CL1-5 Cells Research Articles

Abstract 192: The extracellular vesicles from lung cancer cells activates Stat3 and tumor angiogenesis

Abstract Current research suggests that on tumor-related extracellular vesicles (EV) play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms including angiogenesis. Our previous study demonstrated that IL6 induced secretion of VEGF and contributing angiogenesis via activation of Stat3. To further clarify the biological activities of the lung cancer derived EVs, we analyzed the content of IL6 and VEGF in exosome using ELISA kit. We found that IL-6 and VEGF were more enriched in EVs than non-EVs in AS2 and CL1-5 cells. And the isolated EV from AS2 and CL1-5 cells increased vascular permeability in miles assay and promotes angiogenesis in tubular formation assay. We further investigated if the inhibition of exosome secretion affects the autocrine IL6/Stat3/VEGF pathway. GW4869 inhibits secretion of exosome by playing an inhibitor of neutral sphingomyelinase (nSMase). Treatment of either cell with GW4869 significantly decreased the levels of released exosomes in AS2 but not in CL1-5 cell. Consistent with the result, the treatment GW4869 dampened autocrine IL6/Stat3/VEGF pathway in AS2 but not in CL1-5 cell. Bevacizumab, the recombinant anti- VEGF monoclonal antibody, were approved for treating patients with advanced NSCLC, has been shown to be efficient in suppressing the accumulation of pleural fluid (malignant pleural effusion). To investigate if the exosomal VEGF has a different affinity for bevacizumab, we treated AS2, CL1-5 and MPE-derived cancer cells with bevacizumab and EV or non-EV part from cell-free medium. We found VEGF dimer form were more abundant in EV part and cannot be neutralized by bevacizumab. Conversely, VEGF mono-form was more abundant non-EV part and can be neutralized by bevacizumab. In animal model, AS2 was injected intraperitoneal and then malignant ascites developed as follow. We found the administration of bevacizumab after AS2 injection i.p. inhibited peritoneal seeding and ascites formation compared to control group. But the inhibition was reversed when we injected EV as well as bevacizumab in combination group. In breast cancer cell, microvesicles (MVs) activates VEGF receptors through a unique 90 kDa form of VEGF (VEGF90K) which has a weakened affinity for Bevacizumab, causing Bevacizumab to be ineffective in blocking MV-dependent VEGF receptor. And an Hsp90 inhibitor (17-AAG) was demonstrated to release VEGF90K from MVs without interaction with VEGF restoring the sensitivity of VEGF90K to Bevacizumab. However, our study found 17-AAG alone inhibited VEGF secretion in EV as well as the activity of upstream Stat3, and Akt pathway in lung cancer. Our preliminary data implied EV-VEGF and the existence of different isoform of VEGF in EV contribute to tumor angiogenesis and limit the effectiveness of bevacizumab therapy which is not dependent on Hsp90. Citation Format: Chien-Chung Lin, Yu-Ting Huang, Chun-Hua Hung, Mei-Ling Tsai, Wu-Chou Su. The extracellular vesicles from lung cancer cells activates Stat3 and tumor angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 192.

Abstract 4599: PD-1/PD-L1 axis in cancer cells contributes to cellular EMT in lung adenocarcinoma

Abstract Background: Since intrinsic PD-1 receptor functions promote tumor growth was reported, we will investigate the role of PD-L1 in lung adenocarcinoma cells, and their impact on clinical outcome. Materials and methods: Lung adenocarcinoma CL1-5 cells, derived from CL1-0 cells have more invasive ability. We prepared PDL1-overexpression human lung adenocarcinoma cell line, derived from CL1-0 cells (CL1-0-PD1) and from PC14PE6 cells (PC14PE6-PD-L1). We observed the cellular morphology, and invasiveness; epithelial-mesenchymal transition (EMT) markers and regulators were also evaluated. To explore interaction between PD-1 and PD-L1, we treated the CL1-0, CL1-5, CL1-0-PDL1 cells, PC14PE6 cells, and PC14PE6-PD-L1 cells with anti-PD-1 antibody, and then evaluated cellular invasion. We also suppressed PD-1 to test whether PD-1/PDL-1 interaction contributed to the EMT change. Further, we evaluated cellular proliferation and chemosensitivity by MTT assay and colony formation assay. Finally, we correlated PD-L1 expression with clinical outcome in patients’ tumor specimen. Results: CL1-5 cells possessed higher PD-L1 expression than parental CL1-0 cells. CL1-0 cells with PD-L1 overexpression had more expression of EMT regulators and mesenchymal markers. Overexpression of PD-L1 in another lung adenocarcinoma PC14PE cells (PC14PE6-PD-L1 cells) become more aggressive. On the contrary, down-regulation of PD-L1 in CL1-5 cells and PC14-PE6-PD-L1 cells became indolent. Besides through the observation from confocal microscopy, PD-L1 expression was more co-localized with vimentin and slug, individually in CL1-5, CL1-0-PD-L1 and PC14PE6-PD-L1 cells. Therefore, PD-L1 promoted EMT in human lung adenocarcinoma cells. After adding anti-PD-1 antibody in CL1-5, CL1-0, and CL1-0-PDL1 cells, migration and invasion ability were decreased. Similar phenomenon was found when PD-1 antibody treatment in PC14PE6 cells, and PC14PE6-PD-L1 cells. Silencing PD-1 by siRNA also disrupt the cellular aggressivenss in CL1-0-PD-L1 and PC14PE6-PD-L1 cells. These results indicated PD-1/PD-L1 axis regulated cancer cells migration and invasiveness. PD-L1 expression also decreased cellular proliferation and had little influence on chemsensitivity. Finally, we found that higher PD-L1 expression was correlated with lymph node metastasis in clinical specimen. Conclusion: Lung adenocarcinoma cells with higher PD-L1 expression promote EMT via PD-1 receptor. PD-L1 expression lowers proliferation rate, but has little impact on chemosensitivity. Lung cancer patients with high PD-L1 expression in tumor have higher lymph node metastasis. Citation Format: Wen-Pin Su, Li-Chan Chang, Jing-Jou Yan, Wu-Chou Su. PD-1/PD-L1 axis in cancer cells contributes to cellular EMT in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4599.

