Cultured Lung Cancer Cells Research Articles

The induction of MIG6 under hypoxic conditions is critical for dormancy in primary cultured lung cancer cells with activating EGFR mutations.

The biologic activity of individual cancer cells is highly heterogeneous. Hypoxia, one of the prominent features of a tumor microenvironment, is thought to be causal in generating this cellular heterogeneity. In this study, we revealed that primary lung cancer cells harboring activating epidermal growth factor receptor (EGFR) mutations generally entered a dormant state when hypoxic. We found that heterodimer formation of the ERBB family receptor tyrosine kinases (RTKs), and their subsequent downstream signaling, was diminished under hypoxic conditions, although phosphorylation of the EGFR was retained. Dormant lung cancer cells were found to be resistant to EGFR tyrosine kinase inhibitor (TKI) treatment. In terms of mechanism, we found that a negative regulator of ERBB signaling, MIG6/ERRFI1/RALT/Gene33, was induced by hypoxia both in vitro and in vivo. MIG6 expression prevented heterodimer formation of ERBB family RTKs, and suppressed their downstream signaling. Knockdown of MIG6 enhanced tumor cell growth under hypoxic conditions, and promoted the phosphorylation of ERK and AKT via increased EGFR-HER3 binding. Critically, sensitivity to an EGFR-TKI, as well as to irradiation under hypoxic conditions, was increased in MIG6 knockdown cells. The expression of MIG6 was partly correlated with a pS6 negative zone in patient tumors. Analyses of tumor sections from 68 patients with activating EGFR mutations showed that patients with high MIG6 expression showed significantly shorter survival after EGFR-TKI treatment than other groups. Collectively, our data suggest that dormant cancer cells with a high MIG6 expression level might be one of the causes of EGFR-TKI resistance in EGFR mutant lung cancer cells.

Evaluation of cytotoxic effect of the combination of a pyridinyl carboxamide derivative and oxaliplatin on NCI-H1299 human non-small cell lung carcinoma cells.

Even with all improvements in both diagnostic and therapeutic techniques, lung cancer remains as the most lethal and prevalent cancer in the world. Therefore, new therapeutic drugs and new strategies of drug combination are necessary to provide treatments that are more efficient. Currently, standard therapy regimen for lung cancer includes platinum drugs, such as cisplatin, oxaliplatin, and carboplatin. Besides of the better toxicity profile of oxaliplatin when compared with cisplatin, peripheral neuropathy remains as a limitation of oxaliplatin dose. This study presents LabMol-12, a new pyridinyl carboxamide derivative with antileishmanial and antichagasic activity, as a new hit for lung cancer treatment, which induces apoptosis dependent of caspases in NCI-H1299 lung cancer cells both in monolayer and 3D culture. Moreover, LabMol-12 allows a reduction of oxaliplatin dose when they are combined, thereby, it is a relevant strategy for reducing the side effects of oxaliplatin with the same response. Molecular modeling studies corroborated the biological findings and suggested that the combined therapy can provide a better therapeutically profile effects against NSCLC. All these findings support the fact that the combination of oxaliplatin and LabMol-12 is a promising drug combination for lung cancer.

Electroneutral composite polymersomes self-assembled by amphiphilic polyphosphazenes for effective miR-200c invivo delivery to inhibit drug resistant lung cancer.

MiR-200c has been confirmed to display remarkable effects on proliferation inhibition and apoptosis induction of certain cancer cells, but the main challenge for its successful translation into the clinic remains its effective delivery to the action site invivo. In this study, a novel composite polyphosphazene vesicle system composed of amphiphilic [NP(PEG)0.3(EAB)1.7]n (PEEP) and weakly cationic [NP(PEG)0.5(DPA)1.5]n (PEDP) was prepared via a very simple dialysis method. The loading of miR-200c was accomplished with high efficiency by taking advantage of the combination effect of physical encapsulation and electrostatic interaction between vectors and miR-200c. The resultant miR-200c-loaded PEEP-PEDP polymersome (Nano-ED-200c) displayed suitable particle size, electric neutrality, excellent Ribonuclease stability and hemocompatibility. We also evaluated its subsequent miR-200c function in paclitaxel resistance human lung cancer (A549/T) cells in culture and tumor xenografts in nude mice. The results showed that Nano-ED-200c could achieve a higher miR-200c level and the enhanced antitumor efficacy with 68% tumor inhibition ratio at a very low dose of 1.0mg/kg than PEEP nanoparticle, PEDP nanoparticle, even than Lipo2000. All these evidences indicated that this miR-200c delivery via polyphosphazene vesicles could act as a potential new therapeutic option for paclitaxel resistant human lung cancer.

