The Comparison of Serum Exosome Protein Profile in Diagnosis of NSCLC Patients.

A thorough study of the exosomal proteomic cargo may enable the identification of proteins that play an important role in cancer development. The aim of this study was to compare the protein profiles of the serum exosomes derived from non-small lung cancer (NSCLC) patients and healthy volunteers (control) using the high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) method to identify potentially new diagnostic and/or prognostic protein biomarkers. Proteins exclusively identified in NSCLC and control groups were analyzed using several bioinformatic tools and platforms (FunRich, Vesiclepedia, STRING, and TIMER2.0) to find key protein hubs involved in NSCLC progression and the acquisition of metastatic potential. This analysis revealed 150 NSCLC proteins, which are significantly involved in osmoregulation, cell-cell adhesion, cell motility, and differentiation. Among them, 3 proteins: Interleukin-34 (IL-34), HLA class II histocompatibility antigen, DM alpha chain (HLA-DMA), and HLA class II histocompatibility antigen, DO beta chain (HLA-DOB) were shown to be significantly involved in the cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) infiltration processes. Additionally, detected proteins were analyzed according to the presence of lymph node metastasis, showing that differences in frequency of detection of protein FAM166B, killer cell immunoglobulin-like receptor 2DL1, and olfactory receptor 52R1 correlate with the N feature according to the TNM Classification of Malignant Tumors. These results prove their involvement in NSCLC lymph node spread and metastasis. However, this study requires further investigation.

Targeting SOX18 Transcription Factor Activity by Small-Molecule Inhibitor Sm4 in Non-Small Lung Cancer Cell Lines.

The transcription factor SOX18 has been shown to play a crucial role in lung cancer progression and metastasis. In this study, we investigated the effect of Sm4, a SOX18 inhibitor, on cell cycle regulation in non-small cell lung cancer (NSCLC) cell lines LXF-289 and SK-MES-1, as well as normal human lung fibroblast cell line IMR-90. Our results demonstrated that Sm4 treatment induced cytotoxic effects on all three cell lines, with a greater effect observed in NSCLC adenocarcinoma cells. Sm4 treatment led to S-phase cell accumulation and upregulation of p21, a key regulator of the S-to-G2/M phase transition. While no significant changes in SOX7 or SOX17 protein expression were observed, Sm4 treatment resulted in a significant upregulation of SOX17 gene expression. Furthermore, our findings suggest a complex interplay between SOX18 and p21 in the context of lung cancer, with a positive correlation observed between SOX18 expression and p21 nuclear presence in clinical tissue samples obtained from lung cancer patients. These results suggest that Sm4 has the potential to disrupt the cell cycle and target cancer cell growth by modulating SOX18 activity and p21 expression. Further investigation is necessary to fully understand the relationship between SOX18 and p21 in lung cancer and to explore the therapeutic potential of SOX18 inhibition in lung cancer.

Genetic variations in the ATP-binding cassette transporter ABCC10 are associated with neutropenia in Japanese patients with lung cancer treated with nanoparticle albumin-bound paclitaxel.

ABCC10/MRP7, an ATP-binding cassette (ABC) transporter, has been implicated in the extracellular transport of taxanes. Our group reported that the ABCC10 single nucleotide polymorphism (SNPs), rs2125739, influences docetaxel cytotoxicity in lung cancer cell lines as well as its side effects in clinical practice. In this study, we investigated whether the rs2125739 variant could affect paclitaxel (PTX) cytotoxicity in lung cancer cell lines. We also investigated the effect of rs2125739 on the efficacy and safety of nanoparticle albumin-bound PTX (nab-PTX) in clinical practice. The association between rs2125739 genotypes and the 50% inhibitory concentration (IC50) of PTX was investigated in 18 non-small cell lung cancer (NSCLC) cell lines, HeLa cells, and genome-edited HeLa cells. Next, blood samples from 77 patients with NSCLC treated with carboplatin plus nab-PTX were collected and analyzed for six SNPs, including rs2125739. The clinical outcomes among the different genotype groups were evaluated. In NSCLC cell lines, HeLa cells, and genome-edited HeLa cells, the IC50 was significantly higher in the ABCC10 rs2125739 T/T group than in the T/C and C/C groups. In 77 patients with NSCLC, there were no significant differences in clinical outcomes between the T/T and T/C groups. However, the rs2125739 T/T genotype was associated with a higher frequency of Grades 3/4 neutropenia. In contrast, there was no association between other SNPs and clinical efficacy or neutropenia. Our results indicate that the ABCC10 rs2125739 variant is associated with neutropenia in response to nab-PTX treatment.