MiR-200c in extracellular vesicles as a circulating biomarker of EGFR-TKI response and a mediator of EGFR-TKI sensitivity in heterogeneous NSCLC.

e20547 Background: Through intercellular transfer of EV(extracellular vesicles) miRNA, tumor cells can confer drug resistance to each other and contribute to tumor heterogeneity. However, the mechanisms why EGFR-TKI can be effective in heterogeneous NSCLC with low abundances of EGFR mutations remain unknown. The aim of the study is to investigate the significance of EV miRNA in mediating the efficacy of EGFR-TKI in heterogeneous NSCLC and serving as the biomarker of response to EGFR-TKI. Methods: We first evaluate if EVs from EGFR mutant cell (PC9) can affect EGFR-TKI sensitivity of EGFR wild type cell (CL1-5, H1299) in vitro co-culture system and i n vivo. We then identified the differential miRNA panel by comparing EVs from PC9 to those from CL1-5. Finally, we verified if the expressions level of these miRNA are different in blood EVs from patient with good response compared to those with poor response to EGFR-TKI. Results: We first verified that CL1-5 can take up labelled EVs from PC9 under time-lapse microscope and EGFR mutant DNA can be detected in recipient EGFR wild-type cell using digital PCR. We found EVs from PC9 enhanced gefitinib sensitivity of CL1-5. And co-culturing PC9 with CL1-5 enhanced CL1-5 gefitinib sensitivity which was reversed by adding GW4789, the inhibitor of exosome secretion. In subcutaneous CL1-5 animal model, in comparison to treating with gefitinib or PC9 EVs alone, only the combination treatment with gefitinib and PC9 EVs delayed cancer growth. Using small RNA sequencing, we identified a unique miRNA profile allowing discriminating EV from PC9 cells to those from CL1-5; MiRNA 200 family including 200a, 200b, 200c and mir429 were up-regulated significantly in PC9 EV. From Aug 2015 to Sep 2017, sixteen patients with good response (PFS > 12M) or poor response (PFS < 6M) to EGFR-TKI were enrolled and blood were collected for EV miRNA isolation. Ten of these blood samples were qualified for miRNA analysis and mir200a and 200c were found up-regulated in good responder to EGFR-TKI. The transfection of mir200c in CL1-5 cells not only inhibited the oncogenic pathway contributing to EGFR-TKI resistance including Stat3, Akt, EMT and BIM pathway but also enhanced gefitinib sensitivity of CL1-5 cells. Conclusions: Our data suggested mir200c from blood EV serve as a biomarker of response to EGFR-TKI and mir200c may mediate EGFR-TKI sensitivity in heterogenous EGFR mutant NSCLC.

High levels of EGFR prevent sulforaphane-induced reactive oxygen species-mediated apoptosis in non-small-cell lung cancer cells

BackgroundSulforaphane (SFN) has been shown to induce the production of reactive oxygen species (ROS) and inhibit epidermal growth factor receptor (EGFR)–mediated signaling in non-small-cell lung cancer (NSCLC). NSCLC cells harboring constitutively active EGFR mutations are more sensitive to SFN treatment than cells with wild-type EGFR, but whether NSCLC cells with high levels of EGFR expression are more resistant or sensitive to SFN treatment is not known. Study designWe employed a pair of cell lines, CL1-0 and CL1-5, which have the same genetic background but different levels of EGFR expression, to examine the effects of high EGFR level in the sensitivity to SFN. MethodsThe effect of SFN on cell viability and tumorigenicity was examined by trypan blue dye-exclusion assay, clonogenic assays, flow cytometry, and immunoblotting in vitro as well as tumorigenicity study in vivo. ROS levels in cells were assessed by flow cytometry using the ROS-reactive fluorescent indicator CM-H2DCFDA. Knockdown of EGFR in CL1-5 cells was infected with an EGFR-targeting small hairpin (interfering) RNA (shRNA)–containing lentivirus. ResultsWe present evidence that cells with high-level EGFR expression (CL1-5) are more resistant to SFN treatment than those with low-level expression (CL1-0). SFN treatment produced a similar increase in ROS and caused arrest of a cell population at S-phase accompanied by the induction of γH2AX, a DNA damage-response marker, in both cell sublines. However, SFN induced apoptosis only in the high-EGFR-expressing CL1-0 subline. Pretreatment with the antioxidant N-acetyl-L-cysteine prevented SFN-induced apoptosis in CL1-0 cells and production of γH2AX in both CL1-0 and CL1-5 cells. shRNA-mediated knockdown of EGFR in CL1-5 cells rendered the cells susceptible to SFN-induced apoptosis. ConclusionThe cellular effects produced by SFN in NSCLC cells are largely mediated by SFN-induced production of ROS. Cells with higher levels of EGFR were more resistant to SFN treatment and showed resistance to SFN-induced apoptosis, suggesting that high EGFR levels protect cells from SFN-induced apoptosis. Despite this, we found that SFN retained the ability to inhibit the growth of NSCLC tumors with high-level EGFR expression in vivo.