Morphometric and cytomorphologic characterization of EGFR-mutated cancer cells-comparison between cultured lung cancer cell lines and lung adenocarcinoma clinical samples.

Recently molecular targeting therapies such as inhibition of enzyme activities associated with gene mutations responsible for lung carcinogenesis have been demonstrating promising outcomes, increasing the importance of gene analysis using clinical samples. Cytomorphologic findings with predictive value toward specific gene mutation such as EGFR mutation could be a useful tool to select appropriate gene analyses using limited clinical samples. Morphometrical and cytomorphological evaluations were performed in 7 cultured lung cancer cell lines and 51 lung adenocarcinoma clinical samples to identify specific cytomorphologic characterization of EGFR-mutated cancer cells compared to the wild type. Morphometry demonstrated that the EGFR mutated cell lines had significantly smaller nuclear area and perimeter and more circular nuclei compared to the wild type. In contrast, EGFR-mutated clinical samples had significantly greater nuclear area and perimeter compared to the wild type EGFR samples. There were no clear differences in cytomorphologic parameters assessing nuclear atypicality between EGFR mutated cells and wild type EGFR cells in either cultured cell lines or clinical samples. Although our study suggested that EGFR mutation may have specific effects on nuclear morphology, no consistent characteristics of EGFR-mutated cells were identified in the clinical samples, probably due to various factors such as different pathologic stages and various incidences of lepidic growth. Further assessment of morphological characterization of EGFR-mutated cells in lung adenocarcinoma is warranted, increasing the number of samples and considering the effects of polyploidy, other gene mutations, pathology stage and tumor subtypes such as lepidic growth. Diagn. Cytopathol. 2016;44:717-724. © 2016 Wiley Periodicals, Inc.

A Fhit-mimetic peptide suppresses annexin A4-mediated chemoresistance to paclitaxel in lung cancer cells.

We recently reported that Fhit is in a molecular complex with annexin A4 (ANXA4); following to their binding, Fhit delocalizes ANXA4 from plasma membrane to cytosol in paclitaxel-resistant lung cancer cells, thus restoring their chemosensitivity to the drug. Here, we demonstrate that Fhit physically interacts with A4 through its N-terminus; molecular dynamics simulations were performed on a 3D Fhit model to rationalize its mechanism of action. This approach allowed for the identification of the QHLIKPS heptapeptide (position 7 to 13 of the wild-type Fhit protein) as the smallest Fhit sequence still able to preserve its ability to bind ANXA4. Interestingly, Fhit peptide also recapitulates the property of the native protein in inhibiting Annexin A4 translocation from cytosol to plasma membrane in A549 and Calu-2 lung cancer cells treated with paclitaxel. Finally, the combination of Tat-Fhit peptide and paclitaxel synergistically increases the apoptotic rate of cultured lung cancer cells and blocks in vivo tumor formation.Our findings address to the identification of chemically simplified Fhit derivatives that mimic Fhit tumor suppressor functions; intriguingly, this approach might lead to the generation of novel anticancer drugs to be used in combination with conventional therapies in Fhit-negative tumors to prevent or delay chemoresistance.