Regioselective Synthesis of 6-O-Acetyl Dieckol and Its Selective Cytotoxicity against Non-Small-Cell Lung Cancer Cells.

Dieckol, a phlorotannin from Ecklonia cava, has shown potential for use as an anticancer agent that selectively kills cancer cells. However, it is necessary to amplify its potency without damaging its inherent safety in order to develop it as a competitive chemotherapeutic. Here, we explored the controlled O-acylations of dieckol. Acyl groups could be consistently introduced to the 6-O position of dieckol with a high regioselectivity, which was confirmed by NOESY, HMBC and HSQC spectroscopies. In cytotoxicity studies on the newly synthesized 6-O-acetyl, 6-O-benzoyl dieckols and previously synthesized 6-O-alkyl dieckols against A549 vs. normal cells, all of the derivatives showed low cytotoxicity in normal cells with an IC50 of 481-719 µM, and highly structure-dependent cytotoxicity in A549 cells with an IC50 of 7.02 (acetyl)-842.26 (benzyl) µM. The selectivity index also showed a large structure dependency in the range of 0.67 (benzyl)-68.58 (acetyl). An analysis of the structure-activity relationship indicated that the activity was dramatically reduced in the presence of a benzene ring and was highly increased in the presence of small polar substituents. Conclusions: Controlled mono-O-modifications of dieckol could be a powerful tool to enhance the anticancer activity of dieckol, thus contributing to the development strategy for dieckol-based chemotherapeutics.

miR-145-5p Targets Sp1 in Non-Small Cell Lung Cancer Cells and Links to BMI1 Induced Pemetrexed Resistance and Epithelial-Mesenchymal Transition.

Pemetrexed is a folic acid inhibitor used as a second-line chemotherapeutic agent for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC), which accounts for 85% of lung cancers. However, prolonged treatment with pemetrexed may cause cancer cells to develop resistance. In this study, we found increased expressions of BMI1 (B Lymphoma Mo-MLV insertion region 1 homolog) and Sp1 and a decreased expression of miR-145-5p was found in pemetrexed-resistant A400 cells than in A549 cells. Direct Sp1 targeting activity of miR-145-5p was demonstrated by a luciferase based Sp1 3'-UTR reporter. Changed expression of miR-145-5p in A400 or A549 cells by transfection of miR-145-5p mimic or inhibitor affected the sensitivity of the cells to pemetrexed. On the other hand, the overexpression of Sp1 in A549 cells caused the decreased sensitivity to pemetrexed, induced cell migratory capability, and epithelial-mesenchymal transition (EMT) related transcription factors such as Snail Family Transcriptional Repressor 1 and Zinc Finger E-Box Binding Homeobox 1. In addition, the overexpression of BMI1 in the A549 cells resulted in an increase in Sp1 and a decrease in miR-145-5p accompanied by the elevations of cell proliferation and EMT transcription factors, which could be reduced by the overexpression of miR-145-5p or by treatment with the Sp1 inhibitor of mithramycin A. In conclusion, the results of this study suggest that the downregulation of miR-145-5p by BMI1 overexpression could lead to the enhanced expression of Sp1 to induce the EMT process in pemetrexed-resistant NSCLC cells. These results suggest that increasing miR-145-5p expression by delivering RNA drugs may serve as a sensitizing agent for pemetrexed-resistant NSCLC patients.

Akt/mTOR Targeting Activity of Resveratrol Derivatives in Non-Small Lung Cancer.

