Lung Cancer Cell Line H1299 Research Articles

Knockdown of HSDL2 inhibits lung adenocarcinoma progression via down-regulating AKT2 expression.

The aims of the present study are to investigate the role of hydroxysteroid dehydrogenase-like 2 (HSDL2) in the progression of lung adenocarcinoma and illuminate the underlying molecular mechanisms. ShRNA targeting HSDL2 gene (siHSDL2) was utilized to knockdown (KD) HSDL2 expression. In vitro and in vivo experiments were carried out to investigate the effect of siHSDL2 on the progression of lung adenocarcinoma. Microarray hybridization and gene expression analysis were used to investigate effect of siHSDL2 on mRNA expression profile in lung cancer cell line H1299. Our data demonstrated that HSDL2 was up-regulated in lung adenocarcinoma tissue samples (P<0.001). Patients with high HSDL2 expression in cancer tissues had a worse overall survival (P<0.001). HSDL2 KD not only inhibited the proliferation, cell cycle, apoptosis, clone-formation, invasion and migration of lung adenocarcinoma cells in vitro (P<0.05), but also suppressed the growth and metastasis in vivo (P<0.05). HSDL2 KD resulted in up-regulation of 681 genes and down-regulation of 276 genes. HSDL2 KD down-regulated the protein expression and phosphorylation of protein kinase B β (AKT2) (P<0.001 and P<0.001, respectively) and protein expression of baculoviral IAP repeat-containing 3 (BIRC3; P=0.001), and up-regulated the phosphorylation of ERK (P<0.001). Rescue experiments showed that AKT2 overexpression reversed the suppression effect of siHSDL2 on cell proliferation (P<0.001), invasion (P<0.001) and migration (P<0.001) significantly. HSDL2 functions as an oncogene to promote the growth and metastasis of lung adenocarcinoma via promoting the expression of AKT2.

Mitochondrial division inhibitor (mdivi-1) decreases oxidative metabolism in cancer

BackgroundPrevious studies suggested that mdivi-1 (mitochondrial division inhibitor), a putative inhibitor of dynamin-related protein (DRP1), decreased cancer cell proliferation through inducing mitochondrial fusion and altering oxygen consumption. However, the metabolic reprogramming underlying the DRP1 inhibition is still unclear in cancer cells.MethodsTo better understand the metabolic effect of DRP1 inhibition, [U-13C]glucose isotope tracing was employed to assess mdivi-1 effects in several cancer cell lines, DRP1-WT (wild-type) and DRP1-KO (knockout) H460 lung cancer cells and mouse embryonic fibroblasts (MEFs).ResultsMitochondrial staining confirmed that mdivi-1 treatment and DRP1 deficiency induced mitochondrial fusion. Surprisingly, metabolic isotope tracing found that mdivi-1 decreased mitochondrial oxidative metabolism in the lung cancer cell lines H460, A549 and the colon cancer cell line HCT116. [U-13C]glucose tracing studies also showed that the TCA cycle intermediates had significantly lower enrichment in mdivi-1-treated cells. In comparison, DRP1-WT and DRP1-KO H460 cells had similar oxidative metabolism, which was decreased by mdivi-1 treatment. Furthermore, mdivi-1-mediated effects on oxidative metabolism were independent of mitochondrial fusion.ConclusionsOur data suggest that, in cancer cells, mdivi-1, a putative inhibitor of DRP1, decreases oxidative metabolism to impair cell proliferation.

Overexpression of Krüppel-Like Factor 4 Suppresses Migration and Invasion of Non-Small Cell Lung Cancer Through c-Jun-NH2-Terminal Kinase/Epithelial-Mesenchymal Transition Signaling Pathway.

