Circulating tumor cell methylation profiles reveal the classification and evolution of non-small cell lung cancer.

Background: The ability of circulating tumor cells (CTCs) to identify lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) could improve pathological diagnosis and the selection of treatments for non-small cell lung cancer (NSCLC). Previous studies have shown that deoxyribonucleic acid (DNA) methylation exhibits cell and tissue specificity. Thus, we aimed to explore the methylation status of CTCs in LUAD and LUSC and identify the potential biomarkers. Methods: We first analyzed Infinium 450K methylation profiles obtained from The Cancer Genome Atlas and Gene Expression Omnibus. We then performed whole-genome sequencing of CTCs in tumor and matched normal lung tissues and white blood cells from 6 NSCLC patients. Results: The bioinformatics analysis revealed a NSCLC-specific DNA methylation marker panel, which could accurately distinguish between LUAD and LUSC with high diagnostic accuracy. The whole-genome sequencing of CTCs in NSCLC patients also showed 100% accuracy for distinguishing between LUAD and LUSC based on the CTC methylation profiles. To investigate the function of CTCs, we further analyzed similar and different methylation profiles between the CTCs and their primary tumors, and found very high similarities between the CTCs and their primary tumor tissues, indicating that these cells inherit information from primary tumors. However, the CTCs also displayed some characteristics that differed to those of primary tumor tissues, which suggest that CTCs acquire some unique characteristics after migrating from the primary tumor; these characteristics may partly explain the ability of tumor cells to evade immune surveillance. Conclusions: Our findings provide insights into the potential use of CTCs in the pathological classification of NSCLC patients. Our findings also show how CTC primary tumor inheritance and CTC evolution affect metastasis and immune escape.

USP7 overexpression predicts a poor prognosis in lung squamous cell carcinoma and large cell carcinoma.

In non-small cell lung cancer (NSCLC), both USP7 expression and p53 gene status were reported to be an indicator of poor prognosis in adenocarcinoma patients; however, its roles and mechanisms in lung squamous cell carcinoma and large cell carcinoma need to be clarified. The USP7 expression was examined in NSCLC tumors (excluding adenocarcinoma), their corresponding non-tumorous tissues, and NSCLC cells. Then, the prognostic role of USP7 was analyzed in 110 NSCLC samples (excluding the adenocarcinoma). Finally, the roles and mechanisms of USP7 in the proliferation, metastasis, and invasion of a NSCLC cell were assessed using a specific vshRNA. The USP7 expression was higher in NSCLC tissues compared to non-tumorous samples, accordingly, the high level of USP7 was detected in NSCLC cell lines compared with HBE cell. After the USP7 downregulation, the H460 cells exhibited decreased metastasis/invasion in vitro and in vivo. The preliminary mechanism study indicated overexpression of USP7 might regulate the p53-MDM2 pathway by inducing the MDM2 de-ubiquitination and subsequent stabilization, which resulted in the upregulation of the Bad phosphorylation. Additionally, we also found that USP7 might induce cell epithelial-mesenchymal transition to enhance the cell invasive ability. Clinically, USP7 overexpression significantly correlated with malignant phenotype. Furthermore, the 5-year overall survival in patients with USP7(low) was higher than that of USP7(high). Multivariate analysis showed USP7 overexpression was an independent prognostic marker for these cancers. USP7 overexpression may regulate the survival and invasive properties of squamous cell carcinoma and large cell carcinoma cells, and may serve as a molecular target.

Expression and prognostic roles of TRPV5 and TRPV6 in non-small cell lung cancer after curative resection.

PURPOSE: We investigated the expression of epithelial Ca2+ channel transient receptor potential vanilloid (TRPV) 5 and 6 in non-small-cell lung cancer (NSCLC) and assessed their prognostic role in patients after surgical resection. MATERIALS AND METHODS: From January 2008 to January 2009, 145 patients who had undergone surgical resection of NSCLCs were enrolled in the study. Patient clinical characteristics were retrospectively reviewed. Fresh tumor samples as well as peritumor tissues were analyzed for TRPV5/6 expression using immune-histochemistry (IHC) and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Patients were grouped based on their TRPV5 and TRPV6 levels in the tumor tissues, followed up after surgery, and statistically analyzed to examine the prognostic roles of TRPV5 and TRPV6 on patients' survival after surgical resection of NSCLCs. RESULTS: Using IHC, among the 145 patients who had undergone surgical resection of NSCLCs, strong protein expression (grade ≥ 2) of TRPV5 and TRPV6 was observed in a lower percentage of primary tumor tissues than in non-tumor tissues of same patients. Similar findings were obtained with the RT-PCR test for mRNA levels. Decreased overall mRNA levels of TRPV5 and TRPV6 were associated with a worse overall survival rate (p=0.004 and p=0.003 respectively) and shorter recurrence-free survival (p?0.001 and p?0.001 respectively). The combining effect of TRPV5 and TRPV6 on survival was further investigated using multivariate analysis. The results showed that a combination of low expression of TRPV5 and TRPV6 could be an independent predictor of poor recurrence-free survival (p=0.002). CONCLUSIONS: Decreased expression of TRPV5/6 in tumor tissues was observed in NSCLC patients and was associated with shorter median survival time after surgical resection. Combined expression of TRPV5 and TRPV6 in tumor tissues demonstrated promising prognostic value in NSCLC patients.

