Immune microenvironment analysis and novel biomarkers of early-stage lung adenocarcinoma evolution.

Background: Lung cancer is the deadliest and most diagnosed type of cancer worldwide. The 5-year survival rate of lung adenocarcinoma (LUAD) dropped significantly when tumor stages advanced. Patients who received surgically resecting at the pre-invasive stage had a 5-year survival rate of nearly 100%. However, the study on the differences in gene expression profiles and immune microenvironment among pre-invasive LUAD patients is still lacking. Methods: In this study, the gene expression profiles of three pre-invasive LUAD stages were compared using the RNA-sequencing data of 10 adenocarcinoma in situ (AIS) samples, 12 minimally invasive adenocarcinoma (MIA) samples, and 10 invasive adenocarcinoma (IAC) samples. Results: The high expression levels of PTGFRN (Hazard Ratio [HR] = 1.45; 95% Confidence Interval [CI]: 1.08-1.94; log-rank P = 0.013) and SPP1 (HR = 1.44; 95% CI: 1.07-1.93; log-rank P = 0.015) were identified to be associated with LUAD prognosis. Moreover, the early LUAD invasion was accompanied by the enhancement of antigen presentation ability, reflected by the increase of myeloid dendritic cells infiltration rate (Cuzick test P < 0.01) and the upregulation of seven important genes participating in the antigen presentation, including HLA-A (Cuzick test P = 0.03), MICA (Cuzick test P = 0.01), MICB (Cuzick test P = 0.01), HLA-DPA1 (Cuzick test P = 0.04), HLA-DQA2 (Cuzick test P < 0.01), HLA-DQB1 (Cuzick test P = 0.03), and HLA-DQB2 (Cuzick test P < 0.01). However, the tumor-killing ability of the immune system was inhibited during this process, as there were no rising cytotoxic T cell activity (Cuzick test P = 0.20) and no increasing expression in genes encoding cytotoxic proteins. Conclusion: In all, our research elucidated the changes in the immune microenvironment during early-stage LUAD evolution and may provide a theoretical basis for developing novel early-stage lung cancer therapeutic targets.

Beneficial effect of the mitochondrial ATP­sensitive potassium channel­specific opener nicorandil on the collapsed lung via inhibition of apoptosis in clinical thoracic surgery.

With the use of thoracoscopic surgery technology, one­lung ventilation (OLV) is becoming more crucial as a basic requirement for enhanced recovery after surgery; however, it can lead to severe pulmonary injury, which is an issue for anesthesiologists. Therefore, it is important to protect pulmonary function during thoracic surgery anesthesia, particularly to protect the function of the collapsed lung. Our previous study on rabbits reported that nicorandil, a US Food and Drug Administration­approved mitochondrial ATP­sensitive potassium channel­specific opener, can protect against lung injury in the collapsed lung. Therefore, the beneficial effect of nicorandil on OLV­induced pulmonary injury in clinical thoracic surgery was further evaluated in the present study. Nicorandil was infused at 2 mg/h for 2 h from induction to 1 h after OLV in the nicorandil group. Trends in arterial oxygen desaturation (SaO2), arterial partial pressure for oxygen (PaO2) and the lung microstructure were assessed. ELISA was used to assess the levels of TNF­α and malondialdehyde (MDA), and the activity of superoxide dismutase (SOD). A TUNEL assay was performed to evaluate apoptosis. Western blotting was used to analyze the relative expression levels of signaling proteins associated with apoptosis. Western blotting was performed to evaluate the protein expression levels of hypoxia­inducible factor 1α (HIF­1α), PI3K, Akt and NF­κB, and reverse transcription­quantitative PCR was used to detect HIF­1α mRNA expression levels in the lungs of patients infused with nicorandil and nitroglycerin. Nicorandil treatment was associated with higher SaO2 and PaO2 compared with nitroglycerin treatment in OLV. The levels of MDA and TNF­α in the operated lung of the nicorandil group were significantly lower compared with those in the control group. In addition, nicorandil was associated with higher SOD activity compared with nitroglycerin. The nicorandil­treated lung, similar to the sham group, exhibited improved microstructure and less apoptosis in the experimental group. The protein expression levels of PI3K, phosphorylated Akt and HIF­1α were significantly increased, whereas NF­κB was significantly decreased in the nicorandil­treated lung compared with the control group. Overall, nicorandil demonstrated beneficial effects by decreasing apoptosis in the operated lung, which was collapsed and then re­expanded during OLV in thoracic surgery anesthesia. Nicorandil may serve a vital role by decreasing the overloading of calcium in mitochondria, shutting off the mitochondrial membrane permeability transition pore, reducing the release of cytochrome c, simultaneously triggering activation of the PI3K/Akt signaling pathway around the cell membrane, downregulating NF­κB, upregulating HIF­1α, and then reducing Bax/Bcl­2, caspase­3 and apoptosis. The trial registration was ChiCTR­IOR­17014061 (registered on December 20, 2017).