Krüppel-like factor 4 (KLF4) is a transcription factor and plays a vital role in cancer initiation and development. However, the role of Krüppel-like factor 4 in the metastasis of non-small cell lung cancer (NSCLC) is not clear. Here, we demonstrated that the expression of Krüppel-like factor 4 was significantly decreased in human non-small cell lung cancer tissues compared with that in normal tissues using Western blot. We performed immunohistochemical staining and observed the decreased expression of Krüppel-like factor 4 in human lung cancer tissues, and metastatic tumor tissues located in the trachea and main bronchus. We also found that the E-cadherin expression was decreased, while vimentin expression was increased in human NSCLC tissues and metastatic tumor tissues located in the trachea and main bronchus. Additionally, enforced expression of Krüppel-like factor 4 in mouse lungs significantly inhibited the metastasis of circulating Lewis lung carcinoma cells to the lungs by attenuating mesenchymal-epithelial transition (MET). Furthermore, cell scratch assays and Matrigel invasion assays revealed that overexpression of Krüppel-like factor 4 inhibited the migration and invasion of non-small cell lung cancer cell lines A549, H1299, H226, and H1650 cells. Moreover, overexpression of Krüppel-like factor 4 attenuated TGF-β1-induced epithelial-mesenchymal transition (EMT) in A549, and inhibited the phosphorylation of c-Jun-NH2-terminal kinase (JNK), an important pathway in metastasis in non-small cell lung cancer. Our in vivo and in vitro findings illustrate that Krüppel-like factor 4 inhibited metastasis and migration of non-small cell lung cancer, and indicate that Krüppel-like factor 4 could be a potential therapeutic target for the treatment of non-small cell lung cancer.

Aquaporin 5 promotes tumor migration and angiogenesis in non-small cell lung cancer cell line H1299.

Non-small cell lung cancer (NSCLC) constitutes the majority of all lung-cancer cases. Aquaporin 5 (AQP5) may be involved in NSCLC by promoting lung-cancer initiation and progression. The present study aimed to determine the role of AQP5 in migration and angiogenesis using NSCLC cells and HUVECs. AQPs 1, 3, 4, 5, 8 and 9 were screened in the NSCLC cell line H1299, and the present results showed that AQP5 mRNA was upregulated compared with the other AQP genes. At the protein level, AQP5 was significantly increased in H1299 cells compared with 16HBE cells. AQP5 knockdown in H1299 cells significantly decreased cell migration compared with untransfected cells, as demonstrated by both Transwell and wound closure assays. The present study further investigated H1299 ability to promote HUVEC vascularisation. The supernatants of both transfected and untransfected H1299 cells were used as conditioned medium for HUVECs, and tube formation was measured. The supernatant of AQP5-downregulated cells exhibited significantly low tube formation potential compared with untransfected cells. Similarly, vascular endothelial growth factor was significantly increased in control cells (si-NC) compared with cells transfected with small interfering RNA targeting AQP5. The present study found that AQP5 downregulation significantly decreased the phosphorylation level of epidermal growth factor receptor and the activity of the ERK1/2 pathway. In summary, the present study suggested that AQP5 influenced migration and angiogenesis in NSCLCs in vitro and may potentially exhibit similar in vivo effects.

Pharmacophore modeling, 3D-QSAR, synthesis, and anti-lung cancer evaluation of novel thieno[2,3-d][1,2,3]triazines targeting EGFR.

Two series of thieno[2,3-d][1,2,3]triazine derivatives were designed, synthesized, and biologically evaluated as potential epidermal growth factor receptor (EGFR) inhibitors targeting the non-small-cell lung cancer cell line H1299. Most of the synthesized compounds displayed IC50 values ranging from 25 to 58 nM against H1299, which are superior to that of gefitinib (40 µM). 3-(5,6,7,8-Tetrahydro-7H-cyclohexa[4:5]thieno[2,3-d]-1,2,3-triazin-4-ylamino)benzene-1,3-diamine (6b) achieved the highest cytotoxic activity against H1299 with an IC50 value of 25 nM; it had the ability to decrease the EGFR concentration in H1299 cells from 7.22 to 2.67 pg/ml. In vitro, the IC50 value of compound 6b was 0.33 nM against EGFR, which is superior to that of gefitinib at 1.9 nM and erlotinib at 4 nM. The three-dimensional quantitative structure-activity relationships and molecular modeling studies revealed comparable binding modes of compound 6b, gefitinib, and erlotinib in the EGFR active site. The in silico ADME (absorption, distribution, metabolism, and excretion) prediction parameters of this compound revealed promising pharmacokinetic and physicochemical properties. Moreover, DFT (density functional theory) calculations showed the high reactivity of compound 6b toward the EGFR compared with other compounds. The designed compound 6b might serve as an encouraging lead compound for the discovery of promising anti-lung cancer agents targeting EGFR.