Two microRNA panels to discriminate three subtypes of lung carcinoma in bronchial brushing specimens.

RATIONALE: Effective treatment for lung cancer requires accuracy in subclassification of carcinoma subtypes. OBJECTIVES: To identify microRNAs in bronchial brushing specimens for discriminating small cell lung cancer (SCLC) from non-small cell lung cancer (NSCLC) and for further differentiating squamous cell carcinoma (SQ) from adenocarcinoma (AC). METHODS: Microarrays were used to screen 723 microRNAs in laser-captured, microdissected cancer cells from 82 snap-frozen surgical lung specimens. Quantitative reverse-transcriptase polymerase chain reaction was performed on 153 macrodissected formalin-fixed, paraffin-embedded (FFPE) surgical lung specimens to evaluate seven microRNA candidates discovered from microarrays. Two microRNA panels were constructed on the basis of a training cohort (n = 85) and validated using an independent cohort (n = 68). The microRNA panels were applied as differentiators of SCLC from NSCLC and of SQ from AC in 207 bronchial brushing specimens. MEASUREMENTS AND MAIN RESULTS: Two microRNA panels yielded high diagnostic accuracy in discriminating SCLC from NSCLC (miR-29a and miR-375; area under the curve [AUC], 0.991 and 0.982 for training and validation data set, respectively) and in differentiating SQ from AC (miR-205 and miR-34a; AUC, 0.977 and 0.982 for training and validation data set, respectively) in FFPE surgical lung specimens. Moreover, the microRNA panels accurately differentiated SCLC from NSCLC (AUC, 0.947) and SQ from AC (AUC, 0.962) in bronchial brushing specimens. CONCLUSIONS: We found two microRNA panels that accurately discriminated between the three subtypes of lung carcinoma in bronchial brushing specimens. The identified microRNA panels may have considerable clinical value in differential diagnosis and optimizing treatment strategies based on lung cancer subtypes.

Prevention of lung ischemia-reperfusion injury by short hairpin RNA-mediated caspase-3 gene silencing.

BACKGROUND: Lung ischemia-reperfusion injury remains a significant problem after lung transplantation. Caspase-mediated apoptotic pathways play an important role in lung ischemia-reperfusion injury, and caspase-3 is presumed to be the "effector" protease in the apoptotic cascade. Silencing gene expression of caspase-3 by short hairpin RNA (shRNA) can downregulate the caspase cascade. Therefore, we evaluated the therapeutic efficacy of caspase-3 shRNA in a rat model of lung ischemia-reperfusion injury. METHODS: Lung ischemia-reperfusion injury was induced in rats by clamping the hilum of the left lung for 1 hour. In vivo delivery of caspase-3 shRNA was performed by intratracheal administration 48 hours before ischemia. As controls, animals received either scrambled shRNA or RNase-free 5% dextrose in water solution. Real-time polymerase chain reaction, Western blotting, and immunohistochemistry were used to assess the gene silencing efficacy. The therapeutic effects of shRNA were evaluated by lung function analysis and the ratio of wet/dry weight. RESULTS: In this study, we have shown that ischemia-reperfusion injury is associated with an increased level of lung caspase-3 messenger RNA. Animals treated with caspase-3 shRNA showed a significant downregulation in lung expression of caspase-3 at transcripts and protein levels. Lung function was protected by caspase-3 shRNA therapy, inasmuch as levels of partial pressure of oxygen and carbon dioxide were significantly increased and reduced, respectively. CONCLUSIONS: In summary, we have demonstrated the therapeutic potential of shRNA to knock down the expression of caspase-3 and prevent lung apoptotic injury. Our findings may have some potential therapeutic relevance for treating lung ischemia-reperfusion injury after transplantation.

The expression of core fucosylated E-cadherin in cancer cells and lung cancer patients: prognostic implications.

It is well documented that the glycosylation of E-cadherin is correlated with cancer metastasis, but whether E-cadherin could be core fucosylated remains largely unknown. We found that E-cadherin was core fucosylated in highly metastatic lung cancer cells while absent in lowly metastatic lung cancer cells. Since alpha-1,6 Fucosyltransferase (alpha-1,6 FucT) is known to catalyze the reaction of core fucosylation, we investigated the biological function of core fucosylation on E-cadherin by alpha-1,6 FucT targeted RNAi and transfecting alpha-1,6 FucT expression vector. As a result, calcium dependent cell-cell adhesion mediated by E-cadherin was strengthened with the reduction of core fucosylation on E-cadherin after RNAi and was weakened with the elevated core fucosylation on E-cadherin after alpha-1,6 FucT over expression. Our data indicated that alpha-1,6 FucT could regulate E-cadherin mediated cell adhesion and thus play an important role in cancer development and progression. Computer modeling showed that core fucosylation on E-cadherin could significantly impair three-dimensional conformation of N-glycan on E-cadherin and produce conformational asymmetry so as to suppress the function of E-cadherin. Furthermore, the relationship between the expression of core fucosylated E-cadherin and clinicopathological background of lung cancer patients was explored in lung cancer tissue of patients. It turns out to demonstrate that core fucosylated E-cadherin could serve as a promising prognostic indicator for lung cancer patients.