GLI pathogenesis-related 1 functions as a tumor-suppressor in lung cancer

BackgroundGLI pathogenesis-related 1 (GLIPR1) was originally identified in glioblastomas and its expression was also found to be down-regulated in prostate cancer. Functional studies revealed both growth suppression and proapoptotic activities for GLIPR1 in multiple cancer cell lines. GLIPR1’s role in lung cancer has not been investigated. Protein arginine methyltransferase 5 (PRMT5) is a protein arginine methyltransferase and forms a stoichiometric complex with the WD repeat domain 77 (WDR77) protein. Both PRMT5 and WDR77 are essential for growth of lung epithelial and cancer cells. But additional gene products that interact genetically or biochemichally with PRMT5 and WDR77 in the control of lung cancer cell growth are not characterized.MethodsDNA microarray and immunostaining were used to detect GLIPR1 expression during lung development and lung tumorigenesis. GLIPR1 expression was also analyzed in the TCGA lung cancer cohort. The consequence of GLIPR1 on growth of lung cancer cells in the tissue culture and lung tumor xenografts in the nude mice was observed.ResultsWe found that GLIPR1 expression is negatively associated with PRMT5/WDR77. GLIPR1 is absent in growing epithelial cells at the early stages of mouse lung development and highly expressed in the adult lung. Expression of GLIPR1 was down-regulated during lung tumorigenesis and its expression suppressed growth of lung cancer cells in the tissue culture and lung tumor xenografts in mice. GLIPR1 regulates lung cancer growth through the V-Erb-B avian erythroblastic leukemia viral oncogene homolog 3 (ErbB3).ConclusionsThis study reveals a novel pathway that PRMT5/WDR77 regulates GLIPR1 expression to control lung cancer cell growth and GLIPR1 as a potential therapeutic agent for lung cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0508-4) contains supplementary material, which is available to authorized users.

Nitrilase 1 modulates lung tumor progression in vitro and in vivo.

Uncovering novel growth modulators for non-small cell lung cancer (NSCLC) may lead to new therapies for these patients. Previous studies suggest Nit1 suppresses chemically induced carcinogenesis of the foregut in a mouse model. In this study we aimed to determine the role of Nit1 in a transgenic mouse lung cancer model driven by a G12D Kras mutation. Nit1 knockout mice (Nit1−/−) were crossed with KrasG12D/+ mice to investigate whether a G12D Kras mutation and Nit1 inactivation interact to promote or inhibit the development of NSCLC. We found that lung tumorigenesis was suppressed in the Nit1-null background (Nit1−/−:KrasG12D/+). Micro-CT scans and gross tumor measurements demonstrated a 5-fold reduction in total tumor volumes compared to Nit1+/+KrasG12D/+ (p<0.01). Furthermore, we found that Nit1 is highly expressed in human lung cancer tissues and cell lines and use of siRNA against Nit1 decreased overall cell survival of lung cancer cells in culture. In addition, cisplatin response was enhanced in human lung cancer cells when Nit1 was knocked down and Nit1−/−:KrasG12D/+ tumors showed increased sensitivity to cisplatin in vivo. Together, our data indicate that Nit1 may play a supportive role in the modulation of lung tumorigenesis and represent a novel target for NSCLCs treatment.

SiRNA-Loaded Polyion Complex Micelle Decorated with Charge-Conversional Polymer Tuned to Undergo Stepwise Response to Intra-Tumoral and Intra-Endosomal pHs for Exerting Enhanced RNAi Efficacy.

Small interfering RNA (siRNA) needs an efficient delivery vehicle to reach the cytoplasm of target cells for successful RNA interference (RNAi) therapy. This study aimed to develop an siRNA-loaded polyion complex (PIC) micelle equipped with a smart polymeric shell featuring tumor targetability and endosome escapability for enhanced RNAi activity in cancer cells. To this end, an acidic pH-responsive polypeptide was designed to exert a stepwise change in its charged state from negative to modestly positive and highly positive in response to slightly acidic environment of tumor (pH ∼6.7) and further lowered-pH condition of late endosomal compartments (pH ∼5.0), respectively, for selective binding to cancer cell surface and subsequent endosome disruption. This polypeptide, termed PAsp(DET-CDM/DBCO), was synthesized by introducing acid-labile carboxydimethyl maleate (CDM) and dibenzylcyclooctyne (DBCO) moieties into a polyaspartamide derivative bearing two-repeated aminoethylene side chains (PAsp(DET)). Then, PAsp(DET-CDM/DBCO) was installed on the surface of disulfide cross-linked PIC micelles prepared from cholesterol-modified siRNA (Chol-siRNA) and azide-poly(ethylene glycol)-b-poly[(3-mercaptopropylamidine)-L-lysine] (N3-PEG-b-PLys(MPA)) through the copper-free click reaction. Successful PAsp(DET-CDM/DBCO) coverage of PIC micelles was confirmed by a significant decrease in ζ-potential as well as a narrowly distributed size of 40 nm. The PAsp(DET-CDM/DBCO)-installed micelles significantly improved the gene-silencing efficiency in cultured lung cancer cells, compared with nonmodified control micelles, especially after incubation at pH 6.7. This improved silencing activity was nicely correlated with the facilitated cellular uptake of siRNA payloads at the acidic pH and the efficient endosomal escape. These results demonstrate that the acidic pH-responsive polypeptide shell is a promising design strategy for tumor-targeted siRNA delivery.