ITGB1‐DT and ITGB1 enhance lung cancer cell metastatic abilities and are associated with poor prognoses in NSCLC patients

Non‐small cell lung cancer (NSCLC) is one of the major leaders in lethal cancer for decades. It has two major types: lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). In the molecular analysis, the genomic backgrounds were different among two sub‐types. But most deaths from these two types of NSCLC were due to the metastatic cells which present different molecular characteristics compared to the primary cells. The differences may provide a key for novel diagnosis and/or therapeutic strategy to treat NSCLC. Here in this study, we have found a long non‐coding RNA, ITGB1‐DT (RP11‐462L8.1) and its neighboring gene, ITGB1 were found associated with poor prognosis of overall survival and recurrence‐free survival analysis from TCGA lung cancer cohort in both LUAD and LUSC. We determined the expression levels with our own clinical cohort and confirmed the findings. We also found that the ITGB1‐DT RNA expression levels were correlated to the higher invasion and migration abilities in CL1‐5 lung cancer cells compared to lower invasion and migration ability parental CL1‐0 cells. It is also involved in epithelial‐mesenchymal‐transition in A549 after TGF‐beta treatment. ShRNA induced inhibition of the ITGB1‐DT gene expression was found to reduce the invasion and migration ability of the highly aggressive NSCLC cells with endogenous ITGB1‐DT overexpression. Knockdown of IGTB1‐D1 in lung cancer cells also significantly suppressed cancer cell invasions and metastases in in‐situ human lung cancer established NSG mouse model. The putative molecular mechanisms of ITGB1_DT and ITGB1 in promoting lung cancer cell metastasis were elucidated. Together, our results identified a new long non‐coding RNA ITGB1‐DT who may interact with its neighbor coding gene ITGB1 to promote lung cancer cell metastasis. Inhibition of ITGB1‐DT and ITGB1 expressions and interactions may provide a novel treatment strategy to combat lung cancer patients with metastasis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Arecoline Promotes Migration of A549 Lung Cancer Cells through Activating the EGFR/Src/FAK Pathway.

Arecoline is the primary alkaloid in betel nuts, which are known as a risk factor for oral submucosal fibrosis and oral cancer. Lung cancer is a severe type of carcinoma with high cell motility that is difficult to treat. However, the detailed mechanisms of the correlation between Arecoline and lung cancer are not fully understood. Here, we investigated the effect of Arecoline on migration in lung cancer cell lines and its potential mechanism through the muscarinic acetylcholine receptor 3 (mAChR3)-triggered EGFR/Src/FAK pathway. Our results indicate that different concentrations of Arecoline treatment (10 µM, 20 µM, and 40 µM) significantly increased the cell migration ability in A549 and CL1-0 cells and promoted the formation of the filamentous actin (F-actin) cytoskeleton, which is a crucial element for cell migration. However, migration of H460, CL1-5, and H520 cell lines, which have a higher migration ability, was not affected by Arecoline treatment. The EGFR/c-Src/Fak pathway, which is responsible for cell migration, was activated by Arecoline treatment, and a decreased expression level of E-cadherin, which is an epithelial marker, was observed in Arecoline-treated cell lines. Blockade of the EGFR/c-Src/Fak pathway with the inhibitors of EGFR (Gefitinib) or c-Src (Dasatinib) significantly prevented Arecoline-promoted migration in A549 cells. Gefitinib or Dasatinib treatment significantly disrupted the Arecoline-induced localization of phospho-Y576-Fak during focal adhesion in A549 cells. Interestingly, Arecoline-promoted migration in A549 cells was blocked by a specific mAChR3 inhibitor (4-DAMP) or a neutralizing antibody of matrix metalloproteinase (MMP7 or Matrilysin). Taken together, our findings suggest that mAChR3 might play an essential role in Arecoline-promoted EGFR/c-Src/Fak activation and migration in an A549 lung cancer cell line.