Self-assembly of a robust, reduction-sensitive camptothecin nanotube.

The self-assembly and covalent crosslinking of a camptothecin (CPT) tetrapeptide nanotube is reported. Intermolecular disulfide bond formation of a self-assembled CPT-peptide reversibly stabilized the nanotubes toward dissociation at low concentrations, resulting in inhibited release of CPT. In the presence of dithiothreitol (DTT), the release of CPT was significantly accelerated. The crosslinked nanotubes also exhibited in vitro cytotoxicity against human non-small cell lung cancer cell lines A549 and H460.

Proapoptotic Index Evaluation of Two Synthetic Peptides Derived from the Coneshell Californiconus californicus in Lung Cancer Cell Line H1299.

Lung cancer is one of the most common types of cancer, accounting for approximately 15% of all cancer cases worldwide. Apoptosis is the dominant defense mechanism against tumor development. The balance between pro- and antiapoptotic members of the Bcl-2 protein family can determine cellular fate. The venom of predatory marine snails Conus is estimated to have 100–400 toxins called conotoxins. The family of α-conotoxins is known to consist of selective antagonists of nicotinic acetylcholine receptors (nAChRs). Lung cancer cells overexpress several subunits of nAChRs and are considered as an excellent target for new anticancer drugs. We compared the cytotoxic effect of two synthetic peptides derived from Californiconus californicus, Cal14.1a, and Cal14.1b, which only differ by one amino acid in their sequence, and compared their proapoptotic balance by Bax and Bcl-2 mRNA expression. We determined the caspase-3 and -7 activation to demonstrate apoptosis induction. Results showed that Cal14.1a induces a high Bax/Bcl-2 ratio in H1299 (lung cancer cells). Although Cal14.1b has a cytotoxic effect on H1299 cells, reducing cell viability by 30%, it does not increase the Bax/Bcl-2 ratio, which could be explained by the Glu in the 15th residue, which is crucial for the ability of Cal14.1a to induce apoptosis.

Micellization, anti-proliferative activity and binding study of cationic gemini surfactants with calf thymus DNA

Aggregation behaviour of two cation gemini surfactants i.e., 1,3-bis (1-decylbenzimidazolium) propane bromide BG10 and 1,3-bis (1-octylbenzimidazolium) propane bromide BG8 was studied using conductivity and dynamic light scattering (DLS). The enthalpy (∆Hmо), entropy (∆Smо) and Gibbs free energy of micellization (∆Gmо) have been observed from the conductivity method at various temperatures. The anti-proliferative potential of these novel gemini surfactants against human non-small lung cancer cell line-H1299 has been investigated. Both gemini surfactants, BG8 and BG10 showed a considerable inhibition in cancer cell proliferation and IC50 values are observed to reduce with chain length. The ctDNA binding was also observed by UV–visible, transmission electron microscopy (TEM), ethidium bromide (EtBr) exclusion experiments, agarose gel electrophoresis, circular dichroism (CD) and viscometry techniques. These results were further validated by in-silico studies. The binding capability of BG surfactants to ctDNA can be observed from their ability to remove EtBr from the intercalating complex of DNA resulting in fluorescence quenching.

Investigate The Different Effect Of Nicotine On H460 And H441 Lung Cells Viability