Association of PTPRD/PTPRT Mutation With Better Clinical Outcomes in NSCLC Patients Treated With Immune Checkpoint Blockades.

The common gamma receptor-dependent cytokines and their JAK-STAT pathways play important roles in T cell immunity and have been demonstrated to be related with response to immune checkpoint blockades (ICBs). PTPRD and PTPRT are phosphatases involved in JAK-STAT pathway. However, their clinical significance for non-small cell lung cancer (NSCLC) treated with ICBs is still unclear. Genomic and survival data of NSCLC patients administrated with anti-PD-1/PD-L1 or anti-CTLA-4 antibodies (Rizvi2015; Hellmann2018; Rizvi2018 Samstein2019) were retrieved from publicly accessible data. Genomic, survival and mRNA data of 1007 patients with NSCLC were obtained from The Cancer Genome Atlas (TCGA). PTPRD/PTPRT mutation was significantly associated with better progression-free survival (PFS) in three independent Rizvi2015, Hellmann2018 and Rizvi2018 cohorts. The median PFS for PTPRD/PTPRT mutant-type vs. wild-type NSCLC patients were not reached vs. 6.3 months (Rizvi2015, HR = 0.16; 95% CI, 0.02-1.17; P=0.03), 24.0 vs. 5.4 months (Hellmann2018, HR, 0.49; 95% CI, 0.26-0.94; P=0.03), 5.6 vs. 3.0 months (Rizvi2018, HR = 0.64; 95% CI, 0.44-0.92; P=0.01) and 6.8 vs. 3.5 months (Pooled cohort, HR, 0.54; 95% CI, 0.39-0.73; P<0.0001) respectively. PTPRD/PTPRT mutation was an independent predictive factor for PFS in pooled cohort (P = 0.01). Additionally, PTPRD/PTPRT mutation associated with better overall survival (OS) in Samstein2019 cohort (19 vs. 10 months, P=0.03). While similar clinical benefits were not observed in patients without ICBs treatment (TCGA cohort, P=0.78). In the further exploratory analysis, PTPRD/PTPRT mutation was significantly associated with increased tumor mutation burden and higher mRNA expression of JAK1 and STAT1. Gene Set Enrichment Analysis revealed prominent enrichment of signatures related to antigen processing and presentation in patients with PTPRD/PTPRT mutation. This work suggested that PTPRD/PTPRT mutation might be a potential positive predictor for ICBs in NSCLC. These results need to be further confirmed in future.

Creation of a Prognostic Risk Prediction Model for Lung Adenocarcinoma Based on Gene Expression, Methylation, and Clinical Characteristics.