A microarray study of radiation-induced transcriptional responses and the role of Jagged 1 in two closely-related lung cancer cell lines

Background: Lung cancer is the most lethal and common cancer in the world. Non-small cell lung cancer accounts for 80% lung cancer cases and its five-year survival rate after radiotherapy is approximately 5%. Patients with lung cancer in the advanced stages are suitable for radiotherapy rather than surgical resection. Unfortunately, radiotherapy alone or in combination with chemotherapy does not significantly improve 5-year survival rates of lung cancer patients. The mechanism by which lung cancer responds to radiation is unclear and needed to be investigated. Methods: Two lung adenocarcinoma cell lines, CL1-0 and CL1-5, that have exhibited different metastasis capacities in previous research, were utilized herein to investigate cellular radio-sensitivity. Clonogenic assay was used to evaluate the radio-sensitivity of CL1-0 and CL1-5. Flow cytometry experiments were performed on both cell lines to understand the change in the cell cycle profile that followed radiation. Gene expression data with the untreated and radiation treated lung cancer cell samples was obtained using Illumina microarrays. Tight hierarchical clustering was conducted to group genes with similar expression profiles. Clonogenic assay and cell cycle distribution were performed with Jagged 1-overexpressed CL1-0 to investigate the role of Jagged 1 in the radio-sensitivity of the lung cancer cells. Results: Clonogenic assay results showed that CL1-5 was more radio-sensitive than CL1-0 after exposure to 10 Gy radiation. The proportion of the CL1-5 cell population in the G2/M phase was found to be markedly higher than that with the CL1-0 cell population following exposure to radiation. Based on the microarray experimental data, 1,595 genes were identified as differentially expressed and sub-grouped into six clusters by hierarchical clustering. The functions of the identified genes in the tight clusters were associated with cell death, cell cycle, cell growth and proliferation, cellular function and maintenance. Moreover, our microarray data suggested that the gene expression levels of Jagged 1 , a Notch ligand, differed significantly between CL1-0 and CL1-5. This finding was further verified by real-time PCR and western blot. Clonogenic assay results further revealed a lower survival rate of Jagged 1-overexpressed CL1-0 than that of CL1-0 following exposure to radiation. Conclusions: CL1-5 was shown to be more radio-sensitive than CL1-0 from our clonogenic assay data. The radiation-induced transcriptional responses in lung cancer cells were evaluated with gene expression microarrays in this work. Jagged 1, the Notch receptor ligand, may have an important role in increasing the radio-sensitivity of lung adenocarcinomas.

The chemopreventive isothiocyanate sulforaphane reduces anoikis resistance and anchorage-independent growth in non-small cell human lung cancer cells

The capacity of cancer cells to resist detachment-induced apoptosis, i.e. anoikis, as well as anchorage-independent growth are crucial prerequisites for tumor metastasis. Therefore, agents interfering these properties may provide novel anti-metastatic strategies. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is known as a potent chemopreventive agent, but its effect on anoikis resistance has not been investigated. In this study, two non-small cell lung cancer (NSCLC) cell lines, A549 and CL1-5 cells, were treated with SFN under either suspension or adhesion conditions. SFN exhibited more potent cytotoxicity against suspending rather than adherent cancer cells. The selective cytotoxicity was due to the induction of anoikis, as evident by chromatin condensation, Annexin V binding, and activation of the mitochondrial apoptotic pathway. SFN also inhibited NSCLC cell to form spherical colonies, suggesting that anchorage-independent growth was prevented by SFN. Consistently, SFN treatment led to inactivation of FAK and Akt, down-regulation of β-catenin, and up-regulation of the cyclin-dependent kinase inhibitor p21. Because A549 cells with wild-type p53 are more sensitive to SFN than p53-mutant CL1-5 cells, p53 dependency of SFN responses were determined in p53-knockdown A549 cells. Knockdown of p53 attenuated the ability of SNF to inhibit anoikis resistance and sphere formation in A549 cancer cells, suggesting that the presence of p53 in NSCLC cancer cells is involved in the sensitivity to SFN. These results provide new insight into mechanisms underlying the chemopreventive ability of SFN and suggest a potential benefit of SFN to interfere with tumor metastasis.

Protease-activated receptor 2 induces migration and promotes Slug-mediated epithelial-mesenchymal transition in lung adenocarcinoma cells

Protease-activated receptor 2 (PAR2), a G protein-coupled receptor for trypsin, contributes to growth, anti-apoptosis, and migration in lung cancer. Given that PAR2 activation in airway epithelial cells compromises the airway epithelium barrier by disruption of E-cadherin adhesion, PAR2 may be involved in epithelial-mesenchymal transition (EMT) in lung adenocarcinoma cells. Although PAR2 is known to promote the migration of lung cancer cells, the detailed mechanism of this event is still not clear. Here, we found that PAR2 is highly expressed in several lung adenocarcinoma cell lines. In two lung adenocarcinoma cell lines, CL1-5 and H1299 cells, activation of PAR2 induces migration and Slug-mediated EMT. The underlying mechanisms involved in PAR2-induced migration and EMT in CL1-5 cells were further investigated. We showed that PAR2-induced migration of CL1-5 cells is mediated by the Src/p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. β-arrestin 1, not G protein, is involved in this PAR2-mediated Src/p38 MAPK signaling pathway. PAR2-induced EMT in CL1-5 cells is dependent on the activation of extracellular-signal-regulated kinase 2 (ERK2). The activation of ERK2 further mediates Slug stabilization through suppressing the activity of glycogen synthase kinase 3β. In addition, a poor prognosis was observed in lung adenocarcinoma patients with a high expression of PAR2. Thus, PAR2 regulates migration through β-arrestin 1-dependent activation of p38 MAPK and EMT through ERK2-mediated stabilization of Slug in lung adenocarcinoma cells. Our finding also suggests that PAR2 might serve as a therapeutic target for metastatic lung adenocarcinoma and a potential biomarker for predicting the prognosis of lung adenocarcinoma.

