Screening anlotinib responders via blood-based proteomics in non-small cell lung cancer.

Anlotinib has been demonstrated to be effective in advanced non-small cell lung cancer (NSCLC) patients. The response stratification of anlotinib remains unclear. In this study, plasma samples from 28 anlotinib-treated NSCLC patients (discovery cohort: 14 responders and 14 non-responders) were subjected to proteomic analysis, and plasma samples from 35 anlotinib-treated NSCLC patients (validation cohort) were subjected to validation analysis. Liquid chromatography-tandem mass spectrometry analysis was performed on samples with different time points, namely baseline (BL), best response (BR), and progression disease (PD). Bioinformatics analysis was performed to screen for the underlying differential proteins. Enzyme-linked immunosorbent assay was performed to detect plasma ARHGDIB, FN1, CDH1, and KNG1 levels respectively. The Kaplan-Meier survival analysis was used for biomarker-based responsive stratification. Our results indicated that differential proteins between responders and non-responders showed that proteomic technology potentially contributes to biomarker screening in plasma samples at BL. Furthermore, our results suggested that the detection of plasma ARHGDIB, FN1, CDH1, and KNG1 levels have potential predictive value for anlotinib response both in the discovery cohort and validation cohort. Collectively, this study offers novel insights into the value of plasma biomarker screening via proteomic examination and suggests that plasma ARHGDIB, FN1, CDH1, and KNG1 levels could be used as biomarkers for anlotinib stratification in NSCLC patients.

Circular RNA EPB41 expression predicts unfavorable prognoses in NSCLC by regulating miR-486-3p/eIF5A axis-mediated stemness.

Dysregulation of circular RNAs (circRNAs) has recently been found to play an important role in the progression and development of cancers such as non-small cell lung cancer (NSCLC). Yet the functions of many circRNAs in NSCLC remain unclear. In this study, the circRNA expression profiles in NSCLC tumor tissues and adjacent non-tumorous tissues were detected by high-throughput sequencing. Bioinformatics analyses, the dual-luciferase reporter system, fluorescence in situ hybridization (FISH) and miRNA/mRNA high-throughput sequencing were used to identify circ-EPB41 and its downstream target. The subcutaneous tumor/caudal vein transfer mouse model was used for tumor growth and invasion analysis. The results show that the circ-EPB41 was upregulated in NSCLC tissues and cell lines. Increased circ-EPB41 expression in NSCLC was significantly correlated with malignant characteristics, and positive to post-surgical overall survival of NSCLC patients. Reduced circ-EPB41 expression in NSCLC decreased cell proliferation and invasion in both in vitro and in vivo experiments. The miRNA/mRNA high-throughput sequencing suggested that downregulation of circ-EPB41 promoted microRNA (miR)-486-3p and suppressed eukaryotic translation initiation factor 5A (eIF5A) expression. Luciferase reporter experiments confirmed that miR-486-3p/eIF5A were downstream targets of circ-EPB41. In addition, we also found that downregulation of circ-EPB41 suppressed self-renewal and decreased expression of stemness markers SOX2, OCT-4, Nanog and CD133 by sponging miR-486-3p to enhance eIF5A expression. Taken togeter, these data revealed the important role of circ-EPB41 in regulating NSCLC cell invasion and proliferation by modifying miR-486-3p/eIF5A axis-mediated stemness. We believe our study provides a novel perspective regarding the role of circRNAs in NSCLC progression.

Equivalent efficacy assessment of QL1101 and bevacizumab in nonsquamous non-small cell lung cancer patients: A two-year follow-up data update.