The Akt-mTOR signal is important for the survival and proliferation of cancer cells and has become an interesting drug target. In this study, five resveratrol derivatives were evaluated for anticancer activity and Akt/mTOR targeting activity in non-small lung cancer cell lines. The effects of resveratrol derivatives on cell proliferation were assessed by 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, nucleus staining, and colony formation assay. Furthermore, the effect of resveratrol derivatives on proliferation-related protein expression was analyzed by immunofluorescence and Western blotting. For the structure-activity relationship (SAR), results reveal that two derivatives of resveratrol which are 4,4'-(ethane-1,2-diyl) bis(2-methoxyphenol) (RD2) and the 4-(3-hydroxy-4-methoxyphenethyl)-2-methoxyphenol (RD3) had very similar structures but exerted different cytotoxicity. The IC50 of RD2 and RD3 were 108.6 ± 10.82 and more than 200 µM in the A549 cell line and 103.5 ± 6.08 and more than 200 µM in H23 cells, respectively. RD2 inhibited cell proliferation and induced apoptosis when compared with the control, while RD3 caused minimal effects. Cells treated with RD2 exhibited apoptotic nuclei in a concomitant with the reduction of cellular p-Akt and p-mTOR. RD3 had minimal effects on such proteins. According to these results, molecular docking analysis revealed a high-affinity interaction between RD2 and an Akt molecule at the ATP-binding and the allosteric sites, indicating this RD2 as a potential Akt inhibitor. This study provides useful information of resveratrol derivatives RD2 for treating lung cancer via Akt/mTOR inhibition.

Reviving up dendritic cells can run cancer immune wheel in non-small cell lung cancer: a prospective two-arm study.

BACKGROUND AND AIM: Lung cancer is the number one cause of cancer-related deaths. Dendritic cells (DCs) are heterogeneous components of innate immunity that play a crucial role in the anti-tumor T cell immunity and may represent a promising approach for tumor immunotherapy. In this study, we aimed to evaluate the frequency of the two major subsets of DCs; plasmacytoid dendritic cells (pDCs) and monocytic dendritic cells (mDCs) in non-small cell lung cancer (NCSLC) and correlating them with different clinicopathologic features and survival outcomes. PATIENTS AND METHODS: This study was a case-controlled one, included 50 patients with denovo pathologically confirmed NSCLC and 20 healthy controls of comparable age and gender. After diagnosis and staging of patients, the frequency of DCs was evaluated using flow cytometry. RESULTS: We unveiled significantly reduced levels of pDCs (P = 0.024), and mDCs (P = 0.013) in NSCLC patients compared to controls. Furthermore, there was a significant accumulation of pDCs in non-metastatic patients compared to metastatic ones (P < 0.0001), while there was no significant (P = 0.6) differences in mDCs, and mDCs/pDCs ratio (P = 0.9). There was a Significant negative correlation (r = - 0.3, P = 0.04) between OS and mDCs. On the other hand, there was a significantly higher OS with pDCs ≥ 0.82 compared to patients with pDCs < 0.82, log rank Ch2 = 12.128, P < 0.0001. CONCLUSION: Despite the controversy about the prognostic role of pDCs not only in NSCLC but also in other solid tumors, our study sheds light on the possible prognostic impact of pDCs and mDCs on treatment outcomes of NSCLC patients.

[ANXA8 Regulates Proliferation of Human Non-Small Lung Cancer Cells A549 via EGFR-AKT-mTOR Signaling Pathway].