BACKGROUND This study aimed to identify important marker genes in lung adenocarcinoma (LACC) and establish a prognostic risk model to predict the risk of LACC in patients. MATERIAL AND METHODS Gene expression and methylation profiles for LACC and clinical information about cases were downloaded from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases, respectively. Differentially expressed genes (DEGs) and differentially methylated genes (DMGs) between cancer and control groups were selected through meta-analysis. Pearson coefficient correlation analysis was performed to identify intersections between DEGs and DMGs and a functional analysis was performed on the genes that were correlated. Marker genes and clinical factors significantly related to prognosis were identified using univariate and multivariate Cox regression analyses. Risk prediction models were then created based on the marker genes and clinical factors. RESULTS In total, 1975 DEGs and 2095 DMGs were identified. After comparison, 16 prognosis-related genes (EFNB2, TSPAN7, INPP5A, VAMP2, CALML5, SNAI2, RHOBTB1, CKB, ATF7IP2, RIMS2, RCBTB2, YBX1, RAB27B, NFATC1, TCEAL4, and SLC16A3) were selected from 265 overlapping genes. Four clinical factors (pathologic N [node], pathologic T [tumor], pathologic stage, and new tumor) were associated with prognosis. The prognostic risk prediction models were constructed and validated with other independent datasets. CONCLUSIONS An integrated model that combines clinical factors and gene markers is useful for predicting risk of LACC in patients. The 16 genes that were identified, including EFNB2, TSPAN7, INPP5A, VAMP2, and CALML5, may serve as novel biomarkers for diagnosis of LACC and prediction of disease prognosis.

Prognostic impact of micropapillary component in patients with node-negative subcentimeter lung adenocarcinoma: A Chinese cohort study.

BACKGROUND: In this study, we investigated the prognostic significance of a micropapillary (MP) component in patients with subcentimeter lung adenocarcinoma. METHODS: A total of 311 patients with subcentimeter lung adenocarcinoma who underwent surgical resection between January 2009 to December 2012 from seven medical centers were included. Recurrence-free survival (RFS) and overall survival (OS) were analyzed. RESULTS: The five-year RFS was 79.8% in 97 (97/311, 31%) cases of adenocarcinoma with a MP component and 93.5% in the 214 (214/311, 69%) cases without. In multivariate analysis, MP was an independent risk factor for worse RFS (hazard ratio [HR], 3.73; 95% confidence interval [CI]: 1.87-7.42; P < 0.001) and OS (HR, 5.84; 95% CI: 2.20-15.49; P < 0.001). There was no significant difference among wedge resection, segmentectomy and lobectomy on RFS (P = 0.256) and OS (P = 0.103) in patients without MP. Regarding patients with a MP component, lobectomy achieved equivalent prognosis than segmentectomy, and both were better than wedge resection (P = 0.001). CONCLUSIONS: A MP component still suggest a poor prognosis in subcentimeter lung adenocarcinoma. Patients with subcentimeter lung adenocarcinoma with a MP component of 5% or greater treated with wedge resection were at higher risk of recurrence than patients treated with anatomical resection.

Impact of histological components on selecting limited lymphadenectomy for lung adenocarcinoma ≤ 2 cm.

OBJECTIVES: There is still some dispute regarding the performance of limited mediastinal lymphadenectomy (LML) even for lung adenocarcinoma ≤ 2 cm. We aimed to recognize the potential candidates who can benefit from LML based on the percentage of histological components (PHC). METHODS: We analyzed 1160 surgical patients with invasive lung adenocarcinoma ≤ 2 cm from seven institutions between January 2012 and December 2015. All histological subtypes were listed in 5% increments by pathological slices. To test the accuracy of frozen section in judging PHC, frozen section slides from 140 cases were reviewed by three pathologists. RESULTS: There were 882 patients with systematic mediastinal lymphadenectomy (SML) and 278 with LML. Multivariable analysis indicated that, the total percentage of micropapillary and solid components (PHCMIP+S) > 5 % was the independent predictor of N2 metastasis (P < 0.001). Overall, recurrence-free survival (RFS) and overall survival (OS) favored SML compared with LML, but the subgroup analysis revealed LML and SML had similar prognosis in the group of PHCMIP+S ≤ 5 %. Moreover, multivariable Cox analysis showed LML (vs. SML) was independently associated with worse prognosis for patients with PHCMIP+S > 5 % (RFS, HR = 2.143, P < 0.001; OS, HR=1.963, P < 0.001), but not for those with PHCMIP+S ≤ 5 % (RFS, P = 0.398; OS, P = 0.298). The sensitivity and specificity of frozen section to intraoperatively identify PHCMIP+S ≤ 5 % were 97.6 % and 84.2 %, respectively. CONCLUSIONS: PHCMIP+S showed the predictive value for N2 metastasis and procedure-specific outcome (LML vs. SML). It may serve as a feasible indicator for identifying proper candidates of LML by using intraoperative frozen section.

