Expression of Cripto‐1 predicts poor prognosis in stage I non‐small cell lung cancer

Cripto‐1 (CR‐1) is related to the biological behaviour and prognosis of carcinomas. The purpose of this study was to investigate the significance of CR‐1 expression in surgically resected stage I non‐small cell lung cancer (NSCLC). One hundred and forty‐eight patients with completely resected stage I NSCLC and available clinical follow‐up data were assessed. The protein expression of CR‐1 in the tumours was detected by immunohistochemistry. CR‐1 was highly expressed in 64 of 148 tumours. Among patients with high CR‐1 expression, progression‐free survival and overall survival rate were significantly lower than those of patients with low CR‐1 levels (P = .013 and P = .019, respectively). The incidence of distant metastasis in patients with high CR‐1 expression was significantly higher than that of in patients with low CR‐1 expression (57.13% vs 21.43%, P = .001). The results of the multivariate analysis confirmed that a high CR‐1 was a significant factor for poor prognosis. In conclusion, CR‐1 could be a useful prognostic factor in patients with stage I NSCLC, likely as an indicator of the metastatic propensity of the tumour.     

microRNA‐145‐3p inhibits non‐small cell lung cancer cell migration and invasion by targeting PDK1 via the mTOR signaling pathway

The mammalian target of rapamycin (mTOR) pathway is dysregulated in more than 50% of all human malignancies and is a major target in cancer treatment. In this study, we explored the underlying mechanism involving microRNA‐145‐3p (miR‐145‐3p) in the development and progression of non‐small cell lung cancer (NSCLC) by targeting PDK1 via the mTOR signaling pathway. NSCLC tissues and adjacent normal tissues were obtained from 83 NSCLC patients. miR‐145‐3p, PDK1, and mTOR levels were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) and immunohistochemistry. Human NSCLC cell lines A549 and H1299 were transfected with miR‐145‐3p and siPDK1 to confirm the effect of miR‐145‐3p and PDK1 on NSCLC cells in vitro. Cell growth was evaluated by a CCK8 assay. Cell motility and chemotaxis analysis were determined by the scratch test and chemotaxis assay, respectively. The protein levels of PDK1 and mTOR were measured using the western blotting. Results showed lower level of miR‐145‐3p and higher levels of PDK1 and mTOR in NSCLC tissues compared to the adjacent normal tissues. In vitro results showed that cell growth, cell motility, and chemotaxis were all inhibited in cells transfected with miR‐145‐3p and those transfected with siPDK. Additionally, dual luciferase reporter gene assay helped confirmed that PDK1 is a target of miR‐145. Finally, levels of PDK1, mTOR, and phosphorylated‐mTOR were lower in cells transfected with miR‐145‐3p as well as those with siPDK1. These findings indicate that miR‐145‐3p may inhibit cell growth, motility, and chemotaxis in NSCLC by targeting PDK1 through suppressing the mTOR pathway.     

CiRS‐7 targeting miR‐7 modulates the progression of non‐small cell lung cancer in a manner dependent on NF‐κB signalling

The purpose of this study was to figure out the effect of ciRS‐7/miR‐7/NF‐κB axis on the development of non‐small cell lung cancer (NSCLC). In response, the expressions of ciRS‐7, miR‐7 and NF‐κB subunit (ie RELA) within NSCLC tissues and cell lines were determined with real‐time polymerase chain reaction (RT‐PCR) and Western blot. Moreover, the NSCLC cells were transfected with pcDNA3‐ciRS‐7‐ir, pcDNA3‐ciRS‐7, miR‐NC and miR‐7 mimic. Furthermore, the targeted relationships between ciRS‐7 and miR‐7, as well as between miR‐7 and RELA, were confirmed by luciferase reporter assay. The proliferation, migration and apoptosis of NSCLC cells were, successively, measured using CCK‐8 assay, wound‐healing assay and flow cytometry test. Consequently, ciRS‐7, miR‐7, histopathological grade, lymph node metastasis and histopathological stage could independently predict the prognosis of patients with NSCLC (all P < .05). Moreover, remarkably up‐regulated ciRS‐7 and RELA expressions, as along with down‐regulated miR‐7 expressions, were found within NSCLC tissues and cells in comparison with normal ones (P < .05). Besides, overexpressed ciRS‐7 and underexpressed miR‐7 were correlated with increased proliferation, migration and invasion, yet reduced apoptosis rate of NSCLC cells (P < .05). More than that, ciRS‐7 specifically targeted miR‐7 to reduce its expressions (P < .05). Ultimately, the NSCLC cells within miR‐7 + RELA group were observed with superior proliferative, migratory and invasive capabilities than those within miR‐7 group (P < .05), and RELA expression was also significantly modified by both ciRS‐7 and miR‐7 (P < .05). In conclusion, the ciRS‐7/miR‐7/NF‐kB axis could exert pronounced impacts on the proliferation, migration, invasion and apoptosis of NSCLC cells.     