Annexin A8 (ANXA8) is a member of the annexin family, which had been reported to regulate multiple cancer cellular processes including proliferation, metastasis and inflammation. However, the specific role of ANXA8 in lung cancer cell biology remains unknown. Our previous transcriptome study revealed that ANXA8 mRNA was downregulated in curcumin analog (MHMD) -treated human non-small lung cancer cells (A549 cell line). Here, we continued to study the ANXA8 expression in A549 cells using reverse transcription-quantitative PCR and Western blotting, compared with that in human normal bronchial epithelium cells (BE-AS-2B cell line). Overexpression of ANXA8 via transfection of pEGFP-ANXA8 recombinant vector contributed to the proliferation and migration of A549 cells. Moreover, the cell cycle protein cyclin E1 was upregulated in ANXA8-transfected A549 cells. Knockdown of ANXA8 using an RNA interference technique decreased A549 cell viability and restrained their migration in vitro. The expression levels of multiple cellular factors, including EGFR, PI3K, Akt, mTOR, p70S6K and 4EBP1, in the epidermal growth factor receptor (EGFR) signaling pathway were also altered by ANXA8 knockdown or overexpression in A549 cells, which confirmed the activation of the EGFR/Akt/mTOR signaling pathway by ANXA8. The present results provided evidence to support further investigation of the functional identification of ANXA8 in lung cancer cells in the future.

Correlation study between flash dual source CT perfusion imaging and regional lymph node metastasis of non-small cell lung cancer.

BACKGROUND: To explore the correlation of flash dual source computed tomography perfusion imaging (CTPI) and regional lymph node metastasis of non-small cell lung cancer (NSCLC), and to evaluate the value of CT perfusion parameters in predicting regional lymph node metastasis of NSCLC. METHODS: 120 consecutive patients with NSCLC confirmed by postoperative histopathology were underwent flash dual source CT perfusion imaging in pre-operation. The CT perfusion parameters of NSCLC, such as blood flow (BF), blood volume (BV), mean transit time (MTT) and permeability (PMB) were obtained by the image post-processing. Then microvessel density (MVD), luminal vascular number (LVN), luminal vascular area (LVA) and luminal vascular perimeter (LVP) of NSCLC were counted by immunohistochemistry. These cases were divided into group A (patients with lymph node metastasis, 58 cases) and group B (patients without lymph node metastasis, 62 cases) according to their pathological results. The CT perfusion parameters and the microvessel parameters were contrastively analysed between the two groups. Receiver operating characteristic (ROC) curve was used to assess the diagnostic efficiency of CT perfusion parameters in predicting regional lymph node metastasis of NSCLC in pre-operation. RESULTS: Group A presented significantly lower LVA, BF and higher MTT, PMB than Group B (P < 0.05), while BV, LVN, LVP and MVD were no significant difference (P > 0.05). Correlation analysis showed that BF was correlated with LVA and LVP (P < 0.05), while BV, MTT and PMB were not correlated with LVN, LVA and LVP (P > 0.05). All the perfusion parameters were not correlated with MVD. According to the ROC curve analysis, when BF < 85.16 ml/100 ml/min as a cutoff point to predict regional lymph node metastasis of NSCLC, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value were 60.8, 81.7, 71.5, 75.6 and 69.5% respectively. CONCLUSION: Flash dual source CT perfusion imaging can non-invasively indicate the luminal vascular structure of tumor and BF can be used as one of the important indexes in predicting regional lymph node metastasis of NSCLC in pre-operation.

One novel curcumin derivative ZYX01 induces autophagy of human non-small lung cancer cells A549 through AMPK/ULK1/Beclin-1 signaling pathway.

Presently, curcumin derivatives had been paid more attention in view of their high bioavailability or water solubility, which herein possibly replaced the curcumin for their functional applications in future. Here, one novel chemically synthesized curcumin derivative, ZYX01, was used to identify anti-proliferation activity of human non-small lung cancer cells A549 and its anti-proliferative mechanism. Our study showed that ZYX01 could induce autophagic death of A549 cells by morphological observation, MTT assay, acridine orange staining and MDC assay, which possess a dose-and time-dependent manner. ZYX01-treated A549 cells possessed an increase in LC3-II/LC3-I ratio, upregulation of beclin-1 and downregulation of p62 expression. We further confirmed the cellular AMPK/ULK1/Beclin-1 signaling pathway in A549 cells after ZYX01 treatment. The anti-migration effect of ZYX01 in A549 cells was also explored by wound healing assay and transwell experiment. Current results had confirmed that ZYX01 induced A549 cells autophagy through AMPK/ULK1/Beclin-1 pathway and shed light on the future study on the anti-cancer molecular mechanism.