The Effect of Cigarette Smoke on the Translocator Protein (TSPO) in Cultured Lung Cancer Cells.

Lung cancer is prevalent in cigarette smokers. The mitochondrial membrane translocator protein (TSPO), is thought to protect cells from free radical damage. We examined the effect of cigarette smoke (CS) (containing free radicals) alone and in the presence of saliva (containing redox active free iron), on survival of H1299 lung cancer cells and on their mitochondrial characteristics, and whether TSPO binding was influenced by CS and by saliva. We exposed H1299 cells to CS in the presence/absence of saliva and also characterized TSPO binding in the cells using [3H]PK 11195 as a radioligand. CS induced a significant drop in mitochondrial potential (ΔΨm), while addition of saliva did not lead to further loss of ΔΨm (42.5% vs. 39.85%). Scatchard analysis of the saturation curve of [3H]PK 11195 binding (0.2-6 nM final concentration) yielded a straight-line plot (R = 0.9). Average Bmax value was 3274 ± 787 fmol/mg of protein, and average Kd value was 9.2 ± 1.3 nM. Benzodiazepine diazepam partially prevented decrease in cell survival following exposure to CS and redox active iron containing media (saliva) while benzodiazepine clonazepam did not, indicating that this effect is TSPO-specific. Exposure of cells to CS resulted in alternation of biomolecules expressed by CLs peroxidation, reduction of TSPO binding, and depletion of the mitochondrial potential. This irreversible damage was enhanced in the presence of saliva. All these modulations may result in cellular death increase following CS exposure, enhanced in the presence of saliva.

Histone deacetylase inhibitors promote the expression of ATP2A3 gene in breast cancer cell lines.

Recent studies have shown that expression of Sarco(endo)plasmic Reticulum Ca(2+) -ATPase 2 (SERCA2) is decreased in oral cancer; whereas expression of SERCA3 is considerably decreased or absent in human colon, gastric, breast, and lung cancers. The ATP2A2 and ATP2A3 genes encode SERCA2 and SERCA3 isoforms, respectively. Promoter methylation on CpG islands was responsible for the repression of ATP2A2 gene in human oral cancer samples. On the other hand, histone deacetylase inhibitors (HDACi) up-regulate ATP2A3 expression in gastric, colon, and lung cancer cells in culture, however, the molecular mechanism is unknown. In this study, we investigate whether HDACi and DNA methylation regulate ATP2A2 and ATP2A3 expression in human breast cancer cell lines. Results show a marked induction of SERCA3a and pan-SERCA3 mRNA expression in human MCF-7 and MDA-MB-231 cells treated with sodium butyrate (NaB) or trichostatin A (TSA); whereas SERCA2b mRNA expression did not change significantly. ChIP assays show that NaB or TSA treatment of MDA-MB-231 cells increases H3K9 acetylation on ATP2A3 promoter. NaB also decreases H3K9 trimethylation; suggesting that these modifications stimulate ATP2A3 gene expression, through a chromatin remodeling mechanism. In contrast, NaB or TSA do not increase H3K9-acetylation of ATP2A2 proximal promoter. In addition, treatment with 5-aza-2'-deoxycytidine did not affect SERCA2b and SERCA3a expression, suggesting that promoter methylation status does not alter their expression in these cell lines. We propose that alteration of SERCA2b/SERCA3a isoform expression ratio could affect calcium management within the cell, and thus, the cellular pathways regulated by calcium could be compromised, such as cellular proliferation or apoptosis. © 2015 Wiley Periodicals, Inc.

Improving Cancer Chemoradiotherapy Treatment by Dual Controlled Release of Wortmannin and Docetaxel in Polymeric Nanoparticles.