Objective: Anti-vascular endothelial growth factor (VEGF) monoclonal antibodies are an effective means of treating non-small cell lung cancer (NSCLC). Here, we aim to update the equivalent efficacy assessment between QL1101 and bevacizumab based on two-year follow-up data. Methods: In total, 535 eligible NSCLC patients were enrolled in this randomized controlled trial. Patients were randomly assigned 1:1 to the QL1101 group and the bevacizumab group. The full end time of this study was defined as 24 months after the last enrolled patient was randomized. The primary endpoint was the objective response rate (ORR); equivalence was confirmed if the two-sided 90% confidence interval (90% CI) of the relative risk was within the range of 0.75-1.33. The secondary endpoints were progression-free survival (PFS) and overall survival (OS). Results: The two-year updated data showed similar ORR (QL1101 vs. bevacizumab: 53.1% vs. 54.3%; relative risk=0.977; 90% CI: 0.838-1.144), PFS (235 d vs. 254 d, log-rank P=0.311), and OS (577 d vs. 641 d, log-rank P=0.099) results between the QL1101 group and the bevacizumab group. The mean shrinkage ratio of targeted lesions was also similar between the QL1101 group and the bevacizumab group (22.5% vs. 23.5%). For patients who received QL1101 maintenance therapy, similar results were shown between the QL1101 group (n=157) and the bevacizumab group (n=148) (PFS: 253 d vs. 272 d, log-rank P=0.387; OS: 673 d vs. 790 d, log-rank P=0.101; mean tumor shrinkage rate: 26.6% vs. 27.5%). Conclusions: This study reported that QL1101 had similar efficacy in treating nonsquamous NSCLC in terms of ORR, PFS and OS based on two-year updated data, providing a basis for the clinical application of QL1101.

PlGF knockdown attenuates hypoxia-induced stimulation of cell proliferation and glycolysis of lung adenocarcinoma through inhibiting Wnt/ß-catenin pathway.

BACKGROUND: Angiogenic placental growth factor (PlGF) plays a role in hypoxia-induced angiogenesis. Here, we aimed to investigate the biological roles of PlGF in cell proliferation and glycolysis of lung adenocarcinoma (LUAD) and the underlying molecular mechanisms. METHODS: PlGF was knocked down in H358 and H1975 cells by lentiviruses, which were then cultured under hypoxia (90% N2, 5%CO2 and 5%O2) for 24 h. PlGF was overexpressed in PC9 cells treated with XAV939, inhibitor of Wnt/ß-catenin signaling pathway. PlGF-silencing H1975 cells were implanted into mice, and tumor xenografts were harvested and analyzed. RESULTS: Hypoxia treatment led to up-regulation of PlGF, C-myc, lactate dehydrogenase A (LDHA), and ß-catenin, promotion of cell proliferation and glycolysis in H358 and H1975 cells, which were obviously reversed by knocking down PlGF. In tumors, PlGF knockdown significantly prohibited cell proliferation and glycolysis, and decreased expression of C-myc, LDHA, and ß-catenin. PlGF overexpression markedly strengthened cell proliferation, which was inhibited by ß-catenin knockdown. Consistently, XAV939, inhibitor of Wnt/ß-catenin pathway, also inhibited PlGF-induced cell proliferation, glycolysis, and ß-catenin expression in PC9 cells. CONCLUSION: PlGF knockdown inhibited the stimulatory effect of hypoxia on cell proliferation and glycolysis of LUAD through deactivating Wnt/ß-catenin pathway.

Chemometric QSAR modeling of acute oral toxicity of Polycyclic Aromatic Hydrocarbons (PAHs) to rat using simple 2D descriptors and interspecies toxicity modeling with mouse.

The information of the acute oral toxicity for most polycyclic aromatic hydrocarbons (PAHs) in mammals are lacking due to limited experimental resources, leading to a need to develop reliable in silico methods to evaluate the toxicity endpoint. In this study, we developed the quantitative structure-activity relationship (QSAR) models by genetic algorithm (GA) and multiple linear regression (MLR) for the rat acute oral toxicity (LD50) of PAHs following the strict validation principles of QSAR modeling recommended by OECD. The best QSAR model comprised eight simple 2D descriptors with definite physicochemical meaning, which showed that maximum atom-type electrotopological state, van der Waals surface area, mean atomic van der Waals volume, and total number of bonds are main influencing factors for the toxicity endpoint. A true external set (554 compounds) without rat acute oral toxicity values, and 22 limit test compounds, were firstly predicted along with reliability assessment. We also compared our proposed model with the OPERA predictions and recently published literature to prove the prediction reliability. Furthermore, the interspecies toxicity (iST) models of PAHs between rat and mouse were also established, validated and employed for filling data gap. Overall, our developed models should be applicable to new or untested or not yet synthesized PAHs falling within the applicability domain (AD) of the models for rapid acute oral toxicity prediction, thus being important for environmental or personal exposure risk assessment under regulatory frameworks.