The Water Extract of Juniperus communis L. Induces Cell Death and Sensitizes Cancer Cells to Cytostatic Drugs through p53 and PI3K/Akt Pathways.

Juniper (Juniperus communis L.) is a northern coniferous plant generally used as a spice and for nutritional purposes in foods and drinks. It was previously reported that juniper extract (JE) affects p53 activity, cellular stress, and gene expression induced cell death in human neuroblastoma cells. Therefore, the effects of juniper on p53 and Akt signaling was examined further in A549 lung, 22RV1 and DU145 prostate, and HepG2 liver cancer cells using Western blot, confocal microscopy, and MTT analysis. We found that juniper simultaneously decreased cell viability, activated the p53 pathway, and inactivated the PI3K/Akt pathway. The p53 activation was associated with increased nuclear p53 level. Akt was dephosphorylated, and its inactivation was associated with increased levels of PHLPP1 and PHLPP2 phosphatases. Parallel increases of PARP suggest that JE decreased cell viability by activating cell death. In adtion, JE potentiated the effects of gemcitabine and 5-fluorouracil anticancer drugs. Thus, JE can activate cell death in different cancer cell lines through p53 and Akt pathways.

Excellence in non-small cell lung cancer staging by endobronchial-TBNA: Comparison with PET-CT and surgery.

Objective: To determine the correlation and/or discrepancies between positron emission tomography (PET-CT) findings, endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and surgery in the staging of non-small cell lung carcinoma. Material and methods: Data were evaluated retrospectively from a prospective interventional endoscopy database. Positive results with EBUS-TBNA was the first end point and all cytology negatives were confirmed with mediastinoscopy/surgery. Results: Four hundred and eighty three patients were included and 1017 lymph nodes (LNs) were sampled in the study. One hundred and twenty eight LNs were excluded (positive with EBUS-TBNA). Four hundred and sixty five LN (52.3%) were found benign with EBUS-TBNA; however, only 15 of these were confirmed to be malignant by surgery (1.7%). The sensitivity, specificity, PPV, NPV and diagnostic accuracy of EBUS-TBNA were 96.5, 100, 100, 96.7 and 98.3%, respectively. The sensitivity, specificity, PPV, NPV and diagnostic accuracy of PET-CT for maximum standardized uptake value (SUVmax) 2.5 were 90.1, 29.2, 55.3, 75.4, 59.2%, respectively. A cut-off SUVmax of 5.2 was detected with 74.8% sensitivity, 84% specificity, 82.0% PPV, 77.5% NPV and 79.5% accuracy (area under the curve (AUC) of 0.818, 95% CI 0.791-0.843, p<.001). Conclusion: EBUS is a reliable, repeatable and safe technique with a high diagnostic accuracy and should be performed quickly to avoid superfluous time loss in the staging of lung cancer. Abbreviations PET-CT F-18 fluorodeoxyglucose positron emission computed tomography NSCLC Non-small cell lung cancer EBUS-TBNA Endobronchial ultrasound-guided transbronchial needle aspiration SUVmax Maximum standardized uptake value LNs Lymph nodes TTF-1 Thyroid transcription factor-1 H&E Hematoxylin and eosin; Med: Mediastinoscopy VATS Video associated thoracic surgery AUC Area under curve OR Odds ratio CI Confidence intervals.

[Silencing Effect of APE1 on the Radiosensitivity of Non-small Cell Lung Cancer Cells].