Synergistic Anticancer Effect of a Combination of Paclitaxel and 5-Demethylnobiletin Against Lung Cancer Cell Line In Vitro and In Vivo.

Lung cancer remains a highly prevalent disease and a leading cause of cancer-related deaths worldwide. Currently, exploring antitumor drugs derived from herbs used in traditional Chinese medicine is increasingly becoming an attractive area of research. Paclitaxel (PTX), a highly effective chemotherapeutic drug, is widely used for treating different cancers; however, the clinical use of PTX is dose limited because of its adverse side effects. Chemotherapeutic agents are being developed to enhance the anticancer activity of PTX, particularly for use in combination therapy. 5-Demethylnobiletin (5-DMN), a natural, active compound isolated from orange peel, has been reported to induce apoptosis in several cancer cell lines. In this study, we tested the synergistic anticancer antiproliferative effects of combinations of PTX and 5-DMN on CL1-5 lung cancer cells through the MTT and propidium iodide assays. After low-dose combination treatments (PTX and 5-DMN), a reduction in cell viability and a concomitant increase in apoptosis were observed in the CL1-5 cells. We propose that 5-DMN cooperates with PTX to induce apoptosis via the caspase pathway (by modulating caspase-3, caspase-8, and caspase-9 activities). Furthermore, we observed that the combination treatment significantly suppressed tumor growth in the nude mouse xenograft model. The results suggest that the synergistic effects of PTX and 5-DMN in lung cancer cells deserve particular attention and indicate the possibility of developing additional new strategies for treating lung cancer.

Transglutaminase 2 Promotes Migration and Invasion of Lung Cancer Cells.

Lung cancer is the leading cause of cancer deaths worldwide. Given that the major threat of cancer is metastasis, delineation of the molecular mechanism underlying it would help devise therapeutic strategies. Transglutaminase 2 (TG2), belonging to the transglutaminase superfamily, is a versatile protein with enzymatic and nonenzymatic functions. It mainly localizes inside the cell, but also appears extracellularly. Recent findings have demonstrated the involvement of TG2 in cancer development. Here we examine the role of TG2 in metastasis of lung cancer using a lung cancer cell line CL1-0, which exhibits low invasiveness, and its invasive subline CL1-5. Our results show that CL1-5 cells express a higher amount of TG2 than CL1-0 cells. Overexpression of TG2 in CL1-0 enhances cell migration and invasion, and lowering TG2 expression in CL1-5 cells reduces their ability to do so. The transamidase activity of TG2 is not required since cells expressing the inactive TG2 mutant or treated with a TG2 inhibitor are still able to migrate and invade. TG2-stimulated migration and invasion are, at least in part, mediated by Rac, as inhibition of Rac activity suppresses cell migration and invasion. Lastly, exogenous application of recombinant TG2 protein to CL1-0 cells substantially augments cell migration and invasion, suggesting the significance of extracellular TG2 in promoting these events. Collectively, our results show that TG2 plays a positive role in cell migration and invasion, and this might help metastasis of lung cancer cells.

Autophagy regulates vinorelbine sensitivity due to continued Keap1-mediated ROS generation in lung adenocarcinoma cells

Autophagy is one of the induced mechanisms in metastatic cancer to escape death due to starvation, hypoxia, metabolic stresses, chemotherapy, and radiation. Some publications have revealed that chemotherapy combined with autophagy inhibitor will overcome drug resistance. We modified AS2 cells with PTEN overexpression, mTOR knockdown, or Keap1 knockdown, and made modification of A549 cells with PTEN knockdown, Atg5 knockdown, and Keap1 overexpression. Our study was aimed toward an exploration of how autophagy modulates Keap1, ROS generation, and vinorelbine-induced apoptosis in these cell lines. We found that lung cancer PC14PE6/AS2 (AS2) had higher mTOR and Akt and also lower PTEN expression than A549 cells. Descended autophagy was demonstrated with more decreased p62 accumulation and LC3 II conversion in AS2 cells as compared to A549 cells. The A549 cells had lower Keap1/Nrf2 and more active anti-oxidant response element (ARE) activity than the AS2 cells. We modified AS2 cells with PTEN overexpression, mTOR knockdown, Keap1 knockdown, and revealed amplified p62 and LC3 expression accompanied with decreased Akt, Keap1, ROS, and vinorelbine-induced apoptosis. Declined p62, LC3 expression were accompanied with increased Akt, Keap1, ROS, and vinorelbine-induced apoptosis after modification of A549 cells with PTEN knockdown, Atg5 knockdown, and Keap1 overexpression. Keap1 overexpression lowered ARE levels in A549 cells, and ARE level exhibited up-growth in Keap1 knockdown AS2 cells. The autophagy inhibitor caused more ROS generation and vinorelbine-induced apoptosis in the A549 and CL1-5 cells. According to these findings, autophagy regulates vinorelbine sensitivity by continuing Keap1-mediated ROS generation in lung adenocarcinoma cells.