Serum TNFRII: A promising biomarker for predicting the risk of subcentimetre lung adenocarcinoma.

Early diagnosis of lung adenocarcinoma requires effective risk predictors. TNFRII was reported to be related to tumorigenesis, but remained unclear in lung cancer. This research set out to investigate the relationship between the sTNFRII (serum TNFRII) level and the risk of lung adenocarcinoma less than 1 cm in diameter. Seventy-one pairs of subcentimetre lung adenocarcinoma patients and healthy controls were analysed through multiplex bead-based Luminex assay and found a significantly lower expression of sTNFRII in patients with subcentimetre lung adenocarcinoma than that in the healthy controls (P < .001), which was further verified through ONCOMINE database analysis. Increased levels of sTNFRII reduced the risk of subcentimetre lung adenocarcinoma by 89% (P < .001). Patients with a higher level of BLC had a 2.70-fold (P < .01) higher risk of subcentimetre adenocarcinoma. Furthermore, a higher BLC/TNFRII ratio was related to a 35-fold higher risk of subcentimetre adenocarcinoma. TNFRII showed good specificity, sensitivity and accuracy (0.72, 0.75 and 0.73, respectively), with an AUC of 0.73 (P < .001). In conclusion, the present study assessed the value of sTNFRII as a potential biomarker to predict the risk of subcentimetre lung adenocarcinoma and provided evidence for the further use of TNFRII as an auxiliary marker in the diagnosis of subcentimetre lung adenocarcinoma.

Racial differences in characteristics and prognoses between Asian and white patients with nonsmall cell lung cancer receiving atezolizumab: An ancillary analysis of the POPLAR and OAK studies.

This study aimed to compare the differences in characteristics and prognoses between Asian and white patients receiving immunotherapy for nonsmall cell lung cancer (NSCLC). We studied 390 patients who received atezolizumab as part of the POPLAR or OAK trial, and analyzed the differences in baseline characteristics, outcomes and genetic mutations in blood samples between Asian and white patients. Overall survival (OS) was longer in Asian compared to white patients (median OS: 18.7 vs. 11.1 months; p = 0.005). Race was identified as an independent prognostic factor for OS (Asian vs. white: hazard ratio 0.647, 95% confidence interval 0.447-0.936, p = 0.021), together with performance status, histology, baseline sum of the longest tumor diameters (BLSLD) and number of metastatic sites. The two groups also differed in terms of characteristics including smoking history, BLSLD, epidermal growth factor receptor (EGFR) mutation frequency, programmed death-ligand 1 expression and blood-based tumor-mutation burden. Blood mutations of STK11, EGFR, KEAP1, POLE, GRM3, ATM and STAG2 were associated with treatment response, and TP53, KEAP1, APC, RB1, CREBBP, EPHA5 and STAG2 mutations were associated with OS. The blood-based mutation profiles differentiated between Asian and white patients, especially in relation to EGFR (23.8 vs. 8.5%), TP53 (30.2 vs. 46.9%) and STK11 (1.6 vs. 12.3%) mutations (all p < 0.05). The different clinicopathological features and mutation profiles in Asian and white patients may explain the superior outcome following atezolizumab treatment in Asian patients with NSCLC. The results of this study have important implications for further studies on racial disparities in relation to immunotherapy.

Erlotinib as Neoadjuvant Therapy in Stage IIIA (N2) EGFR Mutation-Positive Non-Small Cell Lung Cancer: A Prospective, Single-Arm, Phase II Study.