Combining molecularly targeted agents and chemotherapeutics is an emerging strategy in cancer treatment. We engineered sub-50 nm diameter diblock copolymer nanoparticles (NPs) that can sequentially release wortmannin (Wtmn, a cell signaling inhibitor) and docetaxel (Dtxl, genotoxic anticancer agent) to cancer cells. These NPs were studied in chemoradiotherapy, an important cancer treatment paradigm, in the preclinical setting. We demonstrated that Wtmn enhanced the therapeutic efficacy of Dtxl and increased the efficiency of radiotherapy (XRT) in H460 lung cancer and PC3 prostate cells in culture. Importantly, we showed that NPs containing both Wtmn and Dtxl release the drugs in a desirable sequential fashion to maximize therapeutic efficacy in comparison to administering each drug alone. An in vivo toxicity study in a murine model validated that NPs containing both Dtxl and Wtmn do not have a high toxicity profile. Lastly, we demonstrated that Dtxl/Wtmn-coencapsulated NPs are more efficient than each single-drug-loaded NPs or a combination of both single-drug-loaded NPs in chemoradiotherapy using xenograft models. Histopathological studies and correlative studies support that the improved therapeutic efficacy is through changes in signaling pathways and increased tumor cell apoptosis. Our findings suggest that our nanoparticle system led to a dynamic rewiring of cellular apoptotic pathways and thus improve the therapeutic efficiency.

Graphene oxide stabilized by PLA–PEG copolymers for the controlled delivery of paclitaxel

PurposeTo investigate the application of water-dispersible poly(lactide)–poly(ethylene glycol) (PLA–PEG) copolymers for the stabilization of graphene oxide (GO) aqueous dispersions and the feasibility of using the PLA–PEG stabilized GO as a delivery system for the potent anticancer agent paclitaxel. MethodsA modified Staudenmaier method was applied to synthesize graphene oxide (GO). Diblock PLA–PEG copolymers were synthesized by ring-opening polymerization of dl-lactide in the presence of monomethoxy-poly(ethylene glycol) (mPEG). Probe sonication in the presence of PLA–PEG copolymers was applied in order to reduce the hydrodynamic diameter of GO to the nano-size range according to dynamic light scattering (DLS) and obtain nano-graphene oxide (NGO) composites with PLA–PEG. The composites were characterized by atomic force microscopy (AFM), thermogravimetric analysis (TGA), and DLS. The colloidal stability of the composites was evaluated by recording the size of the composite particles with time and the resistance of composites to aggregation induced by increasing concentrations of NaCl. The composites were loaded with paclitaxel and the in vitro release profile was determined. The cytotoxicity of composites against A549 human lung cancer cells in culture was evaluated by flow cytometry. The uptake of FITC-labeled NGO/PLA–PEG by A549 cells was also estimated with flow cytometry and visualized with fluorescence microscopy. ResultsThe average hydrodynamic diameter of NGO/PLA–PEG according to DLS ranged between 455 and 534nm, depending on the molecular weight and proportion of PLA–PEG in the composites. NGO/PLA–PEG exhibited high colloidal stability on storage and in the presence of high concentrations of NaCl (far exceeding physiological concentrations). Paclitaxel was effectively loaded in the composites and released by a highly sustained fashion. Drug release could be regulated by the molecular weight of the PLA–PEG copolymer and its proportion in the composite. The paclitaxel-loaded composites exhibited cytotoxicity against A549 cancer cells which increased with incubation time, in conjunction with the increasing with time uptake of composites by the cancer cells. ConclusionGraphene oxide aqueous dispersions were effectively stabilized by water-dispersible, biocompatible and biodegradable PLA–PEG copolymers. The graphene oxide/PLA–PEG composites exhibited satisfactory paclitaxel loading capacity and sustained in vitro drug release. The paclitaxel-loaded composites could enter the A549 cancer cells and exert cytotoxicity. The results justify further investigation of the suitability of PLA–PEG stabilized graphene oxide for the controlled delivery of paclitaxel.

MKL1 potentiates lung cancer cell migration and invasion by epigenetically activating MMP9 transcription.