Abstract 217: The role of exosomes and exosomal miRNA in mediating EGFR-TKI sensitivity in heterogeneous NSCLC

Abstract Tumor heterogeneity has impacts on tumor biopsy strategy, characterization of actionable targets and drug resistance. At lung cancer, recent study demonstrated that the discordance rates of EGFR mutation implying tumor heterogeneity in metachronous and synchronous settings were 14.3% and 9.1% respectively. And other study further showed intratumoral EGFR mutational heterogeneity was correlated with the response and prognosis of EGFR-TKIs. Extracellular vesicles (EVs) such as exosomes serve as the transporter of bioactive molecules between cells and become one of the major mechanisms contributing intratumoral heterogeneity via intercellular transference of genetic information. Since most patients harboring EGFR mutation showed excellent response, we hypothesized that EVs such as exosome mediating the crosstalk between EGFR mutant cell and EGFR wild type cell contributing the change of sensitivity of EGFR wild type cell to EGFR-TKI in heterogeneous NSCLC. We used ultrafiltration (UF) method to isolate the exosomes. The conditioned media were centrifuged and then the supernatants were passed through 0.22 μm filters and then Vivaspin nanomembrane concentrators step by step. We first incubated EGFR wild type lung cancer cell (CL1-5) with medium containing PKH26 labeled exosomes derived from PC9 cell (EGFR Del 19) for 24 h. The uptake of exosomes from PC9 in CL1-5 cell can be observed under immunofluorescence microscope and recorded by time-lapse microscope. And the EGFR Del19 DNA and specific protein can be detected in recipient CL1-5 cells by digital PCR and Western blotting respectively. Then EGFR wild type lung cancer cell (CL1-0, CL1-5 and H1299) were first treated with exosome from themselves or PC9 cell respectively for 48h and then subjected to gefitinib (0.1 μM) for 72h. We found only PC9 cell derived EV sensitized EGFR wild type cell to gefitinib. To mimic tumor heterogeneity, we next tested the significance of exosome on EGFR-TKI sensitivity in co-culture system with pretreatment with or without GW4869, which blocks exosome secretion by inhibiting neutral sphingomyelinase. We first verified that treating lung cancer cell with GW4869 at concentrations of 2.5, 5 and 10μM inhibited exosome secretion without cytotoxicity. We demonstrated that EGFR wild type lung cancer cell became sensitive to EGFR-TKI after co-culture with PC9 cell for 48h and then subjected to gefitinib for 72h. However, the pretreatment with GW4869 for 48 hours reversed the sensitivity to EGFR-TKI in co-culture system with PC9. We further compared the exosomal miRNAs from PC9 to those from CL1-5 and identified a panel of discriminative miRNAs. Some miRNA among the panel such as miR-200 family have been identified associated with resistance to EGFR-TKI. Our study, at least in part, suggests that exosome release from EGFR mutant cell might be a possible mechanism for excellent EGFR-TKI sensitivity even in heterogeneous NSCLC. Citation Format: Chien-Chung Lin, Wu-Chou Su, Chin-You Wu. The role of exosomes and exosomal miRNA in mediating EGFR-TKI sensitivity in heterogeneous NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 217.

Lobocrassin B Induces Apoptosis of Human Lung Cancer and Inhibits Tumor Xenograft Growth.

Lobocrassin B, a natural cembrane-type compound isolated from the soft coral Lobophytum crassum, has been shown to have significant biological effects, including anticancer activity. As the most common cause of cancer mortality worldwide, lung cancer remains a major concern threatening human health. In the current study, we conducted in vitro experiments to demonstrate the inhibiting effect of Lobocrassin B on CL1-5 and H520 human lung cancer cells growth and to explore the underlying mechanisms, as well as in nude mice bearing CL1-5 tumor xenografts. Lobocrassin B exerted cytotoxic effects on lung cancer cells, as shown by decreasing cell viability, and inducing apoptosis, oxidative stress and mitochondrial dysfunction. In addition, the increased level of Bax, cleaved caspase-3, -9 and -8, and the suppression of Bcl-2 were observed in the Lobocrassin B treated cells. Moreover, in vivo assays verified the significance of these results, revealing that Lobocrassin B inhibited CL1-5 tumor xenograft growth and that inhibitory effects were accompanied by a marked increase in tumor cell apoptosis. In conclusion, the results suggested that Lobocrassin B could be a potential anticancer compound for its propensity to inhibit growth and induce apoptosis in human lung cancer cells.

The anti-cancer effects and mechanisms of Scutellaria barbata D. Don on CL1-5 lung cancer cells.

Lung cancer, with a poor prognosis and resistance to chemotherapy, is the most common malignant tumor and has the highest mortality rate worldwide. Scutellaria barbata D. Don (SB), which is derived from the dried whole plant of Labiatae, is a well-known anti-inflammatory and anti-cancer herb. The aim of this study was to examine the anti-cancer effects and precise regulatory mechanisms of SB in CL1-5 lung cancer cells. In an in vitro assay, we found that the anti-tumor mechanism of SB was due to P38/SIRT1-regulated cell apoptosis through G2/M phase arrest and ER stress-, intrinsic mitochondrial-, and extrinsic FAS/FASL-mediated pathways. Autophagy also plays a key role in SB-induced CL1-5 cell cytotoxicity. In addition, SB exerts additive effects with etoposide or cisplatin in lung cancer cells. In an in vivo assay, we found that SB significantly reduces tumor size with decreased proliferation and angiogenesis, as well as increased apoptosis and autophagy in CL1-5 tumor-bearing mice. These findings provided experimental evidence for the application of SB in the treatment of lung cancer.