OBJECTIVE: To determine the effect of apurinic/apyrimidinic endonuclease-1(APE1) on the radiosensitivity of non-small cell lung cancer cells. METHODS: The expression of APE1 was detected by immunohistochemistry in 20 surgical specimen of non-small cell lung cancer tissues and 5 cases of pericarcinous lung tissues.The expressions of APE1 protein and mRNA in non-small cell lung cancer cell lines(A549, H460, H1299)and normal lung epithelial cell lines was detected by Western blot and qRT-PCR. The A549 and H460 stable cell lines with silencing of APE1 were constructed.The expression of APE1 in the silenced and non-silenced cells after exposure to 0-6 Gy radiation was detected by Western blot. The effect of silencing of APE1 was measured by colony formation assay, the proliferation of non-small cell lung cancer cells detected by CCK-8 assay, and the apoptosis of non-small cell lung cancer cells detected by Annexin Ⅴ/PI flow cytometry. RESULTS: The expression of APE1 was upregulated in non-small cell lung cancer tissues and cell lines.Radiation induced the expression of APE1 in non-small cell lung cancer cells in a dose responsive way. The silencing of APE1 inhibited the expression of APE1 induced by radiation in A549 and H460 cells. Exposure to radiation of the cells with silenced APE1 further inhibited cell colony formation and cell proliferation, and promoted the apoptosis of non-small cell lung cancer cells ( P<0.05). CONCLUSION: Silencing of APE1 enhances the radiosensitivity of non-small cell lung cancer cells.

Combined effect of cabozantinib and gefitinib in crizotinib-resistant lung tumors harboring ROS1 fusions.

The ROS1 tyrosine kinase inhibitor (TKI) crizotinib has shown dramatic effects in patients with non-small cell lung cancer (NSCLC) harboring ROS1 fusion genes. However, patients inevitably develop resistance to this agent. Therefore, a new treatment strategy is required for lung tumors with ROS1 fusion genes. In the present study, lung cancer cell lines, HCC78 harboring SLC34A2-ROS1 and ABC-20 harboring CD74-ROS1, were used as cell line-based resistance models. Crizotinib-resistant HCC78R cells were established from HCC78. We comprehensively screened the resistant cells using a phosphor-receptor tyrosine kinase array and RNA sequence analysis by next-generation sequencing. HCC78R cells showed upregulation of HB-EGF and activation of epidermal growth factor receptor (EGFR) phosphorylation and the EGFR signaling pathway. Recombinant HB-EGF or EGF rendered HCC78 cells or ABC-20 cells resistant to crizotinib. RNA sequence analysis by next-generation sequencing revealed the upregulation of AXL in HCC78R cells. HCC78R cells showed marked sensitivity to EGFR-TKI or anti-EGFR antibody treatment in vitro. Combinations of an AXL inhibitor, cabozantinib or gilteritinib, and an EGFR-TKI were more effective against HCC78R cells than monotherapy with an EGFR-TKI or AXL inhibitor. The combination of cabozantinib and gefitinib effectively inhibited the growth of HCC78R tumors in an in vivo xenograft model of NOG mice. The results of this study indicated that HB-EGF/EGFR and AXL play roles in crizotinib resistance in lung cancers harboring ROS1 fusions. The combination of cabozantinib and EGFR-TKI may represent a useful alternative treatment strategy for patients with advanced NSCLC harboring ROS1 fusion genes.

Levels of serum bilirubin in small cell lung cancer and non-small cell lung cancer patients.

Elevated bilirubin has been associated with protection of cardiovascular and kidney systems, whereas decreased bilirubin may predispose respiratory diseases. However, whether serum bilirubin levels are associated with lung cancer remains unclear. Here, clinical and pathologic data of a cohort of 363 lung cancer patients along with 363 age-and gender-matched healthy subjects were collected. The association of serum bilirubin levels with lung cancer was analyzed. The levels of serum bilirubin were significantly lower in lung cancer patients. The aspartate transaminase and alkaline phosphatase levels were significantly higher in lung cancer. Multi-classification logistics regression analysis revealed low total bilirubin level [OR (95%CI), 1.12 (1.02-1.23)], aspartate transaminase [OR (95%CI), 1.12 (1.02-1.23)], and alanine transaminase [OR (95%CI), 1.12 (1.02-1.23)] were risk factors in lung cancer. Serum bilirubin levels were significantly changed among small cell lung cancer (SCLC), lung adenocarcinoma (LAC) and lung squamous cell carcinoma (LSC). Total bilirubin level, smoke history and heart disease were risk factors for subtypes. Compared with LSC, patients with smoke history had significant higher risk in LAC [OR (95% Confidence Interval, CI), 4.49 (1.70, 11.96)]. Compared with LSC, patients with smoke history [OR (95%CI), 4.49 (1.70, 11.96)] and heart disease [OR (95%CI), 4.49 (1.70, 11.96)] had significant higher risk in SCLC. Compared with SCLC, patients with low total bilirubin [OR (95%CI), 1.12 (1.02-1.23)] and heart disease [OR (95%CI), 3.52 (1.01-12.23)] had significant higher risk in LAC. Taken together, these results suggested low serum bilirubin levels are tightly associated with lung cancer, especially with LAC. Serum bilirubin levels might serve as a predictor for lung cancer patients clinically.

