Integrin beta1 over-expression associates with resistance to tyrosine kinase inhibitor gefitinib in non-small cell lung cancer

The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) such as gefitinib and erlotinib have been widely used in treating patients with advanced non-small cell lung cancer (NSCLC). However, acquired resistance to EGFR TKI almost occurs in every patient eventually. To identify its potential mechanism, we established a human NSCLC cell line PC9/AB2 which was 576-fold decrease in gefitinib sensitivity compared with its parental PC9 cell lines. No EGFR-T790M mutation or abnormal expression of c-Met protein was found in PC9/AB2 cells. Over-expression of integrin β1 was found, accompanied with increase of the cells' adhesion and migration. To further confirm the role of integrin β1 in gefitinib acquired resistance, we transferred its siRNA-expressing plasmid and its whole cDNA expressing plasmid into PC9/AB2 and into PC9 cells, respectively. The sensitivity of NSCLC cells to gefitinib was negatively correlated with integrin β1 expression levels. All these data suggest that up-regulation of integrin β1 might be an important factor for gefitinib resistance in NSCLC cell line PC9/AB2.     

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.     

Prognostic value of S-100 immunostaining in tumour cells of non-small cell lung cancer

S-100 protein expression is present in various malignant tissues, yet its prognostic relevance is debatable. The aim was to assess in non-small cell lung cancer (NSCLC) patients’ prognostic value of S-100 protein considered alone or in relation with other variables. Tumour samples taken from 86 NSCLC patients during resection were assayed for S-100 protein expression with the use of polyclonal DAKO ZO311 antibody. S-100 expression was found in 32 cases (37%). Positive staining was not correlated with clinical characteristics including age, sex, pathology type of tumour, stage and cigarette smoking. There was a tendency for simultaneous expression of S-100 and P53 protein (p=0.06). A median survival rate for the entire group was 2.3 years (95% CI, 0.9–3.6 years). The median and 5-year survival of patients with positive staining for S-100 protein was 1.5 years and 25%, respectively, compared with 3.0 years and 35%, respectively, in the S-100 negative group (p=0.17). In the final model of a multivariate analysis, S-100 protein expression in tumour cells was associated with significantly decreased survival (p=0.005). S-100 protein expression in tumour cells seems to be an independent predictor of poor prognosis in NSCLC patients.     

p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells

This study examined the effects of p53 gene status on DNA damage-induced cell death and chemosensitivity to various chemotherapeutic agents in non-small cell lung cancer (NSCLC) cells. A mutant p53 gene was introduced into cells carrying the wild-type p53 gene and also vice versa to introduce the wild-type p53 gene into cells carrying the mutant p53 gene. Chemosensitivity and DNA damage-induced apoptosis in these cells were then examined. This study included five cell lines, NCI-H1437, NCI-H727, NCI-H441 and NCI-H1299 which carry a mutant p53 gene and NCI-H460 which carries a wild-type p53 gene. Mutant p53-carrying cells were transfected with the wild-type p53 gene, while mutant p53 genes were introduced into NCI-H460 cells. These p53 genes were individually mutated at amino acid residues 143, 175, 248 and 273. The representative cell line NCI-H1437 cells transfected with wild-type p53 gene (H1437/wtp53) showed a dramatic increase in susceptibility to three anticancer agents (7-fold to cisplatin, 21-fold to etoposide, and 20-fold to camptothecin) compared to untransfected or neotransfected H1437 cells. An increase in chemosensitivity was also observed in wild-type p53 transfectants of H727, H441, H1299 cells. The results of chemosensitivity were consistent with the observations on apoptotic cell death. H1437/wtp53 cells, but not H1437 parental cells, exhibited a characteristic feature of apoptotic cell death that generated oligonucleosomal-sized DNA fragments. In contrast, loss of chemosensitivity and lack of p53-mediated DNA degradation in response to anticancer agents were observed in H460 cells transfected with mutant p53. These observations suggest that the increase in chemosensitivity was attributable to wild-type p53 mediation of the process of apoptosis. In addition, our results also suggest that p53 gene status modulates the extent of chemosensitivity and the induction of apoptosis by different anticancer agents in NSCLC cells.     