Active fraction (HS7) from Taiwanofungus camphoratus inhibits AKT-mTOR, ERK and STAT3 pathways and induces CDK inhibitors in CL1-0 human lung cancer cells

BackgroundThe non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. In NSCLC, the oncogenic AKT-mTOR, ERK and STAT3 pathways are commonly dysregulated and have emerged as attractive targets for therapeutic developments. In a relatively limited subset of NSCLC, these pathways driven by mutant EGFR can be treated by the tyrosine kinase inhibitors (TKIs)-mediated targeted therapy. However, for the most NSCLC, more novel targeted agents are imperatively needed. Therefore, we investigated the inhibitory effects of the active fraction HS7 from Taiwanofungus camphoratus, a unique medicinal fungus in Taiwan, on these pathways in CL1-0 EGFR wild-type human NSCLC cells.MethodsThe active fraction HS7 was prepared by n-hexane extraction of T. camphoratus followed by silica gel chromatography. Its effects on the cell viabilities were determined by sulforhodamine B colorimetric assay. Flow cytometry was used to analyze cell-cycle regulation and apoptosis induction. The changes in cellular protein levels were examined by Western blot.ResultsThe active fraction HS7 vigorously inhibits AKT-mTOR, ERK and STAT3 signaling pathways in CL1-0 cells. At dose of 25 μg/mL, these signaling pathways were almost completely inhibited by HS7, accompanied with induction of cyclin-dependent kinase inhibitors such as p15, p21 and p27. Accordingly, the AKT-mTOR downstream targets p-p70S6K and HIF-1α were also suppressed as well. At this dose, the cell proliferation was profoundly suppressed to 23.4% of control and apoptosis induction was observed.ConclusionsThe active fraction HS7 from n-hexane extract of T. camphoratus exerts multi-targeting activity on the suppression of AKT-mTOR, ERK and STAT3 pathways and induction of p15, p21 and p27 in EGFR wild-type NSCLC cells. This multi-targeting activity of HS7 suggests its potential as an alternative medicine for the treatment of EGFR TKIs resistant NSCLC.

Toxicological effects of NCKU-21, a phenanthrene derivative, on cell growth and migration of A549 and CL1-5 human lung adenocarcinoma cells.

BackgroundChemotherapy insensitivity continues to pose significant challenges for treating non-small cell lung cancer (NSCLC). The purposes of this study were to investigate whether 3,6-dimethoxy-1,4,5,8-phenanthrenetetraone (NCKU-21) has potential activity to induce effective toxicological effects in different ethnic NSCLC cell lines, A549 and CL1-5 cells, and to examine its anticancer mechanisms.MethodsMitochondrial metabolic activity and the cell-cycle distribution were analyzed using an MTT assay and flow cytometry in NCKU-21-treated cells. NCKU-21-induced cell apoptosis was verified by Annexin V-FITC/propidium iodide (PI) double-staining and measurement of caspase-3 activity. Western blotting and wound-healing assays were applied to respectively evaluate regulation of signaling pathways and cell migration by NCKU-21. Molecular interactions between target proteins and NCKU-21 were predicted and performed by molecular docking. A colorimetric screening assay kit was used to evaluate potential regulation of matrix metalloproteinase-9 (MMP-9) activity by NCKU-21.ResultsResults indicated that NCKU-21 markedly induced cytotoxic effects that reduced cell viability via cell apoptosis in tested NSCLC cells. Activation of AMP-activated protein kinase (AMPK) and p53 protein expression also increased in both NSCLC cell lines stimulated with NCKU-21. However, repression of PI3K-AKT activation by NCKU-21 was found in CL1-5 cells but not in A549 cells. In addition, increases in phosphatidylserine externalization and caspase-3 activity also confirmed the apoptotic effect of NCKU-21 in both NSCLC cell lines. Moreover, cell migration and translational levels of the gelatinases, MMP-2 and MMP-9, were obviously reduced in both NSCLC cell lines after incubation with NCKU-21. Experimental data obtained from molecular docking suggested that NCKU-21 can bind to the catalytic pocket of MMP-9. However, the in vitro enzyme activity assay indicated that NCKU-21 has the potential to increase MMP-9 activity.ConclusionsOur results suggest that NCKU-21 can effectively reduce cell migration and induce apoptosis in A549 and CL1-5 cells, the toxicological effects of which may be partly modulated through PI3K-AKT inhibition, AMPK activation, an increase in the p53 protein, and gelatinase inhibition.

