. Notably, a total of 1,617 genes were identified as being differentially expressed in gefitinib- and erlotinib-resistant cells. Indeed, Gene ontology analysis revealed altered expression of genes involved in the regulation of cellular proliferation, apoptosis, and the cell cycle in EGFR TKI-resistant cells. The present results demonstrate distinctive gene expression patterns of EGFR TKI-resistant lung cancer cells with the EGFR T790M mutation. The present study can provide key insights into gene expression profiles involved in conferring resistance to EGFR TKI therapy in lung cancer cells.

Identification and characterization of RASSF1C piRNA target genes in lung cancer cells.

RASSF1C up-regulates important genes involved in lung cancer cell growth, including a stem cell self-renewal gene, piwil1. In this article, we report the identification of small noncoding PIWI-interacting RNAs (piRNAs) in lung cancer cells over-expressing RASSF1C. A piRNA microarray screen was performed using RNA isolated from the lung cancer cell line H1299 stably over-expressing RASSF1C and corresponding control. The piRNA microarray screen identified several piRNAs that are regulated by RASSF1C and we have validated the expression of two up-regulated piRNAs (piR-34871 and piR-52200) and two down-regulated piRNAs (piR-35127 and piR-46545) in lung cancer cells with silenced and over-expressed RASSF1C using RT-PCR. We also assessed the expression of these four piRNAs in lung tumor and matched normal tissues (n = 12). We found that piR-34871 and piR-52200 were up-regulated in 58% and 50%, respectively; while piR-35127 and piR-46545 were down-regulated in 50% in lung tumor tissues tested. The expression of piR-35127 was inversely correlated with RASSF1C expression in 10/12 tumor tissues. Over-expression of piR-35127 and piR-46545 and knock-down of piR-34871 and piR-52200 significantly reduced normal lung and breast epithelial cell proliferation and cell colony formation as well as proliferation of lung cancer cell lines (A549 and H1299) and breast cancer cell lines (Hs578T and MDA-MB-231). This suggests that these novel piRNAs may potentially be involved in regulating lung cell transformation and tumorigenesis. RASSF1C may potentially modulate the expression of its piRNA target genes through attenuation of the AMPK pathway, as over-expression of RASSF1C resulted in reduction of p-AMPK, p21, and p27 protein levels.

Hypoxia promotes mitochondrial glutamine metabolism through HIF1α-GDH pathway in human lung cancer cells

Drug-resistance is common in human lung cancer therapy. Hypoxia remarkably contributes to drug-resistance in lung cancer but the underlying mechanism remains elusive. Here we demonstrate that hypoxia-induced glutamine metabolism is involved in drug resistance in lung cancer cells. Hypoxia increases glutamine up-take, glutamate to α-ketoglutarate flux and the generation of ATP in lung cancer cells by up-regulating the expression of glutamate dehydrogenase (GDH). Hypoxia-induced expression of GDH relies on the up-regulation of HIF1α but not HIF2α. HIF1α binds the promoter of GDH and promotes the transcription of GDH gene in lung cancer cells. Finally, we show that GDH represses cisplatin-induced cell apoptosis and repression of colony formation, indicating that GDH contributes to drug-resistance in lung cancer cells. In conclusion, HIF1α-GDH pathway regulates glutamine metabolism and ATP production upon hypoxia stress and contributes to drug-resistance in human lung cancer cells.

Peperomin E reactivates silenced tumor suppressor genes in lung cancer cells by inhibition of DNA methyltransferase.

Advanced lung cancer has poor prognosis owing to its low sensitivity to current chemotherapy agents. Therefore, discovery of new therapeutic agents is urgently needed. In this study, we investigated the antitumor effects of peperomin E, a secolignan isolated from Peperomia dindygulensis, a frequently used Chinese folk medicine for lung cancer treatment. The results indicate that peperomin E has antiproliferative effects, promoting apoptosis and cell cycle arrest in non‐small‐cell lung cancer (NSCLC) cell lines in a dose‐dependent manner, while showing lower toxicity against normal human lung epidermal cells. Peperomin E inhibited tumor growth in A549 xenograft BALB/c nude mice without significant secondary adverse effects, indicating that it may be safely used to treat NSCLC. Furthermore, the mechanisms underlying the anticancer effects of peperomin E have been investigated. Using an in silico target fishing method, we observed that peperomin E directly interacts with the active domain of DNA methyltransferase 1 (DNMT1), potentially affecting its genome methylation activity. Subsequent experiments verified that peperomin E decreased DNMT1 activity and expression, thereby decreasing global methylation and reactivating the epigenetically silenced tumor suppressor genes including RASSF1A,APC,RUNX3, and p16INK4, which in turn activates their mediated pro‐apoptotic and cell cycle regulatory signaling pathways in lung cancer cells. The observations herein report for the first time that peperomin E is a potential chemotherapeutic agent for NSCLC. The anticancer effects of peperomin E may be partly attributable to its ability to demethylate and reactivate methylation‐silenced tumor suppressor genes through direct inhibition of the activity and expression of DNMT1.