Prognostic value of S-100 immunostaining in tumour cells of non-small cell lung cancer

S-100 protein expression is present in various malignant tissues, yet its prognostic relevance is debatable. The aim was to assess in non-small cell lung cancer (NSCLC) patients' prognostic value of S-100 protein considered alone or in relation with other variables. Tumour samples taken from 86 NSCLC patients during resection were assayed for S-100 protein expression with the use of polyclonal DAKO ZO311 antibody. S-100 expression was found in 32 cases (37%). Positive staining was not correlated with clinical characteristics including age, sex, pathology type of tumour, stage and cigarette smoking. There was a tendency for simultaneous expression of S-100 and P53 protein (p=0.06). A median survival rate for the entire group was 2.3 years (95% CI, 0.9-3.6 years). The median and 5-year survival of patients with positive staining for S-100 protein was 1.5 years and 25%, respectively, compared with 3.0 years and 35%, respectively, in the S-100 negative group (p=0.17). In the final model of a multivariate analysis, S-100 protein expression in tumour cells was associated with significantly decreased survival (p=0.005). S-100 protein expression in tumour cells seems to be an independent predictor of poor prognosis in NSCLC patients.     

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.     

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.     

PRIMA-1 cytotoxicity correlates with nucleolar localization and degradation of mutant p53 in breast cancer cells

PRIMA-1 has been identified as a compound that restores the transactivation function to mutant p53 and induces apoptosis in cells expressing mutant p53. Studies on subcellular distribution of the mutant p53 protein upon treatment with PRIMA-1^Met, a methylated form of PRIMA-1, have suggested that redistribution of mutant p53 to nucleoli may play a role in PRIMA-1 induced apoptosis. Here, we specifically investigated the influence of PRIMA-1 on cellular localization of mutated p53-R280K endogenously expressed in tumour cells. By using immunofluorescence staining, we found a strong nucleolar redistribution of mutant p53 following PRIMA-1 treatment. This subcellular localization was associated to p53 degradation via ubiquitylation. When cells were treated with adriamycin, neither nucleolar redistribution nor mutant p53 down modulation and degradation were observed. Interestingly, cells where p53-R280K was silenced were more sensitive to PRIMA-1 than the parental ones. These results indicate that in some cellular context, the cell sensitivity to PRIMA-1 could depend on the abolition of a gain-of-function activity of the mutated p53, through a protein degradation pathway specifically induced by this compound.     

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.     

Programmed cell death ligand-1 expression and survival in a cohort of patients with non-small cell lung cancer receiving first-line through third-line therapy in Denmark

BackgroundPD-L1 expression on tumor cells (TCs) or immune cells (ICs) may be used as a prognostic marker for survival in patients with NSCLC. We characterized PD-L1 expression on TCs or ICs in a patient cohort with NSCLC to determine associations between PD-L1 expression and overall survival (OS), according to EGFR and KRAS mutation status.MethodsDanish patients aged >18 years diagnosed with NSCLC before 2014 on first- (N = 491), second- (N = 368), or third-line (N = 498) therapy were included. Data were extracted from population-based medical registries. Tumor samples from pathology archives were tested for biomarkers. High PD-L1 expression was defined as expression on ≥25 % of TCs or ICs based on first diagnostic biopsy or surgical resection. KRAS and EGFR mutation status were tested using PCR-based assays. Cox regression analysis was used to compute adjusted HRs and associated 95 % CIs.ResultsPD-L1 TC and IC ≥ 25 % were observed in 24.3 %–31.0 % and 11.7–14.7 % of patients, respectively. EGFR and KRAS mutations were detected in 4.7 %–8.8 % and 26.5 %–30.7 % of patients, respectively. PD-L1 TC ≥ 25 % was not associated with survival advantage in first- (HR = 0.96, 95 % CI: 0.75–1.22), second- (1.08, 0.81–1.42), or third-line (0.94, 0.74–1.20) therapy. PD-L1 IC ≥ 25 % was associated with survival advantage in second-line (HR = 0.56, 95 % CI: 0.36–0.86) and third-line (0.69, 0.49–0.97) but not first-line (1.00, 0.70–1.41) therapy.ConclusionNo association was observed between PD-L1 TC ≥ 25 % and OS in any therapy line. PD-L1 IC ≥ 25 % may confer survival benefit among some patients who reach second-line therapy.     

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.     