Clinical relevance and functional implications for human leucocyte antigen‐g expression in non‐small‐cell lung cancer

HLA‐G has been documented both in establishment of anti‐tumour immune responses and in tumour evasion. To investigate the clinical relevance of HLA‐G in non‐small‐cell lung cancer (NSCLC), expression status and potential significance of HLA‐G in NSCLC were analysed. In this study, HLA‐G expression in 101 NSCLC primary lesions and plasma soluble HLA‐G (sHLA‐G) from 91 patients were analysed with immunohistochemistry and ELISA, respectively. Correlations between HLA‐G status and various clinical parameters including survival time were evaluated. Meanwhile, functional analysis of transfected cell surface HLA‐G expression and plasma sHLA‐G form NSCLC patients on natural killer (NK) cell cytolysis were performed. Data revealed that HLA‐G was expressed in 41.6% (42/101) NSCLC primary lesions, while undetectable in adjacent normal lung tissues. HLA‐G expression in NSCLC lesions was strongly correlated to disease stages (P= 0.002). Plasma sHLA‐G from NSCLC patients was markedly higher than that in normal controls (P= 0.004), which was significantly associated with the disease stages (I versus IV, P= 0.025; II versus IV, P= 0.029). Patient plasma sHLA‐G level (≥median, 32.0 U/ml) had a significantly shorter survival time (P= 0.044); however, no similar significance was observed for the lesion HLA‐G expression. In vitro data showed that both cell surface HLA‐G and patient plasma sHLA‐G could dramatically decrease the NK cell cytolysis. Our findings indicated that both lesion HLA‐G expression and plasma sHLA‐G in NSCLC is related to the disease stage and can exert immunosuppression to the NK cell cytolysis, indicating that HLA‐G could be a potential therapeutic target. Moreover, plasma sHLA‐G in NSCLC patients could be used as a prognosis factor for NSCLC.     

Dickkopf‐1‐promoted vasculogenic mimicry in non‐small cell lung cancer is associated with EMT and development of a cancer stem‐like cell phenotype

To characterize the contributions of Dickkopf‐1 (DKK1) towards the induction of vasculogenic mimicry (VM) in non‐small cell lung cancer (NSCLC), we evaluated cohorts of primary tumours, performed in vitro functional studies and generated xenograft mouse models. Vasculogenic mimicry was observed in 28 of 205 NSCLC tumours, while DKK1 was detected in 133 cases. Notably, DKK1 was positively associated with VM. Statistical analysis showed that VM and DKK1 were both related to aggressive clinical course and thus were indicators of a poor prognosis. Moreover, expression of epithelial‐mesenchymal transition (EMT)‐related proteins (vimentin, Slug, and Twist), cancer stem‐like cell (CSC)‐related proteins (nestin and CD44), VM‐related proteins (MMP2, MMP9, and vascular endothelial‐cadherin), and β‐catenin‐nu were all elevated in VM‐positive and DKK1‐positive tumours, whereas the epithelial marker (E‐cadherin) was reduced in the VM‐positive and DKK1‐positive groups. Non‐small cell lung cancer cell lines with overexpressed or silenced DKK1 highlighted its role in the restoration of mesenchymal phenotypes and development of CSC characteristics. Moreover, DKK1 significantly promotes NSCLC tumour cells to migrate, invade and proliferate. In vivo animal studies demonstrated that DKK1 enhances the growth of transplanted human tumours cells, as well as increased VM formation, mesenthymal phenotypes and CSC properties. Our results suggest that DKK1 can promote VM formation via induction of the expression of EMT and CSC‐related proteins. As such, we feel that DKK1 may represent a novel target of NSCLC therapy.     