In Silico Selection Approach to Develop DNA Aptamers for a Stem-like Cell Subpopulation of Non-small Lung Cancer Adenocarcinoma Cell Line A549.

BACKGROUND: Detection of circulating lung cancer cells with cancer-stem like characteristics would represent an improved tool for disease prognosis. However, current antibodies based methods have some disadvantages and therefore cell SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was used to develop DNA aptamers, recognizing cell surface markers of non-small lung carcinoma (NSLC) cells. MATERIALS AND METHODS: The human adenocarcinoma cell line A549 was used for selection in seven cell SELEX cycles. We used human blood leukocytes for negative selection, and lung stem cell protein marker CD90 antibody binding A549 cells for positive selection. RESULTS: The obtained oligonucleotide sequences after the seventh SELEX cycle were subjected to in silico selection analysis based on three independent types of bioinformatics approaches, selecting two closely related aptamer candidates in terms of consensus sequences, structural motifs, binding affinity (Kd) and stability (ΔG). We selected and identified the aptamer A155_18 with very good binding characteristics to A459 cells, selected for CD90 antibody binding. The calculated phylogenetic tree showed that aptamers A155_18 and the known A549 cell aptamer S6 have a close structural relationship. MEME sequence analysis showed that they share two unique motifs, not present in other sequences. CONCLUSIONS: The novel aptamer A155_18 has strong binding affinity for A549 lung carcinoma cell line subpopulation that is expressing stem cell marker CD90, indicating a possible stemness, characteristic for the A459 line, or a subpopulation present within this cell line. This aptamer can be applied as diagnostic tool, identifying NSLC circulating cells.

MiR-509-5p suppresses the proliferation, migration, and invasion of non-small cell lung cancer by targeting YWHAG.

Dysregulation of microRNAs (miRNAs) expression is prevalent in human malignancy development and progression. As previously reported, miR-509-5p has been identified as a tumor suppressor in various cancers, but its role and functional mechanism in non-small lung cancer (NSCLC) remains unknown. In the present study, we found that miR-509-5p was significantly downregulated in NSCLC tissues and cell lines using Quantitative real-time PCR (qRT-PCR). In addition, overexpression of miR-509-5p markedly inhibited the proliferation, migration, invasion and phosphorylation of Akt of NSCLC cells. Moreover, we identified tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) as a direct target of miR-509-5p. Knockdown of YWHAG mimicked the inhibitory effects of miR-509-5p on NSCLC cells for proliferation and motility. Finally, the negative correlation between miR-509-5p and YWHAG expression in NSCLC specimens was demonstrated. Taken together, our present study revealed the tumor suppressive role of miR-509-5p in NSCLC by targeting YWHAG, suggesting that miR-509-5p/YWHAG axis might be considered as a novel and potential target for clinical diagnosis and therapeutics of NSCLC.

Prediction of skeletal-related events in patients with non-small cell lung cancer.