LncRNA RMRP Promotes Cell Proliferation and Invasion Through miR-613/NFAT5 Axis in Non-Small Cell Lung Cancer.

BACKGROUND: The abnormal expression of RMRP and miR-613 was respectively associated with the pathogenesis of lung cancer, but the role of the RMRP/miR-613 axis in NSCLC has not been studied. METHODS: In this report, we measured the levels of RMRP in clinical NSCLC samples and cell lines. The target gene of RNA was predicted by online tools and verified by Luciferase reporter assay. Moreover, the function and regulatory mechanism of RMRP in the progression of cancer were further investigated. RESULTS: Our data showed that the expression of RMRP in NSCLC tissues and cell lines was both up-regulated. Functionally, RMRP promoted the proliferation and metastasis of A549 and H1299 cells. Luciferase reporter assay confirmed that RMRP was the sponger of miR-613, and NFAT5 is the direct target of miR-613. Functional acquisition and loss-of-function strategies further confirmed that RMRP induces the up-regulation of NFAT5 expression through competitive binding with miR-613, leading to promote the progression and metastasis potential of lung cancer cells. CONCLUSION: Collectively, our findings emphasized the importance of RMRP in the development of NSCLC, which may provide a new therapeutic target and potential diagnostic biomarker for NSCLC therapy.

Procedure-specific prognostic impact of micropapillary subtype may guide resection strategy in small-sized lung adenocarcinomas: a multicenter study.

BACKGROUND: Limited resection has gradually become an acceptable treatment for lung adenocarcinomas (ADCs) presenting as ground-glass nodules (GGNs). However, its role in lung ADCs presenting as pure solid nodules (PSN) remains unclear. In this study, we aimed to identify potential candidates for limited resection in lung ADCs presenting as PSN. METHODS: We retrospectively reviewed 772 patients from seven hospitals with lung ADCs ⩽2 cm, presenting as PSN on computed tomography scans, who had undergone surgery between 2009 and 2013. Histological subtypes were listed in 5% increments. To investigate the value of histological subtypes in surgical decision making, five pathologists prospectively evaluated the feasibility of identifying histological subtypes using frozen section (FS) in two cohorts. RESULTS: The percentage of micropapillary (MIP) subtype had a striking impact on recurrence-free survival (RFS) and overall survival (OS) for lung ADCs ⩽2 cm presenting as PSNs. In multivariable Cox analysis, segmentectomy was significantly associated with worse RFS and OS in patients with MIP >5% than lobectomy, but not in those with MIP ⩽5%. With wedge resection, worse RFS and OS were observed in patients with MIP >5% and those with MIP ⩽5% than lobectomy. The sensitivity and specificity for detecting MIP by FS were 74.2% and 85.6%, respectively, with substantial inter-rater agreement. CONCLUSION: Segmentectomy and lobectomy had similar oncological outcomes in patients with lung ADCs ⩽2 cm presenting as PSN with MIP ⩽5%. Randomized trials are necessary to validate the feasibility of intraoperative FS to choose candidates for segmentectomy.

Lepidic component identifies a subgroup of lung adenocarcinoma with a distinctive prognosis: a multicenter propensity-matched analysis.

BACKGROUND: Our aim was to investigate the prognostic impact of the lepidic component on T stage in patients with lung adenocarcinoma (LUAD). METHODS: A retrospective data set including 863 cases of LUAD with lepidic component and 856 cases without lepidic component was used to identify matched lepidic-positive and lepidic-negative cohorts (n = 376 patients per group) using a propensity-score matching. Primary outcome variables included recurrence-free survival (RFS) and overall survival (OS). Prognostic factors were assessed by Cox regression analysis and Kaplan-Meier estimates. RESULTS: Multivariate analysis revealed that lepidic component presence was an independent prognostic factor for prolonged RFS (p < 0.001) and OS (p < 0.001). Furthermore, lepidic ratio (LR) >25% or ⩽25% were confirmed to be independent prolonged survival predictors. No survival differences were observed between patients with LUAD with LR >25% or ⩽25% (RFS p = 0.333; OS p = 0.078). The 5-year OS rates of patients with LUAD with a lepidic component were 90% regardless of the T stage, and these survival rates were significantly better than those of patients with LUAD without a lepidic component in the corresponding T stage. Multivariate analysis confirmed that T stage was associated with survival only in patients with LUAD without a lepidic component. CONCLUSIONS: Lepidic component presence identifies a LUAD subgroup with an excellent prognosis independent of the LR, pathological T classification. Considering the lepidic component presence may improve prognostic predictions for patients with LUAD.