Background: Nicotine is the foremost chemical constituent responsible for addiction in tobacco products, in the non-ionized condition can be easily absorbed via epithelial tissue of the lung, the mouth, the nose and across the skin&#x0D; Objective:The study examines the harmful effect of the nicotine which is an important component of cigarette in vitro.&#x0D; Type of the study: Cross-sectional study.&#x0D; Methods: Examines the harmful effect of the nicotine which is an important component of cigarette in vitro by using two types of lung cancer cell lines (H460 TP53+/+, H441 TP53-/-).&#x0D; Results: The results showed the total count of H460( TP53+/+) cancer lung cell lines was (5.2 ×106 cells /ml) , the number (4.9 ×106 cells /ml) of them were alive and (3.6×105 cells /ml) of them were dead, with percentage of viability (93.15%), while the total count of H441( TP53-/-) lung cancer cells was (5.1 ×106 cells /ml), the number (4.1 ×106 cells /ml) of them were alive, and the number (9.9×105 cells /ml) of them were dead with percentage of viability (80.55%). And it revealed that the nicotine inhibited viability of H460 lung cancer cell lines in all concentrations (1, 10, 500 and 1000 μ M) and the cells were completely abolished by treatment with (1000 μ M) when the viability reached to minimum percentage (3.43%), while nicotine induced proliferation in H441 lung cancer cell lines even in lowest concentration (1 μ M), whereas the viability reached to maximum percentage (41.04%) at concentration (1000 μ M). Also it noticed that the treatment with nicotine at concentrations (1,10, 500 and 1000 μ M ) for 24 and 48 hr induced the apoptosis but not necrosis in H460 lung cancer cell lines, especially at the highest concentrations (1000 μ M) for 48hr when the percentage of apoptosis reached to the maximum value (55.5%), however it was induced proliferation in H441 lung cancer cell lines with highest proliferation at concentration (1000 μ M), when the percentage apoptosis value reached to the minimum percentage (1.5%) when compared to un treated cells (control).&#x0D; Conclusions: the cells lack to TP53(H441 TP53-/-) will proliferate when exposed to nicotine and some of these cells will suffer from necrosis as a replacement of apoptosis.&#x0D;

Mutated p53 Promotes the Symmetric Self-Renewal of Cisplatin-Resistant Lung Cancer Stem-Like Cells and Inhibits the Recruitment of Macrophages.

It has been proposed that mutant p53 is correlated with the recurrence of lung cancer. Recently, a small population of cells with asymmetric or symmetric self-renewal potential has been identified in lung cancer, which was termed as cancer stem-like cells (CSCs) and was speculated to be the reason for cancer recurrence after chemotherapy. In this study, we used lung cancer cell lines with different TP53 backgrounds to elucidate the potential role of mutant p53 in regulating lung CSC self-renewal and on lung cancer recurrence. Cisplatin-resistant lung cancer cells with different TP53 backgrounds were generated in vitro by exposing A549, H460, and H661 lung cancer cell lines repeatedly to cisplatin. CD44+/CD90+ stem-like cells were identified in above cisplatin-resistant lung cancers (termed as cisplatin-resistant lung cancer stem-like cells, (Cr-LCSCs)) and stained with PKH26 dye which was used to define the self-renewal pattern. The proportion of symmetric divisions was significantly higher in Cr-LCSCs with mutant (mt) p53 compared with Cr-LCSCs with wild-type (wt) p53, and forced expression of mt p53 promoted the symmetric division of Cr-LCSCs. Furthermore, fewer macrophages accumulated in subcutaneously implanted xenografts consisting of mt p53 Cr-LCSCs compared with wt p53 Cr-LCSCs. These results indicated that mt p53 might accelerate the recurrence of lung cancer by regulating the self-renewal kinetics of Cr-LCSCs as well as the recruitment of macrophages.

Synthesis of gadolinium oxide nanocuboids for in vitro bioimaging applications

Undoped and Eu-doped gadolinium oxide (GGNCs and EGNCs) nanocuboids functionalized with D-gluconic acid (GA) were synthesised by a simple yet unique scheme using all the benign solvents and temperature. Samples were characterized and presented with properties like good dispersity, biocompatibility, and stability required for standard contrast agent used in magnetic resonance imaging (MRI) and optical coherence tomography (OCT). Biological assays such as 3-(4, 5-dimethyl-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) assay, flow cytometry and confocal microscopy were used to determine its biocompatibility, cellular internalization and optical cellular imaging using A549 and H1299 lung cancer cell lines. EGNCs treated with cell lines emitted red fluorescence which was used to track the internalization of EGNCs within the cells. GGNCs sample showed ∼20% enhanced MRI relaxivity as compared to EGNCs; whereas EGNCs revealed better contrast in doctor scans of OCT. Samples could be used as promising candidate for other biomedical applications such as drug delivery when equipped with well functionalised drug molecules.