Ring finger protein 19A is overexpressed in non-small cell lung cancer and mediates p53 ubiquitin-degradation to promote cancer growth

The expression pattern, biological functions and the related mechanisms of the ring finger protein 19A (RNF19A) in non-small cell lung cancer (NSCLC) remain poorly understood. This study aimed to explore the role of RNF19A, as well as the underlying potential mechanism, in the development of NSCLC. Here, we found that RNF19A was overexpressed in NSCLC tissues, and RNF19A expression in NSCLC tissue samples was associated with NSCLC carcinogenesis and poor outcome. RNF19A promoted the proliferation of NSCLC cells and inhibited apoptosis. RNF19A reduced p53, p21 and BAX expression and induced Cyclin D1, CDK4, CDK6 and BCL2 expression. The inhibitory effect of RNF19A knockdown on proliferation was partially rescued by p53 silencing. RNF19A interacted with p53, shortened p53 half-life and mediated p53 ubiquitin-degradation. Collectively, we suggest that RNF19A plays a critical oncogenic role in lung carcinogenesis by disrupting the function of p53. RNF19A may serve as a new biomarker and/or target for NSCLC management.     

Metformin and tenovin‐6 synergistically induces apoptosis through LKB1‐independent SIRT1 down‐regulation in non‐small cell lung cancer cells

Sirtuin 1 (SIRT1) is known to play a role in a variety of tumorigenesis processes by deacetylating histone and non‐histone proteins; however, antitumour effects by suppressing SIRT1 activity in non‐small cell lung cancer (NSCLC) remain unclear. This study was designed to scrutinize clinicopathological significance of SIRT1 in NSCLC and investigate effects of metformin on SIRT1 inhibition. This study also evaluated new possibilities of drug combination using a SIRT1 inhibitor, tenovin‐6, in NSCLC cell lines. It was found that SIRT1 was overexpressed in 300 (62%) of 485 formalin‐fixed paraffin‐embedded NSCLC tissues. Its overexpression was significantly associated with reduced overall survival and poor recurrence‐free survival after adjusted for histology and pathologic stage. Thus, suppression of SIRT1 expression may be a reasonable therapeutic strategy for NSCLC. Metformin in combination with tenovin‐6 was found to be more effective in inhibiting cell growth than either agent alone in NSCLC cell lines with different liver kinase B1 (LKB1) status. In addition, metformin and tenovin‐6 synergistically suppressed SIRT1 expression in NSCLC cells regardless of LKB1 status. The marked reduction in SIRT1 expression by combination of metformin and tenovin‐6 increased acetylation of p53 at lysine 382 and enhanced p53 stability in LKB1‐deficient A549 cells. The combination suppressed SIRT1 promoter activity more effectively than either agent alone by up‐regulating hypermethylation in cancer 1 (HIC1) binding at SIRT1 promoter. Also, suppressed SIRT1 expression by the combination synergistically induced caspase‐3‐dependent apoptosis. The study concluded that metformin with tenovin‐6 may enhance antitumour effects through LKB1‐independent SIRT1 down‐regulation in NSCLC cells.     

Cx31.1 acts as a tumour suppressor in non-small cell lung cancer (NSCLC) cell lines through inhibition of cell proliferation and metastasis

Reduced connexin expression and loss of gap junction function is a characteristic of many cancers, including lung cancer. However, there are little reports about the relation between Cx31.1 and lung cancer. This study was conducted to investigate the effect of Cx31.1 on non-small cell lung cancer (NSCLC). We found that the Cx31.1 was down-regulated in NSCLC cell lines, and the expression levels were reversely related with their metastatic potential. We ectopically expressed Cx31.1 in H1299 NSCLC cell line to examine the influence of Cx31.1 overexpression. The results showed that overexpression of Cx31.1 in H1299 cells reduced cell proliferation, induced a delay in the G(1) phase, inhibited anchorage-independent growth and suppressed cell migration and invasion. The cell cycle delay and cell migration and invasion suppressive effects of Cx31.1 were partially reversed by siRNA targeting mRNA of Cx31.1. Moreover, xenografts of Cx31.1 overexpressing H1299 cells showed reduced tumourigenicity. These results suggested that Cx31.1 has tumour-suppressive properties. Further investigation indicated that cyclin D3 may be responsible for Cx31.1-induced G(1) phase delay. Importantly, Cx31.1 increased the expression of epithelial markers, such as cytokeratin 18, and decreased expression of mesenchymal markers, such as vimentin, indicating a Cx31.1-mediated partial shift from a mesenchymal towards an epithelial phenotype. We concluded that Cx31.1 inhibit the malignant properties of NSCLC cell lines, the mechanisms under this may include regulation of EMT.