LESSONS LEARNED: The findings of this prospective, single-arm, phase II study showed that neoadjuvant erlotinib was well tolerated and might improve the radical resection rate in patients with stage IIIA-N2 epidermal growth factor receptor mutation-positive non-small cell lung cancer (NSCLC).Erlotinib shows promise as a neoadjuvant therapy option in this patient population.Next-generation sequencing may be useful for predicting outcomes with preoperative tyrosine kinase inhibitors (TKIs) in patients with NSCLC.Large-scale randomized controlled trials investigating the role of TKIs in perioperative therapy, combining neoadjuvant and adjuvant treatments to enhance personalized therapy for patients in this precision medicine era, are warranted. BACKGROUND: Information on epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) as neoadjuvant therapy in non-small cell lung cancer (NSCLC) is scarce. We evaluated whether neoadjuvant erlotinib improves operability and survival in patients with stage IIIA-N2 EGFR mutation-positive NSCLC. METHODS: We conducted a prospective, single-arm, phase II study. Patients received erlotinib 150 mg per day for 56 days in the neoadjuvant period. The primary endpoint was the radical resection rate. RESULTS: Nineteen patients were included in the final analysis. After erlotinib treatment, 14 patients underwent surgery. The radical resection rate was 68.4% (13/19) with a 21.1% (4/19) rate of pathological downstaging. The objective response rate was 42.1%; 89.5% (17/19) of patients achieved disease control, with a 10.3-month median disease-free survival among patients who underwent surgery. Among all 19 patients who received neoadjuvant therapy, median progression-free survival (PFS) and overall survival were 11.2 and 51.6 months, respectively. Adverse events (AEs) occurred in 36.8% (7/19) of patients, with the most common AE being rash (26.3%); 15.8% experienced grade 3/4 AEs. Quality of life (QoL) improvements were observed after treatment with erlotinib for almost all QoL assessments. Effects of TP53 mutation on prognosis were evaluated in eight patients with adequate tissue samples. Next-generation sequencing revealed that most patients had a TP53 gene mutation (7/8) in addition to an EGFR mutation. No TP53 mutation, or very low abundance, was associated with longer PFS (36 and 38 months, respectively), whereas high abundance was associated with short PFS (8 months). CONCLUSION: Neoadjuvant erlotinib was well tolerated and may improve the radical resection rate in this patient population. Next-generation sequencing may predict outcomes with preoperative TKIs.

Role of anlotinib-induced CCL2 decrease in anti-angiogenesis and response prediction for nonsmall cell lung cancer therapy.

BACKGROUND: Anlotinib has been demonstrated in clinical trials to be effective in prolonging the progression-free survival (PFS) and overall survival (OS) of refractory advanced nonsmall cell lung cancer (NSCLC) patients. However, the underlying molecular mechanisms and predictive biomarkers of anlotinib are still unclear. METHODS: A retrospective analysis of anlotinib administered to 294 NSCLC patients was performed to screen for underlying biomarkers of anlotinib-responsive patients. Transcriptome and functional assays were performed to understand the antitumour molecular mechanisms of anlotinib. Changes in serum CCL2 levels were analysed to examine the correlation of the anlotinib response between responders and nonresponders. RESULTS: Anlotinib therapy was beneficial for prolonging OS in NSCLC patients harbouring positive driver gene mutations, especially patients harbouring the epithelial growth factor receptor (EGFR)T790M mutation. Moreover, anlotinib inhibited angiogenesis in an NCI-H1975-derived xenograft model via inhibiting CCL2. Finally, anlotinib-induced serum CCL2 level decreases were associated with the benefits of PFS and OS in refractory advanced NSCLC patients. CONCLUSIONS: Our study reports a novel anti-angiogenesis mechanism of anlotinib via inhibiting CCL2 in an NCI-H1975-derived xenograft model and suggests that changes in serum CCL2 levels may be used to monitor and predict clinical outcomes in anlotinib-administered refractory advanced NSCLC patients using third-line therapy or beyond.

Adjuvant Chemotherapy Candidates in Stage I Lung Adenocarcinomas Following Complete Lobectomy.