Protective effect of nicorandil on collapse­induced lung injury in rabbits by inhibiting apoptosis.

The one­lung ventilation (OLV) technique is vital in thoracic surgery. However, it can result in severe lung injury, which is difficult to manage. The main solution at present is the use of ventilation strategies, including continuous positive oxygen pressure, low tidal volume and high frequency ventilation, and the administering of drugs, including phenylephrine, dexmedetomidine and morphine. However, the protective effect of these methods on the lungs is not sufficient to improve the prognosis of patients. Therefore, how to develop a novel protective drug remains an open question. Nicorandil, a mitochondrial (mito)KATP­specific opener, serves an important role in cardioprotection, although its effect on lung injury remains unclear. The present study examined the protective role of nicorandil against collapse­induced lung injury in rabbits undergoing OLV. Changes in arterial oxygen saturation (SaO2), arterial partial pressure for oxygen (PaO2), wet/dry weight ratio, and the microstructure of tissues and cells were observed. Enzyme­linked immunosorbent assays were used to determine the concentrations of malondialdehyde (MDA) and tumor necrosis factor (TNF)­α, and the activity of superoxide dismutase (SOD) in rabbits treated with nicorandil. Terminal deoxynucleotidyl transferase transfer­mediated dUTP nick end­labeling was used to detect apoptosis and western blotting was used to analyze the relative proteins involved in apoptosis. Western blotting and reverse transcription­quantitative polymerase chain reaction analysis were used to examine the expression of hypoxia inducible factor 1α (HIF­1α), phosphatidylinositol­3­kinase (PI3K), protein kinase B (Akt) and nuclear factor (NF)­κB in the lungs of rabbits treated with nicorandil. The SaO2 and PaO2 in the high­dose group were significantly higher than those in the control group in the process of OLV. The wet/dry weight ratio, and the concentrations of MDA and TNF­α in the collapsed lung of the high­dose group were significantly lower than those in the control group. The activity of SOD in the high­dose group was significantly higher than that in the control group. The lung had improved microstructure and less apoptosis, which was determined by the Bax/Bcl2 ratio in the high­dose group. The expression levels of PI3K, phosphorylated Akt and HIF­1α were upregulated, whereas the expression of NF­κB was downregulated. In conclusion, nicorandil had a protective effect via inhibiting apoptosis in non­ventilated lung collapsed and re­expansion during OLV in the rabbit. It acted on mitoKATP through the PI3K/Akt signaling pathway.

Overexpression of RCN1 correlates with poor prognosis and progression in non-small cell lung cancer.

We investigated the expression of reticulocalbin-1 (RCN1) and its prognostic significance in non-small cell lung cancer (NSCLC). RCN1 expression was evaluated by immunohistochemical analysis with tissue microarrays in NSCLC tissues and matched adjacent noncancerous tissues. Furthermore, quantitative polymerase chain reaction and Western blot were also used to examine the expression of RCN1. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, clone formation, and transwell assays were used to measure cell proliferation, migration, and invasion. Lastly, we used the Kaplan-Meier method and log-rank test to compare overall survival rates between the RCN1-high expression group and the RCN1-low expression group. In this study, immunohistochemistry by tissue microarray at RCN1 expression was significantly up-regulated in NSCLC tissues compared with adjacent noncancerous tissues. We further confirmed the up-regulation of RCN1 by quantitative polymerase chain reaction and Western blot assay. RCN1 expression level was closely related to lymph node metastasis (P < .001) and TNM stage (P = .012). Kaplan-Meier analysis showed that high RCN1 expression was remarkably associated with poor prognosis with NSCLC patients. A suppression of cell proliferation, migration, and invasion was obtained in A549 cells treated with RCN1 small interfering RNA. Our data indicate that RCN1 expression may have an vital role at promoting the occurrence of NSCLC, and it may be a vital molecular marker in the diagnosis and prognosis of NSCLC patients.