Phospholipase C ε‐1 inhibits p53 expression in lung cancer

The pathogenesis of lung cancer is to be further investigated. Recent reports indicate that phospholipase C ε‐1 (PLCE1) is a critical molecule involved in tumour growth. This study aims to investigate the role of PLCE1 in the regulation of apoptosis in lung cancer cells. In this study, the surgically removed non‐small‐cell lung cancer (NSCLC) tissue was collected from 36 patients. Single NSCLC cells were prepared from the tissue, in which immune cells of CD3⁺, CD11c⁺, CD19⁺, CD68⁺ and CD14⁺ were eliminated by magnetic cell sorting. The expression of PLCE1 and p53 was assessed by quantitative real‐time polymerase chain reaction and Western blotting. Apoptosis of NSCLC cells was analysed by flow cytometry. The results showed that, in cultured NSCLC cells, high levels of PLCE1 and low levels p53 were detected; the two molecules showed a negative correlation (p < 0.01). The addition of anti‐PLCE1 antibody increased the expression of p53 in NSCLC cells, which increased the frequency of apoptotic NSCLC cells. We conclude that NSCLC cells express high levels of PLCE1, which suppresses the expression of p53 in NSCLC cells. PLCE1 can be a therapeutic target of NSCLC. Copyright © 2013 John Wiley & Sons, Ltd.     

Long non‐coding RNAs in non‐small cell lung cancer as biomarkers and therapeutic targets

Lung cancer‐associated mortality is the most common cause of cancer death worldwide. Non‐coding RNAs (ncRNAs), with no protein‐coding ability, have multiple biological roles. Long non‐coding RNAs (lncRNAs) are a recently characterized class of ncRNAs that are over 200 nucleotides in length. Many lncRNAs have the ability of facilitating or inhibiting the development and progression of tumours, including non‐small cell lung cancer (NSCLC). Because of their fundamental roles in regulating gene expression, along with their involvement in the biological mechanisms underlying tumourigenesis, they are a promising class of tissue‐ and/or blood‐based cancer biomarkers. In this review, we highlight the emerging roles of lncRNAs in NSCLC, and discuss their potential clinical applications as diagnostic and prognostic markers and as therapeutic targets.     

LncRNA DUXAP9‐206 directly binds with Cbl‐b to augment EGFR signaling and promotes non‐small cell lung cancer progression

Long noncoding RNAs (lncRNAs) are involved in the pathology of various tumours, including non‐small cell lung cancer (NSCLC). However, the underlying molecular mechanisms of their specific association with NSCLC have not been fully elucidated. Here, we report that a cytoplasmic lncRNA, DUXAP9‐206 is overexpressed in NSCLC cells and closely related to NSCLC clinical features and poor patient survival. We reveal that DUXAP9‐206 induced NSCLC cell proliferation and metastasis by directly interacting with Cbl‐b, an E3 ubiquitin ligase, and reducing the degradation of epidermal growth factor receptor (EGFR) and thereby augmenting EGFR signaling in NSCLC. Notably, correlations between DUXAP9‐206 and activated EGFR signaling were also validated in NSCLC patient specimens. Collectively, our findings reveal the novel molecular mechanisms of DUXAP9‐206 in mediating the progression of NSCLC and DUXAP9‐206 may serve as a potential target for NSCLC therapy.     

Classification of early‐stage non‐small cell lung cancer by weighing gene expression profiles with connectivity information

Pathway‐based feature selection algorithms, which utilize biological information contained in pathways to guide which features/genes should be selected, have evolved quickly and become widespread in the field of bioinformatics. Based on how the pathway information is incorporated, we classify pathway‐based feature selection algorithms into three major categories—penalty, stepwise forward, and weighting. Compared to the first two categories, the weighting methods have been underutilized even though they are usually the simplest ones. In this article, we constructed three different genes’ connectivity information‐based weights for each gene and then conducted feature selection upon the resulting weighted gene expression profiles. Using both simulations and a real‐world application, we have demonstrated that when the data‐driven connectivity information constructed from the data of specific disease under study is considered, the resulting weighted gene expression profiles slightly outperform the original expression profiles. In summary, a big challenge faced by the weighting method is how to estimate pathway knowledge‐based weights more accurately and precisely. Only until the issue is conquered successfully will wide utilization of the weighting methods be impossible.     