BACKGROUND: This study aimed to explore adjuvant chemotherapy (ACT) candidates based on a recurrence risk-scoring model in completely lobectomized stage I patients with lung adenocarcinoma (LAD). METHODS: A retrospective study was performed on 4606 patients (non-ACT group: n = 3514; ACT group: n = 1092) who underwent complete lobectomy for LAD at Shanghai Chest Hospital from 2008 to 2014. The nomogram predicting recurrence-free survival (RFS) was developed in the non-ACT group using Cox proportional hazards regression. The nomogram-based risk score was calculated in the entire cohort. Differences of RFS between the non-ACT and ACT groups were compared as stratified by the risk score. The score cut-off points were determined using the X-tile software. RESULTS: Six independent predictors, including age, sex, tumor size, pathological subtype, visceral pleural invasion (VPI), and lymphovascular invasion (LVI) were associated with RFS. The nomogram more accurately predicted RFS than the 8th TNM staging {C-index: 0.784 [95% confidence interval (CI) 0.756-0.812] vs. 0.719 (95% CI 0.689-0.749), p = 0.0017}. A significant RFS difference was observed among the low-, intermediate- and high-risk groups (p < 0.0001), as divided by the optimal cut-points of risk score (203 and 244). ACT did not improve RFS for patients at intermediate-risk, or was even detrimental for low-risk patients; however, improved RFS was observed in ACT receivers at high-risk (p = 0.0416). ACT candidates with a risk score ≥ 245 constituted 2.6% of stage I patients. CONCLUSIONS: The nomogram provided an individual prediction of RFS for stage I LAD following lobectomy. High-risk patients (score ≥ 245) may benefit from postoperative ACT.

Genomic Landscape Survey Identifies SRSF1 as a Key Oncodriver in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is an aggressive disease with poor survival. A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, however, much still remains unknown, particularly in the Asian patient population. Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Dysregulation of tumor suppressor genes TP53 and RB1 was observed in 82% and 62% of SCLC patients, respectively, and more than half of the SCLC patients (62%) harbored TP53 and RB1 mutation and/or copy number loss. Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts. Functional studies in vitro and in vivo demonstrate that SRSF1 is important for tumorigenicity of SCLC and may play a key role in DNA repair and chemo-sensitivity. These results strongly support SRSF1 as a prognostic biomarker in SCLC and provide a rationale for personalized therapy in SCLC.

Pretreatment direct bilirubin and total cholesterol are significant predictors of overall survival in advanced non-small-cell lung cancer patients with EGFR mutations.

This study was designed to examine the prediction of pretreatment circulating bilirubin and cholesterol for overall survival in 459 advanced non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations. Circulating total bilirubin, direct bilirubin (DB), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were measured at baseline. The mean age (standard deviation) of all study patients was 58.7 (10.5) years, and 42.9% of them was males. Ever smokers accounted for 27.0% and lung adenocarcinoma for 90.4%. The median follow-up time and survival time were 29.5 and 34.9 months, respectively. Patients with higher DB had a 1.68-fold increased risk of death compared with patients with lower DB (hazard ratio [HR] = 1.68, 95% confidence interval [CI]: 1.22-2.30, p = 0.001), while patients with higher TC were at a 63% reduced risk of death compared with patients with lower TC (HR = 0.37, 95% CI: 0.20-0.67, p = 0.001). As for HDL-C, patients with higher levels had the risk of death reduced by 46% (HR = 0.54, 95% CI: 0.29-1.00, p = 0.049) compared with patients with lower levels. After the Bonferroni correction, only DB and TC were significantly associated with NSCLC survival. Our findings demonstrate for the first time that pretreatment DB was identified as a significant risk factor, yet TC as a protective factor, for overall survival in NSCLC patients with EGFR mutations.

Combination of chemotherapy and gefitinib as first-line treatment for patients with advanced lung adenocarcinoma and sensitive EGFR mutations: A randomized controlled trial.

To explore the optimal treatment strategy for patients who harbor sensitive EGFR mutations, a head-to-head study was performed to compare chemotherapy and gefitinib in combination or with either agent alone as first-line therapy, in terms of efficacy and safety. A total of 121 untreated patients with advanced lung adenocarcinoma who harbored sensitive EGFR mutations were randomly assigned to receive gefitinib combined with pemetrexed and carboplatin, pemetrexed plus carboplatin or gefitinib alone. The progression-free survival (PFS) of patients in the combination group (17.5 months, 95% CI, 15.3-19.7) was longer than that of patients in the chemotherapy group (5.7 months, 95% CI, 5.2-6.3) or gefitinib (11.9 months, 95% CI, 9.1-14.6) group. The (hazard ratios) HRs of PFS for the combination group vs. chemotherapy and gefitinib groups were 0.16 (95% CI, 0.09-0.29, p < 0.001) and 0.48 (95% CI, 0.29-0.78, p = 0.003), respectively. The overall response rate (ORR) in the combination therapy group, chemotherapy group and the gefitinib group was 82.5%, 32.5% and 65.9%, respectively. The combinational strategy resulted in longer overall survival (OS) than chemotherapy (HR = 0.46, p = 0.016) or gefitinib (HR = 0.36, p = 0.001) alone. Our finding suggested that treatment with pemetrexed plus carboplatin combined with gefitinib could provide better survival benefits for patients with lung adenocarcinoma harboring sensitive EGFR mutations.