Unique genomic profiles obtained from cerebrospinal fluid cell-free DNA of non-small cell lung cancer patients with leptomeningeal metastases.

BACKGROUND: Leptomeningeal metastases (LM), associated with poor prognosis, are frequent complications of advanced non-small cell lung cancer (NSCLC) patients, especially in patients with epidermal growth factor receptor (EGFR) mutations. Due to limited access to leptomeningeal lesions, the mutational landscape of LM has not been comprehensively investigated in large cohorts and the underlining biology of LM remains elusive. Some studies have explored the potential of cerebrospinal fluid (CSF) in reflecting the molecular profile of LM but with limited number of patients enrolled. METHODS: In this study, we performed capture-based targeted sequencing using a panel consisting of 168 lung cancer-related genes on matched CSF and plasma samples from 72 advanced NSCLC patients with confirmed LM to interrogate the potential of CSF as a source of liquid biopsy. RESULTS: We revealed a rate of detection of 81.5% and 62.5% for CSF and plasma, respectively (p = 0.008). The maximum allelic fraction (MaxAF) was also significantly higher in CSF (43.6% vs. 4.6%) (p < 0.001). CSF, harboring a unique genomic profile by having a significant number of CSF-specific mutations, primarily copy number variations, is superior to plasma in reflecting the mutational profile of LM. Further pathway enrichment analysis revealed that most of CSF-specific mutations participated in pathways relevant to the tumorigenesis and the development of metastases. Moreover, our data also revealed that TP53 loss of heterozygosity (LOH) predominantly existed in CSF (p < 0.001). CONCLUSIONS: Collectively, we demonstrated that CSF provides a more comprehensive profile of LM than plasma in a large cohort, thus can be used as an alternative source of liquid biopsy for LM patients.

The expression level and prognostic value of HIPK3 among non-small-cell lung cancer patients in China.

BACKGROUND: Lung cancer is one of the most common malignancies in the world and is at the forefront of causes of all cancer deaths. Identification of new prognostic predictors or therapeutic targets might improve a patient's survival rate. PURPOSE: The Homeodomain interacting protein kinases (HIPKs) function as modulators of cellular stress responses and regulate cell differentiation, proliferation and apoptosis, but the function of HIPK3 is remain unknown. PATIENTS AND METHODS: We used quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting methods to detective the expression of HIPK3. A total of 206 samples were obtained from patients and Immunochemical evaluation to determine HIPK3 protein expression. HIPK3 protein levels in in non-small cell lung cancer (NSCLC) were correlated with the clinical characteristics of patients and their 5-year survival rate. In addition, HIPK3 knockdown by specific RNAi promoted cell proliferation, migration, and invasion in A549 and HCC827 cancer cell lines. RESULTS: The quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting methods to demonstrate that HIPK3 expression was significantly down-regulated in non-small cell lung cancer (NSCLC) tissues compared with that in normal lung tissues. At the same time, the results of immunohistochemistry assays showed that low expression of HIPK3 was significantly associated with pathology grade; tumor, node, and metastases (TNM) stage; lymph node metastasis; Ki-67 expression; and the 5-year survival rate in NSCLC patients. Univariate analysis revealed that HIPK3 expression, Ki-67 expression, tumor diameter, TNM stage, and age were significantly associated with a poor prognosis. The multivariable analysis illustrated that HIPK3, tumor diameter, TNM, Ki-67 expression, and age had effects on the overall survival of NSCLC patients independently. Kaplan-Meier survival curves revealed that NSCLC patients with a lower HIPK3 expression had a poorer prognosis. In addition, in vivo results also confirmed that HIPK3 over-expression could inhibit tumor growth. CONCLUSION: Our findings confirmed that low expression of HIPK3 in NSCLC tissues was significantly correlated with poor survival rates after curative resection. HIPK3 could potentially be used as a valuable biomarker in the prognosis of the survival of NSCLC patients.