Malignant tumors are exemplified by excessive proliferation and aggressive migration/invasion contributing to increased mortality of cancer patients. Matrix metalloproteinase 9 (MMP9) expression is positively correlated with lung cancer malignancy. The mechanism underlying an elevated MMP9 expression is not clearly defined. We demonstrate here that the transcriptional modulator megakaryocytic leukemia 1 (MKL1) was activated by hypoxia and transforming growth factor (TGF-β), two prominent pro-malignancy factors, in cultured lung cancer cells. MKL1 levels were also increased in more invasive types of lung cancer in humans. Depletion of MKL1 in lung cancer cells attenuated migration and invasion both in vitro and in vivo. Overexpression of MKL1 potentiated the induction of MMP9 transcription by hypoxia and TGF-β, whereas MKL1 silencing diminished MMP9 expression. Of interest, MKL1 knockdown eliminated histone H3K4 methylation surrounding the MMP9 promoter. Further analyses revealed that MKL1 recruited ASH2, a component of the H3K4 methyltransferase complex, to activate MMP9 transcription. Depletion of ASH2 ameliorated cancer cell migration and invasion in an MMP9-dependent manner. Together our data indicate that MKL1 potentiates lung cancer cell migration and invasion by epigenetically activating MMP9 transcription.

In vitro cultured lung cancer cells are not suitable for animal-based breath biomarker detection

In vitro cultured lung cancer cell lines were investigated regarding the possible identification of volatile organic compounds as potential biomarkers. Gas samples from the headspace of pure culture medium and from the cultures of human lung adenocarcinoma cell lines A549 and Lu7466 were exposed to polypropylene fleece in order to absorb odour components. Sniffer dogs were trained with loaded fleeces of both cell lines, and honey bees were trained with fleeces exposed to A549. Afterwards, their ability to distinguish between cell-free culture medium odour and lung cancer cell odour was tested. Neither bees nor dogs were able to discriminate between odours from the cancer cell cultures and the pure culture medium. Solid phase micro extraction followed by gas chromatography with mass selective detection produced profiles of volatiles from the headspace offered to the animals. The profiles from the cell lines were largely similar; distinct differences were based on the decrease of volatile culture medium components due to the cells’ metabolic activity. In summary, cultured lung cancer cell lines do not produce any biomarkers recognizable by animals or gas chromatographic analysis.

Reactivation of epigenetically silenced miR-512 and miR-373 sensitizes lung cancer cells to cisplatin and restricts tumor growth.

MicroRNAs (miRs) regulate a variety of cellular processes, and their impaired expression is involved in cancer. Silencing of tumor-suppressive miRs in cancer can occur through epigenetic modifications, including DNA methylation and histone deacetylation. We performed comparative miR profiling on cultured lung cancer cells before and after treatment with 5'aza-deoxycytidine plus Trichostatin A to reverse DNA methylation and histone deacetylation, respectively. Several tens of miRs were strongly induced by such 'epigenetic therapy'. Two representatives, miR-512-5p (miR-512) and miR-373, were selected for further analysis. Both miRs were secreted in exosomes. Re-expression of both miRs augmented cisplatin-induced apoptosis and inhibited cell migration; miR-512 also reduced cell proliferation. TEAD4 mRNA was confirmed as a direct target of miR-512; likewise, miR-373 was found to target RelA and PIK3CA mRNA directly. Our results imply that miR-512 and miR-373 exert cell-autonomous and non-autonomous tumor-suppressive effects in lung cancer cells, where their re-expression may benefit epigenetic cancer therapy.

Study on inhibition effect of alkaloid monomers from Sophora alopecuroides on in vitro cultured lung cancer cells

Objective To investigate in vitro anti-tumor activity and the related mechanism of four alkaloid monomers from Sophora alopecuroides,and to evaluate their toxicity on normal cells,thereby to screen the drugs with high efficiency and low toxicity anti-tumor effects.Methods Methyl thiazolyl tetrazolium (MTT) assay was employed to determine the effects of four alkaloid monomers on the proliferation activity of human lung cancer cells and hamster lung cells.Morphology of apoptotic lung cancer cell nucleus was observed by laser scanning confocal microscopy with Hochest33342 staining.Cell apoptosis and necrosis were measured by Annexin Ⅴ-FITC/PI double staining.Results The four alkaloid monomers showed different degree of proliferation inhibition on lung cancer cells with dose dependent manner.Oxysophoridine had the strongest inhibition effect on lung cancer cells and the minimun toxicity on normal lung cells.Hochest33342 staining and flow cytometry analysis demonstrated that oxysophoridine could induce the apoptosis and necrosis of A549 cells.Conclusions All four alkaloid monomers have certain anti-tumor effect on lung cancer cells.Oxysophoridine is the most active one with high efficiency and low toxicity characteristics,which is suitable for use as anti-tumor drug candidate for further development. Key words: Alkaloid monoers; Lung cancer cells; Anti-tumor activity; Action mechanism