Comparing the Microdosimetric Kinetic Model-Predicted Proton RBE Values Using Constant and Variable Quadratic Beta Coefficients

In the current microdosimetric kinetic model (MKM) model for predicting the biological effects of therapeutic particles, the quadratic beta coefficient is assumed to be a constant independent of radiation types. However, many cell irradiation experiments have shown the variable beta coefficients for different beam qualities. The purpose of this study is to compare the predicted relative biological effectiveness (RBE) of protons using the MKM model with a constant beta value and beam quality (i.e., the microdosimetric quantity lineal energy) dependent beta values. We have spatially mapped the biological effects along the path of a mono-energetic 81.4 MeV proton beam using the high-throughput method. Clonogenic cell survival curves with twelve different beam qualities (dose mean lineal energy, yD) were obtained for the non-small cell lung cancer (NSCLC) cell line H460. The Cs-137 photon source was used as the reference. The microdosimetric data were obtained from the track-structure Monte Carlo simulations using Geant4-DNA with a water sphere (d = 2 μm) as the target. The alpha and beta coefficients for protons and reference photons were obtained from the linear-quadratic fit results of each survival curve. The beta coefficient of photons was selected as the constant beta value for MKM predictions. We calculated the alpha coefficient using the constant beta first and then using the variable beta for each proton beam quality. The MKM model predicted RBE values as a function of beam quality were then compared with the experimental RBE. The twelve evenly sampled yD values of the proton beam ranges from 1.38 to 21.77 keV/μm with the value of 12.93 keV/μm at the Bragg peak. The alpha and beta coefficients from Cs-137 photon source are 0.23 Gy-1 and 0.10 Gy-2. Our experiments have shown a general trend that the alpha and beta values of protons from experimental survival curves increase with the increase of yD. The experimental RBE increases from 0.99 (yD = 1.38 keV/μm) to 2.85 (yD = 21.77 keV/μm) in a rather non-linear way. Using the constant beta based MKM model, the predicted RBE (cell surviving fraction = 0.1) only varies from 1.00 to 1.12, nearly independent of the beam quality. In contrast, using the variable beta based MKM model, the predicted proton RBE increases as a function of yD and varies from 1.00 to 2.73. The relative difference between variable beta based MKM model prediction and the experimental RBE is within ±10%. From the results in this study, we conclude that using the experimentally determined variable beta values, MKM model can better predict the biological effects of protons. However, more experiments for different beam qualities and different cell lines are needed to further consolidate the conclusion made here.

The role of miR-130a-3p and SPOCK1 in tobacco exposed bronchial epithelial BEAS-2B transformed cells: Comparison to A549 and H1299 lung cancer cell lines

ABSTRACTIn the pathogenesis of human lung cancer induced by tobacco smoke decreased expression levels of microRNAs (miRNAs) are known to occur. At present, the specific miRNAs expression levels reduced by tobacco smoke and subsequent lung cellular transformation remain to be determined. The aim of this study was thus to identify the miRNAs affected following cigarette-smoke exposure in bronchial epithelial BEAS-2B cells that were malignantly transformed into S30 cells. In addition, the miRNAs in S30 transformed cells were compared to human lung cancer cell lines A549 and H1299. Our results identified miR-130a-3p which was down-regulated in S30 cells as well as A549 and H1299 lung cancer cell lines. Using miRNA mimic, a correlation between elevated miR-130a-3p expression levels and reduced migration in A549 and H1299 cell lines and S30 cells was noted as evidenced by transwell and wound healing assays accompanied by enhanced apoptosis. Further, two online target genes prediction programs TargetScan and miRDB were employed to identify the miRNA target gene SPOCK1 in all three cell types. SPOCK1 expression was higher in unexposed bronchial epithelial BEAS-2B cells. It is of interest that however silencing SPOCK1 in transformed S30 cells exposed to cigarette-smoke a marked depression in cell migration was noted. Our findings demonstrate that upregulated miR-130a-3p was associated with reduced SPOCK1 expression in transformed S30 as well as lung cancer A549 and H1299 cell lines indicating that cigarette transformed cells behave similar to lung cancer cells and this process involves diminished lung cancer cells migration.

FRK plays an oncogenic role in non-small cell lung cancer by enhancing the stemness phenotype via induction of metabolic reprogramming.