Transcriptional profiling revealed the anti-proliferative effect of MFN2 deficiency and identified risk factors in lung adenocarcinoma.

Mitofusin-2 (MFN2) was initially identified as a hyperplasia suppressor in hyper-proliferative vascular smooth muscle cells (VSMCs) of hypertensive rat arteries, which has also been implicated in various cancers. There exists a controversy in whether it is an oncogene or exerting anti-proliferative effect on tumor cells. Our previous cell cycle analysis and MTT assay showed that cell proliferation was inhibited in MFN2 deficient A549 human lung adenocarcinoma cells, without investigating the changes in regulatory network or addressing the underlying mechanisms. Here, we performed expression profiling in MFN2 knockdown A549 cells and found that cancer-related pathways were among the most susceptible pathways to MFN2 deficiency. Through comparison with expression profiling of a cohort consisting of 61 pairs of tumor-normal matched samples from The Cancer Genome Atlas (TCGA), we teased out the specific pathways to address the impact that MFN2 ablation had on A549 cells, as well as identified a few genes whose expression level associated with clinicopathologic parameters. In addition, transcriptional factor target enrichment analysis identified E2F as a potential transcription factor that was deregulated in response to MFN2 deficiency. Although bioinformatics analysis usually entail further verification, our study provided considerable information for future scientific inquiries in related areas as well as a paradigm for characterizing perturbation in regulatory network.

The association between the polymorphisms in a sodium channel gene SCN7A and essential hypertension: a case-control study in the Northern Han Chinese.

Nax , an α-subunit of the sodium channel encoded by the SCN7A gene, has been deemed to be a sensor of the concentration of sodium in the brain and may be involved in salt intake behavior. We inferred that Nax /SCN7A may participate in the regulation of blood pressure and the pathogenesis of essential hypertension (EH). The present case-control study involving 615 hypertensives and 617 normotensives was performed to investigate the association between SCN7A polymorphisms and EH in the Northern Han Chinese population. The three common single nucleotide polymorphisms (SNPs) (rs3791251, rs6738031, rs7565062) in the exons of SCN7A were genotyped with the TaqMan assay. Significant association between SNP rs7565062 and EH was found under the addictive and dominant genetic models (P = 0.024, OR = 1.283, 95%CI [1.033-1.592]; P = 0.013, OR = 1.203, 95%CI [1.040-1.392]; respectively). The three SNPs were in close pair-wise linkage disequilibrium with each other and the haplotype analyses indicated that haplotype G-A-T was significantly associated with increased risk of EH (P = 0.023, OR = 1.290). In conclusion, our data showed that SNP rs7565062 of SCN7A was significantly associated with EH and the allele T of rs7565062 or the related haplotype G-A-T will be a genetic risk factor for EH in the Northern Han Chinese population.

Placental growth factor promotes metastases of non-small cell lung cancer through MMP9.

BACKGROUND/AIMS: Neovascularization and invasion coordinate cancer metastases in non-small cell lung cancer (NSCLC). However, the underlying molecular mechanisms are poorly understood. Recently, a substantial role of placental growth factor (PLGF) in cancer cell invasion has been acknowledged in several types of cancer, whereas a possible involvement of PLGF in the metastases of NSCLC has not been studied. METHODS: Here, we analyzed the levels of PLGF and matrix metalloproteinase 9 (MMP9) in NSCLC specimens. We modified either PLGF or MMP9 levels in a NSCLC cell line A549, and examined the effects on the levels of MMP9 and PLGF. The cell invasiveness was quantified in a transwell cell migration assay. Pathway inhibitors were applied to determine the molecular mechanisms underlying the control of MMP9 by PLGF. RESULTS: We found that PLGF and MMP9 levels both significantly increased in the NSCLC specimens and were strongly correlated. Overexpression of PLGF in NSCLC cells increased the levels of MMP9 and cell invasiveness, while inhibition of PLGF in NSCLC cells decreased the levels of MMP9 and cell invasiveness. However, modification of MMP9 levels in NSCLC cells did not alter the levels of PLGF. These data suggest that PLGF may regulate MMP9 in NSCLC cells, but not vice versa. Moreover, inhibition of MMP9 in PLGF-overexpressing NSCLC cells abolished the effects of PLGF on cell invasiveness, suggesting that PLGF increases cell invasion via MMP9. Furthermore, suppression of MAPK-p38, but not suppression of either MAPK-p42/p44, or PI3k, or JNK signaling, substantially abolished the effect of PLGF on MMP9, suggesting that PLGF may activate MMP9 via MAPK-p38 signaling pathway. CONCLUSION: PLGF-stimulated cancer invasion may be mediated through its effects on MMP9 activation in NSCLC cells.