Stanniocalcin-2 (STC2): A potential lung cancer biomarker promotes lung cancer metastasis and progression

The homodimeric glycoprotein, stanniocalcin 2 (STC2) is previously known to be involved in the regulation of calcium and phosphate transport in the kidney and also reported to play multiple roles in several cancers. However, its function and clinical significance in lung cancer have never been reported and still remain uncertain. Here, we investigated the possibility of STC2 as a lung cancer biomarker and identified its potential role in lung cancer cell growth, metastasis and progression. Proteomic analysis of secretome of primary cultured lung cancer cells revealed higher expression of STC2 in cancers compared to that of adjacent normal cells. RT-PCR and Western blot analyses showed higher mRNA and protein expressions of STC2 in lung cancer tissues compared to the adjacent normal tissues. Knockdown of STC2 in H460 lung cancer cells slowed down cell growth progression and colony formation. Further analysis revealed suppression of migration, invasion and delayed G0/G1 cell cycle progression in the STC2 knockdown cells. STC2 knockdown also attenuated the H202-induced oxidative stress on H460 cell viability with a subsequent increase in intracellular ROS levels, which suggest a protective role of STC2 in redox regulatory system of lung cancer. These findings suggest that STC2 can be a potential lung cancer biomarker and plays a positive role in lung cancer metastasis and progression. This article is part of a Special Issue entitled: Medical Proteomics.

Abstract 99: Wnt signaling promotes tumor propagation in a mouse model of lung adenocarcinoma

Abstract The considerable variability within tissue microenvironments as well as the multiclonality of cancers leads to heterogeneity within tumors, increasing the probablility of cellular states that promote resistance to therapy and eventually lead to reconstitution of the tumor by treatment-resistant cancer cells, which in some case have properties of normal tissue stem cells. Wnt signals are important in the maintenance of stem cells in various epithelial tissues, including in lung development and regeneration. We hypothesized that Wnt signals would also contribute to hierarchial organization of KrasG12D and KrasG12D; tp53Δ/Δ mouse lung adenocarcinomas. Indeed, rare cells with nuclear βeta-catenin were detected in mouse tumors in vivo. Stimulating KP mouse lung adenocarcinoma cells with recombinant Wnt3a promoted their spheroid-forming ability in a 3-dimensional Matrigel culture system, whereas RNAi or small-molecule inhibitors targeting Porcupine, an enzyme required for post-translational palmitoylation of Wnt, suppressed tumor initiation in vitro and in vivo. Interestingly, either the stimulation or inhibition of the Wnt signaling pathway had no effect on growth of KP lung cancer cells in standard 2-dimensional cell culture, suggesting that Wnt signaling qualitatively modulates the cellular phenotype instead of controlling cell proliferation. We propose that targeting the Wnt signaling pathway can stimulate re-differentiation of lung adenocarcinoma cells, which may render them more sensitive to conventional anti-tumor therapies. Citation Format: Tuomas Tammela, Natanya Kerper, Wen Xue, Nikhil Joshi, Pekka Katajisto, Tyler Jacks. Wnt signaling promotes tumor propagation in a mouse model of lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 99. doi:10.1158/1538-7445.AM2014-99