PURPOSE: Advanced lung cancer frequently causes bone metastasis which can be associated with skeletal-related events (SREs) that may cause significant deterioration of the patient's quality of life (QoL). The Spinal Instability Neoplastic Score (SINS) can be used to assist in standardizing evaluations of neoplastic spinal instability between spinal and non-spine surgeons. This research investigated the association between SREs and SINS for patients with non-small cell lung cancer (NSCLC). METHODS: Between 2009 and 2013, 47 patients with NSCLC who were diagnosed with bone metastasis were classified using SINS into either a stable group (SINS, 0-6 points) or unstable group (SINS, 7-18 points). The primary endpoint was time from diagnosis of metastasis to SREs. Secondary endpoints included tumor type and epidermal growth factor receptor (EGFR) mutational status. SREs were defined as spinal compression, pathologic fracture, spinal surgery, and hypercalcemia. RESULTS: Patients included 37 cases of adenocarcinoma and 10 cases of squamous cell carcinoma. Mean follow-up time was 10.2 ± 13.7 months. SRE incidence was 15.0 % (3/20) in the stable group versus 44.4 % (12/27) in the unstable group (p = 0.048). A Cox regression model revealed that an EGFR-positive mutational status (hazard ratio [HR] = 0.15, 95 % CI, 0.030.71; p = 0.017) or good spinal stability (HR = 0.49; 0.08-0.99; p = 0.049) were favorable prognostic factors. CONCLUSION: The incidence of SREs was significantly lower in NSCLC patients with better spinal stability as determined by SINS, which was a good prediction tool for SREs from bone metastasis. The lower incidence of SREs in EGFR-positive patients suggests tumor biology should be considered when predicting SREs.

A two kinase-gene signature model using CDK2 and PAK4 expression predicts poor outcome in non-small cell lung cancers.

Risk classification on the basis of specific genomic features can lead to more precise tailoring of treatment for cancer patients. Kinases are potential therapeutic targets and survival factors, but the predictive prognostic potentials of multi-kinase genes have seldom been investigated. In this study, with publicly available microarray data of non-small cell lung cancers (NSCLC), we identified two kinase genes cyclin-dependent kinase 2 (CDK2) and p21 protein (Cdc42/Rac)-activated kinase 4 (PAK4) significantly associated with poor outcome. Then we present a combined gene signature model using CDK2 and PAK4 that can stratify disease poor outcome independently of standard clinical prognostic factors. Next, the predictive robustness of this 2-gene classifier was in silico confirmed in an independent microarray dataset, and experimentally validated in a lung cancer cohort by immunohistochemistry. Therefore, in this study, we demonstrated that the CDK2-PAK4 kinase signature may be a useful prognostic indicator and potential target for NSCLC. We also propose that poor outcome subgroup stratified by this classifier may benefit from the recently developed CDK2 and PAK4 inhibitors.

E-cadherin mediates adhesion of Aspergillus fumigatus to non-small cell lung cancer cells.

A spergillus fumigatus is a widely distributed microorganism, and recently, A. fumigatus culture filtrate has been shown to trigger apoptotic cell death in several human cancer cell lines, including non-small lung cancer (NSCLC) cells, A549. Nevertheless, the molecular adhesion of A. fumigatus to these cancer cells to trigger cell death remains unknown. Here, we knocked down E-cadherin in A549 cells and examined its effects on A. fumigatus. The blastospores of A. fumigatus were incubated with the complete protein extracts from A549 cells, using an affinity purification procedure. Preliminary exploration of E-cadherin-interacting protein on the surface of Aspergillus fumigates was done by immunoprecipitation and mass spectrometry analysis. We found that the adhesion of the blastospores to A549 cells was significantly reduced by E-cadherin suppression in A549 cells, suggesting that E-cadherin of A549 cells may mediate the surface adhesion of A. fumigatus blastospore. Mass spectrometry (MS) analysis predicted two binding proteins for E-cadherin on A. fumigatus, AfA24A6.130c and XP_747789. Finally, the growth of E-cadherin-depleted A549 cells significantly increased by infection of A. fumigatus in vivo. Thus, our study suggests that E-cadherin mediates adhesion of A. fumigatus to NSCLC cells to trigger cell death and provides molecular evidence for the treatment of NSCLC with controlled A. fumigatus infection.