Association of cholesteryl ester transfer protein (CETP) gene polymorphism, high density lipoprotein cholesterol and risk of coronary artery disease: a meta-analysis using a Mendelian randomization approach.

BACKGROUND: Recent randomized controlled trials have challenged the concept that increased high density lipoprotein cholesterol (HDL-C) levels are associated with coronary artery disease (CAD) risk reduction. The causal role of HDL-C in the development of atherosclerosis remains unclear. To increase precision and to minimize residual confounding, we exploited the cholesteryl ester transfer protein (CETP)-TaqIB polymorphism as an instrument based on Mendelian randomization. METHODS: The Mendelian randomization analysis was performed by two steps. First, we conducted a meta-analysis of 47 studies, including 23,928 cases and 27,068 controls, to quantify the relationship between the TaqIB polymorphism and the CAD risk. Next, the association between the TaqIB polymorphism and HDL-C was assessed among 5,929 Caucasians. We further employed Mendelian randomization to evaluate the causal effect of HDL-C on CAD based on the findings from the meta-analysis. RESULTS: The overall comparison of the B2 allele with the B1 allele yielded a significant risk reduction of CAD (P < 0.0001; OR = 0.88; 95% CI: 0.84-0.92) with substantial between-study heterogeneity (I² = 55.2%; P(heterogeneity) <0.0001). The result was not materially changed after excluding the Hardy-Weinberg Equilibrium (HWE)-violation studies. Compared with B1B1 homozygotes, Caucasian carriers of the B2 allele had a 0.25 mmol/L increase in HDL-C level (95% CI: 0.20-0.31; P <0.0001; I² = 0; P(heterogeneity) =0.87). However, a 1 standard deviation (SD) elevation in HDL-C levels due to the TaqIB polymorphism, was marginal associated with CAD risk (OR =0.79; 95% CI: 0.54-1.03; P =0.08). CONCLUSIONS: Taken together, our results lend support to the concept that increased HDL-C cannot be translated into a reduction in CAD risk.

MMP9 activation triggered by epidermal growth factor induced FoxO1 nuclear exclusion in non-small cell lung cancer.

The molecular mechanism underlying activation of matrix metallopeptidase 9 (MMP9) in non-small cell lung cancer (NSCLC) cells, which controls cancer invasiveness and metastasis, remains elusive. Here, we reported a strong correlation of epidermal growth factor receptor (EGFR) and MMP9 levels in NSCLC patients. Thus, we used a human NSCLC line, A549, to examine whether there is a causal link between EGFR and MMP9 activation. We found that EGF-induced activation of EGFR in A549 cells activated MMP9, resulting in an increase in cancer invasiveness. An EGFR inhibitor efficiently blocked this EGF-induced activation of MMP9 and, consequently, increased cancer invasiveness. Moreover, an inhibitor for phosphatidylinositol 3-kinase (PI3K)/Akt, but not an inhibitor for mitogen-activated protein kinase, or an inhibitor for Jun N-terminal kinase, significantly inhibited the epidermal growth factor (EGF)-induced activation of MMP9, suggesting that PI3K/Akt signaling cascades may be responsible for EGF-activated MMP9. We further dissected the pathway and found that nuclear exclusion of a major Akt downstream target, FoxO1, occurred by EGF-induced Akt activation, which could be inhibited by either EGFR inhibitor or by PI3K/Akt inhibitor. In a loss of function, expression of a constitutive nuclear form of FoxO1 significantly inhibited MMP9 activation induced by EGF. Taken together, these findings suggest that EGF/EGFR signaling activates downstream PI3K/Akt to induce FoxO1 nuclear exclusion, which activates MMP9 to promote NSCLC invasiveness. Thus, Akt and FoxO1, in addition to the well-known EGFR, appear to be promising therapeutic targets for preventing the metastasis of NSCLC.