Abstract 538: The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) anti-apoptosis ability

Abstract Emerging evidence has indicated that proteins of a disintergrin and metalloprotease (ADAM) family contribute to cancer progression and metastasis. One member of this family, ADAM15, has been shown to be upregulated in multiple cancers, including gastric, lung, breast, and prostate cancers, and the enzymatic activities of its extracellular metalloprotrease domain promote breast cancer proliferation and migration through mediating ErbB signaling pathway. The patients with ADAM15 high-expressing lung tumors have shorter survival time and ADAM15 has been proved to enhance synovial fibroblasts anti-apoptosis ability via focal adhesion kinase signaling pathway. We firstly demonstrated other than extracellular enzymetic activity, the longest isoform of ADAM15 (ADAM15 i6), which contains the most cytoplasmic Src homology 3 (SH3) binding motifs, significantly upregulated in primary lung cancer tissues and promoted NSCLC proliferation via growth factor receptor-bound protein 2 (Grb2) and Src homolog 2 domain containing (Shc) association. In this study, we further explore the roles of ADAM15 cytosolic domain in NSCLC apoptosis resistance. Overexpression of ADAM15 i6 promoted CL1-0 cell anti-apoptosis ability according to the trypan blue inclusion assay. Ablation of nephrocystin (NPHP1) attenuated the ADAM15 i6-promoted anti-apoptosis ability. Thus, we identified a novel mechanism of the ADAM15 cytoplasmic domain in NSCLC tumor progression, which will shed light on the molecular mechanisms of ADAM proteins, and facilitate development of novel therapy in NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Hsin-Han Hou, Chong-Jen Yu. The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) anti-apoptosis ability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 538. doi:10.1158/1538-7445.AM2017-538

Increased S100A15 expression and decreased DNA methylation of its gene promoter are involved in high metastasis potential and poor outcome of lung adenocarcinoma.

PurposeThis study aims to determine the functional role of S100A15 and its promoter DNA methylation patterns in lung cancer progression.Experimental DesignWe analyzed 178 formalin-fixed paraffin embedded specimens from lung cancer patients, including 24 early stage and 91 advanced stage adenocarcinoma. S100A15 protein expression was evaluated by immunohistochemistry stain, and its DNA methylation levels were measured by pyrosequencing.ResultsS100A15 nuclear staining was increased in lung adenocarcinoma patients with distant metastasis versus those without distant metastasis. There was reduced one/three-year overall survival in adenocarcinoma patients receiving first line target therapy and harboring high nuclear expressions of S100A15. Both DNA methylation levels over -423 and -248 CpG sites of the S100A15 gene promoter were decreased in adenocarcinoma patients with distant metastasis, and the former was associated with lower one-year overall survival. The highly invasive CL1-5 cell lines display decreased DNA methylation over −412/−248/−56 CpG sites of the S100A15 gene promoter and increased S100A15 gene/protein expressions as compared with the less invasive CL1-0 cell lines. Knockdown of S100A15 in CL1-5 cell line inhibited cell proliferation, migration, and invasion, while over-expression of S100A15 in CL1-0 cell line promoted cell proliferation, migration, and invasion. RNA sequencing analysis revealed potential biological effects of S100A15 over-expression and knock-down with CTNNB1, ZEB1, CDC42, HSP90AA1, BST2, and PCNA being the pivotal down-stream mediators.ConclusionsIncreased S100A15 expression and decreased DNA methylation of its gene promoter region were associated with high metastasis potential and poor outcome in lung adenocarcinoma, probably through triggering CTNNB1 -centered pathways.

Cancer cells increase endothelial cell tube formation and survival by activating the PI3K/Akt signalling pathway

BackgroundAngiogenesis is a hallmark of cancer and plays a critical role in lung cancer progression, which involves interactions between cancer cells, endothelial cells and the surrounding microenvironment. However, the gene expression profiles and the changes in the biological phenotype of vascular endothelial cells after interactions with lung cancer cells remain unclear.MethodsAn indirect transwell co-culture system was used to survey the interaction between human umbilical vein endothelial cells (HUVECs) and human lung adenocarcinoma CL1-5 cells, as well as to investigate the morphological and molecular changes of HUVECs. The differentially expressed genes (DEGs) in HUVECs after co-culture with cancer cells were identified by microarray. Moreover, a publicly available microarray dataset of 293 non-small-cell lung cancer (NSCLC) patients was employed to evaluate the prognostic power of the gene signatures derived from HUVECs.ResultsThe interaction between HUVECs and lung cancer cells changes the morphology of HUVECs, causing them to have a mesenchymal-like morphology and alter their cytoskeleton organization. Furthermore, after co-culture with lung cancer cells, HUVECs showed increased cell motility and microvessel tube formation ability and a decreased apoptotic percentage. Transcriptomic profiling of HUVECs revealed that many survival-, apoptosis- and angiogenesis-related genes were differentially expressed after interactions with lung cancer cells. Further investigations showed that the PI3K/Akt signalling pathway and COX-2 are involved in endothelial tube formation under the stimulation of lung cancer cells. Moreover, Rac-1 activation might promote endothelial cell motility through the increased formation of lamellipodia and filopodia. The inhibitors of PI3K and COX-2 could reverse the increased tube formation and induce the apoptosis of HUVECs. In addition, the gene signatures derived from the DEGs in HUVECs could predict overall survival and disease-free survival in NSCLC patients and serve as an independent prognostic factor.ConclusionsIn this study, we found that cancer cells can promote endothelial cell tube formation and survival, at least in part, through the PI3K/Akt signalling pathway and thus change the microenvironment to benefit tumour growth. The gene signatures from HUVECs are associated with the clinical outcome of NSCLC patients.