The role of Fyn-related kinase (FRK) in malignant tumors remains controversial. Our study investigated the function of FRK in lung cancer. Immunohistochemistry staining and generating a knockout of FRK by CRISPR/Cas9 in H1299 (FRK-KO-H1299) cells were strategies used to explore the role of FRK. Immunohistochemistry staining indicated that FRK expression was elevated in 223 lung cancer tissues compared to 26 distant normal lung tissues. FRK contributed to poor survival status in lung cancer patients and acted as a predictor for poor prognosis of lung cancer. Knockout of FRK by CRISPR/Cas9 markedly inhibited proliferation, invasion, colony formation and epithelial-mesenchymal transition (EMT) process in the lung cancer cell line H1299. Further exploration indicated that FRK-KO damaged the stemness phenotype of H1299 by inhibiting CD44 and CD133 expression. Seahorse detection and a U-13 C flux assay revealed that FRK-KO induced metabolism reprogramming by inhibiting the Warburg effect and changing the energy type in H1299 cells. Epidermal growth factor stimulation recovered the expression of FRK and biological functions, metabolic reprogramming and stemness phenotype of H1299 cells. FRK plays an oncogenic role in lung cancer cells via a novel regulation mechanism of enhancing the stemness of H1299 cells by inducing metabolism reprogramming, which finally promotes EMT and metastasis. Our study also indicates that FRK could be used as a potential therapeutic target for drug development.

Development of c‑MET‑specific chimeric antigen receptor‑engineered natural killer cells with cytotoxic effects on human liver cancer HepG2 cells.

In recent years, cellular immunotherapy has served an important role in the combined treatment of hepatocellular carcinoma. The possibility of specific cell therapies for the treatment of solid tumours has been further explored following the success of chimeric antigen receptor (CAR)-T cell therapy in the treatment of haematological tumours. The present study aimed to evaluate the specificity and efficiency of c-MET-targeted CAR-NK cell immunotherapy on human liver cancer in vitro. A CAR structure that targeted and recognised a c-MET antigen was constructed. c-MET-CAR was transferred into primary NK cells using lentiviral infection. c-MET-positive HepG2 cells were used as an in vitro study model. The cytotoxicity assay results revealed that c-MET-CAR-NK cells exhibited more specific cytotoxicity for HepG2 cells with high c-MET expression compared with the lung cancer cell line H1299, which has low levels of c-MET expression. The results of the present study demonstrated that c-MET may be a specific and effective target for human liver cancer cell CAR-NK immunotherapy. Based on these results, CAR-NK cell-based immunotherapy may provide a potential biotherapeutic approach for liver cancer treatment in the future.

Effects of Cigarette Smoke on TSPO-related Mitochondrial Processes.

The 18 kDa translocator protein (TSPO) is an initiator of the mitochondrial apoptosis cascade. Cigarette smoke (CS) exposure provokes alterations in TSPO expression as well as upregulation of its related functions such as mitochondrial membrane potential (ΔψM) and reactive oxygen species generation, which are associated with cell death. In the current study, H1299 lung cancer cell line exposed to CS for various time periods (30 mins, 60 mins and 120 mins) and TSPO expression and cell death processes were studied. CS exposure for 30 mins resulted in a non-significant increase in TSPO expression by 24% (p > 0.05 vs. control). CS exposure for 60 mins and 120 mins resulted in a significant increase by 43% (p < 0.05 vs. control) and by 47% (p < 0.01 vs. control), respectively. Furthermore, TSPO-related mitochondrial functions were upregulated at the 120 mins time point following CS exposure. TSPO expression is upregulated by CS, suggesting that TSPO plays a role in cell death processes induced by CS exposure. Alterations in TSPO-related cell death processes suggest that TSPO may be involved in the tissue damage caused by CS.

The α7 nicotinic receptor silent agonist R-47 prevents and reverses paclitaxel-induced peripheral neuropathy in mice without tolerance or altering nicotine reward and withdrawal