Expression of PFKFB3 and Ki67 in lung adenocarcinomas and targeting PFKFB3 as a therapeutic strategy.

Phosphofructokinase-2/fructose-2, 6-bisphosphatase 3 (PFKFB3) catalyzes the synthesis of F2,6BP, which is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1): the rate-limiting enzyme of glycolysis. During tumorigenesis, PFKFB3 increases glycolysis, angiogenesis, and tumor progression. In this study, our aim was to investigate the significance of PFKFB3 and Ki67 in human lung adenocarcinomas and to target PFKFB3 as a therapeutic strategy. In this study, we determined the expression levels of PFKFB3 mRNA and proteins in cancerous and normal lung adenocarcinomas by quantitative reverse transcription PCR (qRT-PCR), Western blot analysis, and tissue microarray immunohistochemistry analysis, respectively. In human adenocarcinoma tissues, PFKFB3 and Ki67 protein levels were related to the clinical characteristics and overall survival. Both PFKFB3 mRNA and protein were significantly higher in lung adenocarcinoma cells (all P < 0.05). A high expression of PFKFB3 and Ki67 were associated with the degree of differentiation, TNM staging, lymph node metastasis, and survival. A high expression of PFKFB3 protein was an independent prognostic marker in lung adenocarcinoma. Subsequently, 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15) was used as a selective antagonist of PFKFB3. Glycolytic flux was determined by measuring glucose uptake, F2,6BP, and lactate production. Cell viability, cell cycle, cell apoptosis, cell migration, and invasion were analyzed by MTT, flow cytometry, Western blot analysis, wound healing assay, and transwell chamber assay. By targeting PFKFB3, it inhibited cell viability and glycolytic activity. It also caused apoptosis and induced cell cycle arrest. Furthermore, the migration and invasion of A549 cells was inhibited. We conclude that PFKFB3 bears an oncogene-like regulatory element in lung adenocarcinoma progression. In the treatment of lung adenocarcinoma, targeting PFKFB3 would be a promising therapeutic strategy.

CXCL6 promotes non-small cell lung cancer cell survival and metastasis via down-regulation of miR-515-5p.

Chemokine plays an important role in lung cancer and CXCL6 is one of chemokine, however, its effect on miRNAs profile and its roles in non-small cell lung cancer cell (NSCLC) is not elucidated. This study is purposed to explore the influence of CXCL6 on miRNA expression profile and found that CXCL6 could reduce the expression of miR-515-5p in NSCLC cells. MiR-515-5p in NSCLC cells could inhibit NSCLC survival and metastasis. MiR-515-5p acted as a tumor suppressor by targeting CXCL6 in NSCLC cells. These data highlighted a novel molecular interaction between miR-515-5p and CXCL6. MiR-515-5p may constitute a potential therapy target for NSCLC.

Anterior fissureless uniport vs. posterior intra-fissure triple-port thoracoscopic right upper lobectomy: a propensity-matched study.

BACKGROUND: This study aimed to assess the efficiency of anterior fissureless uniport (AFU) thoracoscopic lobectomy for early stage right upper non-small cell lung cancer (NSCLC). METHODS: Between June 2014 and Dec 2016, 162 consecutive NSCLC patients who underwent thoracoscopic right upper lobectomy (RUL) by AFU approach (AFU group, n=65) or posterior intra-fissure triple-port dissection (PIFT group, n=97) were enrolled. A propensity-matched analysis was used to compare perioperative outcomes, safety and efficiency between the two groups. RESULTS: Propensity matching produced 40 pairs in this retrospective study. During the operation, lobectomy took less time in the AFU group compared with the PIFT group, while no statistical differences in mediastinal lymphadenectomy time, intraoperative blood loss, and total of lymph nodes harvested were found between the two groups. Postoperatively, length of hospital stay (LOS) and time of postoperative air leak were significantly reduced in AFU group than in PIFT group. However, the overall complication rate and volume of pleural effusion drainage within 48 h were similar. Compared with the PIFT group, visual analogue scale (VAS) of 3 postoperative days in AFU group was slighter. CONCLUSIONS: In RUL, AFU thoracoscopic approach is safe, efficient and easily maneuverable, which would reduce the duration of lobectomy, LOS and time of postoperative air leak. Postoperative pain is also mild.

