The downstream of tyrosine kinase 7 is reduced in lung cancer and is associated with poor survival of patients with lung cancer

The downstream of tyrosine kinase 7 (DOK7) is an adaptor protein mediating signalling transduction between receptors and intracellular downstream molecules. Reduced expression of DOK7 has been observed in breast cancer. The present study aimed to investigate the role played by DOK7 in lung cancer. The expression of DOK7 at both mRNA and protein levels was evaluated in human lung cancer. A reduced expression of DOK7 transcripts was seen in lung cancers compared with normal lung tissues. Kaplan-Meier analyses showed that the reduced expression of DOK7 was associated with poorer overall survival and progression-free survival of patients with lung cancer. A further western blot analysis revealed a predominant expression of DOK7 isoform 1 (DOK7V1) in normal lung tissues, which was reduced in lung cancer. Forced overexpression of DOK7V1 in lung cancer cell lines, A549 and H3122 resulted in a decrease of in vitro cell proliferation and migration, while adhesion to extracellular matrix was enhanced following the expression. In conclusion, DOK7 was reduced in lung cancer and reduced DOK7 expression was associated with poorer survival. DOK7 isoform 1 plays an inhibitory role on the proliferation and migration of lung cancer cells in which Akt pathway may be involved

Designing a novel high-throughput AlphaLISA assay to quantify plasma NHERF1 as a non-small cell lung cancer biomarker

NHERF1 might play a significant role in biological processes including oncogenic transformation and metastasis. Owing to the lack of highly sensitive and quantitative methods of NHERF1 in human plasma, there have been few reports on the plasma levels of NHERF1 and its correlation with cancer. Here, a novel amplified luminescent proximity homogeneous immunoassay (AlphaLISA) has been developed and validated for the quantification of NHERF1 in human plasma. This assay was based on an AlphaScreen detection technique with two different anti-NHERF1 antibodies coupled to donor and acceptor beads, respectively. The developed AlphaLISA assay was further optimized and validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), precision, recovery, selectivity and interferences. The linear range of NHERF1 in human plasma was 5.00–100 ng mL−1, with an LOD of 2.00 ng mL−1. This AlphaLISA assay has been successfully applied to the quantification of NHERF1 in the plasma from 75 patients with non-small cell lung cancer (NSCLC). The levels of NHERF1 protein in plasma from patients with NSCLC were significantly higher than those in the healthy group (p = 0.0004). Based on the evaluation of the ROC curves, measuring the content of NHERF1 in human plasma could provide a potential diagnostic tool for NSCLC

Differential expression and functions of Ehm2 transcript variants in lung adenocarcinoma

Ehm2 [also known as erythrocyte membrane protein band 4.1‑like protein 4B (EPB41L4B)] is a member of the NF2/ERM/4.1 superfamily. The overexpression of Ehm2 has been observed in metastatic cancer cells. Through alternative splicing, the Ehm2 gene produces two transcript variants that encode the two different isoforms, Ehm2/1 and Ehm2/2. The biological functions of these different Ehm2 transcript variants remain unclear. The present study aimed to determine the expression of the Ehm2 variants in lung adenocarcinoma and their involvement in the disease progression of the patients. The expression of Ehm2 transcript variants in human lung adenocarcinoma tissues was analyzed using immunohistochemistry and western blot analysis. Ehm2 variants were overexpressed or knocked down in A549 human lung adenocarcinoma cells. The consequent effects of the genetic modifications on the cellular functions of lung cancer cells were then examined using in vitro cell viability, invasion and migration assays. The expression of epithelial‑mesenchymal transition (EMT)‑related markers was evaluated by western blot analysis in the cell models. The association of Ehm2 variant expression with patient survival was analyzed using Kaplan‑Meier survival analysis. The expression of Ehm2/1 was significantly decreased in lung cancers compared with the paired normal lung tissues (P<0.05), while the Ehm2/2 protein levels were higher in the tumors than in the paired normal lung tissues, although this was not statistically significant. The overexpression of Ehm2/1 exerted inhibitory effects, while the knockdown of Ehm2/1 promoted the growth, invasion and migration of A549 cells in vitro. Ehm2/2 was expressed at low levels in the A549 cells and the enforced expression of Ehm2/2 significantly increased the invasiveness and migration of the A549 cells. Immunofluorescence staining revealed that Ehm2/1 was confined to the plasma membrane, while Ehm2/2 was observed at both the plasma membrane and cytoplasm. The overexpression of Ehm2/1 resulted in the upregulation of the epithelial marker, E‑cadherin, and in the decreased expression of the mesenchymal markers, N‑cadherin and Snail1, while the knockdown of Ehm2/1 and the enforced expression of Ehm2/2 had the opposite effects on the protein levels of EMT‑related markers. Kaplan‑Meier survival analysis revealed that higher Ehm2/1 transcript levels were associated with the longer survival of patients with lung adenocarcinoma, while the lower expression of Ehm2/2 exhibited a similar association with patient survival. Taken together, the two Ehm2 variants appear to be differentially expressed in lung adenocarcinoma. Ehm2/1 may function as a putative tumor suppressor in the disease progression of lung adenocarcinoma, while Ehm2/2 may have an opposite function

OPN promotes the aggressiveness of non-small-cell lung cancer cells through the activation of the RON tyrosine kinase

Osteopontin (OPN) is identified as a diagnostic and prognostic biomarker of tumor progression and metastasis. In non-small-cell lung cancer (NSCLC), the functions of OPN have not been well characterized. The current study sought to investigate the clinical implications of OPN expression in NSCLC and the role of OPN in the aggressiveness of the lung cancer cells. Using a proteomics approach, we identified that phospho-RON (p-RON) was one of the most remarkably up-regulated proteins in OPN-overexpressing cells. The levels of OPN and RON transcripts were unveiled as independent prognostic indicators of survival in NSCLC (p = 0.001). Higher levels of OPN, RON and p-RON proteins were observed in tumor tissues. Knock down of the OPN gene suppressed the migration and invasion abilities of the A549 lung cancer cells which endogenously expresses OPN. While ectopic expression of OPN in the SK-MES-1 lung cancer cells increased levels of cellular invasion and migration. In addition, these changes were accompanied by a phosphorylated activation of RON. Small-molecule inhibition of RON or siRNA silencing of RON significantly reduced OPN-induced migration and invasion of lung cancer cells and had an inhibitory effect on the OPN-mediated cell epithelial-mesenchymal transition. Our study suggests that in NSCLC, the aberrant expression of OPN can be considered as an independent survival indicator and is associated with disease progression. OPN plays a crucial role in promoting migration and invasion properties of lung cancer cells through its phosphorylation activation of the RON signaling pathway, implying its potential as a therapeutic target in the treatment of NSCLC