Various antitumor drugs, including paclitaxel, frequently cause chemotherapy-induced peripheral neuropathy (CIPN) that can be sustained even after therapy has been completed. The current work was designed to evaluate R-47, an α7 nAChR silent agonist, in our mouse model of CIPN. R-47 was administered to male C57BL/6J mice prior to and during paclitaxel treatment. Additionally, we tested if R-47 would alter nicotine's reward and withdrawal effects. The H460 and A549 non-small cell lung cancer (NSCLC) cell lines were exposed to R-47 for 24–72 h, and tumor-bearing NSG mice received R-47 prior to and during paclitaxel treatment. R-47 prevents and reverses paclitaxel-induced mechanical hypersensitivity in mice in an α7 nAChR-dependent manner. No tolerance develops following repeated administration of R-47, and the drug lacks intrinsic rewarding effects. Additionally, R-47 neither changes the rewarding effect of nicotine in the Conditioned Place Preference test nor enhances mecamylamine-precipitated withdrawal. Furthermore, R-47 prevents paclitaxel-mediated loss of intraepidermal nerve fibers and morphological alterations of microglia in the spinal cord. Moreover, R-47 does not increase NSCLC cell viability, colony formation, or proliferation, and does not interfere with paclitaxel-induced growth arrest, DNA fragmentation, or apoptosis. Most importantly, R-47 does not increase the growth of A549 tumors or interfere with the antitumor activity of paclitaxel in tumor-bearing mice. These studies suggest that R-47 could be a viable and efficacious approach for the prevention and treatment of CIPN that would not interfere with the antitumor activity of paclitaxel or promote lung tumor growth.

Abstract 1114: Upregulation of ALCAM is a marker for non-small-cell lung cancer brain metastases

Abstract Non-small-cell lung cancer (NSCLC) is the most common cause of cancer-related death with still a 5-year survival rate of &amp;lt;10%. Every second patient with advanced NSCLC will develop brain metastases and in 50% of those the brain is the only site of tumor relapse (oligo-metastasis). Cancer cells need to have specific properties to access and colonize the brain microenvironment leading to the question which metastatic factors foster brain metastasis formation. Different cell adhesion molecules (CAMs) have been shown to be involved in the process of metastasis in different tumor entities. The activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein of the immunoglobulin superfamily and involved in the maintenance of cell-cell contacts and has a possible role in the “homing” of metastatic cells in distant organs. ALCAM is also involved in the leukocyte transmigration across the blood-brain barrier. Here we therefore investigated the role of ALCAM in NSCLC brain metastases formation. Our studies on patient material revealed that the ALCAM protein expression in brain metastases (n=71) is significantly increased (50.7% positive, p=0.023) compared to the primary tumor (21.7% positive, n=47). Analysis of matched pairs of primary tumors and brain metastases confirmed this observation, as in 33% a de novo ALCAM expression in the matching brain metastases could be observed. Additionally, patients with a strong ALCAM expression in either their primary tumors or brain metastases showed a significantly shortened overall survival (p=0.017 and p=0.035 respectively). In vitro analysis of the effect of ALCAM knock out in the H460 lung cancer cell line showed no effect on proliferation, migration or colony-forming behavior. However, cell adhesion was severely hampered in the knock out cells. Currently in vivo mice experiments are running. Together these findings indicate an important role for ALCAM in brain metastasis formation. Citation Format: Desiree Loreth, Justine Münsterberg, Cecile Maire, Stefan Werner, Monja Gandrass, Christian Bernreuther, Jolanthe Kropidlowski, Manfred Westphal, Stefan Steurer, Katrin Lamszus, Markus Glatzel, Klaus Pantel, Harriet Wikman. Upregulation of ALCAM is a marker for non-small-cell lung cancer brain metastases [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 1114.