Abstract 466: LMO1 is a novel oncogene in neuroendocrine lung cancer

Abstract Introduction. The aim of the study is to characterize the role of LMO1 in tumorigenesis of neuroendocrine lung cancer. LMO1 is a transcription factor that belongs to the LMO protein family containing the cysteine-rich LIM domains. LMO1 has been demonstrated to function as an important oncogene in the development of T-lineage leukemia and lymphoma. A recent study in neuroblastoma suggests that aberrant overexpression of LMO1 may play a critical role in the development of neuroendocrine cancers. The role of LMO1 in neuroendocrine lung cancer, however, was not investigated. Experimental Approaches. By analyzing a large panel of cell lines including lung cancer cell lines and normal lung epithelial cells, we compared the relative expression of LMO1 in different subtypes of lung cancers, and examined the correlation of LMO1 expression levels with neuroendocrine differentiation markers. Using in vitro approaches, we investigated the function of LMO1 in regulating lung cancer cell growth. To investigate the clinical significance of LMO1 dysregulation in lung cancer patients, we analyzed the correlation of LMO1 tumor levels with lung cancer patient survival. Results. We show that LMO1 is significantly overexpressed in small cell lung cancer (SCLC), an aggressive and major subtype of neuroendocrine lung cancers, relative to non-small cell lung cancer (NSCLC) and normal lung cells. In addition, in NSCLC cells, LMO1 mRNA levels are significantly correlated with expression of neuroendocrine differentiation markers. Functional in vitro investigations indicate that LMO1 overexpression significantly promotes growth of cultured lung cancer cells, suggesting its oncogenic function in lung cancer. More strikingly, our investigations of two independent lung cancer patient populations indicate that high tumor LMO1 mRNA level is an independent predictor of poor patient survival. Conclusions. Altogether, our findings strongly suggest that LMO1 is a neuroendocrine-specific oncogene in lung cancer that plays an important role in determining the cancer aggressiveness. Further studies are certainly warranted to define the mechanisms underlying the oncogenic function of LMO1 in neuroendocrine lung cancer and to further evaluate the clinical significance of LMO1 as a diagnostic and prognostic marker for neuroendocrine lung cancers in prospective studies. Citation Format: Zhenze Zhao, Xiuye Ma, Xiaojie Yu, Tzu-Hung Hsiao, Yidong Chen, Milind Suraokar, Ignacio Wistuba, John D. Minna, Alexander Pertsemlidis, Liqin Du. LMO1 is a novel oncogene in neuroendocrine lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 466. doi:10.1158/1538-7445.AM2014-466

Abstract 3452: N-glycosylation on lung cancer cell-secreted IL-6 prolongs its activation on JAK/STAT pathway

Abstract Interleukin-6 (IL-6) is overexpressed in various cancer cells, intriguingly contributing to both tumor growth and modulating anti-tumor immunity. Cell-type-specific glycosylations on IL-6 have been deduced in early studies; however, the glycosylation pattern and biological function of cancer cell-secreted IL-6 remain unclear. Here, we describe the glycosylation pattern of lung cancer cell-secreted IL-6 and its impact on the activation of JAK/STAT pathway. IL-6 molecules with different molecular weights were detected in the conditional media of IL-6-overexpressed lung cancer cell lines by immunoblot. Because in NetNGlyc 1.0 Server, one possible N-glycosylation site has been predicted at N73 on IL-6, we used treatment of N-glycosylation-specific inhibitors and site-directed mutagenesis on N73 to demonstrate that lung cancer cell-secreted IL-6 is modified by N-glycosylation. To examine which glycotransferases may participate in the modification, we had screened the expression of glycosyltransferases using qPCR and found that the expression of fucosyltransferase 8 (FUT8), responsible for core fucosylation on N-glycosylated proteins, was higher in lung cancer cells compared to normal bronchial cell. We then reduced core fucosylation on lung cancer cell-secreted IL-6 by silencing FUT8 with shRNA transduction. Subsequently, cells were treated with conditional media containing fully-glycosylated or core fucose-depleted IL-6 to uncover the potential influences on cellular signaling from glycosylation on IL-6. The fully-gycosylated IL-6 induced prolonged STAT3Y705 phosphorylation and distinct gene population compared to core fucose-depleted IL-6. The nuclear retention of STAT3 was concordant with the prolonged STAT3 activation in cells treated with fully-gycosylated IL-6. In paired normal (N) and tumor (T) tissues from lung cancer patients, higher FUT8 mRNA was detected in tumor part than normal part. Besides, we found similar glycosylation pattern in the secreted IL-6 of short-term cultured lung cancer cells derived from malignant pleural effusions. Together, we report the presence of specific IL-6 glycoforms secreted from lung cancer cell lines and lung cancer cells from clinical samples. Moreover, the glycosylation on IL-6 changes its activity on the regulation of JAK/STAT pathway. Citation Format: Chun-Hua Hung, Hsuan-Heng Yeh, Hao-Chen Wang, Chien-Chung Lin, Tsung-Lin Tsai, Wei-Lun Huang, Chuan-Fa Chang, Wu-Chou Su. N-glycosylation on lung cancer cell-secreted IL-6 prolongs its activation on JAK/STAT pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3452. doi:10.1158/1538-7445.AM2014-3452