Vitamin D inhibition of lung adenocarcinoma cell proliferation in vitro.

Vitamin D has the capability to inhibit tumor cell proliferation and promote tumor cell apoptosis but whether this mechanism exists in lung adenocarcinoma cells remains to be studied. Our objective is to explore whether vitamin D has the capability to inhibit lung adenocarcinoma cell proliferation and synergize with cisplatin. Our method was to explore the effect of different concentrations of 1,25(OH)2D3 with or without cisplatin on lung adenocarcinoma cells by detecting cell proliferation rates at different time points. 1,25(OH)2D3 was capsulated with nanomaterial before acting on lung adenocarcinoma cells, and cell proliferation rates at different time points were detected with the CCK-8 method. When vitamin D was applied at a concentration of 1 × 10(-7) and 1 × 10(-6) mol/L on A549, PC9, SPC-A1, and H1650 cells for 72 h, no inhibition occurred on cell proliferation. Between the concentrations of 1 × 10(-5) and 0.5 × 10(-5) mol/L, inhibition on cell proliferation increased with drug action time. Between the concentration of 2.5 × 10(-5) and 0.03 × 10(-5) mol/L, inhibition on cell proliferation increased with increasing drug concentration. Analysis using bivariate correlations showed that the correlation coefficient of the proliferation inhibition rate and drug content was 0.580 (p < 0.0001). The correlation coefficient of proliferation inhibition rate and the drug action time was 0.379 (p = 0.01). The combined use of vitamin D and dichlorodiammine-platinum(II) (DDP) significantly increased the inhibition rate on A549 cell proliferation, which peaked after culturing for 96 h (Table 4). Further analysis using bivariate correlations showed that the correlation coefficient between proliferation inhibition rate and DDP concentration was 0.319 (p < 0.0001). The correlation coefficient of the proliferation inhibition rate and vitamin D concentration was 0.269 (p < 0.0001). The correlation coefficient of proliferation inhibition and drug action time was 0.221(p = 0.003). Vitamin D capsulated with nanomaterial (5 ng/ml) on PC-9 cells for 72 h did not inhibit cell proliferation, while after 10 days, the content of crystal violet dissolved decreased by 6.3 ± 3.2% for the nonleaded nanomaterial group and decreased by 45.8 ± 10.9% for the nanomaterial-capsulated vitamin D group (p < 0.0001). Vitamin D has the capability to inhibit the proliferation of lung adenocarcinoma cells, synergistically inhibit the proliferation of lung adenocarcinoma cells with DDP, and when capsulated with nanomaterial can significantly inhibit the proliferation of lung adenocarcinoma cells.

IGF-1R and Bmi-1 expressions in lung adenocarcinoma and their clinicopathologic and prognostic significance.

IGF-1R and Bmi-1 play a critical role in cancer growth and survival. We explored the correlation between IGF-1R and Bmi-1, as well as their relationship with clinicopathological parameters and their impacts on outcomes in patients with lung adenocarcinoma resected. Tumors from 178 surgical lung adenocarcinoma patients were evaluated for IGF-1R and Bmi-1 expression by means of immunohistochemistry. The clinicopathological implications of these molecules were analyzed statistically. There was a significant correlation between the expression of IGF-1R and Bmi-1 (p = 0.011). The 5-year survival rate of patients with Bmi-1 positive was only 31.2%, but patients with Bmi-1 negative had a survival rate of 50.7% (p = 0.004). The pattern of survival curves showed that Bmi-1 was a significant prognostic factor of poor overall survival in lung adenocarcinoma patients. However, there was no obvious correlation between IGF-1R expression and patient survival. The results of multivariate Cox analysis revealed that the pathological stages and Bmi-1 expression were independent prognostic factors. Therefore, Bmi-1 may be a good biomarker to predict the prognosis of patients with completely resected lung adenocarcinoma.