Spondin2 is a new prognostic biomarker for lung adenocarcinoma.

Spondin 2 (SPON2) is a member of the F-spondin superfamily of genes that encode an extracellular matrix protein. SPON2 has been identified by mRNA differential display screening of cancerous and noncancerous lung cell lines in vitro [1], however, its role in pulmonary adenocarcinoma (ADC) patients remains unclear. In our study, we evaluated whether SPON2 can be used as a biomarker for the diagnosis of pulmonary ADC and any association between SPON2 protein levels and clinicopathological characteristics. Firstly, the mRNA levels of SPON2 in pulmonary ADCs and normal adjacent tissue samples were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 60) assay and the expression of SPON2 protein were detected by tissue microarray immunohistochemistry analysis (TMA-IHC) (n = 280). Overexpression of SPON2 protein in cancerous tissues was associated with the clinical characteristics of ADC patients and their overall survival. Levels of SPON2 mRNA and protein were significantly expressed higher in ADC tissues than in adjacent normal tissues. Finally, through univariate and multivariate regression analysis, we found that overexpression of SPON2 protein levels correlates with differentiation, positive lymph nodes metastasis, higher serum carcinoembryonic antigen (CEA) level and poor overall survival. Overexpression of SPON2 protein is an independent prognostic biomarker in ADC patients. Our data revealed that SPON2 played an oncogene role in ADC development and progression. Inhibiting SPON2 might represent a new strategy for pulmonary ADC.

MiR-129 regulates MMP9 to control metastasis of non-small cell lung cancer.

The molecular mechanism underlying activation of MMP9 in non-small cell lung cancer (NSCLC) cells, which controls cancer invasiveness and metastasis, remains elusive. Here, we reported significant decrease in miR-129 and significant increases in phosphorylated EGFR and MMP9 in the resected NSCLC from the patients, compared with adjacent normal tissue. Moreover, strong correlations were detected among these three factors, the relationship of which was examined in two human NSCLC lines, A549 and H460. We found that EGF-induced EGFR phosphorylation in A549 or H460 cells activated MMP9 and, consequently, cancer invasiveness. The EGF-induced activation of MMP9 was efficiently inhibited either by an EGFR inhibitor or by an Akt inhibitor. However, miR-129 level was not affected by EGF stimulation. In addition, overexpression of miR-129 antagonized EGF-induced MMP9 activation without affecting EGFR phosphorylation in A549 or H460 cells. Taken together, our data suggest that miR-129 inhibits EGFR signaling through PI3K signal transduction cascades to regulate MMP9 expression in NSCLC. Thus, miR-129, EGFR, and MMP9 appear to be promising therapeutic targets for preventing the metastasis of NSCLC.

Improved three-tubing approach for the reoperation of perforation suturing anastomotic fistula after radical radiotherapy of upper thoracic esophgeal squamous cell carcinoma.

BACKGROUND: As we all know that traditional three tubes treatment for the anastomotic fistula after esophagectomy is easy and efficient, but there are still so many problems such as pain, intubation discomfort, psychological pressure and prognosis slowly. Our report would introduce a new method to cure the anastomotic fistula for the patient after radical radiotherapy. METHODS: CT and DSA-guided percutaneous cervical gastric wall puncture were performed for the patient while the Flocare tube was introduced entering the mediastinum via the anastomotic fistula under the guidance of contrast medium followed by the procedure of persistent vacuum-suction for 24 hours. Meanwhile, the patient underwent the treatments such as fasting, gastrointestinal decompression, anti-inflammation and nutritional support. RESULTS: Four months later, the patient was discharged after the healing of gastroesophageal anastomotic fistula, significant shrink of tracheo-mediastinal fistula and no discomfort when taking semi-liquid diet. CONCLUSIONS: With the benefits of long-term tube retention, improved three-tubing will lead to positive and effective treatment, especially for the patient who has the healing capacity of the tissue decreases due to radical radiotherapy.