Abstract 2568: Investigation of Fanconi Anemia pathway downstream genes

Abstract Introduction: A key response mechanism to DNA damage is the Fanconi Anemia repair pathway (FA), which involves homologous recombination DNA repair and is activated through mono- ubiquitination of FANCD2. FA deficiency is considered to increase the sensitivity of tumors to particular DNA-targeted agents, and may prove to be a target of cancer treatment. We hypothesize that FA deficient tumors have a low growth rate and reduced ability for DNA repair compared to FA functioning tumors. Given that genetic modifications can interfere with FA functionality, we aim to explore the association between the FA pathways and downstream genes that influence tumor growth. To date, few studies have examined gene expression associated with FA deficiency in lung cancer cells. Identification of the FA downstream genes may provide insight on DNA repair networks that impact cancer treatment. Methods: To generate FANCD2 knockdown cells, human lung cancer cell lines A549 and H1299 were transduced with FANCD2-specific short hairpin RNA expressing and puromycin-resistant lentiviral particles or control shRNA lentiviral particles. The cells were cultured in growth medium, and successful FANCD2 knockdown was confirmed by western immunoblot analysis. RNA deep sequencing was completed with Illumina RNA-Seq. We compared gene expression between knockdown FANCD2 and control samples across three cell lines and ranked significant gene expression changes, defined as a five-fold change in upregulation or downregulation. The fold change was calculated by dividing FANCD2 deficient expression by FANCD2 efficient expression. Results and discussion: 13436 genes were evaluated across three cell lines and 17 genes demonstrated gene expression change by at least 5-fold with FANCD2 knockdown in three cell lines. FANCD2 knockdown resulted in 14 downregulated genes and 3 upregulated genes. The downregulated genes RP11-618G20.1, RP5-1021I20.4, RP11-219A15.1, XXbac-BPG32J3.20, and BMS1P17 demonstrated significant expression change across three cell lines. Of the 14 downregulated genes, 13 genes had literature supporting oncogenic function. FA downstream genes confers oncogenic function. As FANCD2 is considered to promote cell proliferation, downregulation of oncogenic genes expression was expected with FANCD2 knockdown. However, the literature suggested that the 3 upregulated genes with FANCD2 knockdown also have oncogenic function. These genes may have functioning beyond the scope of carcinogenesis which may explain gene upregulation with FANCD2 knockdown. Pinpointing genes related to FA pathway deficiency may provide insight into genetic phenomena that drive cancer. Our results provide a starting point for developing targets to specific downstream genes associated with FA deficient tumors, which may prove to limit cancer progression. Further investigation is needed to determine how FANCD2 interacts with these genes to promote cell proliferation. Citation Format: Bianca Nguyen, Li Gao, Abeer Almiman, Shirley Tang, Kathleen Dotts, Miguel A. Villalona-Calero, Wenrui Duan. Investigation of Fanconi Anemia pathway downstream genes [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 2568.

Abstract 3935: PLK4 inhibition as a strategy for non-small cell lung cancer radiosensitization

Abstract Background/Purpose: Lung cancer remains the leading cause of cancer-related death. Concurrent chemoradiation is the standard of care for locally advanced non-small cell lung cancer (NSCLC). Despite advancements in treatment, local control remains an issue and survival is suboptimal, highlighting the need for novel therapeutic approaches. Polo-like kinase 4 (PLK4) is a serine/threonine kinase that controls centriole duplication. PLK4 is highly expressed in multiple malignancies including lung cancer. CFI-400945 is a specific PLK4 inhibitor currently undergoing clinical trial evaluation. As radiation causes cell death primarily via a mitotic death, we hypothesized that disruption of the mitotic machinery by inhibition of PLK4 would enhance the effects of radiation. Methods: The effects of CFI-400945 on cell survival and radiation sensitivity were examined using the clonogenic survival assay in two KRAS-mutant lung cancer cell lines (H460 and A549). Confocal microscopy was utilized to determine the effects of PLK4 inhibition on radiation-induced mitotic catastrophe (defined as the presence of 2 or more distinct nuclear lobes within a single cell). Cell cycle distribution by flow cytometry was determined after treatment with CFI-400945 and radiation. Results: Exposure of H460 and A549 lung cancer cell lines to 10nM CFI-400945 for 48h reduced clonogenic survival to 63.9% and 83.1% respectively. CFI-400945 treatment of H460 and A549 cells resulted in radiosensitization of both cell lines with dose enhancement factors (DEF) of 1.60 and 1.31. To understand the mechanism of enhanced radiosensitivity with CFI-400945 treatment the effects on radiation-induced mitotic catastrophe were determined. In both H460 and A549 cells the combination of radiation and PLK4 inhibition enhanced the proportion of cells undergoing mitotic catastrophe. The combination of CFI-400945 and radiation resulted in increased G2/M arrest and appearance of &amp;gt;4N populations by flow cytometric analysis when compared to drug or radiation treatment alone. Conclusion: PLK4 inhibition with CFI-400945 enhances the radiosensitivity of lung cancer cell lines via mitotic catastrophe and G2/M arrest and these data support additional pre-clinical testing. Citation Format: Thomas J. Hayman, Mark R. Bray, Jacqueline M. Mason, Tak W. Mak, Joseph N. Contessa. PLK4 inhibition as a strategy for non-small cell lung cancer radiosensitization [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 3935.