Neuropilin 1 expression correlates with the Radio‐resistance of human non‐small‐cell lung cancer cells

The purpose of this study was to determine the correlation between over‐expression of the neuropilin 1 (NRP1) gene and growth, survival, and radio‐sensitivity of non‐small cell lung carcinoma (NSCLC) cells. 3‐[4,5‐dimethylthylthiazol‐2‐yl]‐2,5 diphenyltetrazolium broide (MTT) and colony assays were then performed to determine the effect of NRP1 inhibition on the in vitro growth of NSCLC cells. The Annexin V‐Fluorescein Isothiocyanate (FITC) apoptosis detection assay was performed to analyse the effect of NRP1 enhancement on apoptosis of NSCLC cells. Transwell invasion and migration assays were employed to examine the metastatic ability of A549 cells post X‐ray irradiation. In addition, Western blot assays were carried out to detect the protein level of VEGFR2, PI3K and NF‐κB. Finally, to examine the effect of shNRP1 on proliferation and radio‐sensitivity in vivo, a subcutaneous tumour formation assay in nude mice was performed. Microvessel density in tumour tissues was assessed by immunohistochemistry. The stable transfected cell line (shNRP1‐A549) showed a significant reduction in colony‐forming ability and proliferation not only in vitro, but also in vivo. Moreover, shRNA‐mediated NRP1 inhibition also significantly enhanced the radio‐sensitivity of NSCLC cells both in vitro and in vivo. The over‐expression of NRP1 was correlated with growth, survival and radio‐resistance of NSCLC cells via the VEGF‐PI3K‐ NF‐κB pathway, and NRP1 may be a molecular therapeutic target for gene therapy or radio‐sensitization of NSCLC.     

Sanguinarine exhibits antitumor activity via up‐regulation of Fas‐associated factor 1 in non‐small cell lung cancer

Lung cancer is the most common type of malignancy and one of the leading causes of cancer‐related deaths in the world. Non‐small cell lung carcinomas (NSCLC) account for 85% cases of lung cancer. Sanguinarine (SNG) is a benzophenanthridine alkaloid isolated from plants of the Papaveraceae family that possess diverse biological activities. SNG exhibits antitumor effects in several cancer cells. However, the effects of SAN on NSCLC proliferation, invasion, and migration and the mechanisms remain to be clarified. We showed that SNG concentration‐ and time‐dependently decreased the cell proliferation, viability, and induced a marked increase in cell death in A549 cells. SNG inhibited invasion and migration and induced S phase cell cycle arrest and apoptosis. SNG resulted in a significant increase of E‐cadherin expression and a marked decrease of the expression of N‐cadherin, Vimentin, Smad2/3, and Snail and the phosphorylation of Smad2. SNG increased Fas‐associated factor 1 (FAF1) expression and upregulation of FAF1 inhibited cell proliferation, invasion, and migration and induced cell cycle arrest and apoptosis in NSCLC cells. Knockdown of FAF1 suppressed SNG‐induced inhibition of cell proliferation, invasion, and migration and induction of cell cycle arrest and apoptosis in NSCLC cells. SNG also inhibited implanted tumor growth and increased FAF1 expression in tumors in vivo. Our findings highlight FAF1 as a novel therapeutic target and provide a new insight in the potential use of SNG for the inhibition of NSCLC.     

Long non‐coding RNA SNHG15 promotes CDK14 expression via miR‐486 to accelerate non‐small cell lung cancer cells progression and metastasis

Long non‐coding RNAs (lncRNAs) have been validated to play important role in multiple cancers, including non‐small cell lung cancer (NSCLC). In present study, our team investigate the biologic role of SNHG15 in the NSCLC tumorigenesis. LncRNA SNHG15 was significantly upregulated in NSCLC tissue samples and cells, and its overexpression was associated with poor prognosis of NSCLC patients. In vitro, loss‐of‐functional cellular experiments showed that SNHG15 silencing significantly inhibited the proliferation, promoted the apoptosis, and induced the cycle arrest at G0//G1 phase. In vivo, xenograft assay showed that SNHG15 silencing suppressed tumor growth of NSCLC cells. Besides, SNHG15 silencing decreased CDK14 protein expression both in vivo and vitro. Bioinformatics tools and luciferase reporter assay confirmed that miR‐486 both targeted the 3′‐UTR of SNHG15 and CDK14 and was negatively correlated with their expression levels. In summary, our study conclude that the ectopic overexpression of SNHG15 contribute to the NSCLC tumorigenesis by regulating CDK14 protein via sponging miR‐486, providing a novel insight for NSCLC pathogenesis and potential therapeutic strategy for NSCLC patients.